History of S. aureus Skin Infection Significantly Associates with History of Eczema Herpeticum in Patients with Atopic Dermatitis.

INTRODUCTION: Patients with atopic dermatitis (AD) are uniquely susceptible to a number of serious viral skin complications, including eczema herpeticum (EH), caused by herpes simplex virus. This study explored the associations between biomarkers of epithelial barrier dysfunction, type 2 immunity, Staphylococcus aureus infection, and S. aureus-specific immunoglobulin responses in a cohort of AD subjects with and without a history of EH (EH+ and EH-, respectively). METHODS: A total of 112 subjects with AD (56 EH+, 56 EH-), matched by age and AD severity, were selected from a registry of over 3000 AD subjects. Logistic regression was used to test the association between history of S. aureus skin infection and history of EH, while controlling for a number of confounders. RESULTS: Compared to those without a history of S. aureus skin infection, subjects with a history of S. aureus skin infection were found to have more than sixfold increased odds of having a history of EH (6.60, 95% confidence interval [CI]: 2.00-21.83), after adjusting for history of other viral skin infections (molluscum contagiosum virus, human papillomavirus), serum total IgE, and IgG against the S. aureus virulence factor SElX. CONCLUSIONS: These findings indicate an important relationship between S. aureus skin infections and EH.

Arrayed Imaging Reflectometry monitoring of anti-viral antibody production throughout vaccination and breakthrough Covid-19.

Immune responses to COVID-19 infection and vaccination are individual and varied. There is a need to understand the timeline of vaccination efficacy against current and yet to be discovered viral mutations. Assessing immunity to SARS-CoV-2 in the context of immunity to other respiratory viruses is also valuable. Here we demonstrate the capability of a fully automated prototype Arrayed Imaging Reflectometry system to perform reliable longitudinal serology against a 34-plex respiratory array. The array contains antigens for respiratory syncytial virus, seasonal influenza, common human coronaviruses, MERS, SARS-CoV-1, and SARS-CoV-2. AIR measures a change in reflectivity due to the binding of serum antibodies to the antigens on the array. Samples were collected from convalescent COVID-19 donors and individuals vaccinated with a two-dose mRNA vaccine regimen. Vaccinated samples were collected prior to the first dose, one week after the first dose, one week after the second dose, and monthly thereafter. Information following booster dose and/or breakthrough infection is included for a subset of subjects. Longitudinal samples of vaccinated individuals demonstrate a rise and fall of SARS-CoV-2 spike antibodies in agreement with general knowledge of the adaptive immune response and other studies. Linear Regression analysis was performed to understand the relationship between antibodies binding to different antigens on the array. Our analysis identified strong correlations between closely related influenza virus strains as well as correlations between SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. A small test of using diluted whole blood from a fingerstick provided clean arrays with antibody binding comparable to serum. Potential applications include assessing immunity in the context of exposure to multiple respiratory viruses, clinical serology, population monitoring to facilitate public health recommendations, and vaccine development against new viruses and virus mutations.

StaphAIR: A Label-Free Antigen Microarray Approach to Detecting Anti-Staphylococcus aureus Antibody Responses in Orthopedic Infections.

Arrayed imaging reflectometry (AIR) is an optical biosensor platform for simple, multiplex measurement of antigen-specific antibody responses in patient blood samples. Here, we report the development of StaphAIR, an 8-plex Staphylococcus aureus antigen array on the AIR platform for profiling antigen-specific anti-S. aureus humoral immune responses. Initial validation experiments with mouse and humanized monoclonal antibodies against the S. aureus autolysin glucosaminidase (Gmd) domain, and subsequent testing with dilution series of pooled positive human serum confirmed analytically robust behavior of the array, with all antigens displaying Langmuir-type dose-response curves. Testing a cohort of 82 patients with S. aureus musculoskeletal infections (MSKI) and 30 healthy individuals enabled discrimination of individual patient responses to different S. aureus antigens, with statistical significance between osteomyelitis patients and controls obtained overall for four individual antigens (IsdA, IsdB, Gmd, and SCIN). Multivariate analyses of the antibody titers obtained from StaphAIR revealed its utility as a potential diagnostic tool for detecting S. aureus MSKI (area under the receiver operating characteristic curve (AUC) > 0.85). We conclude that StaphAIR has utility as a high-throughput immunoassay for studying and diagnosing osteomyelitis in patients.

Disposable photonics for cost-effective clinical bioassays: application to COVID-19 antibody testing.

Decades of research have shown that biosensors using photonic circuits fabricated using CMOS processes can be highly sensitive, selective, and quantitative. Unfortunately, the cost of these sensors combined with the complexity of sample handling systems has limited the use of such sensors in clinical diagnostics. We present a new "disposable photonics" sensor platform in which rice-sized (1 × 4 mm) silicon nitride ring resonator sensor chips are paired with plastic micropillar fluidic cards for sample handling and optical detection. We demonstrate the utility of the platform in the context of detecting human antibodies to SARS-CoV-2, both in convalescent COVID-19 patients and for subjects undergoing vaccination. Given its ability to provide quantitative data on human samples in a simple, low-cost single-use format, we anticipate that this platform will find broad utility in clinical diagnostics for a broad range of assays.

Label-Free, Multiplex Glycan Microarray Biosensor for Influenza Virus Detection.

Newly emerging influenza viruses adapted from animal species pose significant pandemic threats to public health. An understanding of hemagglutinin (HA) receptor-binding specificity to host receptors is key to studying the adaptation of influenza viruses in humans. This information may be particularly useful for predicting the emergence of a pandemic outbreak. Therefore, high-throughput sensing technologies able to profile HA receptor binding can facilitate studies of influenza virus evolution and adaptation in humans. As a step toward this goal, we have prepared glycan-based receptor analogue microarrays on the Arrayed Imaging Reflectometry (AIR) platform. These arrays demonstrate label-free, multiplex detection and discrimination between human and avian influenza viruses. Microarrays consisting of glycan probes with 2,6 and 2,3 linkages were prepared. After first confirming their ability to capture lectins (carbohydrate-binding proteins) with known specificities, we observed that the arrays were able to discriminate between and quantify human pandemic influenza A/California/07/2009 (H1N1pdm) and avian A/Netherlands/1/2000 (H13N8) influenza viruses, respectively. As the method may be expanded to large numbers of glycans (>100) and virus subtypes (H1-H18), we anticipate it can be applied to systematically evaluate influenza virus adaptation in humans. In turn, this will facilitate global influenza surveillance and serve as a new tool enabling health organizations, governments, research institutes, and laboratories to react quickly in the face of a pandemic outbreak.

Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients.

Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We report a multiplex label-free antigen microarray on the Arrayed Imaging Reflectometry (AIR) platform for detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43) and three strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects, and provides quantitative information about total Ig, as well as IgG- and IgM-specific responses.

A Stable Biotin-Streptavidin Surface Enables Multiplex, Label-Free Protein Detection by Aptamer and Aptamer-Protein Arrays Using Arrayed Imaging Reflectometry.

While label-free multiplex sensor technology enables "mixing and matching" of different capture molecules in principle, in practice this has been rarely (if ever) demonstrated. To fill this gap, we developed protocols for the preparation of mixed aptamer-protein arrays on the arrayed imaging reflectometry (AIR) sensing platform using streptavidin as a common attachment point for both biotinylated proteins and aptamers. Doing so required overcoming the noted instability of dried streptavidin monolayers on surfaces. After characterizing this degradation, stable surfaces were obtained using a commercial microarray product. Microarraying through the layer of stabilizer then provided mixed aptamer-antibody arrays. We demonstrate that sensor arrays prepared in this manner are suitable for several probes (thrombin and TGF-ß1 aptamers; avi-tagged protein) and targets.

Selective Sexual Dimorphisms in Musculoskeletal and Cardiopulmonary Pathologic Manifestations and Mortality Incidence in the Tumor Necrosis Factor-Transgenic Mouse Model of Rheumatoid Arthritis.

OBJECTIVE: To examine and quantify the sexual dimorphism in pathologic features manifested in the musculoskeletal and cardiopulmonary systems and incidence of mortality in the tumor necrosis factor-transgenic (TNF-Tg; Tg3647 strain) mouse model of inflammatory erosive arthritis. METHODS: Kaplan-Meier survival estimates were determined in male and female Tg3647 mice and sex-matched wild-type (WT) littermate mice. Longitudinal and cross-sectional pathologic outcomes in the musculoskeletal and cardiopulmonary systems were assessed via ultrasound, micro-computed tomography, grip strength measurements, histologic and serologic analyses, flow cytometry, and skeletal muscle physiologic measures. RESULTS: Compared to male Tg3647 mice (n = 30), female Tg3647 mice (n = 34) had significantly shorter lifespans (P < 0.001) and exhibited the following pathologic features (n = 4-6 per group; P < 0.05 versus male Tg3647 littermates): gross deficits in body mass and muscle weight, early-onset inflammatory arthritis with severity of end-stage arthritis that was as severe as that seen in male transgenic mice, and early onset and increased severity of inflammatory interstitial lung disease (ILD). Histologically, the ILD observed in Tg3647 mice was characterized by inflammatory cell accumulation and pulmonary arteriole thickening, which was concomitant with the presence of right ventricular hypertrophy, a feature that was also more severe in the female compared to male Tg3647 mice (P < 0.05). No sexual dimorphisms in TNF-induced deficient grip strength, axial skeletal growth, or bone loss were found. Globally, the extent of the pathologic changes observed in female Tg3647 mice was greater than that observed in male Tg3647 mice when each group was compared to their sex-matched WT littermates. CONCLUSION: These findings indicate that TNF selectively drives the early onset of arthritis and progression of pathologic changes in the cardiopulmonary system in female Tg3647 mice. These results in the Tg3647 mouse identify it as a suitable model to better understand the mechanisms underlying sexual dimorphism and cardiopulmonary disease in the setting of inflammatory arthritis and other connective tissue diseases.

Prepubertal skeletal muscle growth requires Pax7-expressing satellite cell-derived myonuclear contribution.

The functional role of Pax7-expressing satellite cells (SCs) in postnatal skeletal muscle development beyond weaning remains obscure. Therefore, the relevance of SCs during prepubertal growth, a period after weaning but prior to the onset of puberty, has not been examined. Here, we have characterized mouse skeletal muscle growth during prepuberty and found significant increases in myofiber cross-sectional area that correlated with SC-derived myonuclear number. Remarkably, genome-wide RNA-sequencing analysis established that post-weaning juvenile and early adolescent skeletal muscle have markedly different gene expression signatures. These distinctions are consistent with extensive skeletal muscle maturation during this essential, albeit brief, developmental phase. Indelible labeling of SCs with Pax7CreERT2/+ ; Rosa26nTnG/+ mice demonstrated SC-derived myonuclear contribution during prepuberty, with a substantial reduction at puberty onset. Prepubertal depletion of SCs in Pax7CreERT2/+ ; Rosa26DTA/+ mice reduced myofiber size and myonuclear number, and caused force generation deficits to a similar extent in both fast and slow-contracting muscles. Collectively, these data demonstrate SC-derived myonuclear accretion as a cellular mechanism that contributes to prepubertal hypertrophic skeletal muscle growth.

Castration induces satellite cell activation that contributes to skeletal muscle maintenance.

BACKGROUND: Sarcopenia, the age-related loss of skeletal muscle, is a side effect of androgen deprivation therapy (ADT) for prostate cancer patients. Resident stem cells of skeletal muscle, satellite cells (SCs), are an essential source of progenitors for the growth and regeneration of skeletal muscle. Decreased androgen signaling and deficits in the number and function of SCs are features of aging. Although androgen signaling is known to regulate skeletal muscle, the cellular basis for ADT-induced exacerbation of sarcopenia is unknown. Furthermore, the consequences of androgen deprivation on SC fate in adult skeletal muscle remain largely unexplored. METHODS: We examined SC fate in an androgen-deprived environment using immunofluorescence and fluorescence-activated cell sorting (FACS) with SC-specific markers in young castrated mice. To study the effects of androgen deprivation on SC function and skeletal muscle regenerative capacity, young castrated mice were subjected to experimental regenerative paradigms. SC-derived-cell contributions to skeletal muscle maintenance were examined in castrated Pax7CreER/+; ROSA26mTmG/+ mice. SCs were depleted in Pax7CreER/+; ROSA26DTA/+ mice to ascertain the consequences of SC ablation in sham and castrated skeletal muscles. Confocal immunofluorescence analysis of neuromuscular junctions (NMJs), and assessment of skeletal muscle physiology, contractile properties, and integrity were conducted. RESULTS: Castration led to SC activation, however this did not result in a decline in SC function or skeletal muscle regenerative capacity. Surprisingly, castration induced SC-dependent maintenance of young skeletal muscle. The functional dependence of skeletal muscles on SCs in young castrated mice was demonstrated by an increase in SC-derived-cell fusion within skeletal muscle fibers. SC depletion was associated with further atrophy and functional decline, as well as the induction of partial innervation and the loss of NMJ-associated myonuclei in skeletal muscles from castrated mice. CONCLUSION: The maintenance of skeletal muscles in young castrated mice relies on the cellular contributions of SCs. Considering the well-described age-related decline in SCs, the results in this study highlight the need to devise strategies that promote SC maintenance and activity to attenuate or reverse the progression of sarcopenia in elderly androgen-deprived individuals.

Loss of adult skeletal muscle stem cells drives age-related neuromuscular junction degeneration.

Neuromuscular junction degeneration is a prominent aspect of sarcopenia, the age-associated loss of skeletal muscle integrity. Previously, we showed that muscle stem cells activate and contribute to mouse neuromuscular junction regeneration in response to denervation (Liu et al., 2015). Here, we examined gene expression profiles and neuromuscular junction integrity in aged mouse muscles, and unexpectedly found limited denervation despite a high level of degenerated neuromuscular junctions. Instead, degenerated neuromuscular junctions were associated with reduced contribution from muscle stem cells. Indeed, muscle stem cell depletion was sufficient to induce neuromuscular junction degeneration at a younger age. Conversely, prevention of muscle stem cell and derived myonuclei loss was associated with attenuation of age-related neuromuscular junction degeneration, muscle atrophy, and the promotion of aged muscle force generation. Our observations demonstrate that deficiencies in muscle stem cell fate and post-synaptic myogenesis provide a cellular basis for age-related neuromuscular junction degeneration and associated skeletal muscle decline.

Smad4 restricts differentiation to promote expansion of satellite cell derived progenitors during skeletal muscle regeneration.

Skeletal muscle regenerative potential declines with age, in part due to deficiencies in resident stem cells (satellite cells, SCs) and derived myogenic progenitors (MPs); however, the factors responsible for this decline remain obscure. TGFß superfamily signaling is an inhibitor of myogenic differentiation, with elevated activity in aged skeletal muscle. Surprisingly, we find reduced expression of Smad4, the downstream cofactor for canonical TGFß superfamily signaling, and the target Id1 in aged SCs and MPs during regeneration. Specific deletion of Smad4 in adult mouse SCs led to increased propensity for terminal myogenic commitment connected to impaired proliferative potential. Furthermore, SC-specific Smad4 disruption compromised adult skeletal muscle regeneration. Finally, loss of Smad4 in aged SCs did not promote aged skeletal muscle regeneration. Therefore, SC-specific reduction of Smad4 is a feature of aged regenerating skeletal muscle and Smad4 is a critical regulator of SC and MP amplification during skeletal muscle regeneration.

Inducible depletion of adult skeletal muscle stem cells impairs the regeneration of neuromuscular junctions.

Skeletal muscle maintenance depends on motor innervation at neuromuscular junctions (NMJs). Multiple mechanisms contribute to NMJ repair and maintenance; however muscle stem cells (satellite cells, SCs), are deemed to have little impact on these processes. Therefore, the applicability of SC studies to attenuate muscle loss due to NMJ deterioration as observed in neuromuscular diseases and aging is ambiguous. We employed mice with an inducible Cre, and conditionally expressed DTA to deplete or GFP to track SCs. We found SC depletion exacerbated muscle atrophy and type transitions connected to neuromuscular disruption. Also, elevated fibrosis and further declines in force generation were specific to SC depletion and neuromuscular disruption. Fate analysis revealed SC activity near regenerating NMJs. Moreover, SC depletion aggravated deficits in reinnervation and post-synaptic morphology at regenerating NMJs. Therefore, our results propose a mechanism whereby further NMJ and skeletal muscle decline ensues upon SC depletion and neuromuscular disruption.