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Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50 10 μM. There was no toxicity to any of the host cell lines at 10-100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.

Background The presence of a foreign body in the common bile duct (CBD) is a rare phenomenon. Thus, the route and mechanism of its migration remain difficult to fully clarify, especially for cases that occur after gastrectomy with Roux-en-Y reconstruction. Herein, we present a case of a CBD stone that formed around a fish bone as a nidus subsequent to distal gastrectomy with Roux-en-Y reconstruction. Case presentation A 70-year-old man was admitted to our hospital due to repeated episodes of epigastralgia. He had undergone distal gastrectomy with Roux-en-Y reconstruction for gastric cancer approximately 10 years prior. Blood tests revealed obstructive jaundice, hepatobiliary dysfunction, and inflammation. Multi-plane reconstructed computed tomography (CT) revealed a CBD stone with a needle-shaped calcification density at the center, oriented along the length of the CBD. Surgery was performed using an upper median laparotomy approach. Lithotomy with choledochotomy was performed to remove one fragile bilirubin stone that had formed around a 3-cm, needle-shaped fish bone. A choledochoduodenal fistula was not detected intraoperatively. A review of the imaging of a prior examination revealed that the formation of the CBD stone around the fish bone was observable on a follow-up CT performed approximately 2 years prior. However, no clinical symptoms associated with the migration of the fish bone to the CBD were reported and the fish bone was not detected at that time. Conclusion In this case, transpapillary migration of the fish bone could only be speculated in the absence of an observable fistula, choledochostomy, or any clinical symptoms. Our case is clinically relevant as cholangitis developed after CBD stone formation around the fish bone that acted as a nidus.

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, 50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.SIGNIFICANCEThe virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.

This chapter investigates the differences and similarities in students’ attitudes towards school subjects and the use of information and communication technology (ICT) in them. It focuses on the study of geography by secondary school students in Japan and New Zealand as a case study. The teaching methods and achievement goals for this subject are different in the two countries. Geography education in Japan is focused mainly on memorisation of the world’s topographical, economic, and political facts and data, while in New Zealand, it is centred on sustainability education and the acquisition of key geographic concepts/skills. This study conducted a survey of students’ understanding and feedback based on their self-evaluation. The results indicate that students’ evaluation of their comprehension of the subject is lower in Japan, but its usefulness for their future is higher compared to New Zealand. Moreover, the advantages of ICT in geography learning are equally recognised by students in both countries, but their attitudes towards its use in geography learning are more positive in New Zealand. It is not possible to conclude that the geography education in either country is better than the other, but in both cases it can still be improved for better quality and achievement.

Gallinamide A, originally isolated with a modest antimalarial activity, was subsequently reisolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs. Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (10, Ki = 0.0937 ± 0.01 nM and kinact/Ki = 8 730 000). From a protein structure and substrate preference perspective, cruzain, an essential Trypanosoma cruzi cysteine protease, is highly homologous. Our investigations revealed that gallinamide and its analogs potently inhibit cruzain and are exquisitely toxic toward T. cruzi in the intracellular amastigote stage. The most active compound, 5, had an IC50 = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect stage) and the host cell, and thus represents a new candidate for the treatment of Chagas disease.

Gallinamide A, originally isolated with modest antimalarial activity, was subsequently re-isolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs., Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (10, K(i) = 0.0937 ± 0.01 nM and k(inact) /K(i) = 8,730,000). From a protein structure and substrate preference perspective, cruzain, an essential Trypanosoma cruzi cysteine protease, is a highly homologous. Our investigations revealed that gallimamide and its analogs potently inhibit cruzain and are exquisitely toxic towards T. cruzi in the intracellular amastigote stage. The most active compound, 5, had an IC(50) = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect stage) and the host cell, and thus represents a new candidate for treatment of Chagas disease.

The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.

Background Chagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice. Methodology/Principal findings Both acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart. Conclusions/Significance The positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life., Author summary Chagas disease is a parasitic disease caused by the Trypanosoma cruzi. The infection may result in gastrointestinal manifestations and cardiomyopathy. Benznidazole, the current treatment, has limited efficacy and often leads to serious side effects. Aiming to develop new treatments, our group has identified new inhibitors that block the synthesis of parasitic lipids, resulting in parasite death. In this work, we evaluated the safety and efficacy of two of these compounds, 9 and 12, in mouse models of T. cruzi infection. Both compounds were well-tolerated by animals throughout the 28-day administration. In acutely infected mice, the compounds drastically reduced bloodstream parasites and increased survival. When treatment was initiated during the chronic phase, parasitemia dropped to background levels and remained undetectable for 4 weeks after drug withdrawal; parasites were re-activated by chemically-induced immunosuppression. Thus, the experimental compounds tested in these studies had an acceptable safety profile, achieved a marked reduction in parasite load and prevented heart injury due to inflammation, even in the absence of sterile cure. We conclude that the development of non-toxic medications capable of slowing the progression of cardiomyopathy is a valuable treatment option for Chagas disease patients because it could enhance the quality of life.

Toxoplasma gondii is an obligate intracellular parasite of all vertebrates, including man. Successful invasion and replication requires the synchronized release of parasite proteins, many of which require proteolytic processing. Unlike most parasites, T. gondii has a limited number of Clan CA, family C1 cysteine proteinases with one cathepsin B (TgCPB), one cathepsin L (TgCPL) and three cathepsin Cs (TgCPC1, 2, 3). Previously, we characterized toxopain, the only cathepsin B enzyme, which localizes to the rhoptry organelle. Two cathepsin Cs are trafficked through dense granules to the parasitophorous vacuole where they degrade peptides. We now report the cloning, expression, and modeling of the sole cathepsin L gene and the identification of two new endogenous inhibitors. TgCPL differs from human cathepsin L with a pH optimum of 6.5 and its substrate preference for leucine (vs. phenylalanine) in the P2 position. This distinct preference is explained by homology modeling, which reveals a non-canonical aspartic acid (Asp 216) at the base of the predicted active site S2 pocket, which limits substrate access. To further our understanding of the regulation of cathepsins in T. gondii, we identified two genes encoding endogenous cysteine proteinase inhibitors (ICPs or toxostatins), which are active against both TgCPB and TgCPL in the nanomolar range. Over expression of toxostatin-1 significantly decreased overall cysteine proteinase activity in parasite lysates, but had no detectable effect on invasion or intracellular multiplication. These findings provide important insights into the proteolytic cascades of T. gondii and their endogenous control.

A series of oxyguanidine analogues of the cysteine protease inhibitor WRR-483 were synthesized and evaluated against cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi. Kinetic analyses of these analogues indicated that they have comparable potency to previously prepared vinyl sulfone cruzain inhibitors. Co-crystal structures of the oxyguanidine analogues WRR-666 (4) and WRR-669 (7) bound to cruzain demonstrated different binding interactions with the cysteine protease, depending on the aryl moiety of the P1' inhibitor subunit. Specifically, these data demonstrate that WRR-669 is bound noncovalently in the crystal structure. This represents a rare example of noncovalent inhibition of a cysteine protease by a vinyl sulfone inhibitor.

Cysteine proteinases are key virulence factors of the protozoan parasiteEntamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of theE. histolyticagenome project has revealed more than 40 genes encoding cysteine proteinases. We have focused onE. histolyticacysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasiveE. histolyticaand is highly expressed and released. Recombinant EhCP1 was expressed inEscherichia coliand refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

症例は65歳女性，下血，直腸脱を主訴に来院した．Hb6.8g⁄dlと高度の貧血を認め，排便時に手拳大の完全直腸脱を認めた．下部消化管内視鏡検査では肛門縁より５～15cmの直腸に全周性の粘膜浮腫，発赤，ポリープ状の隆起性病変が認められた．病理組織では異型性の無い腺管の増生，固有層間質に細血管の増生拡張・平滑筋組織の樹枝状増生が認められ臨床症状とあわせて完全直腸脱に伴った粘膜脱症候群（Mucosal Prolapse Syndrome: MPS）と診断した．MPSの治療の基本は排便時のいきみの制限，緩下剤による便秘の予防などの保存的治療である．本症例では高度な直腸脱を認めた為，経腹的直腸固定術を施行した．術後経過良好で直腸脱の再発なく，粘膜病変の改善も見られ，MPSに対しても良好な治療効果が得られた．また，MPSと直腸脱の関係を考える上でも重要な症例と考えられたため報告する．

近年，上部消化管穿孔症例（胃潰瘍穿孔，十二指腸潰瘍穿孔）に対する穿孔部閉鎖術として開腹術に比し低侵襲な腹腔鏡下手術が導入されてきている．しかし，腹腔鏡下手術は初心者には手技が困難であり手術時間が延長する．腹腔鏡補助下手術における低侵襲度と手術困難度は明らかでないので従来の開腹手術と比較検討した．腹腔鏡補助下手術（12例）では穿孔部直上に約５cmの小開腹下に，開腹手術（13例）では上腹部正中切開下に穿孔部を直接閉鎖し，有茎大網で被覆した．両群間で背景因子に差はなかった．腹腔鏡補助下群で入院期間，経口摂取までの期間は有意に短縮し，術後７日目のWBC，CRPは有意に低値であった．出血量，手術時間には有意差がなく，術後合併症，再発は両群共になかった．従って腹腔鏡補助下手術は開腹手術と比較して手術困難度は増大せず，低侵襲な術式であると考えられた．

In October 2012, a man in his 60s was hospitalized for the examination of ascites, and was diagnosed with gastric cancer and carcinomatous peritonitis. S-1+cisplatin (CDDP) therapy was administered as neoadjuvant chemotherapy; however, during therapy, he developed cerebral infarction. We suspected that the cerebral infarction was caused by a coagulation disorder owing to Trousseau's syndrome. After completing 5 courses of chemotherapy, total gastrectomy was performed. Thereafter, the treatment was changed to S-1+docetaxel(DTX), followed by nab-paclitaxel (PTX). Although cerebral infarction did not relapse after the administration of an anticoagulant agent, the patient died of gastric cancer 1 year and 5 months after the operation. Trousseau's syndrome is a coagulation disorder in cancer patients. Cerebral infarctions caused by Trousseau's syndrome have a high tendency to relapse, and the prognosis is poor. This syndrome should be kept in mind when treating cancer patients.

症例は54歳,男性.主訴は下腹部痛. 2005年2月に,左尿管結石症で当院泌尿器科を受診し,その際に施行された腹部CTで,骨盤内腫瘍を指摘された.泌尿器科で,経直腸的に生検を施行された.生検後に下腹部痛を認めるようになった.腫瘍切除の目的で, 2005年4月に当科紹介となった.開腹時所見は,腹膜翻転部が発赤しており,炎症性変化で硬化していた.摘出標本は肥厚した壁に囲まれ,内部には角化物様の物質を認めた.また壁内側は表皮様組織で覆われていた.病理組織学的診断は,角化物と表皮を認め,表皮には毛髪や皮脂腺などの付属器,および軟骨成分などは認めず, epidermoid cystと診断された.術後経過は良好で,術後13日目に軽快退院した.仙骨前部に発生したepidermoid cystは,無症状のことが多く発見されにくいものの,感染や悪性化の可能性があるため,診断後は摘出術を施行するべきであると考えられた.

症例は56歳,男性,呼吸困難を主訴とし入院.右胸腔に巨大ブラがあり,健常肺虚脱,縦隔左方偏位がみられ, O221投与下でPaO271.1mmHg, PaCO258.6mmHg, VC1.751, FEV1.00.681, %VC 47.0%, FEV1.0%38.8%であった.右下葉bullaを穿刺し持続吸引後,症状の改善が得られ残存肺機能の評価ができ,安全に手術が可能であった.右腋窩中線第7肋間より胸腔鏡を挿入し第5肋間後側方小開胸下,ブラ壁境界健常肺に4-0 prolene SH水平マットレス縫合を置き,その直上で自動縫合器を掛け嚢胞を切除した.術後肺再膨張は良好でなり室内気でPaO276.4mmHg, PaCO245.9mmHg, VC3.021, FEV1.02.431, %VC 81.2, FEV1.0%85.3%に改善,術後13病日に退院した.巨大ブラのため呼吸困難に陥った症例に対する持続脱気は残存肺の機能評価,術後の再拡張後肺水腫の軽減に有用で,胸腔鏡補助下のブラ切除術は呼吸機能の保持に有用と考えられる.

症例は52歳, 男性, 左小胸筋下から第一肋間を貫通し胸腔内に発育した腫瘤の切除目的で入院となった.左襟状切開に第3肋間までの胸骨正中部分切開を加え前胸壁側と胸腔内から剥離を進めて腫瘍を完全切除した.病理組織診断はInfiltrating typeのintramuscular lipomaであり, 砂時計型経胸壁型成人脂肪腫と診断した.砂時計型経胸壁型成人脂肪腫は本邦で3例の報告が認められるのみである.本腫瘍は再発の可能性があるため脂肪腫といえども完全切除が必要である.また, 今回用いたアプローチは前胸部上部の胸壁内外に存在する腫瘍切除に有用と考えられた.

Background and Aims: Local autologous bone marrow cell implantation (BMCI) can induce therapeutic angiogenesis. We evaluated the safety and feasibility of this treatment for ischemic heart disease. Methods: The safety of BMCI was preclinically confirmed in dogs by giving a direct injection of either 2×107 bone marrow cells (n = 4) suspended in 0.1 mL phosphate-buffered saline (PBS), or 0.1 ml PBS only (n = 4, PBS group), into the myocardium of the left ventricle at six points. Electrocardiograph (ECG), echocardiography, and systemic biochemistry indexes were recorded 1, 7, 30, and 240 days after treatment, and histological change was examined 240 days after treatment. We also evaluated the clinical safety and feasibility of BMCI in six patients with ischemic heart disease, who were given BMCI treatment in combination with coronary artery bypass grafting (CABG), by recording Holter ECG, echocardiography, computed tomography, and systemic biochemistry indexes in the acute and chronic phases after treatment. Results: No significant changes in systemic biochemistry indexes were found after BMCI treatment in comparison with control groups, in either the experimental investigation or the clinical trial. No cardiac damage related to BMCI was detected by ECG or echocardiography, and histological examination showed minimal fibrotic change within the myocardium after BMCI or PBS injection in the dogs. Local calcification was not detected on histological slices 240 days after treatment in the dogs, or on computed tomography 1 year after treatment in the patients. Conclusions: BMCI treatment appears to be a safe and feasible method of inducing angiogenesis to treat ischemic heart disease. (J CARD SURG 2003;18 (Suppl 2):S69-S75)

Background. Local bone marrow cell implantation can induce angiogenesis. In the present study we investigated whether angiogenesis induced by bone marrow cell implantation improves deteriorated cardiac function in a rat heart model of hypoperfusion. Methods. A hypoperfusion heart model was created in Dark Agouti rats by ligating the left anterior descending artery placed against a copper wire (275 m), then pulling out the wire immediately. The left ventricular (LV) anterior wall was injected directly at six points, each with 1 10 7 bone marrow cells in 10 Lo f phosphate-buffered saline or with phosphate-buffered saline only, respectively. Echocardiography was performed to evaluate the cardiac function 7, 30, 60, and 90 days after treatment. Microvessel density and blood flow in the LV anterior wall were estimated 60 days after treatment. Results. Both the increase of LV end-systolic diameter and the decrease of percent of fractional shortening caused by myocardial ischemia were attenuated effectively by bone marrow cell implantation treatment. Bone marrow cell implantation treatment also increased the levels of angiopoietin-1 and vascular endothelial growth factor in the LV anterior wall. The microvessel density, blood flow, and thickness of the LV anterior wall significantly also increased after bone marrow cell implantation treatment compared with those after phosphatebuffered saline injection. Conclusions. The local implantation of autologous bone marrow cells induced angiogenesis and improved the perfusion of ischemic myocardium, thereby preventing LV remodeling and improving deteriorated cardiac function caused by myocardial hypoperfusion.

Background. Peripheral leukocytes and platelets (LAPs) contain many kinds of cells with the ability to secrete several growth factors and cytokines. We attempted to induce therapeutic angiogenesis by injecting self-LAPs into a rat ischemic hindlimb model. Materials and methods. Supernatants from cultured LAPs were used for the endothelial cell (EC) proliferation assay, and LAPs were used in a cornea model to evaluate angiogenic potency. LAPs were injected directly into the male Dark Agouti rat ischemic hindlimb model, after which a microangiogram was done and the capillary/muscle fiber ratio was examined histologically. ELISA revealed the levels of contributing growth factors and cytokines present in the ischemic muscles. Results. The EC proliferation assay showed that the supernatants of LAPs accelerated proliferation and that the LAPs induced angiogenesis in the cornea model. The microangiograms and histological evaluation revealed that angiogenesis was induced more effectively in the rats injected with LAPs (LAP group) than in the those injected with phosphate-buffered saline (PBS group). The levels of basic fibroblast growth factor (bFGF) in the ischemia, PBS, and LAP groups were significantly increased compared to those in the sham group. The level of interleukin-1β (IL-1β) in the LAP group was significantly more elevated than in the other groups. Conclusions. The injection of self-LAPs induced angiogenesis in a rat ischemic hindlimb model. Ischemia caused an elevation in the level of bFGF and also in IL-1β derived from LAPs, which contributed to angiogenesis. This is a novel, yet simple and safe method of inducing therapeutic angiogenesis.

Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence.

開心術における虚血/再灌流障害に好中球が関与しているか否かを知る目的で,大動脈遮断前後で冠循環での顆粒球エラスターゼおよびミエロパーオキシダーゼ産生量を測定した.再灌流後,冠循環での顆粒球エラスターゼおよびミエロパーオキシダーゼ産生量は有意に上昇した.さらに再灌流後の冠循環での顆粒球エラスターゼ,ミエロパーオキシダーゼ産生量は大動脈遮断時間と正の相関を示した.これらの結果から,開心術における心虚血/再灌流障害に好中球が関与していることが示唆された.

体外循環中の肺障害に好中球が関与しているか否かを知る目的で, 体外循環前後で肺循環での顆粒球エラスターゼ, ミエロパーオキシダーゼ産生量および respiratory index を測定した. 体外循環後, 肺循環での顆粒球エラスターゼ, ミエロパーオキシダーゼ産生量および respiratory index 値は有意に上昇した. さらに体外循環後の肺循環での顆粒球エラスターゼ, ミエロパーオキシダーゼ産生量は術前肺動脈圧および respiratory index の変化率と正の相関を示した. これらの結果から, 体外循環中の肺機能障害に好中球が強く関与しており, 肺高血圧症例でそれがより顕著であることが示された.

Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

Cysteine proteinases have a number of important functions in the life cycle of protozoan parasites. Based on our previous studies demonstrating the role of cysteine proteinases in invasion by Entamoeba histolytica, we evaluated the cysteine proteinases of E. invadens, a related protozoan which causes invasive disease of reptiles. E. invadens readily encysts in axenic culture and provides a model to investigate the role of cysteine proteinases in encystation. Broad bands of approximately 130-230, 55, and 35 kDa were detected on gelatin substrate gels and were inhibited with specific cysteine proteinase inhibitors. Maximal enzymatic activity was detected with peptide substrates containing arginine in the P2 position. A 567-bp fragment containing the active site of an E. invadens cysteine proteinase gene was amplified by PCR and had 37.7, 79.1, and 67.9% identity to the derived amino acid sequences of the acp 1, 2, and 3 genes, respectively, of E. histolytica. The PCR product hybridized with a single band of 1.1 kb on a Southern blot of EcoRI-restricted E. invadens genomic DNA. Long-term inhibition of cysteine proteinase activity during encystation resulted in significantly fewer cysts (P < 0.02); however, this effect appeared to be secondary to decreased trophozoite cell division. No difference in chitin synthase activity was detected between controls and encysting cells with inhibited cysteine proteinases, suggesting that these proteinases are not critical for activation of a zymogen form of chitin synthase. These studies demonstrate that cysteine proteinases may be critical for the survival of E. invadens, and specific inhibition may ultimately interrupt transmission.

The incidence of delayed bleeding after endoscopic submucosal dissection (ESD) for gastric neoplasms is reported to be approximately 5 %. We examined whether postprocedural combined treatment using the coagulation plus artery-selective clipping (2C) method is a useful measure for preventing delayed bleeding after ESD. A total of 234 gastric epithelial neoplasms were treated from June 2007 to May 2012. Post-ESD coagulation (PEC) and clipping for part of the vessels was performed for 154 lesions from June 2007 to June 2010. A total of 80 lesions were treated using the 2C method from July 2010 to May 2012. During ESD, the locations of the arteries were recorded on a schematic diagram of the lesion. Arteries were defined as regions of arterial bleeding that required coagulation or apparent arteries in which preventive coagulation was performed. When ESD was completed, soft coagulation was performed for arteries in the resection area using hemostatic forceps, followed by arterial clipping for additional strength. Coagulation also was performed continuously for visible vessels in the resection area. This was a retrospective study. The incidence rates of delayed bleeding after ESD, as evidenced by hematemesis or melena, or the presence of anemia (decline in Hb >2 g/dl) that required emergency endoscopy were recorded. Delayed bleeding occurred in 7 (4.5 %) of the 154 cases treated using PEC and in 1 (1.3 %) of the 80 cases treated using the 2C method. The mean time required for the 2C method was 15.0 ± 7.0 min (range, 5–44 min). The mean number of clippings per lesion was 3.8 ± 2.8 (range, 0–13). Almost all clips fell off within 2 months of the procedure. Coagulation plus artery-selective clipping (the 2C method) of post-ESD ulcers might effectively reduce the incidence of delayed bleeding after ESD for gastric neoplasms.

Aim. Delayed bleeding after endoscopic submucosal dissection (ESD) for gastric epithelial neoplasms is a major complication. We investigated factors related to post-ESD bleeding to identify preventive measures.Methods. The study included 161 gastric epithelial neoplasms in 142 patients from June 2007 to September 2010. Post-ESD bleeding was defined as an ulcer with active bleeding or apparent exposed vessels diagnosed by an emergency endoscopy or a planned follow-up endoscopy. We analyzed associations between bleeding and the following factors: age, sex, morphology, pathology, tumor depth, ulcer presence/absence, location, size of the resected lesion, duration of the procedure, the number of times bleeding occurred during ESD, and the use of anticoagulants and/or antiplatelet drugs. Subsequently, we examined characteristics of bleeding cases.Results. Post-ESD bleeding occurred in 21 lesions. Univariate analysis of these cases showed that ulcer presence/absence (P<0.001), middle or lower third lesions (P=0.036), circumference (P=0.014), and a post-ESD ulcer with an extended lesser curve (P=0.009) were significant predictors of bleeding. Multivariate analysis showed that ulcer presence/absence (OR 9.73, 95% CI 2.28–41.53) was the only significant predictor.Conclusion. Ulcer presence/absence was considered the most significant predictor of post-ESD bleeding.

The enteric protozoan parasite Entamoeba histolytica is the cause of potentially fatal amebic colitis and liver abscesses. E. histolytica trophozoites colonize the colon, where they induce inflammation, penetrate the mucosa, and disrupt the host immune system. The early establishment of E. histolytica in the colon occurs in the presence of antimicrobial human (LL-37) and murine (CRAMP [cathelin-related antimicrobial peptide]) cathelicidins, essential components of the mammalian innate defense system in the intestine. Studying this early step in the pathogenesis of amebic colitis, we demonstrate that E. histolytica trophozoites or their released proteinases, including cysteine proteinase 1 (EhCP1), induce intestinal cathelicidins in human intestinal epithelial cell lines and in a mouse model of amebic colitis. Despite induction, E. histolytica trophozoites were found to be resistant to killing by these antimicrobial peptides, and LL-37 and CRAMP were rapidly cleaved by released amebic cysteine proteases. The cathelicidin fragments however, did maintain their antimicrobial activity against bacteria. Degradation of intestinal cathelicidins is a novel function of E. histolytica cysteine proteinases in the evasion of the innate immune system in the bowel. Thus, early intestinal epithelial colonization of invasive trophozoites involves a complex interplay in which the ultimate outcome of infection depends in part on the balance between degradation of cathelicidins by amebic released cysteine proteinases and upregulation of proinflammatory mediators which trigger the inflammatory response.

We report 2 cases of early gastric cancer arising in hyperplastic polyps. The first case was 6mm in diameter, tub1, in an intestinal phenotype. The second case was 10mm in diameter, pap, in a gastric phenotype. Before resection, their biopsies had been diagnosed as atypical hyperplastic polyps. These cases suggest that small gastric hyperplastic polyps possess a certain malignant potential. If a hyperplastic polyp shows atypia in the biopsy, a complete resection should be considered.

A new therapy for severe ischemic heart disease has been developed; therapeutic angiogenesis induced by the local implantation of autologous bone marrow cells (BMC). After confirming that no detrimental changes were induced by this treatment in a canine heart model, a clinical trial was commenced in 1999. Thus far, 5 patients have been given this new treatment concomitant with coronary artery bypass grafting and all have been followed up for at least 1 year. Autologous BMC were implanted into the ungraftable area and postoperative cardiac scintigraphy showed specific improvement in coronary perfusion in 3 of the 5 patients. Postoperative chest radiography, electrocardiography, echocardiography and blood tests did not reveal any detrimental changes. In conclusion, this new therapy appears to be safe and could provide a treatment option for patients with otherwise untreatable ischemic heart disease.

Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.

Cysteine proteinases are key virulence factors of Entamoeba histolytica that are released during the process of invasion. We used a chemical mutant of E. histolytica strain HM-1:IMSS, clone L6, which is deficient in virulence, phagocytosis, and cysteine proteinase activity to help define the mechanisms of cysteine proteinase release. All cysteine proteinase genes of wild type HM-1 were present in the L6 mutant genome, but three of the major expressed proteinases, ehcp1, ehcp2, and ehcp5 were both transcribed, translated, and released at lower levels in L6. We hypothesized that a central protein such as the calcium binding protein 1, EhCaBP1, which is required for both phagocytosis and exocytosis might be deficient in this mutant. We found that both mRNA and proteinase levels of EhCaBP1 were decreased in L6. These findings provide an important link between phagocytosis, passive release of multiple cysteine proteinases, and attenuated virulence of this E. histolytica mutant.

Peroxiredoxins are an important class of antioxidant enzymes found from Archaea to humans, which reduce and thereby detoxify peroxides and peroxynitrites. The major thiol-containing surface antigen of the invasive ameba, Entamoeba histolytica, is a peroxiredoxin and is likely to be important during the transition from the anaerobic environment of the large intestine to human tissues. The closely related species, Entamoeba dispar, is incapable of invasion and more sensitive to hydrogen peroxide, yet also has a peroxiredoxin. We cloned and expressed the two active recombinant enzymes and found that their activity was similar by a fluorometric stopped-flow assay, giving a Km of

Background . Therapeutic angiogenesis was induced by local autologous bone marrow cell implantation (BMCI) in ischemic hindlimb or ischemic heart models in rats. This study was designed to investigate the toxicity and therapeutic potency of local BMCI using a chronic coronary occlusion model in dogs. Methods . The canine chronic coronary occlusion model was created by ligating of the left anterior descending artery (LAD). The myocardium in the left ventricle was divided into distinct normal, marginal, and infarction areas 30 days after LAD ligation. Each area was injected at two locations, with either 2 × 10 7 bone marrow cells (n = 7, BMCI group) or 0.1 mL phosphate-buffered saline (PBS) only (n = 7, PBS group), respectively. Hemodynamics were evaluated by a single ultrasonic transducer and echocardiography before and 30 days after the treatment. Angiogenesis was evaluated by vessel count 30 days after the treatment. The toxicity of BMCI treatment was also evaluated in 8 normal dogs by following changes in electrocardiography (ECG), echocardiography, local histology, and systemic biochemistry indexes. Results . There was a significantly higher percentage of wall thickening in the marginal area in the BMCI group than in the PBS group 30 days after treatment (14.5 ± 2.28 versus 8.1 ± 3.00, p=0.002). Significantly more microvessels were observed in the marginal area in the BMCI group than in the PBS group 30 days after treatment (127.7 ± 20.1 versus 88.0 ± 10.2/field, p=0.0007). No systemic or local toxicity was found following BMCI treatment in the acute or chronic phases. Conclusions . BMCI treatment improved local wall thickening dynamics, presumably due to the angiogenesis induced by the treatment. This indicates that it might be a safe and effective therapy for ischemic heart disease.