Masamichi Oshima

Churg-Strauss syndrome (allergic granulomatous antiitis) with multiple perforating ulcers of the small intestine, multiple ulcers of the colon, and mononeuritis multiplex

A case of Churg-Strauss syndrome with multiple perforations of the small intestine is described. A 31-year-old woman was admitted with a complaint of epigastric pain. She had a history of bronchial asthma. One week before admission, white blood cell count was 20 800/mm3 with 59% eosinophils. Neurological examination on admission disclosed mononeuritis multiplex with paresthesia in both the lower and upper extremities. At colonoscopy, there were scattered aphthous ulcers in the colon. Ophthalmological examination revealed allergic conjunctivitis. After admission, hypereosinophilia increased to as high as 36 000/mm3. Oral administration of prednisolone (60 mg/day) was begun. On the 3rd day of the treatment, the eosinophil count decreased dramatically, to 400/mm3, while severe abdominal pain developed. Since abdominal X-ray film revealed free air in the abdominal cavity, emergency laparotomy was performed and multiple intestinal ulcers with perforations were found. Partial ileectomy was performed. Pathological findings of the resected specimen were interpreted as a necrotizing angiitis with extravascular granuloma. Since the operation, the patient has been asymptomatic, except for neurological symptoms. Hypereosinophilia has decreased without treatment to counts averaging 270/mm3, within 3 months. On the basis of the clinical features and histopathological findings, a diagnosis of Churg-Strauss syndrome was established.

Contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection

Because the pathogenesis of acute respiratory distress syndrome (ARDS) induced by influenza virus infection remains unknown, we can only improve on existing therapeutic interventions. To approach the subject, we investigated immunological etiology focused on cytokines and an acute lung damage factor in influenza‐induced ARDS by using a PR‐8 (A/H1N1)‐infected mouse model. The infected mouse showed fulminant severe pneumonia with leukocyte infiltration, claudin alteration on tight junctions, and formation of hyaline membranes. In addition to interferon (IFN)‐α, plenty of keratinocyte‐derived chemokines (KC), macrophage inflammatory protein 2 (MIP‐2), regulated on activation normal T‐cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP‐1) were significantly released into bronchoalveolar lavage fluid (BALF) of the model. We focused on neutrophil myeloperoxidase (MPO) as a potent tissue damage factor and examined its contribution in influenza pneumonia by using mice genetically lacking in MPO. The absence of MPO reduced inflammatory damage with suppression of leakage of total BALF proteins associated with alteration of claudins in the lung. MPO−/− mice also suppressed viral load in the lung. The present study suggests that MPO‐mediated OCl− generation affects claudin molecules and leads to protein leakage and viral spread as a damage factor in influenza‐induced ARDS.

A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV.

Abstract In response to SARS-CoV infection, neutralizing antibodies are generated against the Spike (S) protein. Determination of the active regions that allow viral escape from neutralization would enable the use of these antibodies for future passive immunotherapy. We immunized mice with UV-inactivated SARS-CoV to generate three anti-S monoclonal antibodies, and established several neutralization escape mutants with S protein. We identified several amino acid substitutions, including Y442F and V601G in the S1 domain and D757N and A834V in the S2 region. In the presence of each neutralizing antibody, double mutants with substitutions in both domains exhibited a greater growth advantage than those with only one substitution. Importantly, combining two monoclonal antibodies that target different epitopes effected almost complete suppression of wild type virus replication. Thus, for effective passive immunotherapy, it is important to use neutralizing antibodies that recognize both the S1 and S2 regions.

Reversal by Dithiothreitol Treatment of the Block in Murine Leukemia Virus Maturation Induced by Disulfide Cross-Linking

ABSTRACT We previously reported that if murine leukemia virus particles are produced in the presence of the mild oxidizing agent disulfide-substituted benzamide-2, they fail to undergo the normal process of virus maturation. We now show that treatment of these immature particles with a reducing agent (dithiothreitol) induces their maturation in vitro, as evidenced by proteolytic cleavage of Gag, Gag-Pol, and Env proteins and by their morphology. The identification of partial cleavage products in these particles suggests the sequence with which the cleavages occur under these conditions. This may be a useful experimental system for further analysis of retroviral maturation under controlled conditions in vitro.

Leucomycin A3, a 16-membered macrolide antibiotic, inhibits influenza A virus infection and disease progression.

Severe respiratory disease arising from influenza virus infection has a high fatality rate. Neutrophil myeloperoxidase (MPO) has been implicated in the pathogenesis of severe influenza-induced pneumonia because extracellularly released MPO mediates the production of hypochlorous acid, a potent tissue injury factor. To search for candidate anti-influenza compounds, we screened leucomycin A3 (LM-A3), spiramycin (SPM), an erythromycin derivative (EM900, in which anti-bacterial activity has been eliminated), and clarithromycin (CAM), by analyzing their ability to inhibit MPO release in neutrophils from mice and humans. When each candidate was injected into mice infected with a lethal dose of A/H1N1 influenza virus (PR-8), LM-A3 produced the highest survival rate (80.9%). We found that LM-A3 induced beneficial effects on lung pathology and viral proliferation involved in the regulatory activity of MPO release, pro-inflammatory cytokines and interferon-α production in the lung. SPM and EM900 also induced positive survival effects in the infected mice, whereas CAM did not. We further found that these compounds inhibit virus proliferation in human pneumonia epithelial A549 cells in vitro. LM-A3 showed effective action against influenza A virus infection with high anti-viral activity in human host cells, indicating the possibility that LM-A3 is a prospective lead compound for the development of a drug for human influenza. The positive survival effect induced by EM900 suggests that pharmacological architectures between anti-bacterial and anti-influenza virus activities can be dissociated in macrolide derivatives. These observations provide valuable evidence for the potential development of novel macrolide derivatives that have strong anti-viral but no anti-bacterial activity.

Reduction of MPO-ANCA epitopes in SCG/Kj mice by 15-deoxyspergualin treatment restricted by IgG2b associated with crescentic glomerulonephritis

OBJECTIVES The MPO-specific antineutrophil cytoplasmic antibodies (MPO-ANCA) are associated with renal failure. Epitopes of MPO-ANCA and immunoglobulin G (IgG) subclass and cytokine levels in the recovery phase were analysed by the administration of 15-deoxyspergualin (DSG) to SCG/Kj mice, which show spontaneous crescentic GN (CrGN). METHODS We treated SCG/Kj mice by using DSG and MPO deletion mutants to investigate epitopes of MPO-ANCA associated with renal failure in SCG/Kj mice. After DSG treatment for 30 days, we observed histological changes in a crescentic formation and infiltration of neutrophils and lymphocytes into kidney, cytokines/chemokines and MPO-ANCA epitopes by deletion mutants. RESULTS MPO-ANCA were reduced by the administration of DSG, and epitopes of MPO-ANCA, mainly H-6, decreased. Moreover, the IgG2b subclass of the H-6 epitope of MPO-ANCA was greatly decreased by DSG treatment. These observations correlated with a decrease in renal failure and proteinuria, infiltration of neutrophils and lymphocytes into glomeruli, and crescent formation. The CD4/CD8 ratio of splenocytes ranged from 1.68 (0.24) in the non-treated group to 0.90 (0.12) at 100 microg/mouse/day in the DSG-treated group. In addition, elevated levels of IL-12p40, IL-10 and IL-13 in the active phase of CrGN clearly decreased with DSG treatment but not with TNF-alpha. In contrast, the IL-1alpha level increased, and IL-17 and IL-12p70 slightly increased with DSG treatment. CONCLUSION These results strongly suggest that DSG treatment of SCG/Kj mice leads to the reduction of risk antibodies in IgG2b and normalization of B-cell clones accompanied by recovery of the cytokine and chemokine balance.

Detection of a 5′ end subgenome of hepatitis C virus terminating at nucleotide 384 in patients’ plasma and liver tissues

Summary.  Quadri and Negro [Dig Liver Dis 2001; 33: 480] reported greater distribution of 5′ end genomic RNA of hepatitis C virus (HCV) over its 3′ end in the liver of patients with recurrent hepatitis C after liver transplantation. We not only confirmed their results by quantifying the 5′ end subgenomes in various specimens by using dilution and real‐time polymerase chain reaction methods, but also discovered that such subgenomes terminated at nucleotide (nt) 384 of the viral genome or in its immediate upstream. The subgenomes in the plasma uniformly, with a few exceptions, ended at this position, while those in the liver more heterogeneously at various points upstream of nt 384. Subgenome populations ending some points in the downstream of nt 384 were not detected. The amount of the 5′ end subgenome, while fluctuating during the clinical course of the patients, exceeded that of the longer sized HCV genomes which included the intact genome, and, when the relative ratio of the 5′ end subgenome increased, the amount of longer sized HCV RNA tended to decrease, suggesting a suppressive effect of the 5′ end subgenome on viral replication.

Isolation of human monoclonal antibodies to the envelope e2 protein of hepatitis C virus and their characterization.

We isolated and characterized two human monoclonal antibodies to the envelope E2 protein of hepatitis C virus (HCV). Lymphoblastoid cell lines stably producing antibodies were obtained by immortalizing peripheral blood mononuclear cells of a patient with chronic hepatitis C using Epstein-Barr virus. Screening for antibody-positive clones was carried out by immunofluorescence with Huh7 cells expressing the E2 protein of HCV strain H (genotype 1a) isolated from the same patient. Isotype of resulting antibodies, #37 and #55, was IgG1/kappa and IgG1/lambda, respectively. Epitope mapping revealed that #37 and #55 recognize conformational epitopes spanning amino acids 429 to 652 and 508 to 607, respectively. By immunofluorescence using virus-infected Huh7.5 cells as targets both antibodies were reactive with all of the nine different HCV genotypes/subtypes tested. The antibodies showed a different pattern of immuno-staining; while #37 gave granular reactions mostly located in the periphery of the nucleus, #55 gave diffuse staining throughout the cytoplasm. Both antibodies were shown by immuno-gold electron microscopy to bind to intact viral particles. In a neutralization assay (focus-forming unit reduction using chimeric infectious HCV containing structural proteins derived from genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a, and 7a), #55 inhibited the infection of all HCV genotypes tested but genotype 7a to a lesser extent. #37 did not neutralize any of these viruses. As a broadly cross-neutralizing human antibody, #55 may be useful for passive immunotherapy of HCV infection.

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5′‐end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap‐dependent DsRed2 and IRES‐dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AGs signal to DsRed2s in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type‐specific manner, and that a synthetic micro‐RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti‐IRES drugs.