Development of broadly neutralizing antibodies targeting the cytomegalovirus subdominant antigen gH.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that increases morbidity and mortality in immunocompromised individuals including transplant recipients and newborns. New anti-HCMV therapies are an urgent medical need for diverse patient populations. HCMV infection of a broad range of host tissues is dependent on the gH/gL/gO trimer and gH/gL/UL28/UL130/UL131A pentamer complexes on the viral envelope. We sought to develop safe and effective therapeutics against HCMV by generating broadly-neutralizing, human monoclonal antibodies (mAbs) from VelocImmune® mice immunized with gH/gL cDNA. Following high-throughput binding and neutralization screening assays, 11 neutralizing antibodies were identified with unique CDR3 regions and a high-affinity (KD 1.4-65 nM) to the pentamer complex. The antibodies bound to distinct regions within Domains 1 and 2 of gH and effectively neutralized diverse clinical strains in physiologically relevant cell types including epithelial cells, trophoblasts, and monocytes. Importantly, combined adminstration of mAbs with ganciclovir, an FDA approved antiviral, greatly limited virus dissemination. Our work identifies several anti-gH/gL mAbs and sheds light on gH neutralizing epitopes that can guide future vaccine strategies.

Distinct cord blood C-peptide, adipokine, and lipidomic signatures by in utero HIV exposure.

BACKGROUND: Early-life metabolic derangements in HIV-exposed uninfected (HEU) infants have been reported. METHODS: Pregnant women with HIV and HIV-uninfected pregnant women were enrolled with their newborns in a US cohort from 2011 to 2015. We measured cord insulin, C-peptide, and metabolic cytokines of HEU and HIV-unexposed uninfected (HUU) newborns using ELISA and metabolites, lipid subspecies, and eicosanoids via liquid chromatography/mass spectrometry. Linear regression was employed to assess the association of intrauterine HIV/ART with insulin and C-peptide. Graphical lasso regression was used to identify differences between metabolite/lipid subspecies networks associated with C-peptide. RESULTS: Of 118 infants, 56 were HEU, ART exposed. In adjusted analyses, mean cord insulin (ß = 0.295, p = 0.03) and C-peptide (ß = 0.522, p < 0.01) were significantly higher in HEU vs. HUU newborns. HEU neonates exhibited primarily positive associations between complex lipids and C-peptide, indicative of fuel storage, and augmented associations between cord eicosanoids and cytokines. HUU neonates exhibited negative associations with lipids and C-peptide indicative of increased fuel utilization. CONCLUSION: Higher cord insulin and C-peptide in HEU vs. HUU newborns as well as differences in cord metabolites, metabolic-related cytokines, and eicosanoids may reflect a propensity for fuel storage and an inflammatory milieu suggestive of fetal metabolic changes associated with in utero HIV/ART exposure. IMPACT: There is a paucity of studies assessing cord blood and neonatal metabolic health in HIV-exposed uninfected (HEU) newborns, an increasing population worldwide. Compared to HIV-unexposed uninfected (HUU) newborns, HEU newborns exhibit alterations in fuel homeostasis and an inflammatory milieu associated with in utero HIV/antiretroviral therapy (ART) exposure. The long-term implications of these neonatal findings are as yet unknown, but merit continued evaluation as this important and growing population ages into adulthood.

Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera.

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week.IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.

Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera.

The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVΔG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

CD46 facilitates entry and dissemination of human cytomegalovirus.

Human cytomegalovirus (CMV) causes a wide array of disease to diverse populations of immune-compromised individuals. Thus, a more comprehensive understanding of how CMV enters numerous host cell types is necessary to further delineate the complex nature of CMV pathogenesis and to develop targeted therapeutics. To that end, we establish a vaccination strategy utilizing membrane vesicles derived from epithelial cells to generate a library of monoclonal antibodies (mAbs) targeting cell surface proteins in their native conformation. A high-throughput inhibition assay is employed to screen these antibodies for their ability to limit infection, and mAbs targeting CD46 are identified. In addition, a significant reduction of viral proliferation in CD46-KO epithelial cells confirms a role for CD46 function in viral dissemination. Further, we demonstrate a CD46-dependent entry pathway of virus infection in trophoblasts, but not in fibroblasts, highlighting the complexity of CMV entry and identifying CD46 as an entry factor in congenital infection.

Open-label, add-on trial of cetirizine for neuromyelitis optica.

OBJECTIVE: This pilot study preliminarily examined the efficacy and tolerability of cetirizine as an add-on to standard therapy for neuromyelitis optica (NMO). METHODS: Eligible participants met the Wingerchuk 2006 diagnostic criteria or had a single typical episode along with positive NMO immunoglobulin G. After baseline clinical and laboratory assessments, participants began treatment with cetirizine 10 mg orally daily, in addition to their usual disease-modifying therapy for NMO, and continued for 1 year. The primary end point was the annualized relapse rate (ARR) while on the same disease-modifying therapy before starting cetirizine compared with after taking cetirizine. Additional end points included disability (Expanded Disability Status Scale [EDSS]), relapse severity, tolerability, especially with respect to drowsiness measured by the Epworth Sleepiness Scale (ESS), and laboratory parameters. RESULTS: The ARR before cetirizine was 0.4 ± 0.80 and after cetirizine was 0.1 ± 0.24 (p = 0.047). There was no statistically significant difference in the EDSS (mean 3.9 ± 2.18 before the start of the study and 3.2 ± 2.31 at the conclusion of the study, p = 0.500). The ESS remained fairly consistent throughout the study (mean 6.5 ± 5.33 at baseline and 6.9 ± 4.50 at month 12, p = 0.740). Laboratory studies were unrevealing. CONCLUSIONS: In this pilot study, cetirizine was well tolerated, and the prespecified primary efficacy end point was satisfied. However, the open-label design and the small sample size of this pilot study preclude definitive conclusions. Further research is needed. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that in patients with NMO, the addition of cetirizine to standard therapy is safe, well tolerated, and reduces relapses.

Human cytomegalovirus modulates monocyte-mediated innate immune responses during short-term experimental latency in vitro.

UNLABELLED: The ability of human cytomegalovirus (HCMV) to establish lifelong persistence and reactivate from latency is critical to its success as a pathogen. Here we describe a short-term in vitro model representing the events surrounding HCMV latency and reactivation in circulating peripheral blood monocytes that was developed in order to study the immunological consequence of latent virus carriage. Infection of human CD14(+) monocytes by HCMV resulted in the immediate establishment of latency, as evidenced by the absence of particular lytic gene expression, the transcription of latency-associated mRNAs, and the maintenance of viral genomes. Latent HCMV induced cellular differentiation to a macrophage lineage, causing production of selective proinflammatory cytokines and myeloid-cell chemoattractants that most likely play a role in virus dissemination in the host. Analysis of global cellular gene expression revealed activation of innate immune responses and the modulation of protein and lipid synthesis to accommodate latent HCMV infection. Remarkably, monocytes harboring latent virus exhibited selective responses to secondary stimuli known to induce an antiviral state. Furthermore, when challenged with type I and II interferon, latently infected cells demonstrated a blockade of signaling at the level of STAT1 phosphorylation. The data demonstrate that HCMV reprograms specific cellular pathways in monocytes, most notably innate immune responses, which may play a role in the establishment of, maintenance of, and reactivation from latency. The modulation of innate immune responses is likely a viral evasion strategy contributing to viral dissemination and pathogenesis in the host. IMPORTANCE: HCMV has the ability to establish a lifelong infection within the host, a phenomenon termed latency. We have established a short-term model system in human peripheral blood monocytes to study the immunological relevance of latent virus carriage. Infection of CD14(+) monocytes by HCMV results in the generation of latency-specific transcripts, maintenance of viral genomes, and the capacity to reenter the lytic cycle. During short-term latency in monocytes the virus initiates a program of differentiation to inflammatory macrophages that coincides with the modulation of cytokine secretion and specific cellular processes. HCMV-infected monocytes are hindered in their capacity to exert normal immunoprotective mechanisms. Additionally, latent virus disrupts type I and II interferon signaling at the level of STAT1 phosphorylation. This in vitro model system can significantly contribute to our understanding of the molecular and inflammatory factors that initiate HCMV reactivation in the host and allow the development of strategies to eradicate virus persistence.

Differential profiles of immune mediators and in vitro HIV infectivity between endocervical and vaginal secretions from women with Chlamydia trachomatis infection: a pilot study.

Chlamydia trachomatis infection is one of the most prevalent bacterial STIs in the USA and worldwide, and women with C. trachomatis infection are at increased risk of acquiring HIV. Because immune activation at the genital mucosa facilitates HIV/SIV infection, C. trachomatis-mediated cytokine induction may contribute to increased HIV transmission in asymptomatic women. To begin to elucidate the mechanisms, we longitudinally analyzed profiles of innate immune factors and HIV infectivity in genital secretions from anatomically specific sites in asymptomatic women during C. trachomatis infection and post-antibiotic treatment. We found higher levels of cytokines and chemokines in endocervical secretions than vaginal secretions. Compared with the convalescent state, G-CSF, IL-1α, and RANTES were elevated in endocervical secretions, IFN-γ and TNF-α were elevated in vaginal secretions, and IFNγ, IL-1ß, and MIP1-α were elevated in cervicolavage fluid (CVL), before adjustment of multiple comparisons. Elevated endocervical levels of IP-10 and MCP-1 were associated with the use of hormonal contraception in infected women after successful treatment, suggesting the role of hormonal contraception in inflammation independent of STIs. Importantly, soluble factors found in endocervical secretions during infection enhanced HIV infectivity while no difference in HIV infectivity was found with vaginal secretions or CVL during infection or at convalescence. Taken together, the profiles of immune mediators and in vitro HIV infectivity indicate that the endocervical and vaginal mucosa are immunologically distinct. Our results underscore the importance of considering anatomical site and local sampling methodology when measuring mucosal responses, particularly in the presence of C. trachomatis infection.

Development of a high-throughput assay to measure the neutralization capability of anti-cytomegalovirus antibodies.

Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169IE2-YFP) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.

Localized mucosal response to intranasal live attenuated influenza vaccine in adults.

BACKGROUND: Influenza virus infection is a major public health burden worldwide. Available vaccines include the inactivated intramuscular trivalent vaccine and, more recently, an intranasal live attenuated influenza vaccine (LAIV). The measure of successful vaccination with the inactivated vaccine is a systemic rise in immunoglobulin G (IgG) level, but for the LAIV no such correlate has been established. METHODS: Seventy-nine subjects were given the LAIV FluMist. Blood was collected prior to vaccination and 3 days and 30 days after vaccination. Nasal wash was collected 3 days and 30 days after vaccination. Responses were measured systemically and in mucosal secretions for cytokines, cell activation profiles, and antibody responses. RESULTS: Only 9% of subjects who received LAIV seroconverted, while 33% of patients developed at least a 2-fold increase in influenza virus-specific immunoglobulin A (IgA) antibodies in nasal wash. LAIV induced a localized inflammation, as suggested by increased expression of interferon-response genes in mucosal RNA and increased granulocyte colony-stimulating factor (G-CSF) and IP-10 in nasal wash. Interestingly, patients who seroconverted had significantly lower serum levels of G-CSF before vaccination. CONCLUSIONS: Protection by LAIV is likely provided through mucosal IgA and not by increases in systemic IgG. LAIV induces local inflammation. Seroconversion is achieved in a small fraction of subjects with a lower serum G-CSF level.

Estrogen mediates innate and adaptive immune alterations to influenza infection in pregnant mice.

Pregnancy is a leading risk factor for severe complications during an influenza virus infection. Women infected during their second and third trimesters are at increased risk for severe cardiopulmonary complications, premature delivery, and death. Here, we establish a murine model of aerosolized influenza infection during pregnancy. We find significantly altered innate antiviral responses in pregnant mice, including decreased levels of IFN-ß, IL-1α, and IFN-γ at early time points of infection. We also find reduced cytotoxic T cell activity and delayed viral clearance. We further demonstrate that pregnancy levels of the estrogen 17-ß-estradiol are able to induce key anti-inflammatory phenotypes in immune responses to the virus independently of other hormones or pregnancy-related stressors. We conclude that elevated estrogen levels result in an attenuated anti-viral immune response, and that pregnancy-associated morbidities occur in the context of this anti-inflammatory phenotype.

The influence of pregnancy on systemic immunity.

Adaptations in maternal systemic immunity are presumed to be responsible for observed alterations in disease susceptibility and severity as pregnancy progresses. Epidemiological evidence as well as animal studies have shown that influenza infections are more severe during the second and third trimesters of pregnancy, resulting in greater morbidity and mortality, although the reason for this is still unclear. Our laboratory has taken advantage of 20 years of experience studying the murine immune response to respiratory viruses to address questions of altered immunity during pregnancy. With clinical studies and unique animal model systems, we are working to define the mechanisms responsible for altered immune responses to influenza infection during pregnancy and what roles hormones such as estrogen or progesterone play in these alterations.

Immunogenicity of trivalent inactivated influenza vaccination received during pregnancy or postpartum.

OBJECTIVE: To estimate the effects of gestational age and other maternal factors on immunologic responses to influenza vaccination. METHODS: Antepartum and postpartum women receiving influenza vaccination as part of routine clinical care were enrolled through four consecutive vaccination seasons (starting October 2006 through January 2010). Immunologic responses to trivalent inactivated influenza vaccine and monovalent H1N1 were assessed as well as factors influencing vaccine responsiveness. Serum samples were obtained at baseline and 4-8 weeks postvaccination. RESULTS: Two hundred thirty-nine participants were included in the current analysis. Seroconversion rates to trivalent inactivated influenza vaccine strains were lowest in the first trimester (54.8%) and immediately postpartum (54.8%) and were highest in the late third trimester (69.6%) and late postpartum (69.4%); these differences were not statistically significant (P=.23). In a multivariable model, higher baseline antibody levels (P<.001) and prior year flu vaccination (P=.03) were both significantly associated with reduced odds of seroconversion. Overall, results were consistent when comparing trivalent inactivated influenza vaccine and monovalent pandemic H1N1 responses. Although there was overall no significant association between gestational age at vaccination (P=.23) or prepregnancy body mass index (P=.16), we observed somewhat lower rates of seroconversion for women vaccinated in the first trimester and for obese women. CONCLUSION: Adequate immunologic responses to inactivated influenza vaccines were demonstrated during pregnancy and the postpartum period. No diminution of immunogenicity was observed in the third trimester, a time of increased clinical vulnerability to influenza.

Characterizing the pregnancy immune phenotype: results of the viral immunity and pregnancy (VIP) study.

PURPOSE: The increased risk of morbidity and mortality from certain microbial infections and the demonstrated improvements in the clinical course of some autoimmune diseases support the existence of pregnancy-related alterations in immune status. Elucidating the changes in innate and adaptive immunity during gestation may improve pregnancy outcomes and facilitate the development of targeted therapies for autoimmune diseases. METHOD: The Viral Immunity and Pregnancy (VIP) study evaluated over 50 subjects longitudinally at three time points during pregnancy and at two time points post-delivery. Leukocyte enumeration was performed; functional responses of NK cells and CD4 T cells were analyzed, and soluble factors such as cytokines, defensins, and steroid hormones were measured in maternal blood. RESULTS: In comparison to the post-partum period, the latter part of pregnancy was characterized by significant increases in blood phagocytes and pDCs and decreases in the number and activity of NK and T cells. Alterations were found in antimicrobial proteins and serum cytokines. CONCLUSIONS: These data show that pregnancy is not a period of immunosuppression but an alteration in immune priorities characterized by a strengthening of innate immune barriers and a concomitant reduction in adaptive/inflammatory immunity in the later stages of pregnancy.

Peripheral blood cytokine profiling during pregnancy and post-partum periods.

PROBLEM: Pregnancy requires that the maternal immune system adapt to prevent rejection of the fetal semi-allograft. This immunologic adaptation may contribute to pregnancy-related alterations in disease susceptibility and severity of infections from viral pathogens such as influenza virus. METHOD OF STUDY: As part of a larger study investigating the maternal systemic immune response during pregnancy, peripheral blood was collected three times during pregnancy and twice post-partum to measure serum levels of 23 cytokines, chemokines, and growth factors. This longitudinal study design allowed each woman's post-partum blood draw to serve as her own comparison, thus controlling for interpersonal variability in expression levels. RESULTS: When compared to the post-partum samples, significant pregnancy-related changes in IFNγ, TNFα, VEGF, GCSF, Eotaxin, and MCP-1 expression were observed. These changes have significant immunologic effects in vivo and in culture. CONCLUSION: Pregnancy-associated changes to steady state serum cytokines may have important immunologic consequence.

Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

Parainfluenza virus 5 (PIV5 or SV5) infects several mammalian species but is restricted from efficient replication in mice. In humans, PIV5 evades IFN signaling by targeting STAT1 for proteasomal degradation in a STAT2-dependent reaction. In contrast, cell culture experiments have demonstrated that the divergent murine STAT2 protein fails to support STAT1 targeting. Expression of human STAT2 in mouse cells can overcome the species restriction to enable PIV5-induced STAT1 degradation and subsequent IFN antagonism. Here, we describe a transgenic mouse that ubiquitously expresses human STAT2. PIV5 infection induces STAT1 degradation leading to enhanced virus replication and protein expression in the cells from the transgenic mouse but not from the non-transgenic littermates. Importantly, intranasal inoculation with PIV5 results in increased viral load in the lungs of the transgenic mice compared to wild-type littermates. These transgenic mice provide a small animal model to study the role of innate immune evasion in paramyxovirus pathogenesis.

A novel role for viral-defective interfering particles in enhancing dendritic cell maturation.

Dendritic cell (DC) maturation is a crucial event in the development of adaptive immune responses that confer long-lasting protection against reinfection with the same virus. Sendai virus strain Cantell has a particularly strong ability to mature DCs independently of type I IFNs and TLR signaling, currently the best-described pathways for the induction of DC maturation. In this study, we demonstrate that defective-interfering (DI) particles present in Sendai virus-Cantell stocks are required for its robust DC maturation ability. DI particles contain incomplete genomes that are unable to replicate unless the viral polymerase is supplied by coinfection with complete virus. Accordingly, the improvement in the virus-induced maturation of DCs provided by DI particles requires standard virus coinfection and likely results from increased production of dsRNA replication intermediaries. This unique ability of DI particles to stimulate DC maturation cannot be mimicked by simply increasing the dose of standard virus. Furthermore, viruses with weak DC maturation abilities can be converted into potent DC stimulators with the addition of DI particles, supporting a potential application for DI particles as a novel natural adjuvant for viral immunizations.

Evidence for a genetic defect in oral tolerance induction in inflammatory bowel disease.

BACKGROUND: Previous studies have suggested that there may be a defect in the control of immune responses locally in the intestines of patients with inflammatory bowel disease (IBD). Recently, we documented a failure to induce oral tolerance to a fed soluble protein antigen, keyhole limpet hemocyanin (KLH), in IBD patients. Both Crohn's disease (CD) and ulcerative colitis (UC) appear to be multigenic disorders with evidence of familial segregation. In this study, we analyzed multiplex IBD families to determine whether the defect in oral tolerance induction is genetically regulated. METHODS: Patients and first-degree relatives from 6 multiplex families were fed KLH 50 mg on days 0 to 5 and 10 to 15, followed by subcutaneous immunizations on days 26 and 35. Blood was obtained and analyzed for KLH-specific T cell proliferative responses and cytokine production. Intestinal permeability was also assessed. RESULTS: In contrast to normal controls, all IBD patients, save 1 (10 patients out of 11 tested P<.0001 versus normal controls), failed to develop oral tolerance to KLH. Furthermore, in 3 of the 4 CD families, at least 1 unaffected family member (total of 5/14 unaffected individuals, P=.002 versus normal controls) also failed to tolerize. This is in sharp contrast to unaffected individuals with no family history of IBD (1/31 tested to date). CONCLUSIONS: This failure of tolerance induction could not be attributed to increased intestinal permeability. In the UC families, the defect in tolerance segregated with disease. These data support a genetic defect in tolerance induction in CD.

Oral tolerance and inflammatory bowel disease.

PURPOSE OF REVIEW: Oral tolerance refers to the ability of the mucosal immune system to actively inhibit systemic immune responses to fed antigens. Recently, clinical trials have used oral tolerance as a therapy for certain chronic inflammatory and autoimmune diseases such as multiple sclerosis and type I diabetes. Inflammatory bowel disease is now widely thought to be caused by the breakdown of oral tolerance through a combination of genetic and environmental factors. Therefore, it seems incongruous that clinicians would try to use oral tolerance therapy to alleviate the symptoms of inflammatory bowel disease. Yet, armed with the results of select animal models, trials have begun for oral tolerance therapy for Crohn's disease. This review will outline the recent advances in understanding oral tolerance, explore the relation between oral tolerance and inflammatory bowel disease, and comment on the likelihood of successful oral tolerance therapy for inflammatory bowel disease. RECENT FINDINGS: The results of an oral tolerance trial in Crohn's disease patients in Israel have shown some promising results, whereas the results of studies of experimentally induced oral tolerance in patients with inflammatory bowel disease from the authors' laboratory have shown that feeding a neoantigen in an attempt to induce oral tolerance is not successful in patients with inflammatory bowel disease. SUMMARY: The fundamental difference in the mechanisms of oral tolerance in mice and humans requires a more focused effort to understand the human mucosal immune system before oral tolerance therapy for autoimmune and chronic inflammatory disorders reaches its full potential.

Induction of mucosal tolerance in Peyer's patch-deficient, ligated small bowel loops.

To explore the requirement for M cells and the Peyer's patch (PP) in induction of oral tolerance and address the potential in vivo role of intestinal epithelial cells as nonprofessional APCs, we have attempted to induce tolerance in mice with ligated small bowel loops without M cells and Peyer's patches. A 2-centimeter section of vascularized small bowel was spliced away from the gut without disruption of the mesenteric attachments. We introduced OVA directly into the lumen of the loop prior to footpad immunization. By excising segments of bowel that contain PPs in some mice and segments without patches in others, we could study the necessity of the M cell and the underlying patch versus epithelial cells in induction of mucosal tolerance. We show that OVA-specific T cell proliferation and serum antibody responses are reduced in mice that have previously been given OVA both in PP-containing loops and in loops without patches. Furthermore, both high- and low-dose tolerance could be induced in the absence of PPs. Low-dose tolerance is associated with bystander suppression and requires IL-10, which indicates active suppression and the induction of regulatory cells. These data suggest that there is a critical role for components of the mucosal immune system other than PPs in antigen sampling and induction of oral tolerance.