The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.

CLDN1 knock out keratinocytes as a model to investigate multiple skin disorders.

The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.

The effectiveness of brief non-pharmacological interventions in emergency departments and psychiatric inpatient units for people in crisis: A systematic review and narrative synthesis.

OBJECTIVE: Heterogeneous brief non-pharmacological interventions and guidelines exist to treat the burgeoning presentations to both emergency department and inpatient settings, for those in a crisis of mental ill-health. We systematically reviewed the literature to create a taxonomy of these brief non-pharmacological interventions, and review their evaluation methods and effectiveness. METHOD: We conducted a systematic review across Cochrane, CINAHL, DARE, Embase, MEDLINE, PsycINFO databases. Studies meeting quality criteria, using Joanna Briggs Institute tools, were eligible. Interventions were categorised, and outcomes synthesised. RESULTS: Thirty-nine studies were included: 8 randomised controlled trials, 17 quasi-experimental, 11 qualitative studies, and 3 file audits. Taxonomy produced six coherent intervention types: Skills-focussed, Environment-focussed, Special Observation, Psychoeducation, Multicomponent Group and Multicomponent Individual. Despite this, a broad and inconsistent range of outcome measures reflected different outcome priorities and prevented systematic comparison of different types of intervention or meta-analysis. Few brief non-pharmacological interventions had consistent evidential support: sensory modulation rooms consistently improved distress in inpatient settings. Short admissions may reduce suicide attempts and readmission, if accompanied by psychotherapy. Suicide-specific interventions in emergency departments may improve depressive symptoms, but not suicide attempt rates. There was evidence that brief non-pharmacological interventions did not reduce incidence of self-harm on inpatient wards. We found no evidence for frequently used interventions such as no-suicide contracting, special observation or inpatient self-harm interventions. CONCLUSION: Categorising brief non-pharmacological interventions is feasible, but an evidence base for many is severely limited if not missing. Even when there is evidence, the inconsistency in outcomes often precludes clinicians from making inferences, although some interventions show promise.

An increase in glycoprotein concentration on extracellular virions dramatically alters vaccinia virus infectivity and pathogenesis without impacting immunogenicity.

The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L, results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity.

Meta-analysis of clinical risk factors for suicide among people presenting to emergency departments and general hospitals with suicidal thoughts and behaviours.

BACKGROUND: Suicidal thoughts and behaviours (STB) are a common reason for presentation to emergency departments and general hospitals. A meta-analysis of the strength of clinical risk factors for subsequent suicide might aid understanding of suicidal behaviour and help suicide prevention. METHODS: We conducted a meta-analysis of cohort and controlled studies on clinical risk factors and later suicide among people presenting to emergency departments and general hospitals with STB. Data were extracted from papers meeting inclusion criteria, published in Medline, PsycINFO, and Embase between 1 January 1960 and 10 October 2022 using papers located with the search terms ((suicide*).m_titl AND (emergency* OR accident and emergency OR casualty OR general hospital OR toxicology service).mp) or were indexed in PubMed and had titles located with the search terms (suicide* OR self-harm OR self-harm OR self-injury OR self-injury OR self-poisoning OR self-poisoning OR overdose OR para-suicide OR parasuicide [title/abstract]) AND (Emergency department OR emergency room OR Casualty OR general hospital OR toxicology OR accident and emergency [all fields]). Data about the association between clinical risk factors and suicide extracted from three or more studies were included in a random-effects meta-analysis of the odds of later death by suicide. The study was registered in PROSPERO and conducted according to MOOSE and PRISMA guidelines. RESULTS: Seventy-five studies reported on 741,624 people, of which 19,649 died by suicide (2.65%). Male sex (odds ratio (OR) = 1.99) and age (OR = 2.01) were the most consistently reported risk factors. The strongest associations with subsequent death by suicide related to violent self-harm methods at the hospital presentation, including: unspecified violent method (OR = 4.97), any violent method (OR = 4.57) and the specific violent methods of drowning (OR = 4.32), hanging (OR = 4.26), and use of firearms (OR = 10.08). Patients categorised as higher risk using suicide prediction scales or any other method that combined risk factors had moderately increased odds of suicide (OR = 2.58). Younger age, Black and Hispanic ethnicity, overdose, a diagnosis of adjustment disorder, and the absence of any psychiatric diagnosis were protective against suicide. CONCLUSIONS: Most risk factors for suicide among people who have presented with STB are not strongly associated with later suicide. The strongest risk factors relate to self-harm methods. In the absence of clear indicators of future suicide, all people presenting with suicidality warrant a thorough assessment of their needs, and further research is needed before we can meaningfully categorise people with STB according to suicide risk.

Degradation Reduces Microbial Richness and Alters Microbial Functions in an Australian Peatland.

Peatland ecosystems cover only 3% of the world's land area; however, they store one-third of the global soil carbon (C). Microbial communities are the main drivers of C decomposition in peatlands, yet we have limited knowledge of their structure and function. While the microbial communities in the Northern Hemisphere peatlands are well documented, we have limited understanding of microbial community composition and function in the Southern Hemisphere peatlands, especially in Australia. We investigated the vertical stratification of prokaryote and fungal communities from Wellington Plains peatland in the Australian Alps. Within the peatland complex, bog peat was sampled from the intact peatland and dried peat from the degraded peatland along a vertical soil depth gradient (i.e., acrotelm, mesotelm, and catotelm). We analyzed the prokaryote and fungal community structure, predicted functional profiles of prokaryotes using PICRUSt, and assigned soil fungal guilds using FUNGuild. We found that the structure and function of prokaryotes were vertically stratified in the intact bog. Soil carbon, manganese, nitrogen, lead, and sodium content best explained the prokaryote composition. Prokaryote richness was significantly higher in the intact bog acrotelm compared to degraded bog acrotelm. Fungal composition remained similar across the soil depth gradient; however, there was a considerable increase in saprotroph abundance and decrease in endophyte abundance along the vertical soil depth gradient. The abundance of saprotrophs and plant pathogens was two-fold higher in the degraded bog acrotelm. Soil manganese and nitrogen content, electrical conductivity, and water table level (cm) best explained the fungal composition. Our results demonstrate that both fungal and prokaryote communities are shaped by soil abiotic factors and that peatland degradation reduces microbial richness and alters microbial functions. Thus, current and future changes to the environmental conditions in these peatlands may lead to altered microbial community structures and associated functions which may have implications for broader ecosystem function changes in peatlands.

JAK Signaling Is Critically Important in Cytokine-Induced Viral Susceptibility of Keratinocytes.

Little is known about whether type 1 (IFNγ), 2 (IL-4/IL-13), or 3 (IL-17A/IL-22) cytokines affect the susceptibility of keratinocytes (KC) to viruses. These immune pathways predominate in various skin diseases: lupus, atopic dermatitis (AD), and psoriasis, respectively. Janus kinase inhibitors (JAKi) are approved to treat both AD and psoriasis, and are in clinical development for lupus. We evaluated whether these cytokines alter viral susceptibility of KC and determined if this effect is modulated by treatment with JAKi. Viral susceptibility to vaccinia virus (VV) or herpes simplex virus-1 (HSV-1) ± JAKi was assessed in immortalized and primary human KC pretreated with cytokines. Exposure to type 2 (IL-4 + IL-13) or the type 3 (IL-22) cytokines significantly increased KC viral susceptibility. Specifically, there was a peak increase of 12.2 ± 3.1-fold (IL-4 + IL-13) or 7.7 ± 2.8-fold (IL-22) in VV infection as measured by plaque number. Conversely, IFNγ significantly reduced susceptibility to VV (63.1 ± 64.4-fold). The IL-4 + IL-13-induced viral susceptibility was reduced (44 ± 16%) by JAK1 inhibition, while the IL-22-enhanced viral susceptibility was diminished (76 ± 19%) by TYK2 inhibition. IFNγ-mediated resistance to viral infection was reversed by JAK2 inhibition (366 ± 294% increase in infection). Cytokines expressed in AD skin (IL-4, IL-13, IL-22) increase KC viral susceptibility while IFNγ is protective. JAKi that target JAK1 or TYK2 reversed cytokine-enhanced viral susceptibility, while JAK2 inhibition reduced the protective effects of IFNγ.

Web-Based Exercise Program Increases Cervical Strength in Adolescent Athletes.

ABSTRACT: Wilson, JC, Levek, C, Daoud, AK, Brewer, M, Brooks, K, Sochanska, A, Randall, M, and Provance, AJ. Web-based exercise program increases cervical strength in adolescent athletes. J Strength Cond Res 35(4): 1149-1155, 2021-This cohort study aimed to evaluate the efficacy of a web-based 6-week cervical strengthening program on cervical strength in adolescent athletes. It was hypothesized that subjects completing the program would demonstrate significant increase in cervical muscle strength compared with baseline strength. Eighty-three high school soccer athlete subjects were recruited from 2 local nationally competitive soccer clubs. Teams were assigned to either control or intervention groups to minimize information crossover. Fifty subjects were recruited to the intervention group (29 male, 21 female; average age 15.1 years). Thirty-three subjects were recruited to the control group (21 male, 12 female; average age 15.1 years). Intervention group subjects completed a web-based progressive cervical strengthening program over 6 weeks. Cervical strength in flexion, extension, right and left lateral flexion (LLF) was measured in Newton (N) of force at 3 time points during the competitive season for both control and intervention groups. Intervention group subjects significantly increased cervical strength [mean difference (95% confidence interval)] in LLF [24.1 (15.9-32.4)], extension [27.9 (18.4-37.5)], right lateral flexion [18.8 (11.6-26.1)], and flexion [mean ratio: 1.2 (1.1-1.2)] at follow-up testing; whereas control subjects did not see significant changes in strength. A web-based progressive cervical strengthening program improves cervical muscular strength in a population of adolescent athletes over a period of 6 weeks. Such a program could be used by researchers in future studies evaluating the influence on concussion risk and by practitioners as a means of reducing sport-related head and neck injuries.

Vaccinia Virus Phospholipase Protein F13 Promotes Rapid Entry of Extracellular Virions into Cells.

The vaccinia virus protein F13, encoded by the F13L gene, is conserved across the subfamily Chordopoxvirinae and is critical among orthopoxviruses to produce the wrapped form of virus that is required for cell-to-cell spread. F13 is the major envelope protein on the membrane of extracellular forms of virus; however, it is not known if F13 is required in steps postwrapping. In this report, we utilize two temperature-sensitive vaccinia virus mutants from the Condit collection of temperature-sensitive viruses whose small plaque phenotypes have been mapped to the F13L gene. Despite the drastic reduction in plaque size, the temperature-sensitive viruses were found to produce levels of extracellular virions similar to those of the parental strain, Western Reserve (WR), at the permissive and nonpermissive temperatures, suggesting that they are not defective in extracellular virion formation. Analyses of extracellular virions produced by one temperature-sensitive mutant found that those produced at the nonpermissive temperature had undetectable levels of F13 and bound cells with efficiency similar to that of WR but displayed delayed cell entry kinetics. Additionally, low-pH treatment of cells bound by extracellular virions produced at the nonpermissive temperature by the temperature-sensitive reporter virus was unable to overcome a block in infection by bafilomycin A1, suggesting that these virions display increased resistance to dissolution of the extracellular virion envelope. Taken together, our results suggest that F13 plays a role both in the formation of extracellular virions and in the promotion of their rapid entry into cells by enhancing the sensitivity of the membrane to acid-induced dissolution.IMPORTANCE Vaccinia virus (VACV) is an orthopoxvirus and produces two infectious forms, mature virions (MV) and extracellular virions (EV). EV are derived from MV and contain an additional membrane that must first be removed prior to cell entry. F13 is critical for the formation of EV, but a postenvelopment role has not been described. Here, two temperature-sensitive VACV mutants whose deficiencies were previously mapped to the F13L locus are characterized. Both viruses produced EV at the nonpermissive temperature at levels similar to those of a virus that has F13L, yet they had a small plaque phenotype and rate of spread similar to that of an F13L deletion virus. F13 was undetectable on the EV membrane at the nonpermissive temperature, and these EV exhibited delayed cell entry kinetics compared to EV containing F13. This study is the first to conclusively demonstrate a novel role for F13 in cell entry of the EV form of the virus.

Use of praziquantel to control platyhelminth parasites of fish.

Fish are common definitive and intermediate hosts for a variety of parasitic flatworms. In unstressed wild populations, parasitic infections often go unnoticed and are perceived to represent a lesser threat to fish health. In contrast, platyhelminth parasitism of captive fish often results in decreased weight gain and increased mortality which often necessitates chemotherapeutic treatment. The presence of platyhelminth parasites in fish tissues is not only unappealing but in some cases also represents a threat to human health. In veterinary medicine, one of the most commonly used agents with anti-flatworm activity is praziquantel; yet, no praziquantel products are labeled for use in fish in the United States. Veterinarians may use praziquantel preparations approved for other vertebrate species under the Animal Medicinal Drug Use Clarification Act (AMDUCA). However, such extra-label use should be informed by scientific evidence including efficacy and tissue residue studies. Herein, we review studies testing the efficacy of praziquantel for treatment of platyhelminthes along with an assessment of routes of administration, pharmacokinetics, and toxicity information.

Antagonistic Effects of IL-4 on IL-17A-Mediated Enhancement of Epidermal Tight Junction Function.

Atopic dermatitis (AD) is the most common chronic and relapsing inflammatory skin disease. AD is typically characterized by skewed T helper (Th) 2 inflammation, yet other inflammatory profiles (Th1, Th17, Th22) have been observed in human patients. How cytokines from these different Th subsets impact barrier function in this disease is not well understood. As such, we investigated the impact of the canonical Th17 cytokine, IL-17A, on barrier function and protein composition in primary human keratinocytes and human skin explants. These studies demonstrated that IL-17A enhanced tight junction formation and function in both systems, with a dependence on STAT3 signaling. Importantly, the Th2 cytokine, IL-4 inhibited the barrier-enhancing effect of IL-17A treatment. These observations propose that IL-17A helps to restore skin barrier function, but this action is antagonized by Th2 cytokines. This suggests that restoration of IL-17/IL-4 ratio in the skin of AD patients may improve barrier function and in so doing improve disease severity.

Efficacy of injectable praziquantel for elimination of trematode metacercariae in bluegills (Lepomis macrochirus) and quantification of parasite death by propidium iodide staining.

Digenean trematodes have complex life cycles and control of these flatworms can be accomplished by eliminating immature parasite stages from intermediate hosts. In aquaculture systems, presence of trematode metacercariae can negatively impact fish health and lead to economic losses. Posthodiplostomum minimum is a parasite of birds that uses bluegill sunfish (Lepomis macrochirus) as the intermediate host and is commonly found in fish used to stock waterways for recreational purposes. In this study, we evaluated killing of P. minimum metacercariae by injectable praziquantel in naturally infected bluegills. Using propidium iodide staining and motility assessment, we found that 5 mg/kg administered intramuscularly was effective for parasite killing. However, metacercarial death was not apparent until day 7 post-treatment. Our results demonstrated that propidium iodide staining is an effective method for detecting death in metacercariae recovered from treated fish. This method was at least as sensitive as objective motility scoring and provided quantitative assessment of parasite death. Future studies involving treatment of metacercariae in fish with praziquantel may need to be carried out over a period of weeks in order to accurately assess parasite killing and would benefit from using the propidium iodide method.

Molecular Epidemiology of Ascaris Infection Among Pigs in Iowa.

Ascaris is a large roundworm parasite that infects humans and pigs throughout the world. Molecular markers have been used to study parasite transmission in Ascaris-endemic and -nonendemic regions of the world. In the United States, ascariasis still persists in commercial swine and has been designated a neglected disease of poverty in humans. However, relatively few data are available for evaluation of zoonotic transmission. In the present study, we obtained adult worms from abattoirs and characterized each worm on the basis of the gene encoding nuclear internal transcribed sequence (ITS) and mitochondrial cox1 Restriction fragment-length polymorphism analysis of ITS revealed swine, human, and hybrid genotypes. cox1 sequences were compared to all complete sequences available in GenBank, and haplotype analysis demonstrated 92 haplotypes worldwide. Sequences from the parasites in this study represented 10 haplotypes, including 6 new haplotypes that have not been previously described. Our results indicate that anthropozoonotic transmission has occurred in the past, resulting in the presence of human genotypes in pigs and supporting further investigation of zoonotic Ascaris transmission in the United States.

Assessment of in vitro killing assays for detecting praziquantel-induced death in Posthodiplostomum minimum metacercariae.

Control of parasitic infections may be achieved by eliminating developmental stages present within intermediate hosts, thereby disrupting the parasite life cycle. For several trematodes relevant to human and veterinary medicine, this involves targeting the metacercarial stage found in fish intermediate hosts. Treatment of fish with praziquantel is one potential approach for targeting the metacercaria stage. To date, studies investigating praziquantel-induced metacercarial death in fish rely on counting parasites and visually assessing morphology or movement. In this study, we investigate quantitative methods for detecting praziquantel-induced death using a Posthodiplostomum minimum model. Our results revealed that propidium iodide staining accurately identified praziquantel-induced death and the level of staining was proportional to the concentration of praziquantel. In contrast, detection of ATP, resazurin metabolism, and trypan blue staining were poor indicators of metacercarial death. The propidium iodide method offers an advantage over simple visualization of parasite movement and could be used to determine EC50 values relevant for comparison of praziquantel sensitivity or resistance.

Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

Differentiation of keratinocytes or exposure to type 2 cytokines diminishes S. aureus internalization.

Staphylococcus aureus is a leading cause of skin and soft tissue infections. Colonization by this bacterium is increased in individuals with chronic cutaneous diseases such as atopic dermatitis, psoriasis, and bullous pemphigoid. The greater abundance of S. aureus on the skin of subjects with atopic dermatitis in particular has been linked to recurrent cutaneous infections. The primary cell type of the epidermal layer of the skin is the keratinocyte, and it is thought that S. aureus internalized in keratinocytes associates with an increased incidence of skin infections. This study addresses whether keratinocyte differentiation and/or inflammation, two important characteristics altered in cutaneous diseases, influence bacterial internalization. To do this, S. aureus internalization was measured in immortalized and primary keratinocytes that were differentiated using high Ca2+-containing media and/or exposed to cytokines characteristic of atopic dermatitis (IL-4 and IL-13) or psoriasis (IL-17A and IL-22) skin. Our results indicate that S. aureus internalization is uniquely decreased upon keratinocyte differentiation, since this was not observed with another skin-resident bacterium, S. epidermidis. Additionally, treatment with IL-4 + IL-13 diminished bacterial internalization. We interpret this decrease as a mechanism of keratinocyte-based bacterial killing since a similar number of bacterial genomes were detected in cytokine-treated cells, but less viable internalized S. aureus was recovered. Finally, of the receptors reported for S. aureus binding/internalizing into keratinocytes, expression of the α5 component of the α5ß1 integrin was in greatest accordance with the number of internalized bacteria in the context of keratinocyte differentiation.IMPORTANCEIndividuals with chronic cutaneous diseases demonstrate heightened susceptibility for severe and recurrent infections from Staphylococcus aureus. What drives this altered susceptibility remains poorly understood. Previous publications have detected S. aureus as deep as the dermal layer of skin in subjects with atopic dermatitis, suggesting that the cutaneous environment of this disease enables deeper bacterial infiltration than occurs in healthy individuals. This observation indicates that S. aureus has greater opportunity to interact with multiple skin cell types in individuals with chronic inflammatory skin diseases. Identifying the characteristics of the skin that influence bacterial internalization, a common method to establish reservoirs and evade the immune response, is critical for our understanding of S. aureus pathogenesis. The significance of this research is the novel identification of epidermal characteristics that influence S. aureus internalization. With this knowledge, methods can be developed to identify patient populations at greater risk for cutaneous infections.

Electrical Impedance Spectroscopy Quantifies Skin Barrier Function in Organotypic In Vitro Epidermis Models.

Three-dimensional human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in preclinical investigative dermatology and regulatory toxicology. In this study, we investigated the utility of electrical impedance spectroscopy (EIS) for noninvasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for 7 consecutive days did not impact epidermal morphology, and readouts showed comparable trends with HEEs measured only once. We determined 2 frequency ranges in the resulting impedance spectra: a lower frequency range termed EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EISSC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9-engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR, or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EISdiff. Exposure to proinflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EISdiff. This cytokine-associated decrease in EISdiff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a noninvasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects, and repair.

Diagnosis of Anaplasma marginale in cattle at the Iowa State University veterinary diagnostic laboratory 2003-2021.

Anaplasma marginale is a blood-borne rickettsia-like organism that infects cattle erythrocytes and causes anaplasmosis. This study reviews diagnostic data of all A. marginale diagnostics performed from 2003 to August 2021 in the Iowa State Veterinary Diagnostic Laboratory. Typically, the referring veterinarian's initial tentative diagnosis was based on presenting clinical signs or necropsy findings. Confirmatory testing at the ISU-VDL consisted of light microscopy evaluation of stained blood smears or molecular diagnostic procedures. A total of 94 cases were submitted with tissue samples from deceased animals, of which 79 were from Iowa and 15 were from other states. The most typical gross lesions were widespread yellow adipose tissue and splenomegaly. Typical histopathological lesions included marked bile stasis and hemosiderin-laden macrophages in the liver and spleen, respectively. Starting in 2013, when PCR was implemented to confirm cases of anaplasmosis, 315/1125 (28%) were positive to A. marginale, and 810 were negative, using a cut-off of 35.0 Ct. The average (±SD) of the positive PCR Ct was 19.5 (±6.0), and the first and third quartiles were 14.9 and 23.4. Most cases occurred between August and November, peaking in September, whether from necropsies or positive blood samples by PCR. The most common tick observed in Iowa, Dermacentor variabilis, is likely the main vector for transmission. Further surveys should be conducted to estimate seroprevalence by geographical location, the density of cattle populations, distribution of known vectors according to season, and strains of A. marginale.

S. aureus virulence factors decrease epithelial barrier function and increase susceptibility to viral infection.

Individuals with atopic dermatitis (AD) are highly colonized by Staphylococcus aureus and are more susceptible to severe viral complications. We hypothesized that S. aureus secreted virulence factors may alter keratinocyte biology to enhance viral susceptibility through disruption of the skin barrier, impaired keratinocyte differentiation, and/or inflammation. To address this hypothesis, human keratinocytes were exposed to conditioned media from multiple S. aureus strains that vary in virulence factor production (USA300, HG003, and RN4220) or select purified virulence factors. We have identified the S. aureus enterotoxin-like superantigen SElQ, as a virulence factor of interest, since it is highly produced by USA300 and was detected on the skin of 53% of AD subjects (n = 72) in a study conducted by our group. Treatment with USA300 conditioned media or purified SElQ resulted in a significant increase in keratinocyte susceptibility to infection with vaccinia virus, and also significantly decreased barrier function. Importantly, we have previously demonstrated that keratinocyte differentiation influences susceptibility to viral infection, and our qPCR observations indicated that USA300 S. aureus and SElQ alter differentiation in keratinocytes. CRISPR/Cas9 was used to knock out CD40, a potential enterotoxin receptor on epithelial cells. We found that CD40 expression on keratinocytes was not completely necessary for SElQ-mediated responses, as measured by proinflammatory cytokine expression and barrier function. Together, these findings support that select S. aureus virulence factors, particularly SElQ, enhance the susceptibility of epidermal cells to viral infection, which may contribute to the increased cutaneous infections observed in individuals with AD. IMPORTANCE Staphylococcus aureus skin colonization and infection are frequently observed in individuals with atopic dermatitis. Many S. aureus strains belong to the clonal group USA300, and these strains produce superantigens including the staphylococcal enterotoxin-like Q (SElQ). Our studies highlight that SElQ may play a key role by altering keratinocyte differentiation and reducing barrier function; collectively, this may explain the AD-specific enhanced infection risk to cutaneous viruses. It is unclear what receptor mediates SElQ's effects on keratinocytes. We have shown that one putative surface receptor, CD40, was not critical for its effects on proinflammatory cytokine production or barrier function.