Search

Insomnia is highly prevalent and challenging to treat. We typically regard insomnia as a disorder of the modern world, but physicians and patients have been struggling with this malady for millennia. Here we present the curious historical practice of using electrization or faradization to treat insomnia. We present methods of application, hypotheses regarding mechanism of action, and historical case reports and case series to better understand this phenomenon. We put faradization for insomnia in the context of the modern use of electrical therapies to support and facilitate human health in multiple different health care arenas. Last, we examine current efforts to use these antiquated concepts to address insomnia through transcranial direct current stimulation and cranial electrical stimulation. CITATION: Watson NF, McCall C, Doherty M. Faradization for insomnia: a sleep neurology history. J Clin Sleep Med. 2021;17(2):249–254.

Narcolepsy is a central nervous system hypersomnia disorder characterized by uncontrollable episodes of daytime sleep, sleep state instability, and cataplexy (sudden loss of muscle tone precipitated by emotion). Individuals with narcolepsy report more frequent sleep-related crashes, near crashes, and drowsy driving than drivers with other sleep disorders. As such, evaluating risk of sleep-related crashes is of great importance for this patient population. There are no established guidelines for ensuring driving safety in patients with narcolepsy; however, many providers currently use a combination of subjective report, report of prior crashes or near-misses, report of previously falling asleep while driving, sleepiness screening tools, and maintenance of wakefulness testing (MWT) to determine risk. Driving simulator tests, though often unavailable to the clinician, provide data to support the use of MWT for evaluation of alertness in drivers with narcolepsy. Treatments such as modafinil may improve driving performance; however, the impact of other treatments such as stimulants and sodium oxybate on driving has not been extensively studied. Behavioral and lifestyle modifications may also reduce risk, including scheduled naps, driving only short distances, and avoiding driving after meals, sedating medications, and alcohol intake. Even with effective treatment, alertness in patients with narcolepsy may never reach that of normal drivers; however, studies have suggested that narcolepsy patients may be able to drive safely with appropriate limitations.

In-person visits with a trained therapist have been standard care for patients initiating continuous positive airway pressure (CPAP). These visits provide an opportunity for hands-on training and an in-person assessment of mask fit. However, to improve access, many health systems are shifting to remote CPAP initiation with equipment mailed to patients. While there are potential benefits of a mailed approach, relative patient outcomes are unclear. Specifically, many have concerns that a lack of in-person training may contribute to reduced CPAP adherence. To inform this knowledge gap, we aimed to compare treatment usage after in-person or mailed CPAP initiation.Our medical center shifted from in-person to mailed CPAP dispensation in March 2020 during the COVID-19 pandemic. We assembled a cohort of patients with newly diagnosed obstructive sleep apnea (OSA) who initiated CPAP in the months before (n = 433) and after (n = 186) this shift. We compared 90-day adherence between groups.Mean nightly PAP usage was modest in both groups (in-person 145.2, mailed 140.6 min/night). We did not detect between-group differences in either unadjusted or adjusted analyses (adjusted difference - 0.2 min/night, 95% - 27.0 to + 26.5).Mail-based systems of CPAP initiation may be able to improve access without reducing CPAP usage. Future work should consider the impact of mailed CPAP on patient-reported outcomes and the impact of different remote setup strategies.

STUDY OBJECTIVES: Incorporating registered nurses (RN-level) into obstructive sleep apnea (OSA) management decisions has the potential to augment the workforce and improve patient access, but the appropriateness of such task-shifting in typical practice is unclear. METHODS: Our medical center piloted a nurse triage program for sleep medicine referrals. Using a sleep specialist-designed decision-making tool, nurses triaged patients referred for initial sleep studies to either home sleep apnea test (HSAT) or in-laboratory polysomnography (PSG). During the first 5 months of the program, specialists reviewed all nurse triages. We compared agreement between specialists and nurses. RESULTS: Of 280 consultations triaged by nurses, nurses deferred management decisions to sleep specialists in 6.1% (n = 17) of cases. Of the remaining 263 cases, there was 88% agreement between nurses and specialists (kappa 0.80, 95% confidence interval 0.74–0.87). In the 8.8% (n = 23) of cases where supervising specialists changed sleep study type, specialists changed from HSAT to PSG in 16 cases and from PSG to HSAT in 7. The most common indication for change in sleep study type was disagreement regarding OSA pretest probability (n = 14 of 23). Specialists changed test instructions in 3.0% (n = 8) of cases, with changes either related to the use of transcutaneous carbon dioxide monitoring (n = 4) or adaptive servo-ventilation (n = 4). CONCLUSIONS: More than 80% of sleep study triages by registered nurses in a supervised setting required no sleep specialist intervention. Future research should focus on how to integrate nurses into the sleep medicine workforce in a manner that maximizes efficiency while preserving or improving patient outcomes. CITATION: Donovan LM, Fernandes LA, Williams KM, et al. Agreement of sleep specialists with registered nurses’ sleep study orders in supervised clinical practice. J Clin Sleep Med. 2020;16(2):279–283.

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.

STUDY OBJECTIVES: The field of sleep medicine has been an avid adopter of telehealth, particularly during the COVID-19 pandemic. The goal of this study was to assess patients’ experiences receiving sleep care by telehealth. METHODS: From June 2019 to May 2020, the authors recruited a sample of patients for semi-structured interviews, including patients who had 1 of 3 types of telehealth encounters in sleep medicine: in-clinic video, home-based video, and telephone. Two analysts coded transcripts using content analysis and identified themes that cut across patients and categories. RESULTS: The authors conducted interviews with 35 patients and identified 5 themes. (1) Improved access to care: Patients appreciated telehealth as providing access to sleep care in a timely and convenient manner. (2) Security and privacy: Patients described how home-based telehealth afforded them greater feelings of safety and security due to avoidance of anxiety-provoking triggers (eg, crowds). Patients also noted a potential loss of privacy with telehealth. (3) Personalization of care: Patients described experiences with telehealth care that either improved or hindered their ability to communicate their needs. (4) Patient empowerment: Patients described how telehealth empowered them to manage their sleep disorders. (5) Unmet needs: Patients recognized specific areas where telehealth did not meet their needs, including the need for tangible services (eg, mask fitting). CONCLUSIONS: Patients expressed both positive and negative experiences, highlighting areas where telehealth can be further adapted. As telehealth in sleep medicine continues to evolve, the authors encourage providers to consider these aspects of the patient experience. CITATION: Donovan LM, Mog AC, Blanchard KN, et al. Patient experiences with telehealth in sleep medicine: a qualitative evaluation. J Clin Sleep Med. 2021;17(8):1645–1651.

Sleep and sleep disorders are complex phenotypes with genetic, epigenetic, and environmental influences. Twin studies allow researchers to parse out how these factors influence variability in sleep outcomes such as sleep duration and quality, chronotype, and disorders such as insomnia, hypersomnia, sleep apnea, sleep-related movement disorders, and parasomnias. Twin studies assess the overlap in genetic influences for sleep variance and other medical and psychiatric disorders and allow exploration of gene-environment interactions. Longitudinal twin studies demonstrate how these interactions change over the course of a lifetime. In general, twin studies demonstrate that the heritability of common sleep measures such as duration, quality, and chronotype is about 30-50%; however, this can vary widely between samples according to age, sex, comorbidities, and geographic location. Similarly, the heritability of sleep disorders including insomnia, obstructive sleep apnea, and parasomnias is also around 30-50%, with higher estimates for disorders known to run in families, such as sleepwalking. Heritability estimates for medical and psychiatric problems including obesity, depression, post-traumatic stress disorder, and mortality are higher with short sleep duration (typically < 7 h/n), suggesting that short sleep activates disease-related gene expression. Significant genetic overlap exists between insomnia and issues such as obesity, chronic pain, depression, and psychosis. In this review, we describe the major findings of twin studies related to sleep and how they impact our understanding of this critical component of health and disease.

BackgroundThe COVID-19 pandemic has greatly affected front line health care workers (HCW) and first responders (FR). The infection risk from SARS CoV-2, the high mortality of hospitalized COVID-19 patients and the duration of the pandemic have created sustained and often traumatic stressors affecting HCW and FR.ObjectivesTo assess the relationship of COVID-19 stressor frequency scores to psychiatric rating scale scores amongst HCW and FR. To determine if psychiatric rating scale scores mediate stressor effects on perceived work function and likelihood of remaining in current occupation.DesignObservational, self-report in a convenience sample.Participants118 HCW and FR caring for COVID-19 patients in the United States.Main MeasureCOVID-19 related stressor frequencies were assessed using a 17-item questionnaire. Psychiatric symptoms were assessed with the PTSD Checklist 5 (PCL5), the Patient Health Questionnaire 9 (for depression) (PHQ9), the Insomnia Severity Index (ISI), and the General Anxiety Disorder 7 (GAD7).Key ResultsStressor frequency scores correlated significantly with PCL5 scores (R=.57, pConclusionsThese results direct attention to recognizing potentially treatable psychiatric symptoms, particularly those of PTSD, in HCW and FR experiencing COVID-19 related stressors. They also suggest that mitigating COVID-19 related stressors when possible, such as by providing adequate personal protective equipment, can improve HCW and FR mental health, work function and retention in the health care work force.Strengths and Limitations of this StudyDetailed assessments of participants’ exposure to covid-19 related occupational stressors, current psychiatric symptoms, and self-reported occupational functioning and likelihood of remaining in their current field (functional outcomes).Assessment of the dose-response relationship between exposure to covid-19 related occupational stressors and current psychiatric symptoms and functional outcomes.Mediation analysis quantifying the potential for current psychiatric symptoms to mediate the relationship between exposure to covid-19 related occupational stressors and functional outcomes.Limitations: convenience sample, limited numbers of first responder participants

Narcolepsy is a central nervous system hypersomnia disorder characterized by uncontrollable episodes of daytime sleep, sleep state instability, and cataplexy (sudden loss of muscle tone precipitated by emotion). Individuals with narcolepsy report more frequent sleep-related crashes, near crashes, and drowsy driving than drivers with other sleep disorders. As such, evaluating risk of sleep-related crashes is of great importance for this patient population. There are no established guidelines for ensuring driving safety in patients with narcolepsy; however, many providers currently use a combination of subjective report, report of prior crashes or near-misses, report of previously falling asleep while driving, sleepiness screening tools, and maintenance of wakefulness testing (MWT) to determine risk. Driving simulator tests, though often unavailable to the clinician, provide data to support the use of MWT for evaluation of alertness in drivers with narcolepsy. Treatments such as modafinil may improve driving performance; however, the impact of other treatments such as stimulants and sodium oxybate on driving has not been extensively studied. Behavioral and lifestyle modifications may also reduce risk, including scheduled naps, driving only short distances, and avoiding driving after meals, sedating medications, and alcohol intake. Even with effective treatment, alertness in patients with narcolepsy may never reach that of normal drivers; however, studies have suggested that narcolepsy patients may be able to drive safely with appropriate limitations.

Introduction Initiating treatment with continuous positive airway pressure (CPAP) traditionally relies on in-person visits with trained therapists to provide hands-on instruction regarding CPAP usage and mask fit. To overcome geographic barriers and reduce COVID-19 transmission, health systems increasingly rely on remote set-ups of mailed equipment. Despite a strong rationale for the mailed approach, relative effectiveness is unclear. Methods Our VA medical center shifted from in-person to mailed CPAP dispensation during the COVID-19 pandemic in March 2020. Using VA administrative and wireless CPAP usage data, we assembled a cohort of patients with newly diagnosed obstructive sleep apnea (OSA) who initiated CPAP for the first time from July 2019 to August 2020. Our primary outcome was mean nightly usage over the first 90 days. We compared patients with in-person vs. mailed CPAP dispensation using generalized linear models adjusted for age, gender, race, and Charlson Comorbidity Index. Among patients with >1 hour of overall usage, we compared secondary outcomes of leak, apnea hypopnea index (AHI), and obstructive/central apnea indices. Results We identified 693 patients with newly diagnosed OSA whose CPAP was provided in-person and 296 who had CPAP mailed. Nightly usage in the first 90 days was modest in both groups (in-person: 149.7, mailed: 152.9 min/night), and we did not detect a difference in adjusted models (+7.6 min/night, 95%CI -13.6–28.8). We also did not detect a difference in 95th percentile leak (-1.2 liter/minute, 95%CI -3.3-0.9). Device-detected AHI was relatively low overall (in-person: 3.2, mailed: 4.1 events/hour), but was greater in the mailout group (+1.0/hour, 95%CI 0.2–1.7). AHI differences appeared to be driven by obstructive (+0.5/hour, 95%CI 0.2–0.8) but not central events (-0.1, 95% CI -0.2–0.4). Risk of AHI>5 was comparable between groups (in-person: 17.3%, mailed: 19.0%, OR 1.2, 95%CI 0.8–1.7). Conclusion We were able to switch from an in-person to a mail-based system of CPAP initiation without a change in CPAP adherence or mask leak. While AHI was slightly greater in the mailed group, the clinical significance of this finding is unclear. Future work will need to evaluate the impact of remote CPAP dispensation on patient-centered outcomes. Support (if any) VA Health Services Research and Development, CDA 18–187

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.

Study ObjectivesLong and short sleep duration are associated with greater risk of developing post-traumatic stress disorder (PTSD); however, it is unknown how genetic and environmental influences affect this relationship. Thus, we investigated the association between sleep duration and PTSD symptoms using twin models.MethodsData were obtained from 1865 monozygotic and 758 dizygotic twin pairs enrolled in the community-based Washington State Twin Registry. PTSD symptoms were assessed using the Impact of Events Scale (IES). A classical twin model decomposed the variances of sleep duration and IES score into additive genetic, shared environmental, and unique environmental components. We used correlated factor models to examine the moderation of variance components of sleep duration and IES.ResultsShorter and longer sleep duration were associated with higher IES scores with a quadratic association (p < 0.001). The heritability of sleep duration was 36%, and IES 31%. Variance in sleep duration attributable to shared (b1C1 = 2.91, 95% CI = 1.40 to 4.43; p < 0.001) and unique (b1E1 = 0.18, 95% CI = 0.10 to 0.27; p < 0.001) environment was moderated by IES score. Similarly, but to a lesser extent, variance in IES attributable to additive genetics (b1A2 = −0.23, 95% CI = −0.45 to 0.00; p = 0.048) was moderated by sleep duration.ConclusionsGreater PTSD symptom severity was associated with short and long sleep duration. Increasing PTSD symptoms increased variability in sleep duration primarily via shared environmental factors, whereas decreasing sleep duration increased variability in PTSD symptoms primarily via additive genetic factors. This suggests childhood experiences affect variability of sleep duration and genetic factors affect the variability of PTSD symptoms in trauma-exposed individuals.

Sleep disorders and chronically insufficient sleep are common and often debilitating conditions that are associated with numerous adverse health outcomes and enormous economic costs. Many patients with sleep problems are not routinely screened in primary care clinics, and access to sleep medicine providers as well as diagnostic equipment is limited in many parts of the world. Screening tools offer a means to identify patients with potential sleep disorders who would benefit from further evaluation. As consumer technologies become more available and affordable, many patients are also turning to wearable devices that claim to track sleep duration and quality. In this chapter, we review the more frequently used screening tools and consumer sleep technologies available for identifying patients at risk for sleep disorders.

STUDY OBJECTIVES: Respiratory-related leg movements (RRLMs) may contribute to the cardiovascular risk associated with obstructive sleep apnea (OSA). Selective serotonin reuptake inhibitors (SSRIs), but not bupropion, increase periodic leg movements in sleep. This study examines whether patients with OSA using SSRIs have more RRLMs than those taking bupropion or no antidepressant. METHODS: Patients with an apnea-hypopnea index (AHI) of at least 10 events/h during a full-night diagnostic study or split-night study, who were taking bupropion (n = 32), an SSRI (n = 31), or no antidepressant (n = 31), were selected from a database of prestudy questionnaires. RRLMs were scored according to World Association of Sleep Medicine 2016 standards. RESULTS: Patients using SSRIs had significantly greater overall RRLM% (defined as the percentage of respiratory events associated with a leg movement, including apneas, hypopneas, and respiratory effort-related arousals), RRLM index, and periodic limb movement index relative to patients using bupropion and control patients. The difference between the RRLM% in the SSRI and bupropion groups was limited to patients undergoing split-night studies, and that of the SSRI and control groups was limited to patients undergoing full-night diagnostic studies. CONCLUSIONS: The greater number of RRLMs and PLMs in the SSRI group may contribute to treatment-emergent insomnia often seen with SSRI use. Fragmented sleep and elevated autonomic nervous system activation associated with increased RRLMs in patients with OSA taking SSRIs might also limit the tolerability of antidepressant treatment, as well as increase the risk for cardiovascular disease. CITATION: McCall CA, Winkelman JW. Respiratory-related leg movements of sleep are associated with serotonergic antidepressants but not bupropion. J Clin Sleep Med. 2018;14(9):1569–1576.

Sleep problems are common in the elderly, with a prevalence of close to 50% in those older than age 65 years. Older adults may suffer from disturbances of sleep that are related to medical problems, to psychiatric illness, to primary sleep disorders, to use or withdrawal from drugs/medications, and/or to poor sleep habits. Loss of sleep in older adults is associated with significant morbidity, low quality of life, and mortality. When approaching the diagnosis and management of sleep disorders in elderly patients, it is important to evaluate and treat comorbid conditions. Pharmacologic treatments for sleep, although increasingly common, present significant challenges due to age-related changes in drug metabolism and clearance, side effects, and drug-drug interactions. When possible, it is preferable to manage sleep problems with nonpharmacologic approaches. This review discusses current pharmacologic and nonpharmacologic treatments for sleep problems in the elderly. [ Psychiatr Ann . 2015;45(7):342–347.]

In humans with atopic dermatitis (AD), the epicutaneous application of allergens (atopy patch tests or APT) to which the patients are sensitized often results in the development of inflammation resembling that of spontaneous skin lesions. Dogs are affected with a natural homologue of human AD, but information on the induction of positive patch testing reactions is limited. The objectives of this pilot study were to determine the nature and cellular dynamics of inflammation occurring after APT in dogs hypersensitive to house dust mite and flea allergens. Laboratory Beagles were sensitized experimentally to Dermatophagoides farinae house dust mites (two dogs), Ctenocephalides felis flea saliva (one dog) or both (two dogs). Two other dogs served as nonsensitized controls. Both allergens and saline were applied epicutaneously. Macroscopic evaluations and skin biopsies were performed at 4, 24, 48 and 96 h after starting allergenic challenge. Biopsies were evaluated histologically and immunohistochemically with a panel of monoclonal antibodies specific for canine leucocyte antigens. Positive macroscopic reactions consisted of erythema, oedema and induration, and they occurred between 24 and 96 h after allergen application. Macroscopic and microscopic APT reactions developed only whenever serum IgE was present against tested allergens. Microscopically, positive APT was associated with epidermal hyperplasia, Langerhans' cell hyperplasia, and eosinophil and lymphocyte epidermotropism. Dermal inflammation was mixed and arranged in a superficial perivascular to interstitial pattern. Numerous IgE+-CD1+ dendritic cells and gamma-delta T-lymphocytes were observed. Macroscopically and microscopically, APT reactions in these experimentally sensitized animals resembled those seen in lesional biopsy specimens of dogs and humans with spontaneous AD. Therefore, APT in hypersensitive dogs provides a relevant experimental model to investigate the pathogenesis and treatment of both canine and human AD skin lesions.

We used 2 approaches to investigate possible mechanisms underlying positive responses of chironomids to fragmentation of resources in streambeds. We manipulated arrays of leaf patches on a sandy streambed and asked whether responses of chironomids to patch arrangement and leaf species in patches were genus or instar specific. We manipulated densities of Chironomus riparius instars I and III in laboratory microcosms and assessed density dependent dispersal and settlement behaviors. In streambed arrays, chironomid densities in leaf packs were ∼2× higher in fragmented than in aggregated arrays, but density was not affected by leaf species. Density of larvae in sand cores did not differ with respect to patch arrangement or leaf species. Genus and instar composition differed between sand cores and leaf packs, but did not differ with respect to leaf patch arrangement or leaf species in arrays. Resource partitioning in streambed arrays appeared to occur among congeneric instars rather than among genera....

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.

In human patients with atopic dermatitis, the development of inflammation after epicutaneous application of allergens is dependent upon the presence of IgE and Langerhans’ cells. The purpose of this study was to investigate the role of allergen-specific IgE in the generation of positive patch test reactions in experimentally sensitized IgE-hyperresponsive beagle dogs. Patch tests were performed on seven dogs with Dermatophagoides farinae (Df) and flea saliva (FS) allergens or saline control. Two of the dogs had elevated serum IgE against Df, one against FS, two against both and two were not hypersensitive to either allergen. Positive macroscopic reactions consisted of erythema, oedema and induration, and developed between 24 and 96 h after allergen application. Macroscopic and microscopic patch test reactions developed only when serum IgE was present against tested allergens. Serum IgE levels were positively correlated with dermal cell counts at 48 h (Spearman r = 0.59; P = 0.03) and 96 h (r = 0.68; P

Objective—To determine the prevalence of late-phase reactions to intradermal testing with Dermatophagoides farinae in healthy dogs and dogs with atopic dermatitis and an immediate reaction to D farinae. Animals—6 healthy dogs and 20 dogs with atopic dermatitis and immediate reactions to D farinae. Procedure—Intradermal tests were performed with D farinae at 1:1,000 wt/vol and 1:50,000 wt/vol concentrations, and skin reactivity was evaluated after 0.25, 6, and 24 hours. Serum D farinae-specific IgE antibodies were assayed. Extent of lesions (atopy index) and pruritus (visual analogue scale) were evaluated in dogs with atopic dermatitis. Results—Late-phase reactions were observed in healthy dogs at 6 hours (n = 2 dogs) and 24 hours (1) with the 1:1,000 wt/vol concentration, and at 6 hours (1) and 24 hours (1) with the 1:50,000 wt/vol concentration of allergen. Late-phase reactions in healthy dogs were only observed in dogs with an immediate reaction to D farinae. Late-phase reactions were observed in 11 of 20 dogs with atopic dermatitis at 6 and 24 hours with the 1:1,000 wt/vol concentration and in 10 of 20 at 6 and 24 hours with the 1:50,000 wt/vol concentration of allergen. There was no difference in mean atopy index, mean visual analogue scale of pruritus, or mean serum D farinae-specific IgE concentration of dogs with a late-phase reaction, compared to dogs without a late-phase reaction. Conclusion and Clinical Relevance—Late-phase reactions may be observed after an immediate reaction to intradermal skin testing in healthy and allergic dogs but are more commonly observed in dogs with atopic dermatitis. (Am J Vet Res 2002;63:69–73)

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.

A biological response modifier called ImuVert, derived from the bacterium Serratia marcescens, produced long-lasting elevation of peritoneal exudate cell (PEC) numbers after intraperitoneal injection into mice. These cells had enhanced ability to phagocytose both latex beads and opsonized Listeria monocytogenes. PEC harvested 2-14 days after a single injection of ImuVert killed L. monocytogenes, and ImuVert protected mice from infection by L. monocytogenes, measured both by LD50 and bacterial growth in vivo. Cells harvested 7 and 14 days after ImuVert injection also were tumoricidal, measured as killing of P815 mastocytoma cells, and ImuVert induced macrophages to express tumoricidal properties in vitro. These data suggest that ImuVert has a unique ability to induce a chronic inflammatory response, as other agents do not induce such a long-lasting influx of bactericidal inflammatory cells that also show tumoricidal activity. The consequences of this response appear to include protection from infection by certain bacteria.

Background: House dust mites of the Dermatophagoides genus are the most important cause of perennial allergic disease in both humans and companion animals. Although the major mite allergens for humans are proteins of relatively low molecular weight, this is not the case for dogs. Western blotting shows that canine anti-mite IgE responses are directed primarily toward proteins in the molecular weight range of 50 to 120 kd. Objective: The objectives of this study were to characterize a D farinae allergen with a molecular weight of approximately 60 kd and to isolate the cDNA coding for this allergen. Methods: A protein of apparent molecular weight of 60 kd was identified by Western blotting by using canine serum IgE from house dust mite-sensitized atopic dogs. The protein was purified from homogenized D farinae mite bodies by ammonium sulfate precipitation, followed by gel filtration and cation exchange HPLC. The presence of IgE directed to the 60-kd protein in sera from humans and dogs with dust mite allergy was measured by FcϵRIα-based ELISA. A cDNA encoding a full-length 60-kd protein was isolated from a D farinae cDNA library by a combination of both PCR amplification and hybridization screening. A panel of mAbs specific for the 60-kd protein was generated and used to localize the protein in whole body sections of D farinae mites. Results: ELISA showed that the purified protein bound IgE in 54% of the sera from patients with D farinae allergy. In addition, the 60-kd protein was able to bind IgE in 57% to 77% of D farinae -sensitized dogs. A cDNA was isolated that encoded a protein of 462 amino acids, consisting of a 25 amino acid signal sequence and a 437 amino acid mature protein. The calculated molecular weight of the mature protein is 50 kd, and the amino acid sequence contains a single N-glycosylation site. A protein database search showed homology with multiple chitinases. A mAb specific for the 60-kd chitinase recognized the allergen in the mite digestive system, but fecal pellets did not stain positively for this allergen. Conclusions: A 60-kd D farinae protein (Der f 18), with homology to chitinase, is a major allergen for humans and dogs sensitive to house dust mites. (J Allergy Clin Immunol 2003;112:79-86.)

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcvarepsilonRIalpha). Biotinylated FcvarepsilonRIalpha shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73-146pg/ml. Comparison of assay signals using the labeled FcvarepsilonRIalpha and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcvarepsilonRIalpha is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.

An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.

Mammalian epithelium is a tissue with a very high turnover rate. It consists of a rapidly proliferating compartment comprising basal and suprabasal keratinocytes, from which cells move upwards while differentiating into granular keratinocytes. The end product is shed as an enucleate corneocyte, which has a mechanically rigid, chemically resistant cross-linked keratinous envelope. The loss of the nucleus occurs specifically in the granular keratinocyte layer; here, cells with the classical apoptotic morphology of clumped and marginated condensed chromatin may be observed. This morphology is characteristic of "programmed" cell death in other systems, of which the lymphocyte has been most extensively studied, and is associated with the cleavage of nuclear DNA into nucleosome-sized fragments. In the present investigation we separated newborn mouse skin into basal and granular keratinocyte fractions and examined the state of the DNA in each fraction. Our results indicate that cells in the basal layer, while their DNA is perfectly intact, are preparing to die. DNA fragmentation is initiated in the granular keratinocyte layer and is identical in pattern to that seen in other examples of programmed cell death.

Alopecia (hair loss) is among the most distressing side effects of cancer chemotherapy. Little progress has been made, however, in its prevention or treatment, partly because of the lack of suitable experimental model. In recent work on the treatment of myelogenous leukemia in the rat, the following observations were made: (i) treatment of 8-day-old rats with cytosine arabinoside consistently produced alopecia, and (ii) ImuVert, a biologic response modifier derived from the bacterium Serratia marcescens, uniformly produced complete protection against the alopecia. In subsequent experiments, both cyclophosphamide and doxorubicin also produced alopecia in this model, and the doxorubicin-induced alopecia was prevented by treatment with ImuVert. The potential relevance of these observations to chemotherapy-induced alopecia in the clinical setting should be examined.

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.