Dipeptidyl peptidase-1 inhibition in patients hospitalised with COVID-19: a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial

Background Neutrophil serine proteases are involved in the pathogenesis of COVID-19 and increased serine protease activity has been reported in severe and fatal infection. We investigated whether brensocatib, an inhibitor of dipeptidyl peptidase-1 (DPP-1; an enzyme responsible for the activation of neutrophil serine proteases), would improve outcomes in patients hospitalised with COVID-19. Methods In a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial, across 14 hospitals in the UK, patients aged 16 years and older who were hospitalised with COVID-19 and had at least one risk factor for severe disease were randomly assigned 1:1, within 96 h of hospital admission, to once-daily brensocatib 25 mg or placebo orally for 28 days. Patients were randomly assigned via a central web-based randomisation system (TruST). Randomisation was stratified by site and age (65 years or ≥65 years), and within each stratum, blocks were of random sizes of two, four, or six patients. Participants in both groups continued to receive other therapies required to manage their condition. Participants, study staff, and investigators were masked to the study assignment. The primary outcome was the 7-point WHO ordinal scale for clinical status at day 29 after random assignment. The intention-to-treat population included all patients who were randomly assigned and met the enrolment criteria. The safety population included all participants who received at least one dose of study medication. This study was registered with the ISRCTN registry, ISRCTN30564012. Findings Between June 5, 2020, and Jan 25, 2021, 406 patients were randomly assigned to brensocatib or placebo; 192 (47·3%) to the brensocatib group and 214 (52·7%) to the placebo group. Two participants were excluded after being randomly assigned in the brensocatib group (214 patients included in the placebo group and 190 included in the brensocatib group in the intention-to-treat population). Primary outcome data was unavailable for six patients (three in the brensocatib group and three in the placebo group). Patients in the brensocatib group had worse clinical status at day 29 after being randomly assigned than those in the placebo group (adjusted odds ratio 0·72 [95% CI 0·57–0·92]). Prespecified subgroup analyses of the primary outcome supported the primary results. 185 participants reported at least one adverse event; 99 (46%) in the placebo group and 86 (45%) in the brensocatib group. The most common adverse events were gastrointestinal disorders and infections. One death in the placebo group was judged as possibly related to study drug. Interpretation Brensocatib treatment did not improve clinical status at day 29 in patients hospitalised with COVID-19

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

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Vascular endothelial growth factor blockade induces dermal endothelial cell apoptosis in a clinically relevant skin organ culture model

Vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, plays a key role in physiological processes and is a major contributor to several diseases including cancer and psoriasis. Anti-VEGF therapies are widely used as cancer and ophthalmological treatments. There is some evidence that VEGF blockade may have utility in the management of psoriasis, although their potential has been largely unexplored. We hypothesized that a human skin organ culture could provide a stable ex vivo model in which the cutaneous microvascular network could be studied and experimentally manipulated. Punch biopsies (3 mm) of skin, donated by healthy individuals (39-72 years old, n = 5), were incubated with monoclonal antibody (mAb) to human VEGF (bevacizumab) at doses based on data from animal and clinical studies. After 3-day culture, cell death and proliferation as well as vascular endothelial cell changes were assessed using quantitative immunohistomorphometry. Anti-VEGF mAb at 0.8 mg/mL induced a significant increase in cleaved caspase-3 expression in CD31+ cells (p < 0.05). None of the doses tested increased TUNEL or decreased Ki-67 expression in the basal layer of the epidermis, confirming the model's viability. In addition, the lactate dehydrogenase (LDH) assay showed no increase in LDH activity in treated samples compared to untreated control. The highest anti-VEGF mAb dose (0.8 mg/mL) induced an increase in TUNEL expression in the upper epidermis, which did not correlate with caspase-3 immunoreactivity. Further investigation revealed that anti-VEGF mAb did not change the expression of markers of terminal differentiation such as keratin 10, filaggrin, and involucrin, suggesting that VEGF depletion does not affect keratinocyte terminal differentiation. In contrast to the control group, levels of VEGF protein were undetectable in the culture supernatant of samples treated with 0.8 mg/mL of anti-VEGF mAb, suggesting sufficient dose. Our pilot study provides the first evidence that anti-VEGF therapy promotes endothelial cell apoptosis in human skin ex vivo. Our pragmatic human skin organ culture assay offers a valuable tool for future preclinical endothelial cell and translational microvascular network/anti-angiogenesis research in human skin

The Polyamine Regulator AMD1 Upregulates Spermine Levels to Drive Epidermal Differentiation

Maintaining tissue homeostasis depends on a balance of cell proliferation, differentiation and apoptosis. Within the epidermis the levels of the polyamines putrescine, spermidine and spermine are altered in many different skin conditions yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, AMD1, as a crucial regulator of keratinocyte differentiation. AMD1 protein is upregulated on differentiation and highly expressed in the suprabasal layers of the human epidermis. During keratinocyte differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown/inhibition of AMD1 results in reduced spermine levels and inhibition of keratinocyte differentiation. Supplementing AMD1-knockdown keratinocytes with exogenous spermidine/spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signalling proteins that drive keratinocyte differentiation including KLF4 and ZNF750. These findings demonstrate that human keratinocytes use controlled changes in polyamine levels to modulate gene expression to drive cellular behaviour changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or be used in the treatment of hyper-proliferative skin disorders

Germline intergenic duplications at Xq26.1 underlie Bazex-Dupré-Christol basal cell carcinoma susceptibility syndrome

BACKGROUND: Bazex-Dupré-Christol syndrome (BDCS; MIM301845) is a rare X-linked dominant genodermatosis characterized by follicular atrophoderma, congenital hypotrichosis and multiple basal cell carcinomas (BCCs). Previous studies have linked BDCS to an 11.4 Mb interval on chromosome Xq25-q27.1. However, the genetic mechanism of BDCS remains an open question. OBJECTIVES: To investigate the genetic etiology and molecular mechanisms underlying BDCS. METHODS: We ascertained multiple individuals, from eight unrelated families, affected with BDCS (F1-F8). Whole exome (F1 and F2) and genome sequencing (F3) were performed to identify putative disease-causing variants within the linkage region. Array-comparative genomic hybridization and quantitative PCR were used to explore copy number variations (CNV), followed by long-range gap-PCR and Sanger sequencing to amplify the duplication junctions and to define the head-tail junctions. Hi-C was performed on dermal fibroblasts from two affected individuals with BDCS and one control. Public datasets and tools were used to identify regulatory elements and transcription factor binding sites within the minimal duplicated region. Immunofluorescence was performed in hair follicles, BCCs and trichoepitheliomas from BDCS patients and sporadic BCCs. The ACTRT1 variant (p.Met183Asnfs*17), previously proposed to cause BDCS, was evaluated with allele frequency calculator. RESULTS: In eight BDCS families, we identified overlapping 18-135kb duplications (six inherited and two de novo) at Xq26.1, flanked by ARHGAP36 and IGSF1. Hi-C showed the duplications didn't affect the topologically associated domain (TAD), but may alter the interactions between flanking genes and putative enhancers located in the minimal duplicated region. We detected ARHGAP36 expression near the control hair follicular stem cells compartment, and found increased ARHGAP36 levels in hair follicles in telogen, BCCs and trichoepitheliomas from patients with BDCS. ARHGAP36 was also detected in sporadic BCCs from individuals without BDCS. Our modelling showed the predicted ACTRT1 variants maximum tolerated minor allele frequency in control populations to be orders of magnitude higher than expected for a high-penetrant ultra-rare disorder, suggesting loss-of-function of ACTRT1 variants to be an unlikely cause for BDCS. CONCLUSIONS: Noncoding Xq26.1 duplications cause BDCS., The BDCS duplications most likely lead to dysregulation of ARHGAP36. ARHGAP36 is a potential therapeutic target for both inherited and sporadic BCCs

Germline intergenic duplications at Xq26.1 underlie Bazex–Dupré–Christol basal cell carcinoma susceptibility syndrome

BACKGROUND: Bazex-Dupré-Christol syndrome (BDCS; MIM301845) is a rare X-linked dominant genodermatosis characterized by follicular atrophoderma, congenital hypotrichosis and multiple basal cell carcinomas (BCCs). Previous studies have linked BDCS to an 11.4 Mb interval on chromosome Xq25-q27.1. However, the genetic mechanism of BDCS remains an open question. OBJECTIVES: To investigate the genetic etiology and molecular mechanisms underlying BDCS. METHODS: We ascertained multiple individuals, from eight unrelated families, affected with BDCS (F1-F8). Whole exome (F1 and F2) and genome sequencing (F3) were performed to identify putative disease-causing variants within the linkage region. Array-comparative genomic hybridization and quantitative PCR were used to explore copy number variations (CNV), followed by long-range gap-PCR and Sanger sequencing to amplify the duplication junctions and to define the head-tail junctions. Hi-C was performed on dermal fibroblasts from two affected individuals with BDCS and one control. Public datasets and tools were used to identify regulatory elements and transcription factor binding sites within the minimal duplicated region. Immunofluorescence was performed in hair follicles, BCCs and trichoepitheliomas from BDCS patients and sporadic BCCs. The ACTRT1 variant (p.Met183Asnfs*17), previously proposed to cause BDCS, was evaluated with allele frequency calculator. RESULTS: In eight BDCS families, we identified overlapping 18-135kb duplications (six inherited and two de novo) at Xq26.1, flanked by ARHGAP36 and IGSF1. Hi-C showed the duplications didn't affect the topologically associated domain (TAD), but may alter the interactions between flanking genes and putative enhancers located in the minimal duplicated region. We detected ARHGAP36 expression near the control hair follicular stem cells compartment, and found increased ARHGAP36 levels in hair follicles in telogen, BCCs and trichoepitheliomas from patients with BDCS. ARHGAP36 was also detected in sporadic BCCs from individuals without BDCS. Our modelling showed the predicted ACTRT1 variants maximum tolerated minor allele frequency in control populations to be orders of magnitude higher than expected for a high-penetrant ultra-rare disorder, suggesting loss-of-function of ACTRT1 variants to be an unlikely cause for BDCS. CONCLUSIONS: Noncoding Xq26.1 duplications cause BDCS., The BDCS duplications most likely lead to dysregulation of ARHGAP36. ARHGAP36 is a potential therapeutic target for both inherited and sporadic BCCs

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