The intestinal microbiota influences the development and function of myeloid lineages such as neutrophils, but the underlying molecular mechanisms are unresolved. Using gnotobiotic zebrafish, we ...identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization. Saa is a conserved secreted protein produced in the intestine and liver with described effects on neutrophils in vitro, however its in vivo functions remain poorly defined. We engineered saa mutant zebrafish to test requirements for Saa on innate immunity in vivo. Zebrafish mutant for saa displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. At baseline, saa mutants exhibited moderate neutrophilia and altered neutrophil tissue distribution. Molecular and functional analyses of isolated neutrophils revealed that Saa suppresses expression of pro-inflammatory markers and bactericidal activity. Saa's effects on neutrophils depended on microbiota colonization, suggesting this protein mediates the microbiota's effects on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we over-expressed saa in the intestine or liver and found that sufficient to partially complement neutrophil phenotypes observed in saa mutants. These results indicate Saa produced by the intestine in response to microbiota serves as a systemic signal to neutrophils to restrict aberrant activation, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to wounds.
Mortality in the first months of antiretroviral therapy (ART) is a significant clinical problem in sub-Saharan Africa. To date, no post-mortem study has investigated the causes of mortality in these ...patients.
HIV-positive adults who died as in-patients at a Johannesburg academic hospital underwent chart-review and ultrasound-guided needle autopsy for histological and microbiological examination of lung, liver, spleen, kidney, bone marrow, lymph node, skin and cerebrospinal fluid. A clinico-pathologic committee considered all available data and adjudicated immediate and contributing causes of death.
Thirty-nine adults were enrolled: 14 pre-ART, 15 early-ART (7-90 days), and 10 late-ART (>90 days). Needle sampling yielded adequate specimen in 100% of kidney, skin, heart and cerebrospinal fluid samples, 97% of livers and lungs, 92% of bone marrows, 87% of spleens and 68% of lymph nodes. Mycobacterial infections were implicated in 69% of deaths (26 of 27 of these due to M. tuberculosis), bacterial infections in 33%, fungal infections in 21%, neoplasm in 26%, and non-infectious organ failure in 26%. Immune reconstitution inflammatory syndrome (IRIS) was implicated in 73% of early-ART deaths. Post-mortem investigations revealed previously undiagnosed causes of death in 49% of cases. Multiple pathologies were common with 62% of subjects with mycobacterial infection also having at least one other infectious or neoplastic cause of death.
Needle biopsy was efficient and yielded excellent pathology. The large majority of deaths in all three groups were caused by M. tuberculosis suggesting an urgent need for improved diagnosis and expedited treatment prior to and throughout the course of antiretroviral therapy. Complex, unrecognized co-morbidities pose an additional challenge.
To determine the incidence, clinical manifestations, risk factors and outcome of immune reconstitution inflammatory syndrome (IRIS) in South Africa.
Prospective surveillance cohort and nested ...case-control study in a large, University hospital-based antiretroviral therapy (ART) clinic.
A total of 423 ART-naive HIV-infected South African patients were followed for signs and symptoms IRIS during the first 6 months of ART. We also performed a nested case-control study with controls matched to IRIS cases on ART duration.
During the first 6 months of ART, 44 (10.4%) patients experienced IRIS for an overall incidence rate of 25.1 cases per 100 patient-years. Diagnoses included tuberculosis (18/44, 41%), abscess formation and suppurative folliculitis (8/44, 18.2%), varicella zoster (6/44, 13.6%), herpes simplex (4/44, 9.1%), cryptococcal meningitis (3/44, 6.8%), molluscum contagiosum (3/44, 6.8%), and Kaposi's sarcoma (2/44, 4.5%). Median IRIS onset was 48 days (interquartile range, 29-99) from ART initiation. In comparison with controls, IRIS cases had significantly lower CD4 cell counts at baseline (79 versus 142 cells/microl; P = 0.02) and at IRIS diagnosis (183 versus 263 cells/microl; P = 0.05), but similar virological and immunological response to ART. In multivariable analyses, higher baseline CD4 cell count was protective of developing IRIS (HR 0.72 per 50 cells/microl increase). Most IRIS cases were mild, with ART discontinued in three (6.8%) patients, corticosteroids administered to four (9.1%) patients, and hospitalization required in 12 (27.3%) patients. Two deaths were attributable to IRIS.
IRIS may affect 10% of patients initiating ART in Africa, particularly those with advanced immunosuppression, but severe, life-threatening IRIS is uncommon.
The immune reconstitution inflammatory syndrome (IRIS) in HIV-infected patients initiating antiretroviral therapy (ART) results from restored immunity to specific infectious or non-infectious ...antigens. A paradoxical clinical worsening of a known condition or the appearance of a new condition after initiating therapy characterizes the syndrome. Potential mechanisms for the syndrome include a partial recovery of the immune system or exuberant host immunological responses to antigenic stimuli. The overall incidence of IRIS is unknown, but is dependent on the population studied and its underlying opportunistic infectious burden. The infectious pathogens most frequently implicated in the syndrome are mycobacteria, varicella zoster, herpesviruses, and cytomegalovirus (CMV). No single treatment option exists and depends on the underlying infectious agent and its clinical presentation. Prospective cohort studies addressing the optimal screening and treatment of opportunistic infections in patients eligible for ART are currently being conducted. These studies will provide evidence for the development of treatment guidelines in order to reduce the burden of IRIS. We review the available literature on the pathogenesis and epidemiology of IRIS, and present treatment options for the more common infectious manifestations of this diverse syndrome and for manifestations associated with a high morbidity.
For centuries, Bermuda has been challenged with wastewater management for the protection of human and environmental health. By quantifying the δ15N of the common sea fan Gorgonia ventalina sampled ...from 30 sites throughout Bermuda we show that sewage-derived nitrogen is detectable on nearshore coral reefs and declines across the lagoon to the outer rim. We also sampled gorgonians from two museum collections representing a 50y time-series (1958–2008). These samples revealed an increase in δ15N of >4.0‰ until the mid-1970s, after which δ15N values slowly declined by ~2.0‰. A δ15N chronology from a gorgonian skeleton exhibited a similar decline over the last 30–40years of approximately 0.6‰. We conclude that policies have been effective in reducing sewage impacts to Bermudian reefs. However, significant sources of sewage pollution persist and are likely have a strong impact on harbor and nearshore coral communities and human health.
Objectives:
Determine the prevalence of airway disease (e.g., asthma, airflow obstruction, and eosinophilic airway inflammation) in Kenya, as well as related correlates of airway disease and ...health-related quality of life.
Methods:
A three-stage, cluster-randomized cross-sectional study in Uasin Gishu County, Kenya was conducted. Individuals 12 years and older completed questionnaires (including St. George’s Respiratory Questionnaire for COPD, SGRQ-C), spirometry, and fractional exhaled nitric oxide (FeNO) testing. Prevalence ratios with 95% confidence intervals (CIs) were calculated. Multivariable models were used to assess correlates of airflow obstruction and high FeNO.
Results:
Three hundred ninety-two participants completed questionnaires, 369 completed FeNO testing, and 305 completed spirometry. Mean age was 37.5 years; 64% were women. The prevalence of asthma, airflow obstruction on spirometry, and eosinophilic airway inflammation was 21.7%, 12.3% and 15.7% respectively in the population. Women had significantly higher SGRQ-C scores compared to men (15.0 vs. 7.7). Wheezing or whistling in the last year and SGRQ-C scores were strongly associated with FeNO levels >50 ppb after adjusting for age, gender, BMI, and tobacco use.
Conclusion:
Airway disease is a significant health problem in Kenya affecting a young population who lack a significant tobacco use history.
Previous studies suggest that the presence of antigen-specific polyfunctional T cells is correlated with improved pathogen clearance, disease control, and clinical outcomes; however, the molecular ...mechanisms responsible for the generation, function, and survival of polyfunctional T cells remain unknown. The study of polyfunctional T cells has been, in part, limited by the need for intracellular cytokine staining (ICS), necessitating fixation and cell membrane permeabilization that leads to unacceptable degradation of RNA. Adopting elements from prior research efforts, we developed and optimized a modified protocol for the isolation of high-quality RNA (i.e., RIN > 7) from primary human T cells following aldehyde-fixation, detergent-based permeabilization, intracellular cytokines staining, and sorting. Additionally, this method also demonstrated utility preserving RNA when staining for transcription factors. This modified protocol utilizes an optimized combination of an RNase inhibitor and high-salt buffer that is cost-effective while maintaining the ability to identify and resolve cell populations for sorting. Overall, this protocol resulted in minimal loss of RNA integrity, quality, and quantity during cytoplasmic staining of cytokines and subsequent flourescence-activated cell sorting. Using this technique, we obtained the transcriptional profiles of functional subsets (i.e., non-functional, monofunctional, bifunctional, polyfunctional) of CMV-specific CD8+T cells. Our analyses demonstrated that these functional subsets are molecularly distinct, and that polyfunctional T cells are uniquely enriched for transcripts involved in viral response, inflammation, cell survival, proliferation, and metabolism when compared to monofunctional cells. Polyfunctional T cells demonstrate reduced activation-induced cell death and increased proliferation after antigen re-challenge. Further
analysis of transcriptional data suggested a critical role for
transcriptional activity in polyfunctional cell activation. Pharmacologic inhibition of
was associated with a significant reduction in polyfunctional cell cytokine expression and proliferation, demonstrating the requirement of STAT5 activity not only for proliferation and cell survival, but also cytokine expression. Finally, we confirmed this association between CMV-specific CD8+ polyfunctionality with
signaling also exists in immunosuppressed transplant recipients using single cell transcriptomics, indicating that results from this study may translate to this vulnerable patient population. Collectively, these results shed light on the mechanisms governing polyfunctional T cell function and survival and may ultimately inform multiple areas of immunology, including but not limited to the development of new vaccines, CAR-T cell therapies, and adoptive T cell transfer.
A promising strategy to cure HIV-infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, ...reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5%, 11.2%, and 12.1% percentage of cells, respectively. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single-cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found.
Objective
Global medical oxygen security is limited by knowledge gaps in hypoxaemia burden and oxygen access in low-income and middle-income countries. We examined the prevalence and phenotypic ...trajectories of hypoxaemia among hospitalised adults in Kenya, with a focus on chronic hypoxaemia.
Design
Single-centre, prospective cohort study.
Setting
National tertiary referral hospital in Eldoret, Kenya between September 2019 and April 2022.
Participants
Adults (age ≥18 years) admitted to general medicine wards.
Primary and secondary outcome measures
Our primary outcome was proportion of patients who were hypoxaemic (oxygen saturation, SpO2 ≤88%) on admission. Secondary outcomes were proportion of patients with hypoxaemia on admission who had hypoxaemia resolution, hospital discharge, transfer, or death among those with unresolved hypoxaemia or chronic hypoxaemia. Patients remaining hypoxaemic for ≤3 days after admission were enrolled into an additional cohort to determine chronic hypoxaemia. Chronic hypoxaemia was defined as an SpO2 ≤ 88% at either 1-month post-discharge follow-up or, for patients who died prior to follow-up, a documented SpO2 ≤88% during a previous hospital discharge or outpatient visit within the last 6 months.
Results
We screened 4104 patients (48.5% female, mean age 49.4±19.4 years), of whom 23.8% were hypoxaemic on admission. Hypoxaemic patients were significantly older and more predominantly female than normoxaemic patients. Among those hypoxaemic on admission, 33.9% had resolution of their hypoxaemia as inpatients, 55.6% had unresolved hypoxaemia (31.0% died before hospital discharge, 13.3% were alive on discharge and 11.4% were transferred) and 10.4% were lost to follow-up. The prevalence of chronic hypoxaemia was 2.1% in the total screened population, representing 8.8% of patients who were hypoxaemic on admission. Chronic hypoxaemia was determined at 1-month post-discharge among 59/86 patients and based on prior documentation among 27/86 patients.
Conclusion
Hypoxaemia is highly prevalent among adults admitted to a general medicine ward at a national referral hospital in Kenya. Nearly 1 in 11 patients who are hypoxaemic on admission are chronically hypoxaemic.
BACKGROUNDPreclinical studies suggest that cholesterol accumulation leads to insulin resistance. We previously reported that alterations in a monocyte cholesterol metabolism transcriptional network ...(CMTN) - suggestive of cellular cholesterol accumulation - were cross-sectionally associated with obesity and type 2 diabetes (T2D). Here, we sought to determine whether the CMTN alterations independently predict incident prediabetes/T2D risk, and correlate with cellular cholesterol accumulation.METHODSMonocyte mRNA expression of 11 CMTN genes was quantified among 934 Multi-Ethnic Study of Atherosclerosis (MESA) participants free of prediabetes/T2D; cellular cholesterol was measured in a subset of 24 monocyte samples.RESULTSDuring a median 6-year follow-up, lower expression of 3 highly correlated LXR target genes - ABCG1 and ABCA1 (cholesterol efflux) and MYLIP (cholesterol uptake suppression) - and not other CMTN genes, was significantly associated with higher risk of incident prediabetes/T2D. Lower expression of the LXR target genes correlated with higher cellular cholesterol levels (e.g., 47% of variance in cellular total cholesterol explained by ABCG1 expression). Further, adding the LXR target genes to overweight/obesity and other known predictors significantly improved prediction of incident prediabetes/T2D.CONCLUSIONThese data suggest that the aberrant LXR/ABCG1-ABCA1-MYLIP pathway (LAAMP) is a major T2D risk factor and support a potential role for aberrant LAAMP and cellular cholesterol accumulation in diabetogenesis.FUNDINGThe MESA Epigenomics and Transcriptomics Studies were funded by NIH grants 1R01HL101250, 1RF1AG054474, R01HL126477, R01DK101921, and R01HL135009. This work was supported by funding from NIDDK R01DK103531 and NHLBI R01HL119962.
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