The Role of Bcl-2 and Beclin-1 Complex in "Switching" between Apoptosis and Autophagy in Human Glioma Cells upon LY294002 and Sorafenib Treatment.

BACKGROUND: Gliomas are the most malignant tumors of the central nervous system. One of the factors in their high drug resistance is avoiding programmed death (PCD) induction. This is related to the overexpression of intracellular survival pathways: PI3K-Akt/PKB-mTOR and Ras-Raf-MEK-ERK. Apoptosis and autophagy are co-existing processes due to the interactions between Bcl-2 and beclin-1 proteins. Their complex may be a molecular "toggle-switch" between PCD types. The aim of this research was to investigate the role of Bcl-2:beclin-1 complex in glioma cell elimination through the combined action of LY294002 and sorafenib. METHODS: Drug cytotoxicity was estimated with an MTT test. The type of cell death was evaluated using variant microscopy techniques (fluorochrome staining, immunocytochemistry, and transmission electron microscopy), as well as the Bcl-2:beclin-1 complex formation and protein localization. Molecular analysis of PCD indicators was conducted through immunoblotting, immunoprecipitation, and ELISA testing. SiRNA was used to block Bcl-2 and beclin-1 expression. RESULTS: The results showed the inhibitors used in simultaneous application resulted in Bcl-2:beclin-1 complex formation and apoptosis becoming dominant. This was accompanied by changes in the location of the tested proteins. CONCLUSIONS: "Switching" between apoptosis and autophagy using PI3K and Raf inhibitors with Bcl-2:beclin-1 complex formation opens new therapeutic perspectives against gliomas.

Comparison of Selected Immune Parameters in a Single Infection and Co-Infection with Infectious Pancreatic Necrosis Virus with Other Viruses in Rainbow Trout.

Infectious pancreatic necrosis virus (IPNV) often occurs in an aquatic environment in co-infection with other viruses. In this study, we wanted to investigate the effect of this virus on the course of co-infection with other viruses in rainbow trout. For co-infection we used viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV) and salmonid alphavirus (SAV) field strains and infected rainbow trout divided into eight groups; I; IPNV, II; IHNV, III; VHSV, I; SAV, V; IPNV+IHNV, VI; IPNV+VHSV, VII; IPNV+SAV, and the control group. We assessed apoptosis in white blood cells and used a real time RT-PCR to analyze RNA obtained from the internal organs of the fish. During single infection and co-infection the level of expression of immune genes such as interferon and toll-like receptor 3 (TLR-3) was assessed. The highest mortality during the experiment was observed in group III infected by VHSV. The average percentage of apoptotic cells was higher in groups without co-infection, especially in groups II and III. Interferon expression was higher in singly infected groups, the highest being in the heart in group III, while expression of the TLR-3 gene was generally raised in all tested organs in all groups. We found that co-infection with IPNV had a positive impact on the course of infection with the viruses listed because it lowered mortality, reduced apoptosis in co-infected cells, and positively affected fish health.

Emerging Viral Pathogens in Sturgeon Aquaculture in Poland: Focus on Herpesviruses and Mimivirus Detection.

Recently, Poland has become a leading producer of sturgeon meat and caviar in Europe and is one of the largest in the world. The growing importance of this branch of aquaculture means that diseases of these fish, especially viral ones, are becoming the object of interest for ichthyopathologists. In recent years, there have been increasing reports of health problems in the dynamically developing sturgeon farming. The greatest risk appears to be emerging infectious diseases that are caused by viruses and that can become a serious threat to the development of the aquaculture industry and the success of sturgeon restitution programs undertaken in many European countries, including Poland. In this paper, an attempt was made to determine the spread of the two most important groups of viruses in Polish sturgeon farming: These include the herpesviruses and sturgeon nucleocytoplasmic large DNA viruses (sNCLDV), in particular, mimiviruses. In the years 2016-2020, 136 samples from nine farms were collected and tested by using the WSSK-1 cell line, PCR and Real Time PCR methods. All results were negative for herpesviruses. Out of the samples, 26% of the samples have been tested positive for mimiviruses. Sanger sequencing of mimiviruses demonstrated their affiliation with AciV-E. The sequence characterization confirmed the presence of both V1 and V2 lineages in Polish fish facilities, but variant V2 seems to be more widespread, as is observed in other European countries.

Involvement of PI3K Pathway in Glioma Cell Resistance to Temozolomide Treatment.

The aim of the study was to investigate the anticancer potential of LY294002 (PI3K inhibitor) and temozolomide using glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells. Apoptosis, autophagy, necrosis, and granules in the cytoplasm were identified microscopically (fluorescence and electron microscopes). The mitochondrial membrane potential was studied by flow cytometry. The activity of caspases 3, 8, and 9 and Akt was evaluated fluorometrically, while the expression of Beclin 1, PI3K, Akt, mTOR, caspase 12, and Hsp27 was determined by immunoblotting. SiRNA was used to block Hsp27 and PI3K expression. Cell migration and localization of Hsp27 were tested with the wound healing assay and immunocytochemistry, respectively. LY294002 effectively diminished the migratory potential and increased programmed death of T98G and MOGGCCM. Autophagy was dominant in MOGGCCM, while apoptosis was dominant in T98G. LY294002 with temozolomide did not potentiate cell death but redirected autophagy toward apoptosis, which was correlated with ER stress. A similar effect was observed after blocking PI3K expression with siRNA. Transfection with Hsp27 siRNA significantly increased apoptosis related to ER stress. Our results indicate that inhibition of the PI3K/Akt/mTOR pathway sensitizes glioma cells to apoptosis upon temozolomide treatment, which was correlated with ER stress. Hsp27 increases the resistance of glioma cells to cell death upon temozolomide treatment.

The effect of selected molecules influencing the detrimental host immune response on a course of rabies virus infection in a murine model.

Rabies is invariably fatal, when post-exposure prophylaxis is administered after the onset of clinical symptoms. In many countries, rabies awareness is very low and the availability of post-exposure prophylaxis, as recommended by WHO guidelines, is very limited or non-existent, probably as a consequence of high cost. Therefore, new concepts for rabies therapy are needed. Innate immune mechanisms involving the production of pro-inflammatory cytokines and chemokines, activated after rabies infection, are thought to be involved in the neuropathogenesis of rabies. These mechanisms can contribute to a detrimental host response to the rabies virus (RABV) infection. The use of inhibitors of cytokines/chemokines are supposed to extend the survival of a sick individual. Inhibitors of TNF-α, IL-6 and MAPKs were used in RABV inoculated mice to define their influence on the survival time of rabid mice. The study demonstrated that all inhibitors extended mice survival, but at different rates. A log-rank test confirmed the statistically significant survival of mice treated with TNF-α (p = .0087) and MAPKs inhibitors (p = .0024). A delay in the time of onset of rabies was also recorded, in mice given TNF-α and MAPKs inhibitors. The highest virus load was found in the spinal cord and the lowest in the cortex, regardless of the experimental group. Significant TNF-α (p ≤ .0001) and IL-6 (p ≤ .0001) gene upregulation was observed in mice, as a consequence of RABV infection. Regarding MAPKs pathways, there was significant upregulation of the caspase 3 (p = .012, p = .0026) and Mcl-1 (p = .0348, p = .0153) genes, whereas significant downregulation of the cytochrome C (p ≤ .0001), Bcl2 (p = .0002, p = .0007) and JNK3 (p = .042) genes. Rabies pathogenesis is multifactorial, involving both virus and host influences on the course of the infection.

Proteome analysis of sheep B lymphocytes in the course of bovine leukemia virus-induced leukemia.

Presented are the results of a study of the expression pattern of different proteins in the course of bovine leukemia virus-induced leukemia in experimental sheep and I discuss how the obtained data may be useful in gaining a better understanding of the pathogenesis of the disease, diagnosis, and for the selection of possible therapeutic targets. In cattle, the disease is characterized by life-long persistent lymphocytosis leading to leukemia/lymphoma in about 5% of infected animals. In sheep, as opposed to cattle, the course of the disease is always fatal and clinical symptoms usually occur within a three-year period after infection. For this reason, sheep are an excellent experimental model of retrovirus-induced leukemia. This model can be useful for human pathology, as bovine leukemia virus is closely related to human T-lymphotropic virus type 1. The data presented here provide novel insights into the molecular mechanisms of the bovine leukemia virus-induced tumorigenic process and indicate the potential marker proteins both for monitoring progression of the disease and as possible targets of pharmacological intervention. A study of the proteome of B lymphocytes from four leukemic sheep revealed 11 proteins with altered expression. Among them, cytoskeleton and intermediate filament proteins were the most abundant, although proteins belonging to the other functional groups, i.e. enzymes, regulatory proteins, and transcription factors, were also present. It was found that trypsin inhibitor, platelet factor 4, thrombospondin 1, vasodilator-stimulated phosphoprotein, fibrinogen alpha chain, zyxin, filamin-A, and vitamin D-binding protein were downregulated, whereas cleavage and polyadenylation specificity factor subunit 5, non-POU domain-containing octamer-binding protein and small glutamine-rich tetratricopeptide repeat-containing protein alpha were upregulated. Discussed are the possible mechanisms of their altered expression and its significance in the bovine leukemia virus-induced leukemogenic process. Impact statement The submitted manuscript provides new data on the molecular mechanisms of BLV-induced tumorigenic process indicating the potential marker proteins both for monitoring the progression of the disease and as possible targets of pharmacological intervention. This is to my knowledge the first study of the proteome of the transformed lymphocytes in the course of bovine leukemia virus-induced leukemia in susceptible animals. BLV can be considered as useful model for related human pathogen - HTLV-1, another member of the deltaretrovirus genus evolutionary closely related to BLV. Information gathered in this study can be useful to speculate on possible shared mechanisms of deltaretrovirus-induced carcinogenesis.

Quantification of Cell Turnover in the Bovine Leukemia Virus Model.

In a perspective of a comparative virology approach, characterization of the bovine leukemia virus (BLV) model may be helpful to better understand infection by the related human T-lymphotropic virus type 1 (HTLV-1). In this paper, we first provide detailed protocols to inoculate cloned BLV proviruses into sheep or cattle. We also describe methods to quantify apoptosis ex vivo and cell turnover in vivo.

Bovine Leukemia Virus Small Noncoding RNAs Are Functional Elements That Regulate Replication and Contribute to Oncogenesis In Vivo.

Retroviruses are not expected to encode miRNAs because of the potential problem of self-cleavage of their genomic RNAs. This assumption has recently been challenged by experiments showing that bovine leukemia virus (BLV) encodes miRNAs from intragenomic Pol III promoters. The BLV miRNAs are abundantly expressed in B-cell tumors in the absence of significant levels of genomic and subgenomic viral RNAs. Using deep RNA sequencing and functional reporter assays, we show that miRNAs mediate the expression of genes involved in cell signaling, cancer and immunity. We further demonstrate that BLV miRNAs are essential to induce B-cell tumors in an experimental model and to promote efficient viral replication in the natural host.

Recent Advances in BLV Research.

Different animal models have been proposed to investigate the mechanisms of Human T-lymphotropic Virus (HTLV)-induced pathogenesis: rats, transgenic and NOD-SCID/γcnull (NOG) mice, rabbits, squirrel monkeys, baboons and macaques. These systems indeed provide useful information but have intrinsic limitations such as lack of disease relevance, species specificity or inadequate immune response. Another strategy based on a comparative virology approach is to characterize a related pathogen and to speculate on possible shared mechanisms. In this perspective, bovine leukemia virus (BLV), another member of the deltaretrovirus genus, is evolutionary related to HTLV-1. BLV induces lymphoproliferative disorders in ruminants providing useful information on the mechanisms of viral persistence, genetic determinants of pathogenesis and potential novel therapies.

Mutation of a Single Envelope N-Linked Glycosylation Site Enhances the Pathogenicity of Bovine Leukemia Virus.

UNLABELLED: Viruses have coevolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field or designed in vitro. In this study, we sought to understand the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability. IMPORTANCE: Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. We show that N-linked glycosylation of the bovine leukemia virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing.

Proteomics in the search for biomarkers of animal cancer.

The prevalence of cancer in companion animals has increased in the recent decade, making this disease one of the major causes of deaths. As in human medicine, veterinary medicine faces the problem of cancer prevention as well as early diagnosis and effective therapy. Early diagnosis of cancer is crucial for the successful treatment of the disease and there is a need for biomarkers that could be used as a diagnostic tool, and to guide a targeted therapy or monitor a therapeutic response. Proteomic technologies that were introduced to human cancer research over a decade ago provide the opportunity to identify distinct protein patterns for cancer diagnosis and therapy monitoring. These also have potential to be utilised in veterinary medicine. The present paper summarises the current knowledge about proteomic studies on animal cancer biomarker research published to date.

Granulomatous pneumonia and hepatitis associated with Providencia rettgeri infection in a crocodile monitor lizard (Varanus salvadorii).

The present report describes a case of granulomatous pneumonia and hepatitis in a male crocodile monitor lizard (Varanus salvadorii). During the necropsy of the monitor lizard, multifocal to coalescing pale yellow lesions were observed in both lung lobes, as well as similar, though milder, changes in the liver, and an ulcerative lesion on the food pad of the right hindlimb. Histopathologically, the presence of multiple necrotising, chronic granulomas containing bacterial clumps were observed in the parenchyma of the lung and the liver. By microbiological examination of the pathologically altered lung tissues, Providencia rettgeri was identified. Altogether, our findings indicate that the bacterial infection resulting in extensive chronic necrotising granulomatous inflammation was the primary cause of the reptile's death. To our knowledge, this is the first report of Providencia rettgeri-associated granulomatous pneumonia and hepatitis in the monitor lizard.

Viral expression directs the fate of B cells in bovine leukemia virus-infected sheep.

The host immune response is believed to tightly control viral replication of deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). However, this assumption has not been definitely proven in vivo. In order to further evaluate the importance of the immune response in the BLV model, we studied the fate of cells in which viral expression was transiently induced. Using a dual fluorochrome labeling approach, we showed that ex vivo induction of viral expression induces higher death rates of B cells in vivo. Furthermore, cyclosporine treatment of these animals indicated that an efficient immune response is required to control virus-expressing cells.

Chromatin disruption in the promoter of bovine leukemia virus during transcriptional activation.

Bovine leukemia virus expression relies on its chromatin organization after integration into the host cell genome. Proviral latency, which results from transcriptional repression in vivo, represents a viral strategy to escape the host immune system and likely allows for tumor progression. Here, we discriminated two types of latency: an easily reactivable latent state of the YR2 provirus and a 'locked' latent state of the L267 provirus. The defective YR2 provirus was characterized by the presence of nuclease hypersensitive sites at the U3/R junction and in the R/U5 region of the 5'-long terminal repeat (5'-LTR), whereas the L267 provirus displayed a closed chromatin configuration at the U3/R junction. Reactivation of viral expression in YR2 cells by the phorbol 12-myristate 13-acetate (PMA) plus ionomycin combination was accompanied by a rapid but transient chromatin remodeling in the 5'-LTR, leading to an increased PU.1 and USF-1/USF-2 recruitment in vivo sustained by PMA/ionomycin-mediated USF phosphorylation. In contrast, viral expression was not reactivated by PMA/ionomycin in L267 cells, because the 5'-LTR U3/R region remained inaccessible to nucleases and hypermethylated at CpG dinucleotides. Remarkably, we elucidated the BLV 5'-LTR chromatin organization in PBMCs isolated from BLV-infected cows, thereby depicting the virus hiding in vivo in its natural host.

Reduced levels of reactive oxygen species correlate with inhibition of apoptosis, rise in thioredoxin expression and increased bovine leukemia virus proviral loads.

BACKGROUND: Bovine Leukemia virus (BLV) is a deltaretrovirus that induces lymphoproliferation and leukemia in ruminants. In ex vivo cultures of B lymphocytes isolated from BLV-infected sheep show that spontaneous apoptosis is reduced. Here, we investigated the involvement of reactive oxygen species (ROS) in this process. RESULTS: We demonstrate that (i) the levels of ROS and a major product of oxidative stress (8-OHdG) are reduced, while the thioredoxin antioxidant protein is highly expressed in BLV-infected B lymphocytes, (ii) induction of ROS by valproate (VPA) is pro-apoptotic, (iii) inversely, the scavenging of ROS with N-acetylcysteine inhibits apoptosis, and finally (iv) the levels of ROS inversely correlate with the proviral loads. CONCLUSION: Together, these observations underline the importance of ROS in the mechanisms of inhibition of apoptosis linked to BLV infection.

Earlier onset of delta-retrovirus-induced leukemia after splenectomy.

Infection by delta-retroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) is mostly asymptomatic. Indeed, only a minority (<5%) of delta-retrovirus infected hosts will develop either lymphoproliferative or neurodegenerative diseases after long latency periods. In fact, the host immune response is believed to tightly control viral replication but this assumption has not been definitely proven in vivo. Here, we provide direct experimental evidence demonstrating that integrity of the spleen is required to control pathogenesis. In the BLV model, we show that asplenia decreases efficiency of the immune response and induces an imbalance in cell dynamics resulting in accelerated onset of leukemia. These observations enlighten a potential threat in splenectomized HTLV-1 carriers and justify a regular preventive evaluation.

[Current views on the mechanism of oncogenic cell transformation in ovine pulmonary adenocarcinoma]. / Aktualne poglady na mechanizm transformacji nowotworowej komórek w gruczolakoraku pluc owiec.

Lung cancer is the most frequently diagnosed of all the human neoplasms leading to death. Because twenty percent of cases are not associated with cigarette smoking, other causes and methods of early diagnosis are being sought. Bronchioloalveolar cancer, which is a subtype of the most common primary lung cancer, adenocarcinoma, is very similar to ovine pulmonary adenocarcinoma (OPA), a naturally occurring lung cancer in sheep. OPA is caused by the virus Jaagsiekte Sheep Retrovirus (JSRV), a member of the genus of beta-retroviruses. The virus induces neoplastic transformation of secretory epithelial cells of the lung, i.e. alveolar type II pneumocytes and Clara cells. JSRV's tropism for these cells is connected with viral LTR regions interacting with cellular factors that play major roles in the expression of lung-specific genes, e.g. those of surfactant proteins. Results of studies on the mechanisms of viral mutagenesis indicate a viral envelope protein (Env) as an oncogenic factor. There are two main enzymatic pathways involved in the cell transformation: PI3K-Akt and Ras-MEK-MAPK, both activated by the cytoplasmic tail of the envelope protein. Tumor development is associated with telomerase activation. Insertional mutagenesis has also been suggested because there is at least one common integration site for JSRV in OPA. Morphological and histological similarities with human bronchioloalveolar cancer and the possibility of experimental induction of the tumor in animals makes OPA a good model for the study of oncogenesis and target therapy of lung adenocarcinoma.

Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human.

In 1871, the observation of yellowish nodules in the enlarged spleen of a cow was considered to be the first reported case of bovine leukemia. The etiological agent of this lymphoproliferative disease, bovine leukemia virus (BLV), belongs to the deltaretrovirus genus which also includes the related human T-lymphotropic virus type 1 (HTLV-1). This review summarizes current knowledge of this viral system, which is important as a model for leukemogenesis. Recently, the BLV model has also cast light onto novel prospects for therapies of HTLV induced diseases, for which no satisfactory treatment exists so far.

Cell dynamics and immune response to BLV infection: a unifying model.

Bovine Leukemia virus (BLV) is the natural etiological agent of a lymphoproliferative disease in cattle. BLV can also be transmitted experimentally to a related ruminant species, sheep, in which the pathogenesis is more acute. Although both susceptible species develop a strong anti-viral immune response, the virus persists indefinitely throughout life, apparently at a transcriptionally silent stage, at least in a proportion of infected cells. Soon after infection, these humoral and cytotoxic activities very efficiently abolish the viral replicative cycle, permitting only mitotic expansion of provirus-carrying cells. Short term cultures of these infected cells initially indicated that viral expression protects against spontaneous apoptosis, suggesting that leukemia is a process of accumulation of long-lived cells. This conclusion was recently reconsidered following in vivo dynamic studies based on perfusions of nucleoside (bromodeoxyuridine) or fluorescent protein markers (CFSE). In sheep, the turnover rate of infected cells is increased, suggesting that a permanent clearance process is exerted by the immune system. Lymphocyte trafficking from and to the secondary lymphoid organs is a key component in the maintenance of cell homeostasis. The net outcome of the immune selective pressure is that only cells in which the virus is transcriptionally silenced survive and accumulate, ultimately leading to lymphocytosis. Activation of viral and/or cellular expression in this silent reservoir with deacetylase inhibitors causes the collapse of the proviral loads. In other words, modulation of viral expression appears to be curative in lymphocytic sheep, an approach that might also be efficient in patients infected with the related Human T-lymphotropic virus type 1. In summary, a dynamic interplay between BLV and the host immune response modulates a complex equilibrium between (i) viral expression driving (or) favoring proliferation and (ii) viral silencing preventing apoptosis. As conclusion, we propose a hypothetical model unifying all these mechanisms.

Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep.

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.