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Objective/BackgroundHealthcare workers have experienced high rates of psychiatric symptom burden and occupational attrition during the COVID-19 pandemic. Identifying contributory factors can inform prevention and mitigation measures. Here, we explore the potential contributions of occupational stressors vs COVID-19 infection to insomnia symptoms in US healthcare workers.Patients/MethodsAn online self-report survey was collected between September 2020 and July 2022 from N=594 US healthcare workers, with longitudinal follow-up up to 9 months. Assessments included the Insomnia Severity Index (ISI), the PTSD Checklist for DSM-5 (PCL-5), and a 13-item scale assessing COVID-19 related occupational stressors.ResultsInsomnia was common (45% of participants reported at least moderate and 9.2% reported severe symptoms at one or more timepoint) and significantly associated with difficulty completing work-related tasks, increased likelihood of occupational attrition, and thoughts of suicide or self-harm (all pConclusionsBoth recent occupational stress and personal history of COVID-19 infection were significantly associated with insomnia in healthcare workers. These results suggest that both addressing occupational stressors and reducing rates of COVID-19 infection are important to protect healthcare workers and the healthcare workforce.

Drowsy driving is common and causes 21% of fatal crashes. Individuals at risk include young men, shift workers, older adults, and people with chronic short sleep duration, untreated obstructive sleep apnea (OSA), and narcolepsy. Untreated OSA is a particular concern in commercial drivers, who are at higher risk for the disorder. Treatment for sleep problems such as sleep extension for chronic short sleep, positive airway pressure (PAP) for OSA, pharmacologic treatments, and drowsy driving countermeasures may reduce the risk of crashes. Implementing screening measures to identify common sleep problems contributing to drowsy driving continues to be of high importance.

Our previous study showed that recombinant canine IL-13 (rcaIL-13) stimulated production of allergen-specific IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs. This has also been demonstrated using human IL-13 (huIL-13) and PBMC isolated from human allergy patients. The stimulatory activity of rcaIL-13 was specifically inhibited by a fusion protein of the extracellular domain of canine IL-13Ralpha2 and the Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). In this communication, we report the construction and expression of a non-fused recombinant extracellular domain of canine IL-13Ralpha2 (rcaIL-13Ralpha2) in an E. coli expression system. The E. coli expressed rcaIL-13Ralpha2 was isolated in inclusion bodies, then solubilized in buffer containing denaturants and reducing agents. After refolding and purification, the biological activity of rcaIL-13Ralpha2 was found in the monomer fraction resulting from gel filtration and ion exchange chromatographies. Biological activity of purified rcaIL-13Ralpha2 was demonstrated by the specific inhibition of rcaIL-13 activity in a TF-1 cell proliferation assay. Additionally, rcaIL-13Ralpha2 was found to be active in neutralizing rcaIL-13 induced upregulation of IgE mRNA levels in PBMCs of "high IgE" dogs, which have been bred to exhibit a predisposition for high IgE production and are used as a model for allergic asthma. The data confirm our previous report that the regulatory effects of IL-13 on IgE production in canine PBMCs are similar to those reported in humans. Thus, allergic dogs, such as the "high IgE" producing dogs, may be excellent models for research on IgE-mediated diseases in humans.

Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae (D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109kDa (98/109kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15.

Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa. Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, approximately 25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.

1 ~ ' ~ g ~ Epicutaneous Exposure of High IgE Responder Beagle PupIUllJl~]l pies to Dfarinae Induces Dermatitis and Anti-Mite IgE and T Cell Responses Catherine McCall, Katie Clarke, Kim Stedman, Steve Dreitz, Karen Sellins Heska Corporation, Fort Collins, CO Atopic dermatitis in dogs has many similarities to the human disease, including genetic predisposition, age of onset and distribution of symptoms. Exposure to house dust mites particularly D. farinae, induces perennial atopic dermatitis in susceptible animals. We have developed a model of atopic dermatitis in a strain of laboratory beagle dogs selected for persistent high IgE responses to perinatal allergen exposure. Puppies were sensitized epicutaneously by painting a small area (maximum 1.0 x 1.0 cm) of intact untreated skin in the inguinal or axillary regions, with a paste made by sonicating D. farinae mites in saline. The D. farinae paste was applied at least once per week until the puppies were 4 months old. 8/10 puppies had measurable D. farinae specific serum IgE at 3 months of age, and all puppies developed dermatitis at the site of allergen exposure within 16 weeks. Patch testing of seven 6 12 month old sensitized dogs elicited reactions ranging from mild induration and erythema to pruritic, erythematous papular rashes at the site of allergen application. In positive patch test dogs, histology of 24 and 48 hour biopsies of the reactive skin revealed a cellular infiltrate of eosinophils and mononuclear cells. In 4/6 dogs with serum IgE to D. farinae, patch testing boosted IgE, measured 7 days after allergen application. In addition, in 5/7 dogs, peripheral blood mononuclear cells obtained at 7 days showed enhanced proliferation to D.farinae extract, compared to cells obtained prior to patch testing. In a separate experiment, treatment of patch test skin with FK-506 ointment (a recently approved treatment for human atopic dermatitis) prior to allergen application in 3 dogs abolished the macroscopically visible response to allergen. High IgE responder beagles may therefore be a useful system for preclinical evaluation of new treatments for human atopic dermatitis.