Activation of alveolar epithelial ER stress by ß-coronavirus infection disrupts surfactant homeostasis in mice: implications for COVID-19 respiratory failure.

COVID-19 syndrome is characterized by acute lung injury, hypoxemic respiratory failure, and high mortality. Alveolar type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and other members of the ß-coronavirus genus induce an endoplasmic reticulum (ER) stress response in vitro; however, the consequences for host AT2 cell function in vivo are less understood. To study this, two murine models of coronavirus infection were used-mouse hepatitis virus-1 (MHV-1) in A/J mice and a mouse-adapted SARS-CoV-2 strain. MHV-1-infected mice exhibited dose-dependent weight loss with histological evidence of distal lung injury accompanied by elevated bronchoalveolar lavage fluid (BALF) cell counts and total protein. AT2 cells showed evidence of both viral infection and increased BIP/GRP78 expression, consistent with activation of the unfolded protein response (UPR). The AT2 UPR included increased inositol-requiring enzyme 1α (IRE1α) signaling and a biphasic response in PKR-like ER kinase (PERK) signaling accompanied by marked reductions in AT2 and BALF surfactant protein (SP-B and SP-C) content, increases in surfactant surface tension, and emergence of a reprogrammed epithelial cell population (Krt8+ and Cldn4+). The loss of a homeostatic AT2 cell state was attenuated by treatment with the IRE1α inhibitor OPK-711. As a proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis. We conclude that lung injury from ß-coronavirus infection results from an aberrant host response, activating multiple AT2 UPR stress pathways, altering surfactant metabolism/function, and changing AT2 cell state, offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and acute respiratory failure.NEW & NOTEWORTHY COVID-19 syndrome is characterized by hypoxemic respiratory failure and high mortality. In this report, we use two murine models to show that ß-coronavirus infection produces acute lung injury, which results from an aberrant host response, activating multiple epithelial endoplasmic reticular stress pathways, disrupting pulmonary surfactant metabolism and function, and forcing emergence of an aberrant epithelial transition state. Our results offer a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and respiratory failure.

Tumor cell E-selectin ligands determine partialefficacy of bortezomib on spontaneous lung metastasis formation of solid human tumors in vivo.

Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.

Invariant natural killer T cells from children with versus without food allergy exhibit differential responsiveness to milk-derived sphingomyelin.

BACKGROUND: A key immunologic feature of food allergy (FA) is the presence of a T(h)2-type cytokine bias. Ligation of the invariant natural killer T cell (iNKT) T-cell receptor (TCR) by sphingolipids presented via the CD1d molecule leads to copious secretion of T(h)2-type cytokines. Major food allergens (eg, milk, egg) are the richest dietary source of sphingolipids (food-derived sphingolipids [food-SLs]). Nonetheless, the role of iNKTs in FA is unknown. OBJECTIVE: To investigate the role of iNKTs in FA and to assess whether food-SL-CD1d complexes can engage the iNKT-TCR and induce iNKT functions. METHODS: PBMCs from 15 children with cow's milk allergy (MA), 12 children tolerant to cow's milk but with allergy to egg, and 13 healthy controls were incubated with α-galactosylceramide (αGal), cow's milk-sphingomyelin, or hen's egg-ceramide. iNKTs were quantified, and their cytokine production and proliferation were assessed. Human CD1d tetramers loaded with milk-sphingomyelin or egg-ceramide were used to determine food-SL binding to the iNKT-TCR. RESULTS: Milk-sphingomyelin, but not egg-ceramide, can engage the iNKT-TCR and induce iNKT proliferation and T(h)2-type cytokine secretion. Children with FA, especially those with MA, had significantly fewer peripheral blood iNKTs and their iNKTs exhibited a greater T(h)2 response to αGal and milk-sphingomyelin than iNKTs of healthy controls. CONCLUSION: iNKTs from children with FA, especially those with MA, are reduced in number and exhibit a T(h)2 bias in response to αGal and milk-sphingomyelin. These data suggest a potential role for iNKTs in FA.

Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer membrane tyrosine kinase.

Mice lacking the membrane tyrosine kinase c-mer have been shown to have altered macro-phage cytokine production and defective phagocytosis of apoptotic cells despite normal phagocytosis of other particles. We show here that c-mer-deficient mice have impaired clearance of infused apoptotic cells and that they develop progressive lupus-like autoimmunity, with antibodies to chromatin, DNA, and IgG. The autoimmunity appears to be driven by endogenous antigens, with little polyclonal B cell activation. These mice should be an excellent model for studying the role of apoptotic debris as an immunogenic stimulus for systemic autoimmunity.

Disrupted Mer receptor tyrosine kinase expression leads to enhanced MZ B-cell responses.

Control of lymphocyte homeostasis is essential to ensure efficient immune responses and to prevent autoimmunity. Splenic marginal zone B cells are important producers of autoantibodies, and are subject to stringent tolerance mechanisms to prevent autoimmunity. In this paper, we explore the role of the Mer tyrosine kinase (Mertk) in regulating autoreactive B cells. This receptor tyrosine kinase serves to bind apoptotic cells, to mediate their phagocytosis, and to regulate subsequent cytokine production. Mice lacking Mertk suffer from impaired apoptotic cell clearance and develop a lupus-like autoimmune syndrome. Here we show that such Mertk-KO mice have expanded numbers of splenic marginal zone B cells. Mertk-KO mice bearing a DNA-specific immunoglobulin heavy-chain transgene (3H9) produced anti-DNA antibodies that appeared to be secreted largely by marginal zone B cells. Finally, Mertk-KO mice developed greater antibody responses after NP-Ficoll immunization than their B6 counterparts. Taken together, our data show that Mertk has a major effect on the development of the marginal zone B-cell compartment. Mertk is also important in establishing DNA-specific B-cell tolerance in 3H9 anti-DNA transgenic mice.

Obstetric admissions to tertiary level intensive care unit - Prevalence, clinical characteristics and outcomes.

BACKGROUND AND AIMS: Obstetric admissions to the intensive care unit (ICU) are a subject of increasing interest, as it is an indirect indicator of maternal morbidity and mortality. The studies from areas reported to have a higher maternal mortality rate are lacking. Thus, we undertook this study to determine the prevalence pattern, clinical characteristics and outcome of obstetric patients admitted to the ICU of a tertiary care hospital. METHODS: All obstetric patients (up till 42 days of delivery) admitted to the ICU from 1st October 2015 to 30th September 2016 and from 1st October 2010 to 30th September 2015 were included. Data collected for our study included demographic characteristics, Acute Physiologic Assessment and Chronic Health Evaluation (APACHE) II score at the time of admission, obstetric and medical history, provisional diagnosis, the reason for ICU admission, interventions required in ICU and the outcome. RESULTS: The third trimester (46.79%) and postpartum period (40.37%) were the most common time of admission with conditions such as severe pre-eclampsia, eclampsia, HELLP syndrome (Haemolysis, elevated liver enzymes, low platelet count), antepartum haemorrhage, postpartum haemorrhage and anaemia. The mean APACHE II score was 16.89 ± 7.48 with a mortality rate of 17.76%. The mean length of stay in ICU was 3.47 ± 3.16 days, and mean length of stay in our hospital was 8.78 ± 6.76 days. CONCLUSION: Obstetric patients recover well if treated early. A good ICU care with monitoring can save a young productive life.

Assessment of malnutrition and enteral feeding practices in the critically ill: A single-centre observational study.

BACKGROUND AND AIMS: Early identification of malnutrition among hospitalised patients is essential to institute appropriate patient-specific nutritional strategies. This study was conducted to evaluate the nutritional status of medical patients at admission to the adult intensive care unit (ICU) and to identify factors which prevent attainment of daily feeding goals in them. METHODS: This was a 1 year prospective, observational study on 200 medical adult ICU patients. The study was carried out based on daily documentation. The primary outcome was the nutritional status of medical Patients at admission to the adult ICU. The tests for statistical analysis used were independent t test, Chi-square test, Fisher's exact test and multivariate logistic regression analysis. RESULTS: Out of the 200 patients in our study, 45%, 48.5% and 9% of patients had mild, moderate and severe malnutrition, respectively, corresponding to subjective global assessment (SGA) rating A,B and C, respectively. The most common reasons for non-attainment of daily feeding goals were delayed feed procurement (17.57%), and feeds being held for procedures (16.36%). The overall mean length of ICU stay was 8.63 ± 7.26 days, and the ICU mortality rate was 47.5% (95/200). Patients with SGA rating B and C at admission had higher risk of mortality in the ICU, with an adjusted odds ratio of 3.54 (95% confidence interval [CI]- 1.71-7.33, P = 0.001) and 11.11 (95% CI-2.26-54.66, P = 0.003), respectively. CONCLUSION: Malnutrition is commonly present at admission among medical ICU patients, and is associated with higher ICU mortality.

Involvement of TIMP-1 in PECAM-1-mediated tumor dissemination.

Platelet endothelial cell adhesion molecule­1 (PECAM­1) is expressed on the vascular endothelium and has been implicated in the late progression of metastatic tumors. The activity of PECAM­1 appears to be mediated by modulation of the tumor microenvironment (TME) and promotion of tumor cell proliferation, rather than through the stimulation of tumor angiogenesis. The present study aimed to extend those initial findings by indicating that the presence of functional PECAM­1 on the endothelium promotes a proliferative tumor cell phenotype in vivo, as well as in tumor cell (B16­F10 melanoma and 4T1 breast cancer cell lines) co­culture assays with mouse endothelial cells (ECs) or a surrogate EC line (REN­MP). The pro­proliferative effects were mediated by soluble endothelial­derived factors that were dependent on PECAM­1 homophilic ligand interactions, but which were independent of PECAM­1­dependent signaling. Further analysis of the conditioned media obtained from tumor/EC and tumor/REN­MP co­cultures identified TIMP metallopeptidase inhibitor­1 (TIMP­1) as a PECAM­1­regulated factor, the targeting of which in the tumor cell/REN­MP system inhibited tumor cell proliferation. In addition, TIMP­1 expression was decreased in metastatic tumors from the lungs of PECAM­1­null mice, thus providing evidence of the in vivo significance of co­culture studies. Taken together, these studies indicated that endothelial PECAM­1, through PECAM­1­dependent homophilic binding interactions, may induce release of TIMP­1 from the endothelium into the TME, thus leading to increased tumor cell proliferation.

Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1.

Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications.

Influence of PECAM-1 ligand interactions on PECAM-1-dependent cell motility and filopodia extension.

Platelet endothelial cell adhesion molecule (PECAM-1) has been implicated in angiogenesis through processes that involve stimulation of endothelial cell motility. Previous studies suggest that PECAM-1 tyrosine phosphorylation mediates the recruitment and then activation of the tyrosine phosphatase SHP-2, which in turn promotes the turnover of focal adhesions and the extension of filopodia, processes critical to cell motility. While these studies have implicated PECAM-1-dependent signaling in PECAM-1-mediated cell motility, the involvement of PECAM-1 ligand binding in cell migration is undefined. Therefore to investigate the role of PECAM-1 binding interactions in cell motility, mutants of PECAM-1 were generated in which either homophilic or heparin/glycosaminoglycan (GAG)-mediated heterophilic binding had been disabled and then expressed in an endothelial cell surrogate. We found that the ability of PECAM-1 to stimulate cell migration, promote filopodia formation and trigger Cdc42 activation were lost if PECAM-1-dependent homophilic or heparin/GAG-dependent heterophilic ligand binding was disabled. We further observed that PECAM-1 concentrated at the tips of extended filopodia, an activity that was diminished if homophilic, but not heparin/GAG-mediated heterophilic binding had been disrupted. Similar patterns of activities were seen in mouse endothelial cells treated with antibodies that specifically block PECAM-1-dependent homophilic or heterophilic adhesion. Together these data provide evidence for the differential involvement of PECAM-1-ligand interactions in PECAM-1-dependent motility and the extension of filopodia.

Ideal anesthetic agents for day-care gynecological procedures: A clinical trial comparing thiopentone with ketamine as adjuncts to propofol.

BACKGROUND: Day-care gynecological procedures require the use of anesthetic agents, which ensure rapid induction and recovery. Although propofol is the gold standard drug in day-care procedures, it has its own side effects like apnea, cardiovascular instability, pain on injection, as well as its cost. The ideal drug combination to achieve this end remains elusive. Therefore, a combination of propofol, thiopentone, and ketamine may be a better alternative. MATERIALS AND METHODS: This prospective, double-blind, randomized study was conducted on 60 women, aged 18-50 years, American Society of Anesthesiologists (ASA) physical status 1 and 2, undergoing day-care gynecological surgeries. The patients were allocated to two groups. Group T received an admixture containing 10 ml of 1% propofol and 10 ml of 1.25% thiopentone. Group K received an admixture containing 10 ml of 1% propofol and 10 ml of 0.5% ketamine. RESULTS: There was less variation in the mean systolic blood pressure of patients in Group K as compared to patients in Group T. The mean total dose of propofol required in Group K (0.85 mg/kg) was significantly less than that required in Group T (1.12 mg/kg) (P = 0.0004). The mean recovery time in Group T (3.67 minutes) was significantly less than in Group K (6.27 minutes; P = 0.0001). However, the mean discharge time in both the groups was similar. (P = 0.7392). The results were analyzed statistically using the Student's t-test and the Fisher's exact test. CONCLUSIONS: Both the propofol-thiopentone and propofol-ketamine admixtures provided adequate anesthesia. Propofol-ketamine proved superior to propofol-thiopentone in terms of hemodynamic stability and requirement of a lesser total dose of propofol. However, the patients in the propofol-thiopentone group had faster recovery.

Premedication dilemmas, is Pregabalin the answer?

OBJECTIVE: Laryngoscopy and intubation are associated with sympathetic stimulation which can prove deleterious in patients with cardiovascular compromise; so, various methods have been tried to obtund this pressor response. In this study, we have assessed the efficacy of pregabalin in attenuating the pressor response to laryngoscopy and intubation. METHODS: This prospective randomized study included 80 patients with American Society of Anesthesiologists physical status grades I-II, in the age group of 18-60 years of age. The patients were randomized into two groups of 40 patients each. Group A received the placebo orally, 90 min prior to surgery. Group B received 150 mg of pregabalin orally, 90 min prior to surgery. These patients were assessed in terms of sedation with Ramsay sedation scale (RSS). In the operation theatre, the heart rate (HR), systolic blood pressure, diastolic blood pressure, mean arterial pressure, and oxygen saturation recorded at baseline and 1, 3, 5, and 10 min after intubation. The rate pressure product (RPP) was calculated for these time intervals. In the postoperative period, patients were assessed for complications like dizziness, nausea, and blurred vision. Statistical analysis was performed using Chi-square and ANOVA tests. FINDINGS: The group receiving 150 mg of pregabalin as premedication was found to be adequately sedated at 1 h post-premedication with 52% patients having a RSS score of 3 compared to 4% with the same RSS score in the placebo group (P < 0.0001). Hemodynamics was more stable post-intubation with significant stability in the HR (P = 0.002) and RPP (P = 0.004) in the pregabalin group. CONCLUSION: Pregabalin when given as a premedication provides adequate sedation and obtunds the pressor response seen with intubation.

Isolation of endothelial cells from mouse lung.

The isolation of endothelial cells (ECs) from knockout and transgenic mouse lines provides the opportunity to study the endothelial-specific activities of a targeted molecule. As a means of pursuing these types of investigations, the protocols described in this unit provide a reliable method for isolating lung microvascular ECs from mouse neonatal pups that can be serially passaged. These protocols are useful in settings where mouse age is irrelevant and a pure population of pulmonary vascular ECs, uncontaminated by other cells, is needed. When a specific source of ECs is not required, these procedures also represent a reliable means of obtaining murine ECs in general.

IL-17A induces Pendrin expression and chloride-bicarbonate exchange in human bronchial epithelial cells.

The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.

Tracheal granulation as a cause of unrecognized airway narrowing.

Tracheostomy is one of the most common elective surgical procedures performed in critically ill patients. The most frequent late complication after tracheostomy is the development of granulation tissue, a complication that may cause airway occlusion or result in airway stenosis. We report the successful management of a patient with tracheal granulation presenting as an unrecognised cause of difficulty breathing.

T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection.

Innate and adaptive defense mechanisms protect the respiratory system from attack by microbes. Here, we present evidence that the bitter taste receptor T2R38 regulates the mucosal innate defense of the human upper airway. Utilizing immunofluorescent and live cell imaging techniques in polarized primary human sinonasal cells, we demonstrate that T2R38 is expressed in human upper respiratory epithelium and is activated in response to acyl-homoserine lactone quorum-sensing molecules secreted by Pseudomonas aeruginosa and other gram-negative bacteria. Receptor activation regulates calcium-dependent NO production, resulting in stimulation of mucociliary clearance and direct antibacterial effects. Moreover, common polymorphisms of the TAS2R38 gene were linked to significant differences in the ability of upper respiratory cells to clear and kill bacteria. Lastly, TAS2R38 genotype correlated with human sinonasal gram-negative bacterial infection. These data suggest that T2R38 is an upper airway sentinel in innate defense and that genetic variation contributes to individual differences in susceptibility to respiratory infection.