Svetlana Kozireva

Down regulation of 3p genes, LTF, SLC38A3 and DRR1, upon growth of human chromosome 3-mouse fibrosarcoma hybrids in severe combined immunodeficiency mice.

We have applied a functional test for tumour antagonizing genes based on human chromosome 3 (chr3)–mouse fibrosarcoma A9 MCHs that were studied in vitro and after growth as tumours in severe combined immunodeficiency (SCID) mice. Previously, we reported that 9 out of the 36 SCID‐tumours maintained the transferred chr3 (“chr3+” tumours), but lost the expression of the known human TSG fragile histidine triad gene (FHIT) in contrast to 14 other 3p‐genes examined. Here we report the results of the duplex RT‐PCR analysis of 9 “chr3+” tumours and 3 parental MCHs. We have examined the expression of 34 human 3p‐genes from known cancer‐related regions of instability, including 13 genes from CER1 defined by us previously at 3p21.33–p21.31 and 10 genes from the LUCA region at 3p21.31. We have found that in addition to FHIT, expression of the LTF gene from CER1 at 3p21.33‐p21.31 was lost in all 9 tumours analyzed. The transcript of the solute carrier family 38 member 3 gene (SLC38A3) gene from LUCA region at 3p21.31 was not found in 8 and was greatly reduced in 1 out of these 9 tumours. Expression of the down‐regulated in renal cell carcinoma gene (DRR1) gene at 3p14.2 was lost in 7 and down regulated in 2 “chr3+” tumours. In the SCID‐tumour derived cell lines treatment with 5‐aza‐2′‐deoxycytidine restored the mRNA expression of LTF, indicating the integrity of DNA sequences. Notably that transcription of the LTF and 2 flanking genes, LRRC2 and TMEM7, as well as transcription of the SLC38A3 gene, were also impaired in all 5 RCC cell lines analyzed. Our data indicate these genes as putative tumour suppressor genes.

Single-tube nested quantitative PCR: a rational and sensitive technique for detection of retroviral DNA. Application to RERV-H/HRV-5 and confirmation of its rabbit origin

It was reported earlier that a few patients suffering from non-Hodgkins lymphoma had low amounts of DNA from the so-called fifth human exogenous retrovirus, HRV-5. A sensitive and rational method for large-scale screening for HRV-5 DNA was therefore developed. It is a single-tube nested quantitative PCR (stnQPCR), which uses two functionally isolated primer pairs and one probe target distinct from related endogenous retroviral sequences, yet encompassing known HRV-5 variation, allowing optimal use of sequence conservation. DNA from lymphoma, myeloma, and follicular dendritic cell lines was tested for HRV-5 positivity, as was DNA from whole blood of blood donors, non-Hodgkins lymphoma and systemic lupus erythematosus patients, as well as DNA from lymph node biopsies of rheumatoid arthritis patients with lymphoma. One blood donor, one systemic lupus erythematosus patient, two previously known positive non-Hodgkins lymphoma patients, and one rheumatoid arthritis lymphoma patient, came out positive. They had 24, 2, 148, 480 and 30 proviral copies per microg of DNA from PBMC or lymphoma tissue, respectively. During the completion of this work it was reported that HRV-5 is a rabbit endogenous retrovirus (RERV-H), and that HRV-5 positivity was due to presence of rabbit DNA. DNA from six RERV-H/HRV-5 positive samples was therefore retested. Three also contained rabbit mitochondrial DNA. A search for HRV-5 antibodies using synthetic peptides was negative in sera from three RERV-H/HRV-5 positive individuals, as well as in 144 other sera, according with a noninfectious origin of the RERV-H/HRV-5 DNA in human samples. A search for possible sources of rabbit DNA contamination was negative. Methods for prevention of PCR contamination were strictly adhered to. Three samples from RERV-H/HRV-5 positive individuals positive at the Uppsala laboratory were retested at one or two other laboratories, and all three were positive. Two other samples, which were positive in the Riga laboratory, were tested also in London and also found positive. One non-Hodgkins lymphoma patient was RERV-H/HRV-5 positive in four consecutive samples, showing that positivity was a property of that patient. It is concluded that the stnQPCR developed to detect and quantify minute amounts of RERV-H/HRV-5 DNA is a principle which can be applied widely and HRV-5 is a RERV-H. Its presence in a few human blood samples could not be explained.

Human retrovirus 5 sequences in peripheral blood cells of patients with B-cell non-Hodgkin's lymphoma

A recently described sequence from a probable 5th human exogenous retrovirus, HRV‐5, is related to type A, B and D retroviruses. It was initially detected in a salivary gland biopsy from a patient with Sjögrens syndrome, but it is not consistently associated with this disease. We searched for the HRV‐5 sequence in DNA extracted from whole blood of 300 blood donors, 81 patients with hematological malignancy and 21 patients with neurological disease using PCR. While samples from none of the blood donors and the neurological patients became positive, 3 of the 81 patients with hematological malignancy were HRV‐5 DNA positive. All 3 had B‐cell non‐Hodgkins lymphoma of low grade. The difference in frequency between NHL and controls is statistically significant. HRV‐5 DNA was found in DNA from whole blood and in plastic‐adherent cells but not in tumor cell DNA. Thus, monocytes/macrophages may be preferred targets for HRV‐5. Our result, together with a previous finding of HRV‐5 DNA in 2 NHL cases, is compatible with an association between HRV‐5 and NHL, whether causal or not. Int. J. Cancer 85:762–770, 2000.

Human Retrovirus Type 5 Sequences in Non-Hodgkin's Lymphoma of T Cell Origin

DNA of a recently described fifth exogenous retrovirus (HRV-5) has been found in blood samples from patients with autoimmune diseases and lymphoma. We analyzed HRV-5 sequence in DNA extracted from whole blood of 17 patients with T cell non-Hodgkins lymphoma (NHL) and 186 patients with hematological malignancies other than NHL, using a sensitive PCR technique. While all samples of patients with hematological malignancies other than NHL were negative, 2 of the 17 patients with T cell NHL were HRV-5 DNA positive. Both HRV-5-positive patients had T cell NHL of high-grade malignancy (stage IV) and diffuse distribution of the lymphoma, including infiltration of bone marrow or lung and pleura. The difference in HRV-5 DNA detection frequency between NHL and control groups is significant (p value of 0.0004 judged by the Fisher exact test). These data, together with our previous finding of HRV-5 DNA in three B cell NHL cases, are compatible with an association between HRV-5 and NHL, of both T cell and B cell origin.

Upregulation of the Chemokine Receptor CCR2B in Epstein‒Barr Virus-Positive Burkitt Lymphoma Cell Lines with the Latency III Program

CCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein‒Barr virus (EBV) infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL) tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments.

Peripheral Blood Mononuclear Cells’ Proliferative Response to Human Parvovirus B19 Antigens in Patients with Rheumatoid Arthritis / Reimatoīdā Artrīta Slimnieku Perifēro Asiņu Mononukleāro Šūnu Proliferativā Atbilde Uz Cilvēka Parvovīrusa B19 Antigēniem

Abstract This study aimed to determine peripheral blood mononuclear cells’ (PBMC) proliferative response to parvovirus B19 (B19) antigens in rheumatoid arthritis (RA) patients and possible changes in proliferative response due to chemotherapy. Serum and blood samples of 52 RA patients and 25 sex and age matched healthy individuals were examined for the presence of anti-B19 IgG and IgM class antibodies and virus specific DNA sequence by the recomLine B19 test and nested polymerase chain reaction, respectively. The PBMC proliferative activity was estimated on the 3rd and 6th day of PBMC cultivation in the presence of virus and B19 VP1/VP2 peptide, using thymidine incorporation assay. On the 3rd day, PBMC response to B19 antigens was detected in 74.1% RA patients with active, in 44.8% - with remote and in 40% - with latent stage of persistent B19 infection, while in the control group the response was observed only in two individuals with active viral infection. On the 6th day, the response was found in 50% RA patients with active, 68.9% - with remote and in 80% - with latent stage of latent persistent infection as well as in 41.1% remotely infected control individuals. The highest PBMC mean stimulation indices were detected in the RA patients and control persons with active infection as well as in RA patients with latent stage of persistent viral infection. On the 3rd and 6th day, strong proliferative response was significantly more frequently observed in RA patients not receiving methotrexate treatment, compared to the patients receiving methotrexate treatment in different combinations with other drugs. RA patients had more frequent and faster response to B19 antigens than apparently healthy persons.

Effect of Human Herpesviruses 6 and 7 Infection on the Clinical Course of Rheumatoid Arthritis / Cilvēka Herpesvīrusa 6 Un 7 Infekcijas Ietekme Uz Reimatoīdā Artrīta Klīnisko Gaitu

Abstract Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease affecting joints and causing symmetrical chronic progressive aseptic synovitis and erosive-destructive changes. Viruses and viral infections are considered to be the main risk factors for autoimmune disease development (especially for individuals with genetic predisposition). The goal of this study was to evaluate the frequency of HHV-6 and HHV-7 persistent infection and its activity phase in RA and osteoarthritis (OA) patients, and healthy persons. We examined also the influence of HHV-6 and -7 infections on RA activity, aggressiveness, radiographical stage, and frequency of complications as well as the presence of HHV-6 infection markers in synovial fluid and synovial tissues of RA joints of affected patients. Despite the lack of significant correlation between frequency of persistent single HHV-6, single HHV-7, and concurrent HHV-6 and HHV-7 infection and RA clinical course, we found that both active and latent HHV-6 and/or HHV-7 infection increased RA activity and progression in several clinical and laboratory parameters. Regarding the severity of the course of RA, we observed also a high prevalence of RA complications in the patient group with active single HHV-6 infection and also a more severe radiographical stage in RA patients with active concurrent HHV-6 and HHV-7 infection. Moreover, viral infection markers were found in synovial fluid and synovial tissues of affected joints of RA patients. This suggests that HHV-6 and/or HHV-7 infection has effect on the disease clinical course, but virus reactivation may be a consequence of immunosuppressive treatment.