Post-mortem studies can provide important information for understanding new diseases and small autopsy case series have already reported different findings in COVID-19 patients.
We evaluated whether ...some specific post-mortem features are observed in these patients and if these changes are related to the presence of the virus in different organs. Complete macroscopic and microscopic autopsies were performed on different organs in 17 COVID-19 non-survivors. Presence of SARS-CoV-2 was evaluated with immunohistochemistry (IHC) in lung samples and with real-time reverse-transcription polymerase chain reaction (RT-PCR) test in the lung and other organs.
Pulmonary findings revealed early-stage diffuse alveolar damage (DAD) in 15 out of 17 patients and microthrombi in small lung arteries in 11 patients. Late-stage DAD, atypical pneumocytes, and/or acute pneumonia were also observed. Four lung infarcts, two acute myocardial infarctions, and one ischemic enteritis were observed. There was no evidence of myocarditis, hepatitis, or encephalitis. Kidney evaluation revealed the presence of hemosiderin in tubules or pigmented casts in most patients. Spongiosis and vascular congestion were the most frequently encountered brain lesions. No specific SARS-CoV-2 lesions were observed in any organ. IHC revealed positive cells with a heterogeneous distribution in the lungs of 11 of the 17 (65%) patients; RT-PCR yielded a wide distribution of SARS-CoV-2 in different tissues, with 8 patients showing viral presence in all tested organs (i.e., lung, heart, spleen, liver, colon, kidney, and brain).
In conclusion, autopsies revealed a great heterogeneity of COVID-19-associated organ injury and the remarkable absence of any specific viral lesions, even when RT-PCR identified the presence of the virus in many organs.
The T cell integrin receptor LFA-1 orchestrates adhesion between T cells and antigen-presenting cells (APCs), resulting in formation of a contact zone known as the immune synapse (IS) which is ...supported by the cytoskeleton. L-plastin is a leukocyte-specific actin bundling protein that rapidly redistributes to the immune synapse following T cell–APC engagement. We used single domain antibodies (nanobodies, derived from camelid heavy-chain only antibodies) directed against functional and structural modules of L-plastin to investigate its contribution to formation of an immune synapse between Raji cells and human peripheral blood mononuclear cells or Jurkat T cells. Nanobodies that interact either with the EF hands or the actin binding domains of L-plastin both trapped L-plastin in an inactive conformation, causing perturbation of IS formation, MTOC docking towards the plasma membrane, T cell proliferation and IL-2 secretion. Both nanobodies delayed Ser
5
phosphorylation of L-plastin which is required for enhanced bundling activity. Moreover, one nanobody delayed LFA-1 phosphorylation, reduced the association between LFA-1 and L-plastin and prevented LFA-1 enrichment at the IS. Our findings reveal subtle mechanistic details that are difficult to attain by conventional means and show that L-plastin contributes to immune synapse formation at distinct echelons.
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EMUNI, NUK, SBMB, SBNM, UL, UM, UPUK
Adult stem cells (SCs) are at high risk of accumulating deleterious mutations because they reside and self-renew in adult tissues for extended periods. Little is known about how adult SCs sense and ...respond to DNA damage within their natural niche. Here, using mouse epidermis as a model, we define the functional consequences and the molecular mechanisms by which adult SCs respond to DNA damage. We show that multipotent hair-follicle-bulge SCs have two important mechanisms for increasing their resistance to DNA-damage-induced cell death: higher expression of the anti-apoptotic gene Bcl-2 and transient stabilization of p53 after DNA damage in bulge SCs. The attenuated p53 activation is the consequence of a faster DNA repair activity, mediated by a higher non-homologous end joining (NHEJ) activity, induced by the key protein DNA-PK. Because NHEJ is an error-prone mechanism, this novel characteristic of adult SCs may have important implications in cancer development and ageing.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Treatment with pembrolizumab, an anti-programmed cell death-1 (PDCD-1) monoclonal antibody for the treatment of non-small cell lung cancers (NSCLCs) requires prior immunohistochemical (IHC) analysis ...of the expression of the programmed death-ligand 1 (PD-L1) (also known as CD274 molecule) which is a heterogeneous and complex marker. The present study aimed to investigate how pathological and technical factors (such as tumor location and sampling type, respectively) may affect the PD-L1 evaluation in patients with NSCLC in the daily practice of pathology laboratories. The current study was retrospective, and included 454 patients with NSCLC, for whom PD-L1 expression analysis by IHC was prospectively performed between November 2016 and January 2018. The association between PD-L1 expression and the clinicopathological characteristics of patients was statistically investigated using either the χ
and Fisher exact tests or the Mann-Whitney and Kruskal-Wallis tests, depending on whether PD-L1 expression was assessed in three large categories (<1, 1-49, ≥50%) or in more precise percentages. Furthermore, the same statistical methodology was used to analyze the heterogeneity of PD-L1 expression according to its sampling type (cytology, biopsy or surgical specimen) and its location (primary tumor, lymph node or distant metastasis). Intra- and inter-observer discrepancies were also studied using double-blind evaluation and concordance analyses based on the weighted κ coefficient. The results demonstrated a significant association between PD-L1 expression and sample location (P=0.005), histological type (P=0.026), total number of mutations (P=0.004) and KRAS proto-oncogene, GTPase mutations (P=0.024). In addition, sampling type did not influence PD-L1 expression. The inter- and intra-observer discrepancies were 15% and between 16 and 17.5%, respectively. The present study confirmed that evaluation of PD-L1 expression by IHC can be performed on all types of samples. In addition, the results from the current study highlighted the heterogeneity of PD-L1 expression among the different types of sample location. In complex cases, a second evaluation of PD-L1 expression by IHC would be performed due to intra- and inter-observer discrepancies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The Mdm2 and Mdm4 oncoproteins are key negative regulators of the p53 tumor suppressor. However, their physiological contributions to the regulation of p53 stability and activity remain highly ...controversial. Here, we combined a p53 knock-in allele, in which p53 is silenced by a transcriptional stop element flanked by loxP sites, with the mdm2-and mdm4-null alleles. This approach allows Cre-mediated conditional p53 expression in tissues in vivo and cells in vitro lacking Mdm2, Mdm4, or both. Using this strategy, we show that Mdm2 and Mdm4 are essential in a nonredundant manner for preventing p53 activity in the same cell type, irrespective of the proliferation/differentiation status of the cells. Although Mdm2 prevents accumulation of the p53 protein, Mdm4 contributes to the overall inhibition of p53 activity independent of Mdm2. We propose a model in which Mdm2 is critical for the regulation of p53 levels and Mdm4 is critical for the fine-tuning of p53 transcriptional activity, both proteins acting synergistically to keep p53 in check.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Dmrt
genes encode a large family of transcription factors characterized by the presence of a DM domain, an unusual zinc finger DNA binding domain. While
Dmrt
genes are well known for their important ...role in sexual development in arthropodes, nematodes and vertebrates, several new findings indicate emerging functions of this gene family in other developmental processes. Here, we provide an overview of the evolution, structure and mechanisms of action of
Dmrt
genes. We summarize recent findings on their function in sexual regulation and discuss more extensively the role played by these proteins in somitogenesis and neural development.
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EMUNI, NUK, SBMB, SBNM, UL, UM, UPUK
Mdm2 and Mdm4 are critical negative regulators of the p53 tumor suppressor. Mdm4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This ...phenotype may indicate a cell-intrinsic function of Mdm4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. Here, we further explore the tissue-specific functions of Mdm2 and Mdm4 in the erythroid lineage by intercrossing conditional Mdm4 and Mdm2 alleles to an erythroid-specific Cre (Er-GFP-Cre) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, Mdm4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, Mdm4 only contributes to p53 regulation at a specific phase of the differentiation program.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Podosomes are cellular structures acting as degradation 'hot-spots' in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, ...including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract only
574
Background: Gene fusions involving one of the 3 neurotrophic tyrosine receptor kinases ( NTRK) have been identified in approximately 1% of solid tumors and inhibitors of TRK (e.g. ...larotrectinib) have been shown to have anti-tumor activity regardless of tumor type. NTRK gene fusions have been previously reported in bilio-pancreatic cancers. It is of interest therefore to determine the incidence and molecular characteristics of NTRK gene fusions in patients with bilio-pancreatic cancers. Methods: Formalin-fixed paraffin-embedded archival blocks from surgical resections, biopsies or cytological samples of biliary tract tumors including intra-hepatic cholangiocarcinoma (IH), extra-hepatic cholangiocarcinoma (EH), perihilar cholangiocarcinoma (PH) and gallbladder tumors (G) were selected/retrieved from the tumor bank of the CUB Hôpital Erasme between January 2010 and July 2019. A two-step diagnostic method incorporating immunohistochemistry (IHC) screening followed by NGS analysis was used. Pan–TRK IHC (monoclonal antibody clone EPR17341 AbCam, Cambridge, MA) was used for the screening method. Staining intensity (negative, weak, moderate or strong) and localization (cytoplasmic or nuclear) were evaluated. The presence of at least weak staining tumor cells led to testing by a RNA-based NGS panel (Oncomine Focus Assay, ThermoFisher scientific). Results: 145 archival tumors samples (81 surgical resections, 48 biopsies and 16 cytology) have been selected, including 61 IH, 32 PH, 26 EH and 26 G (67 female and 78 male). 134 samples were suitable to perform IHC. 17 samples were IHC positive. Intensity of staining was weak in 16 samples and moderate in one. Staining location was cytoplasmic (14/17), nuclear (2/17), and nuclear+cytoplasmic (1/17). NGS testing of the 17 IHC positive samples revealed a single NTRK3 gene fusion ( ETV6(4)- NTRK3(14)). In this case (female patient with a poorly differentiated PH, deceased), IHC had a weak focal cytoplasmic and nuclear staining. Overall in the patients screened by IHC and confirmed by NGS, the percentage of NTRK fusions was 0.75%. Conclusions: NTRK gene fusions are rare in biliary cancers but testing is of high interest due to the emergence of possible treatment with specific TRK inhibitors.
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