Prevalence of fatigue and cognitive impairment after traumatic brain injury.

BACKGROUND: Following traumatic brain injury (TBI) some patients develop lingering comorbid symptoms of fatigue and cognitive impairment. The mild cognitive impairment self-reported by patients is often not detected with neurocognitive tests making it difficult to determine how common and severe these symptoms are in individuals with a history of TBI. This study was conducted to determine the relative prevalence of fatigue and cognitive impairment in individuals with a history of TBI. METHODS: The Fatigue and Altered Cognition Scale (FACs) digital questionnaire was used to assess self-reported fatigue and cognitive impairment. Adults aged 18-70 were digitally recruited for the online anonymous study. Eligible participants provided online consent, demographic data, information about lifetime TBI history, and completed the 20 item FACs questionnaire. RESULTS: A total of 519 qualifying participants completed the online digital study which included 204 participants with a history of TBI of varied cause and severity and 315 with no history of TBI. FACs Total Score was significantly higher in the TBI group (57.7 ± 22.2) compared to non-TBI (39.5 ± 23.9; p<0.0001) indicating more fatigue and cognitive impairment. When stratified by TBI severity, FACs score was significantly higher for all severity including mild (53.9 ± 21.9, p<0.0001), moderate (54.8 ± 24.4, p<0.0001), and severe (59.7 ± 20.9, p<0.0001) TBI. Correlation analysis indicated that more severe TBI was associated with greater symptom severity (p<0.0001, r = 0.3165). Ancillary analysis also suggested that FACs scores may be elevated in participants with prior COVID-19 infection but no history of TBI. CONCLUSIONS: Adults with a history of even mild TBI report significantly greater fatigue and cognitive impairment than those with no history of TBI, and symptoms are more profound with greater TBI severity.

The altered TBI fecal microbiome is stable and functionally distinct.

Introduction: Patients who suffer a traumatic brain injury (TBI) often experience chronic and sometimes debilitating sequelae. Recent reports have illustrated both acute and long-term dysbiosis of the gastrointestinal microbiome with significant alterations in composition and predicted functional consequences. Methods: Working with participants from past research, metagenomic stability of the TBI- associated fecal microbiome (FMB) was evaluated by custom qPCR array comparing a fecal sample from 2015 to one collected in 2020. Metatranscriptomics identified differently expressed bacterial genes and biochemical pathways in the TBI FMB. Microbiota that contributed the largest RNA amounts identified a set of core bacteria most responsible for functional consequences of the TBI FMB. Results: A remarkably stable FMB metagenome with significant similarity (two-tail Spearman nonparametric correlation p < 0.001) was observed between 2015 and 2020 fecal samples from subjects with TBI. Comparing the 2020 TBI FMB metagenome to FMBs from healthy controls confirmed and extended the dysbiotic genera and species. Abundance differences between average TBI and healthy FMBs revealed Bacteroides caccae, B. uniformis, Blautia spp., Collinsella spp., Dialister spp., and Ordoribacter spp. were significantly different. Functionally, the Parabacteroides genus contributed the highest percentage of RNA sequences in control FMBs followed by the Bacteroides genus as the second highest contributor. In the TBI FMB, the Corynebacterium genus contributed the most RNA followed by the Alistipes genus. Corynebacterium and Pseudomonas were distinct in the top 10 contributing genera in the TBI FMB while Parabacteroides and Ruminococcus were unique to the top 10 in controls. Comparing RNA profiles, TBI samples had ∼1.5 fold more expressed genes with almost 700 differently expressed genes (DEGs) mapped to over 100 bacterial species. Bioinformatic analysis associated DEGs with pathways led identifying 311 functions in the average TBI FMB profile and 264 in the controls. By average profile comparison, 30 pathways had significantly different abundance (p < 0.05, t-test) or were detected in >80% of the samples in only one of the cohorts (binary distinction). Discussion: Functional differences between TBI and healthy control FMBs included amino acid metabolism, energy and carbon source usage, fatty acid metabolism, bacterial cell wall component production and nucleic acid synthesis and processing pathways. Together these data shed light on the functional consequences of the dysbiotic TBI FMB decades after injury.

Low growth hormone secretion associated with post-acute sequelae SARS-CoV-2 infection (PASC) neurologic symptoms: A case-control pilot study.

OBJECTIVE: To determine if patients that develop lingering neurologic symptoms of fatigue and "brain fog" after initial recovery from coronavirus disease 2019 (COVID-19) have persistent low growth hormone (GH) secretion as seen in other conditions with similar symptom etiology. DESIGN: In this case-control observational pilot study, patients reporting lingering neurologic post-acute sequelae of SARS-CoV-2 (PASC, n = 10) symptoms at least 6 months after initial infection were compared to patients that recovered from COVID-19 without lingering symptoms (non-PASC, n = 13). We compared basic blood chemistry and select metabolites, lipids, hormones, inflammatory markers, and vitamins between groups. PASC and non-PASC subjects were tested for neurocognition and GH secretion, and given questionnaires to assess symptom severity. PASC subjects were also tested for glucose tolerance and adrenal function. RESULTS: PASC subjects reported significantly worse fatigue, sleep quality, depression, quality of life, and gastrointestinal discomfort compared to non-PASC. Although PASC subjects self-reported poor mental resilience, cognitive testing did not reveal significant differences between groups. Neurologic PASC symptoms were not linked to inflammatory markers or adrenal insufficiency, but were associated with reduced growth hormone secretion. CONCLUSIONS: Neurologic PASC symptoms are associated with gastrointestinal discomfort and persistent disruption of GH secretion following recovery from acute COVID-19. (www. CLINICALTRIALS: gov; NCT04860869).

Efficient assessment of brain fog and fatigue: Development of the Fatigue and Altered Cognition Scale (FACs).

Debilitating symptoms of fatigue and accompanying "brain fog" are observed among patients with various chronic health conditions. Unfortunately, an efficient and psychometrically sound instrument to assess these co-occurring symptoms is unavailable. Here, we report the development and initial psychometric properties of the Fatigue and Altered Cognition Scale (the FACs), a measure of self-reported central fatigue and brain fog. Traumatic brain injury (TBI) was chosen to model and develop the FACs due to research team expertise and established links between TBI and the symptom complex. Potential items were generated by researchers and clinicians with experience treating these symptoms, drawing from relevant literature and review of patient responses to measures from past and current TBI studies. The 20 candidate items for the FACs-ten each to assess altered cognition (i.e., brain fog) and central fatigue-were formatted on an electronic visual analogue response scale (eVAS) via an online survey. Demographic information and history of TBI were obtained. A total of 519 participants consented and provided usable data (average age = 40.23 years; 73% female), 204 of whom self-reported a history of TBI (75% reported mild TBI). Internal consistency and reliability values were calculated. Confirmatory factor analysis (CFA) examined the presumed two-factor structure of the FACs and a one-factor solution for comparison. A measurement invariance test of the two latent constructs (altered cognition, fatigue) among participants with and without TBI was conducted. All items demonstrated normal distribution. Cronbach's alpha coefficients indicated good internal consistency for both factors (α's = .95). Omega reliability values were favorable (α's = .95). CFA supported the presumed two-factor model and item loadings which outperformed the one-factor model. Measurement invariance found the two-factor structure was consistent between the two groups. Implications of these findings, study limitations, and potential use of the FACs in clinical research and practice are discussed.

Traumatic brain injury, abnormal growth hormone secretion, and gut dysbiosis.

The gut microbiome has been implicated in a variety of neuropathologies with recent data suggesting direct effects of the microbiome on host metabolism, hormonal regulation, and pathophysiology. Studies have shown that gut bacteria impact host growth, partially mediated through the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis. However, no study to date has examined the specific role of GH on the fecal microbiome (FMB) or the changes in this relationship following a traumatic brain injury (TBI). Current literature has demonstrated that TBI can lead to either temporary or sustained abnormal GH secretion (aGHS). More recent literature has suggested that gut dysbiosis may contribute to aGHS leading to long-term sequelae now known as brain injury associated fatigue and cognition (BIAFAC). The aGHS observed in some TBI patients presents with a symptom complex including profound fatigue and cognitive dysfunction that improves significantly with exogenous recombinant human GH treatment. Notably, GH treatment is not curative as fatigue and cognitive decline typically recur upon treatment cessation, indicating the need for additional studies to address the underlying mechanistic cause.

Validation of vaginal microbiome proxies for in vitro experiments that biomimic Lactobacillus-dominant vaginal cultures.

The vaginal microbiome includes diverse microbiota dominated by Lactobacillus [L.] spp. that protect against infections, modulate inflammation, and regulate vaginal homeostasis. Because it is challenging to incorporate vaginal microbiota into in vitro models, including organ-on-a-chip systems, we assessed microbial metabolites as reliable proxies in addition to traditional vaginal epithelial cultures (VECs). Human immortalized VECs cultured on transwells with an air-liquid interface generated stratified cell layers colonized by transplanted healthy microbiomes (L. jensenii- or L. crispatus-dominant) or a community representing bacterial vaginosis (BV). After 48-h, a qPCR array confirmed the expected donor community profiles. Pooled apical and basal supernatants were subjected to metabolomic analysis (untargeted mass spectrometry) followed by ingenuity pathways analysis (IPA). To determine the bacterial metabolites' ability to recreate the vaginal microenvironment in vitro, pooled bacteria-free metabolites were added to traditional VEC cultures. Cell morphology, viability, and cytokine production were assessed. IPA analysis of metabolites from colonized samples contained fatty acids, nucleic acids, and sugar acids that were associated with signaling networks that contribute to secondary metabolism, anti-fungal, and anti-inflammatory functions indicative of a healthy vaginal microbiome compared to sterile VEC transwell metabolites. Pooled metabolites did not affect cell morphology or induce cell death (∼5.5%) of VEC cultures (n = 3) after 72-h. However, metabolites created an anti-inflammatory milieu by increasing IL-10 production (p = .06, T-test) and significantly suppressing pro-inflammatory IL-6 (p = .0001), IL-8 (p = .009), and TNFα (p = .0007) compared to naïve VEC cultures. BV VEC conditioned-medium did not affect cell morphology nor viability; however, it induced a pro-inflammatory environment by elevating levels of IL-6 (p = .023), IL-8 (p = .031), and TNFα (p = .021) when compared to L.-dominate microbiome-conditioned medium. VEC transwells provide a suitable ex vivo system to support the production of bacterial metabolites consistent with the vaginal milieu allowing subsequent in vitro studies with enhanced accuracy and utility.

Is there a role for growth hormone replacement in adults to control acute and post-acute COVID-19?

The SARS-CoV-2 pandemic created a multitude of medical crossroads requiring real time adaptations of best practice covering preventative and interventional aspects of care. Among the many discoveries borne from efforts to address the myriad clinical presentations across multiple organ systems was a common impact on tissues with cells that express the ACE-2 receptor. The vast majority of acute infections began and often ended in the respiratory tract, but more recent evaluations have confirmed significant extrapulmonary manifestations including symptom clusters that extend beyond the acute phase of infection collectively referred to as "post-acute sequelae SARS-CoV-2 infection" (PASC) or more commonly as "long (-haul) COVID". Both acute SARS-CoV-2 infection and PASC are associated with gut microbiome dysbiosis and alterations in the gut-brain and HPA-axis in a subset of the infected. Mounting evidence suggests these extrapulmonary manifestations may ultimately lead to reduced growth hormone (GH) secretion as demonstrated following stimulation tests. Disrupted GH secretion could cause or exacerbate long lasting neuropsychological symptoms as seen in other similar manifesting conditions. Ongoing clinical research has shown promising improvement in PASC patients with fatigue and cognition complaints can be achieved via GH replacement therapy. GH stimulation testing should be considered in PASC workups and future research should delve deeper into the mechanistic effects of GH on acute COVID and PASC.

Boosting of vaginal HSV-2-specific B and T cell responses by intravaginal therapeutic immunization results in diminished recurrent HSV-2 disease.

Boosting herpes simplex virus (HSV)-specific immunity in the genital tissues of HSV-positive individuals to increase control of HSV-2 recurrent disease and virus shedding is an important goal of therapeutic immunization and would impact HSV-2 transmission. Experimental therapeutic HSV-2 vaccines delivered by a parenteral route have resulted in decreased recurrent disease in experimental animals. We used a guinea pig model of HSV-2 infection to test if HSV-specific antibody and cell-mediated responses in the vaginal mucosa would be more effectively increased by intravaginal (Ivag) therapeutic immunization compared to parenteral immunization. Therapeutic immunization with HSV glycoproteins and CpG adjuvant increased glycoprotein-specific IgG titers in vaginal secretions and serum to comparable levels in Ivag- and intramuscular (IM)-immunized animals. However, the mean numbers of HSV glycoprotein-specific antibody secreting cells (ASCs) and IFN-γ SCs were greater in Ivag-immunized animals demonstrating superior boosting of immunity in the vaginal mucosa compared to parenteral immunization. Therapeutic Ivag immunization also resulted in a significant decrease in the cumulative mean lesion days compared to IM immunization. There was no difference in the incidence or magnitude of HSV-2 shedding in either therapeutic immunization group compared to control-treated animals. Collectively, these data demonstrated that Ivag therapeutic immunization was superior compared to parenteral immunization to boost HSV-2 antigen-specific ASC and IFN-γ SC responses in the vagina and control recurrent HSV-2 disease. These results suggest that novel antigen delivery methods providing controlled release of optimized antigen/adjuvant combinations in the vaginal mucosa would be an effective approach for therapeutic HSV vaccines. IMPORTANCE HSV-2 replicates in skin cells before it infects sensory nerve cells where it establishes a lifelong but mostly silent infection. HSV-2 occasionally reactivates, producing new virus which is released back at the skin surface and may be transmitted to new individuals. Some HSV-specific immune cells reside at the skin site of the HSV-2 infection that can quickly activate and clear new virus. Immunizing people already infected with HSV-2 to boost their skin-resident immune cells and rapidly control the new HSV-2 infection is logical, but we do not know the best way to administer the vaccine to achieve this goal. In this study, a therapeutic vaccine given intravaginally resulted in significantly better protection against HSV-2 disease than immunization with the same vaccine by a conventional route. Immunization by the intravaginal route resulted in greater stimulation of vaginal-resident, virus-specific cells that produced antibody and produced immune molecules to rapidly clear virus.

Next generation 3D-printed intravaginal ring for prevention of HIV and unintended pregnancy.

Globally, there are 20 million adolescent girls and young women living with HIV who have limited access to long-acting, effective, women-controlled preventative methods. Additionally, although there are many contraceptive methods available, globally, half of all pregnancies remain unintended. Here we report the first 3D-printed multipurpose prevention technology (MPT) intravaginal ring (IVR) for HIV prevention and contraception. We utilized continuous liquid interface production (CLIP™) to fabricate MPT IVRs in a biocompatible silicone-based resin. Etonogestrel (ENG), ethinyl estradiol (EE), and islatravir (ISL) were loaded into the silicone poly(urethane) IVR in a controlled single step drug loading process driven by absorption. ENG/EE/ISL IVR promoted sustained release of drugs for 150 days in vitro and 14 days in sheep. There were no adverse MPT IVR-related findings of cervicovaginal toxicity or changes in vaginal biopsies or microbiome community profiles evaluated in sheep. Furthermore, ISL IVR in macaques promoted sustained release for 28 days with ISL-triphosphate levels above the established pharmacokinetic benchmark of 50-100 fmol/106 PBMCs. The ISL IVR was found to be safe and well tolerated in the macaques with no observed mucosal cytokine changes or alterations in peripheral CD4 T-cell populations. Collectively, the proposed MPT IVR has potential to expand preventative choices for young women and girls.

Microbiota Associated With Cholesteatoma Tissue in Chronic Suppurative Otitis Media.

Otitis media (OM), defined as infection or inflammation of the middle ear (ME), remains a major public health problem worldwide. Cholesteatoma is a non-cancerous, cyst-like lesion in the ME that may be acquired due to chronic OM and cause disabling complications. Surgery is required for treatment, with high rates of recurrence. Current antibiotic treatments have been largely targeted to previous culturable bacteria, which may lead to antibiotic resistance or treatment failures. For this study, our goal was to determine the microbiota of cholesteatoma tissue in comparison with other ME tissues in patients with long-standing chronic OM. ME samples including cholesteatoma, granulation tissue, ME mucosa and discharge were collected from patients undergoing tympanomastoidectomy surgery for chronic OM. Bacteria were profiled by 16S rRNA gene sequencing in 103 ME samples from 53 patients. Respiratory viruses were also screened in 115 specimens from 45 patients. Differences in bacterial profiles (beta-diversity) and the relative abundances of individual taxa were observed between cholesteatoma and ME sample-types. Additionally, patient age was associated with differences in overall microbiota composition while numerous individual taxa were differentially abundant across age quartiles. No viruses were identified in screened ME samples. Biodiversity was moderately lower in cholesteatoma and ME discharge compared to ME mucosal tissues. We also present overall bacterial profiles of ME tissues by sample-type, age, cholesteatoma diagnosis and quinolone use, including prevalent bacterial taxa. Our findings will be useful for fine-tuning treatment protocols for cholesteatoma and chronic OM in settings with limited health care resources.

Inflammatory response elicited by Ureaplasma parvum colonization in human cervical epithelial, stromal, and immune cells.

Ureaplasma parvum is a commensal bacterium in the female reproductive tract but has been associated with pregnancy complications such as preterm prelabor rupture of membranes and preterm birth (PTB). However, the pathologic effects of U. parvum in the cervix, which prevents ascending infections during pregnancy, are still poorly understood. To determine the impact of U. parvum on the cervix, ectocervical (ecto) and endocervical (endo) epithelial and stromal cells were incubated with U. parvum. Macrophages were also tested as a proxy for cervical macrophages to determine the antigenicity of U. parvum. The effects of U. parvum, including influence on cell cycle and cell death, antimicrobial peptide (AMP) production, epithelial-to-mesenchymal transition (EMT), and inflammatory cytokine levels, were assessed. U. parvum colonized cervical epithelial and stromal cells 4 h post-infection. Like uninfected control, U. parvum neither inhibited cell cycle progression and nor caused cell death in cervical epithelial and stromal cells. U. parvum increased the production of the AMPs cathelicidin and human ß-defensin 3 and exhibited weak signs of EMT evidenced by decreased cytokeratin 18 and increased vimentin expression in cervical epithelial cells. U. parvum induced a proinflammatory environment (cytokines) and increased MMP-9 in cervical epithelial cells but promoted pro- and anti-inflammatory response in cervical stromal cells and macrophages. U. parvum may colonize the cervical epithelial layer, but induction of AMPs and anti-inflammatory response may protect the cervix and may prevent ascending infections that can cause PTB. These findings suggest that U. parvum is a weak inducer of inflammation in the cervix.

Longitudinal COVID-19 Surveillance and Characterization in the Workplace with Public Health and Diagnostic Endpoints.

Public health practices and high vaccination rates currently represent the primary interventions for managing the spread of coronavirus disease 2019 (COVID-19). We initiated a clinical study based on frequent, longitudinal workplace disease surveillance to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission among employees and their household members. We hypothesized that the study would reduce the economic burden and loss of productivity of both individuals and small businesses resulting from standard isolation methods, while providing new insights into virus-host dynamics. Study participants (27 employees and 27 household members) consented to provide frequent nasal or oral swab samples that were analyzed by reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 RNA. Two study participants were found to be infected by SARS-CoV-2 during the study. One subject, a household member, was SARS-CoV-2 RNA positive for at least 71 days and had quantifiable serum virus-specific antibody concentrations for over 1 year. One unrelated employee became positive for SARS-CoV-2 RNA over the course of the study but remained asymptomatic, with low associated viral RNA copy numbers, no detectable serum IgM and IgG concentrations, and IgA concentrations that decayed rapidly (half-life: 1.3 days). A COVID-19 infection model was used to predict that without surveillance intervention, up to 7 employees (95% confidence interval [CI] = 3 to 10) would have become infected, with at most 1 of them requiring hospitalization. Our scalable and transferable surveillance plan met its primary objectives and represents a powerful example of an innovative public health initiative dovetailed with scientific discovery. IMPORTANCE The rapid spread of SARS-CoV-2 and the associated COVID-19 has precipitated a global pandemic heavily challenging our social behavior, economy, and health care infrastructure. In the absence of widespread, worldwide access to safe and effective vaccines and therapeutics, public health measures represent a key intervention for curbing the devastating impacts from the pandemic. We are conducting an ongoing clinical study based on frequent, longitudinal workplace disease surveillance to control SARS-CoV-2 transmission among employees and their household members. Our study was successful in surveying the viral and immune response dynamics in two participants with unusual infections: one remained positive for SARS-CoV-2 for 71 days, while the other was asymptomatic, with low associated viral RNA copy numbers. A COVID-19 infection model was used to predict that without surveillance intervention, up to 7 employees would have become infected, with at most 1 of them requiring hospitalization, underscoring the importance of our program.

Progesterone alters human cervical epithelial and stromal cell transition and migration: Implications in cervical remodeling during pregnancy and parturition.

The cervix undergoes extensive remodeling throughout pregnancy and parturition. This process involves both ECM collagen degradation and cellular remodeling, which includes cell proliferation, transition and migration. Progesterone (P4) has been used clinically to delay cervical ripening and prevent preterm birth (PTB). However, the mechanisms by which progesterone affects cell transition and the migration of cervical epithelial and stromal cells are not yet fully known. In this study, we documented the role of a gestational level of P4 in the cellular transition (epithelial-mesenchymal transition [EMT] and mesenchymal-epithelial transition [MET]), cell migration, and inflammatory responses of endocervical epithelial cells (EEC) and cervical stromal cells (CSC). EEC and CSC were treated with LPS and P4 for 6 days. The epithelial:mesenchymal ratio (regular microscopy and cell shape index analysis), shift in intermediate filaments (immunofluorescence microscopy and western blot analyses for cytokeratin [CK]-18 and vimentin), adhesion molecules and transcription factors (western blot analyses for E-cadherin, N-cadherin and SNAIL), were used to determine growth characteristics and EMT and MET changes in EEC and CSC under the indicated conditions. To test cell remodeling, scratch assays followed by cellular analyses as mentioned above were performed. Inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor α [TNFα]) and matrix metallopeptidase 9 (MMP9) were measured by ELISA. LPS promoted EMT (decreased cell shape index, decreased CK-18 and E-cadherin, increased vimentin, N-cadherin, and SNAIL), and increased IL-6 and MMP9 production by EEC. A gestational level of P4 prevented LPS-induced EMT in EEC and exhibited anti-inflammatory effect in both EEC and CSC. LPS slowed down wound healing in CSC but P4 treatment prevented the negative impact of LPS in CSC wound healing. These results may explain the cellular mechanisms by which P4 helps to stabilize the cervical epithelial barrier and preserve the mechanical and tensile strength of the cervical stromal layer, which are important in normal cervical remodeling processes during pregnancy.

Microbiota Detection Patterns Correlate With Presence and Severity of Barrett's Esophagus.

Background: The microbiome has been increasingly associated with different disease processes, but its role in esophagus is largely unknown. Our goal was to determine the associations of the esophageal microbiota with Barrett's esophagus. Methods: A total of 74 patients were included in this prospective study, including 34 patients with Barrett's esophagus and 40 patients without Barrett's esophagus. Esophageal swabs were obtained from the uvula, and mucosal biopsies were obtained from the proximal esophagus and distal esophagus in each patient. The microbiome of each sample was assessed using a customized Esophageal Microbiome qPCR array (EMB). For each clinical sample, we completed a detection/non-detection analysis for each organism in the EMB. The limit of detection (LOD) for each target was established by analysis of plasmid dilutions. Results: Average age was 60.2 years. There were significantly different microbial detection patterns in patients with Barrett's esophagus compared to the control population. There were a greater number of organisms which had different likelihoods of detection in the distal esophagus, compared to the proximal esophagus or uvula. In addition, as the length of the Barrett's column increased, multiple organisms were less likely to be detected. This decreased likelihood occurred only in the distal esophagus. Beside Barrett's esophagus, no other demographic factors were associated with differences in detection patterns. Conclusions: Microbial community structures differ between patients with and without Barrett's esophagus. Certain organisms are less likely to be detected as the severity of Barrett's esophagus worsens. These results suggest that particular organisms may have a protective effect against the development of Barrett's esophagus.

Characterization of the Ovine Vaginal Microbiome and Inflammation Patterns as an Improved Testing Model of Human Vaginal Irritation.

The development of therapies targeted to improve the health of women has utilized direct vaginal delivery as a more effective and less toxic method of protection from HIV and other pathogens. Vaginal applicants and delivery devices that provide sustained effects have been met with increasing acceptability, but the efficacy and toxicity outcomes have not been successfully predicted by preclinical in vitro studies and animal modeling. We have explored the utilization of sheep as a model for testing the safety of vaginal applicants and devices based on spatial and structural similarities to the human vagina. As recently noted by the FDA, an additional safety measure is an impact on the vaginal microbiome (VMB) that is known to contribute to vaginal health and influence pathogen susceptibility and drug metabolism. To advance the utility of the sheep vaginal model, we completed a thorough molecular characterization of the ovine VMB utilizing both next-generation sequencing (NGS) and PCR methods. The process also created a custom PCR array to quantify ovine VMB community profiles in an affordable, higher throughput fashion. The results from vaginal swabs (>475 samples) collected from non-pregnant crossbred Dorset and Merino ewes treated with selected vaginal applicants or collected as sham samples established 16 VMB community types (VMB CTs). To associate VMB CTs with eubiosis or dysbiosis, we also completed custom ELISAs for six cytokines identifying IL1B, IL8, TNFa, and CXCL10 as useful markers to support the characterization of ovine vaginal inflammation. The results indicated that Pasteurella, Actinobacillus, Pseudomonas, Bacteroides, Leptotrichia, and E. coli were common markers of eubiosis (low inflammatory marker expression), and that Haemophilus, Ureaplasma, and Corynebacterium were associated with dysbiosis (high cytokine levels). Utilizing the optimized workflow, we also confirmed the utility of three commonly used vaginal applicants for impact on the VMB and inflammatory state, producing a dataset that supports the recommendation for the use of sheep for testing of vaginal applicants and devices as part of preclinical pipelines.

Development of Gram Stain Scoring System Based on Pro-Inflammatory Cytokines in the Sheep Model for Testing Toxicity of Vaginal Products.

Background: Development of safe, effective products to prevent the sexual transmission of HIV remains a priority. Prior to clinical testing, the products must undergo strict safety evaluations to avoid mucosal drug toxicity, inflammation, and vaginal microbiome (VMB) shifts. Based on the Food and Drug Administration (FDA) guidance, we designed a study to measure the inflammatory markers and VMB changes after intravaginal treatment with products that have been associated with toxicity, with the objective to develop a Gram stain slide scoring system, similar to Nugent scoring, correlated with the proinflammatory cytokines in sheep. Methods: Non-pregnant Dorset ewes (n = 34) were randomized to receive 5 ml intravaginal 4% nonoxynol-9 (N9) contraceptive gel, positive control (0.2% benzalkonium chloride), placebo control [hydroxethyl cellulose (HEC)], or no application daily for 10 days, with 11-day post-treatment follow-up. The vaginal swabs were collected for the cytokines, VMB, and Gram-stained slides. An enzyme-linked immunosorbent assay (ELISA) analysis of cytokines interleukin (IL)-1ß, IL-8, CXCL10, and tumor necrosis factor-α (TNF-α) was used to determine inflammatory state of the sample. Vaginal microbiome community types (CT) were utilized to create five equivalent slide subsets for iterative development of a Gram-stained slide scoring system with comparisons with inflammatory state based on the cytokine levels. Results: Digital images of the Gram-stained slides were scored based on Gram staining and morphology of bacteria, presence of sheep epithelial cells, and immune cells. The scoring system was modified in an iterative fashion with weighting based on cytokine categorization of inflamed samples, with three of four cytokine values above the mean indicating that the sample was inflamed. The parameters in the final version of the scoring system included mature epithelial cells, Gram-negative rods, and Gram-positive diplococci indicating normal and immune cells indicating inflammation. The area under the receiver operator characteristic curve (ROC AUC) was 0.725 (ROC AUCs range between 0.5 and 1.0) with a greater area indicating higher diagnostic ability of a test with a binary outcome: inflamed or normal. Conclusion: The scoring system, derived from the advanced VMB and cytokine analyses, provides a validated, practical method for quantification of Gram-stained slides that can be performed in most laboratories, increasing the potential for standardization. The training plan can assist laboratories to determine the safety of intravaginal products in their sheep studies or the methodological approach can be applied to other animal models where such data are also needed.

Effects of proton pump inhibitor use on the esophageal microbial community.

BACKGROUND: Changes in the esophageal microbiome correlate with esophageal disease, but the effects of proton pump inhibitor (PPI) drugs are incompletely characterized. Our objective was to identify the effects of PPI use on the microbial community of the esophagus. METHODS: Mucosal biopsies of the distal esophagus were analyzed using a customized esophageal microbiome qPCR panel array (EMB). Patient demographics, use of PPIs, duration of use and dose were recorded. RESULTS: Fifty-eight patients were included. Mean age was 60.5 years. Ninety percent (52/58) of patients were on PPIs. Mean dose was 42.7 mg. Mean duration of use was 2.5 years. The use of PPIs led to a significant difference in absolute levels of only one organism, Actinomyces, in the entire array (p < 0.01). Among patients who used proton pump inhibitors, there was no significant association between dose and absolute levels of any organism. Similarly, there was no association between duration of use and absolute levels of any organism. CONCLUSIONS: PPI use does not seem to cause significant changes in the distal esophageal microbial community. Future studies with larger sample sizes and esophageal pH testing should be performed to determine the level of acidity and its relationship to the microbial community.

Alterations of the GH/IGF-I Axis and Gut Microbiome after Traumatic Brain Injury: A New Clinical Syndrome?

CONTEXT: Pituitary dysfunction with abnormal growth hormone (GH) secretion and neurocognitive deficits are common consequences of traumatic brain injury (TBI). Recognizing the comorbidity of these symptoms is of clinical importance; however, efficacious treatment is currently lacking. EVIDENCE ACQUISITION: A review of studies in PubMed published between January 1980 to March 2020 and ongoing clinical trials was conducted using the search terms "growth hormone," "traumatic brain injury," and "gut microbiome." EVIDENCE SYNTHESIS: Increasing evidence has implicated the effects of TBI in promoting an interplay of ischemia, cytotoxicity, and inflammation that renders a subset of patients to develop postinjury hypopituitarism, severe fatigue, and impaired cognition and behavioral processes. Recent data have suggested an association between abnormal GH secretion and altered gut microbiome in TBI patients, thus prompting the description of a hypothesized new clinical syndrome called "brain injury associated fatigue and altered cognition." Notably, these patients demonstrate distinct characteristics from those with GH deficiency from other non-TBI causes in that their symptom complex improves significantly with recombinant human GH treatment, but does not reverse the underlying mechanistic cause as symptoms typically recur upon treatment cessation. CONCLUSION: The reviewed data describe the importance of alterations of the GH/insulin-like growth factor I axis and gut microbiome after brain injury and its influence in promoting neurocognitive and behavioral deficits in a bidirectional relationship, and highlight a new clinical syndrome that may exist in a subset of TBI patients in whom recombinant human GH therapy could significantly improve symptomatology. More studies are needed to further characterize this clinical syndrome.

Methylation Patterns of the HTR2A Associate With Relapse-Related Behaviors in Cocaine-Dependent Participants.

Relapse during abstinence in cocaine use disorder (CUD) is often hastened by high impulsivity (predisposition toward rapid unplanned reactions to stimuli without regard to negative consequences) and high cue reactivity (e.g., attentional bias towards drug reward stimuli). A deeper understanding of the degree to which individual biological differences predict or promote problematic behaviors may afford opportunities for clinical refinement and optimization of CUD diagnostics and/or therapies. Preclinical evidence implicates serotonin (5-HT) neurotransmission through the 5-HT2A receptor (5-HT2AR) as a driver of individual differences in these relapse-related behaviors. Regulation of 5-HT2AR function occurs through many mechanisms, including DNA methylation of the HTR2A gene, an epigenetic modification linked with the memory of gene-environment interactions. In the present study, we tested the hypothesis that methylation of the HTR2A may associate with relapse-related behavioral vulnerability in cocaine-dependent participants versus healthy controls. Impulsivity was assessed by self-report (Barratt Impulsiveness Scale; BIS-11) and the delay discounting task, while levels of cue reactivity were determined by performance in the cocaine-word Stroop task. Genomic DNA was extracted from lymphocytes and the bisulfite-treated DNA was subjected to pyrosequencing to determine degree of methylation at four cytosine residues of the HTR2A promoter (-1439, -1420, -1224, -253). We found that the percent methylation at site -1224 after correction for age trended towards a positive correlation with total BIS-11 scores in cocaine users, but not healthy controls. Percent methylation at site -1420 negatively correlated with rates of delay discounting in healthy controls, but not cocaine users. Lastly, the percent methylation at site -253 positively correlated with attentional bias toward cocaine-associated cues. DNA methylation at these cytosine residues of the HTR2A promoter may be differentially associated with impulsivity or cocaine-associated environmental cues. Taken together, these data suggest that methylation of the HTR2A may contribute to individual differences in relapse-related behaviors in CUD.