Histopathology and genetic susceptibility in COVID-19 pneumonia

The clinical features of COVID-19 range from a mild illness to patients with a very severe illness with acute hypoxemic respiratory failure requiring ventilation and Intensive Care Unit admission. Risk factors for a fatal disease include older age, respiratory disease, diabetes mellitus, obesity and hypertension. Little is known about the mechanisms behind observed episodes of sudden deterioration or the infrequent idiosyncratic clinical demise in otherwise healthy and young subjects. As in other diseases, the answer to some of these questions may in time be provided by genotyping as well careful clinical, serological, radiological and histopathological phenotyping, which enable mechanistic insights into the differences in pathogenesis and underlying immunological and tissue regenerative response patterns. We will aim to provide a brief overview of the existing evidence for such differences in host response and outcome, and generate hypotheses for divergent patterns and avenues for future research, by highlighting similarities and differences in histopathological appearance between COVID19 and influenza as well as previous coronavirus outbreaks, and by discussing predisposition through genetics and underlying disease

Tumor mutational load, CD8+ T cells, expression of PD-L1 and HLA class I to guide immunotherapy decisions in NSCLC patients

Objectives: A minority of NSCLC patients benefit from anti-PD1 immune checkpoint inhibitors. A rational combination of biomarkers is needed. The objective was to determine the predictive value of tumor mutational load (TML), CD8+ T cell infiltration, HLA class-I and PD-L1 expression in the tumor. Materials and methods: Metastatic NSCLC patients were prospectively included in an immune-monitoring trial (NTR7015) between April 2016-August 2017, retrospectively analyzed in FFPE tissue for TML (NGS: 409 cancer-related-genes) and by IHC staining to score PD-L1, CD8+ T cell infiltration, HLA class-I. PFS (RECISTv1.1) and OS were analyzed by Kaplan–Meier methodology. Results: 30 patients with adenocarcinoma (67%) or squamous cell carcinoma (33%) were included. High TML was associated with better PFS (p = 0.004) and OS (p = 0.025). Interaction analyses revealed that patients with both high TML and high total CD8+ T cell infiltrate (p = 0.023) or no loss of HLA class-I (p = 0.026), patients with high total CD8+ T cell infiltrate and no loss of HLA class-I (p = 0.041) or patients with both high PD-L1 and high TML (p = 0.003) or no loss of HLA class-I (p = 0.032) were significantly associated with better PFS. Unsupervised cluster analysis based on these markers revealed three sub-clusters, of which cluster-1A was overrepresented by patients with progressive disease (15 out of 16), with significant effect on PFS (p = 0.007). Conclusion: This proof-of-concept study suggests that a combination of PD-L1 expression, TML, CD8+ T cell infiltration and HLA class-I functions as a better predictive biomarker for response to anti-PD-1 immunotherapy. Consequently, refinement of this set of biomarkers and validation in a larger set of patients is warranted

Copper-Heparin Inhalation Therapy To Repair Emphysema: A Scientific Rationale

Current pharmacotherapy of chronic obstructive pulmonary disease (COPD) aims at reducing respiratory symptoms and exacerbation frequency. Effective therapies to reduce disease progression, however, are still lacking. Furthermore, COPD medications showed less favorable effects in emphysema than in other COPD phenotypes. Elastin fibers are reduced and disrupted, whereas collagen levels are increased in emphysematous lungs. Protease/antiprotease imbalance has historically been regarded as the sole cause of emphysema. However, it is nowadays appreciated that emphysema may also be provoked by perturbations in the sequential repair steps following elastolysis. Essentiality of fibulin-5 and lysyl oxidase-like 1 in the elastin restoration process is discussed, and it is argued that copper deficiency is a plausible reason for failing elastin repair in emphysema patients. Since copper-dependent lysyl oxidases crosslink elastin as well as collagen fibers, copper supplementation stimulates accumulation of both proteins in the extracellular matrix. Restoration of abnormal elastin fibers in emphysematous lungs is favorable, whereas stimulating pulmonary fibrosis formation by further increasing collagen concentrations and organization is detrimental. Heparin inhibits collagen crosslinking while stimulating elastin repair and might therefore be the ideal companion of copper for emphysema patients. Efficacy and safety considerations may lead to a preference of pulmonary administration of copper-heparin over systemic administration

CD16+ monocytes and skewed macrophage polarization toward M2 type hallmark heart transplant acute cellular rejection

Background: During acute heart transplant rejection, infiltration of lymphocytes and monocytes is followed by endothelial injury and eventually myocardial fibrosis. To date, no information is available on monocyte-macrophage-related cellular shifts and their polarization status during rejection. Here, we aimed to define and correlate monocyte-macrophage endomyocardial tissue profiles obtained at rejection and time points prior to rejection, with corresponding serial blood samples in 25 heart transplant recipients experiencing acute cellular rejection. Additionally, 33 healthy individuals served as control. Materials and methods: Using histology, immunohistochemistry, confocal laser scan microscopy, and digital imaging expression of CD14, CD16, CD56, CD68, CD80, and CD163 were explored to define monocyte and macrophage tissue profiles during rejection. Fibrosis was investigated using Sirius Red stainings of rejection, non-rejection, and 1-year biopsies. Expression of co-stimulatory and migration-related molecules on circulating monocytes, and production potential for pro- and anti-inflammatory cytokines were studied using flow cytometry. Results: At tissue level, striking CD16+ monocyte infiltration was observed during rejection (p < 0.001). Significantly more CD68+CD163+ M2 macrophages were documented during rejection compared to barely present CD68+CD80+ M1 macrophages. Rejection was associated with severe fibrosis in 1-year biopsies (p < 0.001). Irrespective of rejection status, decreased frequencies of circulating CD16+ monocytes were found in patients compared to healthy individuals. Rejection was reflected by significantly increased CD54 and HLA-DR expression on CD16+ monocytes with retained cytokine production potential. Conclusion: CD16+ monocytes and M2 macrophages hallmark the correlates of heart transplant acute cellular rejection on tissue level and seem to be associated with fibrosis in the long term

Pharmacological targeting of protease-activated receptor 2 affords protection from bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed whether PAR-2 deficiency persistently reduces bleomycin-induced pulmonary fibrosis or merely delays disease progression and whether pharmacological PAR-2 inhibition limits experimental pulmonary fibrosis. Bleomycin was instilled intranasally into wild-type or PAR-2-deficient mice in the presence/absence of a specific PAR-2 antagonist (P2pal-18S). Pulmonary fibrosis was consistently reduced in PAR-2-deficient mice throughout the fibrotic phase, as evident from reduced Ashcroft scores (29%) and hydroxyproline levels (26%) at d 28. Moreover, P2pal-18S inhibited PAR-2-induced profibrotic responses in both murine and primary human pulmonary fibroblasts (p < 0.05). Once daily treatment with P2pal-18S reduced the severity and extent of fibrotic lesions in lungs of bleomycin-treated wild-type mice but did not further reduce fibrosis in PAR-2-deficient mice. Importantly, P2pal-18S treatment starting even 7 d after the onset of fibrosis limits pulmonary fibrosis as effectively as when treatment was started together with bleomycin instillation. Overall, PAR-2 contributes to the progression of pulmonary fibrosis, and targeting PAR-2 may be a promising therapeutic strategy for treating pulmonary fibrosis

Lack of evidence for a causal role of CALR3 in monogenic cardiomyopathy

The pathogenicity of previously published disease-associated genes and variants is sometimes questionable. Large-scale, population-based sequencing studies have uncovered numerous false assignments of pathogenicity. Misinterpretation of sequence variants may have serious implications for the patients and families involved, as genetic test results are increasingly being used in medical decision making. In this study, we assessed the role of the calreticulin-3 gene (CALR3) in cardiomyopathy. CALR3 has been included in several cardiomyopathy gene panels worldwide. Its inclusion is based on a single publication describing two missense variants in patients with hypertrophic cardiomyopathy. In our national cardiomyopathy cohort (n = 6154), we identified 17 unique, rare heterozygous CALR3 variants in 48 probands. Overall, our patient cohort contained a significantly higher number of rare CALR3 variants compared to the ExAC population (p = 0.0036). However, after removing a potential Dutch founder variant, no statistically significant difference was found (p = 0.89). In nine probands, the CALR3 variant was accompanied by a disease-causing variant in another, well-known cardiomyopathy gene. In three families, the CALR3 variant did not segregate with the disease. Furthermore, we could not demonstrate calreticulin-3 protein expression in myocardial tissues at various ages. On the basis of these findings, it seems highly questionable that variants in CALR3 are a monogenic cause of cardiomyopathy

Defective Connective Tissue Remodeling in Smad3 Mice Leads to Accelerated Aneurysmal Growth Through Disturbed Downstream TGF-β Signaling

Aneurysm-osteoarthritis syndrome characterized by unpredictable aortic aneurysm formation, is caused by SMAD3 mutations. SMAD3 is part of the SMAD2/3/4 transcription factor, essential for TGF-β-activated transcription. Although TGF-β-related gene mutations result in aneurysms, the underlying mechanism is unknown. Here, we examined aneurysm formation and progression in Smad3−/− animals. Smad3−/− animals developed aortic aneurysms rapidly, resulting in premature death. Aortic wall immunohistochemistry showed no increase in extracellular matrix and collagen accumulation, nor loss of vascular smooth muscle cells (VSMCs) but instead revealed medial elastin disruption and adventitial inflammation. Remarkably, matrix metalloproteases (MMPs) were not activated in VSMCs, but rather specifically in inflammatory areas. Although Smad3−/− aortas showed increased nuclear pSmad2 and pErk, indicating TGF-β receptor activation, downstream TGF-β-activated target genes were not upregulated. Increased pSmad2 and pErk staining in pre-aneurysmal Smad3−/− aortas implied that aortic damage and TGF-β receptor-activated signaling precede aortic inflammation. Finally, impaired downstream TGF-β activated transcription resulted in increased Smad3−/− VSMC proliferation. Smad3 deficiency leads to imbalanced activation of downstream genes, no activation of MMPs in VSMCs, and immune responses resulting in rapid aortic wall dilatation and rupture. Our findings uncover new possibil