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Rationale: Current guidelines recommend patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia receive empirical antibiotics for suspected bacterial superinfection on the basis of weak evidence. Rates of ventilator-associated pneumonia (VAP) in clinical trials of patients with SARS-CoV-2 pneumonia are unexpectedly low. Objectives: We conducted an observational single-center study to determine the prevalence and etiology of bacterial superinfection at the time of initial intubation and the incidence and etiology of subsequent bacterial VAP in patients with severe SARS-CoV-2 pneumonia. Methods: Bronchoscopic BAL fluid samples from all patients with SARS-CoV-2 pneumonia requiring mechanical ventilation were analyzed using quantitative cultures and a multiplex PCR panel. Actual antibiotic use was compared with guideline-recommended therapy. Measurements and Main Results: We analyzed 386 BAL samples from 179 patients with SARS-CoV-2 pneumonia requiring mechanical ventilation. Bacterial superinfection within 48 hours of intubation was detected in 21% of patients. Seventy-two patients (44.4%) developed at least one VAP episode (VAP incidence rate = 45.2/1,000 ventilator days); 15 (20.8%) initial VAPs were caused by difficult-to-treat pathogens. The clinical criteria did not distinguish between patients with or without bacterial superinfection. BAL-based management was associated with significantly reduced antibiotic use compared with guideline recommendations. Conclusions: In patients with SARS-CoV-2 pneumonia requiring mechanical ventilation, bacterial superinfection at the time of intubation occurs in

UNLABELLED ESSENTIALS: Many anti-glycoprotein (GP)Ibα antibodies induce platelet clearance in a dimer-dependent manner. Characterization of monoclonal antibodies that bind the mechanosensitive domain (MSD) of GPIbα. An anti-MSD antibody binds two copies of GPIbα in platelets but does not induce platelet clearance. The prevailing clustering model of GPIbα signaling is incorrect or needs revision. BACKGROUND The mechanism of platelet clearance is not clear. Many antibodies binding the membrane-distal ligand-binding domain of glycoprotein (GP)Ibα induce rapid clearance of platelets and acute thrombocytopenia, which requires the bifurcated antibody structure. It was thought that binding of these antibodies induced lateral dimerization or clustering of GPIbα in the plasma membrane, which leads to downstream signaling and platelet clearance. However, many antibodies targeting GPIbβ and GPIX, which are associated with GPIbα in the GPIb-IX complex, do not induce platelet clearance, which is in contradiction to the clustering model. OBJECTIVES To test whether dimerization or clustering of GPIbα is sufficient to transmit the signal that leads to platelet clearance. METHODS We have recently raised several mAbs targeting the mechanosensitive domain (MSD) of GPIbα. Binding of these anti-MSD antibodies was characterized with biochemical methods. Their ability to stimulate platelets and induce platelet clearance in mice was assessed. RESULTS AND CONCLUSION Infusion of anti-MSD antibodies does not cause thrombocytopenia in mice. These antibodies show no detectable effects on platelet activation and aggregation in vitro. Further biochemical investigation showed that the anti-MSD antibody 3D1 binds two copies of GPIbα on the platelet surface. Therefore, lateral dimerization of GPIbα induced by antibody binding is not sufficient to initiate GPIb-IX signaling and induce platelet clearance. Our results suggest that a factor other than or in addition to clustering of GPIbα is required to induce platelet clearance.

Summary Background Ectodomain shedding of glycoprotein Ibα (GPIbα), a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has largely come from studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17. Aim To achieve substrate-specific inhibition of GPIbα shedding. Methods Development of monoclonal antibodies that directly bind the sequence around the GPIbα shedding cleavage site and inhibit GPIbα shedding by blocking ADAM17 access to the cleavage site. Results Six anti-GPIbα monoclonal antibodies with varying binding affinities were obtained. The prototypic clone, designated 5G6, and its monomeric Fab fragment bind specifically purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα. The clone 5G6 showed similar inhibitory potency as a widely used shedding inhibitor GM6001 in both constitutive and induced GPIbα shedding in human platelets. It does not recognize mouse GPIbα or inhibit shedding of other platelet receptors. Finally, 5G6 binding displays no detectable effect on platelet activation and aggregation. Conclusions The clone 5G6 specifically inhibits GPIbα shedding with no detectable effect on platelet functions. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding.

Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/β-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of β-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear β-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis–associated advanced pulmonary fibrosis. Forced activation of β-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-β, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of β-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.

Wnt/β-catenin signaling has been implicated in lung fibrosis, but how this occurs and whether expression changes in Wnt pathway components predict disease progression is unknown.To determine whether the Wnt coreceptor Lrp5 drives pulmonary fibrosis in mice and is predictive of disease severity in humans.We examined mice with impaired Wnt signaling caused by loss of the Wnt coreceptor Lrp5 in models of lung fibrosis induced by bleomycin or an adenovirus encoding an active form of transforming growth factor (TGF)-β. We also analyzed gene expression in peripheral blood mononuclear cells (PBMC) from patients with idiopathic pulmonary fibrosis (IPF).In patients with IPF, analysis of peripheral blood mononuclear cells revealed that elevation of positive regulators, Lrp5 and 6, was independently associated with disease progression. LRP5 was also associated with disease severity at presentation in an additional cohort of patients with IPF. Lrp5 null mice were protected against bleomycin-induced pulmonary fibrosis, an effect that was phenocopied by direct inhibition of β-catenin signaling by the small molecular inhibitor of β-catenin responsive transcription. Transplantation of Lrp5 null bone marrow cells into wild-type mice did not limit fibrosis. Instead, Lrp5 loss was associated with reduced TGF-β production by alveolar type 2 cells and leukocytes. Consistent with a role of Lrp5 in the activation of TGF-β, Lrp5 null mice were not protected against lung fibrosis induced by TGF-β.We show that the Wnt coreceptor, Lrp5, is a genetic driver of lung fibrosis in mice and a marker of disease progression and severity in humans with IPF. Evidence that TGF-β signaling can override a loss in Lrp5 has implications for patient selection and timing of Wnt pathway inhibitors in lung fibrosis.

Computer-assisted sperm analysis (CASA) systems are becoming more widely used. With this spread to technology come more data from toxicology studies, designed to determine if treatment with putative toxicants affects sperm motion parameters. While these CASA methods provide us with more ways to evaluate toxicity and thus perhaps increase our chances of successfully protecting human health, there is also a greater likelihood that different laboratories will use different methods of collecting data on sperm motility. Different systems used with different methods in different laboratories will inevitably generate data that are difficult to compare. In a prospective attempt to address this issue of comparability and limit the problems, a group of individuals using CASA systems to analyze rat sperm motility convened to discuss methodologic issues, share data, and try to reach a consensus about methods for performing these studies. This article shares those meetings and data in the hope that common methods will enhance interlaboratory comparisons.

The Wnt/beta-catenin signaling cascade activates genes that allow cells to adopt particular identities throughout development. In adult self-renewing tissues like intestine and blood, activation of the Wnt pathway maintains a progenitor phenotype, whereas forced inhibition of this pathway promotes differentiation. In the lung alveolus, type 2 epithelial cells (AT2) have been described as progenitors for the type 1 cell (AT1), but whether AT2 progenitors use the same signaling mechanisms to control differentiation as rapidly renewing tissues is not known. We show that adult AT2 cells do not exhibit constitutive beta-catenin signaling in vivo, using the AXIN2(+/LacZ) reporter mouse, or after fresh isolation of an enriched population of AT2 cells. Rather, this pathway is activated in lungs subjected to bleomycin-induced injury, as well as upon placement of AT2 cells in culture. Forced inhibition of beta-catenin/T-cell factor signaling in AT2 cultures leads to increased cell death. Cells that survive show reduced migration after wounding and reduced expression of AT1 cell markers (T1alpha and RAGE). These results suggest that AT2 cells may function as facultative progenitors, where activation of Wnt/beta-catenin signaling during lung injury promotes alveolar epithelial survival, migration, and differentiation toward an AT1-like phenotype.

Summary. Background: Glycoprotein (GP)VI is a key receptor for collagen on the platelet surface. It is a member of the immunoglobulin superfamily and is uniquely expressed on the surface of platelets, where it is assembled with the immunoreceptor tyrosine activation motif subunit, FcR-γ. We have previously reported the generation of a murine model of GPVI deficiency that revealed profound defects in collagen-induced platelet aggregation and in platelet activation following adhesion to collagen. Beyond the hemostasis/thrombosis paradigm, platelet receptors are emerging as significant participants in tumorigenesis and inflammation. Objective: In the current study, we have evaluated a role for platelet GPVI in primary tumor growth and experimental metastasis. Methods: Primary tumor induction and experimental metastasis assays were performed using syngenic immunocompetent animals and tumor cells derived from the C57BL/6J mouse strain in wild-type (C57BL/6J) and N10 C57BL/6J congenic GPVI-deficient mice. Results: Using either a Lewis lung carcinoma (D121) or melanoma (B16F10.1) cell line, we observed an approximately 50% reduction in the number of visible tumor foci in GPVI-deficient mice as compared with control C57BL/6J mice. Additional studies were performed to compare the size of subcutaneously implanted tumor cells, that is, primary tumor growth. Here, we observed no noticeable size difference when comparing the presence or absence of platelet GPVI. Conclusions: The results demonstrate that the presence of platelet GPVI facilitates experimental tumor metastasis but does not contribute to the growth of primary tumors.

Background β-catenin is a dual function adhesion/transcriptional co-activator protein, and both functions are critical for normal tissue homeostasis. Since the transcriptional functions of β-catenin are more often implicated in various disease processes, there is much interest in the development and use of reagents to interrogate spatial and temporal evidence of β-catenin nuclear signaling in cells and tissues. An important study demonstrated that the signaling form of β-catenin is specifically unphosphorylated at residues S37 and T41, and suggested that this form exhibits a propensity for cytosolic/nuclear accumulation relative to the total pool of β-catenin. Results We show that monoclonal antibody, 8E7, which recognizes the signaling form of β-catenin specifically unphosphorylated at S37 and T41 (Active B-Catenin, ABC), also cross-reacts with a widely expressed, variably accessible nuclear antigen that is not β-catenin. In cell types commonly used to study Wnt activation, this non-specific nuclear staining can be robust, obscuring the ABC signal. Definitive detection of nuclear localized ABC can be confirmed through an ability of classical cadherins to sequester ABC to cell junctions. In tissues, milder antigen retrieval methods can reduce the accessibility of mAb 8E7 to this cross-reacting nuclear antigen. Conclusion These findings reveal that interpretation of nuclear, signaling active β-catenin using monoclonal antibody 8E7 should be considered judiciously, and in conjunction with independent methods. Reviewers This article was reviewed by Frank J. T. Staal (nominated by Rachel Gerstein), Jyoti M. Sen (nominated by Avinash Bhandoola) and Manabu Sugai.

This study seeks to evaluate the comprehension of and attitudes toward proper restraint use among women attending prenatal care clinics.Four-hundred and fifty women were asked to complete a survey during prenatal care visits at county health department clinics; the response rate was 92.0%. Women were asked to provide demographic information and report their frequency and knowledge of proper automobile restraint use.Nearly all subjects (95.4%) either maintained or increased their pre-pregnancy frequency of restraint use. Three-hundred (72.5%) subjects demonstrated that they wore their restraints in the correct location, with women who wore restraints more frequently being more likely to report correct placement. Two-hundred and forty-nine (60.1%) of women reported that restraints would protect their baby if they were involved in a collision, while 48 (11.6%) thought the restraints would cause injury to their baby, and 153 (37.0%) were unsure. Women who reported that restraints would protect them and their baby if involved in a collision were significantly more likely to report always wearing restraints compared with those who were unsure or had negative perceptions of restraints (84.4% vs. 64.6%; p0.0001). The most commonly reported reasons for lack of restraint use were lack of comfort (52.8%) and forgetfulness (42.5%). Only 36.9% percent of women reported receiving information regarding restraint use during their current pregnancy.Many gravid women lack information regarding proper seat belt use and their role in injury prevention. Consequently, the frequency of seat belt use and its correct placement are negatively impacted. Health care workers should take an active role in educating pregnant gravid women about proper restraint use.

Background Ectodomain shedding of GPIbα, a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has been obtained from animal studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17. We report herein novel monoclonal antibodies (MAbs) that specifically inhibit shedding of human GPIbα in platelets and may be potentially developed into an additive to improve platelet storage. Methods A synthetic peptide that corresponds to a human GPIbα sequence containing the shedding cleavage site was used as the antigen for mouse immunization. Hybridoma clones obtained from immunized mice were screened in ELISA assays for binding activities to the shedding-site peptide, and purified human GPIb-IX complex. Flow cytometry-based assays and Western blotting were used to screen for direct binding to washed platelets and inhibition of GPIbα shedding in washed platelets. Platelet aggregometry using different agonists was performed on MAb-treated platelets. Results Six MAbs were obtained and characterized for their abilities to bind GPIbα and inhibit its shedding. The purified antibodies bind, with varying affinities, to immobilized monovalent shedding-site peptide, but all exhibit strong binding to ovalbumin-conjugated shedding-site peptide. Similarly, these antibodies bind with varying affinities to immobilized GPIb-IX, but all exhibit strong binding to human platelets. Considering the multivalent nature of ovalbumin-conjugated peptide and the abundance of GPIbα on the platelet surface, these results indicate the divalent binding of GPIbα to these MAbs on the platelet surface. The prototypic clone, designated 5G6, and its monomeric Fab fragment, bind specifically purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα. It is specific to human GPIbα, as it does not bind mouse platelets. 5G6 exhibits similar inhibitory potency as the broad shedding inhibitor GM6001 in both constitutive and induced GPIbα shedding in human platelets. It does not inhibit shedding of other platelet receptors, such as GPVI and GPV. Treatment of washed human platelets with 5G6 or other MAb shedding inhibitors for an hour does not induce platelet activation or aggregation, and does not exhibit detectable effects on agonist-induced platelet aggregation. Consistently, infusion of 5G6 into human transgenic mice does not induce acute thrombocytopenia, unlike other MAbs targeting the N-terminal domain of GPIbα. Conclusion We have obtained, for the first time, reagents that specifically inhibit ectodomain shedding of human GPIbα with little effect on platelet functions. These reagents will be useful to define the functional significance of GPIbα shedding. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding. Disclosures: No relevant conflicts of interest to declare.

Many test compounds used in toxicity studies produce a "systemic" toxicity manifested as reduced body weight gain. While it is known that reduced weight gain during juvenile growth reduces or delays reproductive competence, the effects of adult-onset feed restriction (FR) and inhibited weight gain on the reproductive system of mice are poorly known. To gain some information on the effects of graded body weight reduction, or reduced body weight gain, on commonly used reproductive endpoints, the studies reported below were conducted at two laboratories, using adult mice that were maintained at 90, 80, and 70% of concurrent control body weight (CBW) for up to 21 weeks. Estrous cyclicity and fertility in the females were significantly affected. While male fertility was variably affected, there was a significant decrease in the number of epididymal sperm and in the number of testicular spermatids in the 70% CBW groups. Testis weight was conserved in both studies; relative testis weight increased in all FR groups. These data can improve the interpretation of future studies by helping to separate chemically induced changes from those produced by reduced body weight gain.

A continuous breeding protocol was utilized to examine the reproductive toxicity of three phthalate esters. CD-1 mice were given diets with either di-n-propyl phthalate (DPrP: 0.0, 1.25, 2.5, or 5.0%), di-n-pentyl phthalate (DPP: 0.0, 0.5, 1.25, or 2.5%), or di-n-octyl phthalate (DOP: 0.0, 1.25, 2.5, or 5.0%). Both male and female mice (20 pairs per treatment group, 40 pairs of control animals) were dosed for 7 days prior to and during a 98-day cohabitation period. Reproductive function was evaluated during the cohabitation period by measuring number of litters per pair, live pups per litter, and pup weight. There was no apparent effect on reproductive function in the animals exposed to DOP at dose levels sufficient to cause a significant increase in liver weight. Both DPP and DPrP were toxic to the reproductive system as evidenced by a complete inhibition of fertility at 1.25 and 2.5% DPP or 5.0% DPrP, and reduced fertility (litters/pair and live pups/litter, 0.5% DPP; live pups/litter, 2.5% DPrP). Toxicity of DPP had a strong male component and female component, whereas DPrP was more toxic to the female than the male reproductive system. DPP and DPrP treatment was associated with decreased body weight, increased liver weight, decreased testis and epididymis weights, decreased epididymal sperm concentration, and elevated seminiferous tubule atrophy. A comparison of seven phthalate esters tested using this continuous breeding protocol indicates the relative order of reproductive toxicity as diethylhexyl, dihexyl, dipentyl, dibutyl, dipropyl; diethyl and dioctyl are nontoxic.

Procedures are described for fractionating cells utilizing a universally applicable cellular affinity chromatography matrix. The affinity matrix consists of immunoabsorption purified goat anti-fluorescein isothiocyanate antibody coupled to large derivatized polyacrylamide beads. This matrix may, in principle, be used to isolate any cell subpopulation provided it has a fluorescein-labeled ligand on its surface. In this report the matrix was used to isolate viable purified fractions of mouse surface Ig-positive cells, Lyt1 cells, and mouse lymphocytes that bind the lectin soybean agglutinin. A preliminary experiment using the anti-FITC beads suggested that this technique can provide a fraction of cells enriched in antigen binding cells. Cell populations isolated by this technique retain their ability to respond to in vitro mitogen stimulation, as well as their ability to be maintained in cell culture following fractionation. Additional experiments using a column consisting of goat anti-rabbit Ig antibody coupled to the same support material are also reported.