2019年01月, 北海道整形災害外科学会 学術奨励賞, 北海道整形災害外科学会 Society award　　　　　　　　　　　　　　　
アラーテルカウィ

2018年03月, 第37回北海道大学大学院医学研究院・大学院医学院, 医学部医学科高桑榮松奨学基金奨励賞　　　　　　　　　　　　　　　
アラーテルカウィ

2017年03月, 日本学術振興会 Travel Fellowship Award for the Japan-Brazil Malaria Research Workshop, Travel Fellowship Award for the Japan-Brazil Malaria Research Workshop　　　　　　　　　　　　　　　
アラーテルカウィ

2009年11月, 日本学術振興会外国人特別研究員, JSPS fellowship　　　　　　　　　　　　　　　
アラーテルカウィ

Cellular communication network factor 3 contributes to the pathological process of rheumatoid arthritis through promoting cell senescence and osteoclastogenesis in the joint
Taiki Tokuhiro, Gen Matsumae, Tsutomu Endo, Yuki Ogawa, Takuya Ogawa, Chen Liyile, Yoshio Nishida, Hend Alhasan, Hideyuki Kobayashi, Taku Ebata, Tomohiro Shimizu, Daisuke Takahashi, Tomohiro Onodera, Ken Kadoya, Alaa Terkawi, Norimasa Iwasaki
Journal of Autoimmunity, 2024年12月
英語, 研究論文（学術雑誌）

Chondroprotective functions of neutrophil-derived extracellular vesicles by promoting the production of secreted frizzled-related protein 5 in cartilage.
Keita Kitahara, Taku Ebata, Chen Liyile, Yoshio Nishida, Yuki Ogawa, Taiki Tokuhiro, Junki Shiota, Tatsuya Nagano, Taichi E Takasuka, Tsutomu Endo, Tomohiro Shimizu, Hend Alhasan, Tsuyoshi Asano, Daisuke Takahashi, Kentaro Homan, Tomohiro Onodera, Ken Kadoya, M Alaa Terkawi, Norimasa Iwasaki
Cell communication and signaling : CCS, 22, 1, 569, 569, 2024年11月27日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disease characterized by cartilage degradation and various degrees of inflammation in the synovium. Growing evidence highlights that neutrophil extracellular vesicles (EVs) play a protective role in arthritic joints by promoting the resolution of inflammation and the synthesis of proteoglycans in cartilage. However, this homeostatic function is dependent on the activation state of neutrophils and the surrounding environment/tissues. Hence, we explored the chondroprotective functions of neutrophil-derived EVs under different stimulation conditions and the underlying molecular mechanism. METHODS: Human blood-derived neutrophils, murine bone marrow-derived neutrophils, C-28I2 cells and primary chondrocytes were used. Neutrophils were stimulated with different cytokines, and their EVs were isolated for chondrocyte stimulation and further subjected to RNA-sequencing analysis. Two experimental murine OA models were used, and the treatment was performed by intraarticular injections. RESULTS: Conditioned medium from neutrophils stimulated with TGF-β (N-β) had the greatest inhibitory effect on the expression of catabolic factors in stimulated chondrocytes. These protective effects were not impaired when conditioned medium of N-β from AnxA1-deficient mice was used. Consistent with these results, EVs isolated from N-β significantly reduced the expression of catabolic factors in stimulated chondrocytes. Bulk RNA-seq analysis revealed that secreted frizzled-related protein 5 (SFRP5) is upregulated in N-β-EV-stimulated chondrocytes. Furthermore, recombinant SFRP5 treatment significantly reduced the expression of catabolic factors in vitro and catabolic process in experimental murine OA models. CONCLUSIONS: The current study emphasizes the potential therapeutic application of neutrophils in OA and provides new knowledge on the molecular mechanisms underlying their function.

Dynamic transcriptome analysis of osteal macrophages identifies distinct subset with senescence features in experimental osteoporosis.
Yoshio Nishida, M Alaa Terkawi, Gen Matsumae, Shunichi Yokota, Taiki Tokuhiro, Yuki Ogawa, Hotaka Ishizu, Junki Shiota, Tsutomu Endo, Hend Alhasan, Taku Ebata, Keita Kitahara, Tomohiro Shimizu, Daisuke Takahashi, Masahiko Takahata, Ken Kadoya, Norimasa Iwasaki
JCI insight, 2024年10月31日, ［国際誌］
英語, 研究論文（学術雑誌）, Given the potential fundamental function of osteal macrophages in bone pathophysiology, we study here their precise function in experimental osteoporosis. Gene profiling of osteal macrophages from ovariectomized mice demonstrated the upregulation of genes that were involved in oxidative stress, cell senescence and apoptotic process. A scRNA-seq analysis revealed that osteal macrophages were heterogenously clustered into 6 subsets that expressed proliferative, inflammatory, anti-inflammatory and efferocytosis gene signatures. Importantly, postmenopausal mice exhibited a 20-fold increase in subset-3 that showed a typical gene signature of cell senescence and inflammation. These findings suggest that the decreased production of estrogen due to postmenopause altered the osteal macrophages subsets, resulting in a shift toward cell senescence and inflammatory conditions in the bone microenvironment. Furthermore, adoptive macrophage transfer onto calvarial bone was performed and mice that received oxidative-stressed macrophages exhibited greater osteolytic lesions than control macrophages, suggesting the role of these cells in development of inflammaging in bone microenvironment. Consistently, depletion of senescent cells and oxidative-stressed macrophages subset alleviated the excessive bone loss in postmenopausal mice. Our data provided a new insight into the pathogenesis of osteoporosis and sheds light on a new therapeutic approach for the treatment/prevention of postmenopausal osteoporosis.

Midterm Results of Severe Hip Dysplasia after Using a Cementless Acetabular Component with Bulk Bone Graft in Total Hip Arthroplasty: A Minimum Five-Year Follow-Up Study.
Takuya Konno, Tomohiro Shimizu, Masahiro Inoue, Takeshi Masuda, Mohamad Alaa Terkawi, Norimasa Iwasaki, Daisuke Takahashi
Bioengineering (Basel, Switzerland), 11, 8, 2024年08月19日, ［国際誌］
英語, 研究論文（学術雑誌）, In patients with severe hip dysplasia, total hip arthroplasty (THA) using bulk bone graft (BBG) enhances anatomic cup positioning and provides early structural support. This study assesses the mid-term outcomes of THA with BBG in patients with over 50% graft bone coverage. Among 1951 patients who underwent THA between 2003 and 2007, 183 had BBG. After excluding early dropouts and infections, 151 patients remained. They were classified into uncovered (<50% coverage, 79 patients) and covered (>50% coverage, 72 patients) groups. The efficacy of cup fixation was compared between these groups. After ten years, the survival rate for not needing THA revision was 98% in the uncovered group and 100% in the covered group, while the rate for radiographic stability was 93% versus 99%, respectively. Although the cutoff value for the uncovered portion could not be clarified in this study, the mid-term results for 50% to approximately 70% uncovered were comparable to those for 50% or lesser, which have previously been expected to perform well. Recently, biomechanically advantageous bone grafting techniques have been identified, and based on the results of this study, it may be possible to expand the indications for THA with bone grafting for developmental dysplasia of the hip.

Ossification of the posterior longitudinal ligament is linked to heterotopic ossification of the ankle/foot tendons.
Tsutomu Endo, Masahiko Takahata, Yoshinao Koike, Ryo Fujita, Daisuke Yoneoka, Masahiro Kanayama, Ken Kadoya, Tomoka Hasegawa, Mohamad Alaa Terkawi, Katsuhisa Yamada, Hideki Sudo, Taku Ebata, Misaki Ishii, Norimasa Iwasaki
Journal of bone and mineral metabolism, 2024年06月08日, ［国内誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: Systemic osteogenesis has been speculated to be involved in the pathogenesis of ossification of the posterior longitudinal ligament (OPLL). Our purpose was to compare the radiologic prevalence and severity of heterotopic ossification in foot tendons of Japanese patients with OPLL and to determine their association with systemic heterotopic ossification. MATERIALS AND METHODS: Clinical and radiographic data of 114 patients with OPLL were collected from 2020 to 2022. Control data were extracted from a medical database of 362 patients with ankle radiographs. Achilles and plantar tendon ossification were classified as grades 0-4, and the presence of osteophytes at five sites in the foot/ankle joint was assessed by radiography. Factors associated with the presence and severity of each ossification were evaluated by multivariable logistic regression and linear regression analysis. RESULTS: The prevalence of Achilles and plantar tendon ossification (grade ≥ 2) was 4.0-5.5 times higher in patients with OPLL (40-56%) than in the controls (10-11%). The presence of Achilles tendon ossification was associated with OPLL, age, and coexisting plantar tendon ossification, and was most strongly associated with OPLL (standardized regression coefficient, 0.79; 95% confidence interval, 1.34-2.38). The severity of Achilles and plantar tendon ossification was associated with the severity of ossification of the entire spinal ligament. CONCLUSIONS: The strong association of foot tendon ossification with OPLL suggests that patients with OPLL have a systemic osteogenesis background. These findings will provide a basis for exploring new treatment strategies for OPLL, including control of metabolic abnormalities.

Involvement of Siglec-15 in regulating RAP1/RAC signaling in cytoskeletal remodeling in osteoclasts mediated by macrophage colony-stimulating factor.
Hideyuki Kobayashi, M Alaa Terkawi, Masahiro Ota, Tomoka Hasegawa, Tomomaya Yamamoto, Tomohiro Shimizu, Dai Sato, Ryo Fujita, Toshifumi Murakami, Norio Amizuka, Norimasa Iwasaki, Masahiko Takahata
Bone research, 12, 1, 35, 35, 2024年06月07日, ［国際誌］
英語, 研究論文（学術雑誌）, DNAX-associated protein 12 kD size (DAP12) is a dominant immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptor that activates costimulatory signals essential for osteoclastogenesis. Although several DAP12-associated receptors (DARs) have been identified in osteoclasts, including triggering receptor expressed on myeloid cells 2 (TREM-2), C-type lectin member 5 A (CLEC5A), and sialic acid-binding Ig-like lectin (Siglec)-15, their precise role in the development of osteoclasts and bone remodeling remain poorly understood. In this study, mice deficient in Trem-2, Clec5a, Siglec-15 were generated. In addition, mice double deficient in these DAR genes and FcεRI gamma chain (FcR)γ, an alternative ITAM adaptor to DAP12, were generated. Bone mass analysis was conducted on all mice. Notably, Siglec-15 deficient mice and Siglec-15/FcRγ double deficient mice exhibited mild and severe osteopetrosis respectively. In contrast, other DAR deficient mice showed normal bone phenotype. Likewise, osteoclasts from Siglec-15 deficient mice failed to form an actin ring, suggesting that Siglec-15 promotes bone resorption principally by modulating the cytoskeletal organization of osteoclasts. Furthermore, biochemical analysis revealed that Sigelc-15 activates macrophage colony-stimulating factor (M-CSF)-induced Ras-associated protein-1 (RAP1)/Ras-related C3 botulinum toxin substrate 1 (Rac1) pathway through formation of a complex with p130CAS and CrkII, leading to cytoskeletal remodeling of osteoclasts. Our data provide genetic and biochemical evidence that Siglec-15 facilitates M-CSF-induced cytoskeletal remodeling of the osteoclasts.

Molecular and Cellular Mechanisms of Bone and Cartilage Diseases
M. Alaa Terkawi
Biomedicines, 2023年09月08日
研究論文（学術雑誌）

High whole-body bone mineral density in ossification of the posterior longitudinal ligament.
Ryo Fujita, Tsutomu Endo, Masahiko Takahata, Yoshinao Koike, Daisuke Yoneoka, Ryota Suzuki, Masaru Tanaka, Katsuhisa Yamada, Hideki Sudo, Tomoka Hasegawa, Mohamad Alaa Terkawi, Ken Kadoya, Norimasa Iwasaki
The spine journal : official journal of the North American Spine Society, 23, 10, 1461, 1470, 2023年07月10日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND CONTEXT: Recent studies suggest that ossification of the posterior longitudinal ligament (OPLL) is exacerbated by systemic metabolic disturbances, including obesity. However, although an increase in bone mineral density (BMD) measured at the lumbar spine has been reported in patients with OPLL, no studies have investigated the systemic BMD of patients with OPLL in detail. PURPOSE: We investigated whether patients with OPLL develop increased whole-body BMD. STUDY DESIGN: Single institution cross-sectional study. PATIENT SAMPLE: Data were collected from Japanese patients with symptomatic OPLL (OPLL [+]; n=99). Control data (OPLL [-]; n=226) without spinal ligament ossification were collected from patients who underwent spinal decompression, spinal fusion, or hip replacement surgery. OUTCOME MEASURES: Demographic data, including age, body mass index (BMI), comorbidities, history of treatment for osteoporosis, and history of vertebral and nonvertebral fractures, was obtained from all participants. In addition, whole-body BMD, including the lumbar spine, thoracic spine, femoral neck, skull, ribs, entire upper extremity, entire lower extremity, and pelvis, were measured in all participants using whole-body dual-energy X-ray absorptiometry. METHODS: Patient data were collected from 2018 to 2022. All participants were categorized based on sex, age (middle-aged [<70 years] and older adults [≥70 years]), and OPLL type (localized OPLL [OPLL only in the cervical spine], diffuse OPLL [OPLL in regions including the thoracic spine]), and OPLL [-]) and each parameter was compared. The factors associated with whole-body BMD were evaluated via multivariable linear regression analysis. RESULTS: Compared with the OPLL (-) group, the OPLL (+) group of older women had significantly higher BMD in all body parts (p<.01), and the OPLL (+) group of older men had significantly higher BMD in all body parts except the ribs, forearm, and skull (p<.01). The factors associated with increased BMD of both the femoral neck (load-bearing bone) and skull (nonload-bearing bone) were age, BMI, and coexisting diffuse OPLL in women and BMI and coexisting localized OPLL in men. CONCLUSIONS: Patients with OPLL have increased whole-body BMD regardless of sex, indicating that it is not simply due to load-bearing from obesity. These findings suggested that OPLL is associated with a systemic pathology.

Dyslipidemia as a novel risk for the development of symptomatic ossification of the posterior longitudinal ligament.
Shotaro Fukada, Tsutomu Endo, Masahiko Takahata, Masahiro Kanayama, Yoshinao Koike, Ryo Fujita, Ryota Suzuki, Toshifumi Murakami, Tomoka Hasegawa, Mohamad Alaa Terkawi, Tomoyuki Hashimoto, Kastuhisa Yamada, Hideki Sudo, Ken Kadoya, Norimasa Iwasaki
The spine journal : official journal of the North American Spine Society, 23, 9, 1287, 1295, 2023年05月07日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND CONTEXT: Obesity and visceral fat have been implicated as potential factors in the pathogenesis of the ossification of the posterior longitudinal ligament (OPLL); the details of the factors involved in OPLL remain unclear. PURPOSE: We aimed to determine the association between dyslipidemia and symptomatic OPLL. STUDY DESIGN: Single institution cross-sectional study. PATIENT SAMPLE: Data were collected from Japanese patients with OPLL (n=92) who underwent whole-spine computed tomography scanning. Control data (n=246) without any spinal ligament ossification were collected from 627 Japanese participants who underwent physical examination. OUTCOME MEASURES: Baseline information and lipid parameters, including triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) from fasting blood samples were collected to assess the comorbidity of dyslipidemia. METHODS: Patient data were collected from 2020 to 2022. Patients with dyslipidemia were defined as those who were taking medication for dyslipidemia and who met one of the following criteria: TG ≥150 mg/dL, LDL-C ≥140 mg/dL, and/or HDL-C <40 mg/dL. The factors associated with OPLL development were evaluated using multivariate logistic regression analysis. RESULTS: The comorbidity of dyslipidemia in the OPLL group was more than twice that in the control group (71.7% and 35.4%, respectively). The mean body mass index (BMI) of the OPLL group was significantly higher than that of the control group (27.2 kg/m2 and 23.0 kg/m2). Multivariate logistic regression analysis revealed that dyslipidemia was associated with the development of OPLL (regression coefficient, 0.80; 95% confidence interval, 0.11-1.50). Additional risk factors included age, BMI, and diabetes mellitus. CONCLUSIONS: We demonstrated a novel association between dyslipidemia and symptomatic OPLL development using serum data. This suggests that visceral fat obesity or abnormal lipid metabolism are associated with the mechanisms of onset and exacerbation of OPLL as well as focal mechanical irritation due to being overweight.

Macrophage‐derived extracellular vesicles trigger non‐canonical pyroptosis in chondrocytes leading to cartilage catabolism in osteoarthritis
Taku Ebata, Mohamad Alaa Terkawi, Keita Kitahara, Syunichi Yokota, Junki Shiota, Yoshio Nishida, Gen Matsumae, Hend Alhasan, Masanari Hamasaki, Kazutoshi Hontani, Tomohiro Shimizu, Daisuke Takahashi, Tsutomu Endo, Tomohiro Onodera, Ken Kadoya, Norimasa Iwasaki
Arthritis & Rheumatology, 2023年03月16日, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVES: The severity of osteoarthritis and cartilage degeneration are highly correlated with the development of synovitis, which is mediated by the activity of inflammatory macrophages. A better understanding of intercellular communication between inflammatory macrophages and chondrocytes should aid in the discovery of novel therapeutic targets. Here, we explored the pathological role of inflammatory macrophage extracellular vesicles in cartilage degeneration. METHODS: Macrophages were stimulated by treatment with bacterial lipopolysaccharides to mimic the state of inflammatory macrophages and the resulting extracellular vesicles were harvested for chondrocyte stimulation in vitro and intraarticular injection in a mouse model. The stimulated chondrocytes were further subjected to RNA-seq analysis and other functional assays. The action of caspase-11 was disrupted in vitro using a specific siRNA or wedelolactone, and in experimental OA-murine models by the intraarticular injection of wedelolactone. RESULTS: Stimulated chondrocytes exhibited a significant elevation in the expression of chondrocyte catabolic factors. Consistent with these results, RNA-seq analyses of stimulated chondrocytes indicated that upregulated genes are mainly categorized into apoptotic process and TNF-signaling pathway which suggests the induction of apoptotic process. Moreover, these chondrocytes exhibited a significant elevation in the expression of pyroptosis-related molecules that were correlated with the expression of chondrocyte catabolic factors. The disruption of caspase-11 significantly alleviated pyroptotic and catabolic processes in stimulated chondrocytes and the pathological changes in collagenase-and joint instability-induced OA models. CONCLUSIONS: Our results provide a new insight into the pathological mechanisms of OA and suggest that non-canonical pyroptosis in chondrocytes represents an attractive therapeutic target for future treatment.

Comparison of re-revision rate and radiological outcomes between Kerboull-type plate and metal mesh with impaction bone grafting for revision total hip arthroplasty.
Hotaka Ishizu, Tomohiro Shimizu, Fumio Sasazawa, Daisuke Takahashi, Mohamad Alaa Terkawi, Kaname Takahashi, Yusuke Ohashi, Masahiro Kanayama, Norimasa Iwasaki
BMC musculoskeletal disorders, 24, 1, 134, 134, 2023年02月20日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: This study compared the re-revision rate and radiographic outcomes of revision total hip arthroplasty (THA) using a Kerboull-type acetabular reinforcement device (KT plate) with bulk structural allograft and metal mesh with impaction bone grafting (IBG). METHODS: Ninety-one hips of 81 patients underwent revision THA for American Academy of Orthopedic Surgeons (AAOS) classification type III defects from 2008 to 2018. Of these, seven hips of five patients and 15 hips of 13 patients were excluded due to insufficient follow-up information (< 24 months) and large bone defects with a vertical defect height ≥ 60 mm, respectively. The current study compared the survival and radiographic parameters of 45 hips of 41 patients using a KT plate (KT group) and 24 hips of 24 patients using a metal mesh with IBG (mesh group). RESULTS: Eleven hips (24.4%) in the KT group and 1 hip (4.2%) in the mesh group exhibited radiological failure. Moreover, 8 hips in the KT group (17.0%) required a re-revision THA, while none of the patients in the mesh group required a re-revision. The survival rate with radiographic failure as the endpoint in the mesh group was significantly higher than that in the KT group (100% vs 86.7% at 1-year and 95.8% vs 80.0% at 5-years, respectively; p = 0.032). On multivariable analysis evaluating factors associated with radiographic failure, there were no significant associations with any radiographic measurement. Of the 11 hips with radiographic failure, 1 (11.1%), 3 (12.5%), and 7 (58.3%) hips were of Kawanabe classification stages 2, 3, and 4, respectively. CONCLUSIONS: The findings of this study suggest that revision THA using KT plates with bulk structure allografts could provide poorer clinical outcomes than revision THA using a metal mesh with IBG. Although revision THA using KT plates with bulk structural allografts could set the true hip center, there is no association between a high hip center and clinical outcomes. The relationship between the position of the KT plate and the host bone might be considered more carefully.

Lumbar ossification of the ligamentum flavum reflects a strong ossification tendency of the entire spinal ligament.
Kazuha Nakabachi, Tsutomu Endo, Masahiko Takahata, Ryo Fujita, Yoshinao Koike, Ryota Suzuki, Yuichi Hasegawa, Toshifumi Murakami, Katsuhisa Yamada, Hideki Sudo, Mohamad Alaa Terkawi, Ken Kadoya, Norimasa Iwasaki
Scientific reports, 13, 1, 638, 638, 2023年01月12日, ［国際誌］
英語, 研究論文（学術雑誌）, Patients with ossification of the ligamentum flavum (OLF) in the lumbar spine may be at high risk of developing concomitant ossification of the entire spinal ligament, but the etiology remains unclear. We investigated the propensity for spinal ligament ossification in asymptomatic subjects with lumbar OLF using the data of 595 Japanese individuals receiving medical check-ups, including computed tomography (CT) scanning. The severity of OLF (total number of intervertebral segments with OLF) of the entire spine on CT was quantified using an OLF index. Subjects with OLF were grouped according to this index: localized OLF (n = 138), intermediate OLF (n = 70), and extensive OLF (n = 31). The proportion of subjects with lumbar OLF increased with increasing OLF index (localized 13.7%, intermediate 41.4%, and extensive 70.9%). Multiple regression analysis found that lumbar OLF index was associated with thoracic OLF index, and co-existence of ossification of the posterior longitudinal ligament (OPLL) of the thoracic and lumbar spine. This study showed that subjects with more multilevel lumbar OLF were more likely to develop multilevel thoracic OLF and to have coexisting OPLL. Patients with lumbar OLF may be a distinctive subgroup with a strong tendency to ossification of the entire spinal ligament.

Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma.
Yasuhiro Yamamoto, Ken Kadoya, Mohamad Alaa Terkawi, Takeshi Endo, Kohtarou Konno, Masahiko Watanabe, Satoshi Ichihara, Akira Hara, Kazuo Kaneko, Norimasa Iwasaki, Muneaki Ishijima
Life science alliance, 5, 10, 2022年10月, ［国際誌］
英語, 研究論文（学術雑誌）, Although inflammation is indispensable for the repair process in Wallerian degeneration (WD), the role of neutrophils in the WD repair process remains unclear. After peripheral nerve injury, neutrophils accumulate at the epineurium but not the parenchyma in the WD region because of the blood-nerve barrier. An increase or decrease in the number of neutrophils delayed or promoted macrophage infiltration from the epineurium into the parenchyma and the repair process in WD. Abundant neutrophil extracellular traps (NETs) were formed around neutrophils, and its inhibition dramatically increased macrophage infiltration into the parenchyma. Furthermore, inhibition of either MIF or its receptor, CXCR4, in neutrophils decreased NET formation, resulting in enhanced macrophage infiltration into the parenchyma. Moreover, inhibiting MIF for just 2 h after peripheral nerve injury promoted the repair process. These findings indicate that neutrophils delay the repair process in WD from outside the parenchyma by inhibiting macrophage infiltration via NET formation and that neutrophils, NETs, MIF, and CXCR4 are therapeutic targets for peripheral nerve regeneration.

Inhibitory role of Annexin A1 in pathological bone resorption and its therapeutic implications in periprosthetic osteolysis
Alhasan H, Terkawi MA, Matumae G, Ebata T, Tian Y, Shimizu T, Nishida Y, Yokota S, Garcia-Martin F, Abd Elwakil MM, Takahashi D, Younis MA, Harashima H, Kadoya K, Iwasaki N
Nature Communications, 13, 1, 3919, 3919, 2022年06月, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, There is currently no therapy available for periprosthetic osteolysis, the most common cause of arthroplasty failure. Here, the role of AnxA1 in periprosthetic osteolysis and potential therapeutics were investigated. Reducing the expression of AnxA1 in calvarial tissue was found to be associated with increased osteolytic lesions and the osteolytic lesions induced by debris implantation were more severe in AnxA1-defecient mice than in wild-type mice. AnxA1 inhibits the differentiation of osteoclasts through suppressing NFκB signaling and promoting the PPAR-γ pathway. Administration of N-terminal-AnxA1 (Ac2-26 peptide) onto calvariae significantly reduced osteolytic lesions triggered by wear debris. These therapeutic effects were abrogated in mice that had received the PPAR-γ antagonist, suggesting that the AnxA1/PPAR-γ axis has an inhibitory role in osteolysis. The administration of Ac2-26 suppressed osteolysis induced by TNF-α and RANKL injections in mice. These findings indicate that AnxA1 is a potential therapeutic agent for the treatment of periprosthetic osteolysis.

Low-Grade Inflammation in the Pathogenesis of Osteoarthritis: Cellular and Molecular Mechanisms and Strategies for Future Therapeutic Intervention
M Alaa Terkawi, Taku Ebata, Shunichi Yokota, Daisuke Takahashi, Tsutomu Endo, Gen Matsumae, Tomohiro Shimizu, Ken Kadoya, Norimasa Iwasaki
Biomedicines, 10, 5, 2022年05月10日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Osteoarthritis (OA) is a musculoskeletal disease characterized by cartilage degeneration and stiffness, with chronic pain in the affected joint. It has been proposed that OA progression is associated with the development of low-grade inflammation (LGI) in the joint. In support of this principle, LGI is now recognized as the major contributor to the pathogenesis of obesity, aging, and metabolic syndromes, which have been documented as among the most significant risk factors for developing OA. These discoveries have led to a new definition of the disease, and OA has recently been recognized as a low-grade inflammatory disease of the joint. Damage-associated molecular patterns (DAMPs)/alarmin molecules, the major cellular components that facilitate the interplay between cells in the cartilage and synovium, activate various molecular pathways involved in the initiation and maintenance of LGI in the joint, which, in turn, drives OA progression. A better understanding of the pathological mechanisms initiated by LGI in the joint represents a decisive step toward discovering therapeutic strategies for the treatment of OA. Recent findings and discoveries regarding the involvement of LGI mediated by DAMPs in OA pathogenesis are discussed. Modulating communication between cells in the joint to decrease inflammation represents an attractive approach for the treatment of OA.

IL4 stimulated macrophages promote axon regeneration after peripheral nerve injury by secreting uPA to stimulate uPAR upregulated in injured axons.
Alaa Terkawi
Cellular and molecular life sciences : CMLS, 79, 6, 289, 289, 2022年05月10日, ［国際誌］
英語, 研究論文（学術雑誌）, Accumulating evidences suggest that M2 macrophages are involved with repair processes in the nervous system. However, whether M2 macrophages can promote axon regeneration by directly stimulating axons nor its precise molecular mechanism remains elusive. Here, the current study demonstrated that typical M2 macrophages, which were generated by IL4 simulation, had the capacity to stimulate axonal growth by their direct effect on axons and that the graft of IL4 stimulated macrophages into the region of Wallerian degeneration enhanced axon regeneration and improved functional recovery after PNI. Importantly, uPA (urokinase plasminogen activator)-uPA receptor (uPAR) was identified as the central axis underlying the axon regeneration effect of IL4 stimulated macrophages. IL4 stimulated macrophages secreted uPA, and its inhibition abolished their axon regeneration effect. Injured but not intact axons expressed uPAR to be sensitive to uPA. These results unveil a cellular and molecular mechanism underlying the macrophage related axon regeneration and provide a basis of a novel therapy for PNI.

Finite Element Analysis of Optimal Positioning of Femoral Osteotomy in Total Hip Arthroplasty With Subtrochanteric Shortening.
Daisuke Takahashi, Yoshihiro Noyama, Tomohiro Shimizu, Mohamad Alaa Terkawi, Norimasa Iwasaki
Arthroplasty today, 14, 105, 109, 2022年04月, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Total hip arthroplasty with femoral shortening is frequently recommended for patients with high hip dislocation. However, the possibility of postoperative rotational deviation of the stem presents a challenge for surgeons. We aimed to determine the optimal position for osteotomy in total hip arthroplasty under full weight-bearing and turning torque by using finite element analysis. Methods: Four models of femoral osteotomy with 30-mm transverse shortening at 30% (model 30), 40% (model 40), 50% (model 50), and 60% (model 60) from the proximal end of the full length of the Exeter stem were constructed. Using finite element analysis, the constructs were first analyzed under an axial load of 1500 N and then with an added torsional load of 10°. Results: The analyses under torsional loading conditions revealed that the maximum von Mises stress on the stem in each model occurred at the proximal end of the distal fragment and the distal side of the stem. The maximum stress values at the stem were 819 MPa (model 30), 825 MPa (model 40), 916 MPa (model 50), and 944 MPa (model 60). The maximum stress values at the osteotomy site of the medullary cavity side of the distal bone fragment were 761 MPa (model 30), 165 MPa (model 40), 187 MPa (model 50), and 414 MPa (model 60). Conclusions: The osteotomy level should be around the proximal 40% of the full length of the Exeter stem, which is most suitable for rotation stability in the early postoperative period.

Inflammasome activation in the hip synovium of rapidly destructive coxopathy patients and its relationship with the development of synovitis and bone loss
Shunichi Yokota, Tomohiro Shimizu, Gen Matsumae, Taku Ebata, Hend Alhasan, Daisuke Takahashi, Mohamad Alaa Terkawi, Norimasa Iwasaki
The American Journal of Pathology, 192, 5, 794, 804, Elsevier BV, 2022年03月, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Rapidly destructive coxopathy (RDC), a rare disease of unknown etiology, is characterized by the rapid destruction of the hip joint. In the current study, the potential involvement of inflammasome signaling in the progression of RDC was investigated. Histopathologic changes and the gene expression of inflammasome activation markers in hip synovial tissues collected from patients with RDC were evaluated and compared with those of osteoarthritis and osteonecrosis of the femoral head patients. The synovial tissues of patients with RDC exhibited remarkable increases in the number of infiltrated macrophages and osteoclasts, and the expression of inflammasome activation markers was also increased compared with those of osteoarthritis and osteonecrosis of the femoral head patients. To further understand the histopathologic changes in the joint, a co-culture model of macrophages and synoviocytes that mimicked the joint environment was developed. Remarkably, the gene expression levels of NLRP3, GSDMD, IL1B, TNFA, ADMTS4, ADMTS5, MMP3, MMP9, and RANKL were significantly elevated in the synoviocytes that were co-cultured with activated THP-1 macrophages, suggesting the association between synovitis and inflammasome activation. Consistent with these findings, osteoclast precursor cells that were co-cultured with stimulated synoviocytes exhibited an increased number of tartrate-resistant acid phosphatase-positive cells, compared with cells that were co-cultured with non-stimulated synoviocytes. These findings suggest that the activation of inflammasome signaling in the synovium results in an increase in local inflammation and osteoclastogenesis, thus leading to the rapid bone destruction in RDC.

Interplay between Inflammation and Pathological Bone Resorption: Insights into Recent Mechanisms and Pathways in Related Diseases for Future Perspectives
M A Terkawi, G Matsumae, T Shimizu, D Takahashi, K Kadoya, N Iwasaki
Int. J. Mol. Sci. 2022, 23(3), 1786; https://doi.org/10.3390/ijms23031786, 23, 3, 2022年02月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Bone is a mineralized and elastic connective tissue that provides fundamental functions in the human body, including mechanical support to the muscles and joints, protection of vital organs and storage of minerals. Bone is a metabolically active organ that undergoes continuous remodeling processes to maintain its architecture, shape, and function throughout life. One of the most important medical discoveries of recent decades has been that the immune system is involved in bone remodeling. Indeed, chronic inflammation has been recognized as the most significant factor influencing bone homeostasis, causing a shift in the bone remodeling process toward pathological bone resorption. Bone osteolytic diseases typified by excessive bone resorption account for one of the greatest causes of disability worldwide, with significant economic and public health burdens. From this perspective, we discuss the recent findings and discoveries highlighting the cellular and molecular mechanisms that regulate this process in the bone microenvironment, in addition to the current therapeutic strategies for the treatment of osteolytic bone diseases.

Mature but not developing Schwann cells promote axon regeneration after peripheral nerve injury.
Alaa Terkawi
NPJ Regenerative medicine, 2022年01月28日, ［査読有り］
研究論文（学術雑誌）, Since Schwann cells (SCs) support axonal growth at development as well as after peripheral nerve injury (PNI), developing SCs might be able to promote axon regeneration after PNI. The purpose of the current study was to elucidate the capability of developing SCs to induce axon regeneration after PNI. SC precursors (SCPs), immature SCs (ISCs), repair SCs (RSCs) from injured nerves, and non-RSCs from intact nerves were tested by grafting into acellular region of rat sciatic nerve with crush injury. Both of developing SCs completely failed to support axon regeneration, whereas both of mature SCs, especially RSCs, induced axon regeneration. Further, RSCs but not SCPs promoted neurite outgrowth of adult dorsal root ganglion neurons. Transcriptome analysis revealed that the gene expression profiles were distinctly different between RSCs and SCPs. These findings indicate that developing SCs are markedly different from mature SCs in terms of functional and molecular aspects and that RSC is a viable candidate for regenerative cell therapy for PNI.

Evaluation of biological responses to micro-particles derived from a double network hydrogel
Gen Matsumae, Alaa Terkawi, Takayuki Nonoyama, Takayuki Kurokawa, Daisuke Takahashi, Tomohiro Shimizu, Ken Kadoya, Jian Ping Gong, Kazunori Yasuda, Norimasa Iwasaki
Biomaterials Science, Royal Society of Chemistry ({RSC}), 2022年, ［責任著者］
英語, 研究論文（学術雑誌）, The double network hydrogels (DN gels) composed of poly (2-acrylamido-2-methyl propanesulfonic acid) (PAMPS) as the brittle first network and poly (N,N-dimethylacrylamide) (PDMA) as the ductile second network have been proven...

Determination of optimal concentration of vitamin E in polyethylene liners for producing minimal biological response to prosthetic wear debris
Matsumae G, Kida H, Takahashi D, Shimizu T, Ebata T, Yokota S, Alhasan H, Aly MK, Yutani T, Uetsuki K, Terkawi MA, Iwasaki N
Journal of Biomedical Materials Research: Part B - Applied Biomaterials. Accepted, 110, 7, 1587, 1593, 2022年01月, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The introduction of vitamin E-blended ultra-high molecular weight polyethylene (VE-UHMWPE) for use in prosthetic components of hip implants has resulted in the production of implants that have excellent mechanical properties and substantially less adverse cellular responses. Given the importance of a biological response to wear in the survival of a prosthesis, we generated wear debris from UHMWPE that had been prepared with different concentrations of vitamin E of 0.1, 0.3, 0.5, and 1% and evaluated their biological reaction in vitro and in vivo. All types of VE-UHMWPE debris promoted a significantly lower expression of Tnf-α in murine peritoneal macrophages than that induced by conventional UHMWPE debris. However, levels of Tnf-α were not significantly different among the macrophages that were stimulated with VE-UHMWPE wear at the concentrations tested. The ability of wear debris to induce inflammatory osteolysis was assessed in a mouse calvarial osteolysis model. The expressions of Tnf-α, Il-6, and Rankl in granulomatous tissue formed around the wear debris were significantly reduced in mice that had been implanted with 0.3%VE-UHMWPE debris as compared to the corresponding values for mice that had been implanted with UHMWPE debris. Consistent with this finding, 0.3%VE-UHMWPE debris showed the lowest osteolytic activity, as evidenced by the reduced bone resorption area, the degree of infiltration of inflammatory cells and the TRAP staining area. Our results suggested that a 0.3% vitamin E concentration is the most appropriate concentration for use in prosthetic components with a reduced adverse cellular response for prolonging the life-span of the implant.

末梢神経損傷後早期免疫反応の詳細 好中球由来MIFによる修復機転の阻害
山本 康弘, 角家 健, 市原 理司, 原 章, Terkawi Alaa, 今野 幸太郎, 渡辺 雅彦, 岩崎 倫政, 金子 和夫, 石島 旨章
末梢神経, 32, 2, 268, 268, 日本末梢神経学会, 2021年12月
日本語

炎症性マクロファージ由来細胞外小胞を介した軟骨変性メカニズムの解明
江畑 拓, Terkawi Alaa, 松前 元, 横田 隼一, Alhasan Hend, 清水 智弘, 高橋 大介, 本谷 和俊, 小野寺 智洋, 角家 健, 岩崎 倫政
日本整形外科学会雑誌, 95, 8, S1520, S1520, (公社)日本整形外科学会, 2021年08月
日本語

関節リウマチの局所骨溶解に関与するCCN3の機能解析
松前 元, Alaa Terkawi, 江畑 拓, Hend Alhasan, 横田 隼一, 清水 智弘, 高橋 大介, 角家 健, 岩崎 倫政
日本整形外科学会雑誌, 95, 8, S1649, S1649, (公社)日本整形外科学会, 2021年08月
日本語

急速破壊型股関節症の滑膜におけるインフラマソームの活性化 病態解明の研究
横田 隼一, 清水 智弘, 松前 元, 江畑 拓, Hend Alhasan, 高橋 大介, Alaa Terkawi, 岩崎 倫政
日本整形外科学会雑誌, 95, 8, S1653, S1653, (公社)日本整形外科学会, 2021年08月
日本語

PPARγシグナルを介した炎症性骨溶解制御におけるアネキシンA1(AnxA1)の機能解析
Hend Alhasan, 松前 元, 江畑 拓, 横田 隼一, 清水 智弘, 高橋 大介, Alaa Terkawi, 岩崎 倫政
日本整形外科学会雑誌, 95, 8, S1700, S1700, (公社)日本整形外科学会, 2021年08月
日本語

炎症性マクロファージ由来細胞外小胞を介した軟骨変性メカニズムの解明
江畑 拓, Terkawi Alaa, 松前 元, 横田 隼一, Alhasan Hend, 清水 智弘, 高橋 大介, 本谷 和俊, 小野寺 智洋, 角家 健, 岩崎 倫政
日本整形外科学会雑誌, 95, 8, S1520, S1520, (公社)日本整形外科学会, 2021年08月
日本語

関節リウマチの局所骨溶解に関与するCCN3の機能解析
松前 元, Alaa Terkawi, 江畑 拓, Hend Alhasan, 横田 隼一, 清水 智弘, 高橋 大介, 角家 健, 岩崎 倫政
日本整形外科学会雑誌, 95, 8, S1649, S1649, (公社)日本整形外科学会, 2021年08月
日本語

急速破壊型股関節症の滑膜におけるインフラマソームの活性化 病態解明の研究
横田 隼一, 清水 智弘, 松前 元, 江畑 拓, Hend Alhasan, 高橋 大介, Alaa Terkawi, 岩崎 倫政
日本整形外科学会雑誌, 95, 8, S1653, S1653, (公社)日本整形外科学会, 2021年08月
日本語

PPARγシグナルを介した炎症性骨溶解制御におけるアネキシンA1(AnxA1)の機能解析
Hend Alhasan, 松前 元, 江畑 拓, 横田 隼一, 清水 智弘, 高橋 大介, Alaa Terkawi, 岩崎 倫政
日本整形外科学会雑誌, 95, 8, S1700, S1700, (公社)日本整形外科学会, 2021年08月
日本語

Cardiotrophin like cytokine factor 1 (CLCF1) alleviates bone loss in osteoporosis mouse models by suppressing osteoclast differentiation through activating interferon signaling and repressing the nuclear factor-κB signaling pathway
Shunichi Yokota, Gen Matsumae, Tomohiro Shimizu, Tomoka Hasegawa, Taku Ebata, Daisuke Takahashi, Cai Heguo, Yuan Tian, Hend Alhasan, Masahiko Takahata, Ken Kadoya, Mohamad Alaa Terkawi, Norimasa Iwasaki
Bone, 153, 116140, 116140, Elsevier {BV}, 2021年08月, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, A growing body of evidence suggests that immune factors that regulate osteoclast differentiation and bone resorption might be promising therapeutic agents for the treatment of osteoporosis. The expression of CLCF1, an immune cell-derived molecule, has been reported to be reduced in patients with postmenopausal osteoporosis. This suggests that it may be involved in bone remodeling. Thus, we explored the functional role of CLCF1 in osteoclastogenesis and bone loss associated with osteoporosis. Surprisingly, the administration of recombinant CLCF1 repressed excessive bone loss in ovariectomized mice and prevented RANKL-induced bone loss in calvarial mouse model. Likewise, the addition of recombinant CLCF1 to RANKL-stimulated monocytes resulted in a significant suppression in the number of differentiated osteoclasts with small resorption areas being observed on dentine slices in vitro. At the same dosage, CLCF1 did not exhibit any detectable negative effects on the differentiation of osteoblasts. Mechanistically, the inhibition of osteoclast differentiation by the CLCF1 treatment appears to be related to the activation of interferon signaling (IFN) and the suppression of the NF-κB signaling pathway. Interestingly, the expression of the main components of IFN-signaling namely, STAT1 and IRF1, was detected in macrophages as early as 1 h after stimulation with CLCF1. Consistent with these results, the blockade of STAT1 in macrophages abolished the inhibitory effect of CLCF1 on osteoclast differentiation in vitro. These collective findings point to a novel immunoregulatory function of CLCF1 in bone remodeling and highlight it as a potentially useful therapeutic agent for the treatment of osteoporosis.

Risks vs. benefits of switching therapy in patients with postmenopausal osteoporosis.
Hotaka Ishizu, Kosuke Arita, Mohamad Alaa Terkawi, Tomohiro Shimizu, Norimasa Iwasaki
Expert review of endocrinology & metabolism, 16, 5, 1, 12, 2021年07月26日, ［国際誌］
英語, 研究論文（学術雑誌）, Introduction: Osteoporosis is characterized by the fragility of bones, leading to fractures and, consequently, the deterioration of functional capacity and quality of life. Postmenopausal women, in particular, are prone to osteoporosis and often require anti-osteoporosis treatment. In the last few decades, various anti-osteoporosis drugs have been approved for clinical use. In an aging society, osteoporosis cannot be treated using a single agent; therefore, switching therapy is an important treatment strategy.Areas covered: This review covers switching therapy in patients with postmenopausal osteoporosis. It's extremely important to understand the characteristics of each drug including; limitations on the duration of use, side effects due to long-term use (such as atypical femur fracture and osteonecrosis of the jaw) or discontinuation (such as rebound phenomenon), compliance, and ability to prevent fractures. We review and summarize the risks and benefits of switching therapy.Expert opinion: When switching therapy, the order of drug administration is important. Routine monitoring should be continued after switching treatments. We recommend first using osteoanabolic agents in postmenopausal women with severe osteoporosis. In addition, identifying predictors of the efficacy and side effects of treatment may help prevent the inappropriate use of drugs for the treatment of osteoporosis.

Targeting thymidine phosphorylase as a potential therapy for bone loss associated periprosthetic osteolysis
Gen Matsumae, Tomohiro Shimizu, Yuan Tian, Daisuke Takahashi, Taku Ebata, Hend Alhasan, Shunichi Yokota, Ken Kadoya, Mohamad Alaa Terkawi, Norimasa Iwasaki
Bioengineering & Translational Medicine, 6, 3, e10232, Wiley, 2021年05月19日, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Macrophages are generally thought to play a key role in the pathogenesis of aseptic loosening through initiating periprosthetic inflammation and pathological bone resorption. The aim of this study was to identify macrophage-derived factors that promote osteoclast differentiation and periprosthetic bone destruction. To achieve this, we examined the effects of 12 macrophage-derived factors that were identified by RNA-seq analysis of stimulated macrophages on osteoclast differentiation. Surprisingly, thymidine phosphorylase (TYMP) was found to trigger significant number of osteoclasts that exhibited resorbing activities on dentine slices. Functionally, TYMP knockdown reduced the number of osteoclasts in macrophages that had been stimulated with polyethylene debris. TYMP were detected in serum and synovial tissues of patients that had been diagnosed with aseptic loosening. Moreover, the administration of TYMP onto calvariae of mice induced pathological bone resorption that was accompanied by an excessive infiltration of inflammatory cells and osteoclasts. The RNA-seq for TYMP-induced-osteoclasts was then performed in an effort to understand action mode of TYMP. TYMP stimulation appeared to activate the tyrosine kinase FYN signaling associated with osteoclast formation. Oral administration of saracatinib, a FYN kinase inhibitor, significantly suppressed formation of bone osteolytic lesions in a polyethylene debris-induced osteolysis model. Our findings highlight a novel molecular target for therapeutic intervention in periprosthetic osteolysis.

Flightless I is a catabolic factor of chondrocytes that promotes hypertrophy and cartilage degeneration in osteoarthritis
Taku Ebata, Mohamad Alaa Terkawi, Masanari Hamasaki, Gen Matsumae, Tomohiro Onodera, Mahmoud Khamis Aly, Shunichi Yokota, Hend Alhasan, Tomohiro Shimizu, Daisuke Takahashi, Kentaro Homan, Ken Kadoya, Norimasa Iwasaki
iScience, 24, 6, 102643, 102643, Elsevier BV, 2021年05月, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Synovial macrophages that are activated by cartilage fragments initiate synovitis, a condition that promotes hypertrophic changes in chondrocytes leading to cartilage degeneration in OA. In this study, we analyzed the molecular response of chondrocytes under condition of this type of stimulation to identify a molecular therapeutic target. Stimulated macrophages promoted hypertrophic changes in chondrocytes resulting in production of matrix-degrading enzymes of cartilage. Among the top-upregulated genes, FliI was found to be released from activated chondrocytes and exerted autocrine/paracrine effects on chondrocytes leading to an increase in expression of catabolic and hypertrophic factors. Silencing FliI in stimulated cells significantly reduced expression of catabolic and hypertrophic factors in cocultured chondrocytes. Our further results demonstrated that the FliI-TLR4-ERK1/2 axis is involved in the hypertrophic signaling of chondrocytes and catabolism of cartilage. Our findings provide a new insight into the pathogenesis of OA and identify a potentially new molecular target for diagnostics and therapeutics.

Finite element analysis of double-plate fixation using reversed locking compression-distal femoral plates for Vancouver B1 periprosthetic femoral fractures.
Daisuke Takahashi, Yoshihiro Noyama, Tsuyoshi Asano, Tomohiro Shimizu, Tohru Irie, Mohamad Alaa Terkawi, Norimasa Iwasaki
BMC musculoskeletal disorders, 22, 1, 276, 276, 2021年03月13日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Internal fixation is recommended for treating Vancouver B1 periprosthetic femoral fractures. Although several fixation procedures have been developed with high fixation stability and union rates, long-term weight-bearing constructs are still lacking. Therefore, the aim of the present study was to evaluate the stability of a double-plate procedure using reversed contralateral locking compression-distal femoral plates for fixation of Vancouver B1 periprosthetic femoral fractures under full weight-bearing. METHODS: Single- and double-plate fixation procedures for locking compression-distal femoral plates were analysed under an axial load of 1,500 N by finite element analysis and biomechanical loading tests. A vertical loading test was performed to the prosthetic head, and the displacements and strains were calculated based on load-displacement and load-strain curves generated by the static compression tests. RESULTS: The finite element analysis revealed that double-plate fixation significantly reduced stress concentration at the lateral plate place on the fracture site. Under full weight-bearing, the maximum von Mises stress in the lateral plate was 268 MPa. On the other hand, the maximum stress in the single-plating method occurred at the defect level of the femur with a maximum stress value of 1,303 MPa. The principal strains of single- and double-plate fixation were 0.63 % and 0.058 %, respectively. Consistently, in the axial loading test, the strain values at a 1,500 N loading of the single- and double-plate fixation methods were 1,274.60 ± 11.53 and 317.33 ± 8.03 (× 10- 6), respectively. CONCLUSIONS: The present study suggests that dual-plate fixation with reversed locking compression-distal femoral plates may be an excellent treatment procedure for patients with Vancouver B1 fractures, allowing for full weight-bearing in the early postoperative period.

Local Administration of Low-Dose Nerve Growth Factor Antibody Reduced Pain in a Rat Osteoarthritis Model.
Yuan Tian, Tomohiro Onodera, Mohamad Alaa Terkawi, Koji Iwasaki, Ryosuke Hishimura, Dawei Liang, Takuji Miyazaki, Norimasa Iwasaki
International journal of molecular sciences, 22, 5, 2021年03月04日, ［国際誌］
英語, 研究論文（学術雑誌）, Systemic injection of a nerve growth factor (NGF) antibody has been proven to have a significant relevance in relieving osteoarthritis (OA) pain, while its adverse effects remain a safety concern for patients. A local low-dose injection is thought to minimize adverse effects. In this study, OA was induced in an 8-week-old male Sprague-Dawley (SD) rat joint by monoiodoacetate (MIA) injection for 2 weeks, and the effect of weekly injections of low-dose (1, 10, and 100 µg) NGF antibody or saline (control) was evaluated. Behavioral tests were performed, and at the end of week 6, all rats were sacrificed and their knee joints were collected for macroscopic and histological evaluations. Results showed that 100 µg NGF antibody injection relieved pain in OA rats, as evidenced from improved weight-bearing performance but not allodynia. In contrast, no significant differences were observed in macroscopic and histological scores between rats from different groups, demonstrating that intra-articular treatment does not worsen OA progression. These results suggest that local administration yielded a low effective NGF antibody dose that may serve as an alternative approach to systemic injection for the treatment of patients with OA.

人工関節術後無菌性緩みにおけるチミジンホスホリラーゼの機能と骨吸収メカニズムの解明
松前 元, アラー・テルカウィ, 横田 隼一, 江畑 拓, ヘンド・アルハサン, 清水 智弘, 高橋 大介, 角家 健, 岩崎 倫政
北海道整形災害外科学会雑誌, 63, 139th suppl, 18, 18, 北海道整形災害外科学会, 2021年
日本語

Long-Term Clinical and Radiological Outcomes of Primary Total Hip Arthroplasty with Modular Femoral Stem in Middle-Aged Asian Patients.
Kaname Takahashi, Tomohiro Shimizu, Tsuyoshi Asano, Mohamad Alaa Terkawi, Norimasa Iwasaki, Daisuke Takahashi
The Journal of arthroplasty, 35, 12, 3650, 3655, 2020年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: There is insufficient information regarding the outcome of primary total hip arthroplasty (THA) with the modular femoral stem in middle-aged patients. This study aimed to assess long-term clinical and radiological outcomes of primary THA using the original or modified modular hip system (S-ROM) in middle-aged Asian patients. METHODS: A retrospective review identified 98 primary THAs that used a modular stem and were undertaken between 1997 and 2009 in patients younger than 58 years, for whom at least 5 years of follow-up data were available. Clinical data and radiograph assessments were reviewed to analyze differences between the original and modified modular stem groups. RESULTS: The mean patient follow-up duration was 148.3 months, and the follow-up ratio was 89.1%. The Kaplan-Meier analysis revealed that the survival rate of both stems was 98.9% at 10 years and 89.8% at 15 years. Although no statistically significant differences in the survival rate were observed between the stem designs, the original stem group had increased incidence of thigh pain compared with the modified stem group. In total, 12 and 54 hips showed change in stem alignment and osteolysis, respectively. CONCLUSION: The findings of this study show that the modular stems have a high survival rate, and results suggest positive outcomes among the Asian population over the long term. Although there were very few differences between the stem designs, the results suggest that the modified modular stem could prevent thigh pain and that selection of the implant based on the bone shape is important for THA.

摩耗軟骨片による滑膜炎が誘導する軟骨細胞新規分泌因子「Flightless I」の機能解析
江畑 拓, Terkawi Alaa, 小野寺 智洋, 木田 博朗, 松前 元, 田 園, Alhasan Hend, 濱崎 雅成, 清水 智弘, 高橋 大介, 岩崎 倫政
日本整形外科学会雑誌, 94, 8, S1690, S1690, (公社)日本整形外科学会, 2020年09月
日本語

XCL1/lymphotactin阻害による病的骨吸収抑制効果の検討
Terkawi Alaa, 田 園, 松前 元, 木田 博朗, 高橋 大介, 江畑 拓, Alhasan Hend, 清水 智弘, 岩崎 倫政
日本整形外科学会雑誌, 94, 8, S1706, S1706, (公社)日本整形外科学会, 2020年09月
日本語

人工関節術後無菌性緩みにおけるチミジンホスホリラーゼの機能と骨吸収メカニズムの解明
松前 元, Terkawi Alaa, 木田 博朗, 江畑 拓, 田 園, Alhasan Hend, 清水 智弘, 高橋 大介, 角家 健, 岩崎 倫政
日本整形外科学会雑誌, 94, 8, S1711, S1711, (公社)日本整形外科学会, 2020年09月
日本語

ビタミンE添加超高分子量ポリエチレンインプラントにおける人工関節置換術後無菌性緩みを抑制する至適濃度の検討
木田 博朗, Terkawi Alaa, 松前 元, 江畑 拓, 田 園, Alhasan Hend, 清水 智弘, 高橋 大介, 岩崎 倫政
日本整形外科学会雑誌, 94, 8, S2017, S2017, (公社)日本整形外科学会, 2020年09月
日本語

Transcriptional profiling of murine macrophages stimulated with cartilage fragments revealed a strategy for treatment of progressive osteoarthritis.
Masanari Hamasaki, Mohamad Alaa Terkawi, Tomohiro Onodera, Yuan Tian, Taku Ebata, Gen Matsumae, Hend Alhasan, Daisuke Takahashi, Norimasa Iwasaki
Scientific reports, 10, 1, 7558, 7558, 2020年05月05日, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Accumulating evidence suggests that synovitis is associated with osteoarthritic process. Macrophages play principal role in development of synovitis. Our earlier study suggests that interaction between cartilage fragments and macrophages exacerbates osteoarthritic process. However, molecular mechanisms by which cartilage fragments trigger cellular responses remain to be investigated. Therefore, the current study aims at analyzing molecular response of macrophages to cartilage fragments. To this end, we analyzed the transcriptional profiling of murine macrophages exposed to cartilage fragments by RNA sequencing. A total 153 genes were differentially upregulated, and 105 genes were down-regulated in response to cartilage fragments. Bioinformatic analysis revealed that the most significantly enriched terms of the upregulated genes included scavenger receptor activity, integrin binding activity, TNF signaling, and toll-like receptor signaling. To further confirm our results, immunohistochemical staining was performed to detected regulated molecules in synovial tissues of OA patients. In consistence with RNA-seq results, MARCO, TLR2 and ITGα5 were mainly detected in the intima lining layer of synovial tissues. Moreover, blockade of TLR2 or ITGα5 but not Marco using specific antibody significantly reduced production of TNF-α in stimulated macrophages by cartilage fragments. Our data suggested that blocking TLR2 or ITGα5 might be promising therapeutic strategy for treating progressive osteoarthritis.

Which Contributes to Meniscal Repair, the Synovium or the Meniscus? An In Vivo Rabbit Model Study With the Freeze-Thaw Method.
WooYoung Kim, Tomohiro Onodera, Eiji Kondo, Mohamad Alaa Terkawi, Kentaro Homan, Ryosuke Hishimura, Norimasa Iwasaki
The American journal of sports medicine, 48, 6, 1406, 1415, 2020年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: During meniscal tissue repair, the origin of the reparative cells of damaged meniscal tissue remains unclear. HYPOTHESIS: Comparison of the influence between meniscal and synovial tissues on meniscal repair by the in vivo freeze-thaw method would clarify the origin of meniscal reparative cells. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 48 mature Japanese white rabbits were divided into 4 groups according to the tissue (meniscal or synovial) that received freeze-thaw treatment. The meniscus of each group had a 2 mm-diameter cylindrical defect filled with alginate gel. Macroscopic and histologic evaluations of the reparative tissues were performed at 1, 3, and 6 weeks postoperatively. Additional postoperative measurements included cell density, which was the number of meniscal cells in the cut area per cut area (mm2) of meniscus; cell density ratio, which was the cell density of the sample from each group per the average cell density of the intact meniscus; and cell death rate, which was the number of cells stained by propidium iodide per the number of cells stained by Hoechst 33342 of the meniscal tissue adjacent to the defect. RESULTS: The macroscopic and histologic evaluations of the non-synovium freeze-thaw groups were significantly superior to those of the synovium freeze-thaw groups at 3 and 6 weeks postoperatively. Additionally, the meniscal cell density ratio and cell death rate in the freeze-thaw groups were significantly lower than those in the non-meniscal freeze-thaw groups at 3 and 6 weeks postoperatively. CONCLUSION: The freeze-thawed meniscus recovered few cells in its tissue even after 6 weeks. However, the defect was filled with fibrochondrocytes and proteoglycan when the synovium was intact. On the basis of these results, it is concluded that synovial cells are the primary contributors to meniscal injury repair. CLINICAL RELEVANCE: In meniscal tissue engineering, there is no consensus on the best cell source for meniscal repair. Based on this study, increasing the synovial activity and contribution should be the main objective of meniscal tissue engineering. This study can establish the foundation for future meniscal tissue engineering.

人工関節置換術後無菌性緩みに対するビタミンE添加ポリエチレンインプラントの至適濃度の検討
木田 博朗, Alaa Terkawi, 松前 元, 江畑 拓, 高橋 大介, 岩崎 倫政
北海道整形災害外科学会雑誌, 62, 138th suppl, 5, 5, 北海道整形災害外科学会, 2020年
日本語

First molecular identification of trypanosoma evansi from cattle in Syria
Megasari Marsela, Kyoko Hayashida, Alaa Terkawi, Xuenan Xuan, Chihiro Sugimoto, Junya Yamagishi
Japanese Journal of Veterinary Research, 68, 2, 117, 127, Hokkaido University, 2020年
英語, 研究論文（学術雑誌）, Trypanosoma evansi, the “surra” disease-causing agent, is a blood protozoan parasite that infects a wide range of mammalian species within an unlimited geographical region. It causes anemia, weight loss, and even death of the infected livestock that heavily affect animal husbandry. However, the full epidemiological information of T. evansi is lacking, especially in developing countries, and the risk of the disease is largely underestimated. In this study, 207 samples of blood DNA collected from Holstein Friesian crossbred cattle in the central region of Syria in May 2010 were screened for T. evansi, aiming to determine the prevalence of the parasite. T. evansi was screened by PCR targeting the internal transcribed spacer (ITS) 1 region, and 27 samples were found positive out of 207 (13%), which is relatively high considering that no clinical symptoms were observed. The ITS1 amplicons were later subjected to RoTat1.2-PCR for detection of T. evansi type A. This is the first report of molecular detection of T. evansi in Syria. Our study suggests that advanced investigations in cattle and other domestic animals are necessary in Syria.

Blockade of XCL1/Lymphotactin Ameliorates Severity of Periprosthetic Osteolysis Triggered by Polyethylene-Particles.
Yuan Tian, Mohamad Alaa Terkawi, Tomohiro Onodera, Hend Alhasan, Gen Matsumae, Daisuke Takahashi, Masanari Hamasaki, Taku Ebata, Mahmoud Khamis Aly, Hiroaki Kida, Tomohiro Shimizu, Keita Uetsuki, Ken Kadoya, Norimasa Iwasaki
Frontiers in immunology, 11, 1720, 1720, 2020年, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Periprosthetic osteolysis induced by orthopedic implant-wear particles continues to be the leading cause of arthroplasty failure in majority of patients. Release of the wear debris results in a chronic local inflammatory response typified by the recruitment of immune cells, including macrophages. The cellular mediators derived from activated macrophages favor the osteoclast-bone resorbing activity resulting in bone loss at the site of implant and loosening of the prosthetic components. Emerging evidence suggests that chemokines and their receptors are involved in the progression of periprosthetic osteolysis associated with aseptic implant loosening. In the current study, we investigated the potential role of chemokine C-motif-ligand-1 (XCL1) in the pathogenesis of inflammatory osteolysis induced by wear particles. Expressions of XCL1 and its receptor XCR1 were evident in synovial fluids and tissues surrounding hip-implants of patients undergoing revision total hip arthroplasty. Furthermore, murine calvarial osteolysis model induced by ultra-high molecular weight polyethylene (UHMWPE) particles was used to study the role of XCL1 in the development of inflammatory osteolysis. Mice received single injection of recombinant XCL1 onto the calvariae after implantation of particles exhibited significantly greater osteolytic lesions than the control mice. In contrast, blockade of XCL1 by neutralizing antibody significantly reduced bone erosion and the number of bone-resorbing mature osteoclasts induced by UHMWPE particles. In consistence with the results, transplantation of XCL1-soaked sponge onto calvariae caused osteolytic lesions coincident with excessive infiltration of inflammatory cells and osteoclasts. These results suggested that XCL1 might be involved in the development of periprosthetic osteolysis through promoting infiltration of inflammatory cells and bone resorbing-osteoclasts. Our further results demonstrated that supplementing recombinant XCL1 to cultured human monocytes stimulated with the receptor activator of nuclear factor kappa-B ligand (RANKL) promoted osteoclastogenesis and the osteoclast-bone resorbing activity. Moreover, recombinant XCL1 promoted the expression of inflammatory and osteoclastogenic factors, including IL-6, IL-8, and RANKL in human differentiated osteoblasts. Together, these results suggested the potential role of XCL1 in the pathogenesis of periprosthetic osteolysis and aseptic loosening. Our data broaden knowledge of the pathogenesis of aseptic prosthesis loosening and highlight a novel molecular target for therapeutic intervention.

Babesia microti Confers Macrophage-Based Cross-Protective Immunity Against Murine Malaria.
Artemis Efstratiou, Eloiza May S Galon, Guanbo Wang, Kousuke Umeda, Daisuke Kondoh, Mohamad Alaa Terkawi, Aiko Kume, Mingming Liu, Aaron Edmond Ringo, Huanping Guo, Yang Gao, Seung-Hun Lee, Jixu Li, Paul Franck Adjou Moumouni, Yoshifumi Nishikawa, Hiroshi Suzuki, Ikuo Igarashi, Xuenan Xuan
Frontiers in cellular and infection microbiology, 10, 193, 193, 2020年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Malaria and babesiosis, the two primary intraerythrocytic protozoan diseases of humans, have been reported in multiple cases of co-infection in endemic regions. As the geographic range and incidence of arthropod-borne infectious diseases is being affected by climate change, co-infection cases with Plasmodium and Babesia are likely to increase. The two parasites have been used in experimental settings, where prior infection with Babesia microti has been shown to protect against fatal malarial infections in mice and primates. However, the immunological mechanisms behind such phenomena of cross-protection remain unknown. Here, we investigated the effect of a primary B. microti infection on the outcome of a lethal P. chabaudi challenge infection using a murine model. Simultaneous infection with both pathogens led to high mortality rates in immunocompetent BALB/c mice, similar to control mice infected with P. chabaudi alone. On the other hand, mice with various stages of B. microti primary infection were thoroughly immune to a subsequent P. chabaudi challenge. Protected mice exhibited decreased levels of serum antibodies and pro-inflammatory cytokines during early stages of challenge infection. Mice repeatedly immunized with dead B. microti quickly succumbed to P. chabaudi infection, despite induction of high antibody responses. Notably, cross-protection was observed in mice lacking functional B and T lymphocytes. When the role of other innate immune effector cells was examined, NK cell-depleted mice with chronic B. microti infection were also found to be protected against P. chabaudi. Conversely, in vivo macrophage depletion rendered the mice vulnerable to P. chabaudi. The above results show that the mechanism of cross-protection conferred by B. microti against P. chabaudi is innate immunity-based, and suggest that it relies predominantly upon the function of macrophages. Further research is needed for elucidating the malaria-suppressing effects of babesiosis, with a vision toward development of novel tools to control malaria.

人工関節置換術後の無菌性緩みにおけるマクロファージ由来チミジンホスホリラーゼの機能解析　　　　　　　　　　　　　　　
松前 元, Terkawi Alaa, 江畑 拓, 木田 博朗, 高橋 大介, 岩崎 倫政
日本バイオマテリアル学会大会予稿集, 41回, 180, 180, 日本バイオマテリアル学会, 2019年11月
日本語

人工関節置換術後無菌性緩みに対するビタミンE添加ポリエチレンインプラントの至適濃度の検討　　　　　　　　　　　　　　　
木田 博朗, Terkawi Alaa, 松前 元, 江畑 拓, 高橋 大介, 岩崎 倫政
日本バイオマテリアル学会大会予稿集, 41回, 182, 182, 日本バイオマテリアル学会, 2019年11月
日本語

Cocktail Babesia bovis antigens for global detection of Babesia bovis infection in cattle.
Shimaa El-Sayed, Mohamed Abdo Rizk, MohamadAlaa Terkawi, Ikuo Igarashi
Experimental parasitology, 206, 107758, 107758, 2019年11月, ［国際誌］
英語, 研究論文（学術雑誌）, The diagnostic performance of a cocktail formula consisting of two Babesia (B.) bovis recombinant proteins, including spherical body protein 1 (BbSBP-1) and spherical body protein 4 (BbSBP-4), was evaluated in the present study for the global detection of B. bovis infection in cattle and for the differentiation between B. bovis and B. bigemina infections. The efficacy and the practicality of the rBbSBP-1 and rBbSBP-4 cocktail formula for differentiation between the infection caused by both parasites were assessed using indirect enzyme-linked immunosorbent assay (iELISA) with serum samples collected from cattle experimentally infected by B. bovis (n = 33) or B. bigemina (n = 30). Cocktail antigen exhibited the highest optical density (OD) values with B. bovis-infected sera and the lowest OD values with normal bovine sera or B. bigemina-infected sera in comparison with the single antigen. A total of 581 field serum samples collected from four countries with known B. bovis endemicity: Ghana (n = 154), Egypt (n = 162), Thailand (n = 96), and South Africa (n = 169) were screened also in the current study using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. A cocktail formula (rBbSBP-1 and rBbSBP-4) exhibited the highest concordance rate (89.90%) and kappa value (0.73). The obtained results revealed the reliability of the rBbSBP-1 and rBbSBP-4 cocktail antigen for the detection of specific antibodies to B. bovis in cattle and demonstrated the usefulness of cocktail antigen for differentiation between B. bovis and B. bigemina infections compared with the single antigen in cattle, which will be useful for epidemiological surveys and control of bovine babesiosis.

Minimum Ten-Year Outcomes of Partial Ulnar Nerve Transfer for Restoration of Elbow Flexion in Patients with Upper Brachial Plexus Injury.
Yusuke Nagano, Daisuke Kawamura, Alaa Terkawi, Atsushi Urita, Yuichiro Matsui, Norimasa Iwasaki
The journal of hand surgery Asian-Pacific volume, 24, 3, 283, 288, 2019年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Partial ulnar nerve transfer to the biceps motor branch of the musculocutaneous nerve (Oberlin's transfer) is a successful approach to restore elbow flexion in patients with upper brachial plexus injury (BPI). However, there is no report on more than 10 years subjective and objective outcomes. The purpose of this study was to clarify the long-term outcomes of Oberlin's transfer based on the objective evaluation of elbow flexion strength and subjective functional evaluation of patients. Methods: Six patients with BPI who underwent Oberlin's transfer were reviewed retrospectively by their medical records. The mean age at surgery was 29.5 years, and the mean follow-up duration was 13 years. The objective functional outcomes were evaluated by biceps muscle strength using the Medical Research Council (MRC) grade at preoperative, postoperative, and final follow-up. The patient-derived subjective functional outcomes were evaluated using the Quick Disability of the Arm, Shoulder, and Hand (QuickDASH) questionnaire at final follow-up. Results: All patients had MRC grade 0 (M0) or 1 (M1) elbow flexion strength before operation. Four patients gained M4 postoperatively and maintained or increased muscle strength at the final follow-up. One patient gained M3 postoperatively and at the final follow-up. Although one patient achieved M4 postoperatively, the strength was reduced to M2 due to additional disorder. The mean score of QuickDASH was 36.5 (range, 7-71). Patients were divided into two groups; three patients had lower scores and the other three patients had higher scores of QuickDASH. Conclusions: Oberlin's transfer is effective in the restoration of elbow flexion and can maintain the strength for more than 10 years. Patients with upper BPI with restored elbow flexion strength and no complicated nerve disorders have over ten-year subjective satisfaction.

Effects of femoral bone defect morphology on initial polished tapered stem stability in massive defect model: a biomechanical study.
Tohru Irie, Daisuke Takahashi, Tsuyoshi Asano, Tomohiro Shimizu, Ryuta Arai, Alaa Muhammad Terkawi, Yoichi M Ito, Norimasa Iwasaki
BMC musculoskeletal disorders, 20, 1, 355, 355, 2019年08月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Good outcomes have been reported in revision total hip replacement with massive segmental defects using impaction bone grafting with circumferential metal meshes. However, the morphology of defects that require a mesh is poorly defined. The purpose of this study was to evaluate the effects of a variety of segmental defects on load transmission to the proximal femur under both axial and rotational loads. METHODS: Initial stability of the Exeter stem was investigated in a composite bone model using three medial bone defect morphologies: Long (length 5 cm × width 2 cm), Short (2.5 cm × 2 cm), Square (3.2 cm × 3.2 cm), Square with mesh (3.2 cm × 3.2 cm defect covered with metal mesh), and with no defect as control. Specimens (5 per group) were axially loaded and internally rotated up to 20° or to failure. Strain distributions of the femora were measured using a strain gauge. RESULTS: All Square group specimens failed while rotation was increasing. In the other four groups, failure was not observed in any specimens. Mean torsional stiffness in the Long (4.4 ± 0.3 Nm/deg.) and Square groups (4.3 ± 0.3 Nm/deg.) was significantly smaller than in the Control group (4.8 ± 0.3 Nm/deg.). In the medio-cranial region, the magnitude of the maximum principal strain in the Square group (1176.4 ± 100.9) was significantly the largest (Control, 373.2 ± 129.5, p < 0.001; Long, 883.7 ± 153.3, p = 0.027; Short, 434.5 ± 196.8, p < 0.001; Square with mesh, 256.9 ± 100.8, p < 0.001). Torsional stiffness, and both maximum and minimum principal strains in the Short group showed no difference compared to the Control group in any region. CONCLUSIONS: Bone defect morphology greatly affected initial stem stability and load transmission. If defect morphology is not wide and the distal end is above the lower end of the lesser trochanter, it may be acceptable to fill the bone defect region with bone cement. However, this procedure is not acceptable for defects extending distally below the lower end of the lesser trochanter or defects 3 cm or more in width.

Bilateral subchondral insufficiency fractures of the femoral head in a normally active adolescent patient: A case report.
Hotaka Ishizu, Tomohiro Shimizu, Takuma Kaibara, Tsuyoshi Asano, Mohamad Alaa Terkawi, Daisuke Takahashi, Norimasa Iwasaki
Journal of orthopaedic science : official journal of the Japanese Orthopaedic Association, S0949-2658(19)30148-4, 2, 492, 494, 2019年06月05日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）

Identification of IL-27 as potent regulator of inflammatory osteolysis associated with vitamin E-blended ultra-high molecular weight polyethylene debris of orthopedic implants.
Mohamad Alaa Terkawi, Ken Kadoya, Daisuke Takahashi, Yuan Tian, Masanari Hamasaki, Gen Matsumae, Hend Alhasan, Sameh Elmorsy, Keita Uetsuki, Tomohiro Onodera, Masahiko Takahata, Norimasa Iwasaki
Acta biomaterialia, 89, 242, 251, 2019年04月15日, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Vitamin E-blended ultra-high molecular weight polyethylene (VE-UHMWPE) is a newly introduced material for prosthetic components that has proven a better mechanical performance with lesser adverse cellular responses than conventional polyethylene in experimental animal models. However, the mechanisms by which VE-UHMWPE particles trigger a reduced osteolytic activity are unclear and remain to be investigated. Therefore, the current study aims at exploring a possible anti-osteolytic mechanism associated with VE-UHMWPE particles. Transcriptional profiling and bioinformatic analyses of human macrophages stimulated by VE-UHMWPE particles revealed a distinct transcriptional program from macrophages stimulated with UHMWPE particles. Out of the up-regulated genes, IL-27 was found to be significantly elevated in macrophages cultured with VE-UHMWPE particles as compared to these with UHMWPE particles (p = 0.0084). Furthermore, we studied the potential anti-osteolytic function of IL-27 in osteolysis murine model. Interestingly, administration of recombinant IL-27 onto calvariae significantly alleviated osteolytic lesions triggered by UHMWPE particles (p = 0.0002). Likewise, IL-27 inhibited differentiation of osteoclasts (p = 0.0116) and reduced inflammatory response (p < 0.0001) elicited by conventional UHMWPE particles in vitro. This is the first study demonstrating the involvement of IL-27 in macrophage response to VE-UHMWPE particles and its regulatory role in osteolysis. Our data highlight a novel therapeutic agent for treatment of inflammatory osteolysis induced by polyethylene debris. STATEMENT OF SIGNIFICANCE: Aseptic loosening due to inflammatory osteolysis remains the major cause of arthroplasty failure and represents a substantial economic burden worldwide. Ideal approach to prevent this failure should be directed to minimize inflammatory response triggered by wear particles at the site of implant. Understanding the mechanism by which VE-UHMWPE particles triggers lesser cellular responses and reduced osteolysis as compared to conventional UHMWPE particles may aid in discovery of regulatory factors. In the current study, we reported that IL-27 is a potent regulator of inflammatory osteolysis involved in the reduced biologic activities and osteolytic potentials associated with VE-UHMWPE particles. Initiating the production IL-27 in vivo after total joint arthroplasties might be a novel strategy to prolong the life-spam of implant.

Effects of Ultra-Purified Alginate Gel Implantation on Meniscal Defects in Rabbits.
WooYoung Kim, Tomohiro Onodera, Eiji Kondo, Yasuyuki Kawaguchi, Mohamad Alaa Terkawi, Rikiya Baba, Kazutoshi Hontani, Zenta Joutoku, Shinji Matsubara, Kentaro Homan, Ryosuke Hishimura, Norimasa Iwasaki
The American journal of sports medicine, 47, 3, 640, 650, 2019年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Many tissue-engineered methods for meniscal repair have been studied, but their utility remains unclear. HYPOTHESIS: Implantation of low-endotoxin, ultra-purified alginate (UPAL) gel without cells could induce fibrocartilage regeneration on meniscal defects in rabbits. STUDY DESIGN: Controlled laboratory study. METHODS: Forty-two mature Japanese White rabbits were divided into 2 groups of 21 animals each. In each animal, a cylindrical defect measuring 2 mm in diameter was created with a biopsy punch on the anterior horn of the medial meniscus. In the control group, no treatment was applied on the left medial meniscal defect. In the UPAL gel group, the right medial meniscal defect was injected with the UPAL gel and gelated by a CaCl2 solution. Samples were evaluated at 3, 6, and 12 weeks postoperatively. For biomechanical evaluation, 6 additional samples from intact animals were used for comparison. RESULTS: The macroscopic score was significantly greater in the UPAL gel group than in the control group at 3 weeks (mean ± SE: 5.6 ± 0.82 vs 3.4 ± 0.83, P = .010), 6 weeks (5.9 ± 0.72 vs 2.5 ± 0.75, P = .026), and 12 weeks (5.2 ± 1.21 vs 1.0 ± 0.63, P = .020). The histological score was significantly greater in the UPAL group than in the control group at 3 weeks (2.1 ± 0.31 vs 1.2 ± 0.25, P = .029) and 12 weeks (2.2 ± 0.55 vs 0.3 ± 0.21, P = .016). The mean stiffness of the reparative tissue in the UPAL gel group was significantly greater than that in the control group at 6 weeks (24.325 ± 3.920 N/mm vs 8.723 ± 1.190 N/mm, P = .006) and at 12 weeks (27.804 ± 6.169 N/mm vs not applicable [because of rupture]). CONCLUSION: The UPAL gel enhanced the spontaneous repair of fibrocartilage tissues in a cylindrical meniscal defect in rabbits. CLINICAL RELEVANCE: These results imply that the acellular UPAL gel may improve the repair of traumatic meniscal injuries.

A Novel Cartilage Fragments Stimulation Model Revealed that Macrophage Inflammatory Response Causes an Upregulation of Catabolic Factors of Chondrocytes In Vitro.
Masanari Hamasaki, Mohamad Alaa Terkawi, Tomohiro Onodera, Kentaro Homan, Norimasa Iwasaki
Cartilage, 12, 1947603519828426, 1947603519828426, 1947603519828426, 2019年02月01日, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, OBJECTIVE: Osteoarthritis is a progressive joint disease characterized by cartilage degradation and synovial inflammation. Presence of cartilage fragments in the joint due to degradation of cartilage is thought to be associated with local inflammatory response and progressive osteoarthritic process. Understanding the mechanism by which cartilage fragments elicit this destructive process should aid in designing novel therapeutic approaches. Therefore, objective of current study is to establish an in vitro model to examine the cross-talk between chondrocytes and cartilage fragments-stimulated macrophages. DESIGN: Cartilage fragments were prepared from femoral head cartilages of mice and analyzed using a scanning electron microscope and particle size analyzer. Bone marrow-derived macrophages were co-cultured with cartilage fragments and chondrocytes using transwell co-culture system. Macrophage inflammatory mediators in supernatant of cultures were determined by ELISA and gene expression of macrophages and chondrocyte were quantified by qRT-PCR. RESULTS: Shapes of cartilage fragments were irregular with sizes ranged between 0.54 and 55 μm. Macrophages cultured with cartilage fragments released significantly higher concentrations of TNF-α, IL-6, and NO than those of mock and control. Consistently, gene expressions of TNF-α, IL-6, and MMP-9 were significantly increased in stimulated macrophages. The elevation in production of pro-inflammatory molecules in stimulated macrophages cultures were coincident with an increase in gene expression of chondrocyte MMP-13, iNOS, and IL-6. CONCLUSION: We developed an in vitro co-culture model to study the impact of stimulation of macrophage by cartilage fragments on the expression of chondrocyte carbolic factors. Our results revealed that cartilage fragments triggered macrophages inflammatory response that enhanced the production of chondrocyte catabolic factors.

炎症性骨溶解及び骨吸収におけるNOV(Nephroblastoma overexpressed)の機能解析
松前 元, テルカウィ・アラー, 田 園, 濱崎 雅成, ヘンド・アルハサン, 江畑 拓, 高橋 大介, 角家 健, 岩崎 倫政
北海道整形災害外科学会雑誌, 61, 136th suppl, 29, 29, 北海道整形災害外科学会, 2019年
日本語

Identification of il-27 as potent regulator of osteolysis triggered by of orthopaedic implants debris　　　　　　　　　　　　　　　
Daisuke Takahashi, Mohamad Alaa Terkawi, Gen Matsumae, Yuan Tian, Hend Alhasan, Norimasa Iwasaki.
Transactions of the Annual Meeting of the Society for Biomaterials and the Annual International Biomaterials Symposium, 40, 612, Society for Biomaterials, 2019年
英語, 研究論文（国際会議プロシーディングス）, Statement of Purpose: Aseptic loosening due to inflammatory osteolysis remains the major postoperative complication of total joint arthroplasty that leads to prosthesis failure. Inflammation occurs when macrophages recognize wear debris derived from implant bearing surfaces and release an array of cytokines and chemokines that promote the recruitment of other inflammatory cells and the osteoclastic-bone resorbing activity [1]. Vitamin E-blended ultra-high molecular weight polyethylene (VE-UHMWPE) is a newly developed material for prosthetic components that has proven greater clinical performance with lesser adverse cellular responses than conventional polyethylene in short-term clinical studies as well as in experimental animal models [2]. However, the mechanisms by which VE-UHMWPE debris triggers reduced osteolytic activity are largely unclear and remain to be investigated. Therefore, the current study aims at exploring a possible mechanism by which VE-UHMWPE debris triggers a reduced adverse cellular response. with macrophage response to debris.

Is There an Association Between Borderline-to-mild Dysplasia and Hip Osteoarthritis? Analysis of CT Osteoabsorptiometry.
Tohru Irie, Daisuke Takahashi, Tsuyoshi Asano, Ryuta Arai, Muhammad Alaa Terkawi, Yoichi M Ito, Norimasa Iwasaki
Clinical orthopaedics and related research, 476, 7, 1455, 1465, 2018年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: The definitive treatment of borderline-to-mild dysplasia remains controversial. A more comprehensive understanding of the etiology of osteoarthritis (OA) and clarification of any possible association between borderline-to-mild dysplasia and the pathogenesis of OA are essential. QUESTIONS/PURPOSES: (1) Does the distribution of acetabular subchondral bone density increase according to dysplasia severity? (2) Is there an association between borderline-to-mild dysplasia and OA pathogenesis? METHODS: We evaluated bilateral hips of patients with developmental dysplasia of the hip who underwent eccentric rotational acetabular osteotomy (ERAO) for inclusion in the dysplasia group and contralateral hips of patients with unilateral idiopathic osteonecrosis of the femoral head (ONFH) who underwent curved intertrochanteric varus osteotomy (CVO) for the control group. ERAO was performed in 46 patients and CVO was performed in 32 patients between January 2013 and August 2016 at our institution. All patients underwent bilateral hip CT. The study included 55 hips categorized according to dysplasia severity: (1) borderline-mild, 19 hips (15° ≤ lateral center-edge angle [LCEA] < 25°); (2) moderate, 20 hips (5° ≤ LCEA < 15°); (3) severe, 16 hips (LCEA < 5°); and (4) control, 15 hips. Thirty-seven dysplastic hips (age < 15 or > 50 years old, prior hip surgery, subluxation, aspherical femoral head, cam deformity, and radiographic OA) and 17 control hips (age < 15 or > 50 years old, bilateral ONFH, LCEA < 25° or ≥ 35°, cam deformity, and radiographic OA) were excluded. CT-osteoabsorptiometry (OAM) predicts physiologic biomechanical conditions in joints by evaluating subchondral bone density. We evaluated the distribution of subchondral bone densities in the acetabulum with CT-OAM, dividing the stress distribution map into six segments: anteromedial, anterolateral, centromedial, centrolateral, posteromedial, and posterolateral. We calculated the percentage of high-density area, which was defined as the upper 30% of Hounsfield units values in each region and compared least square means difference estimated by the random intercept model among the four groups. RESULTS: In all regions, the percentage of high-density area did not differ between the borderline-mild group and the control (eg, anterolateral, 16.2 ± 5.6 [95% CI, 13.4 to 18.9] versus 15.5 ± 5.7 [95% CI, 12.4 to 18.5, p = 0.984]; centrolateral, 39.1 ± 5.7 [95% CI, 36.4 to 41.8] versus 39.5 ± 4.7 [95% CI, 36.6 to 42.5, p = 0.995]; posterolateral, 10.9 ± 5.2 [95% CI, 8.0 to 13.8] versus 15.1 ± 6.8 [95% CI, 11.7 to 18.5, p = 0.389]). In the anterolateral region, a smaller percentage of high-density area was observed in the borderline-mild group than in both the moderate group (16.2 ± 5.6 [95% CI, 13.4-18.9] versus 28.2 ± 5.1 [95% CI, 25.5-30.9], p < 0.001) and the severe group (16.2 ± 5.6 [95% CI, 13.4-18.9] versus 22.2 ± 6.8 [95% CI, 19.2-25.2, p = 0.026). CONCLUSIONS: Our results suggest that the cumulative hip stress distribution in borderline-to-mild dysplasia was not concentrated on the lateral side of the acetabulum, unlike severe dysplasia. CLINICAL RELEVANCE: Based on the stress distribution pattern, our results may suggest that there is no association between borderline-to-mild dysplasia and the pathogenesis of OA. Further studies are needed to evaluate the association between borderline-to-mild dysplasia and instability of the hip.

A mathematical model for predicting postoperative leg shortening after curved intertrochanteric varus osteotomy for osteonecrosis of the femoral head.
Tsuyoshi Asano, Daisuke Takahashi, Tomohiro Shimizu, Tohru Irie, Ryuta Arai, Mohamad Alaa Terkawi, Norimasa Iwasaki
PloS one, 13, 12, e0208818, 2018年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Despite good clinical outcomes associated with curved intertrochanteric varus osteotomy for the treatment of osteonecrosis of the femoral head, post-operative leg-length discrepancy is frequently reported and might reduce patient satisfaction. Although previous report showed that varus angulation affected post-operative leg-length discrepancy, sufficient varus angulation is the most important factor for obtaining a lateral intact portion. Therefore, to ensure better postoperative outcomes, detection of other parameters associated with leg shortening may prove useful. This study aimed to detect other factors influencing post-operative leg-length discrepancy and to develop a theory for pre-operative planning. The study included 42 hips of 36 patients with osteonecrosis of the femoral head [25 men and 11 women; mean age at the time of surgery, 33.8 years (range, 17 to 53 years)]. Patients were assessed their clinical and radiological results bofore and after surgery. Additionally, a mathematical model was developed to predict leg shortening after curved intertrochanteric varus osteotomy based on the degree of varus angulation and the distance between the femoral head and osteotomy arc centers. Predicted and actual leg shortening in patients were compared to verify the accuracy of our model. Post-operatively, mean varus angle was 21.7° (range, 15 to 38°) and mean leg shortening was 1.7 mm (range, -5.1 to 11.4 mm). Univariate analysis showed that varus angulation and lateral shift of the osteotomy arc might influence the degree of leg shortening. Furthermore, mathematically predicted leg shortening significantly correlated with actual leg shortening (r = 0.905, p < 0.001), suggesting the usefulness of our model for predicting complications of curved intertrochanteric varus osteotomy. This study indicates the importance of not positioning the center of the osteotomy arc lateral from the center of the femoral head to minimize leg shortening after curved intertrochanteric osteotomy.

Differential Gene Expression Profile of Human Neutrophils Cultured with Plasmodium falciparum-Parasitized Erythrocytes.
Mohamad Alaa Terkawi, Ryo Takano, Kentaro Kato
Journal of immunology research, 2018, ID 6709424, 6709424, 6709424, 2018年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Neutrophils (PMNs) are the most abundant cellular component of our innate immune system, where they play central roles in the pathogenesis of and resistance to a broad range of diseases. However, their roles in malarial infection remain poorly understood. Therefore, we examined the transcriptional gene profile of human PMNs in response to Plasmodium falciparum-parasitized erythrocytes (iRBCs) by using oligonucleotide microarrays. Results revealed that PMNs induced a broad and vigorous set of changes in gene expression in response to malarial parasites, represented by 118 upregulated and 216 downregulated genes. The transcriptional response was characterized by the upregulation of numerous genes encoding multiple surface receptors, proteins involved in signal transduction pathways, and defense response proteins. This response included a number of genes which are known to be involved in the pathogenesis of malaria and other inflammatory diseases. Gene enrichment analysis suggested that the biological pathways involved in the PMN responses to the iRBCs included insulin receptor, Jak-STAT signaling pathway, mitogen-activated protein kinase (MAPK), and interleukin and interferon-gamma (IFN-γ) signaling pathways. The current study provides fundamental knowledge on the molecular responses of neutrophils to malarial parasites, which may aid in the discovery of novel therapeutic interventions.

Transcriptional profile of human macrophages stimulated by ultra-high molecular weight polyethylene particulate debris of orthopedic implants uncovers a common gene expression signature of rheumatoid arthritis.
Mohamad Alaa Terkawi, Masanari Hamasaki, Daisuke Takahashi, Masahiro Ota, Ken Kadoya, Tomoyo Yutani, Keita Uetsuki, Tsuyoshi Asano, Tohru Irie, Ryuta Arai, Tomohiro Onodera, Masahiko Takahata, Norimasa Iwasaki
Acta biomaterialia, 65, 417, 425, Acta Materialia Inc, 2018年01月, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Osteolysis is a serious postoperative complication of total joint arthroplasty that leads to aseptic loosening and surgical revision. Osteolysis is a chronic destructive process that occurs when host macrophages recognize implant particles and release inflammatory mediators that increase bone-resorbing osteoclastic activity and attenuate bone-formation osteoblastic activity. Although much progress has been made in understanding the molecular responses of macrophages to implant particles, the pathways/signals that initiate osteolysis remain poorly characterized. Transcriptomics and gene-expression profiling of these macrophages may unravel key mechanisms in the pathogenesis of osteolysis and aid the identification of molecular candidates for therapeutic intervention. To this end, we analyzed the transcriptional profiling of macrophages exposed to ultra-high molecular weight polyethylene (UHMWPE) particles, the most common components used in bearing materials of orthopedic implants. Regulated genes in stimulated macrophages were involved in cytokine, chemokine, growth factor and receptor activities. Gene enrichment analysis suggested that stimulated macrophages elicited common gene expression signatures for inflammation and rheumatoid arthritis. Among the regulated genes, tumor necrosis factor superfamily member 15 (TNFSF15) and chemokine ligand 20 (CCL20) were further characterized as molecular targets involved in the pathogenesis of osteolysis. Treatment of monocyte cultures with TNFSF15 and CCL20 resulted in an increase in osteoclastogenesis and bone-resorbing osteoclastic activity, suggesting their potential contribution to loosening between implants and bone tissues. STATEMENT OF SIGNIFICANCE: Implant loosening due to osteolysis is the most common mode of arthroplasty failure and represents a great challenge to orthopedic surgeons and a significant economic burden for patients and healthcare services worldwide. Bone loss secondary to a local inflammatory response initiated by particulate debris from implants is considered the principal feature of the pathogenesis of osteolysis. In the present study, we analyzed the transcriptional profiling of human macrophages exposed to UHMWPE particles and identified a large number of inflammatory genes that were not identified previously in macrophage responses to wear particles. Our data provide a new insight into the molecular pathogenesis of osteolysis and highlights a number of molecular targets with prognostic and therapeutic implications.

Molecular identification and antigenic characterization of Babesia divergens Erythrocyte Binding Protein (BdEBP) as a potential vaccine candidate.
Shimaa Abd El-Salam El-Sayed, Mohamed Abdo Rizk, Mohamad Alaa Terkawi, Naoaki Yokoyama, Ikuo Igarashi
Parasitology international, 66, 6, 721, 726, ELSEVIER IRELAND LTD, 2017年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Host cell invasion is the only step where Babesia parasites are extracellular, and their survival is menaced during this step. Therefore, interfering with this critical stage is a target for an anti-Babesia intervention strategy. In this regard, recombinant protein encoding Babesia divergens Erythrocyte Binding Protein (BdEBP) was produced in Escherichia coli in the current study, and its antiserum was prepared in mice for further molecular characterization. Western blotting and indirect fluorescent antibody test (IFAT) revealed the specific reaction of the anti-rBdEBP serum with a corresponding authentic protein of B. divergens. Next, bovine RBCs were incubated with a B. divergens lysate, and anti-rBdEBP serum was produced in mice to detect the ability of BdEBP to bind with host cells. Bands corresponding to 29.6-kDa proteins in the protein-bound erythrocyte lysate were detected by specific immune rBdEBP using Western blotting. These results suggest that BdEBP is functional in the merozoite stage and may be involved in attachment to bovine RBCs. A significant inhibition of the in vitro growth of B. divergens culture treated with anti-rBdEBP serum was observed. Moreover, the efficacy of pre-incubated free merozoites to invade bovine erythrocytes was inhibited by 60% after incubation with 2mg/ml of anti-rBdEBP serum for 6h. The obtained data suggest the possible use of rBdEBP as a vaccine candidate against bovine babesiosis.

Chemotherapeutic efficacies of a clofazimine and diminazene aceturate combination against piroplasm parasites and their AT-rich DNA-binding activity on Babesia bovis.
Bumduuren Tuvshintulga, Mahmoud AbouLaila, Thillaiampalam Sivakumar, Dickson Stuart Tayebwa, Sambuu Gantuya, Khandsuren Naranbaatar, Aki Ishiyama, Masato Iwatsuki, Kazuhiko Otoguro, Satoshi Ōmura, Mohamad Alaa Terkawi, Azirwan Guswanto, Mohamed Abdo Rizk, Naoaki Yokoyama, Ikuo Igarashi
Scientific reports, 7, 1, 13888, 13888, NATURE PUBLISHING GROUP, 2017年10月24日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Recently, we reported that clofazimine (CF) has an anti-piroplasm activity, but it could not completely eliminate parasites in the host. The currently available anti-piroplasm drug, diminazene aceturate (DA), has sometimes been reported to have toxic side effects. In the present study, we evaluated the combination treatment with CF and DA against piroplasms both in vitro and in vivo. Additionally, mRNA level and DNA amounts were analyzed in CF‒ and DA‒treated Babesia bovis by a qPCR. The CF-DA combination had additive effects on Babesia bovis, B. bigemina, and B. caballi and synergistic effects on Theileria equi. The CF-DA combination chemotherapies against B. microti in mice were more potent than their monotherapies. In the CF‒ and DA‒treated B. bovis, CF dose-dependently down-regulated mRNA level and DNA amounts of extranuclear genes (AT-rich featured), whereas DA down-regulated only DNA amounts of extranuclear genes, but those of nuclear genes were slightly down- or up-regulated by CF and DA. In conclusion, the CF-DA combination has a higher efficiency against piroplasms than CF or DA monotherapies. CF and DA might have an AT-rich DNA-binding activity. All results suggest that the CF-DA combination chemotherapy will be a better choice to treat piroplasmosis instead of DA monotherapy.

Evaluation of immunochromatographic test (ICT) strips for the serological detection of Babesia bovis and Babesia bigemina infection in cattle from Western Java, Indonesia.
Azirwan Guswanto, Puttik Allamanda, Euis Siti Mariamah, Tserendorf Munkjargal, Bumduuren Tuvshintulga, Hitoshi Takemae, Thillaiampalam Sivakumar, Mahmoud AbouLaila, Mohamad Alaa Terkawi, Madoka Ichikawa-Seki, Yoshifumi Nishikawa, Naoaki Yokoyama, Ikuo Igarashi
Veterinary parasitology, 239, 76, 79, ELSEVIER SCIENCE BV, 2017年05月30日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Three types of immunochromatographic test (ICT) strips were prepared for the detection of an antibody response against spherical body protein 4 (SBP-4) of Babesia bovis (bovICT), C-terminal-truncated rhoptry-associated protein 1 (rRAP1/CT17) of B. bigemina (bigICT), and the combination of both proteins (dual-ICT). The evaluation of their performance was conducted using a confirmed positive and negative serum panel for B. bovis and B. bigemina. Together with ELISA, the ICT strips were applied to determine the seroprevalence of bovine babesiosis in Western Java, Indonesia. Among 991 serum samples, 28.4%, 25.3%, and 24.5% of cattle were detected to be seropositive to B. bovis infection using ELISA, bovICT, and dual-ICT, respectively. B. bigemina seropositive was detected in 27.1%, 24.2%, and 22.8% of samples using ELISA, bigICT, and dual-ICT, respectively. The comparison of ICT strips and ELISA results using field serum samples showed good agreement with Kappa values >0.7 between all methods The application of ICT strips is preferable in the field situations where rapid diagnosis is required. Furthermore, the data showed the current seroprevalence of bovine babesiosis in Western Java, Indonesia, and efficient control strategies are needed to reduce economic losses due to the disease.

A rare case of multiple phosphaturic mesenchymal tumors along a tendon sheath inducing osteomalacia.
Ryuta Arai, Tomohiro Onodera, Mohamad Alaa Terkawi, Tomoko Mitsuhashi, Eiji Kondo, Norimasa Iwasaki
BMC musculoskeletal disorders, 18, 1, 79, 79, BIOMED CENTRAL LTD, 2017年02月13日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome characterized by renal phosphate wasting, hypophosphatemia, reduction of 1,25-dihydroxyl vitamin D, and bone calcification disorders. Tumors associated with TIO are typically phosphaturic mesenchymal tumors that are bone and soft tissue origin and often present as a solitary tumor. The high production of fibroblast growth factor 23 (FGF23) by the tumor is believed to be the causative factor responsible for the impaired renal tubular phosphate reabsorption, hypophosphatemia and osteomalacia. Complete removal of the tumors by surgery is the most effective procedure for treatment. Identification of the tumors by advanced imaging techniques is difficult because TIO is small and exist within bone and soft tissue. However, systemic venous sampling has been frequently reported to be useful for diagnosing TIO patients. CASE PRESENTATION: We experienced a case of 39-year-old male with diffuse bone pain and multiple fragility fractures caused by multiple FGF23-secreting tumors found in the hallux. Laboratory testing showed hypophosphatemia due to renal phosphate wasting and high levels of serum FGF23. Contrast-enhanced MRI showed three soft tissue tumors and an intraosseous tumor located in the right hallux. Systemic venous sampling of FGF23 revealed an elevation in the right common iliac vein and external iliac vein, which suggested that the tumors in the right hallux were responsible for overproduction of FGF23. Thereafter, these tumors were surgically removed and subjected to histopathological examinations. The three soft tissue tumors were diagnosed as phosphaturic mesenchymal tumors, which are known to be responsible for TIO. The fourth tumor had no tumor structure and was consisting of hyaline cartilage and bone tissue. Immediately after surgery, we noted a sharply decrease in serum level of FGF23, associated with an improved hypophosphatemia and a gradual relief of systematic pain that disappeared within two months of surgery. CONCLUSION: The authors reported an unusual case of osteomalacia induced by multiple phosphaturic mesenchymal tumors located in the hallux. Definition of tumors localization by systemic venous sampling led to successful treatment and cure this patient. The presence of osteochondral tissues in the intraosseous tumor might be developed from undifferentiated mesenchymal cells due to high level of FGF23 produced by phosphaturic mesenchymal tumors.

Involvement of β-defensin 130 (DEFB130) in the macrophage microbicidal mechanisms for killing Plasmodium falciparum.
Mohamad Alaa Terkawi, Ryo Takano, Atsushi Furukawa, Fumi Murakoshi, Kentaro Kato
Scientific Reports, 7, 41772, 41772, 41772, NATURE PUBLISHING GROUP, 2017年02月09日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Understanding the molecular defense mechanism of macrophages and identifying their effector molecules against malarial parasites may provide important clues for the discovery of new therapies. To analyze the immunological responses of malarial parasite-induced macrophages, we used DNA microarray technology to examine the gene profile of differentiated macrophages phagocytizing Plasmodium falciparum-parasitized erythrocytes (iRBC). The transcriptional gene profile of macrophages in response to iRBCs represented 168 down-regulated genes, which were mainly involved in the cellular immune response, and 216 upregulated genes, which were involved in cellular proteolysis, growth, and adhesion. Importantly, the specific upregulation of β-defensin 130 (DEFB130) in these macrophages suggested a possible role for DEFB130 in malarial parasite elimination. Differentiated macrophages phagocytizing iRBCs exhibited an increase in intracellular DEFB130 levels and DEFB130 appeared to accumulate at the site of iRBC engulfment. Transfection of esiRNA-mediated knockdown of DEFB130 into macrophages resulted in a remarkable reduction in their antiplasmodial activity in vitro. Furthermore, DEFB130 synthetic peptide exhibited a modest toxic effect on P. falciparum in vitro and P. yoelii in vivo, unlike scrambled DEFB130 peptide, which showed no antiplasmodial activity. Together, these results suggest that DEFB130 might be one of the macrophage effector molecules for eliminating malarial parasites. Our data broaden our knowledge of the immunological response of macrophages to iRBCs and shed light on a new target for therapeutic intervention.

Efficacy of teriparatide in the treatment of nontraumatic osteonecrosis of the femoral head: a retrospective comparative study with alendronate.
Ryuta Arai, Daisuke Takahashi, Masahiro Inoue, Tohru Irie, Tsuyoshi Asano, Takuya Konno, Mohamad Alaa Terkawi, Tomohiro Onodera, Eiji Kondo, Norimasa Iwasaki
BMC musculoskeletal disorders, 18, 1, 24, 24, BIOMED CENTRAL LTD, 2017年01月19日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Collapse of the femoral head associated with nontraumatic osteonecrosis (NOFH) is one of the most common causes of disability in young adult patients. Excessive bone resorption by osteoclast coincident with the suppression of osteogenesis are believed to be responsible for collapse progression. Alendronate that inhibits bone resorption by inducing osteoclast apoptosis has been traditionally used for treating NOFH; however, several reports documented serious complications by the use of this drug. On the other hand, teriparatide activates osteoblasts leading to an overall increase in bone volume, and is expected to reduce the progression of femoral head collapse in NOFH. Therefore, the present study was undertaken to examine pharmacological effects of teriparatide on collapse progression of NOFH and to compare these effects with alendronate. METHODS: We conducted a retrospective study in our facility for comparing the pharmacological effects of teriparatide and alendronate on 32 NOFH patients diagnosed with osteoporosis. Between 2007 and 2013, patients were treated with daily administration of 20 μg teriparatide (15 patients: 18 hips), or with 35 mg of alendronate once a week (17 patients: 22 hips). The mean period of follow-up was 18.7 months. The progression of collapse was evaluated prior to the administration and later every three months by anteroposterior radiographs. Collapse progression with > 1 mm was defined as advanced collapse, while with < 1 mm was defined as stable radiologic disease. Student's t-test and the chi-square test was used to do compare the pharmacological effects of the two groups. RESULTS: Treatment with terparatide had a tendency to reduce the rate of advanced collapse as compared to that with alendronate (p = 0.105). Kaplan-Meier curves related to stable radiologic disease showed that teriparatide-treated patients had better stable states than these treated with alendronate (p = 0.08, log-rank test). Moreover, treatment with teriparatide resulted in a significant reduction in collapse progression as compared to that with alendronate, noted at the end of follow-up period (p = 0.049). CONCLUSION: The present study suggests that teriparatide has greater pharmacological effects than alendronate for treating NOFH and preventing the collapse of femoral head. TRIAL REGISTRATION: The registration number in UMIN Clinical Trial Registry is UMIN000017582 . The date of registration is May 5, 2015.

Correction: Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules.
Ahmed Abdelmoniem Mousa, Daniel Barry Roche, Mohamad Alaa Terkawi, Kyohko Kameyama, Ketsarin Kamyingkird, Patrick Vudriko, Akram Salama, Shinuo Cao, Sahar Orabi, Hanem Khalifa, Mohamed Ahmed, Mabrouk Attia, Ahmed Elkirdasy, Yoshifumi Nishikawa, Xuenan Xuan, Emmanuel Cornillot
PloS one, 12, 12, e0189383, 2017年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, [This corrects the article DOI: 10.1371/journal.pone.0185372.].

Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules.
Ahmed Abdelmoniem Mousa, Daniel Barry Roche, Mohamad Alaa Terkawi, Kyohko Kameyama, Ketsarin Kamyingkird, Patrick Vudriko, Akram Salama, Shinuo Cao, Sahar Orabi, Hanem Khalifa, Mohamed Ahmed, Mabrouk Attia, Ahmed Elkirdasy, Yoshifumi Nishikawa, Xuenan Xuan, Emmanuel Cornillot
PloS one, 12, 10, e0185372, PUBLIC LIBRARY SCIENCE, 2017年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Human babesiosis is caused by the apicomplexan parasite Babesia microti, which is of major public health concern in the United States and elsewhere, resulting in malaise and fatigue, followed by a fever and hemolytic anemia. In this paper we focus on the characterization of a novel B. microti thrombospondin domain (TSP1)-containing protein (BmP53) from the new annotation of the B. microti genome (locus 'BmR1_04g09041'). This novel protein (BmP53) had a single TSP1 and a transmembrane domain, with a short cytoplasmic tail containing a sub-terminal glutamine residue, but no signal peptide and Von Willebrand factor type A domains (VWA), which are found in classical thrombospondin-related adhesive proteins (TRAP). Co-localization assays of BmP53 and Babesia microti secreted antigen 1 (BmSA1) suggested that BmP53 might be a non-secretory membranous protein. Molecular mimicry between the TSP1 domain from BmP53 and host platelets molecules was indicated through different measures of sequence homology, phylogenetic analysis, 3D structure and shared epitopes. Indeed, hamster isolated platelets cross-reacted with mouse anti-BmP53-TSP1. Molecular mimicry are used to help parasites to escape immune defenses, resulting in immune evasion or autoimmunity. Furthermore, specific host reactivity was also detected against the TSP1-free part of BmP53 in infected hamster sera. In conclusion, the TSP1 domain mimicry might help in studying the mechanisms of parasite-induced thrombocytopenia, with the TSP1-free truncate of the protein representing a potential safe candidate for future vaccine studies.

Immunization with Toxoplasma gondii peroxiredoxin 1 induces protective immunity against toxoplasmosis in mice.
Ragab M Fereig, Yasuhiro Kuroda, Mohamad Alaa Terkawi, Motamed Elsayed Mahmoud, Yoshifumi Nishikawa
PloS one, 12, 4, e0176324, PUBLIC LIBRARY SCIENCE, 2017年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, To develop a vaccine against Toxoplasma gondii, a vaccine antigen with immune-stimulating activity is required. In the present study, we investigated the immunogenicity and prophylactic potential of T. gondii peroxiredoxin 1 (TgPrx1). The TgPrx1 was detected in the ascitic fluid of mice 6 days postinfection, while specific antibody levels were low in the sera of chronically infected mice. Treatment of murine peritoneal macrophages with recombinant TgPrx1 triggered IL-12p40 and IL-6 production, but not IL-10 production. In response to TgPrx1, activation of NF-kB and IL-6 production were confirmed in mouse macrophage cell line (RAW 264.7). These results suggest the immune-stimulating potentials of TgPrx1. Immunization of mice with recombinant TgPrx1 stimulated specific antibody production (IgG1 and IgG2c). Moreover, spleen cell proliferation and interferon-gamma production significantly increased in the TgPrx1- sensitized cells from mice immunized with the same antigen. Immunization with TgPrx1 also increased mouse survival and decreased cerebral parasite burden against lethal T. gondii infection. Thus, our results suggest that TgPrx1 efficiently induces humoral and cellular immune responses and is useful as a new vaccine antigen against toxoplasmosis.

Isolation and co-cultivation of human macrophages and neutrophils with Plasmodium falciparum-parasitized erythrocytes: An optimized system to study the phagocytic activity to malarial parasites.
Mohamad Alaa Terkawi, Ryo Takano, Kentaro Kato
Parasitology international, 65, 5 Pt B, 545, 548, ELSEVIER IRELAND LTD, 2016年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Macrophages and neutrophils are our front line of defense against invading pathogens. They are professional phagocytic cells that play a key role in the clearance of Plasmodium falciparum-parasitized erythrocytes from the host circulation. A stable in vitro culture system for these cells and parasitized erythrocytes would provide the means to study their immunological responses to infection, potentially revealing important clues for new therapeutic interventions for malaria. Here, we present an optimized protocol for cultivating human peripheral blood monocyte-derived macrophages and neutrophils with Plasmodium falciparum-parasitized erythrocytes.

Molecular detection of spotted fever group rickettsiae in Amblyomma variegatum ticks from Benin.
Paul Franck Adjou Moumouni, Mohamad Alaa Terkawi, Charoonluk Jirapattharasate, Shinuo Cao, Mingming Liu, Ryo Nakao, Rika Umemiya-Shirafuji, Naoaki Yokoyama, Chihiro Sugimoto, Kozo Fujisaki, Hiroshi Suzuki, Xuenan Xuan
Ticks and tick-borne diseases, 7, 5, 828, 833, Elsevier GmbH, 2016年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Spotted fever group (SFG) rickettsiae are obligate intracellular, Gram-negative bacteria transmitted by ticks and causing febrile illness in humans. Despite the presence of suitable tick vectors, the occurrence of SFG rickettsiae has never been investigated in the Republic of Benin (West Africa). In the present study, 910 Amblyomma variegatum ticks collected from 8 different locations in North Eastern Benin were tested for SFG rickettsiae. The samples were first screened for the presence of rickettsial bacteria using 16S rDNA PCR and positive samples were subsequently characterized by ompA PCR. Randomly selected samples among those positive for both assays were subjected to sequencing of 16S rDNA and ompA genes for species identification. The 16S rDNA gene was amplified in 63.4% of the samples (585/910) and the SFG rickettsia-specific ompA gene was detected in 29.4% of the samples (267/910). The prevalence of SFG rickettsiae varied according to the location, and tick gender. Sequence analyses demonstrated the presence of Rickettsia africae and/or closely related species in Benin. These findings extend the geographic distribution of R. africae and spotted fever rickettsioses in Africa. Clinicians in Benin and those treating travellers should be aware of the possibility of SFG rickettsiae infection when they are treating patients with febrile illness.

Molecular identification and antigenic characterization of a merozoite surface antigen and a secreted antigen of Babesia canis (BcMSA1 and BcSA1).
Mo Zhou, Shinuo Cao, Yuzi Luo, Mingming Liu, Guanbo Wang, Paul Franck Adjou Moumouni, Charoonluk Jirapattharasate, Aiko Iguchi, Patrick Vudriko, Mohamad Alaa Terkawi, Mario Löwenstein, Angela Kern, Yoshifumi Nishikawa, Hiroshi Suzuki, Ikuo Igarashi, Xuenan Xuan
Parasites & vectors, 9, 1, 257, 257, BioMed Central Ltd., 2016年05月03日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Babesia canis is an apicomplexan tick-transmitted hemoprotozoan responsible for causing canine babesiosis in Europe and west Asia. Despite its importance, there is no known rapid diagnostic kit detection of B. canis infection in dogs. The present study identified two novel antigens of B. canis and used the recombinant antigens to establish a rapid, specific and sensitive serodiagnostic technique for detection of B. canis infection. METHODS: A complementary DNA (cDNA) expression library was constructed from the mRNA of B. canis and immunoscreened using B. canis-infected dog sera. The cDNAs encoding a merozoite surface antigen and a secreted antigen protein were identified and designated as BcMSA1 and BcSA1, respectively. The recombinant BcMSA1 and BcSA1 (rBcMSA1 and rBcSA1) expressed in Escherichia coli were purified and injected into mice for production of anti-sera. The native proteins were characterized by Western blot analysis and immunofluorescence. Furthermore, indirect enzyme-linked immunosorbent assays (iELISA) and rapid immunochromatographic tests (ICT) based on rBcMSA1 or rBcSA1 were established and evaluated to test specific antibodies in consecutive plasma samples from two B. canis-infected dogs. RESULTS: Antiserum raised against rBcMSA1 and rBcSA1 recognized the 39 kDa and 44 kDa native proteins by Western blot analysis, respectively. In addition, immunofluorescence and confocal microscopic observations revealed that BcMSA1 was found on the surface of parasites. However, BcSA1 localized in the matrix of the merozoites. The ELISA and ICT based on rBcMSA1 or rBcSA1 could detect specific antibodies in consecutive plasma samples from two B. canis-infected dogs. They showed no cross-reactions against the serum samples collected from dogs experimentally infected with closely related parasites. CONCLUSION: Taken together, the current results indicated that the rBcMSA1 and rBcSA1 are promising serodiagnostic antigens for developing iELISA and ICT to detect B. canis infection. To our knowledge, this study is the first to report BcMSA1 and BcSA1 as potential antigenic proteins for serodiagnosis of B. canis infection in dogs.

Dextran sulfate inhibits acute Toxoplama gondii infection in pigs.
Kentaro Kato, Yuho Murata, Noriyuki Horiuchi, Atsuko Inomata, Mohamad Alaa Terkawi, Akiko Ishiwa, Yohsuke Ogawa, Shinya Fukumoto, Fumikazu Matsuhisa, Kenji Koyama
Parasites & vectors, 9, 134, 134, 2016年03月09日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Toxoplasma gondii is a highly prevalent protozoan that can infect all warm-blooded animals, including humans. Its definitive hosts are Felidae and its intermediate hosts include various other mammals and birds, including pigs. It is found in the meat of livestock which is a major source of human infection. Hence the control of toxoplasmosis in pigs is important for public health. We previously showed that dextran sulfate (DS), especially DS10 (dextran sulfate MW 10 kDa), is effective against T. gondii infection both in vitro and in mice. In this study, we asked whether DS affects T. gondii infection of pigs, one of the main animal sources of toxoplasmosis transmission to humans. METHODS: Fourteen-day-old male pigs (n = 10) were infected with T. gondii and then immediately treated with different doses of DS10; clinical, pathological, and immunological analyses were performed 5 days post-infection. RESULTS: DS10 had an inhibitory effect on toxoplasmosis in pigs. Intravenous injection of DS10 prevented the symptoms of toxoplasmosis and reduced the parasite burden and inflammation induced by T. gondii infection. High-dose DS10 (500 μg per head) caused reversible hepatocellular degeneration of the liver; middle-dose DS10 (50 μg per head) was effective against toxoplasmosis in pigs without causing this side effect. CONCLUSIONS: Our data suggest that middle-dose DS10 led to minimal clinical symptoms of T. gondii infection and caused little hepatocellular degeneration in our pig model, thereby demonstrating its potential as a new treatment for toxoplasmosis. These data should be very beneficial to those interested in the control of toxoplasmosis in pigs.

Depletion of Phagocytic Cells during Nonlethal Plasmodium yoelii Infection Causes Severe Malaria Characterized by Acute Renal Failure in Mice.
Mohamad Alaa Terkawi, Maki Nishimura, Hidefumi Furuoka, Yoshifumi Nishikawa
Infection and immunity, 84, 3, 845, 55, AMER SOC MICROBIOLOGY, 2016年01月11日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In the current study, we examined the effects of depletion of phagocytes on the progression of Plasmodium yoelii 17XNL infection in mice. Strikingly, the depletion of phagocytic cells, including macrophages, with clodronate in the acute phase of infection significantly reduced peripheral parasitemia but increased mortality. Moribund mice displayed severe pathological damage, including coagulative necrosis in liver and thrombi in the glomeruli, fibrin deposition, and tubular necrosis in kidney. The severity of infection was coincident with the increased sequestration of parasitized erythrocytes, the systematic upregulation of inflammation and coagulation, and the disruption of endothelial integrity in the liver and kidney. Aspirin was administered to the mice to minimize the risk of excessive activation of the coagulation response and fibrin deposition in the renal tissue. Interestingly, treatment with aspirin reduced the parasite burden and pathological lesions in the renal tissue and improved survival of phagocyte-depleted mice. Our data imply that the depletion of phagocytic cells, including macrophages, in the acute phase of infection increases the severity of malarial infection, typified by multiorgan failure and high mortality.

Characterization of Toxoplasma gondii glyoxalase 1 and evaluation of inhibitory effects of curcumin on the enzyme and parasite cultures.
Youn-Kyoung Goo, Junya Yamagishi, Akio Ueno, Mohamad Alaa Terkawi, Gabriel Oluga Aboge, Dongmi Kwak, Yeonchul Hong, Dong-Il Chung, Makoto Igarashi, Yoshifumi Nishikawa, Xuenan Xuan
Parasites & vectors, 8, 1, 654, 654, BioMed Central Ltd., 2015年12月23日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: The glyoxalase pathway, which includes two enzymes, glyoxalase 1 and 2 (Glo1 and Glo2), is a ubiquitous cellular system responsible for the removal of cytotoxic methylglyoxal produced during glycolysis. Protozoan parasites, including Toxoplasma gondii (T. gondii) tachyzoites, produce methylglyoxal because of increased glycolytic fluxes. A Glo1 inhibitor such as curcumin could be considered a drug candidate for anti-protozoan, anti-inflammatory, and anti-cancer therapy. METHODS: The T. gondii Glo1 gene (TgGlo1) was cloned and the recombinant protein was produced. Enzyme kinetics of TgGlo1 and five mutants were evaluated by adding methylglyoxal and glutathione to a reaction mixture. Finally, the inhibitory effects of various concentrations of curcumin on recombinant TgGlo1 were evaluated using in vitro cultures of T. gondii. RESULTS: Active recombinant TgGlo1 was successfully produced and the active sites (E166 and E251) of TgGlo1 were verified by point mutagenesis. Curcumin at the tested doses inhibited the enzymatic activity of recombinant TgGlo1 as well as the parasitic propagation of in vitro-cultured T. gondii. The Ki and IC50 were 12.9 ± 0.5 μM and 38.3 ± 0.9 μM, respectively. CONCLUSION: The inhibitory effect of curcumin on the enzymatic activity of TgGlo1 and parasitic propagation of T. gondii could be explored in the potential development of a potent drug for the treatment of toxoplasmosis. However, considering the fact that curcumin is known to have many effects on other molecules in the micromolar range, further elucidation of curcumin's direct inhibition of the glyoxalase system of T. gondii will be needed.

Cocktail of Theileria equi antigens for detecting infection in equines
Shimaa Abd El-Salam El-Sayed, Mohamed Abdo Rizk, Mohamed Alaa Terkawi, Ahmed Mousa, El Said El Shirbini El Said, Gehad Elsayed, Mohamed Fouda, Naoaki Yokoyama, Ikuo Igarashi
Asian Pacific Journal of Tropical Biomedicine, 5, 12, 977, 981, Hainan Medical University, 2015年12月01日, ［査読有り］
英語, 研究論文（学術雑誌）, Objective: To use two diagnostic antigens belonging to the frequently associated in Theileria domain, Theileria equi (T. equi) protein 82 (Te 82) and T. equi 104 kDa microneme-rhoptry antigen precursor (Te 43), to diagnose T. equi infection in horses as compared with equi merozoite antigen-2 (EMA-2). Methods: In the current study, we applied a cocktail-ELISA containing two antigens (EMA-2 + Te 82) to diagnose T. equi infection either in experimentally infected horses or in field infection. Results: Our findings have revealed that a cocktail formula of EMA-2 + Te 82 provided a more practical and sensitive diagnostic candidate for diagnosing T. equi infection in horses as compared with Te 82 or Te 43 alone. Conclusions: The ELISA technique using a cocktail formula of EMA-2 + Te 82 offers a practical and sensitive diagnostic tool for diagnosing T. equi infection in horses and using of this promising cocktail formula will be applicable for epidemiological surveys and will help control the infection in horses.

Molecular Characterization of Babesia bovis M17 Leucine Aminopeptidase and Inhibition of Babesia Growth by Bestatin.
Gabriel Oluga Aboge, Shinuo Cao, Mohamad Alaa Terkawi, Tatsunori Masatani, Younkyoung Goo, Mahmoud AbouLaila, Yoshifumi Nishikawa, Ikuo Igarashi, Hiroshi Suzuki, Xuenan Xuan
The Journal of parasitology, 101, 5, 536, 41, American Society of Parasitologists, 2015年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 μM, respectively, while the Ki was 2210 ± 358 μM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 μM] than B. gibsoni [IC50 = 460.8 ± 114.45 μM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this.

Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya.
Paul Franck Adjou Moumouni, Gabriel Oluga Aboge, Mohamad Alaa Terkawi, Tatsunori Masatani, Shinuo Cao, Ketsarin Kamyingkird, Charoonluk Jirapattharasate, Mo Zhou, Guanbo Wang, Mingming Liu, Aiko Iguchi, Patrick Vudriko, Adrian Patalinghug Ybanez, Hisashi Inokuma, Rika Shirafuji-Umemiya, Hiroshi Suzuki, Xuenan Xuan
Parasites & vectors, 8, 1, 496, 496, BioMed Central Ltd., 2015年09月30日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Infections with Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale are endemic in Kenya yet there is a lack of adequate information on their genotypes. This study established the genetic diversities of the above tick-borne hemoparasites infecting cattle in Kenya. METHODS: Nested PCR and sequencing were used to determine the prevalence and genetic diversity of the above parasites in 192 cattle blood samples collected from Ngong and Machakos farms. B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, A. marginale major surface protein 5, Theileria spp. 18S rRNA, T. parva p104 and T. orientalis major piroplasm surface protein were used as the marker genes. RESULTS: B. bovis, B. bigemina, T. parva, T. velifera, T. taurotragi, T. mutans and A. marginale were prevalent in both farms, whereas T. ovis, Theileria sp. (buffalo) and T. orientalis were found only in Ngong farm. Co-infections were observed in more than 50 % of positive samples in both farms. Babesia parasites and A. marginale sequences were highly conserved while T. parva and T. orientalis were polymorphic. Cattle-derived T. parva was detected in Machakos farm. However, cattle and buffalo-derived Theileria were detected in Ngong farm suggesting interactions between cattle and wild buffaloes. Generally, the pathogens detected in Kenya were genetically related to the other African isolates but different from the isolates in other continents. CONCLUSIONS: The current findings reaffirm the endemicity and co-infection of cattle with tick-borne hemoparasites, and the role of wildlife in pathogens transmission and population genetics in Kenya.

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.
Mohamed Abdo Rizk, Shimaa Abd El-Salam El-Sayed, Mohamed Alaa Terkawi, Mohamed Ahmed Youssef, El Said El Shirbini El Said, Gehad Elsayed, Sabry El-Khodery, Maged El-Ashker, Ahmed Elsify, Mosaab Omar, Akram Salama, Naoaki Yokoyama, Ikuo Igarashi
PloS one, 10, 4, e0125276, Public Library of Science, 2015年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

Macrophages are the determinant of resistance to and outcome of nonlethal Babesia microti infection in mice.
Mohamad Alaa Terkawi, Shinuo Cao, Maria S Herbas, Maki Nishimura, Yan Li, Paul Franck Adjou Moumouni, Asadullah Hamid Pyarokhil, Daisuke Kondoh, Nobuo Kitamura, Yoshifumi Nishikawa, Kentaro Kato, Naoaki Yokoyama, Jinlin Zhou, Hiroshi Suzuki, Ikuo Igarashi, Xuenan Xuan
Infection and immunity, 83, 1, 8, 16, AMER SOC MICROBIOLOGY, 2015年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In the present study, we examined the contributions of macrophages to the outcome of infection with Babesia microti, the etiological agent of human and rodent babesiosis, in BALB/c mice. Mice were treated with clodronate liposome at different times during the course of B. microti infection in order to deplete the macrophages. Notably, a depletion of host macrophages at the early and acute phases of infection caused a significant elevation of parasitemia associated with remarkable mortality in the mice. The depletion of macrophages at the resolving and latent phases of infection resulted in an immediate and temporal exacerbation of parasitemia coupled with mortality in mice. Reconstituting clodronate liposome-treated mice at the acute phase of infection with macrophages from naive mice resulted in a slight reduction in parasitemia with improved survival compared to that of mice that received the drug alone. These results indicate that macrophages play a crucial role in the control of and resistance to B. microti infection in mice. Moreover, analyses of host immune responses revealed that macrophage-depleted mice diminished their production of Th1 cell cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Furthermore, depletion of macrophages at different times exaggerated the pathogenesis of the infection in deficient IFN-γ(-/-) and severe combined immunodeficiency (SCID) mice. Collectively, our data provide important clues about the role of macrophages in the resistance and control of B. microti and imply that the severity of the infection in immunocompromised patients might be due to impairment of macrophage function.

Cloning and characterization of aspartic protease 3 of Toxoplasma gondii.
Kamyingkird K, Goo YK, Cao S, Adjou Moumouni PF, Aboge GO, Yamagishi J, Terkawi MA, Masatani T, Yu L, Nishikawa Y, Xuan X
Journal of Protozoology Research, 24, 1, 18, 25, National Research Center for Protozoan Diseases, 2014年12月
英語, 研究論文（学術雑誌）

Seroprevalence of Babesia caballi and Theileria equi in horses and mules from Northern Thailand
Kamyingkird K, Yangtara S, Desquesnes M, Cao S, Adjou Moumouni PF, Jittapalapong S, Nimsupan B, Terkawi MA, Masatani T, Nishikawa Y, Igarashi I, Xuan X
Journal of Protozoology Research, 24, 1, 11, 17, National Research Center for Protozoan Diseases, 2014年12月
英語, 研究論文（学術雑誌）

Plasmodium berghei circumsporozoite protein encapsulated in oligomannose-coated liposomes confers protection against sporozoite infection in mice.
Mohamad Alaa Terkawi, Yasuhiro Kuroda, Shinya Fukumoto, Sachi Tanaka, Naoya Kojima, Yoshifumi Nishikawa
Malaria journal, 13, 426, 426, BIOMED CENTRAL LTD, 2014年11月05日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: The design and development of an effective malaria vaccine against the pre-erythrocytic and erythrocytic-stages of infection present a great challenge. METHODS: In the present study, protective efficacy of oligomannose-coated liposome (OML)-entrapped merozoite and sporozoite antigens against Plasmodium berghei challenge infection in BALB/c mice was evaluated. RESULTS: Subcutaneous immunization with truncated merozoite surface protein 1 entrapped with OML (OML-PbMSP1) prolonged survival, but failed to protect the mice from erythrocytic-stage infection, despite the antigen-specific antibody responses induced by the immunization regimen. In contrast, immunization with circumsporozoite protein entrapped with OML (OML-PbCSP) elicited antigen-specific humoral and cellular responses, which correlated with substantial protection against sporozoite challenge infections. CONCLUSIONS: The current results represent the use of an oligomannose-coated liposome-based vaccine against pre-erythrocytic and erythrocytic stages malaria infection. This approach may offer a new vaccination strategy against malaria infection.

Molecular and seroepidemiological survey of Babesia bovis and Babesia bigemina infections in cattle and water buffaloes in the central region of Vietnam.
Yan Li, Yuzi Luo, Shinuo Cao, Mohamad Alaa Terkawi, Dinh Thi Bich Lan, Phung Thang Long, Longzheng Yu, Mo Zhou, Haiyan Gong, Houshuang Zhang, Jinlin Zhou, Naoaki Yokoyama, Hiroshi Suzuki, Xuenan Xuan
Tropical biomedicine, 31, 3, 406, 13, Malaysian Society for Parasitology, 2014年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In the present study, a total of 137 blood samples were collected from cattle and water buffaloes in central region of Vietnam and tested using nested polymerase chain reaction (nPCR), enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT) to determine the molecular and serological prevalence of Babesia bovis and Babesia bigemina. In cattle, the prevalence of B. bovis and B. bigemina was 21.3% and 16.0% by nPCR, 73.4% and 42.6% by ELISA and 60.6% and 59.6% by IFAT, respectively, whereas those of water buffalos were 23.3% and 0% by nPCR, 37.2% and 9.3% by ELISA and 27.9% and 18.6% by IFAT, respectively. IFAT and ELISA detected a higher number of infected cattle and water buffaloes than nPCR totally. Statistically significant differences in the prevalence of the two infections were observed on the basis of age. Overall, the current data suggest high incidence of B. bovis and B. bigemina infections in the central region of Vietnam, which is needed to develop comprehensive approach to the modern surveillance, diagnosis and control of bovine babesiosis.

Mycophenolic acid, mycophenolate mofetil, mizoribine, ribavirin, and 7-nitroindole inhibit propagation of Babesia parasites by targeting inosine 5'-monophosphate dehydrogenase.
Shinuo Cao, Gabriel Oluga Aboge, Mohamad Alaa Terkawi, Mo Zhou, Ketsarin Kamyingkird, Paul Franck Adjou Moumouni, Tatsunori Masatani, Ikuo Igarashi, Yoshifumi Nishikawa, Hiroshi Suzuki, Xuenan Xuan
The Journal of parasitology, 100, 4, 522, 6, American Society of Parasitologists, 2014年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The resistance of Babesia parasites to current anti-babesiosis drugs is an issue of major concern. The inosine 5'-monophosphate dehydrogenase (IMPDH) of Babesia gibsoni has been identified and characterized as a molecular drug target in our previous studies. In the present study, inhibitory effects of IMPDH inhibitors (mycophenolate mofetil, mizoribine, ribavirin, 7-nitroindole, and mycophenolic acid) were evaluated in vitro or in vivo. In the inhibition assay of recombinant B. gibsoni IMPDH activity, mycophenolate mofetil was the most potent inhibitor (IC(50) = 2.58 ± 1.32 μM) while ribavirin was the least potent. The inhibitory effects of mycophenolate mofetil, mizoribine, ribavirin, and 7-nitroindole on the in vitro growths of B. gibsoni and Babesia bovis were also assessed. The results revealed that mycophenolate mofetil was the most potent inhibitor of the multiplications of both B. gibsoni (IC(50) = 0.13 ± 0.05 μM) and B. bovis (IC(50) = 0.97 ± 0.49 μM). Ribavirin was also the least potent for both B. gibsoni and B. bovis in vitro. Mycophenolic acid, a metabolite of mycophenolate mofetil, caused an inhibition of Babesia microti in mice with noticeable improvement in hematological parameters of the infected mice (ED(50) = 44.15 ± 12.53 mg/kg). Although the report provides a non-exhaustive view of potential treatment strategy without addressing the potential adverse effect of immune suppression on infections, these results indicated that the IMPDH might be a molecular target of MPA for B. microti . Altogether, we provide a basis for development of antibabesia prodrugs by targeting IMPDH of the parasites in the treatment of babesiosis.

Babesia bovis dihydroorotate dehydrogenase (BboDHODH) is a novel molecular target of drug for bovine babesiosis.
Ketsarin Kamyingkird, Shinuo Cao, Tatsunori Masatani, Paul Franck Adjou Moumouni, Patrick Vudriko, Ahmed Abd El Moniem Mousa, Mohamad Alaa Terkawi, Yoshifumi Nishikawa, Ikuo Igarashi, Xuenan Xuan
The Journal of veterinary medical science, 76, 3, 323, 30, Japanese Society of Veterinary Science, 2014年03月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The emergence of drug resistance and adverse side effects of current bovine babesiosis treatment suggest that the search of new drug targets and development of safer and effective compounds are required. This study focuses on dihydroorotate dehydrogenase (DHODH), the fourth enzyme of pyrimidine biosynthesis pathway as a potential drug target for bovine babesiosis. Recombinant Babesia bovis DHODH protein (rBboDHODH) was produced in Escherichia coli and used for characterization and measurement of enzymatic activity. Furthermore, the effects of DHODH inhibitors were evaluated in vitro. The recombinant B. bovis DHODH histidine fusion protein (rBboDHODH) had 42.4-kDa molecular weight and exhibited a specific activity of 475.7 ± 245 Unit/mg, a Km = 276.2 µM for L-dihydroorotate and a Km= 94.41 µM for decylubiquinone. A 44-kDa band of native BboDHODH was detected by Western blot analysis and found in parasites mitochondria using a confocal microscope. Among DHODH inhibitors, atovaquone (ATV) and leflunomide (LFN) significantly inhibited the activity of rBboDHODH as well as the growth of B. bovis in vitro. The half maximal inhibitory concentration (IC50) of ATV and LFN was 2.38 ± 0.53 nM and 52.41 ± 11.47 µM, respectively. These results suggest that BboDHODH might be a novel target for development of new drug for treatment of B. bovis infection.

Inhibitory effect of allicin on the growth of Babesia and Theileria equi parasites.
Akram Ahmed Salama, Mahmoud AbouLaila, Mohamad Alaa Terkawi, Ahmed Mousa, Ahmed El-Sify, Mahmoud Allaam, Ahmed Zaghawa, Naoaki Yokoyama, Ikuo Igarashi
Parasitology research, 113, 1, 275, 83, SPRINGER, 2014年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Allicin is an active ingredient of garlic that has antibacterial, antifungal, antiviral, and antiprotozoal activity. However, the inhibitory effects of allicin on Babesia parasites have not yet been examined. In the present study, allicin was tested as a potent inhibitor against the in vitro growth of bovine and equine Babesia parasites and the in vivo growth of Babesia microti in a mouse model. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia caballi, or Theileria equi was inhibited by allicin in a dose-dependent manner and had IC50 values of 818, 675, 470, and 742 μM, respectively. Moreover, allicin significantly inhibited (P < 0.001) invasion of B. bovis, B. bigemina, B. caballi, and T. equi into the host erythrocyte. Furthermore, mice treated with 30 mg/kg of allicin for 5 days significantly (P < 0.05) reduced the parasitemia of B. microti over the period of the study. To further examine the potential synergism of allicin with diminazene aceturate, growth inhibitory assays were performed in vitro and in vivo. Interestingly, combinations of diminazene aceturate with allicin synergistically potentiated its inhibitory effects in vitro and in vivo. These results indicate that allicin might be beneficial for the treatment of babesiosis, particularly when used in combination with diminazene aceturate.

Cloning and characterization of a 2-Cys peroxiredoxin from Babesia gibsoni
Tatsunori Masatani, Masahito Asada, Madoka Ichikawa-Seki, Miho Usui, Mohamad A. Terkawi, Kei Hayashi, Shin-Ichiro Kawazu, Xuenan Xuan
Journal of Veterinary Medical Science, 76, 1, 139, 143, 2014年01月, ［査読有り］
英語, Peroxiredoxins (Prxs) are a family of antioxidant enzymes. Here, we cloned a 2-Cys Prx, BgTPx-1, from the canine Babesia parasite B. gibsoni. Sequence identity between BgTPx-1 and 2-Cys Prx of B. bovis was 81% at the amino acid level. Enzyme activity assay by using recombinant BgTPx-1 (rBgTPx-1) indicated that BgTPx-1 has antioxidant activity. Antiserum from a mouse immunized with rBgTPx-1 reacted with parasite lysates and detect a protein with a monomeric size of 22 kDa and also a 44 kDa protein, which might be an inefficiently reduced dimer. BgTPx-1 was expressed in the cytoplasm of B. gibsoni merozoites. These results suggest that the BgTPx-1 may play a role to control redox balance in the cytoplasm of B. gibsoni. © 2014 The Japanese Society of Veterinary Science.

Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target.
Patrick Vudriko, Tatsunori Masatani, Shinuo Cao, Mohamad Alla Terkawi, Ketsarin Kamyingkird, Ahmed A Mousa, Paul F Adjou Moumouni, Yoshifumi Nishikawa, Xuenan Xuan
Drug target insights, 8, 8, 31, 8, Libertas Academica Ltd., 2014年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Babesia microti is an emerging zoonotic protozoan organism that causes "malaria-like" symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection.

Prime-boost vaccination with plasmid DNA followed by recombinant vaccinia virus expressing BgGARP induced a partial protective immunity to inhibit Babesia gibsoni proliferation in dogs.
Shinuo Cao, Ahmed Abdelmoniem Mousa, Gabriel Oluga Aboge, Ketsarin Kamyingkird, Mo Zhou, Paul Franck Adjou Moumouni, Mohamad Alaa Terkawi, Tatsunori Masatani, Yoshifumi Nishikawa, Hiroshi Suzuki, Shinya Fukumoto, Xuenan Xuan
Acta parasitologica, 58, 4, 619, 23, 2013年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs.

Molecular and serological prevalence of Babesia bigemina and Babesia bovis in cattle and water buffalos under small-scale dairy farming in Beheira and Faiyum Provinces, Egypt.
Hany M Ibrahim, Paul F Adjou Moumouni, Khaled Mohammed-Geba, Sherin K Sheir, Ihab S Y Hashem, Shinuo Cao, Mohamad A Terkawi, Ketsarin Kamyingkird, Yoshifumi Nishikawa, Hiroshi Suzuki, Xuenan Xuan
Veterinary parasitology, 198, 1-2, 187, 92, 2013年11月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In order to determine the molecular and serological prevalence of Babesia bigemina and Babesia bovis, a total of 247 blood samples were collected from cattle and water buffalos in Beheira and Faiyum Provinces in Egypt and examined by nested polymerase chain reaction (nPCR) and enzyme-linked immunosorbent assay (ELISA). In cattle, the prevalence of B. bigemina and B. bovis was 5.30% and 3.97% by nPCR and 10.60% and 9.27% by ELISA, respectively, whereas those of water buffalos were 10.42% and 4.17% by nPCR and 15.63% and 11.46% by ELISA, respectively. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and health status. Sequencing analysis revealed two genotypes for B. bovis spherical body protein-4. In conclusion, the current data provide valuable information regarding the epidemiology of B. bigemina and B. bovis infections in cattle and water buffalos from Egypt, which can be employed in developing future strategies for disease management and control.

Specific antibody to a conserved region of Babesia apical membrane antigen-1 inhibited the invasion of B. bovis into the erythrocyte.
Akram Ahmed Salama, Mohamad Alaa Terkawi, Satoru Kawai, Mahmoud Aboulaila, Mohamed Nayel, Ahmed Mousa, Ahmed Zaghawa, Naoaki Yokoyama, Ikuo Igarashi
Experimental parasitology, 135, 3, 623, 8, 2013年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Apical membrane antigen-1 (AMA-1) is a microneme protein that exists in all apicomplexan parasites and plays an indispensable role in the invasion into host cell. Central region of ectodomains I and II of Babesia bovis apical membrane antigen-1 (BbAMA-1P) is highly conserved with these of Babesia species and may be beneficial for vaccine development against babesiosis. In the present study, recombinant protein encoding the central region of B. bovis AMA-1 (rBbAMA-1P) was produced in Escherichia coli and its antiserum was prepared in mice for further molecular characterization. Anti-rBbAMA-1P serum specifically reacted with corresponding authentic protein of B. bovis as determined by Western blotting and IFAT. Cultured B. bovis treated with anti-rBbAMA-1P serum showed significant reduction in the in vitro growth of the parasites. Moreover, preincubated free merozoites with 1mg/ml anti-rBbAMA-1P serum inhibited their efficiency in the invasion into erythrocytes (RBCs) by 61% and 70% at 3h and 6h, respectively. Our data suggest that the central region of domains I and II of BbAMA-1 may serve as a vaccine candidate against babesiosis.

Identification of a highly antigenic region of subtilisin-like serine protease 1 for serodiagnosis of Neospora caninum infection.
Rochelle Haidee D Ybañez, Mohamad Alaa Terkawi, Kyohko Kameyama, Xuenan Xuan, Yoshifumi Nishikawa
Clinical and vaccine immunology : CVI, 20, 10, 1617, 22, 2013年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Neospora caninum is an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that the N. caninum subtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool for Neospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) to identify the antigenic regions. The serodiagnostic performances of NcSUB1t and NcSUB1tr were compared with that of N54, which contains a single copy of the repeats (aa 649 to 784), and with the truncated NcSAG1 (NcSAG1t), which lacks a signal peptide and C-terminal hydrophobic regions, as a positive reference. Serum samples from N. caninum experimentally infected cattle and mice and cattle from a farm with confirmed cases of Neospora abortion were tested by enzyme-linked immunosorbent assay (ELISA) with the four antigens. In the N. caninum experimentally infected cattle, the highest IgG1 antibody titers were detected against NcSUB1t, while specific IgG1 antibodies were detectable from 16 days postinfection (dpi), with levels peaking at 36 dpi for all of the antigens. On the other hand, the levels of anti-NcSUB1 IgG2 antibodies were lower than those of anti-SAG1t IgG2 antibodies. The ELISA with NcSUB1t and NcSUB1tr had good sensitivity (94.59 to 95.95%) and specificity (80 to 100%) with bovine serum field samples compared to NcSAG1t and showed no cross-reactions with sera from Toxoplasma gondii experimentally infected mice. Moreover, IgG antibodies against NcSUB1t were detected during parturition in the NcSAG1t antibody-positive cattle, and NcSUB1t-specific antibody transfer was observed from a mother to her calf. Our results show that the NcSUB1 tandem repeat is potentially useful for serodiagnosis of N. caninum.

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).
Ahmed Abdelmoniem Mousa, Shinuo Cao, Gabriel Oluga Aboge, Mohamad Alaa Terkawi, Ahmed El Kirdasy, Akram Salama, Mabrouk Attia, Mahmoud Aboulaila, Mo Zhou, Ketsarin Kamyingkird, Paul Franck Adjou Moumouni, Tatsunori Masatani, Sami Ahmed Abd El Aziz, Waheed Mohammed Moussa, Bayin Chahan, Shinya Fukumoto, Yoshifumi Nishikawa, Salah Sayed El Ballal, Xuenan Xuan
Experimental parasitology, 135, 2, 414, 20, 2013年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.

Actin polymerization mediated by Babesia gibsoni aldolase is required for parasite invasion.
Youn-Kyoung Goo, Akio Ueno, Mohamad Alaa Terkawi, G Oluga Aboge, Yamagishi Junya, Makoto Igarashi, Jung-Yeon Kim, Yeon-Chul Hong, Dong-Il Chung, Yoshifumi Nishikawa, Xuenan Xuan
Experimental parasitology, 135, 1, 42, 9, 2013年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Host cell invasion by apicomplexan parasites driven by gliding motility and empowered by actin-based movement is essential for parasite survival and pathogenicity. The parasites share a conserved invasion process: actin-based motility led by the coordination of adhesin-cytoskeleton via aldolase. A number of studies of host cell invasion in the Plasmodium species and Toxoplasma gondii have been performed. However, the mechanisms of host cell invasion by Babesia species have not yet been studied. Here, we show that Babesia gibsoni aldolase (BgALD) forms a complex with B. gibsoni thrombospondin-related anonymous protein (BgTRAP) and B. gibsoni actin (BgACT), depending on tryptophan-734 (W-734) in BgTRAP. In addition, actin polymerization is mediated by BgALD. Moreover, cytochalasin D, which disrupts actin polymerization, suppressed B. gibsoni parasite growth and inhibited the host cell invasion by parasites, indicating that actin dynamics are essential for erythrocyte invasion by B. gibsoni. This study is the first molecular approach to determine the invasion mechanisms of Babesia species.

TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network.
Tatsunori Masatani, Tomohide Matsuo, Tetsuya Tanaka, Mohamad Alaa Terkawi, Eung-Goo Lee, Youn-Kyoung Goo, Gabriel Oluga Aboge, Junya Yamagishi, Kei Hayashi, Kyohko Kameyama, Shinuo Cao, Yoshifumi Nishikawa, Xuenan Xuan
Parasitology international, 62, 4, 372, 9, 2013年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV.

Identification, cloning and characterization of BmP41, a common antigenic protein of Babesia microti.
Tatsunori Masatani, Hideo Ooka, Mohamad A Terkawi, Shinuo Cao, Yuzi Luo, Masahito Asada, Kei Hayashi, Yoshifumi Nishikawa, Xuenan Xuan
The Journal of veterinary medical science, 75, 7, 967, 70, 2013年07月31日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Babesia microti is a rodent tick-borne blood parasite and the major causative agent of emerging human babesiosis. Here, we identified a candidate of common antigenic protein BmP41 of B. microti by serological screening of cDNA library of human-pathogenic Gray strain with antisera against rodent Munich strain. Immunofluorescent antibody test using mouse anti-recombinant BmP41 (rBmP41) serum revealed that native BmP41 was expressed in each of the developmental stages of B. microti merozoites. An enzyme-linked immunosorbent assay (ELISA) using rBmP41 detected specific antibodies in sera from hamsters infected with B. microti Gray strain and mice infected with B. microti Munich strain. Taken together, BmP41 could be a promising universal serological marker for diagnosis of human babesiosis.

Epidemiological survey of Babesia bovis and Babesia bigemina infections of cattle in Philippines.
Longzheng Yu, Mohmad Alaa Terkawi, Mary Jane Cruz-Flores, Florencia G Claveria, Gabriel Oluga Aboge, Junya Yamagishi, Youn-Kyoung Goo, Shinuo Cao, Tatsunori Masatani, Yoshifumi Nishikawa, Xuenan Xuan
The Journal of veterinary medical science, 75, 7, 995, 8, 2013年07月31日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, A total of 250 blood samples were collected from clinically healthy cattle in five provinces of Philippines. DNA was extracted from the samples and analyzed by nested PCR assays for an epidemiological survey of Babesia bovis and Babesia bigemina infections. Out of the 250 samples, 27 (10.8%) and 16 (6.4%) were positive for B. bovis infection and B. bigemina infection, respectively. Mixed infections were detected in a total of 4 samples (1.6%). Our data provide baseline information regarding the epidemiology of B. bovis and B. bigemina infections in cattle in Philippines, which can be utilized in developing proper strategies for disease control and management.

Prevalence and genetic diversity of equine piroplasms in Tov province, Mongolia.
Tserendorj Munkhjargal, Thillaiampalam Sivakumar, Badgar Battsetseg, Tserendorj Nyamjargal, Mahmoud Aboulaila, Byambaa Purevtseren, Dorj Bayarsaikhan, Badarch Byambaa, Mohamad Alaa Terkawi, Naoaki Yokoyama, Ikuo Igarashi
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 16, 178, 85, 2013年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Equine piroplasmosis represents a serious problem in horse industry. Although, researchers suggested the possible use of sub-unit vaccines to control equine piroplasmosis, the genetic diversity of vaccine candidate antigens was not properly investigated. In the present study, we screened 250 horses reared in three different districts of Tov province, Mongolia, for Babesia caballi and Theileria equi using ELISA and nested PCR (nPCR) assays. Among these animals, piroplasms were detected in 128 (51.2%) horses by nPCR assays (B. caballi, 42.4%; T. equi, 6.4%; and mixed infections, 2.4%), while 204 (81.6%) were positive by ELISA (B. caballi, 51.6%; T. equi, 19.6%; and mixed infections, 10.4%). Male and middle-aged horses showed higher positive rates than female and younger or older horses. The findings also suggested that a combination of nPCR and ELISA techniques might be useful to detect horses that were chronically or subclinically infected with piroplasms. B. caballi-BC48 and T. equi-EMA-1 gene sequences, in addition to 18S rRNA, were subjected to phylogenetic analyses, and the findings suggested the presence of genetically diverse populations of equine piroplasms in Mongolia. BC48 sequences were separated into four clades in phylogram, and all the Mongolian sequences determined in the present study were found in a single clade. However, a single BC48 sequence previously isolated from a tick in Mongolia formed a separate branch. Similarly, EMA-1 sequences formed four clades, and Mongolian sequences were observed in two different clades, one of which was formed only of Mongolian sequences and is suggested as a new clade. This is the first report that describes the genotypes of equine piroplasms in Mongolia. The findings also emphasized the need for further investigations to study the effect of genetic diversity observed among BC48 as well as EMA-1 sequences on host's immune responses.

Development of an immunochromatographic assay based on dense granule protein 7 for serological detection of Toxoplasma gondii infection.
Mohamad Alaa Terkawi, Kyohko Kameyama, Nazim Hamza Rasul, Xuean Xuan, Yoshifumi Nishikawa
Clinical and vaccine immunology : CVI, 20, 4, 596, 601, 2013年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Dense granule antigen proteins derived from Toxoplasma gondii (TgGRAs) are potential antigens for the development of diagnostic tools. TgGRA7 and TgGRA14 were detected in the peritoneal fluid of T. gondii-infected mice, suggesting that TgGRAs may be highly antigenic proteins. Here, TgGRA7 and TgGRA14 were evaluated as candidates for the development of a marker for a rapid diagnostic test. The specificity and sensitivity of purified recombinant proteins of TgGRA7 and TgGRA14 were compared in an indirect enzyme-linked immunosorbent assay (iELISA) using a series of serum samples from T. gondii-experimentally infected mice and using recombinant T. gondii major surface antigen 2 (TgSAG2) as a reference control. The iELISA with TgGRA7 showed the greatest diagnostic accuracy and could detect anti-TgGRA7 antibody in acute and chronic infections. A total of 59 field samples from pigs were also examined by the iELISAs, and the results compared with those of the latex agglutination test (LAT). Among the three recombinant antigens, TgGRA7 had the highest rates of positivity, with significant concordance (88.14) and kappa value (0.76) in comparison with the results using LAT. Furthermore, an immunochromatographic test (ICT) based on recombinant TgGRA7 was developed for rapid detection of antibodies to the infection. The ICT differentiated clearly between sera from T. gondii-infected mice and uninfected or Neospora caninum-infected mice. Pig sera were examined with the ICT, and the results compared favorably with those of LAT and iELISA for TgGRA7, with kappa values of 0.66 and 0.70 to 0.79, respectively. These data suggest that the ICT based on TgGRA7 is a promising diagnostic tool for routine testing in the clinic and mass screening of samples in the field.

Farsenyl pyrophosphate synthase is a potential molecular drug target of risedronate in Babesia bovis.
Akio Ueno, Mohamad Alaa Terkawi, Miki Yokoyama, Shinuo Cao, Gabriel Aboge, Mahmoud Aboulaila, Yoshifumi Nishikawa, Xuenan Xuan, Naoaki Yokoyama, Ikuo Igarashi
Parasitology international, 62, 2, 189, 92, 2, 2013年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011bp with predicted 336 amino acids and molecular mass of 38kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the Km value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophosphate was 2.494±1.536μM. Risedronate inhibited the activity of BbFPPS yielding IC50 value of 8.4±1.2nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar concentration of risedronate (IC50=4.02±0.91μM). No regrowth of B. bovis was observed at 10μM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis.

Cloning, characterization and validation of inosine 5'-monophosphate dehydrogenase of Babesia gibsoni as molecular drug target.
Shinuo Cao, Gabriel Oluga Aboge, Mohamad Alaa Terkawi, Mo Zhou, Yuzi Luo, Longzheng Yu, Yan Li, Younkyoung Goo, Ketsarin Kamyingkird, Tatsunori Masatani, Hiroshi Suzuki, Ikuo Igarashi, Yoshifumi Nishikawa, Xuenan Xuan
Parasitology international, 62, 2, 87, 94, 2013年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The inosine monophosphate dehydrogenase (IMPDH) enzyme has been characterized and validated as a molecular drug target in other apicomplexans but not in the genus Babesia. Subsequently, we cloned and expressed a Babesia gibsoni IMPDH (BgIMPDH) cDNA in Escherichia coli. We also determined the inhibitory effect of mycophenolic acid (MPA) on recombinant BgIMPDH (rBgIMPDH) activity and the Babesia-growths in vitro. The translated BgIMPDH peptide contained thirteen amino acid residues responsible for substrate and cofactor binding in its catalytic domain with Gly374 in BgIMPDH being replaced by Ser388 in mammalian IMPDH. The native BgIMPDH enzyme in the parasite was approximately 54-kDa a mass similar to His-tag rBgIMPDH protein. The Km values of the rBgIMPDH were 8.18±0.878 (mean±standard error of the mean) μM and 360.80±43.41μM for IMP and NAD(+), respectively. MPA inhibited the rBgIMPDH activity yielding a Ki value of 20.93±1.83μM with respect to NAD(+). For Babesia growths, the IC50s were 0.95±0.21 and 2.88±0.49μM for B. gibsoni and B. bovis, respectively. Therefore, our results suggest that MPA may inhibit the replication of Babesia parasites by targeting IMPDH enzyme of the purine pathway.

Identification and characterization of an interspersed repeat antigen of Babesia microti (BmIRA).
Shinuo Cao, Yuzi Luo, Gabriel Oluga Aboge, Mohamad Alaa Terkawi, Tatsunori Masatani, Hiroshi Suzuki, Ikuo Igarashi, Yoshifumi Nishikawa, Xuenan Xuan
Experimental parasitology, 133, 3, 346, 52, 2013年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In this report, a novel gene encoding an interspersed repeat antigen from Babesia microti (BmIRA) was identified and described. The full-length cDNA containing an open reading frame of 1,947 bp was obtained by immunoscreening a B. microti cDNA expression library. The full-length of BmIRA gene was expressed as a GST fusion recombinant BmIRA (rBmIRA) in Escherichia coli. Sera of mice immunized with the rBmIRA detected a native parasite protein with a molecular mass of 76 kDa on Western blot analysis. The same protein was detected in the parasites by immunofluorescent antibody test (IFAT). An enzyme-linked immunosorbent assay (ELISA) using rBmIRA detected specific antibodies as early as 11 days post-infection in sera from a hamster experimentally infected with B. microti Gray stain (US type). Furthermore, a rapid immunochromatographic test (ICT) using rBmIRA detected specific antibodies in a hamster experimentally infected with B. microti from day 11 to at least day 180 post-infection. The results indicate the antibody response against the rBmIRA was maintained during the chronic stage of infection. On the other hand, an immunoprotective property of rBmIRA as a subunit vaccine was evaluated in hamsters against B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmIRA could be a useful serodiagnostic antigen for screening of B. microti infection.

Evaluation of in vitro and in vivo inhibitory effects of fusidic acid on Babesia and Theileria parasites.
Akram Ahmed Salama, Mahmoud Aboulaila, Ahmed A Moussa, Mohamed A Nayel, Ahmed El-Sify, Mohamad A Terkawi, Hany Y Hassan, Naoaki Yokoyama, Ikuo Igarashi
Veterinary parasitology, 191, 1-2, 1, 10, 1-2, 2013年01月16日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Fusidic acid known to has antibacterial, antifungal, and antimalarial activities. Fusidic acid blocks translation elongation factor G gene in Plasmodium falciparum. In the present study, the inhibitory effects of fusidic acid on the in vitro growth of bovine and equine Babesia parasites were evaluated. The inhibitory effect of fusidic acid on the in vivo growth of Babesia microti was also assessed. The in vitro growth of four Babesia species that were tested was significantly inhibited (P<0.05) by micromolar concentrations of fusidic acid (IC(50) values=144.8, 17.3, 33.3, and 56.25 μM for Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, respectively). Combinations of fusidic acid with diminazene aceturate synergistically potentiated its inhibitory effects in vitro on B. bovis and B. caballi. In B. microti-infected mice, fusidic acid caused significant (P<0.05) inhibition of the growth of B. microti at the dose of 500 mg/kg BW relative to control group. These results indicate that fusidic acid might be incorporated in treatment of babesiosis.

Molecular characterization of a new Babesia bovis thrombospondin-related anonymous protein (BbTRAP2).
Mohamad Alaa Terkawi, Jadsada Ratthanophart, Akram Salama, Mahmoud AbouLaila, Masahito Asada, Akio Ueno, Hend Alhasan, Azirwan Guswanto, Tatsunori Masatani, Naoaki Yokoyama, Yoshifumi Nishikawa, Xuenan Xuan, Ikuo Igarashi
PloS one, 8, 12, e83305, 2013年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite's growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

Molecular detection of Theileria species in sheep from northern China.
Shinuo Cao, Shoufa Zhang, Lijun Jia, Shujiang Xue, Longzheng Yu, Ketsarin Kamyingkird, Paul Franck Adjou Moumouni, Ahmed Abd El Moniem Moussa, Mo Zhou, Yuanming Zhang, Mohamad Alaa Terkawi, Tatsunori Masatani, Yoshifumi Nishikawa, Xuenan Xuan
The Journal of veterinary medical science, 75, 9, 1227, 30, 2013年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Ovine theileriosis is a tick-borne disease that restricts the development of small ruminant husbandry in northern China. In this study, we report on a molecular epidemiological survey of ovine Theileria spp. in 198 blood samples taken from sheep in northern China. The DNA samples were screened by a nested polymerase chain reaction (PCR) targeting the 18S rRNA gene of ovine Theileria spp. The prevalence of ovine Theileria spp. in Yanji, Nongan, Longjing, Toudao and Jinchang was 80%, 40%, 37%, 24% and 32%, respectively. The sequencing analyses approved the present of the T. orientalis and/or T. luwenshuni in these regions. Taken together, we have demonstrated a high incidence of Theileria spp. in northern China that calls for the need to design effective control programs for ovine theileriosis.

Expression and immunological characterization of the heat shock protein-70 homologue from Babesia bigemina.
Mahmoud AbouLaila, Mohamad Alaa Terkawi, Faasoa Junior Seuseu, Naomi Ota, Alan Caine Costa de Macedo, Naoaki Yokoyama, Xuenan Xuan, Ikuo Igarashi
Veterinary parasitology, 190, 3-4, 401, 10, 3-4, 2012年12月21日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The Babesia bigemina heat shock protein-70 gene (BbigHSP-70) was cloned from cDNA by polymerase chain reaction (PCR) and sequenced. The length of the gene is 1947 bp and the predicted polypeptide is 649 amino acids long with a calculated molecular weight of 70.85 kDa. BbigHSP-70 has a signal peptide of 15 amino acids. Phylogenetic analysis of the amino acid sequence of BbigHSP-70 showed that B. bigemina was most closely related to B. caballi and B. bovis and lies within a phylogenetic cluster with Theileria. rBbigHSP-70 was expressed in E. coli as a soluble GST-fusion protein with a molecular mass of 96.8-kDa. The serum raised in mice against rBbigHSP-70 detected the native protein in B. bigemina, B. bovis, B. caballi, B. gibsoni, and B. microti lysates and also reacted with B. bigemina, B. bovis, and B. caballi merozoites in the IFAT. Mice vaccinated with rBbigHSP-70 showed lower parasitemia against the challenge infection with B. microti than GST-vaccinated and non-vaccinated controls. These results added a new member of Babesia heat shock proteins70 that is well conserved among intraerythrocytic protozoa and demonstrated its protective effects in an experimental model of rodent babesiosis.

Development of a rapid immunochromatographic test using a recombinant thrombospondin-related adhesive protein of Babesia gibsoni.
Youn-Kyoung Goo, Naeun Lee, Mohamad Alaa Terkawi, Yuzi Luo, Gabriel Oluga Aboge, Yoshifumi Nishikawa, Hiroshi Suzuki, Suk Kim, Xuenan Xuan
Veterinary parasitology, 190, 3-4, 595, 8, 2012年12月21日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We developed an immunochromatographic test (ICT) with the full-length of thrombospondin-related adhesive protein of Babesia gibsoni expressed by the modified expression method. The developed ICT showed high sensitivity, specificity, and kappa value with a reference test (100%, 93.78%, and 0.8976, respectively), indicating that the ICT could be a new practical diagnostic test for B. gibsoni infection.

Molecular and immunological characterization of a novel 32-kDa secreted protein of Babesia microti.
Hideo Ooka, Mohamad A Terkawi, Shinuo Cao, Gabriel Aboge, Youn-Kyoung Goo, Yuzi Luo, Yan Li, Yoshifumi Nishikawa, Ikuo Igarashi, Xuenan Xuan
The Journal of parasitology, 98, 5, 1045, 8, 5, 2012年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A cDNA encoding the Babesia microti 32-kDa protein was identified by serological immunoscreening of a cDNA expression library and designated as BmP32. The full length of BmP32 contains an open reading frame of 918 base pairs consisting of 306 amino acids having a significant homology with B. microti secreted antigen 1. Antiserum raised against recombinant protein (rBmP32) specifically reacted with a 32-kDa native protein of the parasite lysate using western blot analysis. The indirect immunofluorescent antibody test showed a preferable localization of BmP32 in the cytoplasm of the intra- and extracellular parasites. Moreover, BmP32 was secreted in the cytosol of infected erythrocytes, especially during the peak parasitemia and the recovery phase of the infection. Next, the antigenicity of rBmP32 was examined by an enzyme-linked immunosorbent assay (ELISA) and sera from mice experimentally infected with either B. microti or closely related parasites. ELISA was highly specific and sensitive when used for the detection of B. microti antibody in a mouse model. Furthermore, mice immunized with rBmP32 emulsified with Freund's adjuvant were not significantly protected against challenge infection with B. microt i. However, high antibody titer was detected just before the challenge infection. Our data suggest that rBmP32 may be a specific diagnostic antigen but not a subunit vaccine.

Inhibitory effects of pepstatin A and mefloquine on the growth of Babesia parasites.
Tserendorj Munkhjargal, Mahmoud AbouLaila, Mohamad Alaa Terkawi, Thillaiampalam Sivakumar, Madoka Ichikawa, Batdorj Davaasuren, Tserendorj Nyamjargal, Naoaki Yokoyama, Ikuo Igarashi
The American journal of tropical medicine and hygiene, 87, 4, 681, 8, 4, 2012年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We evaluated the inhibitory effects of pepstatin A and mefloquine on the in vitro and in vivo growths of Babesia parasites. The in vitro growth of Babesia bovis, B. bigemina, B. caballi, and B. equi was significantly inhibited (P < 0.05) by micromolar concentrations of pepstatin A (50% inhibitory concentrations = 38.5, 36.5, 17.6, and 18.1 μM, respectively) and mefloquine (50% inhibitory concentrations = 59.7, 56.7, 20.7, and 4 μM, respectively). Furthermore, both reagents either alone at a concentration of 5 mg/kg or in combinations (2.5/2.5 and 5/5 mg/kg) for 10 days significantly inhibited the in vivo growth of B. microti in mice. Mefloquine treatment was highly effective and the combination treatments were less effective than other treatments. Therefore, mefloquine may antagonize the actions of pepstatin A against babesiosis and aspartic proteases may play an important role in the asexual growth cycle of Babesia parasites.

C-Terminal region of 48-kDa rhoptry protein for serological detection of Babesia caballi antibodies in horses.
Mohamad Alaa Terkawi, Hend Alhasan, Akio Ueno, Jadsada Ratthanophart, Yuzi Luo, Shinuo Cao, Ketsarin Kamyingkird, Mahmoud Aboulaila, Goo Youn-Kyoung, Yoshifumi Nishikawa, Naoaki Yokoyama, Xuenan Xuan, Ikuo Igarashi
Parasitology international, 61, 3, 493, 6, 3, 2012年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A recombinant C-terminal antigen derived from Babesia caballi 48-kDa rhoptry protein (rBc48/CT) was made for the development of a serologically diagnostic test. Antiserum raised against the rBc48/CT reacted specifically with the corresponding native protein by Western blotting and the indirect fluorescent antibody test (IFAT). Next, an indirect enzyme-linked immunosorbent assay (Bc48/CT-ELISA) and an immunochromatographic test based on the Bc48/CT (Bc48/CT-ICT) were constructed and employed for the detection of an antibody to B. caballi in a variety of equine sera. The results of Bc48/CT-ELISA and Bc48/CT-ICT were highly concordant with those of IFAT and ELISA, with full-length protein of Bc48 used as the reference tests. Our results demonstrate the success of Bc48/CT as antigen for the serological diagnosis of B. caballi infection in horses.

Molecular detection and identification of Babesia bovis and Babesia bigemina in cattle in northern Thailand.
Shinuo Cao, Gabriel Oluga Aboge, Mohamad Alaa Terkawi, Longzheng Yu, Ketsarin Kamyingkird, Yuzi Luo, Yan Li, Youn-Kyoung Goo, Junya Yamagishi, Yoshifumi Nishikawa, Naoaki Yokoyama, Hiroshi Suzuki, Ikuo Igarashi, Ryuichiro Maeda, Tawin Inpankaew, Sathaporn Jittapalapong, Xuenan Xuan
Parasitology research, 111, 3, 1259, 66, 2012年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in cattle from central region of Syria.
Mohamad Alaa Terkawi, Hend Alhasan, Nguyen Xuan Huyen, Amin Sabagh, Karam Awier, Shinuo Cao, Youn-Kyoung Goo, Gabriel Aboge, Naoaki Yokoyama, Yoshifumi Nishikawa, Abdul Karim Kalb-Allouz, Darem Tabbaa, Ikuo Igarashi, Xuenan Xuan
Veterinary parasitology, 187, 1-2, 307, 11, 1-2, 2012年06月08日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A total of 207 bovine blood samples were collected from clinically healthy cattle bred in central region of Syria and examined by Giemsa-stained blood smears, nested PCR, ELISA, and IFAT to determine the molecular and serological prevalence of Babesia bovis and B. bigemina. All samples were negative to Babesia spp. by microscopic examination of blood smears. On the other hand, the overall prevalence of B. bovis and B. bigemina was 9.18% and 15.46% by nPCR, 15.46% and 18.84% by ELISA, and 18.36% and 21.74% by IFAT, respectively. Mixed infections were detected in a total of 5 samples (2.4%) by nPCR, 16 (7.73%) by ELISA and 27 (13.04%) by IFAT. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location. These data provide valuable information regarding the occurrence and epidemiology of B. bovis and B. bigemina infections in Syrian cattle, which can be employed in developing rational strategies for disease control and management.

Four promising antigens, BgP32, BgP45, BgP47, and BgP50, for serodiagnosis of Babesia gibsoni infection were classified as B. gibsoni merozoite surface protein family.
Youn-Kyoung Goo, Gabriel Oluga Aboge, M Alaa Terkawi, Honglin Jia, Junya Yamagishi, Fujiko Sunaga, Kazuhiko Namikawa, Se-Yeoun Cha, Hyung-Kwan Jang, Suk Kim, Yoshifumi Nishikawa, Xuenan Xuan
Parasitology international, 61, 2, 364, 8, 2012年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We determined the molecular characteristics of four proteins, BgP32, BgP45, BgP47, and BgP50, of Babesia gibsoni. Localization by subcellular fractionations followed by Western blotting revealed that the corresponding native proteins belong to merozoite surface protein family of B. gibsoni (BgMSP). Moreover, antisera against either rBgP45 or rBgP47 cross-reacted with all the proteins of the BgMSP family on ELISA and IFAT analyses. Of the four candidate antigens, ELISA with rBgP45 yielded high sensitivity, and ELISA with rBgP32 resulted in high specificity and in concordance with IFAT results.

A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model.
Yuzi Luo, Mohamad Alaa Terkawi, Honglin Jia, Gabriel Oluga Aboge, Youn-Kyoung Goo, Shinuo Cao, Yan Li, Longzheng Yu, Hideo Ooka, Ketsarin Kamyingkird, Tatsunori Masatani, Shoufa Zhang, Yoshifumi Nishikawa, Ikuo Igarashi, Xuenan Xuan
Experimental parasitology, 130, 2, 178, 82, 2012年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) targeting secreted antigen 1 of Babesia microti (BmSA1) was developed for detection of B. microti infection. The optimized DAS-ELISA was sensitive enough to detect circulating BmSA1 by day 2 post-infection, in sequential sera of a hamster infected with B. microti. This detection was 4 days earlier than antibody detection by indirect ELISA. The kinetics of circulating BmSA1 coincided with the profile of parasitemia. The specificity of this assay was evaluated using sera from animals experimentally infected with different species of Babesia. The DAS-ELISA had a higher sensitivity than the microscopic examination of Giemsa-stained blood smears for detection of the infection in hamsters. Taken together, these results indicated that BmSA1 could be a potential marker for surveillance of human babesiosis.

Macrophages are critical for cross-protective immunity conferred by Babesia microti against Babesia rodhaini infection in mice.
Yan Li, Mohamad Alaa Terkawi, Yoshifumi Nishikawa, Gabriel Oluga Aboge, Yuzi Luo, Hideo Ooka, Youn-Kyoung Goo, Longzheng Yu, Shinuo Cao, Yongfeng Sun, Junya Yamagishi, Tatsunori Masatani, Naoaki Yokoyama, Ikuo Igarashi, Xuenan Xuan
Infection and immunity, 80, 1, 311, 20, AMER SOC MICROBIOLOGY, 2012年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Although primary infection of mice with Babesia microti has been shown to protect mice against subsequent lethal infection by Babesia rodhaini, the mechanism behind the cross-protection is unknown. To unravel this mechanism, we investigated the influence of primary infection of mice with nonlethal B. microti using different time courses on the outcome of subsequent lethal B. rodhaini infection. Simultaneous infections of mice with these parasites resulted in rapid increases in parasitemia, with 100% mortality in BALB/c mice, as observed with control mice infected with B. rodhaini alone. In contrast, mice with acute, resolving, and chronic-phase B. microti infections were completely protected against B. rodhaini, resulting in low parasitemia and no mortalities. Mice immunized with dead B. microti were not protected from B. rodhaini infection, although high antibody responses were induced. Interestingly, the protected mice had significantly decreased levels of antibody response, cytokines (including gamma interferon [IFN-γ], interleukin-2 [IL-2], IL-8, IL-10, and IL-12), and nitric oxide levels after infection with B. rodhaini. SCID mice and IFN-γ-deficient mice with chronic B. microti infections demonstrated protective responses comparable to those of immunocompetent mice. Likewise, in vivo NK cell depletion did not significantly impair the protective responses. Conversely, macrophage depletion resulted in increased susceptibility to B. rodhaini infection associated with changes in their antibody and cytokines profiles, indicating that macrophages contribute to the protection against this challenge infection. We conclude that future development of vaccines against Babesia should include a strategy that enhances the appropriate activation of macrophages.

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa.
Mohamad Alaa Terkawi, Oriel M M Thekisoe, Charles Katsande, Abdalla A Latif, Ben J Mans, Olivier Matthee, Nozipho Mkize, Nomsa Mabogoane, Frances Marais, Naoaki Yokoyama, Xuenan Xuan, Ikuo Igarashi
Veterinary parasitology, 182, 2-4, 337, 42, 2-4, 2011年12月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.

Molecular prevalence of different genotypes of Theileria orientalis detected from cattle and water buffaloes in Thailand.
Khukhuu Altangerel, Thillaiampalam Sivakumar, Tawin Inpankaew, Sathaporn Jittapalapong, Mohamad Alaa Terkawi, Akio Ueno, Xuenan Xuan, Ikuo Igarashi, Naoaki Yokoyama
The Journal of parasitology, 97, 6, 1075, 9, 6, 2011年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Here we report on an epidemiological study regarding the molecular prevalence of different genotypes of Theileria orientalis present among domestic cattle and water buffalo populations bred in Thailand. A phylogenetic analysis based on the parasitic gene encoding a major piroplasm surface protein revealed the presence of 5 genotypes (Types 1, 3, 5, 7, and N-3) in cattle and 7 genotypes (Types 1, 3, 4, 5, 7, N-2, and N-3) in water buffaloes. Types 4, 7, and N-3 of T. orientalis were reported for the first time in water buffaloes. The previously reported C and Thai types from Thailand clustered as types 7 and 6, respectively, in the present analysis. Great similarities were observed among nucleotide sequences of isolates of the same genotype from cattle and water buffaloes, and, therefore, water buffaloes were considered to serve as a reservoir for these genotypes of T. orientalis in Thailand. In conclusion, T. orientalis parasites circulating in Thailand are more diverse in their genetic characters than previously anticipated.

Expression of truncated Babesia gibsoni thrombospondin-related adhesive proteins in Escherichia coli and evaluation of their diagnostic potential by enzyme-linked immunosorbent assay.
S Narantsatsral, Youn-Kyoung Goo, B Battsetseg, P Myagmarsuren, Mohamad Alaa Terkawi, Takehisa Soma, Yuzi Luo, Yan Li, Shinuo Cao, Longzheng Yu, Ketsarin Kamyingkird, Gabriel Oluga Aboge, Yoshifumi Nishikawa, Xuenan Xuan
Experimental parasitology, 129, 2, 196, 202, 2011年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Among the previously established enzyme-linked immunosorbent assays (ELISAs), an ELISA using the full length of a recombinant thrombospondin-related adhesive protein of Babesia gibsoni (rBgTRAPf) is considered as the most sensitive diagnostic method for the detection of an antibody to B. gibsoni in dogs. However, the expression of rBgTRAPf in high concentration is poor and, thus, limits its usefulness as a diagnostic antigen. To improve its expression level, we have truncated BgTRAPf into two fragments having either an N- or a C-terminus (BgTRAPn or BgTRAPc, respectively). The expression of BgTRAPc protein in Escherichia coli yielded adequate recombinant protein. The specificity and sensitivity of ELISAs with the truncated proteins were determined using dog sera experimentally infected with B. gibsoni and specific pathogen-free (SPF) dog sera. A total of 254 field dog sera were examined by the ELISA with rBgTRAPn, rBgTRAPc, and rBgTRAPf as well as by an indirect fluorescent antibody test (IFAT). The specificity of rBgTRAPc was the highest (97.15%), and its kappa value was more (0.8003) than rBgTRAPn (0.7083). With a sufficient level of expression as well as higher specificity and reliable sensitivity, rBgTRAPc appears to be a potential candidate antigen for the serodiagnosis of B. gibsoni infection in dogs.

Secretion of a new spherical body protein of Babesia bovis into the cytoplasm of infected erythrocytes
Mohamad Alaa Terkawi, Faasoa Junior Seuseu, Putut Eko-Wibowo, Nguyen Xuan Huyen, Yuka Minoda, Mahmoud Aboulaila, Satoru Kawai, Naoaki Yokoyama, Xuenan Xuan, Ikuo Igarashi
Molecular and Biochemical Parasitology, 178, 1-2, 40, 45, 1-2, 2011年07月, ［査読有り］
英語, 研究論文（学術雑誌）, A cDNA encoding a new Babesia bovis spherical body protein 4 (BbSBP-4) was reported to have no significant homology to other apicomplexan proteins or previously reported B. bovis spherical body proteins. In the present study, we further examined the molecular characteristics of BbSBP-4 including the expression and cellular localization of the BbSBP-4. An anti-rBbSBP-4 mouse serum specifically reacted to a 41-kDa native protein B. bovis in Western blot analysis. The immunoelectron microscopic examination confirmed the localization of BbSBP-4 in spherical bodies, but not in the nucleus, rhoptries, and micronemes. Interestingly, the protein was found to be localized not only in the spherical body of B. bovis but also in the cytoplasm of infected erythrocytes (iRBC) at the later stage of parasite development. The confocal laser microscopic examination and Western blot analysis demonstrated the increased accumulation of BbSBP-4 in the cytoplasm of iRBC and in the supernatant of cultivated B. bovis during the late developmental stage of the parasite. These results suggest that BbSBP-4 was secreted from spherical body into cytoplasm of iRBC during the late developmental stage of the parasite before the rupture of infected RBC. Taken together, BbSBP-4 might play an important role as a secreted protein in the intracellular development and/or survival of B. bovis. © 2011 Elsevier B.V. All rights reserved.

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the northeast region of Thailand.
Mohamad Alaa Terkawi, Nguyen Xuan Huyen, Cao Shinuo, Tawin Inpankaew, Khuanwalai Maklon, Mahmoud Aboulaila, Akio Ueno, Youn-Kyoung Goo, Naoaki Yokoyama, Sathaporn Jittapalapong, Xuenan Xuan, Ikuo Igarashi
Veterinary parasitology, 178, 3-4, 201, 7, 3-4, 2011年06月10日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and Babesia bigemina and is characterized by significant morbidity and mortality worldwide. The disease is widespread in the northeastern region of Thailand, where an increasingly large part of the livestock is composed of water buffaloes. The present study was therefore conducted to investigate the epidemiological distribution of B. bovis and B. bigemina in water buffaloes in the northeastern region of Thailand. A total of 305 buffalo blood samples were randomly collected from five provinces and simultaneously analyzed by the nested PCR (nPCR) assay, ELISA, and IFAT techniques. The overall prevalence of B. bovis and B. bigemina was 11.2% and 3.6% by nPCR, 14.7% and 5.9% by ELISA, and 16.8% and 5.6% by IFAT, respectively. The high concordance between the molecular and the serological detection tests revealed the specificity and sensitivity of the diagnostic assays used for the detection of infection as well as the endemic stability status of the parasites in the surveyed areas. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location but not gender. Our data provide valuable information regarding the epidemiology of B. bovis and B. bigemina infection in water buffaloes in the northeastern region of Thailand which will likely be very beneficial for management and control programs of this disease.

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test.
Yuzi Luo, Honglin Jia, M Alaa Terkawi, Youn-Kyoung Goo, Suguru Kawano, Hideo Ooka, Yan Li, Longzheng Yu, Shinuo Cao, Junya Yamagishi, Kozo Fujisaki, Yoshifumi Nishikawa, Atsuko Saito-Ito, Ikuo Igarashi, Xuenan Xuan
Parasitology international, 60, 2, 119, 25, 2, 2011年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.

Immunogenicity of orally administrated recombinant Lactobacillus casei Zhang expressing Cryptosporidium parvum surface adhesion protein P23 in mice.
Geriletu, Rihua Xu, Honglin Jia, Mohamad Alaa Terkawi, Xuenan Xuan, Heping Zhang
Current microbiology, 62, 5, 1573, 80, SPRINGER, 2011年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal diseases in both humans and animals. What is more, there is no promising strategy for controlling cryptosporidiosis. In this study, the P23 immunodominant surface protein of C. parvum sporozoites was stably expressed in the Lactobacillus casei Zhang strain and its immunogenicity was evaluated in a mouse model. The molecular weight (23 kDa) and immunogenicity of p23 gene expressed by L. casei Zhang were similar to that of the native P23 protein. Oral immunization with control L. casei Zhang and recombinant L. casei Zhang-p23 activated the mucosal immune system to elicit serum immunoglobulin G (IgG) and mucosal IgA in mice. Furthermore, the expression of cytokines such as IL-4, IL-6, and IFN-γ in splenocytes of mice was detected by real-time PCR after oral immunization. P23-specific immunocyte activation was also verified. These findings indicate that the live L. casei Zhang vector may be a new tool for the production of mucosal vaccines against cryptosporidiosis in animals.

Drug Discovery against Babesia and Toxoplasma
Mohamad Alaa Terkawi, Ikuo Igarashi
Apicomplexan Parasites: Molecular Approaches toward Targeted Drug Development, 453, 467, Wiley-VCH, 2011年02月21日, ［査読有り］
英語, 論文集(書籍)内論文, The Apicomplexa, which consist of numerous genera of pathogenic protozoa, constitute a global medical and economic problem. Toxoplasma gondii and certain Babesia spp. have recently emerged as lethal opportunistic pathogens in immunocompromised patients, and as systemic veterinary pathogens associated with significant economic losses in the animal industry. The widespread parasitic infections and the increase of drug resistance in many areas of the world have exacerbated the problem and demonstrated the need for the development of safe, effective, and affordable agents. The genomics and proteomics of apicomplexan parasites provide comprehensive insights into their biochemistry and cell processes, and highlight possible drug targets. On the other hand, a recombinant protein technique combined with three-dimensional structure elucidation using X-ray crystallography are critical for identifying the structural information of target molecules and obtaining a model of the target binding site, leading to the best design of antiparasitic drugs. However, the efficacy, toxic side effects, pharmacokinetic compatibility, and potential for developing resistance are major parameters in the design of a successful future drug. In this chapter, the biochemical features of Babesia spp. and Toxoplasma gondii are discussed and updated, and possible molecular targets available in the field of drug discovery are highlighted. © 2011 WILEY-VCH Verlag GmbH &
Co. KGaA, Weinheim.

Spherical body protein 4 is a new serological antigen for global detection of Babesia bovis infection in cattle.
Mohamad Alaa Terkawi, Nguyen Xuan Huyen, Putut Eko Wibowo, Faasoa Junior Seuseu, Mahmoud Aboulaila, Akio Ueno, Youn-Kyoung Goo, Naoaki Yokoyama, Xuenan Xuan, Ikuo Igarashi
Clinical and vaccine immunology : CVI, 18, 2, 337, 42, 2, 2011年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Five Babesia bovis recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentally B. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentally B. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions of B. bovis endemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies to B. bovis in cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future.

Babesia microti: molecular and antigenic characterizations of a novel 94-kDa protein (BmP94).
Hideo Ooka, Mohamad Alaa Terkawi, Youn-Kyoung Goo, Yuzi Luo, Yan Li, Junya Yamagishi, Yoshifumi Nishikawa, Ikuo Igarashi, Xuenan Xuan
Experimental parasitology, 127, 1, 287, 93, 1, 2011年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A novel gene, BmP94, encoding 94-kDa protein of Babesia microti was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis.

Artesunate, a potential drug for treatment of Babesia infection.
Youn-Kyoung Goo, M Alaa Terkawi, Honglin Jia, G Oluga Aboge, Hideo Ooka, Bryce Nelson, Suk Kim, Fujiko Sunaga, Kazuhiko Namikawa, Ikuo Igarashi, Yoshifumi Nishikawa, Xuenan Xuan
Parasitology international, 59, 3, 481, 6, 3, 2010年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The effects of artesunate, a water-soluble artemisinin derivative, against Babesia species, including Babesia bovis, Babesia gibsoni and Babesia microti were studied. Cultures of B. bovis and B. gibsoni were treated with 0.26, 2.6, 26 and 260microM artesunate, showing inhibition of parasite growth at concentrations equal to and greater than 2.6microM artesunate by days 3 post-treatment for B. gibsoni and B. bovis in a dose-dependent manner. Consistent with in vitro experiments, artesunate was effective in the treatment of mice infected with B. microti at doses equal to and greater than 10mg/kg of body weight on days 8-10 post-infection. Taken together, these results suggest that artesunate could be a potential drug against Babesia infection.

Serodiagnosis of ovine toxoplasmosis in Mongolia by an enzyme-linked immunosorbent assay with recombinant Toxoplasma gondii matrix antigen 1.
Buyannemekh Tumurjav, Mohamad Alaa Terkawi, Houshuang Zhang, Guohong Zhang, Honglin Jia, Youn-Kyoung Goo, Junya Yamagishi, Yoshifumi Nishikawa, Ikuo Igarashi, Chihiro Sugimoto, Xuenan Xuan
The Japanese journal of veterinary research, 58, 2, 111, 9, 2, 2010年08月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Toxoplasma gondii matrix antigen 1 (TgMAG1), known as the 65-kDa protein, which is abundantly expressed in both bradyzoites and tachyzoites, was evaluated as a candidate for the development of a diagnostic reagent for ovine toxoplasmosis. The TgMAG1 gene was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and the recombinant TgMAG1 (rTgMAG1) was tested in an enzyme-linked immunosorbent assay (ELISA). The ELISA with rTgMAG1 showed a highly specific reaction with sera from mice experimentally infected with T. gondii but not with the closely related Neospora caninum. The antibodies to TgMAG1 were detectable from the acute to the chronic infectious stages in a mouse model. A total of 175 serum samples collected from sheep in 7 provinces of Mongolia were examined for the serodiagnosis of T. gondii infection by the ELISA with rTgMAG1, and the results were compared with those from the commercialized latex agglutination test (LAT). Of 175 serum samples analyzed, 42 (24.00%) and 29 (16.57%) samples were positive by the ELISA and LAT, respectively. Of 29 LAT-positive samples, 27 (93.10%) were positive by the ELISA. These results suggest that rTgMAG1 could be used as a reliable antigen for the detection of T. gondii infection in sheep.

Babesia gibsoni: identification, expression, localization, and serological characterization of a Babesia gibsoni 22-kDa protein.
Youn-Kyoung Goo, Honglin Jia, Mohamad Alaa Terkawi, Gabriel Oluga Aboge, Junya Yamagishi, Yoshifumi Nishikawa, Suk Kim, Hyung-Kwan Jang, Kozo Fujisaki, Xuenan Xuan
Experimental parasitology, 123, 3, 273, 6, 2009年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Babesia gibsoni causes canine babesiosis. Here, we describe the identification and characterization of a novel gene, BgP22, containing an open reading frame of 621bp and encoding a 22-kDa protein from B. gibsoni, as a serodiagnostic candidate. The recombinant BgP22 (rBgP22) was expressed and used as an antigen to produce anti-rBgP22 sera in mice. Using these anti-rBgP22 sera, a native 22-kDa protein was recognized by Western blot analysis and observed in the membrane of the parasites by immunofluorescent antibody tests (IFAT). The enzyme-linked immunosorbent assay (ELISA) using the rBgP22 detected specific antibodies to this protein in the sera of dogs experimentally and naturally infected with B. gibsoni in chronic stage. Furthermore, it did not show a cross reaction with the closely related apicomplexan parasites, indicating that the rBgP22 could be used as a diagnostic antigen for a detection of the chronic carrier stages of B. gibsoni infection.

Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s).
M A Terkawi, A Amornthep, H Ooka, G Aboge, H Jia, Y-K Goo, B Nelson, J Yamagishi, Y Nishikawa, I Igarashi, S-I Kawazu, K Fujisaki, X Xuan
Parasitology, 136, 10, 1147, 60, 10, 2009年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Three cDNAs encoding rhoptry-associated protein 1 (RAP-1) homologues were found in the Babesia gibsoni EST database. Based on similarities to BgRAP-1a, which was identified previously by serological screening of a cDNA merozoite library, the two new genes were designated BgRAP-1b (33.7%) and BgRAP-1c (57%). Mice antiserum raised against each recombinant protein reacted specifically with B. gibsoni parasites as determined by Western blotting, which showed native molecular sizes of the BgRAP-1a (51 kDa), BgRAP-1b (53 kDa) and BgRAP-1c (47 kDa) consistent with predictable molecular weights. Immunofluoresence using these antibodies revealed localization of all BgRAP-1s within the matrix of merozoites; however, BgRAP-1a appeared to diverge from the other two when it was found secreted into the cytoplasm of infected erythrocytes. Apical localization of all 3 BgRAP-1s during the extracellular stage of the parasite combined with their ability to bind a canine erythrocyte membrane fraction was suggestive of a role for these proteins in erythrocyte attachment. Lastly, the ability of these recombinant proteins to be used as diagnostic reagents was tested by ELISA and the sensitivities of BgRAP-1a and BgRAP-1c were found increased through N-terminal truncation. Taken together, our data suggest divergent roles for the 3 BgRAP-1s in the merozoite stage of B. gibsoni.

Detection of Babesia gibsoni by reaction of phage display single chain antibodies with P50 proteins.
Yoshino Hirose, Saeka Matsuhira, Tatiana Alexandrovna Batanova, Youn-Kyoung Goo, Mohamad A Terkawi, Yoshifumi Nishikawa, Katsuya Kitoh, Xuenan Xuan, Yasuhiro Takashima
The Journal of veterinary medical science, 71, 8, 1133, 6, 2009年08月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Babesia gibsoni (B. gibsoni) is a tick-borne hemoprotozoan parasite, which causes piroplasmosis in dogs. Diagnosis of canine babesiosis is commonly carried out using Giemsa-stained thin blood smears. However, at low levels of infection, it is difficult to detect Babesia organisms by observation of Giemsa-stained thin blood smears. We constructed a monoclonal phage display single chain antibody (scFv) against a B. gibsoni merozoite antigen, P50 protein. Intraerythrocytic B. gibsoni organisms are clearly stained using this antibody. The monoclonal scFv facilitated the detection of B. gibsoni organisms in canine blood samples.

Characterization of a leucine aminopeptidase of Babesia gibsoni.
H Jia, M A Terkawi, G O Aboge, Y-K Goo, Y Luo, Y Li, J Yamagishi, Y Nishikawa, I Igarashi, C Sugimoto, K Fujisaki, X Xuan
Parasitology, 136, 9, 945, 52, 9, 2009年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Peptidases of parasitic protozoa are currently under intense investigation in order to identify novel virulence factors, drug targets, and vaccine candidates, except in Babesia. Leucine aminopeptidases in protozoa, such as Plasmodium and Leishmania, have been identified to be involved in free amino acid regulation. We report here the molecular and enzymatic characterization, as well as the localization of a leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from B. gibsoni (BgLAP). A functional recombinant BgLAP (rBgLAP) expressed in Escherichia coli efficiently hydrolysed synthetic substrates for aminopeptidase, a leucine substrate. Enzyme activity of the rBgLAP was found to be optimum at pH 8.0 and at 37 degrees C. The substrate profile was slightly different from its homologue in P. falciprum. The activity was also strongly dependent on metal divalent cations, and was inhibited by bestatin, which is a specific inhibitor for metalloprotease. These results indicated that BgLAP played an important role in free amino acid regulation.

Molecular and immunological characterization of Babesia gibsoni and Babesia microti heat shock protein-70.
M Alaa Terkawi, G Aboge, H Jia, Y-K Goo, H Ooka, J Yamagishi, Y Nishikawa, N Yokoyama, I Igarashi, S-I Kawazu, K Fujisaki, X Xuan
Parasite immunology, 31, 6, 328, 40, 6, 2009年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Serological immunoscreening was used to identify a gene encoding heat shock protein-70 from Babesia gibsoni (BgHSP-70) that showed high homology with HSP-70s from other apicomplexan parasites. This gene corresponded to a full-length cDNA containing an open reading frame of 1968 bp predicted to result in a 70-kDa mature protein consisting of 656 amino acids. Analysis of the expression levels of BgHSP-70 indicated elevated transcription from cultured parasites incubated at 40 degrees C for 1 h, but not at 30 degrees C. Interestingly, antiserum raised against recombinant BgHSP-70 protein reacted specifically not only with a 70-kDa protein of B. gibsoni but also with a corresponding native protein of B. microti (BmHSP-70), indicating the high degree of conservation of this protein. The BmHSP-70 gene was then isolated and characterized and the immunoprotective properties of recombinant BgHSP-70 (rBgHSP-70) and rBmHSP-70 were compared in vitro and in vivo. Both proteins had potent mitogenic effects on murine and canine mononuclear cells as evidenced by high proliferative responses and IFN-gamma production after stimulation. Immunization regimes in BALB/c and C57BL/6 mice using rBgHSP-70 and rBmHSP-70 elicited high antibody levels, with concurrent significant reductions in peripheral parasitaemias. Taken together, these results emphasize the potential of HSP-70s as a molecular adjuvant vaccine.

Identification of secreted antigen 3 from Babesia gibsoni.
Honglin Jia, M Alaa Terkawi, Gabriel Oluga Aboge, Youn-Kyoung Goo, Liqing Ma, Jinlin Zhou, Yoshifumi Nishikawa, Ikuo Igarashi, Kozo Fujisaki, Xuenan Xuan
Clinical and vaccine immunology : CVI, 16, 6, 944, 8, 6, 2009年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A cDNA expression library of Babesia gibsoni was screened with the serum collected from a dog experimentally infected with B. gibsoni. A novel antigen sharing homology with secreted antigen 1 of B. gibsoni was isolated. The genomic analysis indicated that the BgSA3 gene exists as multicopies in the genome of B. gibsoni. The putative peptide encoded by the BgSA3 gene showed some characteristics of secreted proteins. The serum raised in mice immunized with the recombinant BgSA3 expressed in Escherichia coli could recognize a native parasite protein with a molecular mass of 70 kDa. Moreover, a sandwich enzyme-linked immunosorbent assay with anti-BgSA3 antibodies could detect the circulating BgSA3 in the blood plasma of dogs experimentally infected with B. gibsoni. The identification of BgSA3 provided a useful target for the development of a diagnostic test for detecting specific antibodies and circulating antigens.

Genetic diversity of two selected antigen loci in Babesia gibsoni Asian genotype obtained from Japan and Jeju island of South Korea.
Honglin Jia, Gabriel Oluga Aboge, M Alaa Terkawi, Youn-Kyoung Goo, Yoshifumi Nishikawa, Ken Kuriki, Kyoung-Kap Lee, Hyung-Kwan Jang, Suk Kim, Kozo Fujisaki, Xuenan Xuan
Veterinary parasitology, 162, 1-2, 142, 6, 2009年05月26日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Previous reports have shown that the secreted antigen 1 of Babesia gibsoni (BgSA1) and the thrombospondin-related adhesive protein of B. gibsoni (BgTRAP) are promising diagnostic reagents and vaccine candidates. Therefore, we determined the extent of nucleotide sequence variation in the BgSA1 and BgTRAP genes, obtained from eight isolates of B. gibsoni got from clinically infected dogs in geographically distinct areas of Japan and one isolate from Jeju island of South Korea. Sequence analyses have revealed that nucleotide diversity is lower in BgSA1 than that in BgTRAP. The mean number of non-synonymous (dn) nucleotide substitutions was significantly greater than that of synonymous (ds) ones per site in region II of BgTRAP. Overall, the results predict more allele-specific immunity to BgTRAP than that to BgSA1, which could be useful in designing and testing efficacy of diagnostic reagents as well as vaccine candidates for the B. gibsoni isolates from Japan and Jeju island of South Korea.

Characterization of the Babesia gibsoni 12-kDa protein as a potential antigen for the serodiagnosis.
Youn-Kyoung Goo, Honglin Jia, G Oluga Aboge, M Alaa Terkawi, Eung-goo Lee, Junya Yamagishi, Yoshifumi Nishikawa, Hyung-Kwan Jang, Fujiko Sunaga, Kazuhiko Namikawa, Kozo Fujisaki, Xuenan Xuan
Parasitology international, 58, 1, 55, 60, 2009年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A novel gene, BgP12, encoding a 12-kDa protein was identified from Babesia gibsoni. The full-length cDNA of BgP12 contains an open reading frame of 378 bp, corresponding to 126 amino acid (aa) residues consisting of a putative 26 aa signal peptide and a 100 aa mature protein. The recombinant BgP12 (rBgP12) lacking the N-terminal signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein (rBgP12) that produced an anti-rBgP12 serum in mice after immunization. Using this anti-rBgP12 serum, a native 12-kDa protein in B. gibsoni was recognized by Western blot analysis. Immunofluorescent antibody tests (IFAT) revealed that BgP12 was mainly seen during the ring stage of B. gibsoni trophozoite. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP12 detected specific antibodies in the sequential sera of a dog experimentally infected with B. gibsoni beginning 10 days post-infection to 442 days post-infection, even when the dog became chronically infected and showed a low level of parasitemia. Moreover, the antigen did not show cross-reaction with antibodies to the closely related apicomplexan parasites, indicating that the rBgP12 might be an immunodominant antigen for B. gibsoni infection that could be used as a diagnostic antigen for B. gibsoni infection with high specificity and sensitivity.

Cloning, expression, and characterization of Babesia gibsoni dihydrofolate reductase-thymidylate synthase: inhibitory effect of antifolates on its catalytic activity and parasite proliferation.
Gabriel O Aboge, Honglin Jia, Mohamad A Terkawi, Youn-Kyoung Goo, Yoshifumi Nishikawa, Fujiko Sunaga, Kuzuhiko Namikawa, Naotoshi Tsuji, Ikuo Igarashi, Hiroshi Suzuki, Kozo Fujisaki, Xuenan Xuan
Antimicrobial agents and chemotherapy, 52, 11, 4072, 80, 2008年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed K(m) values of 4.70 +/- 0.059 (mean +/- standard error of the mean) and 9.75 +/- 1.64 microM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC(50)] = 68.6 +/- 5.20 nM) than pyrimethamine (IC(50) = 55.0 +/- 2.08 microM) and trimethoprim (IC(50) = 50 +/- 12.5 microM). Moreover, the antifolates' inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, the B. gibsoni DHFR-TS is a molecular antifolate drug target.

C3 contributes to the cross-protective immunity induced by Babesia gibsoni phosphoriboprotein P0 against a lethal B. rodhaini infection
M. A. Terkawi, G. Zhang, H. Jia, G. Aboge, Y. K. Goo, Y. Nishikawa, N. Yokoyama, I. Igarashi, S. I. Kawazu, K. Fujisaki, X. Xuan
Parasite Immunology, 30, 6-7, 365, 370, 6-7, 2008年06月, ［査読有り］
英語, 研究論文（学術雑誌）, We have studied the impact of complement component 3 (C3) deficiency on the progression of lethal Babesia rodhaini infection in immune mice. A B. gibsoni ribosomal phosphoprotein P0 (BgP0) previously reported to be a cross-protective antigen against Babesia infection was used to immunize C57BL/6 wild-type (WT) and C3-deficient (C3-/-) mice. Test mice were immunized intraperitoneally (i.p.) with recombinant BgP0 (rBgP0), while controls either were immunized with PBS or did not receive any immunization. Following the immunization regime, test WT mice induced a specifically strong humoral response consisting of mixed immunoglobulins IgG1 and IgG2 associated with high production of IFN-γ in the supernatant of splenocytes. While test C3-/- mice had significantly decreased total IgG, IgG1 and IgG2b responses, the secretions of IL-12 and IFN-γ tended to be lower than those in WT mice. Furthermore, partial protection was only observed in rBgP0-immunized WT mice but not in C3-/- mice or controls. Indeed, rBgP0-immunized WT mice showed significant reductions in the initiation of parasitaemia correlated with delayed mortalities and considerable survival rates. Taken together, our results indicate that cross-protection was impaired in C3-/- mice in view of the decrease in the antibody responses and cytokine production and the high susceptibility to infection. © 2008 The Authors.

Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP.
Youn-Kyoung Goo, Honglin Jia, G Oluga Aboge, M Alaa Terkawi, Ken Kuriki, Chinatsu Nakamura, Akiko Kumagai, Jinlin Zhou, Eung-goo Lee, Yoshifumi Nishikawa, Ikuo Igarashi, Kozo Fujisaki, Xuenan Xuan
Experimental parasitology, 118, 4, 555, 60, 4, 2008年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.

Babesia gibsoni: Identification of an immunodominant, interspersed repeat antigen
Honglin Jia, M. Alaa Terkawi, Gabriel Oluga Aboge, Youn-Kyoung Goo, Jinlin Zhou, Eung-Goo Lee, Yoshifumi Nishikawa, Ikuo Igarashi, Kozo Fujisaki, Xuenan Xuan
Experimental Parasitology, 118, 1, 146, 149, 1, 2008年01月, ［査読有り］
英語, 研究論文（学術雑誌）, In this report, an immunodominant antigen called BgIRA from Babesia gibsoni is identified and described. A highly repetitive antigen was screened from a cDNA library. The genomic BgIRA gene exists as single cope gene and contains 10 introns. BgIRA plays a dominant role in the immune response in dogs infected with B. gibsoni. The specificity and sensitivity of the rBgIRA in an ELISA indicated that this antigen might be useful in a diagnostic test. © 2007 Elsevier Inc. All rights reserved.

A shared antigen among Babesia species: Ribosomal phosphoprotein P0 as a universal babesial vaccine candidate
M. Alaa Terkawi, Honglin Jia, Aboge Gabriel, Youn-Kyoung Goo, Yoshifumi Nishikawa, Naoaki Yokoyama, Ikuo Igarashi, Kozo Fujisaki, Xuenan Xuan
Parasitology Research, 102, 1, 35, 40, 1, 2007年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Babesia gibsoni ribosomal phosphoprotein P0 (BgP0) was previously identified as a cross-protective antigen against Babesia microti infection in mice. Interestingly, the same protein showed considerable antigenicity when tested with serum samples collected from Babesia-infected animals. Moreover, the polyclonal antibody raised against the recombinant BgP0 (rBgP0) recognized the P0 homologues from other Babesia species either by immunoblotting or by immunoscreening. The P0 genes from Babesia caballi, Babesia equi, and Babesia bigemina were then cloned and sequenced. The phylogenic analyses based on the amino acid sequences indicated that BgP0 has high identities with B. caballi P0 (88.1%), B. bigemina P0 (85.6%), Babesia bovis P0 (81.4%), and B. equi P0 (64.9%). Western blot analyses revealed that the corresponding native proteins ranged between 31 and 34 kDa, consistent with predicated molecular weight of Babesia P0. Furthermore, the immunogenic property of anti-rBgP0 IgG was evaluated against a B. bovis in vitro culture. The growth of B. bovis parasites was restricted by anti-rBgP0 IgG in a concentration-dependent manner, and significant reductions in parasitemia were observed only at 1 mg/ml in the culture. Taken together, these data suggest that P0 is a conserved protective antigen among Babesia species and might be a potentially universal vaccine candidate for babesiosis. © 2007 Springer-Verlag.

A novel 57-kDa merozoite protein of Babesia gibsoni is a prospective antigen for diagnosis and serosurvey of canine babesiosis by enzyme-linked immunosorbent assay.
Gabriel Oluga Aboge, Honglin Jia, Mohamad Alaa Terkawi, Younkyoung Goo, Ken Kuriki, Yoshifumi Nishikawa, Ikuo Igarashi, Hiroshi Suzuki, Xuenan Xuan
Veterinary parasitology, 149, 1-2, 85, 94, 2007年10月21日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We isolated a novel single copy gene encoding a 57-kDa merozoite protein of Babesia gibsoni (BgP57). The nucleotide sequence of the cDNA was 2387 bp with an open reading frame (ORF) of 1644 bp encoding a 57-kDa predicted polypeptide having 547 amino acid residues. The recombinant BgP57 (rBgP57) without a predicted signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein. Western blotting showed that the corresponding native protein was 57-kDa, consistent with molecular weight of predicted mature polypeptide. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP57 detected specific antibodies in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with antibodies to B. canis sub-species and closely related apicomplexan parasites indicating that the rBgP57 was a specific antigen for B. gibsoni antibodies. The diagnostic performance of ELISA based on rBgP57 using 107 sera from B. gibsoni-naturally infected dogs was the same as the previously identified rBgP32 but performed better than the previously studied rBgP50. Although, seminested PCR detected higher proportions (82%) of positive samples than the ELISAs, the Mcnemar's chi-square test showed that there was no significant difference in relative effectiveness of rBgP57-ELISA and seminested PCR (chi(2)=2.70; P=0.1003) in identifying positive samples. The rBgP57-ELISA when used in combination with rBgP32-ELISA and rBgP50-ELISA appeared to improve sensitivity of the rBgP57-ELISA for detection of B. gibsoni antibodies. Overall, the rBgP57-ELISA and seminested PCR when used in combination, could improve epidemiological surveys and clinical diagnosis of B. gibsoni infection.

Babesia gibsoni ribosomal phosphoprotein P0 induces cross-protective immunity against B. microti infection in mice.
M Alaa Terkawi, Honglin Jia, Jinlin Zhou, Eung-goo Lee, Ikuo Igarashi, Kozo Fujisaki, Yoshifumi Nishikawa, Xuenan Xuan
Vaccine, 25, 11, 2027, 35, ELSEVIER SCI LTD, 2007年03月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Babesia gibsoni ribosomal phosphoprotein P0 (BgP0) was identified as an immunodominant cross-reactive antigen with B. microti. The BgP0 gene is a single copy with a predicted open reading frame of 942 bp and 314 amino acids. The BgP0 was expressed as a glutathione S-transferase fusion protein in Escherichia coli. The serum raised in mice with the recombinant BgP0 showed a specific band with a 34-kDa molecular mass in the extracts of B. gibsoni and B. microti merozoites. Furthermore, the intraperitoneal (i.p.) immunization of rBgP0 and Freund's adjuvant induced strong humoral response consisting of mixed immunoglobulins IgG1 and IgG2a in BALB/c mice. Following the challenge with B. microti, these mice delayed the onset of parasites and significantly reduced the peripheral parasitemia. On the other hand, passive-transfer of purified anti-BgP0 IgG into SCID mice showed partial protection against B. microti challenge infection. It was only effective in restricting the initial parasitemia but not later during its progress. Taken together, the immunological response elicited by rBgP0 protected the mice against B. microti challenge infection. These data suggest that BgP0 is a potentially universal vaccine candidate for both B. gibsoni and B. microti infections.

網羅的遺伝子発現解析によるマクロファージに対する軟骨破片の影響の検討
濱崎雅成, TERKAWI Alaa, 小野寺智洋, 宝満健太郎, 菱村亮介, XU Liang, 宮崎拓自, TIAN Yuan, 岩崎倫政, 日本軟骨代謝学会プログラム・抄録集, 32nd, 118, 2019年03月
日本語

超高分子ポリエチレン摩耗粉によるヒトマクロファージの遺伝子発現変化は関節リウマチと類似する　　　　　　　　　　　　　　　
Terkawi Alaa, 濱崎 雅成, 高橋 大介, 角家 健, 浅野 毅, 入江 徹, 小野寺 智洋, 高畑 雅彦, 岩崎 倫政, 日本整形外科学会雑誌, 91, 8, S1722, S1722, 2017年08月
(公社)日本整形外科学会, 日本語

国際獣疫事務局(OIE)馬ピロプラズマ症リファレンスラボラトリーの役割と活動
五十嵐郁男, TERKAWI Alaa, 玄学南, 横山直明, 日本獣医学会学術集会講演要旨集, 156th, 235, 2013年08月30日
日本語

エジプトとケニアの牛および水牛より単離されたBabesia bovisとB.bigeminaの分子学的性状解析(Molecular characterizations of Babesia bovis and B. bigemina isolated from cattle and water buffalo in Egypt and Kenya)　　　　　　　　　　　　　　　
Moumouni Paul, Franck Adjou, Aboge Gabriel, Hany Ibrahim, Terkawi Alaa, 正谷 達謄, 曹 世諾, Kamyingkird Ketsarin, Moussa Ahmed, 西川 義文, 鈴木 宏志, 玄 学南, 日本獣医学会学術集会講演要旨集, 155回, 212, 212, 2013年03月
(公社)日本獣医学会, 英語

寄生虫Babesia M17ロイシンアミノペプチダーゼの分子薬剤標的としての有効性の検証(Validating M17 leucine aminopeptidase of Babesia parasite as a molecular drug target)　　　　　　　　　　　　　　　
Aboge Gabriel Oluga, 曹 世諾, Terkawi Alaa, 正谷 達謄, 具 潤景, 横山 直明, 五十嵐 郁男, 西川 義文, 鈴木 宏志, 玄 学南, 日本獣医学会学術集会講演要旨集, 154回, 216, 216, 2012年08月
(公社)日本獣医学会, 英語

Babesia gibsoni原虫の薬剤標的DHFR-TSの性状解析　　　　　　　　　　　　　　　
Aboge G, Jia Honglin, Terkawi A, Goo Y.K, 西川 義文, 須永 藤子, 並河 和彦, 五十嵐 郁男, 鈴木 宏志, 藤崎 幸蔵, 玄 学南, 獣医寄生虫学会誌, 7, 2, 91, 91, 2008年12月
日本獣医寄生虫学会, 英語

Drug discovery against Babesia and Toxoplasma. (Apicomplexan Parasites: Molecular Approaches Toward Targeted Drug Development Drug Discovery in Infectious Diseases.　　　　　　　　　　　　　　　
Terkawi MA, Igarashi I
Wiley-VCH, Weinheim, 2011年

Macrophages: A double-edged sword in aseptic loosening of total joint arthroplasty　　　　　　　　　　　　　　　
アラーテルカウィ
Asia Pacific Society for Biology and Medical Sciences (APSBMS), 2019年07月29日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

Macrophage and neutrophils, Friend or foe to our health? Journey through our immune system　　　　　　　　　　　　　　　
アラーテルカウィ
帯広畜産大学テニュアトラック研究成果発表会, 2016年07月20日, 英語
［招待講演］, ［国内会議］

2021年10月 - 現在
Japan College of Rheumatology　　　　　　　　　　　　　　　

2017年10月 - 現在
Japanese society for immunology　　　　　　　　　　　　　　　

2016年10月 - 現在
Japanese society for biomaterials　　　　　　　　　　　　　　　

2016年10月 - 現在
一般社団法人 日本骨代謝学会　　　　　　　　　　　　　　　

2016年04月 - 現在
北海道整形災害外科学会　　　　　　　　　　　　　　　

骨粗鬆症治療薬開発のための骨代謝制御を担う骨マクロファージの機能解析
科学研究費助成事業 基盤研究(B)
2023年04月 - 2026年03月
照川 アラー
日本学術振興会, 基盤研究(B), 北海道大学, 23H03025

高齢化社会における運動器疾患に対する治療法の開発　　　　　　　　　　　　　　　
2023年03月 - 2025年03月
公益財団法人小林財団 第11回, 研究代表者

炎症収束性貪食細胞由来細胞外小胞による関節炎収束メカニズムの解明　　　　　　　　　　　　　　　
2020年04月 - 2023年03月
研究計画調書基盤研究(C), 研究分担者

抗炎症性マクロファージ由来因子と骨関連細胞に着目した新規骨粗鬆症メカニズムの解明.　　　　　　　　　　　　　　　
2020年04月 - 2023年03月
令和2年度(2020年度) 基盤研究（Ｃ）（一般）研究計画調書基盤研究(C)., 研究分担者

Communication between gut and osteal macrophages as a novel regulatory axis in bone homeostasis　　　　　　　　　　　　　　　
2021年12月 - 2022年12月
Alaa Terkawi (Terukawa), Tatiana Milena Marques,
MIRAI2.0 – Joint seed funding of Japan-Sweden collaborative projects, Hokkaido University, Örebro University, 研究代表者

骨粗鬆症の新規治療薬として応用可能なOsteomacs (骨マクロファージ)由来因子の網羅的解析　　　　　　　　　　　　　　　
2021年01月 - 2022年12月
公益財団法人ヒロセ財団, 研究代表者

培養細胞上の糖鎖抗原変化と自家細胞移植における免疫応答発生機序の解明　　　　　　　　　　　　　　　
2019年06月 - 2021年03月
挑戦的研究(萌芽), 研究分担者

滑膜マクロファージ由来エクソソームが関節炎の病態進行に及ぼす影響　　　　　　　　　　　　　　　
2018年04月 - 2021年03月
研究計画調書基盤研究(C), 研究代表者

変形性関節症制圧を目指した新規免疫ネットワーク解析手法を用いた治療薬の開発　　　　　　　　　　　　　　　
2017年06月 - 2020年03月
挑戦的研究(萌芽), 研究分担者

複合細胞移植による新規末梢神経再生方法の開発　　　　　　　　　　　　　　　
2017年04月 - 2020年03月
研究計画調書基盤研究(C), 研究分担者

人工股関節置換術後の骨溶解を制御する新規治療法の開発　　　　　　　　　　　　　　　
基盤研究（Ｃ）（一般）
2017年04月 - 2019年03月
アラーテルカウィ
基盤研究（Ｃ）（一般）, 研究代表者, 競争的資金

炎症性骨溶解を制御する新規治療法の開発　　　　　　　　　　　　　　　
2018年 - 2019年
公益財団法人 秋山記念生命科学財団 奨励助成, 研究代表者

免疫制御細胞の原虫破壊機構の解明と抗原虫ペプチドの開発　　　　　　　　　　　　　　　
若手研究 (B)
2015年04月 - 2017年03月
アラーテルカウィ
Japan Society for the Promotion of Science, 研究代表者, 競争的資金

破骨細胞分化抑制因子を含む骨粗鬆症治療剤　　　　　　　　　　　　　　　
特許権, アラーテルカウィ
特願P2018-008, 2018年04月09日

抗原虫能を持つ合成ペプチド　　　　　　　　　　　　　　　
特許権, アラーテルカウィ
特願2015-233632, 2015年10月

RESEARCH INTERESTS
My vision on research is driven by the concept that researchers should aid in addressing challenges and health problems in our society. Our immune system plays a central role in the human health and well-being via the ability to destroy a broad range of invading microbes and to eliminate toxic or allergenic substances. In addition, non-infectious substances that are perceived harmless can be recognized by immune system as invaders and can trigger an inflammatory response associated with harmful reaction in the tissues. This may cause serious complications in patients after organ transplantation, implantation or blood transfusion. Aseptic loosening of joint implants is one of the most common postoperative complications in artificial joint surgery due to inflammatory response. The particulate wear debris generated by motion at the bearing surfaces of implant and bone trigger a local inflammatory response coincident with loosening of implants. However, detailed mechanisms of inflammation and osteolytic events leading to joint failure are not yet understood.
The objective of my research is to clarify the cellular function of immune cells in loosening of joint implants.