Swim bladder as a primary site of mycobacterial infection in Nothobranchius 'belly sliders'.

The swim bladder inflates early after fish hatching via its interconnection with the digestive tract (ductus pneumaticus). This interconnection may serve as a portal to foreign particles, including bacteria, causing deficiencies in primary swim bladder inflation. We histologically examined 134 African annual killifish (genus Nothobranchius) with secondary loss of swim bladder function ('belly sliders'). We demonstrate that these fish lost the ability of air regulation in their swim bladders likely due to Mycobacterium spp. infection at an individual-specific age. Nearly all examined belly sliders had thickened swim bladder walls, and their swim bladder was filled with material containing mycobacteria, cell debris, young monocytic cells and phagocyting macrophages. Mycobacterial infection was restricted to the swim bladder in juveniles, where mycobacteria likely enter the host through the ductus pneumaticus. Infection in adults was systemic and mycobacteria were present in all examined organs. Presence of mycobacteria in the epithelial lining and submucosal layers of the digestive tract of adults suggests that it may also serve as the entrance site of infection. We suspect 2 sources of Mycobacterium contamination: dietary (with bloodworms) and/or contaminated hatching substrate. These sources of contamination may be eliminated by use of laboratory dry feed and egg disinfection prior to hatching.

Barriers in systemic delivery and preclinical testing of synthetic microRNAs in animal models: an experimental study on miR-215-5p mimic.

Mus musculus is the most commonly used animal model in microRNA research; however, little is known about the endogenous miRNome of the animals used in the miRNA-targeting preclinical studies with the human xenografts. In the presented study, we evaluated the NOD/SCID gamma mouse model for the preclinical study of systemic miR-215-5p substitution with a semitelechelic poly[N-(2-hydroxypropyl)-methacrylamide]-based carrier conjugated with miR-215-5p-mimic via a reductively degradable disulfide bond. Murine mmu-miR-215-5p and human hsa-miR-215-5p have a high homology of mature sequences with only one nucleotide substitution. Due to the high homology of hsa-miR-215-5p and mmu-hsa-miR-215-5p, a similar expression in human and NOD/SCID gamma mice was expected. Expression of mmu-miR-215 in murine organs did not indicate tissue-specific expression and was highly expressed in all examined tissues. All animals included in the study showed a significantly higher concentration of miR-215-5p in the blood plasma compared to human blood plasma, where miR-215-5p is on the verge of a reliable detection limit. However, circulating mmu-miR-215-5p did not enter the human xenograft tumors generated with colorectal cancer cell lines since the levels of miR-215-5p in control tumors remained notably lower compared to those originally transfected with miR-215-5p. Finally, the systemic administration of polymer-miR-215-5p-mimic conjugate to the tail vein did not increase miR-215-5p in NOD/SCID gamma mouse blood plasma, organs, and subcutaneous tumors. It was impossible to distinguish hsa-miR-215-5p and mmu-miR-215-5p in the murine blood and organs due to the high expression of endogenous mmu-miR-215-5p. In conclusion, the examination of endogenous tissue and circulating miRNome of an experimental animal model of choice might be necessary for future miRNA studies focused on the systemic delivery of miRNA-based drugs conducted in the animal models.

MicroRNAs as outcome predictors in patients with metastatic colorectal cancer treated with bevacizumab in combination with FOLFOX.

Bevacizumab is a humanized anti-vascular endothelial growth factor monoclonal antibody, used in combination with a oxaliplatin-based chemotherapy in the treatment of metastatic colorectal cancer (mCRC). The aim of the present study was to identify microRNA (miRNA)-based predictive biomarkers of therapy response in order to avoid unnecessary and costly therapy to non-responding patients. High-throughput miRNA microarray profiling (Affymetrix miRNA array) was performed on a discovery cohort of patients with mCRC. The discovery cohort was (n=20) divided into either responding (n=10) or non-responding (n=10) groups of bevacizumab/5-flourouracil, leucovorin, oxaliplatin (FOLFOX) treatment according to Response Evaluation Criteria in Solid Tumors criteria. Validation of candidate miRNAs was performed on an independent cohort of 41 patients with mCRC using quantitative reverse transcription polymerase chain reaction. Normalized data were subjected to receiver operating characteristic and Kaplan-Meier analyses. In total, 67 miRNAs were identified to be differentially expressed when miRNA expression was compared between responding and non-responding patients to bevacizumab/FOLFOX treatment (P<0.05). A total of 7 miRNAs were chosen for independent validation, which confirmed significantly higher expression of miR-92b-3p, miR-3156-5p, miR-10a-5p and miR-125a-5p (P<0.005) in tumor tissue of responding patients compared with non-reponding patients. Using the combination of miRNAs, the present study identified responders to the therapy with sensitivity 82% and specificity 64% (area under the curve = 0.8015). In conclusion, 4 predictive miRNAs associated with progression-free survival (PFS) were identified in patients with mCRC treated with bevacizumab/FOLFOX. Following further independent validations, detection of these miRNA may enable identification of patients with mCRC who may potentially benefit from the therapy.

Reduced inducibility of SOCS3 by interferon gamma associates with higher resistance of human breast cancer lines as compared to normal mammary epithelial cells.

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the antiproliferative effect of interferon gamma in relation to SOCS3 expression in a panel of breast cancer cell lines and normal mammary epithelial cells. Compared to normal cells most breast cancer lines (7/8) were highly resistant to IFN-gamma. Using Northern blot and real time RT-PCR we investigated transcription of SOCS3 genes. All normal epithelial cells (4/4) showed SOCS3 induction (2-14 fold) while most breast cancer lines did not or weakly activated SOCS3 after the interferon gamma treatment. Among the cancer lines, the MDA-MB-468 cells showed increased sensitivity to IFN-gamma and relatively high level of SOCS3 induction (2-3 fold). Together, there was a good correlation

Defective IFNα/γ-induced STAT3 protein activation in human malignant melanoma cells.

Signal transducer and activator of transcription 3 (STAT3) protein has been documented as a significant mediator of interferon (IFN) signaling. Physiological STAT3 phosphorylation involves tyrosine (Y705) and serine (S727) activation. Impairment of STAT3 protein levels and/or of STAT3 phosphorylation after IFN treatment has been found in many pathological conditions such as cancer, immunopathy and inflammatory disease. To analyze tumor-associated defective STAT3 response to IFNs, the induction of S727 and Y705 STAT3 activation after IFN exposure was evaluated in 18 human malignant melanoma cell lines and 68 primary cell cultures established from the lymph node metastases of melanoma patients. STAT3 expression and STAT3 phosphorylated forms were assayed by Western blot analysis employing specific STAT3 antibodies. All melanoma cell lines as well as samples derived from metastatic melanoma patients expressed STAT3 with variable signal intensities depending on the appropriate cell type. Significantly altered IFNγ-induced S727 STAT3 activation was found in both experimental models, with on average 94.1% of patients detected to be non-responders in lymph node cell cultures and 83.3% in melanoma cell lines. Moreover, a deficiency in IFNα-induced S727 induction was detected in 88.9% of melanoma cell lines. Defects in Y705 STAT3 phosphorylation were determined in clinical material (61.8% after IFNγ exposure) as well as in melanoma cell lines (absence of response to IFNα/γ in 83.3 and 55.5%, respectively). Our data clearly confirm STAT3 pathophysiological perturbances in human malignant melanoma cells. Depending on the induction of STAT3-activated phosphoforms by IFNs, three categories of melanoma cells were identified: a) phosphorylation on both the S727 and Y705 amino acid residues; b) STAT3 activation on Y705 only; c) phosphorylation at neither S727 nor Y705. The significance of in vitro STAT3 activation for predicting patient response to immunotherapy will be examined in a prospective clinical study by our group.

Development of IFN-gamma resistance is associated with attenuation of SOCS genes induction and constitutive expression of SOCS 3 in melanoma cells.

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the evolution of an IFN-resistant state in vitro using melanoma cell lines. We found that the cells became less sensitive to antiproliferative effect of IFN-gamma after prolonged cultivation enabling us to isolate sensitive and resistant subclones of the parental line. We investigated transcription of signal transducer and activator of transcription (STAT) 1-6 and suppressor of cytokine signalling (SOCS) 1-3 genes, and phosphorylation of STAT 1 protein. The resistant subline (termed WM 1158R) differed from the sensitive subline (WM 1158S) by a constitutive expression of SOCS 3, lack or weak SOCS 1-3 activation following IFN-gamma, and short duration of cytokine activatory signal. Similar correlations were observed in additional melanoma lines differing in IFN sensitivities. At the protein level, IFN-gamma induced strong and prolonged STAT 1 activation at serine 727 (S727) in WM 1158R while in WM 1158S cells phosphorylation of this amino acid was much less pronounced. On the other hand, phosphorylation of tyrosine 701 (Y701) was stimulated regardless of the sensitivity phenotype. In conclusion, constitutive expression of SOCS 3 is correlated with attenuation of its induction following IFN treatment. These results suggest that progression of melanoma cells from IFN sensitivity to IFN insensitivity associates with changes in SOCS expression.

Transcription protein STAT1: biology and relation to cancer.

Cell homeostasis is controlled and regulated by multiple signalling proteins that operate almost in all cellular compartments. Their common task is to process regulatory signals from both the extracellular and intracellular spaces by triggering a cascade of intracellular events leading to modulation of downstream gene activity. One of the important signalling pathways is represented by the STAT multigene family comprising seven members. In general, various STATs act as potent transcription factors delivering signals of diverse polypeptide ligands (i.e. cytokines and growth factors) into the nucleus. This review summarizes some up-to-date data on the role of STAT1 in maintaining cellular homeostasis with the emphasis on its role in the control of cell growth, proliferation, apoptosis, and immune reactions. Part of the review deals with expression and posttranslational abnormalities of this molecule identified in a variety of human pathological conditions including cancer. The direct or indirect involvement of STAT1 in the process of malignant transformation is highlighted in view of these molecular perturbances that may contribute to oncogenesis and that may be potentially used as novel targets for anticancer therapy.