Functional consequences of cell type-restricted expression of laminin alpha-5 in mouse placental labyrinth and kidney glomerular capillaries

The labyrinth is the highly vascularized part of the rodent placenta that allows efficient transfer of gases, nutrients, wastes, and other molecules between the maternal and embryonic circulations. These two blood compartments are separated by blastocyst-derived trophoblasts and endothelial cells with an intervening basement membrane that contains laminin and other typical basement membrane components. Previously we reported that the labyrinth of laminin α5 knockout (LMα5−/−) embryos exhibits reduced vascularization and detachment of endothelial cells from the basement membrane, which normally contains LMα5. As very little is known about the origin of this vascular basement membrane, we investigated the cellular requirements for LMα5 expression in the mouse placental labyrinth. By fluorescence-activated cell sorting and RT-PCR we confirmed that both endothelial cells and trophoblasts normally express LMα5. Using Cre-loxP technology and doxycycline-mediated gene expression, we generated genetically mosaic placentas in which either the trophoblasts or the endothelial cells, but not both, expressed LMα5. We found that the overall architecture of the labyrinth was normal as long as one of these two cell types expressed LMα5, even if it was transgene-derived human laminin α5. These results suggest that laminin trimers containing α5 that are synthesized and secreted by endothelium or by trophoblasts are capable of integrating into the basement membrane and promoting normal vascularization of the placenta. Additional studies showed that endothelium-expressed human LMα5 can support vascularization of the kidney glomerulus, consistent with previous studies using a tissue grafting approach

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure

<p>Abstract</p> <p>Background</p> <p>Bronchioles are critical zones in cigarette smoke (CS)-induced lung inflammation. However, there have been few studies on the <it>in vivo </it>dynamics of cytokine gene expression in bronchiolar epithelial cells in response to CS.</p> <p>Methods</p> <p>We subjected C57BL/6J mice to CS (whole body exposure, 90 min/day) for various periods, and used laser capture microdissection to isolate bronchiolar epithelial cells for analysis of mRNA by quantitative reverse transcription-polymerase chain reaction.</p> <p>Results</p> <p>We detected enhanced expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) by bronchial epithelial cells after 10 consecutive days of CS exposure. This was mirrored by increases in neutrophils and KC, MIP-2, TNF-α, and IL-1β proteins in the bronchoalveolar lavage (BAL) fluid. The initial inhalation of CS resulted in rapid and robust upregulation of KC and MIP-2 with concomitant DNA oxidation within 1 hr, followed by a return to control values within 3 hrs. In contrast, after CS exposure for 10 days, this initial surge was not observed. As the CS exposure was extended to 4, 12, 18 and 24 weeks, the bronchiolar KC and MIP-2 expression and their levels in BAL fluid were relatively dampened compared to those at 10 days. However, neutrophils in BAL fluid continuously increased up to 24 weeks, suggesting that neutrophil accumulation as a result of long-term CS exposure became independent of KC and MIP-2.</p> <p>Conclusion</p> <p>These findings indicate variable patterns of bronchiolar epithelial cytokine expression depending on the duration of CS exposure, and that complex mechanisms govern bronchiolar molecular dynamics <it>in vivo</it>.</p

The anatomical distribution of genetic associations

Deeper understanding of the anatomical intermediaries for disease and other complex genetic traits is essential to understanding mechanisms and developing new interventions. Existing ontology tools provide functional, curated annotations for many genes and can be used to develop mechanistic hypotheses; yet information about the spatial expression of genes may be equally useful in interpreting results and forming novel hypotheses for a trait. Therefore, we developed an approach for statistically testing the relationship between gene expression across the body and sets of candidate genes from across the genome. We validated this tool and tested its utility on three applications. First, we show that the expression of genes in associated loci from GWA studies implicates specific tissues for 57 out of 98 traits. Second, we tested the ability of the tool to identify novel relationships between gene expression and phenotypes. Specifically, we experimentally confirmed an underappreciated prediction highlighted by our tool: that white blood cell count – a quantitative trait of the immune system – is genetically modulated by genes expressed in the skin. Finally, using gene lists derived from exome sequencing data, we show that human genes under selective constraint are disproportionately expressed in nervous system tissues

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Functional consequences of cell type-restricted expression of laminin α5 in mouse placental labyrinth and kidney glomerular capillaries.

The labyrinth is the highly vascularized part of the rodent placenta that allows efficient transfer of gases, nutrients, wastes, and other molecules between the maternal and embryonic circulations. These two blood compartments are separated by blastocyst-derived trophoblasts and endothelial cells with an intervening basement membrane that contains laminin and other typical basement membrane components. Previously we reported that the labyrinth of laminin α5 knockout (LMα5-/-) embryos exhibits reduced vascularization and detachment of endothelial cells from the basement membrane, which normally contains LMα5. As very little is known about the origin of this vascular basement membrane, we investigated the cellular requirements for LMα5 expression in the mouse placental labyrinth. By fluorescence-activated cell sorting and RT-PCR we confirmed that both endothelial cells and trophoblasts normally express LMα5. Using Cre-loxP technology and doxycycline-mediated gene expression, we generated genetically mosaic placentas in which either the trophoblasts or the endothelial cells, but not both, expressed LMα5. We found that the overall architecture of the labyrinth was normal as long as one of these two cell types expressed LMα5, even if it was transgene-derived human laminin α5. These results suggest that laminin trimers containing α5 that are synthesized and secreted by endothelium or by trophoblasts are capable of integrating into the basement membrane and promoting normal vascularization of the placenta. Additional studies showed that endothelium-expressed human LMα5 can support vascularization of the kidney glomerulus, consistent with previous studies using a tissue grafting approach

Tumor Necrosis Factor-α from Macrophages Enhances LPS-Induced Clara Cell Expression of Keratinocyte-Derived Chemokine

Tumor necrosis factor (TNF)-α is a cytokine produced by alveolar macrophages in response to LPS in the lung. Clara cells are bronchiolar epithelial cells that produce a variety of proinflammatory cytokines in response to LPS but not to TNF-α. In this study, we examined whether TNF-α affects Clara cell cytokine production in the setting of LPS stimulation. Using a transformed murine Clara cell line (C22), we observed that both LPS and TNF-α induced production of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein (MCP)-1. We also found that simultaneous LPS and TNF-α stimulation is synergistic for KC production, but additive for MCP-1 production. By using a Transwell coculture system of RAW264.7 macrophages and Clara cells isolated from C57Bl/6 mice, we found that macrophages produce a soluble factor that enhances Clara cell KC production in response to LPS. Cocultures of Clara cells from mice deficient in TNF-α receptors with RAW264.7 macrophages demonstrated that the effect of macrophages on Clara cells is mediated primarily via TNF-α. To determine whether these findings occur in vivo, we treated wild-type and TNF receptor–deficient mice intratracheally with LPS and examined the expression of KC. LPS-treated, TNF receptor–deficient mice showed much less KC mRNA in airway epithelial cells compared with wild-type mice. In contrast, a similar number of KC-expressing cells was seen in the lung periphery. Thus, upregulation of KC by Clara cells in the setting of LPS stimulation is largely dependent on TNF-α originating from alveolar macrophages. These findings shed light on macrophage–Clara cell interactions in regulating the pulmonary inflammatory response to LPS