The CD34+ human acute myeloid leukemia–derived cell line MUTZ-3 is dependent on hematopoietic growth factors for its proliferation and is able to differentiate into dendritic cells (DCs) in response to the combination of granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-α. This cell line carries human leukocyte antigen (HLA)-A2.1, HLA-A3, and HLA-B44, which cover most of the caucasian population, and it could therefore be used as an off-the-shelf allogeneic DC-based vaccine. Signal transduction and activation of transcription (STAT) 5b is involved in cytokine signal transduction, particularly of cytokines involved in DC precursor growth and differentiation. The constitutively active form of STAT5b induced cytokine-independent growth of MUTZ-3 cells. Furthermore, STAT5b-transduced cells differentiated into mature DCs in 3 to 4 days after stimulation with DC differentiation-inducing cytokines, reducing the culture period to obtain mature DCs with 5 days compared with unmodified MUTZ-3–derived mature DC cultures. Both DC types expressed DC maturation markers and were equally effective in inducing primary T-cell responses. DCs derived from the STAT5b-transduced cells had a more stable mature phenotype after cytokine deprivation, which was reflected in a better performance in functional assays. In conclusion, these results show that STAT5b-transduced MUTZ-3 can be propagated in cytokine-free medium and rapidly differentiated into functional mature DCs that sustain a mature phenotype over a period of 3 to 5 days in the absence of differentiation-inducing cytokines. The simplified propagation and rapid differentiation into mature DCs may facilitate clinical application of this cell line as an allogeneic DC-based vaccine.
Desmoplakin (DP) is an important component of desmosomes, essential in cell-cell connecting structures in stress-bearing tissues. Over the years, many hundreds of pathogenic variants in DSP have been associated with different cutaneous and cardiac phenotypes or a combination, known as a cardiocutaneous syndrome. Of less than 5% of the reported DSP variants, the effect on the protein has been investigated. Here, we describe and have performed RNA, protein and tissue analysis in a large family where DSPc.273+5G>A/c.6687delA segregated with palmoplantar keratoderma (PPK), woolly hair and lethal cardiomyopathy, while DSPWT/c.6687delA segregated with PPK and milder cardiomyopathy. hiPSC-derived cardiomyocytes and primary keratinocytes from carriers were obtained for analysis. Unlike the previously reported nonsense variants in the last exon of DSP that bypassed the nonsense-mediated mRNA surveillance system leading to protein truncation, variant c.6687delA was shown to cause the loss of protein expression. Patients carrying both variants and having a considerably more severe phenotype were shown to have 70% DP protein reduction, while patients carrying only c.6687delA had 50% protein reduction and a milder phenotype. The analysis of RNA from patient cells did not show any splicing effect of the c.273+5G>A variant. However, a minigene splicing assay clearly showed alternative spliced transcripts originating from this variant. This study shows the importance of RNA and protein analyses to pinpoint the exact effect of DSP variants instead of solely relying on predictions. In addition, the particular pattern of inheritance, with simultaneous or separately segregating DSP variants within the same family, strongly supports the theory of a dose-dependent disease severity.
Aims: Inhibition of apoptosis is important in the pathogenesis of lymphomas. c‐FLIP, a regulator of caspase 8‐mediated apoptosis, plays an important role in protecting normal B and T cells from apoptosis and possibly also in lymphomas. Because of contradictory reports about immunohistochemical detection of c‐FLIP expression, the aim was to test the specificity of four antibodies in c‐FLIP‐transfected cells and subsequently to investigate expression of c‐FLIP in different types of lymphoma. Methods and results: Two of four antibodies were specific. In primary lymphomas c‐FLIP expression was restricted to Hodgkin’s lymphomas (> 90%) and diffuse large B‐cell lymphomas (44%). Burkitt lymphomas and indolent B‐cell lymphomas were negative in all cases. No expression was detected in primary T‐cell lymphomas, although expression was observed in one relapsed ALK+ anaplastic large cell lymphoma. Expression of c‐FLIP was inversely correlated with caspase 8 activation. Conclusions: c‐FLIP is important in escape of B cells from apoptosis during normal follicle centre cell reaction and may thus be an important early event in the development of B‐cell‐derived lymphomas. Moreover, non‐specific staining of frequently used antibodies might explain discrepancies in different reports of c‐FLIP expression.
ABSTRACT The role mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes in the heart. However, most EHT systems are unable to model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained fractional shortening of >10%. To do this, 3D EHTs are integrated with an elastic polydimethylsiloxane (PDMS) strip that provides mechanical pre- and afterload to the tissue in addition to enabling contractile force measurements based on strip bending. Our results demonstrate in wild-type EHTs that dynamic loading is beneficial based on the magnitude of the forces, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we use hiPSC–derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy (ACM) due to mutations in desmoplakin. We demonstrate that manifestation of this desmosome-linked disease state requires the dyn-EHT conditioning and that it cannot be induced using 2D or standard 3D EHT approaches. Thus, dynamic loading strategy is necessary to provoke a disease phenotype (diastolic lengthening, reduction of desmosome counts, and reduced contractility), which are akin to primary endpoints of clinical disease, such as chamber thinning and reduced cardiac output. Single Sentence Summary Development of a dynamic mechanical loading platform to improve contractile function of engineered heart tissues and study cardiac disease progression.
Two splice variants of the α6 integrin subunit, α6A and α6B, with different cytoplasmic domains, have previously been described. While α6B is expressed throughout the development of the mouse, the expression of α6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, α6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of α6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of α6A in embryonic stem cells, and the deletion resulted in the expression of α6B in all tissues that normally express α6A. We show that α6A−/− mice develop normally and are fertile. The substitution of α6A by α6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in α6A−/− mice. However, the substitution of α6A by α6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin–ligand interactions, was not affected in the α6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of α6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions.
### A1 Intratumoral immunotherapy. B16-F10 murine melanoma model
#### J. Ženka1, V. Caisova1, O. Uher1, P. Nedbalova1, K. Kvardova1, K. Masakova1, G. Krejcova1, L. Paďoukova1, I. Jochmanova2, K. I. Wolf3, J. Chmelař1, J. Kopecký1
##### 1Department of Medical Biology, Faculty of
It has become apparent that extracellular matrix components and their cellular receptors, the integrins, are important regulators of glomerular development and function. In this rapidly evolving field we studied the production of extracellular matrix components and integrins by rat glomerular visceral epithelial and mesangial cells, using molecular probes and antibodies that have recently become available. Special attention was paid to laminin isoforms and to splice variants of the integrin subunits α3 and α6. Results were compared to the in vivo expression in human fetal, newborn and adult kidneys.The mesangial cells were found to produce laminin-1, nidogen and two as yet unidentified laminin isoforms with putative α chains of about 395 (m) and of 375 kDa (cry), tentatively described before as bovine kidney laminin. Furthermore, they expressed the integrins α1β1, α2β, α3Aβ1, α5β1, αvβ3, αvβ5, and small amounts of α6Aβ1 and α6Bβ1. The glomerular visceral epithelial cells produced the two new laminin isoforms mentioned above, laminin-5, but no laminin-1 or nidogen. The integrins α2β1, αAβ1, α6Aβ4, αBβ4 and the integrin subunit av were found to be expressed.We show that during nephrogenesis, the laminin α1 chain disappears and is replaced by another a chain, possibly one of the two as yet unidentified α chains mentioned above. The laminin β1 chain is replaced by the β2 chain somewhat later in glomerular development. In general, the integrins found to be expressed in glomeruli of adult kidney were consistent with those found in cultured glomerular visceral epithelial and mesangial cells. No splice variant switch of the integrin α3 or α6 subunits could be demonstrated during nephrogenesis.Our results suggest an important role for the mesangial cell in providing nidogen as a crucial component of the supramolecular stucture of the glomerular basement membrane. Furthermore our results indicate that laminin αxβ2γ1 and αβ2γ1 isoforms are important in the glomerulus of adult kidney and that the integrin α3Aβ1 is the main integrin receptor for laminin isoforms on glomerular visceral epithelial and mesangial cells, both in vitro and in vivo.
