Tirtha Raj Koirala

Malignant lymphoma induction of rabbits with oral spray of Epstein-Barr virus-related herpesvirus from Si-IIA cells (HTLV-II-transformed Cynomolgus cell line): A possible animal model for Epstein-Barr virus infection and subsequent virus-related tumors in humans

Malignant lymphoma (ML) was Induced in eight of nine rabbb inoculated by oral spray of the cell‐free pellets from SI‐IIA culture (MLV‐ll‐transformed leukocyte cell line of the Cynomolgus‐producing Epsteln‐Barr virus (EBV)‐related herpesvirus) after 64–141 days. None of the rabbits inoculated with EBV from B‐95–8 cells or HTLV‐II from MOT cells developed ML. Malignant lymphomas were usually of diffuse, large‐cell or mixed type. HTLV‐II infection was excluded by the polymerase chain reaction (PCR) and the particle agglutination test. EBV‐encoded RNA‐1 and EBV‐related DNA were detected in the tumor tissues by In situ hybridization and PCR, respectively. Anti‐viral capsid antigen of EBV antibody (anti‐VCA) was observed 3 weeks after oral inoculation of Si‐IIA cell‐free pellets. Polymerase chain reaction revealed continuous detection of EBV‐related virus DNA in the peripheral blood leukocytes from 3 days after oral inoculation. These results show that ML induced orally wtth SI‐IIA cell‐free pellets was caused by EBV‐related herpesvirus harbored by Si‐IIA cells. Oral spray of EBV from B‐95–8 also induced EBV Infection in rabbits, which was confirmed both by the presence of anti‐VCA and by PCR. These oral infection and mallgnant lymphoma induction systems of rabbit using EBV‐related virus from Si‐IIA or human EBV are useful animal models for the study of EBV infection and EBV‐related lymphomas in humans.

HTLV‐II Non‐integrated Malignant Lymphoma Induction in Japanese White Rabbits Following Intravenous Inoculation of HTLV‐II‐infected Simian Leukocyte Cell Line (Si‐IIA)

Lymphoma induction in rabbits by an unknown factor derived from an HTLV‐II‐producing simian (Cynomolgus) leukocyte cell line (Si‐IIA) is reported. Thirteen of 17 male Japanese white rabbits (76%) inoculated intravenously with Si‐IIA cells developed malignant lymphoma including Hodgkin‐like lymphoma between 62 and 167 days after inoculation. Historically, there was extensive diffuse or nodular infiltration of either large cell type or mixed type lymphoma cells in many organs, frequently involving the spleen, liver, lymph nodes and kidneys, and less frequently the thymus, bone marrow, lungs, heart, skin and gastrointestinal tract. Hodgkin‐like lymphoma was also observed in two rabbits. Chromosomal analysis of five cell lines established from tumor‐bearing rabbits revealed the male rabbit karyotype. The immunophenotype of these tumor cells was usually T‐cell (CD5+or, r RT1+, RT2+or‐, CD45+, CD4−, RABELA− and MHC class II‐DQ+) except for Hodgkin‐like lymphoma cells which expressed only CD45. However, integration of the HTLV‐II provirus genome could not be demonstrated in the tumor tissues or any of the rabbit cell lines by polymerase chain reaction or Southern blot analysis. Moreover, no lymphoma was induced by inoculation of HTLV‐IIC, MOT (other HTLV‐II‐producing human cell lines) or TALL‐1 (control). Two of four rabbits injected with cell‐free pellets from Si‐IIA cultures died of malignant lymphoma (15‐20 days). Five irradiated rabbit cell lines were inoculated but only one (Ra‐SLN) induced lymphoma in 1 of 3 rabbits at 27 days. Neither Herpesvirus saimiri nor Herpesvirus ateles (simian oncogenic viruses) was detected in Si‐IIA cells by immunofluorescence testing. These data suggest that the high rate of lymphoma induction in rabbits may be caused not by only HTLV‐II or well known simian oncogenic viruses, but rather by an unknown passenger agent derived from Si‐IIA or HTLV‐IIA, with which Si‐IIA was established.

Inflammatory pseudotumor of the urinary bladder with an aberrant expression of cytokeratin

A case of inflammatory pseudotumor of the urinary bladder in a 47 year old Japanese male patient is presented. Inflammatory pseudotumor of the urinary bladder is a benign but rare proliferative lesion of the submucosal stroma, easily mistaken for a malignant neoplasm. Based on the clinical diagnosis of bladder cancer by cystoscopy and magnetic resonance imaging (MRI), urologists started chemotherapy before results of the histological report were available which described inflammatory pseudotumor on the biopsy. Biopsied materials showed marked proliferation of irregularly bundled spindle ceils, varied in size and shape and separated in severe loose myxoid stroma with moderate infiltration of the inflammatory cells and capillary proliferations. At a glance, these findings resemble the sarcomatous pattern. However neither severe nuclear atypism nor atypical mitoses were present. Immunohistochemically, these spindle cells, which were positive for vimentin and α‐smooth muscle actin, showed a diffuse aberrant expression of cytokeratin. Some of them were positive for phosphotungstic acid hematoxylin. Electron microscopy revealed only the fibroblasts. No recurrence has been observed for 10 months. These findings indicate that inflammatory pseudotumor is a benign mesenchymal lesion that must be discriminated from true sarcoma to avoid subjecting the patient to unnecessary therapy. Only careful histological examination can enable a successful diagnosis.

HTLV-II-specific antisera raised in rabbits immunized with a synthetic peptide of HTLV-II envelope protein

SummaryIn order to discriminate HTLV-II from HTLV-I, HTLV-II-specific polyclonal antibodies against a synthetic peptide of HTLV-II envelope sequence were raised in rabbits. We immunized two adult rabbits with a KLH-conjugated synthetic peptide corresponding to the amino acid sequence 171–196 of the HTLV-II envelope sequence, which is a specific region for HTLV-II as evaluated with an ELISA method. The resulting rabbit antisera to the synthetic peptide reacted with gp46 of HTLV-II lysates in Western blot analysis, but not with that of HTLV-I. Flow cytometric analysis and immunohistochemical study revealed that these affinity purified antisera recognized some HTLV-II-producing cell lines examined, but not HTLV-I-producing cell lines or other cell lines uninfected by HTLV. These findings indicate that these antisera specifically recognized the envelope glycoprotein (gp46) of HTLV-II and suggest the specificity of this region in the immune response to HTLV-II. Such antisera are useful in distinguishing between HTLV-I and HTLV-II infection and in determining the presence of individual HTLV-II-infected cells both in vivo and in vitro, including non-lymphoid cells. They may also assist in the elucidation of the pathogenesis of HTLV-II.

Protection of rabbits against HTLV-II infection with a synthetic peptide corresponding to HTLV-II neutralization region

SummaryRabbit immune sera raised against synthetic peptides of the HTLV-II envelope gp46 region were examined for HTLV-II neutralization ability by HTLV-vesicular stomatitis virus (VSV) pseudotype assay and syncytium inhibition assay. HTLV-II neutralization activity was detected in the sera against HTLV-II Env gp46, 80–103 but not in those to HTLV-II Env gp46, 171–196. Three rabbits immunized with the synthetic peptide of HTLV-II Env gp46, 80–103 and three non-immunized rabbits were challenged with intravenous inoculation of an HTLV-II-producing human cell line (MOT, 1×107 cells). The non-immunized rabbits showed seroconversion for HTLV-II after 2 weeks and maintained persistent infection but the immunized rabbits were protected from HTLV-II infection. Nested or repeated polymerase chain reaction revealed the presence of HTLV-II provirus sequences in the non-immunized rabbits but not in the immunized rabbits. These results suggest that peptide vaccination with a synthetic peptide corresponding to the HTLV-II neutralization region is useful for preventing HTLV-II infection.