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Developing new tools for studying the intestinal epithelium aids in addressing unanswered questions in developmental biology, physiology, and disease pathology. Primary intestinal epithelial stem cell (ISC) cultures are an invaluable in vitro model system. However, their cultivation remains technically demanding and costly, limiting their accessibility and use. Though commonly utilized, most extant intestinal epithelial cell lines were derived from colon adenomas from various mammals, are largely uncharacterized, and possess a plethora of genetic abnormalities, thus restricting rigor, reproducibility, and physiological relevance. Here we derived immortalized intestinal epithelial cell lines (iIECs) from jejunal and colonic stem cell-enriched spheroid cultures from wild type C57BL/6 mice. We transduced ISCs with a lentiviral vector encoding the SV40 large T antigen, and subsequently selected lines through adaptation to growth on plastic. Additionally, we weaned these lines off conditioned medium including the growth factors Wnt3a, Rspo3, and Noggin. We found that iIECs have growth characteristics similar to conventional plastic adapted cell lines. While we observed transcriptional signatures of epithelial-–mesenchymal transition (EMT), these cells retained several epithelial characteristics including expression of junctional proteins, segment-specific identities, and mounted an innate immune response to viral infection. In addition, we found several fibroblastic clones secreted cytokines in response to LPS stimulation. We further demonstrated the utility of iIECs through transient and stable genetic manipulation, as well as pathogen infection. iIECs occupy an important experimental niche through offering a scalable, practical, and physiologically relevant model system that can function as a discovery platform before transitioning to primary spheroid cultures and/or animal models. We propose that they will be a valuable tool for advancing understanding of intestinal epithelial biology.

Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted reactivators. Granulomas from Mtb/SIV animals displayed a more robust T cell activation profile (IFN-α, IFN-γ, TNF, IL-17, IL-2, IL-10, IL-4 and granzyme B) compared to CD4 depleted animals and controls though these effectors did not protect against reactivation or dissemination, but instead may be related to increased viral and/or Mtb antigens. SIV replication within the granuloma was associated with reactivation, greater overall Mtb growth and reduced Mtb killing resulting in greater overall Mtb burden. These data support that SIV disrupts protective immune responses against latent Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas.

Clinical immunity against Plasmodium falciparum infection develops in residents of malaria endemic regions, manifesting in reduced clinical symptoms during infection and in protection against severe disease but the mechanisms are not fully understood. Here, we compare the cellular and humoral immune response of clinically immune (0-1 episode over 18 months) and susceptible (at least 3 episodes) during a mild episode of Pf malaria infection in a malaria endemic region of Malawi, by analysing peripheral blood samples using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses. In the clinically immune, we find increased proportions of circulating follicular helper T cells and classical monocytes, while the humoral immune response shows characteristic age-related differences in the protected. Presence of memory CD4 + T cell clones with a strong cytolytic ZEB2 + T helper 1 effector signature, sharing identical T cell receptor clonotypes and recognizing the Pf -derived circumsporozoite protein (CSP) antigen are found in the blood of the Pf -infected participants gaining protection. Moreover, in clinically protected participants, ZEB2 + memory CD4 + T cells express lower level of inhibitory and chemotactic receptors. We thus propose that clonally expanded ZEB2 + CSP-specific cytolytic memory CD4 + Th1 cells may contribute to clinical immunity against the sporozoite and liver-stage Pf malaria. It is important to understand why some individuals in endemic regions acquire natural immunity against malaria while others remain susceptible. Here authors show that during episodes of Plasmodium falciparum malaria, circumsporozoite-specific cytolytic memory CD4 + T cells are clonally expanded in patients, and those with clinical immunity demonstrate reduction in the chemotactic and inhibitory receptor expression in ZEB2 + memory CD4 + T cells.

Modulation of immune tone at mucosal surfaces is critical to maintain homeostasis while facilitating the handling of emerging threats. One dynamic component of immune modulation is the phagocytosis and clearance of apoptotic bodies known as efferocytosis that inhibits inflammation by promoting its resolution. Here, we evaluated the effects of apoptotic body phagocytosis by intestinal epithelial stem and progenitor cells (ISCs). Unexpectedly, instead of immunomodulation through efferocytosis, this process elevated local immune system activity. To achieve this result, ISCs actively engaged apoptotic bodies in a unique fashion, leading to their engulfment and ultimate delivery to lysosomes for processing. We found that ISCs were capable of actively recruiting inert material such as apoptotic bodies by using actin-based intrinsic biomechanical processes. Uptake of apoptotic bodies was facilitated by complement factor C3 produced by apoptotic bodies themselves. ISCs in turn generated signals heightening T cell activity that was driven in part by ISC-generated TNF. Taken together, uptake of apoptotic bodies by ISCs produced a local inflammatory alert to specific immune cells. This altered paradigm for the response to phagocytosed apoptotic bodies fits the needs of active mucosal surfaces and demonstrates that efferocytosis as currently defined is not a universal response of all cell types.

Plasmodium falciparum (Pf) malaria causes high rates of morbidity and mortality and lacks a sufficiently effective vaccine. Clinical immunity develops in residents of malaria endemic regions which confers reduced clinical symptoms during infection and protection against severe disease. We hypothesized that understanding the immune mechanisms of clinical immunity could inform vaccine design to improve efficacy. We compared the peripheral blood cellular and humoral immune responses during a mild episode of Pf malaria infection. Participants were classified as either clinically susceptible or clinically protected, based on the number of recurrent clinical infections over an 18-month longitudinal study in a malaria endemic region in Malawi. Susceptible participants had three or more recurrent clinical episodes while clinically immune individuals had one or none. Protected participants exhibited higher plasma immunoglobulin G (IgG) breadth and titers against Pf antigens, and greater antibody (Ab)-dependent Pf opsonization compared to susceptible participants. Using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses, we identified expanded memory CD4+ T cell clones sharing identical T cell receptor clonotypes in the blood of protected participants during malaria infection. These cells express a strong cytolytic T helper 1 effector program with transcripts encoding granzymes (A, B, H, M), granulysin, NKG7 and the Zeb2 master transcriptional regulator of terminally differentiated effector T cells. Memory CD4+ T cells expressing Zeb2+ were CD39hiTIGIThi and expressed multiple chemotactic and checkpoint inhibitory receptors, although the cellular levels of several of these receptors were reduced in protected compared to susceptible individuals. We propose that clonally expanded Zeb2+ cytolytic memory CD4+ Th1 cells could represent essential contributors to clinical immunity against Pf malaria. Competing Interest Statement The authors have declared no competing interest.

Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted reactivators. Granulomas from Mtb/SIV animals displayed a more robust T cell activation profile (IFN-α, IFN-γ, TNF, IL-17, IL-2, IL-10, IL-4 and granzyme B) compared to Mtb/αCD4 animals and controls though these effectors did not protect against reactivation or dissemination, but instead may be related to increased viral and/or Mtb antigens. SIV replication within the granuloma was associated with reactivation, greater overall Mtb growth and Mtb killing resulting in greater overall Mtb burden. These data support that SIV disrupts protective immune responses against latent Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas. Most humans are able to control infection with Mycobacterium tuberculosis (Mtb), the bacteria that causes tuberculosis (TB). Controlled, asymptomatic infection (latent infection) can develop into symptomatic, severe TB (reactivation TB) when the immune system is impaired, and HIV is the most common risk factor. Chronic HIV infection is associated with low CD4 T cells but there are likely other factors involved. Using macaques with latent Mtb infection, we could induce reactivation from either CD4 T cell depletion or SIV infection. We found that SIV induced reactivation was more dramatic with more bacterial dissemination and bacterial growth compared to those with CD4 depletion. While SIV-infected animals had more activated immune responses in the lung granulomas (a collection of immune cells that functions to combat Mtb), they could not prevent bacterial spread of Mtb resulting in more TB pathology, higher bacterial burden and disease throughout the body. These data suggest that the HIV-induced reactivation TB is not solely from the loss of CD4 T cells. Furthermore, our data suggest that SIV and Mtb have a synergistic relationship within granulomas that impairs the ability to kill Mtb and that this relationship exacerbates TB disease.

Background Anaemia and thrombocytopenia adversely affect adolescent HIV outcomes, yet adolescent-specific data from the tenofovir/lamivudine/dolutegravir (TLD) era remain scarce, and access to full blood count (FBC) testing is limited in Cameroon. We evaluated the prevalence, severity, and factors associated with these cytopenias among adolescents living with HIV (ADLHIV) in the TLD era. Methods Multicentre cross-sectional study was conducted among ADLHIV (10–19 years) receiving TLD in the CIPHER-ADOLA cohort in Cameroon. Full blood count, viral load (VL) and CD4-count were performed. Factors associated with anaemia and thrombocytopenia were ascertained. Results A total of 252 ADLHIV was enrolled (50.8% male, 83.3% were vertically infected, and 7.2% were underweighted). ART-duration and TLD-exposure were 10 [6–13] years and 26 [12–33] months, respectively. Concerning virological response, 71.4%, 13.1%, and 15.5% had a VL < 50, 50–999, and ≥ 1000, respectively. Overall, 102 (40.5%) were anaemic, with only 2.9% severe. Anaemia rate was twice higher in females (55.6%, p  < 0.001); 64.1% with VL ≥ 1000 against 35.0% with VL < 50 ( p  = 0.003); 60.0% with CD4 < 200 against 35.4% with CD4 > 500 ( p  = 0.046). Regarding thrombocytopenia, the burden was low (6.7%), but higher among VL ≥ 1000 ( p  = 0.003). Multivariate analyses showed a threefold higher anaemia prevalence in females (aOR [95% CI: 3.406 [1.8952-5.940]), fivefold without formal education (0.191 [0.047–0.776]), threefold in VL ≥ 1000 copies/ml (0.338 [0.156–0.733]). Thrombocytopenia was fourfold more likely in males (aOR: 0.236 [0.072–0.774]) and sevenfold more likely in individuals with VL ≥ 1000 copies/mL (aOR: 0.140 [0.038–0.510]). Conclusion In the TLD era, anaemia remains common but generally mild, and thrombocytopenia is uncommon. Cytopenias were associated with unsuppressed viral load, with a stronger association for anaemia in females. These findings support programmatic targeted haemovigilance prioritising adolescents with unsuppressed viral load, particularly females, in settings where access to FBC testing is limited.

Since the beginning of the cholera epidemic in Haiti 5 years ago, the prevalence of this deadly water-borne disease has fallen far below the initial rates registered during its explosive outset. However, cholera continues to cause extensive suffering and needless deaths across the country, particularly among the poor. The urgent need to eliminate transmission of cholera persists: compared to the same period in 2014, the first 4 months of 2015 saw three times the number of cholera cases. Drawing upon epidemiology, clinical work (and clinical knowledge), policy, ecology, and political economy, and informed by ethnographic data collected in a rural area of Haiti called Bocozel, this paper evaluates the progress of the nation's 10-year Plan for the Elimination of Cholera. Bocozel is a rice-producing region where most people live in extreme poverty. The irrigation network is decrepit, the land is prone to environmental shocks, fertilizer is not affordable, and the government's capacity to assist farmers is undermined by resource constraints. When peasants do have rice to sell, the price of domestically grown rice is twice that of US-imported rice. Canal water is not only used to irrigate thousands of acres of rice paddies and sustain livestock, but also to bathe, wash, and play, while water from wells, hand pumps, and the river is used for drinking, cooking, and bathing. Only one out of the three government-sponsored water treatment stations in the research area is still functional and utilized by those who can afford it. Latrines are scarce and often shared by up to 30 people; open defecation remains common. Structural vulnerabilities cut across all sectors - not just water, sanitation, health care, and education, but agriculture, environment, (global and local) commerce, transportation, and governance as well. These are among the hidden sicknesses that impede Haiti and its partners' capacity to eliminate cholera.

Abstract Purpose A study among Filipinos revealed that only 15% of patients with diabetes achieved glycemic control, and poor response to metformin could be one of the possible reasons. Recent studies demonstrate how genetic variations influence response to metformin. Hence, the present study aimed to determine genetic variants associated with poor response to metformin. Methods Using a candidate variant approach, 195 adult Filipino participants with newly diagnosed type 2 diabetes mellitus (T2DM) were enrolled in a case-control study. Genomic DNA from blood samples were collected. Allelic and genotypic associations of variants with poor response to metformin were determined using exact statistical methods. Results Several polymorphisms were nominally associated with poor response to metformin (Puncorr < 0.05). The most notable is the association of multiple variants in the SLC2A10 gene—rs2425911, rs3092412, and rs2425904—with common additive genetic mode of inheritance. Other variants that have possible associations with poor drug response include rs340874 (PROX-AS1), rs815815 (CALM2), rs1333049 (CDKN2B-AS1), rs2010963 (VEGFA), rs1535435 and rs9494266 (AHI1), rs11128347 (PDZRN3), rs1805081 (NPC1), and rs13266634 (SLC30A8). Conclusion In Filipinos, a trend for the association for several variants was noted, with further observation that several mechanisms may be involved. The results may serve as pilot data for further validation of candidate variants for T2DM pharmacotherapy.

Four full genome scans have been carried out by the partners of the European cluster on coeliac disease as well as follow-up studies of candidate regions. No region outside HLA showed significant linkage to the disease in any single study. We first applied a meta-analysis based on a modification of Genome Screen Meta-Analysis to take into account the different linkage statistics, the arbitrariness of bin cutoff points, as well as the sample size of each study. We then performed a pooled linkage analysis of all families and raw genotypes. Besides the HLA region, already known to harbour a risk factor for coeliac disease, both approaches leave very little doubt on the presence of a genetic risk factor in the 5q31–33 region. This region was suggested by several individual studies, but did not reach statistical values high enough to be conclusive when data sets were analysed separately.