Immunity, Inflammation and Disease

Loss and changes in taste and smell are well-reported symptoms of SARS-CoV-2 infection. The virus targets cells for entry by high affinity binding of its spike protein to cell-surface angiotensin-converting enzyme-2 (ACE2). It was not known whether ACE2 is expressed on taste receptor cells (TRCs) nor if TRCs are infected directly. Using an in-situ hybridization (ISH) probe and an antibody specific to ACE2, it seems evident that ACE2 is present on a subpopulation of specialized TRCs, namely, PLC{beta}2 positive, Type II cells in taste buds in taste papillae. Fungiform papillae (FP) of a SARS-CoV-2+ patient exhibiting symptoms of COVID-19, including taste changes, were biopsied. Based on ISH, replicating SARS-CoV-2 was present in Type II cells of this patient. Therefore, taste Type II cells provide a portal for viral entry that predicts vulnerabilities to SARS-CoV-2 in the oral cavity. The continuity and cell turnover of the FP taste stem cell layer of the patient were disrupted during infection and had not fully recovered 6 weeks post symptom onset. Another patient suffering post-COVID-19 taste disturbances also had disrupted stem cells. These results indicate that a COVID-19 patient who experienced taste changes had replicating virus in their taste buds and that SARS-CoV-2 infection results in deficient stem cell turnover needed for differentiation into TRCs.

ContextMolecular alterations in premalignant lesions of oral cavity are not well known, many reports and have found increased HER2 expression to be correlated with poor prognosis in oral cancer. However, literature on expression of HER2 in premalignant lesions is limited and data is conflicting in nature. Overexpression of HER2 in premalignant lesions may denote its positive contribution in malignant transformation of these lesions. AimsTo evaluate the expression of HER2 in premalignant lesions of oral cavity. Settings and DesignIn this prospective observational study of 2 months, patients attending OPD at Department of ENT and meeting the inclusion criteria were included. Methods and Material23 samples of Leukoplakia and 1 sample of oral lichen planus were stained by routine H&E to confirm clinical diagnosis and assess dysplasia if any, 5 samples of normal mucosa were used as control. Immunohistochemical staining for HER2 was done. ASCO/CAP 2018 guidelines were used for reporting the results. Statistical analysis usedPercentage of lesions expressing cytoplasmic or membranous expression was calculated. Results1 sample of Leukoplakia with severe dysplasia expressed focal membranous staining. 20% leukoplakia lesions expressed cytoplasmic staining. Staining was not observed in oral lichen planus and leucoplakia without dysplasia. ConclusionsMembranous expression in Severe dysplasia and higher expression in oral cancer is in concordance with the multistep theory of carcinogenesis. Larger studies are needed if HER2 is to be proposed as a marker for oral premalignant lesions. Significance of cytoplasmic staining in oral premalignant lesions needs to be elucidated. Key MessagesTo the best of our knowledge, this is the first report of focal membranous expression of HER2 in leucoplakia in India. 20% leucoplakia with dysplasia expressed cytoplasmic staining. The significance of cytoplasmic staining needs to be further explored.

Background Leading to a sustained epidemic spread with >40,000 confirmed human infections, including >10,000 deaths, COVID-19 was caused by 2019-nCov and resulted in acute respiratory distress syndrome (ARDS) and sepsis, which brought more challenges to the patient’s treatment. The S-glycoprotein, which recognized as the key factor for the entry of 2019-nCov into the cell, contains two functional domains: an ACE2 receptor binding domain and a second domain necessary for fusion of the coronavirus and cell membranes. FURIN activity, exposes the binding and fusion domains, is essential for the zoonotic transmission of 2019-nCov. Moreover, it has been reported that ACE2 is likely to be the receptor for 2019-nCoV. In addition, FURIN enzyme and ACE2 receptor were expressed in airway epithelia, cardiac tissue, and enteric canals, which considered as the potential target organ of the virus. However, report about the expression of FURIN and ACE2 in oral tissues was limited.Methods In order to investigate the potential infective channel of new coronavirus in oral cavity, we analyze the expression of ACE2 and FURIN that mediate the new coronavirus entry into host cells in oral mucosa using the public single-cell sequence datasets. Furthermore, immunohistochemical staining experiment was performed to confirm the expression of ACE2 and FURIN in the protein level.Results The bioinformatics results indicated the differential expression of ACE2 and FURIN on epithelial cells of different oral mucosal tissues and the proportion of FURIN-positive cells was obviously higher than that of ACE2-positive cells. IHC experiments revealed that both the ACE2-positive and FURIN-positive cells in the target tissues were mainly positioned in the epithelial layers, partly expressed in fibroblasts, which further confirm the bioinformatics results.Conclusions Based on these findings, we speculated that 2019-nCov could effectively invade oral mucosal cells though two possible routes: binding to the ACE2 receptor and fusion with cell membrane activated by FURIN protease. Our results indicated that oral mucosa tissues are susceptible to 2019-nCov, which provides valuable information for virus-prevention strategy in clinical care as well as daily life.Competing Interest StatementThe authors have declared no competing interest.View Full Text

BackgroundLung transplantation (LUTX) is frequently complicated by Primary Graft Dysfunction (PGD), a heterogeneous form of acute lung injury associated with multi-organ failure and rejection. We hypothesized that early, bedside plasma biomarkers could capture this biological heterogeneity, identifying phenotypes with differential clinical outcomes. MethodsThis two-year prospective single-center observational study enrolled 78 bilateral LUTX recipients. With a real-time point-of-care immunoanalyzer we measured IL-1{beta}, IL-2, IL-6, IFN-{gamma}, TNF, CCL-2, IL-15, Ferritin, and D-dimer 36 hours post-reperfusion. Outcome-agnostic Latent Profile Analysis (LPA) was applied to identify bio-signatures. PGD (grade 3) incidence and clinical outcomes were compared across classes. ResultsLPA identified three distinct classes, interpreted as biological sub-phenotypes. The Adaptive phenotype (68%) had the lowest inflammatory activation, shortest vasoactive support and Invasive Mechanical Ventilation (IMV) (both 1 day) and ICU stay (3 days), and lowest 72-hours PGD (8%) and Acute Kidney Injury (AKI) (28%) rates. The Hyperinflammatory phenotype (18%) showed the highest IL-6 and Ferritin levels with resolving PGD (69% to 23%, from 6 to 72 hours), but prolonged IMV (6 days), vasoactive support (3 days), ICU stay (7 days), with a high AKI rate (69%). The Coagulopathic phenotype (15%), requiring more intraoperative blood products, exhibited the highest TNF and D-dimer with persistent PGD (36% at 24 and 72 hours), intermediate vasoactive support (2 days), and AKI severity. Six-month rejection-free survival was similar across phenotypes. ConclusionsThis preliminary hypothesis-generating study suggests that point-of-care biomarkers after LUTX may identify biological phenotypes with potential clinical relevance. Future studies are needed to confirm such phenotyping.

Antimicrobial peptides (AMPs) are essential components of the innate immune system, exhibiting diverse mechanisms of action. This study investigates the roles of cathelicidin (LL-37), alpha-defensins, and the S100 proteins S100A8 and S100A9 in systemic inflammation associated with sepsis, severe COVID-19, and acute pancreatitis using whole-blood bulk RNA-sequencing data. Gene co-expression network analysis revealed that during septic shock and severe COVID-19, cathelicidin and alpha-defensins act synergistically in innate immune responses, while S100A8 and S100A9 function through distinct pathways related to mitochondrial metabolism and ubiquitin ligase binding. In contrast, the acute pancreatitis network displayed a different pattern, with CAMP co-expressed alongside S100A8 and S100A9, whereas alpha-defensins were downregulated and associated with inhibited mucosal immune responses. These findings suggest that antimicrobial peptides contribute variably to systemic inflammation depending on the underlying insult, underscoring their complex, context-dependent roles in critical illness.

In mice, oral epithelial stem cells (OESCs) are essential for oral mucosal homeostasis and repair. Less is known regarding the role of OESCs in the human oral mucosa. Here, we studied the behaviour of OESCs and their contribution to tissue maintenance and repair in oral lichen planus (OLP). OLP is a chronic T cell-mediated disease characterized by basal keratinocyte degeneration, epithelial atrophy, acanthosis, and hyperkeratosis. Using immunohistological techniques and semi-automated image analysis, we observed that in OLP proliferative activity was increased in the normally largely quiescent basal cell compartment. In areas of OLP mucosa with intact basal cell layer, expression of NGFR, KRT15, and KRT19-markers of slowly cycling reserve OESCs, was strongly reduced or absent. In contrast, expression of CSPG4, a marker for actively cycling stem cells, was increased in OLP basal cells. Tissue compartmentalization, as evaluated by keratin expression, was strongly disturbed. Taken together, our findings indicate that the inflammation in OLP leads to activation and proliferation of OESCs that give rise to a population of cells with an aberrant differentiation programme. Along with the well-documented epithelial up-regulation of anti-apoptotic proteins in OLP, this likely reflects an attempt by the epithelium to avoid overt ulceration.

Pulmonary immunity against SARS-CoV-2 infection has not been well studied. This study investigated the distribution of immune cells int the lungs of 8 rhesus macaques experimentally infected with SARS-CoV-2, and euthanized 11-14 days later. Using immunohistochemistry, inducible bronchus-associated lymphoid tissue was found in all animals. The inducible bronchus-associated lymphoid tissues were composed of B cells, T cells, and follicular dendritic cells with evidence of lymphocyte priming and differentiation. This suggests local immunity plays an important role in the SARS-CoV-2 infection. Further study of local immunity in the lung would benefit our understanding of SARS-CoV-2 pathogenesis and could lead to new interventions to control the SARS-CoV-2 infection and disease.

BackgroundOral lichen planus (OLP) is a chronic, immune mediated interface mucositis of oral mucosa. Though the apoptosis of keratinocytes is a feature of OLP, not much is known about the clearance of cell debris (efferocytosis) resulting from apoptosis. We postulate that there is a defective or delayed efferocytosis in OLP, which may have a role in modulating the immune response in OLP. MethodsPublished mRNA expression of tissue of 14 patients with OLP and 14 cases of normal tissues were subjected to differential analysis (DE) and a list of DE genes identified. From this list, the genes that involved in efferocytosis were collated, compared and their interactions are typed. ResultIn all, two studies fulfilled the inclusion and exclusion criteria. On combining the data, 1486 genes were significantly different between OLP and normal tissues. 28 of these 1486 genes are were associated with efferocytosis of which the suppression of LRP1, LDLR, ANAX2, C2, PBX1, PDCD4, S1PR5, CX3CL1, STAT6 and Wnt3A is indicative of defective or delayed efferocytosis in OLP. The role of pathways and associations were analyzed and is presented here. Discussion and ConclusionThe study revealed that certain key genes mRNAs that are associated with efferocytosis are altered in OLP. They could delay or lead to defective efferocytosis. Studying such genes in detail could provide deeper understanding of the pathogenesis of the disease and the discovery of therapeutic targets.

Antimicrobial peptides (AMPs) are a complex network of 10-100 amino acid sequence molecules, widely distributed in Nature. Even though more than 300 AMPs have been described in mammals, cathelicidins and defensins remain the most investigated to date. Some publications examined the role of AMPs in COVID-19, but the findings are preliminary and in vivo studies are still lacking. Here, we report the plasma levels of five AMPs (LL-37, -defensin 1, -defensin 3, {beta}-defensin 1 and {beta}-defensin 3) and five cytokines (tumor necrosis factor-, interleukin-1{beta}, interleukin-6, interleukin-10, interferon-{gamma} and monocyte chemoattractant protein-1), in 15 healthy volunteers, 36 COVID-19 patients without Acute Kidney Injury (AKI) and 17 COVID-19 patients with AKI, since AKI is a well-known marker of worse prognosis in Sars-CoV-2 infections. We found increased levels of -defensin 1, -defensin 3 and {beta}-defensin 3, but not LL-37 or {beta}-defensin 3, in our COVID-19 population, when compared with the healthy controls, in conjunction with higher levels of interleukin-6, interleukin-10, interferon-{gamma} and monocyte chemoattractant protein-1, putting in evidence that these AMPs and cytokines may have an important role in the systemic inflammatory response and tissue damage that characterizes severe COVID-19. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/23292389v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@f12f98org.highwire.dtl.DTLVardef@6ba5aaorg.highwire.dtl.DTLVardef@14950a1org.highwire.dtl.DTLVardef@4c9455_HPS_FORMAT_FIGEXP M_FIG C_FIG

Gamma delta ({gamma}{delta}) T cells are a subset of T cells that express MHC-independent {gamma}{delta} T cell receptors (TCRs) and can perform the same T-helper functions as CD4+ cells as well as cytotoxic functions like CD8+ T cells. The MHC-independent nature of {gamma}{delta} TCRs allows them to recognize a larger diversity of antigens, including self and non-self-antigens, making them ideal candidates for immunotherapeutic interventions. Both translational science efforts to better understand the physiology of {gamma}{delta} T cells and clinical research concerning the applications of {gamma}{delta} T cell immunotherapy require successful {gamma}{delta} T cell expansion techniques. Building on prior methods and recent innovations, in this study we optimized in vitro methods for rapid and efficient expansion of V{delta}1+ T cells from stimulated PBMCs. CD3-stimulated PBMCs from rhesus macaques in the presence of phospho-vitamin C (pVC) and IL-15 achieved up to a 6561-fold expansion (average 2559-fold) increase of V{delta}1+ T cells after 9-day culture. Comparable expansion was obtained with phytohemagglutinin (PHA)-stimulated PBMCs, achieving up to 7574-fold expansion (average 2040-fold) increase when IL-7 and IL-18 were added alongside IL-15 and pVC. Notably, inclusion of pVC significantly enhanced the expansion of V{delta}1+ T cells in both stimulation conditions. These results provide optimized conditions for scalable in vitro expansion of peripheral blood V{delta}1+ T cells from nonhuman primate models, supporting downstream applications in immunophenotyping, functional assays, and preclinical modeling of {gamma}{delta} T cell-based immunotherapies.

This work aimed to establish a translationally viable, xeno-free, serum-free platform and protocol for the isolation and expansion of human salivary stem/progenitor cells (hS/PCs) suitable for regulatory qualification and future FDA-approved first-in-human autologous regenerative therapy trials for the treatment of hyposalivation disorders. Parotid gland specimens from non-cancerous regions/tissues were collected from consented surgical patients. Primary hS/PCs were isolated from tissue specimens, cultured in animal-component-free conditions, expanded to produce millions of cells, then enriched for CD44+ stem/progenitor cells by magnetic cell sorting. Normal epithelial purity was assessed using cytokeratins 5/14. Anti-CD133/PROM1 (cancer marker) and anti- fibroblast (clone TE-7) antibodies were used to demonstrate a lack of contaminating cells. Phenotype validation was performed by flow cytometry and immunocytochemistry on both CD44+ sorted and unsorted populations. Senescence-associated beta-galactosidase (SA-{beta}-gal) assays were performed across serial passages (P1-P6). Pluripotency was demonstrated by culture under conditions supporting lineage-specific differentiation. Primary hS/PCs demonstrated consistent expansion and epithelial morphology under serum-free conditions. CD44 expression remained high (>95%) throughout expansion, with negligible detection of CD133 or fibroblast markers, confirming epithelial purity and absence of tumorigenic or stromal contamination. Immunocytochemistry corroborated these expression profiles. SA-{beta}-gal staining revealed only a minor, passage-dependent increase (5-16%) in senescent cells from multiple donors, indicating retention of proliferative potential. Our defined, animal-free culture system supports stable expansion of pure low passage hS/PCs under conditions compatible with good manufacturing practice (GMP).

BackgroundT-cell activation and proliferation are critical for understanding immune responses in both healthy and pathological conditions such as acute graft-versus-host disease (aGVHD). Phytohemagglutinin (PHA) and interleukin-2 (IL-2) are commonly used in in vitro assays to study T-cell responses. In view of the discrete response of T cells from aGvHD patients cohorts, our study optimized PHA / IL-2 based T-cell response among healthy individuals versus aGVHD patients. MethodsPeripheral blood was collected from age- and sex-matched healthy individuals (n=10) and aGVHD patients (n=10). CD3+ T-cell were isolated and stimulated with varying concentrations of PHA (1-10g/ml) and IL-2 (50-500 IU/ml). Cell proliferation was assessed using MTS and CFSE assays, while their apoptosis was evaluated with Annexin V/7-AAD staining. ResultsWe observed enhanced proliferation of healthy individuals at higher PHA concentrations (5-10g/ml), whereas aGVHD patients exhibited heightened proliferation even at lower PHA concentrations (1-2.5g/ml) at 48 hours. Prolonged exposure of T cells from GvHD patients to PHA led to decreased proliferation while it increased in the T cells from healthy donors.IL-2 supplementation (50 IU/ml) of T-cells from healthy donors significantly enhanced their proliferation and survival, with the optimal concentration supporting robust proliferation over extended culture periods. ConclusionOur study optimized PHA and IL-2 concentrations required for T-cell proliferation studies among healthy individuals and aGVHD patients. and underscored experimental conditions required for studying T-cell behavior/dysregulation in aGVHD condition. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/611044v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@ab3c47org.highwire.dtl.DTLVardef@2786forg.highwire.dtl.DTLVardef@30c698org.highwire.dtl.DTLVardef@3e4821_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights of the studyO_LILower doses of PHA (1.0g/ml) and IL-2 (50IU/ml) are optimum conditions for aGVHD patients derived CD3+ T-cell proliferation under in vitro conditions. C_LIO_LIThe maximum T-cell proliferation in healthy individuals occurs with 7.5g/ml PHA and 50IU/ml IL-2. C_LIO_LIHigher doses of PHA induce cytotoxicity in both cohorts. C_LIO_LIIL-2 significantly enhances T-cell survival, with 50IU/ml maintaining robust proliferation over extended periods. C_LI

Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of immature, immune suppressive myeloid cells. However, their role in periodontal disease (PD), a microbially induced inflammatory oral disease, remains understudied. Here we show that gingiva (gums) from PD patients exhibit significantly higher levels of MDSC markers including Granulocytic (G)-MDSC and Monocytic (M)-MDSC subsets as well as CD4+ T cells and CD19+ B cells. Gingival MDSC subsets exhibit potent immunoregulatory activity as marked by attenuated autologous CD4+ T cells proliferation and IFN{gamma} production. In a murine model of ligature-induced periodontitis (LIP), we noticed time-dependent gingival MDSC infiltration, which correlates with CD4+ T cell and CD19+ B cell infiltration. To test whether MDSC confer immunoregulatory function in vivo, we adoptively transferred G-MDSCs and M-MDSCs in mice. Interestingly, we observed significant reduction in inflammatory marker expression (IL6, TNF-, and IL-1{beta}), infiltration of CD4+ T cells, and concomitant increase in MDSC-derived immune suppressive molecules, ARG1 and IL-10, and CD4+CD25+FoxP3+ Tregs compared to mock. Conversely, depletion of MDSC using anti-Gr1 antibody resulted in marked induction of periodontal inflammation, reduced Treg population, and significantly higher alveolar bone loss. These findings, for the first time, suggest an anti-inflammatory and osteoprotective function of MDSCs in PD and offer a promising target to treat unresolved periodontal inflammation.

IntroductionCoronavirus disease 2019 was initially detected in China and has been declared a global pandemic by World Health Organization on March 11, 2020. In the majority of patients, SARS{square}CoV{square}2 causes a mild to moderate illness characterized by fever and respiratory symptoms, with or without evidence of pneumonia. The recent studies suggest that anti-cytokine targeted therapies might be associated with benefit for patients with severe COVID-19 especially in improving respiratory failure. Tocilizumab, a monoclonal antibody against interleukin 6 (IL6) receptor, is associated with clinical benefit for COVID-19 patients as it inhibits IL6 and decreases inflammation. MethodsAs Tocilizumab has been an important part of our treatment and a strict criterion was followed to administer Tocilizumab, a retrospective study design used to assess the beneficial effects of Tocilizumab in improvement of ratio partial pressure of arterial Oxygen and fraction of inspired Oxygen (PaO2/FiO2 or P/F ratio) and C-reactive protein (CRP) in COVID19 patients has been done. 60 patients were taken for this study by using convenient sampling technique the data of demographics, laboratory results, and clinical outcomes i.e. improvement of respiratory failure depicted in the form of PF Ratio were obtained from the medical records, Statistical analysis was done with SPSS, version 21.0. ResultsSixty patients (47 males and 13 females) with COVID{square}19 were included in this study, the mean age of patients was 53.83 (14{square}81) years. After administration of Tocilizumab the lab parameters were changed as CRP decreased down to .40 (9.6{square}73) mg/L but other parameters were not affected. The PF ratio improved in COVID-19 patients after administration of Tocilizumab the median of PF Ratio before treatment was 108 (52{square}362) and improved up to 128 (37{square}406) after Tocilizumab therapy. ConclusionIn summary, Tocilizumab appears to be associated with improvement in P/F Ratio and CRP in COVID19 patients but other markers did not improve in response to Tocilizumab therapy in severely ill COVID-19 patients.

BackgroundThe novel human coronavirus, severe acute respiratory syndrome coronavirus-2 (SARs-CoV-2), was declared a global pandemic by the World Health Organization on March 11, 2020. Hence, there is an urgency to find effective treatment. Of those patients afflicted in the United States, many have required treatment with ventilator secondary to acute respiratory distress syndrome (ARDS). Data are needed regarding the benefit of treatment and prevention of the cytokine storms in COVID-19 patients with Tocilizumab. MethodsClinical outcomes data for patients admitted to Orange Regional Medical Center with confirmed COVID-19 from Mar 15, 2020 to Apr 20, 2020 were identified through electronic health record chart review. We conducted a retrospective case-control study in confirmed COVID 19 positive patients with ARDS requiring mechanical ventilation and compared outcome in terms of mortality and length of stay amongst those who received Tocilizumab as treatment modality opposed to those that did not. ResultsA total of 94 patients with COVID-19 ARDS were analyzed. 44 were in the study group and 50 in the control group. We tried to match both group as close as possible in terms of age, sex, BMI and HS score-calculated using inflammatory markers-ferritin, triglycerides, AST and fibrinogen. The median age was 55.5 years in the study group and 66 in the control group, difference was not statistically significant. Average HS score was 114 in the Tocilizumab group and 92 in the control group, difference was statistically significant with P<0.0001. Also, the patients in the study group had elevated levels of IL-6, triglycerides, AST, ferritin which were statistically significant with p < 0.0001 when compared to the control group. Length of stay was longer, average 17.9 days in the Tocilizumab. Survival rate was much lower at 48 % in the control group and 61.36 % in patients who received Tocilizumab with significant P value of < 0.00001. The number needed to treat (NNT) was 7.48, if we treat 8 patients with Tocilizumab, 1 will not die. ConclusionCytokine Release Syndrome (CRS) occurs in a large number of patients with severe COVID-19, which is also an important cause of death. IL-6 is the key molecule of CRS, so IL-6R antagonist Tocilizumab may be of value in improving outcomes. In our study Tocilizumab group seemed to have improved survival outcome. Results have to be interpreted with caution since this is a retrospective study and mortality is affected by multiple, confounding factors. We await the results of ongoing randomized controlled trials to definitely answer the question of whether Tocilizumab improves survival in COVID-19 ARDS patients.

BackgroundIn December 2019, the first cases of severe pneumonia associated with a new coronavirus were reported in Wuhan, China. Severe respiratory failure requiring intensive care was reported in up to 5% of cases. There is, however, limited information available in Mexico. ObjectivesThe purpose of this study was to describe the clinical manifestations, and outcomes in a COVID-19 cohort attended to from March to May 2020 in our RICU. In addition, we explored the association of clinical variables with mortality. MethodsThe first consecutive patients admitted to the RICU from March 3, 2020, to Jun 24, 2020, with confirmed COVID-19 were investigated. Clinical and laboratory data were obtained. Odds ratios (ORs) were calculated using a logistic regression model. The survival endpoint was mortality at discharge from the RICU. ResultsData from 68 consecutive patients were analyzed. Thirty-eight patients survived, and 30 died (mortality: 44.1 %). Of the 16 predictive variables analyzed, only 6 remained significant in the multivariate analysis [OR (95% confidence interval)]: no acute kidney injury (AKI)/AKI 1: [.61 (.001;.192)]; delta lymphocyte count: [.061 (.006;.619)]; delta ventilatory ratio: [8.19 (1.40;47.8)]; norepinephrine support at admission: [34.3 (2.1;550)]; body mass index: [1.41 (1.09;1.83)]; and bacterial coinfection: [18.5 (1.4;232)]. ConclusionsWe report the characteristics and outcome of patients with ARDS and COVID-19. We found six independent factors associated with the mortality risk: delta lymphocyte count, delta ventilatory ratio, BMI, norepinephrine support, no AKI/AKI 1, and bacterial coinfection.

RationalePneumonia is the most common infection in ICU patients and a leading cause for death. Assessment of bronchoalveolar lavage fluid (BALF) cellularity can aid in pneumonia diagnosis. Low percentages (<50%) of BALF neutrophils have a high negative predictive value for bacterial pneumonia in a general medical ICU population. The operating characteristics in patients with immunocompromise and neutropenia are less clear. ObjectiveTo compare BALF % neutrophils operating characteristics in patients with and without immunocompromise or neutropenia. MethodsThis was a single center observational cohort study. Patients were categorized into three groups: (1) patients with neutropenia, (2) patients with underlying immunocompromise, and (3) patients with neither. BAL-level analysis reflected neutropenia and immunocompromise state on day of BAL sampling. Operating characteristics of BALF % neutrophils were calculated using varying thresholds of alveolar neutrophilia. Median [Q1,Q3] are reported for nonparametric data and compared using Mann-Whitney U tests. Results688 mechanically ventilated patients had 1736 BAL samples. Among bacterial pneumonia episodes, no difference was found in BALF % neutrophils between patients with underlying immunocompromise and patients with neither neutropenia nor immunocompromise on day of sampling: 82.0% [61.0, 91.0] vs 81.0% [66.0, 91.0], p=0.859. However, when neutropenic on day of sampling, the median BALF % neutrophils was 35.0% [8.8, 67.5] (p<0.001 compared with other categories). In patients with neutropenia, a BALF % neutrophil threshold of 7% had a sensitivity of 90% for excluding bacterial pneumonia. ConclusionsWe found that among patients with bacterial pneumonia, BALF % neutrophil was not significantly lower in patients with a broad spectrum of immunocompromised states but was significantly lower when measured during acute neutropenia. We found varying thresholds of BALF % neutrophils across the three groups. Patients with neutropenia who mount even a low percent of alveolar neutrophils should raise concern for bacterial pneumonia.

Research with BSL-4 viruses such as Ebola, Marburg, and Nipah presents significant challenges due to their high virulence and the stringent containment measures required. A major limitation in studying viral pathogenesis and developing therapeutic strategies is the absence of suitable animal models that accurately replicate human disease. In this context, 3D cell culture systems offer significant advantages over traditional 2D monolayer cultures, mimicking native physiological conditions including cell polarization and composition. Human airway organoids, derived from pluripotent or adult stem cells, closely replicate the structure and function of the human respiratory system, providing a relevant and accessible environment for studying viral replication and pathogenesis. In contrast to conventional cell lines, airway organoids enable investigation of virus-host interactions within a human tissue context, providing insights that are more directly translatable to human disease. In our study, we generated airway organoids from both clinical donor tissues and commercially available nasal epithelial cells and showed in comparative analyses with whole lung tissue that these organoids are comparable in terms of cell composition. Despite donor-specific variations due to genetic factors, airway organoids derived from different sources and donors exhibit a remarkably similar cellular make-up. We further demonstrated that organoids derived from nasal swabs can effectively replicate BSL-4 viruses, establishing them as a standardized 3D model for broader research applications and advancing our understanding of these pathogens, especially in the absence of reliable animal models. Author SummaryThis study establishes human airway organoids as a robust model for investigating BSL-4 pathogens, such as Ebola, Marburg, and Nipah virus. Airway organoids represent reliable systems due to their ability to replicate the complexity of human respiratory epithelia and support viral infection. These organoids exhibit high susceptibility to these viruses, allowing for subsequent analysis of infection kinetics, immune evasion, and tissue-specific tropism within a controlled environment. This platform provides a powerful tool for antiviral testing and studying virus-host interactions, thus helping bridge critical gaps in high-containment virus research.

We are currently facing a frightening increase in COVID-19 patients admitted to the ICU. Aiming at screening for secondary pneumonia, we collected the data of our first twelve ICU patients who underwent bronchoalveolar lavage (BAL). Surprisingly, four were detected with Pneumocystis jirovecii (Pj) DNA and RNA, resulting in Pj prevalence of 17%. Pj is a ubiquitous ascomycetes fungus that thrives at the surface of type-I pneumocytes, specifically in human alveoli, leading to pneumocystosis in immunocompromised patients. Interestingly, none of our patients was immunocompromised per se before admission, while all presented the recognized risk factors for life-threatening COVID-19 infection. Observing such high prevalence in COVID-infected patients was unexpected. Almost all patients developed ARDS and received high-dose steroids to prevent worsening, as suggested by reports from China. In Pj-positive patients requiring steroids, prophylaxis was given to avoid the risk of pneumocystosis and increased lung inflammation that may compromise the outcome.We are strongly convinced that testing deep lung specimens for Pj in severe COVID-19 patients should be recommended and Pj-positive patients treated with steroids, and given anti-Pj prophylaxis. This message is important, given the high mortality rate of COVID-19 patients in the ICU.

BackgroundAntinuclear antibody (ANA) testing is used to diagnose systemic autoimmune rheumatic disease (SARD). Autoantibodies (Abs) associated with the "homogeneous-like" pattern on ANA HEp-2 cell nuclei can be classified as pathological (e.g., anti-dsDNA, anti-nucleosome, anti-histone, anti-Scl-70 Abs) or non-pathological (e.g., anti-DFS70 Abs). MethodsAnti-neutrophil cytoplasmic anti-antibody (ANCA) testing was used to classify individuals who presented with a homogeneous-like pattern on ANA testing. Enrolled subjects included (1) young individuals with a dense fine speckled pattern on ANA testing (young non-SARD group, n = 62) and patients with (2) systemic lupus erythematosus (SLE) with anti-dsDNA Abs (SLE group, n = 33), (3) rheumatoid arthritis (RA) with anti-nucleosome, anti-histone Abs, and others (RA group, n = 45), and (4) diffuse systemic sclerosis (SSc) with Scl-70 Abs (diffuse SSc group, n = 11). ResultsNegative rates (95% confidence interval) of neutrophil nuclear patterns on ANCA testing were: 96.8% (88.8%-99.6%) of the young non-SARD group, 3.0% (0.1 %-15.8%) of the SLE group, 4.4% (0.5%-15.2%) of the RA group, and 54.5% (23.4%-83.3%) of the diffuse SSc group. The negative rate of the non-SARD group was significantly higher than those of the SARD group (all P < 0.05). ConclusionsANCA testing helps to identify individuals with non-pathological anti-DFS70 Abs who present with homogeneous-like patterns in HEp-2 cell nuclei on ANA testing.