Month: July 2020

Genetic characterization of viruses isolated in the environment can provide interesting information regarding gene prevalence and circulation in habitats used by waterfowl. Such information is critical to assess the possibility of inter-annual persistence in water, long-term circulation of environmentally-isolated virus strains in wild duck populations, and identify XAV939 potential genetic basis for the persistence of IA viruses in water. Previous studies have demonstrated differences in the ability of IA viruses to remain infective in water and have suggested that subtyperelated variations in persistence may exist, especially at low temperatures. Both isolates tested in our study persisted for several months in distilled water at constant temperatures, below 17uC and at neutral pH. Overall, the estimated persistence was higher than reported for 12 different waterfowl derived viruses. Limited data precludes conclusions related to increased environmental persistence associated with viruses recovered from the environment. Future experiments should however consider such a possibility as the demonstration of long-time persistence of water-isolated viruses would suggest the potential for inter-annual persistence in aquatic habitats. Inversely, habitats characterized by deep water may prevent freezing and strong temperature changes affecting viral persistence, but are less likely to be used by dabbling ducks. Viral persistence in frozen pond and lake water has been suggested to be an potential mechanisms for inter-annual persistence and source of contamination for waterfowl in breeding grounds. In high latitude habitats, shallow lakes and ponds may freeze relatively quickly during fall and winter, potentially reducing effects of such temperature changes. The effects of latitudinal-dependent temperature regimes on surface water viruses warrant future experimental work. Because diurnal surface water temperature fluctuations could represent a constraint on viral persistence, we also investigated the effects of temperature variations between 17uC and 23uC. Compared to results from constant temperature, no effects on viral persistence were observed. This suggests that diurnal surface water temperature fluctuations are not likely to significantly affect virus persistence, although as for freeze-thaw cycles additional environmental simulation studies would be required to confirm this trend. Experimental infections provided evidence that both waterisolated IA viruses replicated efficiently in Mallards and, in both cases, shedding patterns were consistent with a previous study performed with duck-isolated LP IA viruses. These results support that viruses isolated from surface water replicate in ducks as well as LP virus isolates that have a wild duck-origin. In this study, we highlighted that water-isolated IA viruses can provide complementary information regarding circulating viruses and viral genes in wild waterfowl populations. With current methodological limitations the recovery of viruses from aquatic habitats should not be considered as a replacement of traditional surveillance approaches utilizing wild bird or fecal sampling.

In conclusion, by mapping DNA methylated viral integration sites in murine leukemias induced by retroviral integration mutagenesis followed by comparative analysis in human AML, we identified PTP4A3 not only as a candidate HIG contributing to leukemogenesis in mice but also as an independent prognostic indicator in human AML. We designed a strategy to identify candidate HIGs in AML using retroviral integration mutagenesis, by mapping DNA methylated proviral integrations. By using HAT, we deliberately aimed at detecting integrations present in the majority of the leukemic cells, which are most likely involved in the early phase of leukemogenesis. At the same time, integrations present in subclones that contribute to later stages of leukemic progression will be missed using this approach. We identified 6 genes that are flanked by methylated viral integrations. Expression analysis showed that Lrmp, Hcls1 and Prkrir ) were up regulated and Ptp4a3, a phosphatase also known as Prl3 was down regulated in the respective murine tumor. These results indicate that a flanking methylated viral integration site does not necessarily lead to transcriptional repression. As 1 out of 4 genes flanked by a mVIS was transcriptionally down regulated and expression of the 2 other genes could not be investigated, the efficiency to detect potential HIGs by identifying mVIS would approximately be 17–25%. However, the number of analysed tumors is too small to allow an accurate estimation of the efficiency. Ptp4a3 expression is controlled by p53 induced after DNA damage in mouse embryonic fibroblasts and its activity is involved in inducing a G1 cell cycle arrest in these cells. Surprisingly however, the same study also demonstrated a cell cycle arrest upon reduction of PTP4A3 expression. Apparently, depending on expression level dosage, PTP4A3 may have both positive and negative effects on cell cycle regulation. Hence, PTP4A3 haplo-insufficiency, but not its complete loss, may lead to an impairment of cell cycle arrest after DNA damage. Dosage effects of PTP4A3 expression in relation to cellular responses may be more complex, particularly in cancer cells. For example, in carcinoma cell lines PTP4A3 expression may lead to down regulation of p53 and it is variably induced by c-irradiation. Finally, high PTP4A3 expression has been linked to increased tumor aggressiveness in different types of solid tumors, e.g., melanoma, gastric cancer, colon cancer, hepatocellular carcinoma and breast cancer, possibly because high PTP4A3 expression leads to increased epithelial-mesenchymal transition. The role of PTP4A3 in hematopoietic malignancies has not been studied as XL-184 extensively as in carcinoma. Only a few studies report differences in expression levels of PTP4A3 in ALL and myeloma subgroups, based on gene expression profiling. Interestingly however, in a recent study, PTP4A3 has been proposed to have a role in drug-resistance in AMLs with internal tandem duplication of FLT3. This finding, together with the observation that high PTP4A3 expression negatively correlates with prognostic outcome, indicates that PTP4A3 might be a potential therapeutic target in AML.

Susceptibility genes related to PCa might function in host immune responses and protection against cell and DNA damage caused by oxidative agents. For example, two suggested susceptibility risk genes for hereditary PCa, RNASEL and MSR1, are both involved with innate immunity. Lymphoblastoid cell lines represent an essential component of the immune response, so this result may indicate that ARLTS1 has a function in immune system processes. Alterations in important molecular pathways involved in PCa have been implicated in proliferative inflammatory atrophy and prostatic intraepithelial neoplasia lesions. Tumor suppressor genes NKX3.1, CDKN1B which encodes p27 and the phosphatase and tensin homologue are highly expressed in normal prostate epithelium and have shown to be down-regulated or absent in PIN and PCa. Since ARLTS1 acts as a tumor suppressor protein and has a decreased expression in PCa, these recent findings are in line with the results from other suggested PCa tumor suppressor proteins. Previously it has been shown that ARLTS1 induces apoptosis in lung cancer cells and in ovarian carcinoma. In the case of prostatic inflammation and antigen engagement, the need for apoptosis increases and the processes of the immune system, together with endogenous inflammatory cells become PD325901 activated. Here we showed that in the lymphoblastoid cell lines of PCa patients, ARLTS1 expression was significantly decreased among the CC carrying patients compared to the wild-type allele T carrying patients. The lymphoblastoid cell lines were derived by Epstein-Barr virus transformation of peripheral mononuclear leucocytes from patients, indicating that these cells have encountered antigen stimuli via viral infection. The CC carrying patients had a low ARLTS1 expression status suggesting that ARLTS1 function is decreased due to this risk genotype and consequently this leads to decreased apoptosis. This may indicate that the Cys148Arg CC genotype contributes to the immune response by diminishing apoptosis rates, decreasing defense mechanisms and finally cancer progression. It has been shown earlier that lung cancer cells carrying the Trp149Stop variant and expressing the truncated protein had a reduced capability to induce apoptosis compared to cells expressing the full-length protein. This reduced apoptosis could also be the causal factor in PCa. Our data suggest for the first time that the predisposing effect of the CC genotype of Cys148Arg is related to reduced expression in immune system cells rather than in tumor cells where ARLTS1 expression is naturally very low. Like all other organs, the normal prostate contains endogenous inflammatory cells and immune response mechanisms. Consequently, the function of ARLTS1 may not exclusively be based on tumor suppression as suggested before, but also on immune response functions that occur either locally in prostate or in peripheral tissues. This is in line with the findings that PCa is a very heterogeneous disease and different mechanisms of cancer progression may occur.

With CLL have a high prevalence of EBV and thus possess EBV-specific memory cells with a high frequency of CD4+ gp350- specific T cells, the incorporation of gp350 into exosomes has a dual function: it confers B-cell tropism and serves as an immunodominant viral CD4 antigen. The cellular immune system of cancer patients is usually impaired but virus-specific immune responses are detectable even in late-stage patients and T lymphocytes specific for herpes viruses are often present at high numbers. Given the fact that the vast majority of CLL patients are seropositive for EBV, gp350 and other EBV proteins have the potential as promising neo-antigens in B-CLL cells exploiting and redirecting the strong anti-viral cellular immune responses to leukemic cells. Based on these results, we propose here a new immunotherapeutic approach for B-CLL, based on the simultaneous targeted transfer of functional CD154 and the EBV protein gp350 onto malignant cells using exosomes. gp350 is the major envelope protein of EBV and confers viral B-cell tropism by interacting with the complement receptor 2, which is highly expressed on B lymphocytes. Here, we generated modified exosomes produced in 293 cells. As a result of gp350 incorporation, these particles have a profound B-cell tropism similar to wild-type EBV, so that gp350- carrying vesicles specifically and efficiently bind to B cells. In addition, gp350 serves as a viral neo-antigen in B-CLL cells. We also found that CD154 on exosomes from HEK293 cells is functionally active as demonstrated by the induction of immune accessory molecules on B target cells probably through the CD40 pathway. Taken together, our experiments suggest that leukemia cells WZ4002 treated with CD154+/gp350+ exosomes are efficiently stimulated and subsequently killed by autologous B-CLL and gp350-specific cytolytic T lymphocytes. In summary, our results demonstrate that modified exosomes carrying the EBV protein gp350 display a distinct tropism to normal and leukemic B cells and efficiently transfer CD154 as a functional protein onto these cells. Leukemic B cells treated with these particles acquire an activated phenotype and become potent stimulators of autologous T lymphocytes. Engineered exosomes can be easily generated and can readily be scaled up for clinical applications. In addition, they can be individually tailored to express additional accessory molecules like OX40L or the Fas ligand or alternative viral molecules to target other classes of cells like macrophages and DCs. The generation of modified exosomes is not limited to 293 cells, which we used in this proof-of-concept. Instead, other cell lines that are approved for human therapy, such as MRC-5 fibroblasts, should also be tested as an optional origin of exosomes to facilitate transition into clinical trials. Modified gp350-carrying exosomes can thus be regarded as powerful and promising tools for various immunotherapeutic approaches. Globins are small heme-proteins that have the ability to reversibly bind molecular oxygen. For a long time only two globin types have been known in vertebrates: hemoglobin and myoglobin. Most likely, Hb and Mb are the best-studied proteins in biological, biochemical, biophysical and medical sciences.

Temozolomide ARLTS1 belongs to the ADP-ribosylation factor -ARF-like family of the Ras protein superfamily. ARFs are guanine-nucleotide-binding proteins which are critical components of several different eukaryotic vesicle trafficking pathways. As with other members of the Ras superfamily, ARFs function as molecular switches by cycling between inactive GDP- and active GTP-bound conformations. ARLTS1 has been characterized as an intracellular protein having tissue specific expression in the lung and leucocytes. ARLTS1 variants, such as the nonsense polymorphism Trp149Stop have been suggested to have a role in different cancers. Prostate cancer is the most frequently diagnosed cancer in males in many countries, including Finland. Aging and improved diagnostics most evidently increase the number of new cases, but the incidence is influenced also by some unknown factors. Growing number of new cases create pressure to health care system and new tools for PCa diagnostics, prognostics and treatment are required, especially to avoid over treatment and unnecessary biopsies. During the last several years there has been extensive research in PCa etiology and genome-wide association studies have revealed several common low penetrance genetic alterations. The association of these variants with clinicopathologic features and prognosis remains unclear and results are lacking clinical implications. We recently showed a significant association withCys148Arg variant and the risk of PCa. Further evaluation of this variant is warranted to increase the power of the association and study the functional role of the variant in PCa. More samples are also needed to evaluate the implication of this variant to clinical outcome and a potential role in predictive biomarker of PCa. Besides the genetic variants, DNA copy number aberrations are one of the most frequently observed genetic changes in familial and sporadic PCa. In most of the cases target genes for the aberrations are not fully identified. Interestingly, allelic imbalance has been detected at 13q14.2-13q14.3, and it is an important event in the progression of localized PCa. Differences of 13q14 loss of heterozygosity in different PCa groups could also be used to distinguish clinically insignificant PCa. In this study we analyzed the role of ARLTS1 in more detail, especially the role of Cys148Arg in PCa risk. Chromosomal aberration in 13q14.3 was analyzed with aCGH to evaluate the ARLTS1 copy number changes in PCa xenografts and cell lines. The expression of ARLTS1 was studied in clinical tumor samples, BPH samples and also co-expression data form previously published data was analyzed. The ARLTS1 gene and ARLTS1 polymorphisms have been shown to have a role in the pathogenesis of many cancers. The nonsense polymorphism at the end of the coding region has been revealed to predispose to familial cancer. Functional analyses of the truncated protein have indicated that the Trp149Stop variant might affect apoptosis and tumor suppression. Another ARLTS1 variant, the missense polymorphism Cys148Arg, and especially the CC genotype, has been found to be significantly associated with high-risk familial breast cancer.