The Role of HIV-1-Encoded microRNAs in Viral Replication.

microRNAs (miRNAs) are small non-coding RNAs (sncRNAs) that play an important role in the life cycle of human viruses. We sought to characterize human immunodeficiency virus 1 (HIV-1)-encoded miRNAs and determine their role in viral replication. Initially, a bioinformatic analysis was used to predict HIV-1-encoded miRNAs. Next, a representative number of these predicted sequences were verified using a miRNA microarray chip, reverse transcription PCR (RT-PCR), and the deep sequencing of RNA extracted from HIV-1-infected cells. Eight HIV-1-encoded sncRNA sequences conforming to the criteria that define miRNAs were identified in HIV-1-infected immortalized T cells and human primary CD4+ lymphocytes; five of the eight sequences have not been previously reported. Deep sequencing validated the presence of these virus-encoded miRNA sequences and uncovered large numbers of atypical sncRNA sequences, lacking characteristics of conventional miRNAs. We named these sequences small RNAs (smRNAs). The overexpression of four candidate HIV-1-encoded miRNAs and silencing of two smRNAs significantly increased HIV-1 viral replication. Our study uncovered novel HIV-1-encoded sncRNAs that, upon deregulated expression, alter viral titers in HIV-1-infected cells, suggesting that miRNAs and smRNAs play an important role in regulating viral replication. Future studies may reveal the function of HIV-1-encoded sncRNAs and their possible implications for diagnosis and treatment.

Motivating school communities towards behavior change and local ownership: a gamification intervention to prevent trachoma at primary schools in southern Ethiopia.

BACKGROUND: Ethiopia alone carries 49% of the global burden of trachoma, associated with a lack of safe water, sanitation and hygiene (WASH) and poor health practices. The aim of this study was to examine whether gamification among schoolchildren and promotion of local ownership of school WASH is associated with healthy behaviors and WASH infrastructure improvements. METHODS: Application of the Accelerate gamification intervention for elimination of trachoma, with an emphasis on gamification among schoolchildren and community involvement in motivating face-washing, handwashing and functional use of latrines, was undertaken. RESULTS: The study was conducted over 9 mo in 223 rural schools from six districts within the intervention area, reaching 93 518 schoolchildren. At baseline, students were observed washing their hands after using latrines in 23 (10.3%) schools. This increased to 132 (59%) schools (p≤0.001) at follow-up. The number of latrines increased from 585 at baseline to 594 at follow-up (p=0.031). The availability of handwashing stations in schools increased from 31 (13.9%) with water access (8%) and soap (5%) to 155 (69.5%) schools with handwashing stations with water access in 153 (98.7%) (p<0.001) and soap in 121 (78%) (p<0.001). CONCLUSIONS: Motivational strategies such as gamification among schoolchildren and promotion of local ownership of school WASH may be associated with healthy behaviors and WASH infrastructure improvements.

High prevalence of soil-transmitted helminths and schistosomiasis among primary schoolchildren in Southwest Ethiopia: the need for health strategies alongside mass drug administration.

BACKGROUND: Soil-transmitted helminths (STH) and schistosomiasis remain widely prevalent in Ethiopia. The aim of this study was to evaluate the prevalence of STH and schistosomiasis among schoolchildren in Gidi Bench district (Southern Nations, Nationalities, and People's Republic, Southwest Ethiopia) and the association with knowledge and health-related behaviors. METHODS: A cross-sectional study was conducted. Stool samples, analyzed by the Kato-Katz technique and a knowledge, attitudes and practices questionnaire, were collected. RESULTS: Out of 611 participants (mean age 12.8±3.1 y), 129 (21.1%) were infected with schistosomiasis and 382 (62.5%) had STH. More than 30% (n=195, 31.9%) were infected with a single intestinal parasite, while 138 (22.6%) and 47 (7.7%) were infected with two or three parasitic infections, respectively. Boys and those who did not participate in school clubs had higher infection rates (p=0.05). Lower parasitic infection was associated with using a latrine when available, washing hands and vegetables and wearing shoes regularly. Higher rates of infection were found among those who reported swimming and washing cloths and utensils in the river regularly. CONCLUSIONS: Schistosomiasis and STH were highly prevalent among schoolchildren in Gidi Bench district. Infection rates were associated with gender, lack of knowledge on parasitic infections and unhealthy behaviors. Findings from this study may assist in decision making regarding disease prevalence and methods of control alongside mass drug administration.

Schistosomiasis Control in Ethiopia: The Role of Snail Mapping in Endemic Communities.

Introduction: Schistosomiasis, a neglected tropical disease (NTD), remains a public health problem in Ethiopia. Freshwater snails, acting as intermediate hosts, release cercariae, the infectious parasite, into the water, which penetrate human skin that encounters infested waters. The objective of this study was to map snail abundance along rivers and study its association with schistosomiasis infection in communities using these rivers. Materials and Methods: A cross-sectional study was carried out at 20 river sites in Mizan Aman city administration, Bench Sheko zone, South West Ethiopia Peoples (SWEP) region, Ethiopia, to study the distribution of host snails and transmission sites for intestinal schistosomiasis. This study used a quantitative database consisting of data on the prevalence of infected snails, the characteristics of rivers and riverbanks, and the prevalence of schistosomiasis in the community, based on stool samples collected from community members near the sampling sites. Results: Aquatic snails were found in 11 of the 20 sites sampled. A total of 598 snails was collected, including Biomphalaria pfeifferi, Biomphalaria sudanica, Radix natalensis and Bulinus globosus species; the most abundant species was Biomphalaria pfeifferi. Stool samples were collected from 206 community members from all 20 sites. Forty-one (19.9%) were positive for Schistosoma mansoni. A positive correlation was found between the presence of snails and positive stool samples (r = 0.60, p = 0.05) and between the presence of infected snails and the prevalence of infection (r = 0.64, p = 0.03). Locations with muddy riverbanks were associated with the presence of snails (r = 0.81, p < 0.001). Conclusions: These results emphasize the importance of mapping snails for the control of schistosomiasis by defining hotspots of infection and identifying factors associated with the presence of infected snails. The results support the need for a continuous mapping of snails and the introduction of snail control as a major element for the successful control of schistosomiasis in endemic communities.

Sustainable Elimination of Schistosomiasis in Ethiopia-A Five-Year Follow-Up Study.

In 2009, Mekele, the capital of the Tigray Region in Ethiopia, presented a mean prevalence of 44.7% of schistosomiasis (S. mansoni) in school children. Termed a public health problem, NALA, an international public health non-governmental organization, and their partners implemented a novel model of intervention, which aimed to compliment mass drug administration (MDA) campaigns with behavioral change (BC) and improved sanitation to achieve sustained elimination of schistosomiasis. The four-year intervention (2009−2012) covered 38 primary schools. The objective of this study was to examine factors associated with control or resurgence of the disease, and the association between the behavioral change program and disease prevalence, ten years after initiation. Eleven primary schools were selected for this follow-up study. All students provided a stool sample and filled in a knowledge, attitude and practice (KAP) questionnaire. In seven out of eleven schools (63.6%) the prevalence of schistosomiasis was maintained below 2% ten years after the initiation of the intervention. In four schools, prevalence returned to pre-intervention levels, defining them as persistent hot spots (PHS). Students from PHS schools scored lower on KAP questionnaires compared to students from responder schools; 3.9 ± 0.9 vs. 4.2 ± 0.9 (p-value < 0.001) for practice questions and 4.4 ± 1.4 vs. 4.6 ± 1.5 (p-value = 0.03) for attitude questions. The prevalence of schistosomiasis correlated positively with age, (p-value = 0.049), sex (relative risk = 1.7, p-value < 0.001), and location. Semi-urban locations (n = 382) had higher disease prevalence than urban locations (n = 242), (22.7% vs. 5.5%, p-value < 0.001). Students residing in semi-urban areas and close to a river (<500 m) were at higher risk of contracting schistosomiasis than those living in urban areas far from the river (RR = 5.95, p-value < 0.001). Finally, a correlation between prevalence and proximity of schools to rivers was found (semi-urban areas; RR = −0.91, p-value = 0.001 vs. urban areas; RR = −0.51, p-value = 0.001). Soil-transmitted-helminths prevalence in 2009 was 8.1% and declined during the intervention years to 0.5%. Prevalence in 2018 was found to be stable at 0.8%. These results demonstrate the long-term success of NALAs' comprehensive model of intervention for elimination of schistosomiasis in school children, combining behavioral change and improved sanitation with MDA.

Biogeographical Landscape of the Human Face Skin Microbiome Viewed in High Definition.

The bacterial community that colonizes the human face imparts physiochemical and physiological effects on the facial skin. These skin-microbe interactions impact dermatological, cosmetic and skincare applications due to the centrality of the human face in daily interactions. However, fine-scale characterization of the human face skin microbiome is lacking. Using 16S rRNA sequencing and 3D cartography, this study plotted and characterized the facial skin microbiome in high- definition, based on 1,649 samples from 12 individuals. Analysis yielded a number of novel insights, including that of the relative uniformity of skin microbiome composition within skin sites, site localization of certain microbes, and the interpersonal variability of the skin microbiome. The results show that high-resolution topographical mapping of the skin microbiome is a powerful tool for studying the human skin microbiome. Despite a decade of skin microbiome research, there is still much to be discovered.

A Placebo-Controlled Double-Blind Randomized Trial with Individualized Homeopathic Treatment Using a Symptom Cluster Approach in Women with Premenstrual Syndrome.

BACKGROUND: In a double-blind placebo-controlled randomized trial with parallel groups, the efficacy of individually prescribed homeopathic medicines was evaluated in women with premenstrual syndrome (PMS). METHODS: In an outpatient department of a university clinic in Jerusalem, Israel (1996-1999), women with PMS, aged 18 to 50 years, entered a 2-month screening phase with prospective daily recording of premenstrual symptoms by the Menstrual Distress Questionnaire (MDQ). They were included after being diagnosed with PMS. A reproducible treatment protocol was used: women received a homeopathic prescription based on symptom clusters identified in a questionnaire. The symptoms were verified during a complementary, structured, interview. Only women whose symptoms matched the symptom profile of one of 14 pre-selected homeopathic medicines were included. Each participant was administered active medicine or placebo via random allocation. Primary outcome measures were differences in changes in mean daily premenstrual symptom (PM) scores by the MDQ. Analysis was by intention-to-treat. RESULTS: A total of 105 women were included: 49 were randomized to active medicine and 56 to placebo. Forty-three women in the active medicine group and 53 in the placebo group received the allocated intervention with at least one follow-up measurement and their data were analyzed. Significantly greater improvement of mean PM scores was measured in the active medicine group (0.443 [standard deviation, SD, 0.32] to 0.287 [SD, 0.20]) compared to placebo (0.426 [SD, 0.34] to 0.340 [SD, 0.39]); p = 0.043. CONCLUSIONS: Individually prescribed homeopathic medicines were associated with significantly greater improvement of PM scores in women with PMS, compared to placebo. Replication, with larger sample size and other refinements, is recommended to confirm the efficacy of this treatment in other settings.

Skin Microbiome Compositional Changes in Atopic Dermatitis Accompany Dead Sea Climatotherapy.

Dead Sea climatotherapy (DSC) is a well-established therapeutic modality for the treatment of several diseases, including atopic dermatitis. Skin microbiome studies have shown that skin microbiome diversity is anticorrelated with both atopic dermatitis severity and concurrent Staphylococcus aureus overgrowth. This study aimed to determine whether DSC induces skin microbiome changes concurrent with clinical improvements in atopic dermatitis. We sampled 35 atopic dermatitis patients and ten healthy controls on both the antecubital and popliteal fossa. High-resolution microbial community profiling was attained by sequencing multiple regions of the 16S rRNA gene. Dysbiosis was observed in both lesional and nonlesional sites, which was partially attenuated following treatment. Severe AD skin underwent the most significant community shifts, and Staphylococcus epidermidis, Streptococcus mitis and Micrococcus luteus relative abundance were significantly affected by Dead Sea climatotherapy. Our study highlights the temporal shifts of the AD skin microbiome induced by Dead Sea climatotherapy and offers potential explanations for the success of climatotherapy on a variety of skin diseases, including AD.

Temporal Stability of the Healthy Human Skin Microbiome Following Dead Sea Climatotherapy.

Dead Sea climatotherapy (DSC) is a therapeutic modality for a variety of chronic skin conditions, yet there has been scarce research on the relationship between the cutaneous microbiota and disease states in response to DSC. We characterized the skin bacterial and fungal microbiome of healthy volunteers who underwent DSC. Bacterial community diversity remained similar before and after treatment, while fungal diversity was significantly reduced as a result of the treatment. Individuals showed greater inter-individual than temporal bacterial community variance, yet the opposite was true for fungal community composition. We further identified Malassezia as the genus driving temporal mycobiome variations. The results indicate that the microbiome remains stable throughout DSC, while the mycobiome undergoes dramatic community changes. The results of this study will serve as an important baseline for future investigations of microbiome and mycobiome temporal phenomena in diseased states.

Endogenous Antimicrobial Peptide Expression in Response to Bacterial Epidermal Colonization.

Bacterial commensal colonization of human skin is vital for the training and maintenance of the skin's innate and adaptive immune functions. In addition to its physical barrier against pathogen colonization, the skin expresses a variety of antimicrobial peptides (AMPs) which are expressed constitutively and induced in response to pathogenic microbial stimuli. These AMPs are differentially effective against a suite of microbial skin colonizers, including both bacterial and fungal residents of the skin. We review the breadth of microorganism-induced cutaneous AMP expression studies and their complementary findings on the efficacy of skin AMPs against different bacterial and fungal species. We suggest further directions for skin AMP research based on emerging skin microbiome knowledge in an effort to advance our understanding of the nuanced host-microbe balance on human skin. Such advances should enable the scientific community to bridge the gap between descriptive disease-state AMP studies and experimental single-species in vitro studies, thereby enabling research endeavors that more closely mimic the natural skin environs.

Effects of Helminth Eradication on the Immune System.

INTRODUCTION: Helminth infection has a profound effect on the immune system. However, the precise nature of the immune changes that are elicited by helminth infection have not been sufficiently characterized. Furthermore, the reversibility of these changes after treatment has not been documented sufficiently. We studied the immune profiles of Ethiopian immigrants to Israel at baseline, that is on arrival and at one-year follow-up and compared individuals who received antihelminth treatment during the study period with those who missed the treatment. METHODS: A longitudinal follow up study involving different groups of subjects was conducted. Baseline data was recorded from the newly arrived Ethiopian immigrants for a series of peripheral blood tests, including: IgE and Eosinophil levels, T-cell populations, T-cell receptor phenotypes, and cytokine measurement. These tests were all repeated after a 1-year interval. Results were compared between the newly arrived Ethiopian immigrants (NEW-Eth-Il), long term Ethiopian immigrants (LT-Eth-Il), and non Ethiopian Israeli controls (NON-Imm-Il). RESULTS: Of the 184 individuals, 111 were NEW-Eth-Il, who had a high prevalence of helminth infection, the immunological changes were elevated IgE levels and eosinophil counts, decreased CD4/CD8 ratio, increased proportion of HLA-DR+CD3+, HLA-DR+CD4+ and HLA-DR+CD8+ cells, decreased proportion of CD45RA+CD4+ (naive) and CD28+CD8+ cells, increased proportion of CD45RO+CD4+ (memory) cells, and increased secretion of IL-4 and IL-5 (Th2 type cytokines). In the 42 LT-Eth-Il participants, who all had negative tests for helminth infection, we did not observe these immune changes and their immune profile did not differ markedly from that of the NON-Imm-Il controls. The follow-up immune profiles of 33 NEW-Eth-Il who received succesful antihelminth treatment, showed a significant normalization in the above-mentioned variables that was not observed in the 19 NEW-Eth-Il who missed and did not receive the antihelminth treatment. CONCLUSIONS: These findings demonstrate that helminth infection is associated with profound immune changes that are normalized within a short time after helminth eradication. They also strengthen the hypothesis that effective antihelminth interventions, in areas endemic for intestinal helminths, may have an impact on AIDS and tuberculosis epidemics.

The Role of Helminth Infection and Environment in the Development of Allergy: A Prospective Study of Newly-Arrived Ethiopian Immigrants in Israel.

Helminth infection may be protective against allergy and account for the low prevalence of allergy in developing countries. We studied prospectively the prevalence of allergy in Ethiopian immigrants with heavy helminth infection on arrival in Israel, and again after a year of adjustment to an urban industrialized setting, to explore the roles of helminth infection, changed environment and background immunity on the manifestations of allergy. 126 newly arrived Ethiopian immigrants were studied at baseline and 115 after a year of follow up in Israel. Allergic symptoms, Skin prick tests (SPT), Tuberculin (PPD) skin tests, stool and blood samples were obtained for determining parasites, blood IgE and eosinophil levels, respectively. Anti-helminthic therapy was offered to the entire infected individuals, but only 50/108 (46.3%) took the medication. At baseline, there was a significant negative association between helminth infection and allergy, 4/18 (22.2%) of uninfected participants were allergic compared to 7/108 (6.5%) of helminth-infected participants (p = 0.028), as well as between helminth infection and SPT reactivity, 12/18 (66.6%) of uninfected participants compared to 43/108 (39.8%) of helminth-infected participants (p = 0.033). After one year, a significant general increase in allergy and SPT was observed. While only 11/126 (8.7%) were allergic at baseline, 30/115 (26.1%) became allergic at follow-up (p<0.0001), and while 55/126 (43.7%) were SPT+ at baseline, 79/115 (68.7%) became SPT+ at follow-up (p<0.001). A twofold increase in allergen sensitization was also observed after one year in Israel, particularly for dust mites, grasses and olive tree (p<0.001). These results show that: a) Helminth infection is significantly associated with low allergy and low SPT reactivity; b) One year after immigration to Israel, allergy and SPT reactivity increased significantly in all immigrants; c) Higher increases in positive SPT and allergy were observed after a year in the group that remained infected with helminths, even though they had a lowered helminth load; d) The reasons for the increased allergy one year after immigration needs further investigation but probably reflects the combined influence of the decreased helminth load and novel environmental factors.

In vivo expression of human cytomegalovirus (HCMV) microRNAs during latency.

Viral encoded microRNAs play key roles in regulating gene expression and the life cycle of human herpes viruses. Latency is one of the hallmarks of the human cytomegalovirus (HCMV or HHV5) life cycle, and its control may have immense practical applications. The present study aims to identify HCMV encoded microRNAs during the latency phase of the virus. We used a highly sensitive real time PCR (RTPCR) assay that involves a pre-amplification step before RTPCR. It can detect HCMV encoded microRNAs (miRNAs) during latency in purified monocytes and PBMCs from HCMV IgG positive donors and in latently infected monocytic THP-1 cell lines. During the latency phase, only eight HCMV encoded microRNAs were detected in PBMCs, monocytes and in the THP-1 cells. Five originated from the UL region of the virus genome and three from the US region. Reactivation of the virus from latency, in monocytes obtained from the same donor, using dexamethasone restored the expression of all known HCMV encoded miRNAs including those that were absent during latency. We observed a shift in the abundance of the two arms of mir-US29 between the productive and latency stages of the viral life cycle, suggesting that the star "passenger" form of this microRNA is preferentially expressed during latency. As a whole, our study demonstrates that HCMV expresses during the latency phase, both in vivo and in vitro, only a subset of its microRNAs, which may indicate that they play an important role in maintenance and reactivation of latency.

Evaluation of a benchtop HIV ultradeep pyrosequencing drug resistance assay in the clinical laboratory.

Detection of low-abundance drug resistance mutations (DRMs) of HIV-1 is an evolving approach in clinical practice. Ultradeep pyrosequencing has shown to be effective in detecting such mutations. The lack of a standardized commercially based assay limits the wide use of this method in clinical settings. 454 Life Sciences (Roche) is developing an HIV ultradeep pyrosequencing assay for their benchtop sequencer. We assessed the prototype plate in the clinical laboratory. Plasma samples genotyped by the standardized TruGene kit were retrospectively tested by this assay. Drug-treated subjects failing therapy and drug-naive patients were included. DRM analysis was based on the International AIDS Society USA DRM list and the Stanford algorithm. The prototype assay detected all of the DRMs detected by TruGene and additional 50 low-abundance DRMs. Several patients had low-abundance D67N, K70R, and M184V reverse transcriptase inhibitor mutations that persisted long after discontinuation of the drug that elicited these mutations. Additional patient harbored low-abundance V32I major protease inhibitor mutation, which under darunavir selection evolved later to be detected by TruGene. Stanford analysis suggested that some of the low-abundance DRMs were likely to affect the resistance burden in these subjects. The prototype assay performs at least as well as TruGene and has the advantage of detecting low-abundance drug resistance mutations undetected by TruGene. Its ease of use and lab-scale platform will likely facilitate its use in the clinical laboratory. The extent to which the detection of low-abundance DRMs will affect patient management is still unknown, but it is hoped that use of such an assay in clinical practice will help resolve this important question.

Finding quasi-modules of human and viral miRNAs: a case study of human cytomegalovirus (HCMV).

BACKGROUND: MicroRNAs (miRNAs) are important regulators of gene expression encoded by a variety of organisms, including viruses. Although the function of most of the viral miRNAs is currently unknown, there is evidence that both viral and host miRNAs contribute to the interactions between viruses and their hosts. miRNAs constitute a complex combinatorial network, where one miRNA may target many genes and one gene may be targeted by multiple miRNAs. In particular, viral and host miRNAs may also have mutual target genes. Based on published evidence linking viral and host miRNAs there are three modes of mutual regulation: competing, cooperating, and compensating modes. RESULTS: In this paper we explore the compensating mode of mutual regulation upon Human Cytomegalovirus (HCMV) infection, when host miRNAs are down regulated and viral miRNAs compensate by mimicking their function. To achieve this, we develop a new algorithm which finds groups, called quasi-modules, of viral and host miRNAs and their mutual target genes, and use a new host miRNA expression data for HCMV-infected and uninfected cells. For two of the reported quasi-modules, supporting evidence from biological and medical literature is provided. CONCLUSIONS: The modules found by our method may advance the understanding of the role of miRNAs in host-viral interactions, and the genes in these modules may serve as candidates for further experimental validation.

Vaccinia virus infection suppresses the cell microRNA machinery.

MicroRNAs are key players in the regulation of gene expression by posttranscriptional suppression. They are involved in physiological processes, and thus their deregulation may contribute to the development of diseases and progression of cancer. Virus-encoded microRNAs and microRNAs of host origin play an important role in controlling the virus life cycle and immunity. The aim of this study was to determine the effect of vaccinia virus (VACV) infection on the expression of host-encoded microRNAs. A marked general suppression of most microRNAs in the infected cells was observed within 24 hours after VACV infection of a number of cell types. We demonstrate that this suppression was associated with abrogation of expression of the Dicer1 enzyme, which is a key enzyme in the generation of microRNAs.

The microRNA Transcriptome of Human Cytomegalovirus (HCMV).

The purpose of the present study was to characterize the microRNA transcriptome (miRNAome) of the human cytomegalovirus (HCMV or HHV5). We used deep sequencing and real time PCR (qPCR) together with bioinformatics to analyze the pattern of small RNA expression in cells infected with low-passage isolates of HCMV as well as in plasma and amniotic fluid. We report here on the discovery of four new precursors and ten new miRNAs as well as eleven microRNA-offset-RNAs (moRs) that are all encoded by HCMV. About eighty percent of the total HCMV reads were perfectly mapped to HCMV miRNAs, strongly suggestive of their important biological role that in large remains still to be defined and characterized. Taken altogether, the results of this study demonstrate the power and usefulness of the combined bioinformatics/biological approach in discovering additional important members of HCMV- encoded small RNAs and can be applied to the study of other viruses as well.

Epithelial microRNAs regulate gut mucosal immunity via epithelium-T cell crosstalk.

Colonic homeostasis entails epithelium-lymphocyte cooperation, yet many participants in this process are unknown. We show here that epithelial microRNAs mediate the mucosa-immune system crosstalk necessary for mounting protective T helper type 2 (T(H)2) responses. Abolishing the induction of microRNA by gut-specific deletion of Dicer1 (Dicer1(Δgut)), which encodes an enzyme involved in microRNA biogenesis, deprived goblet cells of RELMß, a key T(H)2 antiparasitic cytokine; this predisposed the host to parasite infection. Infection of Dicer1(Δgut) mice with helminths favored a futile T(H)1 response with hallmarks of inflammatory bowel disease. Interleukin 13 (IL-13) induced the microRNA miR-375, which regulates the expression of TSLP, a T(H)2-facilitating epithelial cytokine; this indicated a T(H)2-amplification loop. We found that miR-375 was required for RELMß expression in vivo; miR-375-deficient mice had significantly less intestinal RELMß, which possibly explains the greater susceptibility of Dicer1(Δgut) mice to parasites. Our findings indicate that epithelial microRNAs are key regulators of gut homeostasis and mucosal immunity.