All shook up: how the COVID-19 pandemic rocked pediatric asthma care.

PURPOSE OF REVIEW: Asthma management is a crucial aspect of public health. The landscape of asthma management underwent significant change in the wake of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. These changes greatly affected existing patients, individuals suffering with undiagnosed illness, providers, and the healthcare system as a whole. RECENT FINDINGS: Providers had to navigate through the potential risk of exposure while weighing the benefit of office visits for patients. This promoted the rapid uptake of telemedicine and virtual outreach, as well as modifications to acute management and controller therapies. Telehealth allowed for the remote monitoring of these patient populations, increased compliance with home-based self-management, and an emphasis on patient education. Furthermore, the pandemic underscored the importance of proactive asthma management as many individuals were left untreated or undiagnosed for various reasons. It is evident that the SARS-CoV-2 pandemic reshaped the landscape of various components of the healthcare system, including asthma management, necessitating innovative approaches to monitoring and patient education. SUMMARY: Understanding the lessons learned from this time period is crucial for enhancing the resilience of our health system in the wake of future challenges that may be posed against our system.

A fine-tuned yeast surface-display/secretion platform enables the rapid discovery of neutralizing antibodies against Clostridioides difficile toxins.

BACKGROUND: Neutralizing antibody plays a key role in protecting hosts from invasive pathogens and their virulent components. Current high-throughput assays for antibody screening are based on binding activities. However, those antibodies with high affinity may not have neutralizing activities. Subsequent functionality assays are necessary to identify neutralizing antibodies from binders with high affinity to their target antigens, which is laborious and time-consuming. Therefore, a versatile platform that can rapidly identify antibodies with both high binding affinity and neutralizing activity is desired to curb future pandemics like COVID-19. RESULTS: In this proof-of-concept study, we adapted Saccharomyces cerevisiae to either display human antibodies on the yeast surface or secrete soluble antibodies into the cultivation supernatant under a controllable 'switch' through different carbon source induced promoters. Initially, an engineered chimeric-bispecific Fab antibody, derived from humanized nanobodies against both Clostridioides difficile toxin A and B (TcdA and TcdB), was successfully expressed either on the yeast cell surface or in the culture medium with intact bioactivity, suggesting the applicability of our system in antibody display and secretion. Next, a combinatorial Fab library was constructed from B cells isolated from a convalescent patient with a high serological neutralizing titer against TcdB. Following three rounds of magnetic bead enrichment and one round of flow cytometry sorting, antibodies against TcdB were enriched efficiently. We then sorted out single binders with high binding affinity and induced them to express soluble antibodies in culture medium. The neutralizing activity of culture supernatant was analyzed using cell-based assay immediately. This way, we rapidly identified two unique neutralizers (out of seven binders) that can neutralize the cytotoxicity of TcdB. CONCLUSION: The antibody screening platform described here simplifies the neutralizing antibody discovery procedure and will be an attractive alternative for screening functional antibodies against infectious diseases.

Quality improvement for paediatric asthma care in acute settings.

PURPOSE OF REVIEW: This is a summative review of recent trends and novel programming integrated into various clinical settings (i.e. emergency departments, urgent care centres and paediatric clinics) to enhance the quality of care received by paediatric asthma patients Asthma is the most common chronic disease in paediatric patients and despite recognized national management guidelines, implementation and aftercare, especially in the emergency room, remain challenging. RECENT FINDINGS: Outcome-based systematic quality improvement initiatives are described as well as evidence-based recommendations to enhance the education of providers, patients and caregivers. SUMMARY: Many of the care initiatives described in the literature have been integrated into the emergency room. The authors feel some of these process improvements, such as pathway-based care, reducing time to delivery of medications, and personalized asthma education, may also be applicable and add value to clinical practice in additional community-based acute care settings such as urgent care centers and paediatric clinics.

A Novel DNA Vaccine Platform Enhances Neo-antigen-like T Cell Responses against WT1 to Break Tolerance and Induce Anti-tumor Immunity.

Tumor-associated antigens have emerged as important immunotherapeutic targets in the fight against cancer. Germline tumor antigens, such as WT1, Wilms' tumor gene 1, are overexpressed in many human malignancies but have low expression in somatic tissues. Recent vaccination approaches to target WT1 have been hampered by poor in vivo immune potency, likely due to the conserved self-antigen nature of WT1. In this study, we use a novel synthetic micro-consensus SynCon DNA vaccine approach with the goal of breaking tolerance and increasing vaccine immune potency. This approach induced new, neo-antigen-like responses that were superior to those induced by native WT1 DNA immunogens for driving T cell immunity and breaking tolerance. Non-human primates (NHPs) vaccinated with SynCon WT1 antigens elicited immune responses against native rhesus WT1 peptides. When delivered by electroporation (EP) in mice, SynCon-based WT1 constructs elicited strong CD4 and CD8 T cell responses (including IFN-γ, CD107a, and TNF-α) to both native and consensus peptides. In addition, SynCon WT1 vaccine-induced antibodies recognized native WT1 in vitro. Vaccination with the SynCon WT1 immunogens was capable of slowing tumor growth in therapeutic models in vivo. These data support the further study of synthetic consensus DNA vaccines for breaking tolerance to important germline antigens.

Outcomes of neonates born through meconium-stained amniotic fluid pre and post 2015 NRP guideline implementation.

A shift in the Neonatal Resuscitation Program (NRP) guidelines occurred in 2015 from routine intubation and endotracheal suctioning of all meconium-stained non-vigorous infants towards less aggressive interventions based on response to initial resuscitation. This study aims to examine the impact of this change on outcomes of non-vigorous infants born through meconium-stained amniotic fluid at a level III academic NICU encompassing years before and after the change in guideline. This single-center retrospective study compared NICU therapies and clinical outcomes of 117 non-vigorous newborns pre-guideline implementation to 106 non-vigorous newborns post-guideline implementation. Nearly two thirds of infants in the pre-guideline cohort received endotracheal suctioning with recovery of meconium compared to less than a third of infants in the post-guideline cohort (p<0.01). Though a higher proportion of the pre-guideline cohort were admitted to the NICU for respiratory issues compared to the post-guideline cohort, the two groups did not differ significantly with regard to morbidity and therapies. Despite a marked reduction in rates of intubation and endotracheal suctioning, there is no difference in outcomes between pre-guideline implementation vs post-guideline implementation in non-vigorous meconium-stained infants, supporting the recent NRP guideline change and highlighting the benefit of expectant management.

The development of live biotherapeutics against Clostridioides difficile infection towards reconstituting gut microbiota.

Clostridioides difficile is the most prevalent pathogen of nosocomial diarrhea. In the United States, over 450,000 cases of C. difficile infection (CDI), responsible for more than 29,000 deaths, are reported annually in recent years. Because of the emergence of hypervirulent strains and strains less susceptible to vancomycin and fidaxomicin, new therapeutics other than antibiotics are urgently needed. The gut microbiome serves as one of the first-line defenses against C. difficile colonization. The use of antibiotics causes gut microbiota dysbiosis and shifts the status from colonization resistance to infection. Hence, novel CDI biotherapeutics capable of reconstituting normal gut microbiota have become a focus of drug development in this field.

Does Ethnic Identity Moderate the Relationship between Racial Discrimination and Cannabis Use among US- and non-US Born Black Emerging Adults?

Race-based experiences of discrimination (EOD) have been documented as a risk factor for substance use among Black individuals, particularly during emerging adulthood, with ethnic identity serving as a protective influence. Our study extends epidemiologic research on EOD and cannabis use by examining this relation in U.S. and non-U.S. born Black emerging adults across immigrant generations (N = 466, 30% first-generation immigrants, 49% second-generation immigrants, and 21% non-immigrants). Results from self-reported data indicated EOD were associated with an increased likelihood of lifetime cannabis use, while ethnic identity was not significantly related to any odds of lifetime cannabis use. Odds of lifetime use was lower among first-generation immigrants compared to non-immigrants. Although the interaction between ethnic identity and EOD was not significantly associated with cannabis use, the results indicated that for second-generation immigrants, the probability of lifetime use decreased as ethnic identity increased. These findings underscore the importance of ethnic identity as a protective factor for cannabis use, especially among Black immigrants who have been racialized over generations in the United States, providing implications for future study and intervention.

Much Ado about Sleep: Current Concepts on Mechanisms and Predisposition to Pediatric Obstructive Sleep Apnea.

Obstructive Sleep Apnea (OSA) is a form of sleep-disordered breathing characterized by upper airway collapse during sleep resulting in recurring arousals and desaturations. However, many aspects of this syndrome in children remain unclear. Understanding underlying pathogenic mechanisms of OSA is critical for the development of therapeutic strategies. In this article, we review current concepts surrounding the mechanism, pathogenesis, and predisposing factors of pediatric OSA. Specifically, we discuss the biomechanical properties of the upper airway that contribute to its primary role in OSA pathogenesis and examine the anatomical and neuromuscular factors that predispose to upper airway narrowing and collapsibility.

A probiotic yeast-based immunotherapy against Clostridioides difficile infection.

Antibiotic-resistant Clostridioides difficile is an anaerobic Gram-positive bacterium that colonizes the colon and is responsible for more than 29,000 deaths in the United States each year. Hence, C. difficile infection (CDI) poses an urgent threat to public health. Antibody-mediated neutralization of TcdA and TcdB toxins, the major virulence factors of CDI, represents an effective strategy to combat the disease without invoking antibiotic resistance. However, current antitoxin approaches are mostly based on parenteral infusion of monoclonal antibodies that are costly, narrow spectrum, and not optimized against the intestinal disease. Here, we engineered probiotic Saccharomyces boulardii to constitutively secrete a single tetra-specific antibody that potently and broadly neutralized both toxins and demonstrated protection against primary and recurrent CDI in both prophylactic and therapeutic mouse models of disease. This yeast immunotherapy is orally administered, can be used concurrently with antibiotics, and may have potential as a prophylactic against CDI risk and as a therapeutic for patients with CDI.

Selective POTE paralogs on chromosome 2 are expressed in human embryonic stem cells.

POTE is a primate-specific gene family that encodes cancer testis antigens that contain three domains, although the proteins vary greatly in size. The amino-terminal domain is novel and has three cysteine-rich domains of 37 amino acids. The second and third domains are rich in ankyrin repeats and spectrin-like helices respectively. In humans, 13 highly homologous paralogs are dispersed among eight chromosomes. Some members of the POTE gene family have an actin insertion at the carboxyl end of the protein. The expression of the POTE gene in normal adult tissues is restricted, but several POTE paralogs are frequently expressed in many cancers including breast, prostate, and lung cancers. We show here that POTE is expressed in several human embryonic stem (ES) cell lines. We found that UC06, WA01 and ES03 cell lines express mainly a POTE-2gamma transcript but ES02 and ES04 cell lines predominantly express POTE-2alpha. The WA09 cell line expressed both POTE-2gamma and POTE-2alpha. There is no detectable POTE gene expression in fetal tissues (ages 16-36 weeks). The POTE paralogs that are expressed in ES cells may have a specific function during lineage-specific differentiation of ES cells.

POTE paralogs are induced and differentially expressed in many cancers.

To identify new antigens that are targets for the immunotherapy of prostate and breast cancer, we used expressed sequence tag and genomic databases and discovered POTE, a new primate-specific gene family. Each POTE gene encodes a protein that contains three domains, although the proteins vary greatly in size. The NH2-terminal domain is novel and has properties of an extracellular domain but does not contain a signal sequence. The second and third domains are rich in ankyrin repeats and spectrin-like helices, respectively. The protein encoded by POTE-21, the first family member discovered, is localized on the plasma membrane of the cell. In humans, 13 highly homologous paralogs are dispersed among eight chromosomes. The expression of POTE genes in normal tissues is restricted to prostate, ovary, testis, and placenta. A survey of several cancer samples showed that POTE was expressed in 6 of 6 prostate, 12 of 13 breast, 5 of 5 colon, 5 of 6 lung, and 4 of 5 ovarian cancers. To determine the relative expression of each POTE paralog in cancer and normal samples, we employed a PCR-based cloning and analysis method. We found that POTE-2alpha, POTE-2beta, POTE-2gamma, and POTE-22 are predominantly expressed in cancers whereas POTE expression in normal tissues is somewhat more diverse. Because POTE is primate specific and is expressed in testis and many cancers but only in a few normal tissues, we conclude POTE is a new primate-specific member of the cancer-testis antigen family. It is likely that POTE has a unique role in primate biology.

Cysteine Protease-Mediated Autocleavage of Clostridium difficile Toxins Regulates Their Proinflammatory Activity.

BACKGROUND & AIMS: Clostridium difficile toxin A (TcdA) and C difficile toxin toxin B (TcdB), the major virulence factors of the bacterium, cause intestinal tissue damage and inflammation. Although the 2 toxins are homologous and share a similar domain structure, TcdA is generally more inflammatory whereas TcdB is more cytotoxic. The functional domain of the toxins that govern the proinflammatory activities of the 2 toxins is unknown. METHODS: Here, we investigated toxin domain functions that regulate the proinflammatory activity of C difficile toxins. By using a mouse ilea loop model, human tissues, and immune cells, we examined the inflammatory responses to a series of chimeric toxins or toxin mutants deficient in specific domain functions. RESULTS: Blocking autoprocessing of TcdB by mutagenesis or chemical inhibition, while reducing cytotoxicity of the toxin, significantly enhanced its proinflammatory activities in the animal model. Furthermore, a noncleavable mutant TcdB was significantly more potent than the wild-type toxin in the induction of proinflammatory cytokines in human colonic tissues and immune cells. CONCLUSIONS: In this study, we identified a novel mechanism of regulating the biological activities of C difficile toxins in that cysteine protease-mediated autoprocessing regulates toxins' proinflammatory activities. Our findings provide new insight into the pathogenesis of C difficile infection and the design of therapeutics against the disease.

Intravenous adenovirus expressing a multi-specific, single-domain antibody neutralizing TcdA and TcdB protects mice from Clostridium difficile infection.

Clostridium difficile infection (CDI) is the most common cause of antibiotic-associated diarrhea and colitis in developed countries. The disease is mainly mediated via two major exotoxins TcdA and TcdB secreted by the bacterium. We have previously developed a novel, potently neutralizing, tetravalent and bispecific heavy-chain-only single domain (VHH) antibody to both TcdA and TcdB (designated as ABA) that reverses fulminant CDI in mice. Since ABA has a short serum half-life, in this study a replication-deficient recombinant adenovirus expressing ABA was generated and the long-lasting expression of functional ABA was demonstrated in vitro and in vivo Mice transduced with one dose of the adenovirus displayed high levels of serum ABA for more than1 month and were fully protected against systemic toxin challenges. More importantly, the ABA delivered by the adenovirus protected mice from both primary and recurrent CDI. Thus, replication-deficient adenoviral vector may be used to deliver neutralizing antibodies against the toxins in order to prevent CDI and recurrence.

Evolution and expression of chimeric POTE-actin genes in the human genome.

We previously described a primate-specific gene family, POTE, that is expressed in many cancers but in a limited number of normal organs. The 13 POTE genes are dispersed among eight different chromosomes and evolved by duplications and remodeling of the human genome from an ancestral gene, ANKRD26. Based on sequence similarity, the POTE gene family members can be divided into three groups. By genome database searches, we identified an actin retroposon insertion at the carboxyl terminus of one of the ancestral POTE paralogs. By Northern blot analysis, we identified the expected 7.5-kb POTE-actin chimeric transcript in a breast cancer cell line. The protein encoded by the POTE-actin transcript is predicted to be 120 kDa in size. Using anti-POTE mAbs that recognize the amino-terminal portion of the POTE protein, we detected the 120-kDa POTE-actin fusion protein in breast cancer cell lines known to express the fusion transcript. These data demonstrate that insertion of a retroposon produced an altered functional POTE gene. This example indicates that new functional human genes can evolve by insertion of retroposons.