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Despite decades of intense research, our understanding of the correlates of protection against Salmonella Typhi ( S . Typhi) infection and disease remains incomplete. T follicular helper cells (T FH ), an important link between cellular and humoral immunity, play an important role in the development and production of high affinity antibodies. While traditional T FH cells reside in germinal centers, circulating T FH (cT FH ) (a memory subset of T FH ) are present in blood. We used specimens from a typhoid controlled human infection model whereby participants were immunized with Ty21a live attenuated S . Typhi vaccine and then challenged with virulent S . Typhi. Some participants developed typhoid disease (TD) and some did not (NoTD), which allowed us to assess the association of cT FH subsets in the development and prevention of typhoid disease. Of note, the frequencies of cT FH were higher in NoTD than in TD participants, particularly 7 days after challenge. Furthermore, the frequencies of cT FH 2 and cT FH 17, but not cT FH 1 subsets were higher in NoTD than TD participants. However, we observed that ex-vivo expression of activation and homing markers were higher in TD than in NoTD participants, particularly after challenge. Moreover, cT FH subsets produced higher levels of S . Typhi-specific responses (cytokines/chemokines) in both the immunization and challenge phases. Interestingly, unsupervised analysis revealed unique clusters with distinct signatures for each cT FH subset that may play a role in either the development or prevention of typhoid disease. Importantly, we observed associations between frequencies of defined cT FH subsets and anti- S. Typhi antibodies. Taken together, our results suggest that circulating T FH 2 and T FH 17 subsets might play an important role in the development or prevention of typhoid disease. The contribution of these clusters was found to be distinct in the immunization and/or challenge phases. These results have important implications for vaccines aimed at inducing long-lived protective T cell and antibody responses., Competing Interests: ML: Co-inventor of a live attenuated S. Typhi vaccine strain CVD 909 and a S. Paratyphi A vaccine strain CVD 1902 that have been licensed to Bharat Biotech International BBI, Hyderabad, India for clinical development. ML is also a co-inventor of a Trivalent Salmonella Conjugate vaccine that includes S. Enteritidis, S. Typhimurium conjugates core plus O-polysaccharide covalently linked to FliC flagellin subunits of the homologous serovars in combination with BBI’s Vi conjugate Typbar TCV. AP: is chair of the UK department of Health and Social Cares Joint Committee on vaccination and immunisation. He has research grants on typhoid/paratyphoid vaccines from Serum Institute of India, Medical Research Council, Wellcome Trust and Bill & Melinda gates Foundation. Author MM was employed by company Sanofi. Authors SF and CB were employed by company GlaxsoSmithKline. Author RR is presently employed at Moderna Therapeutics and owns shares/options. Her contributions to this project were made prior to her employment at Moderna. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Booth, Rapaka, McArthur, Fresnay, Darton, Blohmke, Jones, Waddington, Levine, Pollard and Sztein.)

The mucosal immune system is a critical first line of defense to infectious diseases, as many pathogens enter the body through mucosal surfaces, disrupting the balanced interactions between mucosal cells, secretory molecules, and microbiota in this challenging microenvironment. The mucosal immune system comprises of a complex and integrated network that includes the gut-associated lymphoid tissues (GALT). One of its primary responses to microbes is the secretion of IgA, whose role in the mucosa is vital for preventing pathogen colonization, invasion and spread. The mechanisms involved in these key responses include neutralization of pathogens, immune exclusion, immune modulation, and cross-protection. The generation and maintenance of high affinity IgA responses require a delicate balance of multiple components, including B and T cell interactions, innate cells, the cytokine milieu (e.g., IL-21, IL-10, TGF-β), and other factors essential for intestinal homeostasis, including the gut microbiota. In this review, we will discuss the main cellular components (e.g., T cells, innate lymphoid cells, dendritic cells) in the gut microenvironment as mediators of important effector responses and as critical players in supporting B cells in eliciting and maintaining IgA production, particularly in the context of enteric infections and vaccination in humans. Understanding the mechanisms of humoral and cellular components in protection could guide and accelerate the development of more effective mucosal vaccines and therapeutic interventions to efficiently combat mucosal infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Carreto-Binaghi, Sztein and Booth.)

Background: An increase in opioid-related overdoses, notably from potent synthetic opioids like fentanyl, prompted this consideration of characteristics of emergency department (ED) patients with evidence for illicit fentanyl use or exposure, the correlation with intentional opioid misuse, and subsequent ED management., Methods: A retrospective review was performed of patients presenting to an urban academic medical center ED with evidence for illicit fentanyl use, determined by positive urine drug screens (UDS), from 6/2021 through 11/2021. Participant demographics, comorbidities, ED chief complaint and disposition, and evidence of intentional opioid misuse were considered. Secondary outcomes included provision of buprenorphine/naloxone and/or naloxone kits at discharge, ED recidivism, and six-month mortality. Bivariate comparisons and logistic regression models were performed., Results: Among 409 unique patients, most were white and male with a mean age of 39.4. Approximately half presented with opioid-related complaints. Evidence of intentional opioid misuse was identified in 72.6 % of patients. Black patients had 79 % lower odds of intentional opioid misuse compared to white patients. Regarding ED management, 28.8 % were discharged with buprenorphine/naloxone and 14.0 % with a naloxone kit. Black patients had 63 % lower odds of receiving buprenorphine/naloxone compared to white patients after controlling for covariates. Nearly 6 % of the study population died within six months of the initial ED visit., Conclusion: This fentanyl-focused review describes patient characteristics which largely mirror the epidemiology of the current opioid epidemic; however, despite evidence of objective exposure, it also suggests that Black patients may be less likely to use fentanyl intentionally. It also highlights potential disparities related to ED-based opioid misuse patient management., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Falls and osteoporosis are risk factors for fragility fractures. Bone mineral density (BMD) assessment is associated with better preventative osteoporosis care, but it is underutilized by those at high fracture risk. We created a novel electronic medical record (EMR) alert-driven protocol to screen patients in the Emergency Department (ED) for fracture risk and tested its feasibility and effectiveness in generating and completing referrals for outpatient BMD testing after discharge. The EMR alert was configured in 2 tertiary-care EDs and triggered by the term "fall" in the chief complaint, age (≥65 years for women, ≥70 years for men), and high fall risk (Morse score ≥ 45). The alert electronically notified ED study staff of potentially eligible patients. Participants received osteoporosis screening education and had BMD testing ordered. From November 15, 2020 to December 4, 2021, there were 2,608 EMR alerts among 2,509 patients. We identified 558 patients at high-risk of fracture who were screened for BMD testing referral. Participants were excluded for: serious illness ( N  = 141), no documented health insurance to cover BMD testing ( N  = 97), prior BMD testing/recent osteoporosis care ( N  = 58), research assistant unavailable to enroll ( N  = 53), concomitant fracture ( N  = 43), bedridden status ( N  = 38), chief complaint of fall documented in error ( N  = 38), long-term care residence ( N  = 34), participation refusal ( N  = 32), or hospitalization ( N  = 3). Of the 16 participants who had BMD testing ordered, 7 scheduled and 5 completed BMD testing. EMR alerts can help identify subpopulations who may benefit from osteoporosis screening, but there are significant barriers to identifying eligible and willing patients for screening in the ED. In our study targeting an innovative venue for osteoporosis care delivery, only about 1% of patients at high-risk of fracture scheduled BMD testing after an ED visit. Adequate resources during and after an ED visit are needed to ensure that older adults participate in preventative osteoporosis care., Competing Interests: None to report., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Salmonella enterica serovar Typhi ( S . Typhi), a human-restricted pathogen, invades the host through the gut to cause typhoid fever. Recent calculations of the typhoid fever burden estimated that more than 10 million new typhoid fever cases occur in low and middle-income countries, resulting in 65,400-187,700 deaths yearly. Interestingly, if not antibiotic-treated, upon the resolution of acute disease, 1%-5% of patients become asymptomatic chronic carriers. Chronically infected hosts are not only critical reservoirs of infection that transmit the disease to naive individuals but are also predisposed to developing gallbladder carcinoma. Nevertheless, the molecular mechanisms involved in the early interactions between gallbladder epithelial cells and S . Typhi remain largely unknown. Based on our previous studies showing that closely related S . Typhi strains elicit distinct innate immune responses, we hypothesized that host molecular pathways activated by S . Typhi strains derived from acutely and chronically infected patients would differ. To test this hypothesis, we used a novel human organoid-derived polarized gallbladder monolayer model, and S . Typhi strains derived from acutely and chronically infected patients. We found that S . Typhi strains derived from acutely and chronically infected patients differentially regulate host mitogen-activated protein kinase (MAPK) and S6 transcription factors. These variations might be attributed to differential cytokine signaling, predominantly via TNF-α and IL-6 production and appear to be influenced by the duration the isolate was subjected to selective pressures in the gallbladder. These findings represent a significant leap in understanding the complexities behind chronic S . Typhi infections in the gallbladder and may uncover potential intervention targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Salerno-Goncalves, Chen, Bafford, Izquierdo, Hormazábal, Lagos, Tettelin, D’Mello, Booth, Fasano, Levine and Sztein.)

Introduction: The opioid epidemic has been exacerbated by the COVID-19 pandemic, resulting in increased acute care opioid-related and overdose visits. We sought to assess how the pandemic may have impacted an obstetric cohort impacted by opioid misuse in the acute care context., Methods: A retrospective review of acute care presentations of patients with concomitant pregnancy (Z33.1) and opioid-related diagnostic codes (T10 codes and/or F11) was conducted over a 24-month period (pre-COVID = March 2019 through February 2020, post-COVID = March 2020 through February 2021). Descriptive statistics and χ2 analysis of pre- versus post-COVID presentations were performed., Results: A total of 193 individuals, 104 (53.9%) pre- and 89 (46.1%) post-COVID, accounting for 292 total encounters, 160 (54.8%) pre- and 132 (45.2%) post-COVID, were seen for acute care visits ( P = 0.84). Age ( P = 0.15), race ( P = 0.59), and insurance status ( P = 0.17) were similar pre- versus post-COVID. The majority of presentations, pre- (40.4%) and post-COVID (44.9%), were for opioid withdrawal ( P = 0.74). Although post-COVID individuals were more likely to lack prenatal care (48.3% versus 39.4% pre-COVID), this trend was not significant ( P = 0.19). Similar proportions of individuals were affected by pregnancy complications (51.9% pre-, 44.9% post-COVID; P = 0.30). Similar proportions of individuals were affected by adverse pregnancy outcomes (44.2% pre-, 48.3% post-COVID; P = 0.64)., Conclusion: The COVID-19 pandemic did not have a statistically significant effect on opioid-related acute care presentations or outcomes for obstetric patients. In this acute care cohort, however, opioid misuse had significant general impact on pregnancy complications and outcomes, suggesting unmet needs in this population., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 American Society of Addiction Medicine.)

Enteric infectious diseases account for more than a billion disease episodes yearly worldwide resulting in approximately 2 million deaths, with children under 5 years old and the elderly being disproportionally affected. Enteric pathogens comprise viruses, parasites, and bacteria; the latter including pathogens such as Salmonella [typhoidal (TS) and non-typhoidal (nTS)], cholera, Shigella and multiple pathotypes of Escherichia coli ( E. coli ). In addition, multi-drug resistant and extensively drug-resistant (XDR) strains (e.g., S. Typhi H58 strain) of enteric bacteria are emerging; thus, renewed efforts to tackle enteric diseases are required. Many of these entero-pathogens could be controlled by oral or parenteral vaccines; however, development of new, effective vaccines has been hampered by lack of known immunological correlates of protection (CoP) and limited knowledge of the factors contributing to protective responses. To fully comprehend the human response to enteric infections, an invaluable tool that has recently re-emerged is the use of controlled human infection models (CHIMs) in which participants are challenged with virulent wild-type (wt) organisms. CHIMs have the potential to uncover immune mechanisms and identify CoP to enteric pathogens, as well as to evaluate the efficacy of therapeutics and vaccines in humans. CHIMs have been used to provide invaluable insights in the pathogenesis, host-pathogen interaction and evaluation of vaccines. Recently, several Oxford typhoid CHIM studies have been performed to assess the role of multiple cell types (B cells, CD8+ T, T regs , MAIT, Monocytes and DC) during S . Typhi infection. One of the key messages that emerged from these studies is that baseline antigen-specific responses are important in that they can correlate with clinical outcomes. Additionally, volunteers who develop typhoid disease (TD) exhibit higher levels and more activated cell types (e.g., DC and monocytes) which are nevertheless defective in discrete signaling pathways. Future critical aspects of this research will involve the study of immune responses to enteric infections at the site of entry, i.e., the intestinal mucosa. This review will describe our current knowledge of immunity to enteric fevers caused by S. Typhi and S. Paratyphi A, with emphasis on the contributions of CHIMs to uncover the complex immunological responses to these organisms and provide insights into the determinants of protective immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sztein and Booth.)

Introduction: Emergency department (ED) boarding, the process of holding patients in the ED due to a lack of inpatient beds after the decision is made to admit, has profound consequences. Increased ED boarding times are associated with adverse patient outcomes, including increased mortality. While previous studies have demonstrated racial disparities with regard to ED boarding, current literature lacks insight into discrepancies that may exist among other demographic groups as it pertains to ED boarding. We sought to review ED boarding times differentiated by demographic characteristics., Methods: We conducted a retrospective review of all ED admissions from an academic ED in the Southeast from April-September 2019. The primary outcome assessed was boarding time, defined as time from decision to admit to ED departure. Patient demographic data including race, gender, and age were collected and analyzed. We performed descriptive statistics and chi-square analyses., Results: The study population included 17,606 patients with a mean age of 56.3. Nearly half (49.8%) of the patients were female. Additionally, 43.8% of patients were Black and 48.6% White. For all admissions, there was no difference in mean boarding time among Black and White patients (5.2 ± 8.8 vs 5.2 ± 8.2 hours, P = 0.11). Among Emergency Severity Index (ESI) level I admissions, Black patients boarded longer than White patients (4.1 ± 0.3 vs 2.7 ± 0.3 hours, P = 0.009). Black patients also boarded significantly longer than White patients for psychiatric admissions (22.7 ± 23.7 vs 18.5 ± 19.4 hours, P <0.05). For all admissions, males boarded longer than females (5.5 ± 8.5 vs 4.9 ± 8.2 hours, P <.0001). Patients older than 75 boarded for less time (3.8 ± 6.2 hours) compared to younger groups (15-24: 6.4 ± 10.8 hours; 25-44: 6.6 ± 10.8; 45-64: 5.0 ± 7.6; and 64-75: 4.7 ± 6.7; all P <.05)., Conclusion: This analysis demonstrated significant differences in ED boarding times between races among psychiatric and ESI I admissions, gender, and age. This data provides insight into differences in ED boarding times among demographic groups and provides a focal point for examining possible factors contributing to the observed differences.

Background: The impact of aging on the immune system is unequivocal and results in an altered immune status termed immunosenescence. In humans, the mechanisms of immunosenescence have been examined almost exclusively in blood. However, most immune cells are present in tissue compartments and exhibit differential cell (e.g., memory T cells -T M ) subset distributions. Thus, it is crucial to understand immunosenescence in tissues, especially those that are exposed to pathogens (e.g., intestine). Using a human model of oral live attenuated typhoid vaccine, Ty21a, we investigated the effect of aging on terminal ileum (TI) tissue resident memory T (T RM ) cells. T RM provide immediate adaptive effector immune responsiveness at the infection site. However, it is unknown whether aging impacts T RM S. Typhi-responsive cells at the site of infection (e.g., TI). Here, we determined the effect of aging on the induction of TI S. Typhi-responsive T RM subsets elicited by Ty21a immunization., Results: We observed that aging impacts the frequencies of TI-lamina propria mononuclear cells (LPMC) T M and T RM in both Ty21a-vaccinated and control groups. In unvaccinated volunteers, the frequencies of LPMC CD103- CD4+ T RM displayed a positive correlation with age whilst the CD4/CD8 ratio in LPMC displayed a negative correlation with age. We observed that elderly volunteers have weaker S. Typhi-specific mucosal immune responses following Ty21a immunization compared to adults. For example, CD103+ CD4+ T RM showed reduced IL-17A production, while CD103- CD4+ T RM exhibited lower levels of IL-17A and IL-2 in the elderly than in adults following Ty21a immunization. Similar results were observed in LPMC CD8+ T RM and CD103- CD8+ T cell subsets. A comparison of multifunctional (MF) profiles of both CD4+ and CD8+ T RM subsets between elderly and adults also showed significant differences in the quality and quantity of elicited single (S) and MF responses., Conclusions: Aging influences tissue resident T M S. Typhi-specific responses in the terminal ileum following oral Ty21a-immunization. This study is the first to provide insights in the generation of local vaccine-specific responses in the elderly population and highlights the importance of evaluating tissue immune responses in the context of infection and aging., Trial Registration: This study was approved by the Institutional Review Board and registered on ClinicalTrials.gov (identifier NCT03970304 , Registered 29 May 2019 - Retrospectively registered).

Many patients with gout flares treated in the Emergency Department (ED) often do not receive optimal continuity of care after an ED visit. Thus, developing methods to identify patients with gout flares in the ED and referring them to appropriate outpatient gout care is required. While Natural Language Processing (NLP) has been used to detect gout flares retrospectively, it is much more challenging to identify patients prospectively during an ED visit where documentation is usually minimal. We annotate a corpus of ED triage nurse chief complaint notes for the presence of gout flares and implement a simple algorithm for gout flare ED alerts. We show that the chief complaint alone has strong predictive power for gout flares. We make available a de-identified version of this corpus annotated for gout mentions, which is to our knowledge the first free text chief complaint clinical corpus available., (©2020 AMIA - All rights reserved.)

B and T cells are key components of the adaptive immune system and coordinate multiple facets of immunity including responses to infection, vaccines, allergens, and the environment. In humans, B- and T-cell immunity has been determined using primarily peripheral blood specimens. Conversely, human tissues have scarcely been studied but they host multiple adaptive immune cells capable of mounting immune responses to pathogens and participate in tissue homeostasis. Mucosal tissues, such as the intestines and respiratory track, are constantly bombarded by foreign antigens and contain tissue-resident memory T (T RM ) cells that exhibit superior protective capacity to pathogens. Also, tissue-resident memory B (B RM ) cells have been identified in mice but whether humans have a similar population remains to be confirmed. Moreover, the immune system evolves throughout the lifespan of humans and undergoes multiple changes in its immunobiology. Recent studies have shown that age-related changes in tissues are not necessarily reflected in peripheral blood specimens, highlighting the importance of tissue localization and subset delineation as essential determinants of functional B and T cells at different life stages. This review describes our current knowledge of the main B- and T-cell subsets in peripheral blood and tissues across age groups.

Objective: There are no vaccines for most of the major invasive Salmonella strains causing severe infection in humans. We evaluated the specificity of adaptive T memory cell responses generated after Salmonella Typhi exposure in humans against other major invasive Salmonella strains sharing capacity for dissemination., Methods: T memory cells from eleven volunteers who underwent controlled oral challenge with wt S . Typhi were characterised by flow cytometry for cross-reactive cellular cytokine/chemokine effector responses or evidence of degranulation upon stimulation with autologous B-lymphoblastoid cells infected with either S . Typhi, Salmonella Paratyphi A (PA), S . Paratyphi B (PB) or an invasive nontyphoidal Salmonella strain of the S . Typhimurium serovar (iNTSTy)., Results: Blood T-cell effector memory (T EM ) responses after exposure to S . Typhi in humans evolve late, peaking weeks after infection in most volunteers. Induced multifunctional CD4 + Th1 and CD8 + T EM cells elicited after S . Typhi challenge were cross-reactive with PA, PB and iNTSTy. The magnitude of multifunctional CD4 + T EM cell responses to S . Typhi correlated with induction of cross-reactive multifunctional CD8 + T EM cells against PA, PB and iNTSTy. Highly multifunctional subsets and T central memory and T effector memory cells that re-express CD45 (T EMRA ) demonstrated less heterologous T-cell cross-reactivity, and multifunctional Th17 elicited after S . Typhi challenge was not cross-reactive against other invasive Salmonella ., Conclusion: Gaps in cross-reactive immune effector functions in human T-cell memory compartments were highly dependent on invasive Salmonella strain, underscoring the importance of strain-dependent vaccination in the design of T-cell-based vaccines for invasive Salmonella ., Competing Interests: AJP is chair of the UK Department of Health's Joint Committee on Vaccination and Immunisation and the European Medicine Agency Scientific Advisory Group on Vaccines, and a member of WHO's Strategic Advisory Group of Experts and an NIHR Senior Investigator. The other authors declare no conflicts of interest., (© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Background: Salmonella enterica serovar Typhi (S. Typhi) is a highly invasive bacterium that infects the human intestinal mucosa and causes ~ 11.9-20.6 million infections and ~ 130,000-223,000 deaths annually worldwide. Oral typhoid vaccine Ty21a confers a moderate level of long-lived protection (5-7 years) in the field. New and improved vaccines against enteric pathogens are needed but their development is hindered by a lack of the immunological correlates of protection especially at the site of infection. Tissue resident memory T (T RM ) cells provide immediate adaptive effector immune responsiveness at the infection site. However, the mechanism(s) by which S. Typhi induces T RM in the intestinal mucosa are unknown. Here, we focus on the induction of S. Typhi-specific CD4+T RM subsets by Ty21a in the human terminal ileum lamina propria and epithelial compartments., Methods: Terminal ileum biopsies were obtained from consenting volunteers undergoing routine colonoscopy who were either immunized orally with 4 doses of Ty21a or not. Isolated lamina propria mononuclear cells (LPMC) and intraepithelial lymphocytes (IEL) CD4+T RM immune responses were determined using either S. Typhi-infected or non-infected autologous EBV-B cell lines as stimulator cells. T-CMI was assessed by the production of 4 cytokines [interferon (IFN)γ, interleukin (IL)-2, IL-17A and tumor necrosis factor (TNF)α] in 36 volunteers (18 vaccinees and 18 controls volunteers)., Results: Although the frequencies of LPMC CD103+ CD4+T RM were significant decreased, both CD103+ and CD103- CD4+T RM subsets spontaneously produced significantly higher levels of cytokines (IFNγ and IL-17A) following Ty21a-immunization. Importantly, we observed significant increases in S. Typhi-specific LPMC CD103+ CD4+T RM (IFNγ and IL-17A) and CD103- CD4+T RM (IL-2 and IL-17A) responses following Ty21a-immunization. Further, differences in S. Typhi-specific responses between these two CD4+T RM subsets were observed following multifunctional analysis. In addition, we determined the effect of Ty21a-immunization on IEL and observed significant changes in the frequencies of IEL CD103+ (decrease) and CD103- CD4+T RM (increase) following immunization. Finally, we observed that IEL CD103- CD4+T RM , but not CD103+ CD4+T RM , produced increased cytokines (IFNγ, TNFα and IL-17A) to S. Typhi-specific stimulation following Ty21a-immunization., Conclusions: Oral Ty21a-immunization elicits distinct compartment specific immune responses in CD4+T RM (CD103+ and CD103-) subsets. This study provides novel insights in the generation of local vaccine-specific responses. Trial registration This study was approved by the Institutional Review Board and registered on ClinicalTrials.gov (identifier NCT03970304, Registered 29 May 2019-Retrospectively registered, http://www.ClinicalTrials.gov/NCT03970304).

Objectives: The objective of this quality improvement (QI) project was to decrease the rate of low-value computed tomography (CT) imaging in established gynecologic oncology patients presenting to the emergency department (ED)., Methods: This was a cohort study with a before and after design that evaluated implementation of a QI project designed to decrease CT utilization in established gynecologic oncology patients in the ED. The pre-intervention cohort included patients admitted through the ED from 4/1/17 to 5/31/18, while the post-intervention cohort was from 6/1/18 to 5/31/19. The intervention included gynecologic oncology consultation before CT on patients who had imaging within the prior 3 weeks. Details regarding CT, ED length of stay (LOS), and oncologic history were abstracted. The value of CT was determined by consensus from 2 reviewers. Prospective data monitoring evaluated for patient safety., Results: Prior to intervention, there were 129 unique ED encounters in gynecologic oncology patients leading to admission. CT scans were performed in 101 (78.3%) encounters, 57.7% of which were deemed to be of low-value. Following implementation, the CT utilization rate decreased significantly from median monthly rate of 75.2% to 49.1% (p < 0.00001), and the ED LOS decreased from 8.1 to 6.9 h (p = 0.0102). The number of CT scans deemed to be low-value in the post-intervention group decreased to 2 (3.8%)., Conclusions: Implementation of an early consultation policy and imaging guidelines led to a significant decrease in unnecessary CT utilization and shorter ED LOS in gynecologic oncology patients presenting to the ED., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tissue-resident memory T cells (T RM ) are newly defined memory T cells (T M ) distinct from circulating T M subsets which have the potential to mount rapid protective immune responses at the site of infection. However, very limited information is available regarding the role and contribution of T RM in vaccine-mediated immune responses in humans at the site of infection. Here, we studied the role and contribution of tissue resident memory T cells (T RM ) located in the terminal ileum (TI) (favored site of infection for S . Typhi) following oral Ty21a immunization in humans. We examined TI-lamina propria mononuclear cells (LPMC) and intra-epithelial lymphocytes (IEL) CD8+ T RM subsets obtained from healthy volunteers undergoing medically-indicated colonoscopies who were either immunized with Ty21a or unvaccinated. No significant differences in the frequencies of LPMC CD8+ T RM and CD8+CD69+CD103- T cells subsets were observed following Ty21a-immunization. However, LPMC CD8+ T RM exhibited significantly higher levels of cytokines (IFN-γ, IL-17A, and TNF-α) ex-vivo in Ty21a-vaccinated than in unvaccinated volunteers. LPMC CD8+ T RM S . Typhi-specific responses were evaluated using S . Typhi-infected targets and found to produce significantly higher levels of S . Typhi-specific IL-17A. In contrast, LPMC CD8+CD69+CD103- T cells produced significantly increased S . Typhi-specific levels of IFN-γ, IL-2, and IL-17A. Finally, we assessed CD8+ T RM in IEL and observed that the frequency of IEL CD8+ T RM is significantly lower following Ty21a immunization. However, ex-vivo IEL CD8+ T RM elicited by Ty21a immunization spontaneously produced significantly higher levels of cytokines (IFN-γ, IL-17A, IL-2, and TNF-α). This study provides the first demonstration of the effect of oral Ty21a vaccination on CD8+ T RM subsets (spontaneous and S . Typhi-specific) responses in the LPMC and IEL compartment of the human terminal ileum mucosa, contributing novel information to our understanding of the generation of mucosal immune responses following oral Ty21a-immunization.

Our current understanding of CD4+ T-cell-mediated immunity (CMI) elicited by the oral live attenuated typhoid vaccine Ty21a is primarily derived from studies using peripheral blood. Very limited data are available in humans regarding mucosal immunity (especially CD4+ T) at the site of infection (e.g. terminal ileum; TI). Here using multiparametric flow cytometry, we examined the effect of Ty21a immunization on TI-lamina propria mononuclear cells (LPMC) and peripheral blood CD4+ T memory (TM) subsets in volunteers undergoing routine colonoscopy. Interestingly, we observed significant increases in the frequencies of LPMC CD4+ T cells following Ty21a immunization, restricted to the T effector/memory (TEM)-CD45RA+ (TEMRA) subset. Importantly, Ty21a immunization elicited Salmonella Typhi-responsive LPMC CD4+ T cells in all major TM subsets [interferon (IFN)γ and interleukin (IL)-17A in TEM; IFNγ and macrophage inflammatory protein (MIP)1β in T central/memory (TCM); and IL-2 in TEMRA]. Subsequently, we analyzed LPMC S. Typhi-responsive CD4+ T cells in depth for multifunctional (MF) effectors. We found that LPMC CD4+ TEM responses were mostly MF, except for those cells exhibiting the characteristics associated with IL-17A responses. Finally, we compared mucosal to systemic responses and observed that LPMC CD4+S. Typhi-specific responses were unique and distinct from their systemic counterparts. This study provides the first demonstration of S. Typhi-specific CD4+ TM responses in the human TI mucosa and provides valuable information about the generation of mucosal immune responses following oral Ty21a immunization., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Japanese Society for Immunology.)

CD4+ and CD8+ T subsets are essential components of the adaptive immune system which act in concert at the site of infections to effectively protect against pathogens. Very limited data is available in humans regarding the relationship between CD4+ and CD8+ S . Typhi responsive cells in the terminal ileum mucosa (TI) and peripheral blood following Ty21a oral typhoid immunization. Here, we compared TI lamina propria mononuclear cells (LPMC) and peripheral blood CD4+ and CD8+ T memory (T M ) subsets responses and their relationship by Spearman's correlation following Ty21a immunization in volunteers undergoing routine colonoscopy. We observed that Ty21a immunization (i) influences the homing and accumulation of both CD4+ and CD8+ T cells in the TI, particularly integrin α4β7+ CCR9+ CD8+ T cells, (ii) elicits significantly higher frequencies of LPMC S . Typhi-responsive CD8+ T multifunctional (CD107a, IFNγ, IL-17A and/or MIP1β) cells than their CD4+ T counterparts, and (iii) results in the correlation of LPMC CD4+ Teffector/memory (T EM ) S . Typhi responses (CD107a, IFNγ, TNFα, IL-17A and/or MIP1β) to their LPMC CD8+ T EM counterparts. Moreover, we demonstrated that these positive correlations between CD4+ and CD8+ T EM occur primarily in TI LPMC but not in PBMC, suggesting important differences in responses between the mucosal and systemic compartments following oral Ty21a immunization. This study provides the first demonstration of the correlation of S . Typhi-specific CD4+ and CD8+ T M responses in the human terminal ileum mucosa and provides valuable information regarding the generation of mucosal and systemic immune responses following oral Ty21a-immunization which might impact future vaccine design and development.

Background & Aims: Systemic cellular immunity elicited by the Ty21a oral typhoid vaccine has been extensively characterized. However, very limited data are available in humans regarding mucosal immunity at the site of infection (terminal ileum [TI]). Here we investigated the host immunity elicited by Ty21a immunization on terminal ileum-lamina propria mononuclear cells (LPMC) and peripheral blood in volunteers undergoing routine colonoscopy., Methods: We characterized LPMC-T memory (T M ) subsets and assessed Salmonella enterica serovar Typhi ( S Typhi)-specific responses by multichromatic flow cytometry., Results: No differences were observed in cell yields and phenotypes in LPMC CD8 + -T M subsets following Ty21a immunization. However, Ty21a immunization elicited LPMC CD8 + T cells exhibiting significant S Typhi-specific responses (interferon-γ, tumor necrosis factor-α, interleukin-17A, and/or CD107a) in all major T M subsets (T-effector/memory [T EM ], T-central/memory, and T EM -CD45RA + ), although each T M subset exhibited unique characteristics. We also investigated whether Ty21a immunization elicited S Typhi-specific multifunctional effectors in LPMC CD8 + T EM . We observed that LPMC CD8 + T EM responses were mostly multifunctional, except for those cells exhibiting the characteristics associated with cytotoxic responses. Finally, we compared mucosal with systemic responses and made the important observation that LPMC CD8 + S Typhi-specific responses were unique and distinct from their systemic counterparts., Conclusions: This study provides the first demonstration of S Typhi-specific responses in the human terminal ileum mucosa and provides novel insights into the generation of mucosal immune responses following oral Ty21a immunization.

Mucosal-associated invariant T (MAIT) cells represent a class of antimicrobial innate-like T cells that have been characterized in human blood, liver, lungs, and intestine. Here, we investigated, for the first time, the presence of MAIT cells in the stomach of children, adults, and the elderly undergoing routine endoscopy and assessed their reactivity to Helicobacter pylori (H. pylori - Hp), a major gastric pathogen. We observed that MAIT cells are present in the lamina propria compartment of the stomach and display a similar memory phenotype to blood MAIT cells. We then demonstrated that gastric and blood MAIT cells are able to recognize H. pylori. We found that CD8(+) and CD4(-)CD8(-) (double negative) MAIT cell subsets respond to H. pylori-infected macrophages stimulation in a MR-1 restrictive manner by producing cytokines (IFN-γ, TNF-α, IL-17A) and exhibiting cytotoxic activity. Interestingly, we observed that blood MAIT cell frequency in Hp(+ve) individuals was significantly lower than in Hp(-ve) individuals. However, gastric MAIT cell frequency was not significantly different between Hp(+ve) and Hp(-ve) individuals, demonstrating a dichotomy between blood and gastric tissues. Further, we observed that the majority of gastric MAIT cells (>80%) expressed tissue-resident markers (CD69(+) CD103(+)), which were only marginally present on PBMC MAIT cells (<3%), suggesting that gastric MAIT cells are readily available to respond quickly to pathogens. These results contribute important new information to the understanding of MAIT cells function on peripheral and mucosal tissues and its possible implications in the host response to H. pylori.

T cells are the main orchestrators of protective immunity in the stomach; however, limited information on the presence and function of the gastric T subsets is available mainly due to the difficulty in recovering high numbers of viable cells from human gastric biopsies. To overcome this shortcoming we optimized a cell isolation method that yielded high numbers of viable lamina propria mononuclear cells (LPMC) from gastric biopsies. Classic memory T subsets were identified in gastric LPMC and compared to peripheral blood mononuclear cells (PBMC) obtained from children, adults, and the elderly using an optimized 14 color flow cytometry panel. A dominant effector memory T (TEM) phenotype was observed in gastric LPMC CD4(+) and CD8(+) T cells in all age groups. We then evaluated whether these cells represented a population of gastric tissue-resident memory T (TRM) cells by assessing expression of CD103 and CD69. The vast majority of gastric LPMC CD8(+) T cells either co-expressed CD103/CD69 (>70%) or expressed CD103 alone (~20%). Gastric LPMC CD4(+) T cells also either co-expressed CD103/CD69 (>35%) or expressed at least one of these markers. Thus, gastric LPMC CD8(+) and CD4(+) T cells had the characteristics of TRM cells. Gastric CD8(+) and CD4(+) TRM cells produced multiple cytokines (IFN-γ, IL-2, TNF-α, IL-17A, MIP-1β) and up-regulated CD107a upon stimulation. However, marked differences were observed in their cytokine and multi-cytokine profiles when compared to their PBMC TEM counterparts. Furthermore, gastric CD8(+) TRM and CD4(+) TRM cells demonstrated differences in the frequency, susceptibility to activation, and cytokine/multi-cytokine production profiles among the age groups. Most notably, children's gastric TRM cells responded differently to stimuli than gastric TRM cells from adults or the elderly. In conclusion, we demonstrate the presence of gastric TRM, which exhibit diverse functional characteristics in children, adults, and the elderly.

Background. While often first treated in the emergency department (ED), identification of sepsis is difficult. Electronic medical record (EMR) clinical decision tools offer a novel strategy for identifying patients with sepsis. The objective of this study was to test the accuracy of an EMR-based, automated sepsis identification system. Methods. We tested an EMR-based sepsis identification tool at a major academic, urban ED with 64,000 annual visits. The EMR system collected vital sign and laboratory test information on all ED patients, triggering a "sepsis alert" for those with ≥2 SIRS (systemic inflammatory response syndrome) criteria (fever, tachycardia, tachypnea, leukocytosis) plus ≥1 major organ dysfunction (SBP ≤ 90 mm Hg, lactic acid ≥2.0 mg/dL). We confirmed the presence of sepsis through manual review of physician, nursing, and laboratory records. We also reviewed a random selection of ED cases that did not trigger a sepsis alert. We evaluated the diagnostic accuracy of the sepsis identification tool. Results. From January 1 through March 31, 2012, there were 795 automated sepsis alerts. We randomly selected 300 cases without a sepsis alert from the same period. The true prevalence of sepsis was 355/795 (44.7%) among alerts and 0/300 (0%) among non-alerts. The positive predictive value of the sepsis alert was 44.7% (95% CI [41.2-48.2%]). Pneumonia and respiratory infections (38%) and urinary tract infection (32.7%) were the most common infections among the 355 patients with true sepsis (true positives). Among false-positive sepsis alerts, the most common medical conditions were gastrointestinal (26.1%), traumatic (25.7%), and cardiovascular (20.0%) conditions. Rates of hospital admission were: true-positive sepsis alert 91.0%, false-positive alert 83.0%, no sepsis alert 5.7%. Conclusions. This ED EMR-based automated sepsis identification system was able to detect cases with sepsis. Automated EMR-based detection may provide a viable strategy for identifying sepsis in the ED.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs signal through TLR9 and activate innate immunity resulting in protection against a variety of parasitic, bacterial and viral pathogens in mouse models. However, few studies have demonstrated protection in humans and large animals. In the present investigations, we evaluated protection by CpG ODN in a parainfluenza-3 (PI-3) virus infection in neonatal lambs. Subcutaneous (SC) injection of CpG ODN induced high levels of 2'5'-A synthetase and significantly reduced PI-3 virus shedding in newborn lambs. Furthermore, pre-treatment of newborn lambs with SC CpG ODN 2 days, but not 6 days prior to the virus challenge was protective. In contrast, intratracheal (IT) administration of CpG ODN induced 2'5'-A synthetase but had no significant impact on PI-3 virus shedding in nasal secretions. We conclude that a systemic administration of CpG ODN and the timing of the treatment are critical for the protection of neonatal lambs against a respiratory viral infection., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) play an important role in the activation of innate and adaptive immune responses. Stimulation with multiple TLR agonists may result in synergistic, complimentary or inhibitory effects on innate immune responses. In this study, we investigated the effects of co-stimulation of sheep peripheral blood mononuclear cells (PBMC) and B cells with agonists for TLR3, 4, 7/8 and 9. Sheep PBMC stimulated with either CpG (TLR9 agonist) or RNA oligoribonucleotides ([ORNs], TLR7/8 agonist) exhibited significant IL-12 production, but only CpG induced IFNalpha, IgM and proliferative responses. In contrast, poly(I:C) (TLR3 agonist) and LPS (TLR4 agonist) did not induce any of these responses. Interestingly, we observed that co-stimulation of PBMC with CpG+ORN or CpG+imiquimod (another TLR7/8 agonist) resulted in significant reduction in CpG-induced IFNalpha production, B cell proliferation and IgM responses. Pre-incubation of cells with CpG prior to exposure of the cells to imiquimod resulted in similar inhibitory responses indicating that the down-regulatory mechanisms are not associated with competition for cellular uptake or for receptors of the two agonists. Sheep B cells constitutively expressed TLR7, TLR8 and TLR9 mRNA transcripts, suggesting a possible role of TLR cross-talk in the down-regulatory mechanisms. Down-regulation of responses by co-stimulation with closely related TLRs may be a regulatory mechanism by which the host prevents overstimulation of innate immune responses., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Intestinal Peyer's patch (PP) regulatory CD21+ B cells (B(regs)) suppress TLR9-induced innate immune responses. However, it is not known whether TLR9 activation is regulated in PP B(regs). Here, we investigated the responses of PP B(regs) to stimulation with the TLR9 agonist CpG oligodeoxynucleotides (ODN). We observed that PP CD21+ B(regs) express high levels of TLR9 mRNA, but fail to proliferate when stimulated with CpG ODN. Furthermore, unlike CD21+ B cells from blood, PP CD21+ B(regs) do not secrete IgM or IL-12 following CpG ODN stimulation. We hypothesized that the unresponsiveness of PP B(regs) to CpG stimulation was due to an inability of the TLR9 agonist to activate the TLR9 signaling pathway in these cells. This was confirmed by kinome analysis which demonstrated dynamic patterns of phosphorylation of key TLR adaptor proteins such as IRAK1, TAK1, IKK and NF-kappaB-p65 in CpG-stimulated blood CD21+ B cells, consistent with activation of the TLR9 pathway. In contrast, stimulation of PP CD21+ B(regs) with CpG ODN resulted in phosphorylation patterns of these adaptor proteins suggestive of inactivation of the TLR9 pathway. The absence of apparent TLR9 signaling events immediately following stimulation indicated that signaling is blocked close to the receptor. Our observations suggest a novel mechanism by which the host regulates TLR responses in TLR-expressing cells with regulatory functions., (2010 S. Karger AG, Basel.)

Influenza A viruses cause significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. Vaccination is the primary method for the prevention of influenza virus infection. Previously, we generated two elastase-dependent mutant SIVs derived from A/Sw/Saskatchewan/18789/02(H1N1): A/Sw/Sk-R345V (R345V) and A/Sw/Sk-R345A (R345A). These two viruses are highly attenuated in pigs, making them good candidates for a live-virus vaccine. In this study, the immunogenicity and the ability of these candidates to protect against SIV infection were evaluated in pigs. We report that intratracheally administrated R345V and R345A induced antigen-specific humoral and cell-mediated immunity characterized by increased production of immunoglobulin G (IgG) and IgA antibodies in the serum and in bronchoalveolar lavage fluid, high hemagglutination inhibition titers in serum, an enhanced level of lymphocyte proliferation, and higher numbers of gamma interferon-secreting cells at the site of infection. Based on the immunogenicity results, the R345V virus was further tested in a protection trial in which pigs were vaccinated twice with R345V and then challenged with homologous A/Sw/Saskatchewan/18789/02, H1N1 antigenic variant A/Sw/Indiana/1726/88 or heterologous subtypic H3N2 A/Sw/Texas/4199-2/9/98. Our data showed that two vaccinations with R345V provided pigs with complete protection from homologous H1N1 SIV infection and partial protection from heterologous subtypic H3N2 SIV infection. This protection was characterized by significantly reduced macroscopic and microscopic lung lesions, lower virus titers from the respiratory tract, and lower levels of proinflammatory cytokines. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs.

Peyer's patches (PPs) play an important role in the induction of immune responses in the intestine, but regulation of Toll-like receptor (TLR)-induced innate immune responses in PPs is not well understood. We investigated the responses of PPs and other immune cells to the TLR9 agonist, CpG oligodeoxynucleotide (ODN). Peripheral blood mononuclear cells and lymph node cells secreted significant amounts of interferon (IFN)-alpha, IFNgamma, and interleukin (IL)-12 following stimulation with CpG ODN. In contrast, PP cells exhibited poor cytokine responses, despite abundant expression of TLR9 mRNA. PP cells spontaneously secreted high levels of IL-10, and the primary source of the IL-10 was resting CD5(-)CD11c(-)CD21(+) B cells. Neutralization of the IL-10 or depletion of CD21(+) B cells resulted in a significant increase in CpG-induced IFNalpha-response in PPs, suggesting that IL-10 from B cells regulate innate responses in PPs. These IL-10-secreting PP B cells may represent a novel subset of the recently proposed regulatory B cells (B(regs)) in the intestine.

CpG ODN signal through Toll-like receptor 9 (TLR9) and trigger a cascade of events that lead to activation of innate and adaptive immune responses. Our current understanding of the immunobiology of host responses to CpG is based largely on studies on peripheral blood mononuclear cells (PBMC) and splenocytes. Little is known regarding CpG-induced responses in other lymphoid tissues. In the present study, we investigated responses induced by CpG in both PBMC and lymph nodes. Cells were isolated from the superficial cervical lymph node (LNC) and blood and then stimulated with CpG ODN (either A-, or B- or C-class ODN). Cytokine production was assayed by ELISA, and lymphocyte proliferation was determined by (3)H-thymidine incorporation. NK-like cytotoxicity was analyzed by lysis of (51)Cr-labelled target cells. All three classes of CpG induced IFNalpha and IFNgamma in LNC. In contrast, only A and C-class ODN induced IFNalpha and IFNgamma in PBMC. Moreover, the IFN levels in LNC were 20-40-fold higher than in PBMC. Furthermore, all classes of ODN induced higher IL-12 levels in LNC (five- to six-fold) than in PBMC. Both B and C-class ODN induced good proliferative responses in PBMC and LNC, but the A-class ODN did not induce proliferation of PBMC and only induced moderate proliferation of LNC. A-class ODN induced significant NK-like activity in LNC. Thus, all three classes of CpG ODN induced similar responses in LNC, and these responses were consistently higher than in PBMC. These observations indicate that CpG ODN-induced responses differ between blood and lymph nodes, and suggest that the functional classification of CpG ODN based on PBMC responses may not be directly applicable to cells from other immune tissues.

Booth, James S. (University of Southern California, Los Angeles) and Milo D. Appleman. Degradation of ergothioneine by cell-free extracts of Alcaligenes faecalis. II. Production of glutamic acid. J. Bacteriol. 85:654-657. 1963.-On the basis of oxidation and paper chromatographic procedures, glutamic acid was identified as the end product of ergothioneine degradation by cell-free extracts of Alcaligenes faecalis. Hydrogen sulfide and ammonia yields were determined. Several differences between the metabolism of whole cells and cell-free extracts were noted. Cleavage of the imidazole ring by cell-free extracts appeared to be hydrolytic rather than oxidative.