miRNA Expression Signatures Induced by Chicken Astrovirus Infection in Chickens.

miRNAs represent ubiquitous regulators of gene expression and play an important and pivotal regulatory role in viral disease pathogenesis and virus-host interactions. Although previous studies have provided basic data for understanding the role of miRNAs in the molecular mechanisms of viral infection in birds, the role of miRNAs in the regulation of host responses to chicken astrovirus (CAstV) infection in chickens is not yet understood. In our study, we applied next-generation sequencing to profile miRNA expression in CAstV-infected chickens and to decipher miRNA-targeted specific signaling pathways engaged in potentially vital virus-infection biological processes. Among the 1354 detected miRNAs, we identified 58 mature miRNAs that were significantly differentially expressed in infected birds. Target prediction resulted in 4741 target genes. GO and KEGG pathway enrichment analyses showed that the target genes were mainly involved in the regulation of cellular processes and immune responses.

Transcriptome Sequencing of the Spleen Reveals Antiviral Response Genes in Chickens Infected with CAstV.

Astrovirus infections pose a significant problem in the poultry industry, leading to multiple adverse effects such as a decreased egg production, breeding disorders, poor weight gain, and even increased mortality. The commonly observed chicken astrovirus (CAstV) was recently reported to be responsible for the "white chicks syndrome" associated with an increased embryo/chick mortality. CAstV-mediated pathogenesis in chickens occurs due to complex interactions between the infectious pathogen and the immune system. Many aspects of CAstV-chicken interactions remain unclear, and there is no information available regarding possible changes in gene expression in the chicken spleen in response to CAstV infection. We aim to investigate changes in gene expression triggered by CAstV infection. Ten 21-day-old SPF White Leghorn chickens were divided into two groups of five birds each. One group was inoculated with CAstV, and the other used as the negative control. At 4 days post infection, spleen samples were collected and immediately frozen at -70 °C for RNA isolation. We analyzed the isolated RNA, using RNA-seq to generate transcriptional profiles of the chickens' spleens and identify differentially expressed genes (DEGs). The RNA-seq findings were verified by quantitative reverse-transcription PCR (qRT-PCR). A total of 31,959 genes was identified in response to CAstV infection. Eventually, 45 DEGs (p-value < 0.05; log2 fold change > 1) were recognized in the spleen after CAstV infection (26 upregulated DEGs and 19 downregulated DEGs). qRT-PCR performed on four genes (IFIT5, OASL, RASD1, and DDX60) confirmed the RNA-seq results. The most differentially expressed genes encode putative IFN-induced CAstV restriction factors. Most DEGs were associated with the RIG-I-like signaling pathway or more generally with an innate antiviral response (upregulated: BLEC3, CMPK2, IFIT5, OASL, DDX60, and IFI6; downregulated: SPIK5, SELENOP, HSPA2, TMEM158, RASD1, and YWHAB). The study provides a global analysis of host transcriptional changes that occur during CAstV infection in vivo and proves that, in the spleen, CAstV infection in chickens predominantly affects the cell cycle and immune signaling.

Novel prognostic molecular markers in lung cancer.

Lung carcinoma, especially in its most commonly diagnosed non-small cell histological form, is a challenge to diagnose and treat worldwide, due to the prognosis in patients with this type of cancer being poor and mortality rates being high. However, a number of patients with this type of lung carcinoma exhibit a longer than average overall survival. The specific molecular background of non-small-cell lung cancer that favors longer survival has not yet been determined. The aim of the current study was to review articles published in the years 2017-2018 and create a list of the most important and strongest non-conventional factors that could be used in the future assessment of the prognosis of patients with adenocarcinoma and squamous cell carcinoma of the lung who cannot undergo current targeted therapy. Analysis identified multiple prognostic factors in non-small cell lung carcinoma, including tumor mutational burden, which was revealed to be independent of the tumor stage or grade as well as other factors, including age, sex or targeted therapy effects. The selected molecular factors exhibit the potential to be used in the treatment of patients with specific problematic lung cancer, and may contribute to setting recommendations for the diagnosis, prognosis and treatment of individual patients with lung cancer.

Prolonged inflammation leads to ongoing damage after spinal cord injury.

The pathogenesis of spinal cord injury (SCI) remains poorly understood and treatment remains limited. Emerging evidence indicates that post-SCI inflammation is severe but the role of reactive astrogliosis not well understood given its implication in ongoing inflammation as damaging or neuroprotective. We have completed an extensive systematic study with MRI, histopathology, proteomics and ELISA analyses designed to further define the severe protracted and damaging inflammation after SCI in a rat model. We have identified 3 distinct phases of SCI: acute (first 2 days), inflammatory (starting day 3) and resolution (>3 months) in 16 weeks follow up. Actively phagocytizing, CD68+/CD163- macrophages infiltrate myelin-rich necrotic areas converting them into cavities of injury (COI) when deep in the spinal cord. Alternatively, superficial SCI areas are infiltrated by granulomatous tissue, or arachnoiditis where glial cells are obliterated. In the COI, CD68+/CD163- macrophage numbers reach a maximum in the first 4 weeks and then decline. Myelin phagocytosis is present at 16 weeks indicating ongoing inflammatory damage. The COI and arachnoiditis are defined by a wall of progressively hypertrophied astrocytes. MR imaging indicates persistent spinal cord edema that is linked to the severity of inflammation. Microhemorrhages in the spinal cord around the lesion are eliminated, presumably by reactive astrocytes within the first week post-injury. Acutely increased levels of TNF-alpha, IL-1beta, IFN-gamma and other pro-inflammatory cytokines, chemokines and proteases decrease and anti-inflammatory cytokines increase in later phases. In this study we elucidated a number of fundamental mechanisms in pathogenesis of SCI and have demonstrated a close association between progressive astrogliosis and reduction in the severity of inflammation.

Myxoma virus derived immune modulating proteins, M-T7 and Serp-1, reduce early inflammation after spinal cord injury in the rat model.

Spinal cord injury (SCI)-initiated inflammation was treated with anti-inflammatory reagents. We compared local spinal cord or intraperitoneal infusion of two Myxoma virus derived immune modulating proteins, Serp-1 and M-T7, with dexamethasone (DEX). Hemorrhage and necrosis after SCI initiate a complex pathogenesis dominated by early, severe and highly destructive inflammatory macrophage infiltration. We examined sustained, 7-day, subdural infusion of either M-T7, a chemokine modulator or Serp-1, a plasminogen activator and factor inhibitor. Mature male rats had epidural balloon crush SCI and sustained subdural infusion of Serp-1, M-T7, DEX or saline for 7 days via the osmotic pump. A separate group of rats with SCI had intra-peritoneal infusion. Clinical evaluation included endpoint monitoring with body weight, hemorrhagic cystitis and bilateral toe pinch response. Sections of the spinal cord were analyzed histologically and macrophage numbers counted by standardized protocol in the cavity of injury (COI). While the rats administered DEX demonstrated substantial body weight loss, dehydration and dermal atrophy consistent with steroid toxicity, rats infused with Serp-1 and M-T7 had no toxicity. Serp-1 improved withdrawal responses. Subdural infusion of Serp-1, M-T7 and DEX significantly reduced numbers of phagocytic, CD68-positive macrophages. With intraperitoneal infusion only M-T7 reduced macrophage counts, Serp-1 showed only a trend. Local infusion of highly active immune modulating proteins; Serp-1 and M-T7, targeting serine protease and chemokine pathways demonstrated excellent potential for neuroprotection after severe SCI in a rat model, without adverse side effects. Sustained subdural infusion offers an alternative route of administration for treatment of SCI.

MMP-2 mRNA Expression in Ovarian Cancer Tissues Predicts Patients' Response to Platinum-Taxane Chemotherapy.

BACKGROUND/AIM: Ovarian cancer is the most frequent cause of death in women among gynecological cancers in Poland. MMP-2 and MMP-9 are frequently dysregulated in cancers and they are considered as potential biomarkers. Our goal was to assess the associations between MMP-2 and MMP-9 mRNA expression, clinicopathological parameters and patients' response to chemotherapy. MATERIALS AND METHODS: We evaluated MMP-2 and MMP-9 mRNA expression in epithelial ovarian cancer (EOC) tissues from 44 untreated patients, four ovarian cancer cell lines, and human skin fibroblasts (HSF). The expression of both MMPs was estimated using qPCR. RESULTS: MMP-2 expression was significantly higher (p=0.020) in EOCs sensitive to chemotherapy compared to resistant and refractory tumors. The highest MMP-2 expression was found in HSF and MMP-9 expression was the highest in EOCs (p<0.001). The expression of neither MMP was significantly associated with patients' overall survival (OS). CONCLUSION: MMP-2 may be engaged in early stages of ovarian carcinogenesis. MMP-2 expression in EOCs may discriminate patients with a favorable response to first line chemotherapy.

Pigmented villonodular synovitis.

Pigmented villonodular synovitis (PVNS) is a benign disease that rarely undergoes malignant transformation. There are two types of disease: localized (nodular tenosynovitis) and di used (pigmented villonodular synovitis/tenosynovitis) with intra- or extra-articular locations. The second one is limited to synovium of the burse (PVNB) or tendon sheath (PVNTS). The intraarticular lesions are usually located in the knee, hip, ankle and elbow joints. Histologically, PVNS is a tenosynovial giant cell tumor, characterized by proliferation of two types of mononuclear cells - predominantly small, histiocyte-like cells and larger cells with dense cytoplasm, reniform or lobulated nucleus, with accompanying multinucleated giant cells and macrophages overloaded with hemosiderin that give typical image on MRI - currently selected as a gold standard for its diagnosis. The classic X-ray and CT are non-specific but similar to ultrasound should be used to evaluate disease progression and treatment response if radiotherapeutic and pharmacological methods were selected for treatment. An open arthroscopic surgery could also be applied in selected cases.

Molecular bases of aberrant miR-182 expression in ovarian cancer.

The molecular bases of miR-182 deregulation in epithelial ovarian cancers (EOCs) remain unknown and its diagnostic or prognostic role in EOCs is still unclear. We performed miR-182 expression analysis using a microarray approach and real-time PCR (qPCR). We also used array comparative genomic hybridization and methylated DNA immunoprecipitation to study copy number changes and methylation aberrations within coding locus/promoter sequences of miR-182 in EOC tissues, respectively. We have found that miR-182 expression is significantly increased in EOC (P < 0.00001) and that higher miR-182 expression in EOC is linked with significantly shorter overall survival (P = 0.026). The methylation of miR-182 promoter was significantly associated with lower miR-182 expression in EOC tissues (P = 0.045). miR-182 over-expression is connected with copy number (CN) gains of this miRNA coding sequences in EOC (P = 0.002), and the number of PRDM5 copies is significantly and inversely correlated with miR-182 expression evaluated by qPCR (R = -0.615, P = 0.009). We conclude that the aberrant miR-182 expression in EOC may be due to CN gains within its coding locus. The miR-182 promoter is rarely methylated in EOC, and its methylation status is associated with lower miR-182 expression. Deletion of the PRDM5 locus may play a supportive role in miR-182 overexpression in EOC. miR-182 is an unfavorable prognostic factor in EOC. © 2016 Wiley Periodicals, Inc.

IL-6, IL-10, c-Jun and STAT3 expression in B-CLL.

Chronic lymphocytic leukemia is characterized by the accumulation of functionally abnormal, monoclonal B lymphocytes in the peripheral blood, bone marrow, lymph nodes and spleen, resulting in a reduction count of normal immunocompetent cells and their impaired immune function. The defect in transmission of signals from various types of extracellular receptors, leading to aberrant cytokines and transcription factors gene expression, may underlie the basis of immune failure in B-CLL. The aim of the study was to assess of IL-6, IL-10, c-Jun, and STAT3 expression. In response to antigenic stimulation IL-6, IL-10, c-Jun, and STAT3 proteins induce mutual activity. The subject of the study was subpopulations of leukemic lymphocytes (CD5+ CD19+) and CD19+ B cells from healthy donors (control group). Our results provide evidence that the regulation of IL-6, IL-10, c-Jun, and STAT3 gene expression in CLL B cells is clearly different from normal B lymphocytes. In B-CLL STAT3 expression in unstimulated lymphocytes is significantly higher (p<0.0001) compared with normal subpopulation of B cell. In contrast, IL-6, IL-10, and c-Jun mRNA expressions are statistically lower in B-CLL in comparison with the control group, in all cases (p<0.0001). When analyzing the relationship between c-Jun expression and B-CLL stage according Rai we revealed decreasing c-Jun expression, both at the mRNA and protein levels, along with advancing stage of disease.

Aberrant TIRAP and MyD88 expression in B-cell chronic lymphocytic leukemia.

TIRAP and Myd88 are adaptor proteins for Toll-like receptors-2 and -4 (TLR2/4) which are engaged in transducing the signal to downstream molecules. Several studies have shown the increased role of infection factors in pathogenesis of B cell chronic lymphocytic leukemia (B-CLL). This prompted us to test whether there is a correlation between MyD88-TIRAP dynamics before and after inflammatory stimuli. We determined the mRNA and protein expression of TIRAP and MyD88 in CD5(+)CD19(+) B-CLL cells and in a subpopulation of normal B CD19(+) lymphocytes. Additionally we determined the influence of lipopolysaccharide Escherichia coli - TLR4-ligand (LPS) and Staphylococcus aureus strain Cowan I - TLR2-ligand (SAC) on TIR-domain-containing adaptor protein, also called MyD88 adaptor-like (TIRAP) and myeloid differentiation primary response protein 88 (MyD88) expression. We have found that the mRNA and protein expression of TIRAP and MyD88 in B-CLL lymphocytes is lower compared with that in normal B lymphocytes. LPS and SAC stimulation in normal lymphocytes significantly altered neither TIRAP nor MyD88 mRNA expression, whereas TIRAP protein level substantially decreased after TLR agonist treatment. We did not observe any changes in MyD88 protein level after B lymphocyte stimulation. There was a significant increase in TIRAP mRNA expression after LPS and SAC stimulation of B-CLL cells. MyD88 mRNA expression levels in B-CLL lymphocytes slightly decreased upon treatment with either stimulator. Stimulation with TLR agonists did not cause changes in TIRAP and MyD88 expression at the protein level in B-CLL lymphocytes. The results of our study suggest that there may exist a, yet unknown, defect of TIRAP and MyD88 proteins in B-CLL lymphocytes.

TLR2 may influence the behavior of the malignant clone in B-CLL.

B-cell receptor (BCR) and Toll-like receptor (TLR) stimulation and integration with signals from the pathogen or immune cells and their products determine the B-cell antibody response. Low expression of BCR is the hallmark of B lymphocytes in CLL, however little is known about the expression and function of TLR in B-CLL. We studied TLR2, TLR4, IL-6 and mIL-6Rα expression on mRNA and protein level in CD19(+) subpopulation of normal lymphocytes and the CD19(+)CD5(+) subpopulation from B-CLL. Experiments were performed on unstimulated and stimulated lymphocytes [Staphylococcus aureus Cowan I (SAC) and lipopolysaccharide (LPS) from Escherichia coli - TLR2- and TLR4-specific agonists, respectively]. We showed undetectable or low IL-6 expression, which seems to be specific for B-CLL lymphocytes. Induction of TLR4 mRNA upon LPS stimulation affected the expression of IL-6, but not of mIL-6Rα. Increased expression of TLR2 (MFI) after LPS and SAC stimulation did not correlate with mIL-6Rα receptor expression. B-CLL CD19(+)CD5(+) lymphocytes showed a significant increase in TLR2 expression at the protein level after stimulation with SAC and LPS compared to normal CD19(+) lymphocytes. TLR2 may influence the behaviour of the malignant clone in B-CLL.

[Expression of podoplanin in ovarian clear cell carcinoma].

INTRODUCTION: Podoplanin is a transmembrane glycoprotein expressed in endothelial lymphatic cells. It was proven to be a predictive marker in a variety of cancers e.g. mesothelioma and head and neck squamous-cell carcinoma. Ovarian clear cell carcinoma (OCCC) is a rare and unique histopathologic subtype of epithelial ovarian cancer (EOC). The molecular basis of that phenomenon remains unknown. OBJECTIVES: The aim of our study was to assess podoplanin expression on the protein level in OCCC. MATERIAL AND METHODS: Immunohistochemistry was performed on paraffin-embedded tissues from 19 patients with diagnosed OCCC. RESULTS: Podoplanin expression was present (moderate or strong) in 52% of OCCC cases (10/19). Nine of eleven (81.2%) postmenopausal and one of eight (12.5%) premenopausal women were podoplanin positive. No differences in podoplanin expression were found in relation to clinical features of the tumor CONCLUSION: The incidence of podoplanin expression is higher in ovarian clear cell adenocarcinoma in postmenopausal patients.

Different expression of CD180, CD284 and CD14 receptors on the CD19+ subpopulation of normal and B-CLL lymphocytes.

Numerous experimental data indicate that B-CLL development and progression are influenced by antigenic pressure. It can not be excluded that these antigens may originate from bacteria and viruses. Toll like receptors (TLRs) interact with pathogen associated molecular patterns as part of innate immunity. TLRs are currently used to target different subclasses of B-cell leukemia, and TLR agonists are being evaluated in clinical trials. It is little known regarding the repertoire and function of TLR in B-CLL. The aim of the study was to assess the CD180, CD284 and mCD14 levels in CD19+ subpopulation of B-CLL peripheral blood lymphocytes and compare them with respective levels in the normal B-cells of adult volunteers, before and after LPS stimulation. We investigated the percentage of the CD19+CD180+, CD19+CD284+, CD19+CD14+ cells and the mean fluorescence intensity (MFI) of CD180, CD284 and CD14 antigens among CD19+ B-CLL as well as in the normal B cells for comparison. MFI analysis revealed that CD180, CD284 and CD14 expression was higher on normal B cells then on CD19+ B-CLL (MFI CD180: 99.16 vs. 25.3, MFI CD284: 7.37 vs. 5.79 and MFI CD14 25.07 vs. 8.32). After 24-hour LPS activation of B-cells, CD180 MFI appeared to decrease, in both healthy and B-CLL patients. CD284 MFI in healthy controls decreased after LPS stimulation while slight increase of MFI was observed in leukemic cells. CD14 MFI in leukemic cells was moderately higher after LPS in comparison to CD14 MFI without LPS stimulation, whereas CD14 MFI in normal CD19+ cells after LPS stimulation decreased over three times. Variations observed in expression of both normal and leukemic receptors may be due to their different sensitivity to antigenic stimulation.