Mouse nidovirus LDV infection alleviates graft versus host disease and induces type I IFN-dependent inhibition of dendritic cells and allo-responsive T cells.

INTRODUCTION: Viruses have developed multiple mechanisms to alter immune reactions. In 1969, it was reported that lactate dehydrogenase-elevating virus (LDV), a single stranded positive sense mouse nidovirus, delays skin allograft rejection and inhibits spleen alterations in graft versus host disease (GVHD). As the underlying mechanisms have remained unresolved and given the need for new therapies of this disease, we reassessed the effects of the virus on GVHD and tried to uncover its mode of action. METHODS: GVHD was induced by transfer of parent (B6) spleen cells to non-infected or LDV-infected B6D2F1 recipients. In vitro mixed-lymhocyte culture (MLC) reactions were used to test the effects of the virus on antigen-presenting cells (APC) and responder T cells. RESULTS: LDV infection resulted in a threefold increase in survival rate with reduced weight loss and liver inflammation but with the establishment of permanent chimerism that correlated with decreased interleukine (IL)-27 and interferon (IFN)γ plasma levels. Infected mice showed a transient elimination of splenic CD11b+ and CD8α+ conventional dendritic cells (cDCs) required for allogeneic CD4 and CD8 T cell responses in vitro. This drop of APC numbers was not observed with APCs derived from toll-like receptor (TLR)7-deficient mice. A second effect of the virus was a decreased T cell proliferation and IFNγ production during MLC without detectable changes in Foxp3+ regulatory T cell (Tregs) numbers. Both cDC and responder T cell inhibition were type I IFN dependent. Although the suppressive effects were very transient, the GVHD inhibition was long-lasting. CONCLUSION: A type I IFN-dependent suppression of DC and T cells just after donor spleen cell transplantation induces permanent chimerism and donor cell implantation in a parent to F1 spleen cell transplantation model. If this procedure can be extended to full allogeneic bone marrow transplantation, it could open new therapeutic perspectives for hematopoietic stem cell transplantation (HSCT).

Modulation of the host microenvironment by a common non-oncolytic mouse virus leads to inhibition of plasmacytoma development through NK cell activation.

Although many cells undergo transformation, few actually develop into tumours, due to successful mechanisms of immunosurveillance. To investigate whether an infectious agent may play a role in this process, the growth of a plasmacytoma was investigated in mice infected by lactate dehydrogenase-elevating virus. Acutely infected animals were significantly protected against tumour development. The mechanisms responsible for this protection were analysed in mice deficient for relevant immune cells or molecules and after in vivo cell depletion. This protection by viral infection correlated with NK cell activation and with IFN-γ production. It might also be related to activation of NK/T-cells, although this remains to be proven formally. Therefore, our results indicated that infections with benign micro-organisms may protect the host against cancer development, through non-specific stimulation of the host's innate immune system and especially of NK cells.

Changes in antibody specificities and cytokine release after infection with lactate dehydrogenase-elevating virus.

Lactate dehydrogenase-elevating virus (LDV) is an apparently innocuous and persistent virus that can modify mouse immune reactions. We have shown that LDV-infected mice immunized with human growth hormone (hGH) showed a deep modification of the specificity of the anti-hGH antibodies (Ab) in CBA/Ht mice but not BALB/c animals. The aim of this work was to extend the previous observations to another mouse strain, C57BL/6, as well as to an antigen unrelated to hGH, ovalbumin (OVA), and to explore at the same time the production of various cytokines at serum and cellular levels. The amount of Ab directed to hGH or OVA native antigenic determinants versus the concentration of Ab to cryptic epitopes was evaluated by ELISA competition experiments. Results indicated that LDV infection affected Ab specificity solely in CBA/Ht mice. In CBA/Ht the virus infection was associated with a reduction of the Ab titers to hGH native epitopes and with a decrease of IL-13 and IL-17 serum levels, but Ab to native OVA epitopes were increased with a simultaneous increase of IL-17. Accordingly, only lymph node cells from infected CBA/Ht mice immunized with OVA were found to produce INF-γ, IL-13 and IL-17. Thus, a correlation of cytokine production with a change in Ab specificity after a viral infection was found, although this phenomenon was restricted to a given antigen and to the genetic background of immunized animals. These observations suggest that an apparent harmless virus can affect some immunological mechanisms, which could lead, for example, to inflammatory or autoimmune disorders.

GSTP1 methylation and polymorphism increase the risk of breast cancer and the effects of diet and lifestyle in breast cancer patients.

Glutathione S-transferases (GSTs) are an important group of isoenzymes that play an essential role in the detoxification of carcinogens. Polymorphism at exon 5 of the GST π family decreases the catalytic activity and affects the detoxification ability of the enzyme, GSTP1. GSTP1 promoter hypermethylation and loss of expression are frequently observed in various types of carcinoma. We hypothesized that somatic epigenetic modification in homozygous mutants increases the degree to which breast cancer risk is affected by lifestyle factors and dietary habits. The present study used tumor biopsies and blood samples from 215 breast cancer patients and 215 blood samples from healthy donors. GSTP1 polymorphism was studied using PCR-restriction fragment length polymorphism, methylation using methylation-specific PCR and loss of expression using immunohistochemistry and western blotting. No significant increase was observed in the breast cancer risk of individuals with the mutant (Val) allele [odds ratio (OR), 1.48; 95% confidence interval (CI), 0.97-2.26 for heterozygotes; OR, 1.42; 95% CI, 0.86-2.42 homozygous mutants]. GSTP1 promoter hypermethylation was detected in one-third of tumor biopsies (74/215) and was found to be associated with a loss of expression. Genotype and tumor methylation associations were not observed. Estrogen (ER) and progesterone (PR) receptor-positive tumors had a higher methylation frequency. GSTP1 polymorphism was not associated with increased promoter hypermethylation. The results suggest that GSTP1 methylation is a major event in breast carcinogenesis and may act as a tumor-specific biomarker.

Characterization of the T-lymphocyte response elicited by mouse immunization with rat platelets.

OBJECTIVE: Immunization of normal CBA mice with rat platelets leads to an autoantibody response directed against mouse platelets. The purpose of this work was to determine the involvement of T lymphocytes in this response. MATERIALS AND METHODS: T-lymphocyte responses were analyzed in vivo by depletion and transfer experiments and ex vivo by proliferation assay and cytokine measurements. RESULTS: Mouse immunization with rat platelets induced production of antibodies reacting with rat and mouse platelets. This response was found to depend on CD4(+) T-helper lymphocytes reacting with rat, but not with mouse platelets. These anti-rat platelet T-helper cells were mainly of the Th1 phenotype. When transferred into naïve mice, they enhanced the anti-mouse platelet antibody response induced by subsequent immunization with rat platelets. In addition, depletion of CD25(+) cells enhanced the thrombocytopenia induced by immunization with rat platelets, whereas adoptive transfer of CD4(+)CD25(+) cells from immunized mice suppressed it. CONCLUSIONS: Our results suggest that activation of anti-rat platelet T-helper cells can bypass the mechanism of tolerance and result in the secretion of autoreactive antibodies, but this response is still controlled by regulatory T cells that develop progressively after immunization.

Detection and relevance of germline genetic polymorphisms in glutathione S-transferases (GSTs) in breast cancer patients from northern Indian population.

INTRODUCTION: Glutathione S-transferases (GSTs) are polymorphic superfamily of detoxification enzymes that detoxify therapeutic drugs and various carcinogens. We undertook a case-control study in northern Indian population based sample consisting of 413 patients and 410 controls to evaluate association of null genotype in GSTM1 and GSTT1 along with polymorphism in GSTP1 (A-->G) with breast cancer risk. METHODS: Genotyping analyses were performed by PCR-based methods, and odds ratios (ORs) were calculated by unconditional logistic regression analysis adjusting for various confounding factors. RESULTS: In this case-control study, we observed a positive correlation between family history of breast cancer and risk of breast cancer (OR = 2.06; 95% CI = 1.47-2.92). We found a significantly increased breast cancer risk associated with GSTM1 null genotype (OR = 2.28; 95% CI = 1.65-2.97) and homozygote mutants in GSTP1 (OR = 2.59; 95% CI = 1.67-4.39). However, we found no association between GSTT1 null genotype with overall breast cancer risk (OR = 1.04; 95% CI = 0.76-1.59). The three-way combination of GSTP1 105AA/AG and null genotypes for both GSTM1 and GSTT1 resulted in fourfold increase in breast cancer risk (OR = 4.02; 95% CI = 1.99-8.51). CONCLUSION: The findings of this study are in line with previously published reports that show an overall association with GSTM1 and GSTP1 polymorphisms with breast cancer risk. Our results suggest that the variants in low penetrance genes such as GSTM1, GSTT1 and GSTP1 are associated with an increased breast cancer risk thereby suggesting their contribution in the etiology of breast cancer.

Hypermethylation analysis of mismatch repair genes (hmlh1 and hmsh2) in locally advanced breast cancers in Indian women.

Alterations in protooncogenes and tumor-suppressor genes at the DNA and/or protein level, which indicate the biological properties of individual breast cancers, led us to design a study encompassing the dilemma of "epigenetic silencing-driven genomic instabilities." In this study, we analyzed the promoter methylation of potent mismatch repair genes (hmlh1 and hmsh2) for the first time in 232 Indian patients with primary breast cancer (using methylation-specific polymerase chain reaction and expressional analysis). The study evaluates the gamut of epigenetic aberrations as well as genomic instabilities (microsatellite instabilities and loss of heterozygosity) and includes analysis of BAT-25, BAT-26, D2S123, D5S346, and D17S250. We observed hypermethylation of the hmlh1 gene in 43.5% of patients with primary breast cancer, of whom 66.9% had locally advanced breast cancer (stage IIIA, IIIB, and IIIC) (P < .0001). Similarly, we also found hypermethylation of the hmsh 2 gene in 16% of primary breast cancer cases. Of these patients, 21.3% had locally advanced breast cancer (P = . 01). To determine the effect of methylation, we also performed expressional studies using reverse transcriptase polymerase chain reaction and Northern blotting, but we were unable to get any significant expression in the presence of hypermethylation of either gene (hmlh1 and hmsh2). Interestingly, statistical analysis revealed that hypermethylation of the hmlh1 gene is one of the peculiar attributes of locally advanced breast cancer. In addition, this study indicates that for more sensitive stage-specific diagnosis or prognosis, both methylation of promoter and expression studies must be considered in the analyses in a reproducible manner. Therefore, pinpointing the methylation fingerprints (5'CpG island methylation) of potent DNA repairing genes not only shows the specific attributes of locally advanced breast cancer but also provides important insight into the mode of therapy to be used by clinical oncologists.

Chemotherapy of leishmaniasis: past, present and future.

Leishmaniasis is a parasitic disease caused by hemoflagellate, Leishmania spp. The parasite is transmitted by the bite of an infected female phlebotomine sandfly. The disease is prevalent throughout the world and in at least 88 countries. Nearly 25 compounds are reported to have anti-leishmanial effects but not all are in use. The pentavalent antimony compounds have remained mainstay for nearly 75 years. Pentavalent antimony is a prodrug that is reduced by glutathione to active trivalent species catalyzed by thiol-dependent-reductase. However, emergence of resistance led to the use of other compounds--amphotericin B, pentamidine, paromomycin, allopurinol etc. Amphotericin B, an antifungal macrolide polyene is characterized by the hydrophilic polyhydroxyl and hydrophobic polyene faces on it long axis. Presently, it is the only drug with highest cure rate. It acts on membrane sterols resulting in parasite cell lysis. Its lipid formulations have been developed to minimize side effects. Other anti-fungals like ketoconazole, fluconazole and terbinafine are found less effective. Recently, anticancer alkylphosphocholines have been found most effective oral compounds. These act as membrane synthetic ether-lipid analogues, and consist of alkyl chains in the lipid portions. Most promising of these are miltefosine (hexadecylphosphocholine), edelfosine (ET-18-OCH(3)) and ilmofosine (BM 41.440). However, the recent focus has been on identifying newer therapeutic targets in the parasite such as DNA topoisomerases. The present review describes the current understanding of different drugs against leishmaniasis, their chemistry, mode of action and the mechanism of resistance in the parasite. Future perspectives in the area of new anti-leishmanial drug targets are also enumerated. However, due to the vastness of the topic main emphasis is given on visceral leishmaniasis.