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Background and Purpose: Neuropsychiatric complications of SARS-CoV-2 infection, also known as neurologic postacute sequelae of SARS-CoV-2 infection (NeuroPASC), affect 10%-60% of infected individuals. There is growing evidence that NeuroPASC is a multi system immune dysregulation disease affecting the brain. The behavioral manifestations of NeuroPASC, such as impaired processing speed, executive function, memory retrieval, and sustained attention, suggest widespread WM involvement. Although previous work has documented WM damage following acute SARS-CoV-2 infection, its involvement in NeuroPASC is less clear. We hypothesized that macrostructural and microstructural WM differences in NeuroPASC participants would accompany cognitive and immune system differences., Materials and Methods: In a cross-sectional study, we screened a total of 159 potential participants and enrolled 72 participants, with 41 asymptomatic controls (NoCOVID) and 31 NeuroPASC participants matched for age, sex, and education. Exclusion criteria included neurologic disorders unrelated to SARS-CoV-2 infection. Assessments included clinical symptom questionnaires, psychometric tests, brain MRI measures, and peripheral cytokine levels. Statistical modeling included separate multivariable regression analyses of GM/WM/CSF volume, WM microstructure, cognitive, and cytokine concentration between-group differences., Results: NeuroPASC participants had larger cerebral WM volume than NoCOVID controls (β = 0.229; 95% CI: 0.017-0.441; t = 2.16; P = .035). The most pronounced effects were in the prefrontal and anterior temporal WM. NeuroPASC participants also exhibited higher WM mean kurtosis, consistent with ongoing neuroinflammation. NeuroPASC participants had more self-reported symptoms, including headache, and lower performance on measures of attention, concentration, verbal learning, and processing speed. A multivariate profile analysis of the cytokine panel showed different group cytokine profiles (Wald-type-statistic = 44.6, P = .046), with interferon (IFN)-λ1 and IFN-λ2/3 levels higher in the NeuroPASC group., Conclusions: NeuroPASC participants reported symptoms of lower concentration, higher fatigue, and impaired cognition compatible with WM syndrome. Psychometric testing confirmed these findings. NeuroPASC participants exhibited larger cerebral WM volume and higher WM mean kurtosis than NoCOVID controls. These findings suggest that immune dysregulation could influence WM properties to produce WM volume increases and consequent cognitive effects and headaches. Further work will be needed to establish mechanistic links among these variables., (© 2024 by American Journal of Neuroradiology.)

Infection by Salmonella enterica serovar Typhi ( S . Typhi), the cause of enteric fevers, is low in high-income countries but persistent in low- and middle-income countries, resulting in 65,400-187,700 deaths yearly. Drug resistance, including in the United States, exacerbates this issue. Evidence indicates that innate lymphocytes (INLs), such as natural killer (NK) cells, and unconventional T lymphocytes ( e.g ., Mucosal-associated invariant T (MAIT) cells and T-cell receptor gamma delta (TCR-γδ) cells) can impact the intestinal epithelial barrier, the primary site of exposure to S . Typhi. Moreover, INL production of IFN-γ is central in controlling S . Typhi infection. However, the impact of epithelial cells (EC) on the secretion of IFN-γ by INLs and the relationship between these events and epigenetic changes remains unknown. Epigenetic modifications in host cells are fundamental for their differentiation and function, including IFN-γ production. Herein, using a human organoid-derived polarized intestinal epithelial cell monolayer, we investigated the role of H3K4me3 and H3K27me3 epigenetic marks in intestinal immunity, focusing on the function of EC, NK, MAIT, and TCR-γδ cells in response to S . Typhi. This study builds on our previous findings that MAIT subsets exhibiting specific IFN-γ pattern signatures were associated with protection against typhoid fever and that S . Typhi infection regulates changes in chromatin marks that depend on individual cell subsets. Here, we show that cultures exposed to S . Typhi without EC exhibit a significant increase in NK and MAIT cells, and, to a lesser extent, TCR-γδ cells, expressing IFN-γ and H3K4me3 but not H3K27me3 marks, contrasting with cultures where EC is present. The influence of EC on INL H3K4me3 marks might be indirectly mediated through the modulation of IL-18 secretion via the Histone Deacetylase 6 gene during S . Typhi infection., 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 and Sztein.)

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.)

Previous work has shown that Secretory-IgA (SIgA) binding to the intestinal microbiota is variable and may regulate host inflammatory bowel responses. Nevertheless, the impact of the SIgA functional binding to the microbiota remains largely unknown in preterm infants whose immature epithelial barriers make them particularly susceptible to inflammation. Here, we investigated SIgA binding to intestinal microbiota isolated from stools of preterm infants <33 weeks gestation with various levels of intestinal permeability. We found that SIgA binding to intestinal microbiota attenuates inflammatory reactions in preterm infants. We also observed a significant correlation between SIgA affinity to the microbiota and the infant's intestinal barrier maturation. Still, SIgA affinity was not associated with developing host defenses, such as the production of mucus and inflammatory calprotectin protein, but it depended on the microbiota shifts as the intestinal barrier matures. In conclusion, we reported an association between the SIgA functional binding to the microbiota and the maturity of the preterm infant's intestinal barrier, indicating that the pattern of SIgA coating is altered as the intestinal barrier matures., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Salmonella enterica serovar Typhi (S. Typhi) causes substantial morbidity and mortality worldwide, particularly among young children. Humans develop an array of mucosal immune responses following S. Typhi infection. Whereas the cellular mechanisms involved in S. Typhi infection have been intensively studied, very little is known about the early chromatin modifications occurring in the human gut microenvironment that influence downstream immune responses. To address this gap in knowledge, cells isolated from human terminal ileum exposed ex vivo to the wild-type S. Typhi strain were stained with a 33-metal-labeled antibody panel for mass cytometry analyses of the early chromatin modifications modulated by S. Typhi. We measured the cellular levels of 6 classes of histone modifications, and 1 histone variant in 11 major cell subsets (i.e., B, CD3 + T, CD4 + T, CD8 + T, NK, TCR-γδ, Mucosal associated invariant (MAIT), and NKT cells as well as monocytes, macrophages, and epithelial cells). We found that arginine methylation might regulate the early-differentiation of effector-memory CD4+ T-cells following exposure to S. Typhi. We also found S. Typhi-induced post-translational modifications in histone methylation and acetylation associated with epithelial cells, NKT, MAIT, TCR-γδ, Monocytes, and CD8 + T-cells that are related to both gene activation and silencing.

Enteric fevers, caused by the Salmonella enterica serovars Typhi (ST), Paratyphi A (PA) and Paratyphi B (PB), are life-threatening illnesses exhibiting very similar clinical symptoms but with distinct epidemiologies, geographical distributions and susceptibilities to antimicrobial treatment. Nevertheless, the mechanisms by which the host recognizes pathogens with high levels of homology, such as these bacterial serovars, remain poorly understood. Using a three-dimensional organotypic model of the human intestinal mucosa and PA, PB, and ST, we observed significant differences in the secretion patterns of pro-inflammatory cytokines and chemokines elicited by these serovars. These cytokines/chemokines were likely to be co-regulated and influenced the function of epithelial cells, such as the production of IL-8. We also found differing levels of polymorphonuclear leukocyte (PMN) migration among various infection conditions that either included or excluded lymphocytes and macrophages (Mϕ), strongly suggesting feedback mechanisms among these cells. Blocking experiments showed that IL-1β, IL-6, IL-8, TNF-α and CCL3 cytokines were involved in the differential regulation of migration patterns. We conclude that the crosstalk among the lymphocytes, Mϕ, PMN and epithelial cells is cytokine/chemokine-dependent and bacterial-serotype specific, and plays a pivotal role in orchestrating the functional efficiency of the innate cells and migratory characteristics of the leukocytes., Competing Interests: The authors declare that the 3-D model of the intestinal mucosa is covered by a US patent (Number: US 9,200,258 B2). Inventors: Rosangela Mezghanni (maiden name Salerno-Goncalves), Alessio Fasano and Marcelo B Sztein

A notable proportion of Salmonella -associated gastroenteritis in the United States is attributed to Salmonella enterica serovar Typhimurium. We have previously shown that live-attenuated S Typhimurium vaccine candidate CVD 1921 (I77 Δ guaBA Δ clpP ) was safe and immunogenic in rhesus macaques but was shed for an undesirably long time postimmunization. In mice, occasional mortality postvaccination was also noted (approximately 1 in every 15 mice). Here we describe a further attenuated vaccine candidate strain harboring deletions in two additional genes, htrA and pipA We determined that S Typhimurium requires pipA to elicit fluid accumulation in a rabbit ileal loop model of gastroenteritis, as an S Typhimurium Δ pipA mutant induced significantly less fluid accumulation in rabbit loops than the wild-type strain. New vaccine strain CVD 1926 (I77 Δ guaBA Δ clpP Δ pipA Δ htrA ) was assessed for inflammatory potential in an organoid model of human intestinal mucosa, where it induced less inflammatory cytokine production than organoids exposed to the precursor vaccine, CVD 1921. To assess vaccine safety and efficacy, mice were given three doses of CVD 1926 (10 9 CFU/dose) by oral gavage, and at 1 or 3 months postimmunization, mice were challenged with 700 or 100 LD 50 (50% lethal doses), respectively, of wild-type strain I77. CVD 1926 was well tolerated and exhibited 47% vaccine efficacy following challenge with a high inoculum and 60% efficacy after challenge with a low inoculum of virulent S Typhimurium. CVD 1926 is less reactogenic yet equally as immunogenic and protective as previous iterations in a mouse model., (Copyright © 2018 American Society for Microbiology.)

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

Gastrointestinal infections by Salmonella enterica serovar Typhi ( S . Typhi) are rare in industrialized countries. However, they remain a major public health problem in the developing world with an estimated 26.9 million new cases annually and significant mortality when untreated. Recently, we provided the first direct evidence that CD8 + MAIT cells are activated and have the potential to kill cells exposed to S . Typhi, and that these responses are dependent on bacterial load. However, MAIT cell kinetics and function during bacterial infections in humans remain largely unknown. In this study, we characterize the human CD8 + MAIT cell immune response to S . Typhi infection in subjects participating in a challenge clinical trial who received a low- or high dose of wild-type S . Typhi. We define the kinetics of CD8 + MAIT cells as well as their levels of activation, proliferation, exhaustion/apoptosis, and homing potential. Regardless of the dose, in volunteers resistant to infection (NoTD), the levels of CD8 + MAIT cells after S . Typhi challenge fluctuated around their baseline values (day 0). In contrast, volunteers susceptible to the development of typhoid disease (TD) exhibited a sharp decline in circulating MAIT cells during the development of typhoid fever. Interestingly, MAIT cells from low-dose TD volunteers had higher levels of CD38 coexpressing CCR9, CCR6, and Ki67 during the development of typhoid fever than high-dose TD volunteers. No substantial perturbations on the levels of these markers were observed in NoTD volunteers irrespective of the dose. In sum, we describe, for the first time, that exposure to an enteric bacterium, in this case S . Typhi, results in changes in MAIT cell activation, proliferation, and homing characteristics, suggesting that MAIT cells are an important component of the human host response to bacterial infection.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA's Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

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.

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, and S. Paratyphi A and B, causative agents of paratyphoid fever, are major public health threats throughout the world. Although two licensed typhoid vaccines are currently available, they are only moderately protective and immunogenic necessitating the development of novel vaccines. A major obstacle in the development of improved typhoid, as well as paratyphoid vaccines is the lack of known immunological correlates of protection in humans. Considerable progress has been made in recent years in understanding the complex adaptive host responses against S. Typhi. Although the induction of S. Typhi-specific antibodies (including their functional properties) and memory B cells, as well as their cross-reactivity with S. Paratyphi A and S. Paratyphi B has been shown, the role of humoral immunity in protection remains undefined. Cell mediated immunity (CMI) is likely to play a dominant role in protection against enteric fever pathogens. Detailed measurements of CMI performed in volunteers immunized with attenuated strains of S. Typhi have shown, among others, the induction of lymphoproliferation, multifunctional type 1 cytokine production, and CD8(+) cytotoxic T-cell responses. In addition to systemic responses, the local microenvironment of the gut is likely to be of paramount importance in protection from these infections. In this review, we will critically assess current knowledge regarding the role of CMI and humoral immunity following natural S. Typhi and S. Paratyphi infections, experimental challenge, and immunization in humans. We will also address recent advances regarding cross-talk between the host's gut microbiota and immunization with attenuated S. Typhi, mechanisms of systemic immune responses, and the homing potential of S. Typhi-specific B- and T-cells to the gut and other tissues.

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.

A common finding when measuring T cell immunity to enteric bacterial vaccines in humans is the presence of background responses among individuals before immunization. Yet the nature of these background responses remains largely unknown. Recent findings show the presence in uninfected individuals of mucosal associated invariant T (MAIT) cells that mount broad spectrum immune responses against a variety of microorganisms including Mycobacterium tuberculosis and enteric bacteria such as Escherichia coli and Salmonella. Therefore, we investigated whether MAIT immune responses to intestinal bacteria might account for the background responses observed before immunization. Here we measured MAIT immune responses to commensal and enteric pathogenic bacteria in healthy individuals with no history of oral immunization with enteric bacteria. We found that MAIT cells were activated by B cells infected with various bacteria strains (commensals and pathogens from the Enterobacteriaceae family), but not by uninfected cells. These responses were restricted by the non-classical MHC-related molecule 1 (MR1) and involved the endocytic pathway. The quality of these responses (i.e., cytokine profile) was dependent on bacterial load but not on the level expression of MR1 or bacterial antigen on B cell surface, suggesting that a threshold level of MR1 expression is required to trigger MAIT activation. These results provide important insights into the role of B cells as a source of antigen-presenting cells to MAIT cells and the gut immune surveillance of commensal microbiota.

T cells are likely to play an important role in the host defense against Salmonella enterica serovar Typhi, the causative agent of typhoid fever. We have shown that HLA-E can function as a restriction element for S. Typhi-specific CD8(+) T cells. Because of the potential importance of HLA-E-restricted CD8(+) responses in resistance to Salmonella infection, we characterized these responses and investigated their kinetics of appearance and persistence in volunteers immunized orally with the licensed attenuated Ty21a strain typhoid vaccine. Cells were obtained from volunteers before and at days 2, 4, 7, 10, 14, 28, 42, 56, 120, 180, 360, and 720 after immunization. An ex vivo multicolor staining panel including antibodies to CD107a and -b, interleukin-2, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) was used to functionally assess memory T-cell subsets by flow cytometry. Increases in cytokine-secreting CD8(+) cells were observed in the T effector/memory (T(EM)) and CD45RA(+) T(EM) (T(EMRA)) subsets as early as 4 days after immunization and persisted, particularly in the T(EMRA) subset, up to 2 years after immunization. The majority of HLA-E-restricted CD8(+) cells 28 to 56 days after immunization coexpressed CD107, IFN-gamma, and TNF-alpha, showing characteristic features of multifunctional T cells. In summary, the multifunctionality and longevity of the HLA-E-restricted CD8 responses observed in this study highlight their significance in adaptive immunity to S. Typhi. Finally, this is the first demonstration, in either animals or humans, of the presence of long-term multifunctional HLA-E-restricted CD8(+) cells after immunization.

Background: The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8(+) T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-gamma secretion. However, how S. Typhi regulates the development of specific CD8(+) responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8(+) T cells., Methodology/principal Findings: We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-alpha, but low levels of IL-12 p70 and IFN-gamma. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells., Conclusions/significance: This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8(+) cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.

Salmonella enterica serovar Typhi (S. typhi) strain Ty21a remains the only licensed attenuated typhoid vaccine. Despite years of research, the identity of the protective immunological mechanisms elicited by immunization with the Ty21a typhoid vaccine remains elusive. The present study was designed to characterize effector T cell responses in volunteers immunized with S. typhi strain Ty21a typhoid vaccine. We determined whether immunization with Ty21a induced specific CTL able to lyse S. typhi-infected cells and secrete IFN-gamma, a key effector molecule against intracellular pathogens. We measured the functional activity of these CTL by a (51)Cr-release assay using 8-day restimulated PBMC from Ty21a vaccinees as effector cells and S. Typhi-infected autologous PHA-activated PBMC as target cells. Most vaccinees exhibited consistently increased CD8-mediated lysis of targets by postimmunization PBMC when compared with preimmunization levels. We also developed an IFN-gamma ELISPOT assay to quantify the frequency of IFN-gamma spot-forming cells (SFC) in PBMC from Ty21a vaccinees using an ex vivo system. Significant increases in the frequency of IFN-gamma SFC following immunization (mean +/- SD, 393 +/- 172; range 185-548 SFC/10(6) PBMC; p = 0.010), as compared with preimmunization levels, were observed. IFN-gamma was secreted predominantly by CD8(+) T cells. A strong correlation was recorded between the cytolytic activity of CTL lines and the frequency of IFN-gamma SFC (r(2) = 0.910, p < 0.001). In conclusion, this work constitutes the first evidence that immunization of volunteers with Ty21a elicits specific CD8(+) CTL and provides an estimate of the frequency of CD8(+) IFN-gamma-secreting cells induced by vaccination.

AY 9944 [AY; trans-1,4-bis(chlorobenzylaminomethyl)-cyclohexane dihydrochloride], an inhibitor of sterol synthesis, was found to help restore the normal mitogenic responses and cytokine profiles of peripheral mononuclear cells (PBMCs) from AIDS patients in vitro. Compared to untreated cells, the human immunodeficiency virus type 1 (HIV-1)-infected PBMCs precultured in the presence of AY exhibited a normal rate of either mitogen-induced or recall- and superantigen-induced proliferation. After 2 weeks in the presence of the drug, the percentage of dead CD4(+) cells in HIV-1-infected cultures was comparable to that observed in uninfected cultures, while over the same time interval it increased by three- to fivefold in HIV-1-infected cultures maintained in the absence of AY. AY also stimulated by 2- to 12-fold interleukin-12 (IL-12) and (gamma interferon production. For IL-12, this effect appears to be related to an increase in corresponding IL-12 p35 and IL-12 p40 mRNA levels. Moreover, AY restored the expression of the IL-2 receptor, which was severely impaired in HIV-1-infected PBMCs. Although the drug has no direct antiviral effect (it does not significantly inhibit reverse transcriptase activity measured in vitro), it might be considered a potential therapeutic agent for HIV-infected patients, in that it may correct viral infection-related immune system defects by indirectly enhancing the level of resistance to HIV and opportunistic infections.

Objective: During HIV-1 infection, CD4+ T lymphocytes migrate to immune-reactive lymphoid organs where they are infected by the virus and/or killed by apoptosis on immunoregulatory stimuli--a potential mechanism underlying fatal CD4+ T-cell depletion observed in AIDS. This study seeks to determine the effects of glucocorticoids (GCC) on the activation-induced T-cell apoptosis triggered by HIV-1., Methods: CD4+ and CD8+ T cells were purified from HIV-negative donor peripheral blood mononuclear cells (PBMC) by positive selection and exposed to HIV-1 (primary isolates). HIV-1-exposed CD4+ and CD8+ T cells as well as PBMC derived from HIV-1-infected patients were cultured with medium alone or anti-CD3 monoclonal antibodies (MAb)/mitogens in the presence or absence of hydrocortisone or prednisolone. Viral infection kinetics were assessed by polymerase chain reaction and viral replication was measured by p24 enzyme-linked immunosorbent assay. Cell survival, apoptosis, T-cell proliferation, blast cell transformation, and interleukin (IL)-2 receptor (CD25) expression were monitored in parallel for each cell population., Results: Fractionated CD4+ T cells acutely infected by HIV-1 underwent apoptotic death on anti-CD3 MAb/mitogen stimulation. This activation-induced apoptotic cell killing was antagonized by pharmacological doses of prednisolone or hydrocortisone added up to 6 h after stimulation. GCC were also found to be capable of inhibiting the accelerated apoptosis in PBMC (including both CD4+ and CD8+ T-cell fractions) from HIV-1-infected patients. This anti-apoptotic action of GCC overbalanced their downregulatory effect on T-cell proliferation, resulting in an overall improvement of CD4+ T-cell survival in patient PBMC. These effects of GCC were abrogated by the anti-GCC RU 486 and were not associated with significant suppression of CD25 expression and IL-2-dependent T-cell blast transformation; moreover, GCC had no impact on viral infection and replication., Conclusion: GCC exert a receptor-mediated anti-apoptotic activity in mature T cells through both activation-induced and HIV-1-triggered pathways and could be potent inhibitors of T-cell apoptosis in HIV-1-infected patients.