Search

Introduction The giant myofilament titin is the third most abundant sarcomeric protein besides actin and myosin (1). A single titin molecule spans half of the sarcomere from the Z-disk to [...], Heterozygous (HET) truncating variant mutations in the TTN gene (TTNtvs), encoding the giant titin protein, are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here, we studied 127 clinically identified DCM human cardiac samples with next- generation sequencing (NGS), high-resolution gel electrophoresis, Western blot analysis, and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene sequence predictions were detected in the majority of the [TTNtv.sup.+] samples. Full-length titin was reduced in [TTNtv.sup.+] compared with [TTNtv.sup.-] samples. Proteomics analysis of washed myofibrils and stimulated emission depletion (STED) super- resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin was structurally integrated into the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape, and intensity analyses pointed at possible structural defects in the I/A junction and the M-band of [TTNtv.sup.+] sarcomeres, which probably contribute, possibly via faulty mechanosensor function, to the development of manifest DCM.

Aims: The progression of primary myelofibrosis is characterised by ongoing extracellular matrix deposition graded based on 'reticulin' and 'collagen' fibrosis, as revealed by Gomori's silver impregnation. Here we studied the expression of the major extracellular matrix proteins of fibrosis in relation to diagnostic silver grading supported by image analysis. Methods and results: By using automated immunohistochemistry, in this study we demonstrate that the expression of both types I and III collagens and fibrillin 1 by bone marrow stromal cells can reveal the extracellular matrix scaffolding in line with myelofibrosis progression as classified by silver grading. 'Reticulin' fibrosis indicated by type III collagen expression and 'collagen' fibrosis featured by type I collagen expression were parallel, rather than sequential, events. This is line with the proposed role of type III collagen in regulating type I collagen fibrillogenesis. The uniformly strong fibrillin 1 immune signals offered the best inter‐rater agreements and the highest statistical correlations with silver grading of the three markers, which was robustly confirmed by automated whole slide digital image analysis using a machine learning‐based algorithm. The progressive up‐regulation of fibrillin 1 during myelofibrosis may result from a negative feedback loop as fibrillin microfibrils sequester TGF‐β, the major promoter of fibrosis. This can also reduce TGF‐β‐induced RANKL levels, which would stimulate osteoclastogenesis and thus can support osteosclerosis in advanced myelofibrosis. Conclusions: Through the in‐situ detection of these extracellular matrix proteins, our results verify the molecular pathobiology of fibrosis during myelofibrosis progression. In particular, fibrillin 1 immunohistochemistry, with or without image analysis, can complement diagnostic silver grading at decent cell morphology. [ABSTRACT FROM AUTHOR]

Introduction EBV is a ubiquitous γ-herpesvirus, persistently infecting more than 90% of the human adult population (1). Despite its growth transforming properties, the immune system of most individuals controls infection [...], Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell trans-formation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immuneevading monomorphic DLBCL-like tumors in NOD/SCID/[ γ.sub.c] -/- mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell-chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell-mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.

Background/Aims: Epithelial–mesenchymal transition of tubular cells into α-smooth muscle actin (SMA)-expressing myofibroblasts is a central mechanism in tubulointerstitial fibrosis. Previously, a ‘two-hit’ model was proposed for epithelial–mesenchymal transition wherein an initial injury of the intercellular contacts and TGF-β1 are both required for SMA protein expression in LLC-PK1 cells. The Rho-Rho kinase-myosin light chain-myocardin-related transcription factor (MRTF)-serum response factor (SRF) pathway and Rac1, p21-activated kinase (PAK) and p38 were described as important regulators of MRTF localization and SMA expression. Cdc42 is another small G protein situated upstream of PAK and p38, and is activated upon cell contact disassembly. Here, we investigated its potential role in the regulation of MRTF nuclear shuttling and in the regulation of the SMA promoter. Results: Transfection of a constitutive active (CA) Cdc42 construct alone induced the activation of the SMA promoter. The dominant negative (DN) Cdc42 construct prevented the activation of the promoter induced by cell contact disassembly, and reduced the combined effect of cell contact disruption and TGF-β1. SRF showed a marked nuclear accumulation in CA Cdc42-transfected cells. Cdc42 induced the nuclear translocation of MRTF, while DN Cdc42 inhibited its nuclear translocation induced by cell contact disassembly. Blocking PAK, MRTF and p38 by the corresponding DN constructs blunted the effects of CA Cdc42 on the SMA promoter. Conclusion: Cdc42 is involved in the regulation of SMA promoter activation through PAK, p38, MRTF and SRF. Cdc42 may be an important regulator of MRTF cellular localization. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In myelofibrosis, pathologically enhanced extracellular matrix production due to aberrant cytokine signalling and clonal megakaryocyte functions result( s ) in impaired hemopoiesis. Disease progression is still determined by detecting reticulin and collagen fibrosis with Gomori's silver impregnation. Here, we tested whether the expression growth related biomarkers L-NGFR/CD271, phospho-ERK1-2 and CXCL12 can be linked to the functional activation of bone marrow stromal cells during primary myelofibrosis progression. Immunoscores for all tested biomarkers showed varying strength of positive statistical correlation with the silver impregnation based myelofibrosis grades. The intimate relationship between spindle shaped stromal cells positive for all three markers and aberrant megakaryocytes was likely to reflect their functional cooperation. L-NGFR reaction was restricted to bone marrow stromal cells and revealed the whole length of their processes. Also, L-NGFR positive cells showed the most intersections, the best statistical correlations with myelofibrosis grades and the strongest interrater agreements. CXCL12 reaction highlighted stromal cell bodies and a weak extracellular staining in line with its constitutive release. Phospho-ERK1-2 reaction showed a similar pattern to CXCL12 in stromal cells with an additional nuclear staining in agreement with its role as a transcription factor. Both p -ERK1-2 and CXCL12 were also expressed at a moderate level in sinus endothelial cells. Connexin 43 gap junction communication channels, known to be required for CXCL12 release to maintain stem cell niche, were also expressed progressively in the myelofibrotic stromal network as a support of compartmental functions. Our results suggest that, diverse growth related pathways are activated in the functionally coupled bone marrow stromal cells during myelofibrosis progression. L-NGFR expression can be a useful biological marker of stromal cell activation which deserves diagnostic consideration for complementing Gomori's silver impregnation., Competing Interests: The authors declare that this study received shared facilities from 3DHISTEC Ltd. AC has been both the employee of 3DHISTECH Ltd. and a PhD student at Semmelweis University under the Co-operative PhD Program funded by the Hungarian Ministry of Innovation. TK is Member of the Editorial Board regularly reviewing manuscripts for POR. TS is a PhD student at Semmelweis University which supports PhD students publications in POR. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Szekely, Krenacs, Maros, Bodor, Daubner, Csizmadia, Vrabely and Timar.)