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.

Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments†.

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (

Surrogate post-coital testing for contraceptive efficacy against human sperm activity in the ovine vaginal model†.

High unintended pregnancy rates are partially due to lack of effective nonhormonal contraceptives; development of safe, effective topical vaginal methods will address this need. Preclinical product safety and efficacy assessment requires in vivo testing in appropriate models. The sheep is a good model for the evaluation of vaginally delivered products due to its close similarities to humans. The study objective was to develop an ovine model for efficacy testing of female nonhormonal contraceptives that target human sperm. Fresh human semen was pooled from male volunteers. Nonpregnant female Merino sheep were treated with control or vaginal contraceptive product (IgG antibody with action against sperm or nonoxynol-9 [N9]). Pooled semen was added to the sheep vagina and mixed with product and vaginal secretions. Microscopic assessment of samples was performed immediately and progressive motility (PM) of sperm was compared between treatments. Cytokines CXCL8 and IL1B were assessed in vaginal fluid after instillation of human semen. No adverse reactions or elevations in proinflammatory cytokines occurred in response to human semen. N9 produced signs of acute cellular toxicity while there were no cellular changes after IgG treatment. N9 and IgG had dose-related effects with the highest dose achieving complete sperm immobilization (no sperm with PM). Surrogate post-coital testing of vaginally administered contraceptives that target human semen was developed in an ovine model established for vaginal product preclinical testing. This expanded model can aid the development of much needed nonhormonal topical vaginal contraceptives, providing opportunities for rapid iterative drug development prior to costly, time-intensive human testing.

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.

Increased Frequency of Virus Shedding by Herpes Simplex Virus 2-Infected Guinea Pigs in the Absence of CD4+ T Lymphocytes.

Reactivation of herpes simplex virus 2 (HSV-2) results in infection of epithelial cells at the neuro-epithelial junction and shedding of virus at the epithelial surface. Virus shedding can occur in either the presence or absence of clinical disease and is usually of short duration, although the shedding frequency varies among individuals. The basis for host control of virus shedding is not well understood, although adaptive immune mechanisms are thought to play a central role. To determine the importance of CD4+ T cells in control of HSV-2 shedding, this subset of immune cells was depleted from HSV-2-infected guinea pigs by injection of an anti-CD4 monoclonal antibody (MAb). Guinea pigs were treated with the depleting MAb after establishment of a latent infection, and vaginal swabs were taken daily to monitor shedding by quantitative PCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed were significantly increased in CD4-depleted compared to control-treated animals. However, there was no difference in the incidence of recurrent disease between the two treatment groups. Serum antibody levels and the number of HSV-specific antibody-secreting cells in secondary lymphoid tissues were unaffected by depletion of CD4+ T cells; however, the frequency of functional HSV-specific, CD8+ gamma interferon-secreting cells was significantly decreased. Together, these results demonstrate an important role for CD4+ T lymphocytes in control of virus shedding that may be mediated in part by maintenance of HSV-specific CD8+ T cell populations. These results have important implications for development of therapeutic vaccines designed to control HSV-2 shedding.IMPORTANCE Sexual transmission of HSV-2 results from viral shedding following reactivation from latency. The immune cell populations and mechanisms that control HSV-2 shedding are not well understood. This study examined the role of CD4+ T cells in control of virus shedding using a guinea pig model of genital HSV-2 infection that recapitulates the shedding of virus experienced by humans. We found that the frequency of virus-shedding episodes, but not the incidence of clinical disease, was increased by depletion of CD4+ T cells. The HSV-specific antibody response was not diminished, but frequency of functional HSV-reactive CD8+ T cells was significantly diminished by CD4 depletion. These results confirm the role of cell-mediated immunity and highlight the importance of CD4+ T cells in controlling HSV shedding, suggesting that therapeutic vaccines designed to reduce transmission by controlling HSV shedding should include specific enhancement of HSV-specific CD4+ T cell responses.

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.

Safety and pharmacokinetics of single, dual, and triple antiretroviral drug formulations delivered by pod-intravaginal rings designed for HIV-1 prevention: A Phase I trial.

BACKGROUND: Intravaginal rings (IVRs) for HIV pre-exposure prophylaxis (PrEP) theoretically overcome some adherence concerns associated with frequent dosing that can occur with oral or vaginal film/gel regimens. An innovative pod-IVR, composed of an elastomer scaffold that can hold up to 10 polymer-coated drug cores (or "pods"), is distinct from other IVR designs as drug release from each pod can be controlled independently. A pod-IVR has been developed for the delivery of tenofovir (TFV) disoproxil fumarate (TDF) in combination with emtricitabine (FTC), as daily oral TDF-FTC is the only Food and Drug Administration (FDA)-approved regimen for HIV PrEP. A triple combination IVR building on this platform and delivering TDF-FTC along with the antiretroviral (ARV) agent maraviroc (MVC) also is under development. METHODOLOGY AND FINDINGS: This pilot Phase I trial conducted between June 23, 2015, and July 15, 2016, evaluated the safety, pharmacokinetics (PKs), and acceptability of pod-IVRs delivering 3 different ARV regimens: 1) TDF only, 2) TDF-FTC, and 3) TDF-FTC-MVC over 7 d. The crossover, open-label portion of the trial (N = 6) consisted of 7 d of continuous TDF pod-IVR use, a wash-out phase, and 7 d of continuous TDF-FTC pod-IVR use. After a 3-mo pause to evaluate safety and PK of the TDF and TDF-FTC pod-IVRs, TDF-FTC-MVC pod-IVRs (N = 6) were evaluated over 7 d of continuous use. Safety was assessed by adverse events (AEs), colposcopy, and culture-independent analysis of the vaginal microbiome (VMB). Drug and drug metabolite concentrations in plasma, cervicovaginal fluids (CVFs), cervicovaginal lavages (CVLs), and vaginal tissue (VT) biopsies were determined via liquid chromatographic-tandem mass spectrometry (LC-MS/MS). Perceptibility and acceptability were assessed by surveys and interviews. Median participant age was as follows: TDF/TDF-FTC group, 26 y (range 24-35 y), 2 White, 2 Hispanic, and 2 African American; TDF-FTC-MVC group, 24.5 y (range 21-41 y), 3 White, 1 Hispanic, and 2 African American. Reported acceptability was high for all 3 products, and pod-IVR use was confirmed by residual drug levels in used IVRs. There were no serious adverse events (SAEs) during the study. There were 26 AEs reported during TDF/TDF-FTC IVR use (itching, discharge, discomfort), with no differences between TDF alone or in combination with FTC observed. In the TDF-FTC-MVC IVR group, there were 12 AEs (itching, discharge, discomfort) during IVR use regardless of attribution to study product. No epithelial disruption/thinning was seen by colposcopy, and no systematic VMB shifts were observed. Median (IQR) tenofovir diphosphate (TFV-DP) tissue concentrations of 303 (277-938) fmol/10(6) cells (TDF), 289 (110-603) fmol/10(6) cells (TDF-FTC), and 302 (177.1-823.8) fmol/10(6) cells (TDF-FTC-MVC) were sustained for 7 d, exceeding theoretical target concentrations for vaginal HIV prevention. The study's main limitations include the small sample size, short duration (7 d versus 28 d), and the lack of FTC triphosphate measurements in VT biopsies. CONCLUSIONS: An innovative pod-IVR delivery device with 3 different formulations delivering different regimens of ARV drugs vaginally appeared to be safe and acceptable and provided drug concentrations in CVFs and tissues exceeding concentrations achieved by highly protective oral dosing, suggesting that efficacy for vaginal HIV PrEP is achievable. These results show that an alternate, more adherence-independent, longer-acting prevention device based on the only FDA-approved PrEP combination regimen can be advanced to safety and efficacy testing. TRIAL REGISTRATION: ClinicalTrials.gov NCT02431273.

A lethal model of disseminated dengue virus type 1 infection in AG129 mice.

The mosquito-borne disease dengue is caused by four serologically and genetically related flaviviruses termed DENV-1 to DENV-4. Dengue is a global public health concern, with both the geographical range and burden of disease increasing rapidly. Clinically, dengue ranges from a relatively mild self-limiting illness to a severe life-threatening and sometimes fatal disease. Infection with one DENV serotype produces life-long homotypic immunity, but incomplete and short-term heterotypic protection. The development of small-animal models that recapitulate the characteristics of the disseminated disease seen clinically has been difficult, slowing the development of vaccines and therapeutics. The AG129 mouse (deficient in interferon alpha/beta and gamma receptor signalling) has proven to be valuable for this purpose, with the development of models of disseminated DENV-2,-3 and -4 disease. Recently, a DENV-1 AG129 model was described, but it requires antibody-dependent enhancement (ADE) to produce lethality. Here we describe a new AG129 model utilizing a non-mouse-adapted DENV-1 strain, West Pacific 74, that does not require ADE to induce lethal disease. Following high-titre intraperitoneal challenge, animals experience a virus infection with dissemination to multiple visceral tissues, including the liver, spleen and intestine. The animals also become thrombocytopenic, but vascular leakage is less prominent than in AG129 models with other DENV serotypes. Taken together, our studies demonstrate that this model is an important addition to dengue research, particularly for understanding the pathological basis of the disease between DENV serotypes and allowing the full spectrum of activity to test comparisons for putative vaccines and antivirals.

Vaginal ecosystem modeling of growth patterns of anaerobic bacteria in microaerophilic conditions.

The human vagina constitutes a complex ecosystem created through relationships established between host mucosa and bacterial communities. In this ecosystem, classically defined bacterial aerobes and anaerobes thrive as communities in the microaerophilic environment. Levels of CO2 and O2 present in the vaginal lumen are impacted by both the ecosystem's physiology and the behavior and health of the human host. Study of such complex relationships requires controlled and reproducible causational approaches that are not possible in the human host that, until recently, was the only place these bacterial communities thrived. To address this need we have utilized our ex vivo human vaginal mucosa culture system to support controlled, reproducible colonization by vaginal bacterial communities (VBC) collected from healthy, asymptomatic donors. Parallel vaginal epithelial cells (VEC)-VBC co-cultures were exposed to two different atmospheric conditions to study the impact of CO2 concentrations upon the anaerobic bacteria associated with dysbiosis and inflammation. Our data suggest that in the context of transplanted VBC, increased CO2 favored specific lactobacilli species defined as microaerophiles when grown as monocultures. In preliminary studies, the observed community changes also led to shifts in host VEC phenotypes with significant changes in the host transcriptome, including altered expression of select molecular transporter genes. These findings support the need for additional study of the environmental changes associated with behavior and health upon the symbiotic and adversarial relationships that are formed in microbial communities present in the human vaginal ecosystem.

New Systems for Studying Intercellular Interactions in Bacterial Vaginosis.

Bacterial vaginosis (BV) affects almost a quarter of US women, making it a condition of major public health relevance. Key questions remain regarding the etiology of BV, mechanisms for its association with poor reproductive health outcomes, and reasons for high rates of treatment failure. New model systems are required to answer these remaining questions, elucidate the complex host-microbe and microbe-microbe interactions, and develop new, effective interventions. In this review, we cover the strengths and limitations of in vitro and in vivo model systems to study these complex intercellular interactions. Furthermore, we discuss advancements needed to maximize the translational utility of the model systems. As no single model can recapitulate all of the complex physiological and environmental conditions of the human vaginal microenvironment, we conclude that combinatorial approaches using in vitro and in vivo model systems will be required to address the remaining fundamental questions surrounding the enigma that is BV.

A lethal murine infection model for dengue virus 3 in AG129 mice deficient in type I and II interferon receptors leads to systemic disease.

UNLABELLED: The mosquito-borne disease dengue (DEN) is caused by four serologically and genetically related viruses, termed DENV-1 to DENV-4. Infection with one DENV usually leads to acute illness and results in lifelong homotypic immunity, but individuals remain susceptible to infection by the other three DENVs. The lack of a small-animal model that mimics systemic DEN disease without neurovirulence has been an obstacle, but DENV-2 models that resemble human disease have been recently developed in AG129 mice (deficient in interferon alpha/beta and interferon gamma receptor signaling). However, comparable DENV-1, -3, and -4 models have not been developed. We utilized a non-mouse-adapted DENV-3 Thai human isolate to develop a lethal infection model in AG129 mice. Intraperitoneal inoculation of six to eight-week-old animals with strain C0360/94 led to rapid, fatal disease. Lethal C0360/94 infection resulted in physical signs of illness, high viral loads in the spleen, liver, and large intestine, histological changes in the liver and spleen tissues, and increased serum cytokine levels. Importantly, the animals developed vascular leakage, thrombocytopenia, and leukopenia. Overall, we have developed a lethal DENV-3 murine infection model, with no evidence of neurotropic disease based on a non-mouse-adapted human isolate, which can be used to investigate DEN pathogenesis and to evaluate candidate vaccines and antivirals. This suggests that murine models utilizing non-mouse-adapted isolates can be obtained for all four DENVs. IMPORTANCE: Dengue (DEN) is a mosquito-borne disease caused by four DENV serotypes (DENV-1, -2, -3, and -4) that have no treatments or vaccines. Primary infection with one DENV usually leads to acute illness followed by lifelong homotypic immunity, but susceptibility to infection by the other three DENVs remains. Therefore, a vaccine needs to protect from all four DENVs simultaneously. To date a suitable animal model to mimic systemic human illness exists only for DENV-2 in immunocompromised mice using passaged viruses; however, models are still needed for the remaining serotypes. This study describes establishment of a lethal systemic DENV-3 infection model with a human isolate in immunocompromised mice and is the first report of lethal infection by a nonadapted clinical DENV isolate without evidence of neurological disease. Our DENV-3 model provides a relevant platform to test DEN vaccines and antivirals.

Symptomatic and asymptomatic respiratory viral infections in the first year of life: association with acute otitis media development.

BACKGROUND: Sensitive diagnostic assays have increased the detection of viruses in asymptomatic individuals. The clinical significance of asymptomatic respiratory viral infection in infants is unknown. METHODS: High-throughput, quantitative polymerase chain reaction assays were used to detect 13 common respiratory viruses from nasopharyngeal specimens collected during 2028 visits from 362 infants followed from near birth up to 12 months of age. Specimens were collected at monthly interval (months 1-6 and month 9) and during upper respiratory tract infection (URTI) episodes. Subjects were followed closely for acute otitis media (AOM) development. RESULTS: Viruses were detected in 76% of 394 URTI specimens and 27% of asymptomatic monthly specimens. Rhinovirus was detected most often; multiple viruses were detected in 29% of the specimens. Generalized mixed-model analyses associated symptoms with increasing age and female sex; detection of respiratory syncytial virus (RSV), influenza, rhinovirus, metapneumovirus, and adenovirus was highly associated with symptoms. Increasing age was also associated with multiple virus detection. Overall, 403 asymptomatic viral infections in 237 infants were identified. Viral load was significantly higher in URTI specimens than asymptomatic specimens but did not differentiate cases of URTI with and without AOM complication. The rate of AOM complicating URTI was 27%; no AOM occurred following asymptomatic viral infections. AOM development was associated with increasing age and infection with RSV, rhinovirus, enterovirus, adenovirus, and bocavirus. CONCLUSIONS: Compared to symptomatic infection, asymptomatic viral infection in infants is associated with young age, male sex, low viral load, specific viruses, and single virus detection. Asymptomatic viral infection did not result in AOM.

Quantification of bacterial uropathogens in preclinical samples using real-time PCR assays.

Uropathogenic Escherichia coli (UPEC) and Staphylococcus saprophyticus (S. saprophyticus) are responsible for the majority of community-acquired urinary tract infections (UTI). Agar plating, a gold standard for detection of bacterial uropathogens, is labor intensive, limited for distinguishing between environmental contaminants and pathogens, and fails to effectively detect mixed infections. A reliable method for specific and sensitive quantitative assessment of infections would allow cost-effective evaluation of large numbers of experimental samples. A methodology such as quantitative PCR (qPCR) addresses the limitations of agar plating. We developed and validated highly specific and sensitive qPCR assays to assist researchers in the evaluation of potential vaccines and interventions in preclinical models of UPEC and S. saprophyticus UTI. The developed UPEC PCR targeted a highly conserved region of the UPEC hemolysin D (hlyD) gene that reproducibly detected type strains CFT073 and J96 over a 9 log range with high precision. To quantify S. saprophyticus genomes, a separate qPCR assay targeting the Trk transport gene was developed with an 8 log range. Neither assay detected bacterial species predicted to be sample contaminants. Using our optimized workflow that includes automated steps, up to 200 urine or tissue samples can be processed in as few as 3 h. Additionally, sequence comparisons of our primers and probe to other UTI bacterial strains indicated the broad applicability of these assays. These optimized qPCR assays provide a cost-effective and time-saving method for quantification of bacterial burdens in tissues and body fluids to assess the effectiveness of candidate vaccines or interventions.

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.

What is the association between the microbiome and cognition? An umbrella review protocol.

INTRODUCTION: Cognitive impairment is reported in a variety of clinical conditions including Alzheimer's disease, Parkinson's and 'long-COVID'. Interestingly, many of these clinical conditions are also associated with microbial dysbiosis. This comanifestation of cognitive and microbiome findings in seemingly unrelated maladies suggests that they could share a common mechanism and potentially presents a treatment target. Although a rapidly growing body of literature has documented this comorbid presentation within specific conditions, an overview highlighting potential parallels across healthy and clinical populations is lacking. The objective of this umbrella review, therefore, is to summarise and synthesise the findings of these systematic reviews. METHODS AND ANALYSIS: On 2 April 2023, we searched MEDLINE (Pubmed), Embase (Ovid), the Web of Science (Core Collection), the Cochrane Library of Systematic Reviews and Epistemonikos as well as grey literature sources, for systematic reviews on clinical conditions and interventions where cognitive and microbiome outcomes were coreported. An updated search will be conducted before completion of the project if the search-to-publication date is >1 year old. Screening, data abstraction and quality assessment (AMSTAR 2, A MeaSurement Tool to Assess systematic Reviews) will be conducted independently and in duplicate, with disagreements resolved by consensus. Evidence certainty statements for each review's conclusions (eg, Grading of Recommendations Assessment, Development and Evaluation (GRADE)) will be extracted or constructed de novo. A narrative synthesis will be conducted and delineated by the review question. Primary study overlap will be visualised using a citation matrix as well as calculated using the corrected covered area method. ETHICS AND DISSEMINATION: No participant-identifying information will be used in this review. No ethics approval was required due to our study methodology. Our findings will be presented at national and international conferences and disseminated via social media and press releases. We will recruit at least one person living with cognitive impairment to collaborate on writing the plain language summary for the review. PROSPERO REGISTRATION NUMBER: CRD42023412903.

Growth hormone treatment for neurologic symptoms of post-acute sequelae of COVID-19.

Following SARS-CoV-2 infection, some patients develop lingering neurologic symptoms of post-acute sequelae of COVID-19 (PASC) that commonly include fatigue and "brain fog." PASC symptoms are also linked with reduced growth hormone (GH) secretion, but GH treatment has not been tested to relieve symptoms. We enrolled 13 adults with neurologic PASC symptoms and peak stimulated GH secretion less than 10 ng/mL (glucagon stimulation) in a pilot study to receive 9 months of daily GH injections and an additional 3 months of off-treatment assessment. We compared peak stimulated GH secretion at baseline and 12 months and assessed measures of cognition, metabolism, body composition, and physical performance over the first 6 months of treatment. Patient-reported outcomes of fatigue, quality of life, sleep, and mood were recorded at baseline and compared with timepoints at 6, 9, and 12 months. GH treatment was associated with significantly improved scores for Brief Fatigue Inventory, Multidimensional Fatigue Symptom Inventory, Quality of Life Assessment of Growth Hormone Deficiency in Adults, Profile of Mood States, and Beck Depression Inventory-II, with no significant change in Pittsburgh Sleep Quality Index. Six months of adjunct GH treatment was not associated with significant changes in cognition, body composition, resting energy expenditure, or physical performance. Peak stimulated GH secretion was not altered at 12 months following 9 months of GH treatment. GH treatment significantly improved neurologic symptoms in PASC patients but cognition, sleep, and physical performance were not significantly altered.

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).

Immunomodulatory and antibacterial effects of cystatin 9 against Francisella tularensis.

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.