Novel tumour suppressor roles for GZMA and RASGRP1 in Theileria annulata-transformed macrophages and human B lymphoma cells.

Theileria annulata is a tick-transmitted apicomplexan parasite that infects and transforms bovine leukocytes into disseminating tumours that cause a disease called tropical theileriosis. Using comparative transcriptomics we identified genes transcriptionally perturbed during Theileria-induced leukocyte transformation. Dataset comparisons highlighted a small set of genes associated with Theileria-transformed leukocyte dissemination. The roles of Granzyme A (GZMA) and RAS guanyl-releasing protein 1 (RASGRP1) were verified by CRISPR/Cas9-mediated knockdown. Knocking down expression of GZMA and RASGRP1 in attenuated macrophages led to a regain in their dissemination in Rag2/γC mice confirming their role as dissemination suppressors in vivo. We further evaluated the roles of GZMA and RASGRP1 in human B lymphomas by comparing the transcriptome of 934 human cancer cell lines to that of Theileria-transformed bovine host cells. We confirmed dampened dissemination potential of human B lymphomas that overexpress GZMA and RASGRP1. Our results provide evidence that GZMA and RASGRP1 have a novel tumour suppressor function in both T. annulata-infected bovine host leukocytes and in human B lymphomas.

Spinal lymphoma and pulmonary filariasis in a pet domestic rabbit (Oryctolagus cuniculus domesticus).

Spinal lymphoma and concurrent pulmonary filariasis are reported in a pet rabbit. The rabbit presented for pelvic limb paralysis resulting from extradural spinal lymphoma, presumably rising from the body of the sixth lumbar vertebra. The neoplasm was subsequently immunophenotyped as a B-cell lymphoma. Pulmonary filariasis was an incidental finding at necropsy.

Molecular pathology of primary intraocular lymphoma.

PURPOSE: To evaluate immunoglobulin heavy chain (IgH) gene rearrangements, cytokines and chemokines, and infectious agents in primary intraocular B-cell lymphoma (PIOL) cells, in order to better diagnose and understand PIOL. METHODS: We studied ocular specimens from 57 patients with PIOL at the National Eye Institute from 1991 to 2001. Specimens were analyzed for IgH gene rearrangements using microdissection and polymerase chain reaction (PCR). We measured vitreal interleukin (IL)-10 and IL-6 levels by enzyme-linked immunosorbent assay. IL-10 mRNA was studied in PIOL cells using microdissection and reverse transcribed (RT)-PCR. Chemokine and chemokine receptor expression was examined by using immunohistochemistry. Infectious DNA of human herpetic virus-8 (HHV-8), Epstein-Bar virus (EBV), and Toxoplasma gondii was detected by using microdissection and PCR and was confirmed with Southern blot hybridization. RESULTS: IgH rearrangement(s) were demonstrated in all 50 tested cases. Cytokine levels were measured in the vitreous of 39 patients. Thirty-one had measurable cytokine levels: 24 of 31 had elevation of IL-10 relative to that of IL-6, and, in contrast, only 7 of 31 had elevation of IL-6 relative to IL-10. IL-10 mRNA was abundant in lymphoma cells of 6 examined cases. Lymphoma cells expressed chemokine receptors of CXCR4 and CXCR5 in three tested cases. HHV-8 DNA was found in 6 of 32 cases (18.8%), EBV DNA in 2 of 21 (9.5%), and T gondii DNA in 2 of 16 (12.5%). CONCLUSIONS: Molecular analyses detecting IgH rearrangements and vitreal levels of IL-10 and IL-6 are useful adjuncts for PIOL diagnosis. A role for specific infectious agents is hypothesized in the pathogenesis of some cases of PIOL. B-cell chemokine is likely involved in attracting PIOL cells into the eye.

Detection of Toxoplasma gondii DNA in primary intraocular B-cell lymphoma.

Primary intraocular lymphoma, a variant of primary central nervous system lymphoma with ocular involvement, is a large B-cell non-Hodgkin's lymphoma. Some cases of primary intraocular lymphoma have been reported to be associated with microorganisms including Epstein-Barr virus (EBV) and human herpes virus-8 (HHV-8), but not parasites. We analyzed 10 cases of primary intraocular lymphoma using microdissection and PCR. Tumor and normal cells were microdissected from ocular tissue on slides and subjected to PCR for genes from Toxoplasma gondii, EBV, and HHV-8. We detected Toxoplasma gondii, not HHV-8 or EBV, DNA in the lymphoma but not in normal cells of two cases that resembled ocular toxoplasmosis clinically. We speculate that Toxoplasma gondii may play a role in some forms of primary intraocular B-cell lymphoma.

Isolation of naturally processed peptides from a Toxoplasma gondii-infected human B lymphoma cell line that are recognized by cytotoxic T lymphocytes.

Naturally processed peptides derived from Toxoplasma gondii (T. gondii) were acid extracted from T. gondii-infected cells and detected by cytotoxic T lymphocytes (CTL) derived from peripheral blood lymphocytes of a patient with chronic toxoplasmosis. The CTL lines were obtained by weekly in vitro stimulation with a T. gondii-infected human B cell lymphoma line, ARH, which shares HLA-A2 and -Cw4 determinants with the patient. The lytic activity of these CTL lines against T. gondii-infected ARH and ARH pulsed with fraction 29 of a reversed-phase high-performance liquid chromatography (HPLC) extract from T. gondii-infected ARH was inhibited by an anti-HLA-A, B, C monoclonal antibody (mAb) and an anti-HLA-A2 mAb. Anti-HLA-DR mAb failed to block the lytic activity. Thus, the presentation of peptides by T. gondii-infected cells for CTL is mediated by HLA-A2 molecules. Interestingly, antigen presentation of ARH pulsed with naturally processed HPLC fraction 29 peptides was not inhibited by treatment with brefeldin A. The amino acid sequence of the HLA-A2-bound peptide in fraction 29 was in part consistent with the predictive algorithm of HLA-A2-binding peptide motifs.