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Summary: The utilization of single-cell resolved spatial transcriptomics to delineate immune responses during SARS-CoV-2 infection was able to identify M1 macrophages to have elevated expression of IFI27 in areas of infection.

Abstract Objectives Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a ‘preeclampsia‐like syndrome’. However, the mechanisms underpinning SARS‐CoV‐2‐induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS‐CoV‐2 infection. Methods We utilised whole‐transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS‐CoV‐2 in the third trimester of their pregnancy (n = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID‐19) pandemic (n = 9). Results Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS‐CoV‐2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH) and preeclampsia (FLT1, EGFR, KISS1, PAPPA2) were enriched with SARS‐CoV‐2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS‐CoV‐2 samples compared to uninfected controls. Conclusions Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS‐CoV‐2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS‐CoV‐2 infection.

Abstract Background The cytokines play an essential role in acute inflammatory processes, and the IL-17 may be responsible for ambiguous aspects, and the correlation with genetic polymorphisms could improve the search for this critical biomarker. Thus, this study aimed to evaluate the IL-17A and IL-17RA tissue expression and the polymorphisms that codified these proteins in a population that died of pandemic Influenza A virus H1N1 subtype compared to a non-pandemic Influenza virus population. Methods Necropsy lung samples immunohistochemistry was performed to assess the presence of IL-17A and IL-17RA in the pulmonary tissue. Eight single nucleotide polymorphisms were genotyped using TaqMan® technology. Results The Influenza A H1N1 pandemic group had higher tissue expression of IL-17A, higher neutrophil recruitment and shorter survival time between admission and death. Three single nucleotide polymorphisms conferred risk for pandemic influenza A H1N1, the AA genotype of rs3819025 G/A, the CC genotype of rs2241044 A/C, and the TT genotype of rs 2,241,043 C/T. Conclusions One IL17A polymorphism (rs381905) and two IL17RA polymorphisms (rs2241044 and rs2241043) represented biomarkers of worse prognosis in the population infected with pandemic influenza A H1N1. The greater tissue expression of IL-17A shows a Th17 polarization and highlights the aggressiveness of the pandemic influenza virus with its duality in the protection and pathogenesis of the pulmonary infectious process.

Abstract Fanconi anemia is a rare autosomal recessive disease. In this disease, cytokine pathways can induce the bone marrow failure that is observed in individuals with Fanconi anemia. Interleukin IL-17 exhibits a protective effect in organisms because it induces neutrophil recruitment and shows a pathological role in several models of autoimmune diseases, periodontal disease, cancer, allograft rejection, and graft versus host disease. Polymorphisms in the IL17A and IL17RA genes were evaluated from DNA in saliva, comparing individuals with or without Fanconi anemia, using models of genotypic transmission (additive, dominant, and recessive). Polymorphisms in the IL17A and IL17RA genes (rs2241044 [C allele], rs879577 [C allele], rs9606615 [T allele], and rs2241043 [C allele]) were risk factors for developing Fanconi anemia. We also performed an analysis of gene markers with clinical variables in the Fanconi group. Polymorphisms in the IL17A gene (rs3819025 [A allele] and rs2275913 [G allele], respectively) were associated with an age of less than 20 years (p = 0.026; RP 0.65) and the female sex (p = 0.043; RP 0.88). The IL17RA gene was also associated with age and the presence of leukoplakia (a potentially malignant oral disorder). An age of less than 20 years was associated with rs917864 (T allele; p = 0.036; RP 0.67). The presence of leukoplakia was associated with rs17606615 (T allele; p = 0.042; RP 0.47). To our knowledge, this is the first study that associates IL17A and IL17RA gene polymorphisms with Fanconi anemia and examines rs2241044 polymorphisms in scientific literature thus far.

Central nervous system tumors, especially astrocytomas, are the solid neoplasms with the highest incidence and mortality rates in childhood. The diagnosis is based on histopathological characteristics, but molecular methods have been increasingly used. Translationally controlled tumor protein (TCTP) protein, encoded by the tumor protein, translationally controlled 1 ( TPT1 ) gene, is a multifunctional protein with an important physiological role in the cell cycle. Expression of this protein has been associated with several neoplasms, including astrocytomas in adults. However, the role of this protein in pediatric astrocytomas is largely unknown. We aim to evaluate in cases of pediatric astrocytomas, the frequency of polymorphisms in the TPT1 gene and other genes associated with its molecular pathways, such as MTOR , MDM2 , TP53 , and CDKN1A , correlating it with protein expression and clinical variables, in formalin-fixed, paraffin-embedded (FFPE) samples. These samples were submitted to genotyping and immunohistochemistry analyses. The most revealing results refer to the MDM2 gene, rs117039649 [G/C], in which C polymorphic allele was observed only in the glioblastomas ( p = .028). The CDKN1A gene, rs3176334 [T/C] presented a homozygous polymorphic genotype only in high-grade astrocytomas, when infiltrating tumors were compared ( p = .039). The immunohistochemical expression of cytoplasmic MDM2 correlated with better survival rates in patients with glioblastoma ( p = .018). The presence of polymorphisms in the MDM2 and CDKN1A genes, as well as a specific correlation between MDM2 expression, suggests a likely association with risk in pediatric astrocytomas. This study sought the probable role involved in the TCTP pathway, and associated proteins, in the tumorigenesis of pediatric astrocytomas, and some could have potential impact as prognostic markers in these patients.

Abstract Parkin, a protein encoded by PRKN, discovered in the context of Parkinson’s disease, controls proteasomal degradation by protein ubiquitination and acts on cell cycle control and mitochondrial homeostasis, among other cellular processes. Parkin has been also implicated in several carcinomas, melanoma and leukemia. In the neoplastic setting, reduced parkin level usually indicates poorer prognosis. Some authors have described the associations between parkin and gliomas. Gliomas are a heterogeneous group of tumors that arise in the central nervous system, astrocytomas being the most common. The aim of this systematic review is to evaluate how parkin behaves in gliomas and the molecular pathways associated in this interaction. A search was conducted in PubMed, EBSCO and Scopus and 8 published articles were identified as eligible studies. The studies were categorized in three groups, according to their main emphasis: PRKN mutation patterns detected in gliomas, parkin effects on tumor growth and survival rates, and molecular interactions between parkin and other proteins. The studies showed higher PRKN mutation rates and lower parkin expression in high grade gliomas. Patients with higher parkin expression had better overall survival. Besides, different molecular pathways associated with parkin were described, some of them regarded as potential therapeutic targets.

Objective: Adaptive changes in DHEA and sulfated-DHEA (DHEAS) production from adrenal zona reticularis (ZR) have been observed in normal and pathological conditions. Here we used three different cohorts to assess timing differences in DHEAS blood level changes and characterize the relationship between early blood DHEAS reduction and cell number changes in women ZR. Materials and methods: DHEAS plasma samples (n = 463) were analyzed in 166 healthy prepubertal girls before pubarche (35% of the females >40 years old and associated with significantly reduced ZR cell number (ba sed on PTEN and hematoxylin signals). ZR cell loss may in part account for lower DHEA/DHEAS expression, but most cells remain alive with lower DHEA/DHEAS biosynthesis. Conclusion: The timely relation between significant reduction of blood DHEAS levels and decreased ZR cell number at the beginning of the 40s suggests that adrenopause is an additional burden for a significant number of middle-aged women, and may become an emergent problem associated with further sex steroids reduction during the menopausal transition.

The COVID-19 pandemic, promoted by the SARS-CoV-2 respiratory virus, has resulted in widespread global morbidity and mortality. The immune response against this pathogen has shown a thin line between protective effects and pathological reactions resulting from the massive release of cytokines and poor viral clearance. The latter is possibly caused by exhaustion, senescence, or both of TCD8+ cells and reduced activity of natural killer (NK) cells. The imbalance between innate and adaptive responses during the early stages of infection caused by SARS-CoV-2 contributes to the ineffective control of viral spread. The present study evaluated the tissue immunoexpression of the tissue biomarkers (Arginase-1, CCR4, CD3, CD4, CD8, CD20, CD57, CD68, CD138, IL-4, INF-α, INF-γ, iNOS, PD-1, Perforin and Sphingosine-1) to understand the cellular immune response triggered in patients who died of COVID-19. We evaluated twenty-four paraffin-embedded lung tissue samples from patients who died of COVID-19 (COVID-19 group) and compared them with ten lung tissue samples from patients who died of H1N1pdm09 (H1N1 group) with the immunohistochemical markers mentioned above. In addition, polymorphisms in the Perforin gene were genotyped through Real-Time PCR. Significantly increased tissue immunoexpression of Arginase, CD4, CD68, CD138, Perforin, Sphingosine-1, and IL-4 markers were observed in the COVID-19 group. A significantly lower immunoexpression of CD8 and CD57 was also found in this group. It is suggested that patients who died from COVID-19 had a poor cellular response concerning viral clearance and adaptive response going through tissue repair.

The inflammasome complex is a key part of chronic diseases and acute infections, being responsible for cytokine release and cell death mechanism regulation. The SARS-CoV-2 infection is characterized by a dysregulated cytokine release. In this context, the inflammasome complex analysis within SARS-CoV-2 infection may prove beneficial to understand the disease’s mechanisms. Post-mortem minimally invasive autopsies were performed in patients who died from COVID-19 (n = 24), and lung samples were compared to a patient control group (n = 11) and an Influenza A virus H1N1 subtype group from the 2009 pandemics (n = 10). Histological analysis was performed using hematoxylin-eosin staining. Immunohistochemical (IHC) staining was performed using monoclonal antibodies against targets: ACE2, TLR4, NF-κB, NLRP-3 (or NALP), IL-1β, IL-18, ASC, CASP1, CASP9, GSDMD, NOX4, TNF-α. Data obtained from digital analysis underwent appropriate statistical tests. IHC analysis showed biomarkers that indicate inflammasome activation (ACE2; NF-κB; NOX4; ASC) were significantly increased in the COVID-19 group (p < 0.05 for all) and biomarkers that indicate cell pyroptosis and inflammasome derived cytokines such as IL-18 (p < 0.005) and CASP1 were greatly increased (p < 0.0001) even when compared to the H1N1 group. We propose that the SARS-CoV-2 pathogenesis is connected to the inflammasome complex activation. Further studies are still warranted to elucidate the pathophysiology of the disease.

Since the beginning of the pandemic, few papers describe the placenta’s morphological and morphometrical features in SARS-CoV-2–positive pregnant women. Alterations, such as low placental weight, accelerated villous maturation, decidual vasculopathy, infarcts, thrombosis of fetal placental vessels, and chronic histiocytic intervillositis (CHI), have been described.ObjectiveTo analyze clinical data and the placental morphological and morphometric changes of pregnant women infected with SARS-CoV-2 (COVID-19 group) in comparison with the placentas of non-infected pregnant women, matched for maternal age and comorbidities, besides gestational age of delivery (Control group).MethodThe patients in the COVID-19 and the Control group were matched for maternal age, gestational age, and comorbidities. The morphological analysis of placentas was performed using Amsterdam Placental Workshop Group Consensus Statement. The quantitative morphometric evaluation included perimeter diameter and number of tertiary villi, number of sprouts and knots, evaluation of deposition of villous fibrin, and deposition of intra-villous collagen I and III by Sirius Red. Additionally, Hofbauer cells (HC) were counted within villi by immunohistochemistry with CD68 marker.ResultsCompared to controls, symptomatic women in the COVID-19 group were more likely to have at least one comorbidity, to evolve to preterm labor and infant death, and to have positive SARS-CoV-2 RNA testing in their concepts. Compared to controls, placentas in the COVID-19 group were more likely to show features of maternal and fetal vascular malperfusion. In the COVID-19 group, placentas of symptomatic women were more likely to show CHI. No significant results were found after morphometric analysis.ConclusionPregnant women with symptomatic SARS-CoV-2 infection, particularly with the severe course, are more likely to exhibit an adverse fetal outcome, with slightly more frequent histopathologic findings of maternal and fetal vascular malperfusion, and CHI. The morphometric changes found in the placentas of the COVID-19 group do not seem to be different from those observed in the Control group, as far as maternal age, gestational age, and comorbidities are paired. Only the deposition of villous fibrin could be more accentuated in the COVID-19 group (p = 0.08 borderline). The number of HC/villous evaluated with CD68 immunohistochemistry did not show a difference between both groups.

COVID-19 is a viral disease associated with an intense inflammatory response. Macrophage Activation Syndrome (MAS), the complication present in secondary hemophagocytic lymphohistiocytosis (sHLH), shares many clinical aspects observed in COVID-19 patients, and investigating the cytolytic function of the responsible cells for the first line of the immune response is important. Formalin-fixed paraffin-embedded lung tissue samples obtained by post mortem necropsy were accessed for three groups (COVID-19, H1N1, and CONTROL). Polymorphisms in MAS cytolytic pathway (PRF1; STX11; STXBP2; UNC13D and GZMB) were selected and genotyping by TaqMan® assays (Thermo Fisher Scientific, MA, USA) using Real-Time PCR (Applied Biosystems, MA USA). Moreover, immunohistochemistry staining was performed with a monoclonal antibody against perforin, CD8+ and CD57+ proteins. Histopathological analysis showed high perforin tissue expression in the COVID-19 group; CD8+ was high in the H1N1 group and CD57+ in the CONTROL group. An association could be observed in two genes related to the cytolytic pathway (PRF1 rs885822 G/A and STXBP2 rs2303115 G/A). Furthermore, PRF1 rs350947132 was associated with increased immune tissue expression for perforin in the COVID-19 group. The genotype approach could help identify patients that are more susceptible, and for this reason, our results showed that perforin and SNPs in the PRF1 gene can be involved in this critical pathway in the context of COVID-19.

The new SARS-CoV-2 virus differs from the pandemic Influenza A virus H1N1 subtype (H1N1pmd09) how it induces a pro-inflammatory response in infected patients. This study aims to evaluate the involvement of SNPs and tissue expression of IL-17A and the neutrophils recruitment in post-mortem lung samples from patients who died of severe forms of COVID-19 comparing to those who died by H1N1pdm09. Twenty lung samples from patients SARS-CoV-2 infected (COVID-19 group) and 10 lung samples from adults who died from a severe respiratory H1N1pdm09 infection (H1N1 group) were tested. The tissue expression of IL-8/IL-17A was identified by immunohistochemistry, and hematoxylin and eosin (H&E) stain slides were used for neutrophil scoring. DNA was extracted from paraffin blocks, and genotyping was done in real time-PCR for two IL17A target polymorphisms. Tissue expression increasing of IL-8/IL-17A and a higher number of neutrophils were identified in samples from the H1N1 group compared to the COVID-19 group. The distribution of genotype frequencies in the IL17A gene was not statistically significant between groups. However, the G allele (GG and GA) of rs3819025 was correlated with higher tissue expression of IL-17A in the COVID-19 group. SARS-CoV-2 virus evokes an exacerbated response of the host’s immune system but differs from that observed in the H1N1pdm09 infection since the IL-8/IL-17A tissue expression, and lung neutrophilic recruitment may be decreased. In SNP rs3819025 (G/A), the G allele may be considered a risk allele in the patients who died for COVID-19.

Molar incisor hypomineralization (MIH) is an enamel condition characterized by lesions ranging in color from white to brown which present rapid caries progression, and mainly affects permanent first molars and incisors. These enamel defects usually occur when there are disturbances during the mineralization or maturation stage of amelogenesis. Both genetic and environmental factors have been suggested to play roles in MIH's development, but no conclusive risk factors have shown the source of the disease. During head and neck development, the interferon regulatory factor 6 (IRF6) gene is involved in the structure formation of the oral and maxillofacial regions, and the transforming growth factor alpha (TGFA) is an essential cell regulator, acting during proliferation, differentiation, migration and apoptosis. In this present study, it was hypothesized that these genes interact and contribute to predisposition of MIH. Environmental factors affecting children that were 3 years of age or older were also hypothesized to play a role in the disease etiology. Those factors included respiratory issues, malnutrition, food intolerance, infection of any sort and medication intake. A total of 1,065 salivary samples from four different cohorts were obtained, and DNA was extracted from each sample and genotyped for nine different single nucleotide polymorphisms. Association tests and logistic regression implemented in PLINK were used for analyses. A potential interaction between TGFA rs930655 with all markers tested in the cohort from Turkey was identified. These interactions were not identified in the remaining cohorts. Associations (p

Mast cells (MCs) have relevant participation in inflammatory and vascular hyperpermeability events, responsible for the action of the kallikrein–kinin system (KKS), that affect patients inflicted by the severe form of COVID-19. Given a higher number of activated MCs present in COVID-19 patients and their association with vascular hyperpermeability events, we investigated the factors that lead to the activation and degranulation of these cells and their harmful effects on the alveolar septum environment provided by the action of its mediators. Therefore, the pyroptotic processes throughout caspase-1 (CASP-1) and alarmin interleukin-33 (IL-33) secretion were investigated, along with the immunoexpression of angiotensin-converting enzyme 2 (ACE2), bradykinin receptor B1 (B1R) and bradykinin receptor B2 (B2R) on post-mortem lung samples from 24 patients affected by COVID-19. The results were compared to 10 patients affected by H1N1pdm09 and 11 control patients. As a result of the inflammatory processes induced by SARS-CoV-2, the activation by immunoglobulin E (IgE) and degranulation of tryptase, as well as Toluidine Blue metachromatic (TB)-stained MCs of the interstitial and perivascular regions of the same groups were also counted. An increased immunoexpression of the tissue biomarkers CASP-1, IL-33, ACE2, B1R and B2R was observed in the alveolar septum of the COVID-19 patients, associated with a higher density of IgE+ MCs, tryptase+ MCs and TB-stained MCs, in addition to the presence of intra-alveolar edema. These findings suggest the direct correlation of MCs with vascular hyperpermeability, edema and diffuse alveolar damage (DAD) events that affect patients with a severe form of this disease. The role of KKS activation in events involving the exacerbated increase in vascular permeability and its direct link with the conditions that precede intra-alveolar edema, and the consequent DAD, is evidenced. Therapy with drugs that inhibit the activation/degranulation of MCs can prevent the worsening of the prognosis and provide a better outcome for the patient.