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SUMMARY A complex microbiota inhabits various microenvironments of the gut, with some symbiotic bacteria having evolved traits to invade the epithelial mucus layer and reside deep within intestinal tissue of animals. Whether these distinct bacterial communities across gut biogeographies exhibit divergent behaviors remains largely unknown. Global transcriptomic analysis to investigate microbial physiology in specific mucosal niches has been hampered technically by overabundance of host RNA. Herein, we employed hybrid selection RNA sequencing (hsRNA-Seq) to enable detailed spatial transcriptomic profiling of a prominent human commensal as it colonizes the colonic lumen, mucus or epithelial tissue of mice. Compared to conventional RNA-Seq, hsRNA-Seq increased reads mapping to the Bacteroides fragilis genome by 48- and 154-fold in mucus and tissue, respectively, allowing for high fidelity comparisons across biogeographic sites. Near the epithelium, B. fragilis up-regulated numerous genes involved in protein synthesis, indicating that bacteria inhabiting the mucosal niche are metabolically active. Further, a specific sulfatase (BF3086) and glycosyl hydrolase (BF3134) were highly induced in mucus and tissue compared to bacteria in the lumen. In-frame deletion of these genes impaired in vitro growth on mucus as a carbon source, as well as mucosal colonization of mice. Mutants in either B. fragilis gene displayed a fitness defect in competing for colonization against bacterial challenge, revealing the importance of site-specific gene expression for robust host-microbial symbiosis. As a versatile tool, hsRNA-Seq can be deployed to explore the in vivo spatial physiology of numerous bacterial pathogens or commensals.

Over the last several years, many advances have been made in understanding the role of bacteria in the pathogenesis of gastrointestinal cancers. Beginning withHelicobacter pyloribeing recognized as the first bacterial carcinogen and the causative agent of most gastric cancers, more recent studies have examined the role of enteric microbes in colorectal cancer. In the digestive tract, these communities are numerous and have a complex interrelationship with local immune/inflammatory responses that impact the health of the host. As modifying the microbiome in the stomach has decreased the risk of gastric cancer, modifying the distal microbiome may decrease the risk of colorectal cancers. To date, very few studies have considered the notion that mucosal lymphocyte-dependent immune memory may confound attempts to change the microbial components in these communities. The goal of this review is to consider some of the factors impacting host-microbial interactions that affect colorectal cancer and raise questions about how immune memory responses to the local microbial consortium affect any attempt to modify the composition of the intestinal microbiome.

Protein S is a potent anticoagulant that downregulates thrombin formation and is a vitamin K-dependent glycoprotein which is primarily synthesized in the liver. A deficiency in this protein or decreased activity, as seen in hereditary protein S deficiency, can lead to life-threatening thrombosis. Hereditary protein S deficiency is a rare disease as listed by the National Organization for Rare Disorders (NORD). It is known to cause venous as well as arterial thromboembolic events commonly occurring in the deep leg and pelvic veins. Dural venous sinus thrombosis is a rare consequence of protein S deficiency and is associated with a risk of increased morbidity and mortality. We report a case of dural venous sinus thrombosis in a patient with a family history of protein S deficiency in nine family members. A 53-year-old female presented to the ED with a three-day history of persistent left-sided headache, left facial numbness with tingling, and photophobia. She denied any visual disturbances, slurring of speech, and/or unilateral weakness. Some 10 years prior to this episode, she was placed on warfarin therapy for deep vein thrombosis (DVT) of lower extremity, but she discontinued it after three years of treatment without consulting her treating physician. She was taking oral contraceptive pills (OCPs) for two years and discontinued one month ago. She has nine family members with protein S deficiency, but the patient was never screened for a hypercoagulable state. On admission, her vital signs were within normal limits. Pupils were round and reactive to light, neck was supple, there was a sensory deficit for pinprick on the left V2-V3 distribution, and remainder of the cranial nerves and neurologic examination was unremarkable. CT scan of the head demonstrated a hyper-density within the left transverse and sigmoid sinus suspicious for dural venous sinus thrombosis. This was confirmed by CT angiogram showing a filling defect throughout the transverse sinus and sigmoid sinus extending below the jugular bulb into the superior aspect of the jugular vein. Intravenous heparin and warfarin were initiated. As the patient had severe trypanophobia and IV heparin required frequent activated partial thromboplastin time (APTT) monitoring, this was later changed to subcutaneous low-molecular-weight heparin and warfarin. Subsequent thrombosis panel showed a reduced protein S activity of 15% and low levels of total and free protein S antigens. She was discharged home with life-long warfarin therapy. In conclusion, cerebral dural venous sinus thrombosis is a rare and potentially life-threatening condition that can be seen in hereditary protein S deficiency. A high degree of suspicion in young females with worsening headache and neurologic signs and symptoms will help with timely diagnosis and management avoiding serious consequences. In a patient with a family history of thrombophilia, as seen in our patient, screening is important in order to confirm an underlying thrombophilic state. Such testing may have helped our patient regarding education on avoiding potential risk factors for thrombophilia and importance of treatment adherence.

Mesenchymal cells in the crypt play indispensable roles in the maintenance of intestinal epithelial homeostasis through their contribution to the preservation of stem cells. However, the acquisition properties of the production of stem cell niche factors by the mesenchymal cells have not been well elucidated, due to technical limitations regarding the isolation and subsequent molecular and cellular analyses of cryptal mesenchymal cells. To evaluate the function of mesenchymal cells located at the large intestinal crypt, we established a novel method through which cells are harvested according to the histologic layers of mouse colon, and we compared cellular properties between microenvironmental niches, the luminal mucosa and crypts. The gene expression pattern in the cryptal mesenchymal cells showed that receptors of the hormone/cytokine leptin were highly expressed, and we found a decrease in Wnt2b expression under conditions of leptin receptor deficiency, which also induced a delay in cryptal epithelial proliferation. Our novel stratified layer isolation strategies thus revealed new microenvironmental characteristics of colonic mesenchymal cells, including the intrinsic involvement of leptin in the control of mucosal homeostasis.

Background/Aims: We aimed to investigate the effect of a moderately high vitamin D dose on lipid parameters and biochemical markers of vascular calcification (VC) in patients with established cardiovascular disease. Methods: We included in this pre-specified secondary analysis of a randomized controlled trial 161 patients with advanced heart failure and 25-hydroxyvitamin D (25OHD) concentrations < 75 nmol/L (vitamin D group: n = 80; placebo group: n = 81), who received a daily vitamin D3 supplement of 4,000 IU for 3 years. We assessed between-group differences of the lipid parameters total-cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and triglycerides, and the VC markers fetuin-A and non-phosphorylated undercarboxylated matrix gla protein (MGP) at study termination, with adjustment for baseline values. Results: Lipid parameters, the percentage of patients with dyslipoproteinemia, and VC markers did not differ significantly between groups at study termination (p values: 0.395–0.939). Likewise, vitamin D achieved no significant treatment effect on these markers in subgroup analyses in patients with 25OHD concentrations < 30 nmol/L, nonusers of lipid-lowering drugs, or diabetic patients (p values: 0.245–0.998). Conclusion: Our data indicate that vitamin D does not improve the lipid profile and does not influence the calcification inhibitors fetuin-A and non-phosphorylated undercarboxylated MGP in patients with advanced heart failure.

Helicobacter pylori is a gram-negative, microaerophilic bacterium that infects the stomach and can lead to, among other disorders, the development of gastric cancer. The inability of the host to clear the infection results in a chronic inflammatory state with continued oxidative stress within the tissue. Reactive oxygen species and reactive nitrogen species produced by the immune and epithelial cells damage the host cells and can result in DNA damage. H pylori has evolved to evoke this damaging response while blunting the hostâs efforts to kill the bacteria. This long-lasting state with inflammation and oxidative stress can result in gastric carcinogenesis. Continued efforts to better understand the bacterium and the host response will serve to prevent or provide improved early diagnosis and treatment of gastric cancer. Keywords: AP Endonuclease, DNA Damage, H pylori, Gastric Cancer, Oxidative Stress

Summary The application of bacteriophages (phages) is proposed as a highly specific therapy for intestinal pathobiont elimination. However, the infectious associations between phages and bacteria in the human intestine, which is essential information for the development of phage therapies, have yet to be fully elucidated. Here, we report the intestinal viral microbiomes (viromes), together with bacterial microbiomes (bacteriomes), in 101 healthy Japanese individuals. Based on the genomic sequences of bacteriomes and viromes from the same fecal samples, the host bacteria-phage associations are illustrated for both temperate and virulent phages. To verify the usefulness of the comprehensive host bacteria-phage information, we screened Clostridioides difficile-specific phages and identified antibacterial enzymes whose activity is confirmed both in vitro and in vivo. These comprehensive metagenome analyses reveal not only host bacteria-phage associations in the human intestine but also provide vital information for the development of phage therapies against intestinal pathobionts.

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected withSalmonella entericaserovar Typhimurium or adherent-invasiveEscherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function.

Background & Aims Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT. Methods The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria–phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated. Results Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented. Conclusions The coordinated action of phages and their host bacteria restored the recipients' intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.

Background & Aims Dysregulation of the microbiome has been associated with development of complex diseases, such as obesity and diabetes. However, no method has been developed to control disease-associated commensal microbes. We investigated whether immunization with microbial antigens, using CpG oligodeoxynucleotides and/or curdlan as adjuvants, induces systemic antigen-specific IgA and IgG production and affects development of diseases in mice. Methods C57BL/6 mice were given intramuscular injections of antigens (ovalbumin, cholera toxin B-subunit, or pneumococcal surface protein A) combined with CpG oligodeoxynucleotides and/or curdlan. Blood and fecal samples were collected weekly and antigen-specific IgG and IgA titers were measured. Lymph nodes and spleens were collected and analyzed by enzyme-linked immunosorbent assay for antigen-specific splenic T-helper 1 cells, T-helper 17 cells, and memory B cells. Six weeks after primary immunization, mice were given a oral, nasal, or vaginal boost of ovalbumin; intestinal lamina propria, bronchial lavage, and vaginal swab samples were collected and antibodies and cytokines were measured. Some mice were also given oral cholera toxin or intranasal Streptococcus pneumoniae and the severity of diarrhea or pneumonia was analyzed. Gnotobiotic mice were gavaged with fecal material from obese individuals, which had a high abundance of Clostridium ramosum (a commensal microbe associated with obesity and diabetes), and were placed on a high-fat diet 2 weeks after immunization with C ramosum. Intestinal tissues were collected and analyzed by quantitative real-time polymerase chain reaction. Results Serum and fecal samples from mice given injections of antigens in combination with CpG oligodeoxynucleotides and curdlan for 3 weeks contained antigen-specific IgA and IgG, and splenocytes produced interferon-gamma and interleukin 17A. Lamina propria, bronchial, and vaginal samples contained antigen-specific IgA after the ovalbumin boost. This immunization regimen prevented development of diarrhea after injection of cholera toxin, and inhibited lung colonization by S pneumoniae. In gnotobiotic mice colonized with C ramosum and placed on a high-fat diet, the mice that had been immunized with C ramosum became less obese than the nonimmunized mice. Conclusions Injection of mice with microbial antigens and adjuvant induces antigen-specific mucosal and systemic immune responses. Immunization with S pneumoniae antigen prevented lung infection by this bacteria, and immunization with C ramosum reduced obesity in mice colonized with this microbe and placed on a high-fat diet. This immunization approach might be used to protect against microbe-associated disorders of intestine.

Despite its direct exposure to huge amounts of microorganisms and foreign and dietary antigens, the gut mucosa maintains intestinal homeostasis by utilizing the mucosal immune system. The gut mucosal immune system protects the host from the invasion of infectious pathogens and eliminates harmful non-self antigens, but it allows the cohabitation of commensal bacteria in the gut and the entry of dietary non-self antigens into the body via the mucosal surface. These physiological and immunological activities are regulated by the ingenious gut mucosal immune network, comprising such features as gut-associated lymphoid tissue, mucosal immune cells, cytokines, chemokines, antimicrobial peptides, secretory IgA, and commensal bacteria. The gut mucosal immune network keeps a fine tuned balance between active immunity (against pathogens and harmful non-self antigens) and immune tolerance (to commensal microbiota and dietary antigens), thus maintaining intestinal healthy homeostasis. Disruption of gut homeostasis results in persistent or severe gastrointestinal infection, inflammatory bowel disease, or allergic inflammation. In this review, we comprehensively introduce current knowledge of the gut mucosal immune system, focusing on its interaction with allergic inflammation. Keywords: Allergy, Microbiota, Mucosal immunity, Oral tolerance, Regulatory T cell

Animal models have been tremendously useful to translational research, but there is a need to maximize their predictive value to human disease. This Comment proposes novel strategies that consider evolutionary history and the presence, absence or modification of molecular networks in one species that are being studied in the other. Mice are generally the ‘go-to’ organism for modeling of the human immune system, but this often leads to inaccurate interpretations. Ernst and Carvunis argue in this Comment that taking into account the evolutionary and environmental context can generate better models of disease.

Low vitamin D status, i.e. circulating 25-hydroxyvitamin D (25OHD) levels50 nmol/l, is independently associated with increased CVD risk. Medication use may influence 25OHD levels. We therefore investigated the association of circulating 25OHD with medication use in patients scheduled for cardiac surgery.A total of 11,256 patients were included in this cross-sectional study. We compared 25OHD levels of medication users (18 groups of continuously used and 5 groups of intermittently used medications) with levels of non-users. Moreover, we assessed variables (medications, demographic and clinical parameters) that were independently associated with 25OHD levels50 nmol/l. The prevalence of 25OHD levels50 nmol/l was 65.7%. The use of statins and immunosuppressive agents was significantly associated with higher 25OHD levels and lower odds ratios of 25OHD levels50 nmol/l. The use of ACE-inhibitors, catecholamines and antibiotics was associated with lower 25OHD levels and higher odds ratios of 25OHD levels50 nmol/l. However, only use of antibiotics, immunosuppressive agents and catecholamines showed clinically relevant differences in 25OHD levels, i.e. differences of more than +4 nmol/l or -4 nmol/l, compared with respective non-users. These medications were prescribed either intermittently (antibiotics, catecholamines) and/or infrequently (2%; immunosuppressive agents, catecholamines) and/or its causal relationship with circulating 25OHD is questionable (antibiotics). Female sex and blood drawing during wintertime were associated with the highest odds ratios of 25OHD levels50 nmol/l.Data indicate that in patients with high cardiovascular risk profile medication use does not substantially contribute to 25OHD levels50 nmol/l.

The gastrointestinal barrier is more than a tissue with physiological function. As reviewed by others, it is now recognized as a complex ecosystem that requires a delicate homeostatic balance in order for the tissue to remain healthy and functional. The niche involves a multifaceted and dynamic mixture of host cells and molecules; microbes and their metabolites; as well as environmental factors that may reflect diet or contamination with toxins. Furthermore, gene expression in the host can modulate transcription in microbes resulting in changes in the metabolic profile and local immune responses that resonate throughout the body. Thus, these biological spheres are highly interconnected. A more recent and important concept is that the microbial-host interactions that begin in the digestive tract not only impact gastrointestinal inflammation and risks of local cancer, but they also affect the hosťs susceptibility to conditions ranging from diabetes, obesity to various neurological conditions. This chapter summarizes the key elements whereby the gut barrier contributes to innate immunity in the digestive tract.

Observational studies indicate a positive association between circulating 25-hydroxyvitamin D (25OHD) and testosterone (T) concentrations. Because low 25OHD concentrations and T deficiency are considered to be a generalized phenomenon in patients with advanced heart failure (HF), we aimed to investigate whether vitamin D supplementation has beneficial effects on T indices in these patients. In a pre-specified secondary analysis of the EVITA (effect of vitamin D on mortality in heart failure) randomized controlled trial, we analyzed in male subjects with 25OHD concentrations

Adenosine is a purine metabolite that can mediate anti-inflammatory responses in the digestive tract through the A2A adenosine receptor (A2AAR). We examined the role of this receptor in the control of inflammation in the adoptive transfer model of colitis. Infection of A2AAR−/− mice with Helicobacter hepaticus increased colonic inflammation scores compared with uninfected A2AAR controls. Comparison of T cell subsets in wild-type and A2AAR−/− mice revealed differences in markers associated with activated helper T (Th) cells and regulatory T (Treg) cells. Previous studies showed that expression of A2AAR on CD45RBHI and CD45RBLO Th cells is essential for the proper regulation of colonic inflammation. Adoptive transfer of CD45RBHI with CD45RBLO from wild-type mice into RAG1−/−/A2AAR−/− mice induced severe disease within 3 wk, although transfer of the same subsets into RAG1−/− mice does not induce colitis. This suggests that the presence of A2AAR on recipient cells is also important for controlling colitis. To investigate the role of A2AAR in myeloid cells, chimeric recipients were generated by injection of bone marrow from RAG1−/− or RAG1−/−/A2AAR−/− mice into irradiated RAG1−/− mice. After adoptive transfer, these recipients did not develop colitis, regardless of A2AAR expression by the donor. Together, our results suggest that the control of inflammation in vivo is dependent on A2AAR signaling through multiple cell types that collaborate in the regulation of colitis by responding to extracellular adenosine.

After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5-fold increase in the binding of apoptotic GECs to THP-1 cells or peripheral blood monocyte-derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP-1 cells to apoptotic cells inhibited IL-6 production from 1340 to

Increased apoptotic death of gastric epithelial cells is a hallmark of H. pylori infection, and altered epithelial cell turnover is an important contributor to gastric carcinogenesis. To address the fate of apoptotic gastric epithelial cells and their role in H. pylori mucosal disease, we investigated phagocyte clearance of apoptotic gastric epithelial cells in H. pylori infection. Human gastric mononuclear phagocytes were analyzed for their ability to take up apoptotic epithelial cells in vivo using immunofluorescence analysis. We then used primary human gastric epithelial cells induced to undergo apoptosis by exposure to live H. pylori to study apoptotic cell uptake by autologous monocyte-derived macrophages. We show that HLA-DR+ mononuclear phagocytes in human gastric mucosa contain cytokeratin-positive and TUNEL-positive apoptotic epithelial cell material, indicating that gastric phagocytes are involved in apoptotic epithelial cell clearance. We further show that H. pylori both increased apoptosis in primary gastric epithelial cells and decreased phagocytosis of the apoptotic epithelial cells by autologous monocyte-derived macrophages. Reduced macrophage clearance of apoptotic cells was mediated in part by H. pylori-induced macrophage TNF-α, which was expressed at higher levels in H. pylori-infected, compared to uninfected, gastric mucosa. Importantly, we show that H. pylori-infected gastric mucosa contained significantly higher numbers of apoptotic epithelial cells and higher levels of non-phagocytosed TUNEL-positive apoptotic material, consistent with a defect in apoptotic cell clearance. Thus, as shown in other autoimmune and chronic inflammatory diseases, insufficient phagocyte clearance may contribute to the chronic and self-perpetuating inflammation in human H. pylori infection.

Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract (GI) for which treatments with immunosuppressive drugs have significant side-effects. Consequently, there is a clinical need for site-specific and non-toxic delivery of therapeutic genes or drugs for CD and related disorders such as inflammatory bowel disease. The aim of this study was to validate a gene delivery platform based on ultrasound-activated lipid-shelled microbubbles (MBs) targeted to inflamed mesenteric endothelium in the CD-like TNFΔARE mouse model. MBs bearing luciferase plasmid were functionalized with antibodies to MAdCAM-1 (MB-M) or VCAM-1 (MB-V), biomarkers of gut endothelial cell inflammation and evaluated in an in vitro flow chamber assay with appropriate ligands to confirm targeting specificity. Following MB retro-orbital injection in TNFΔARE mice, the mean contrast intensity in the ileocecal region from accumulated MB-M and MB-V was 8.5-fold and 3.6-fold greater, respectively, compared to MB-C. Delivery of luciferase plasmid to the GI tract in TNFΔARE mice was achieved by insonating the endothelial cell-bound agents using a commercial sonoporator. Luciferase expression in the midgut was detected 48 h later by bioluminescence imaging and further confirmed by immunohistochemical staining. The liver, spleen, heart, and kidney had no detectable bioluminescence following insonation. Transfection of the microcirculation guided by a targeted, acoustically-activated platform such as an ultrasound contrast agent microbubble has the potential to be a minimally-invasive treatment strategy to ameliorate CD and other inflammatory conditions.

Summary The cytoplasmic Elmo1:Dock180 complex acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac and functions downstream of the phagocytic receptor BAI1 during apoptotic cell clearance, and in the entry of Salmonella and Shigella into cells [1–7]. We discovered an unexpected binding between Elmo1 and the Mediator complex subunit Med31. The Mediator complex is a regulatory hub for nearly all gene transcription via RNA polymerase II, bridging the general transcription machinery with gene-specific regulatory proteins [8–14]. Med31 is the smallest and the most evolutionarily conserved Mediator subunit [15, 16], and knockout of Med31 results in embryonic lethality in mice [17]; however, Med31 function in specific biological contexts is poorly understood. We observed that in primary macrophages, during Salmonella infection, Elmo1 and Med31 specifically affected expression of the cytokine genes Il10 and Il33 among the >25 genes monitored. Although endogenous Med31 is predominantly nuclear localized, Elmo1 increased the cytoplasmic localization of Med31. We identify ubiquitination as a novel posttranslational modification of Med31, with the cytoplasmic monoubiquitinated form of Med31 being enhanced by Elmo1. These data identify Elmo1 as a novel regulator of Med31, revealing a previously unrecognized link between cytoplasmic signaling proteins and the Mediator complex.

Genes and microbes converge in colitis Both host genetics and intestinal microbes probably contribute to a person's overall susceptibility to inflammatory bowel disease (IBD). The human gut microbe Bacteroides fragilis produces immunomodulatory molecules that it releases via outer membrane vesicles (OMVs). These molecules can protect mice from experimentally induced colitis. Chu et al. now find that OMV-mediated protection from colitis requires Atg16l1 and Nod2 genes whose human orthologs are associated with an increased risk for developing IBD. OMVs trigger an ATG16L1 and NOD2–dependent noncanonical autophagy pathway in dendritic cells (DCs). OMV-primed DCs, in turn, induce regulatory T cells in the intestine that protect against colitis. Science , this issue p. 1116

SAMP1/YitFcs mice serve as a model of Crohn's disease, and we have used them to assess gastritis. Gastritis was compared in SAMP1/YitFcs, AKR, and C57BL/6 mice by histology, immunohistochemistry, and flow cytometry. Gastric acid secretion was measured in ligated stomachs, while anti-parietal cell antibodies were assayed by immunofluorescence and enzyme-linked immunosorbent spot assay. SAMP1/YitFcs mice display a corpus-dominant, chronic gastritis with multifocal aggregates of mononuclear cells consisting of T and B lymphocytes. Relatively few aggregates were observed elsewhere in the stomach. The infiltrates in the oxyntic mucosa were associated with the loss of parietal cell mass. AKR mice, the founder strain of the SAMP1/YitFcs, also have gastritis, although they do not develop ileitis. Genetic studies using SAMP1/YitFcs-C57BL/6 congenic mice showed that the genetic regions regulating ileitis had comparable effects on gastritis. The majority of the cells in the aggregates expressed the T cell marker CD3 or the B cell marker B220. Adoptive transfer of SAMP1/YitFcs CD4+ T helper cells, with or without B cells, into immunodeficient recipients induced a pangastritis and duodenitis. SAMP1/YitFcs and AKR mice manifest hypochlorhydria and anti-parietal cell antibodies. These data suggest that common genetic factors controlling gastroenteric disease in SAMP1/YitFcs mice regulate distinct pathogenic mechanisms causing inflammation in separate sites within the digestive tract.

Adenosine is an endogenous metabolite produced during hypoxia or inflammation. Previously implicated as an anti-inflammatory mediator in CD4+ T cell regulation, we report that adenosine acts via dendritic cell (DC) A2B adenosine receptor (A2BAR) to promote the development of Th17 cells. Mouse naive CD4+ T cells cocultured with DCs in the presence of adenosine or the stable adenosine mimetic 5′-(N-ethylcarboximado) adenosine resulted in the differentiation of IL-17– and IL-22–secreting cells and elevation of mRNA that encode signature Th17-associated molecules, such as IL-23R and RORγt. The observed response was similar when DCs were generated from bone marrow or isolated from small intestine lamina propria. Experiments using adenosine receptor antagonists and cells from A2BAR−/− or A2AAR−/−/A2BAR−/− mice indicated that the DC A2BAR promoted the effect. IL-6, stimulated in a cAMP-independent manner, is an important mediator in this pathway. Hence, in addition to previously noted direct effects of adenosine receptors on regulatory T cell development and function, these data indicated that adenosine also acts indirectly to modulate CD4+ T cell differentiation and suggested a mechanism for putative proinflammatory effects of A2BAR.

Background: Helicobacter pylori (H. pylori) is a gram negative bacterium that can cause diseases such as peptic ulcers and gastric cancer. IL-17A, a proinflammatory cytokine that can induce the production of CXC chemokines for neutrophil recruitment, has recently been shown to be elevated in both H. pylori-infected patients and mice. Furthermore, studies in mouse models of vaccination have reported levels significantly increased over infected, unimmunized mice and blocking of IL-17A during the challenge phase in immunized mice reduces protective immunity. Because many aspects of immunity had redundant or compensatory mechanisms, we investigated whether mice could be protectively immunized when IL-17A function is absent during the entire immune response using IL-17A and IL-17A receptor knockout (KO) mice immunized against H. pylori. Materials and Methods: Gastric biopsies were harvested from naive, unimmunized/challenged, and immunized/challenged wild type (WT) and KO mice and analyzed for inflammation, neutrophil, and bacterial levels. Groups of IL-17A KO mice were also treated with anti-IFNγ or control antibodies. Results: Surprisingly, all groups of immunized KO mice reduced their bacterial loads comparably to WT mice. The gastric neutrophil counts did not vary significantly between IL-17A KO and WT mice, whereas IL-17RA KO mice had on average a four-fold decrease compared to WT. Additionally, we performed an immunization study with CXCR2 KO mice and observed significant gastric neutrophils and reduction in bacterial load. Conclusion: These data suggest that there are compensatory mechanisms for protection against H. pylori and for neutrophil recruitment in the absence of an IL-17A-CXC chemokine pathway.

Hypoxia-inducible factor 1 (HIF1) consists of a hypoxia-inducible α subunit and a constitutively expressed β subunit. Reactive oxygen species (ROS) induced by Helicobacter pylori stabilize HIF1α in the human gastric epithelium in normoxia. HIF1α plays crucial role in carcinogenesis and has been associated with malignant progression of gastric cancer. Several genes contain functional hypoxia-response elements (HREs) in their promoters including Bcl2 family member, Mcl1. Cellular ratios of antiapoptotic oncogenic protein, Mcl1, and tumor suppressor proapoptotic protein, Noxa, determine cell fate by regulating normal cellular growth, cell death and oncogenic processes. The aim of the present study was to examine the mechanism of HIF1α induction in the H. pylori -infected gastric epithelium to better understand disease pathogenesis by H. pylori relevant to gastric carcinogenesis. Our data showed that the dose-dependent increase in HIF1α in H. pylori -infected gastric epithelia is mediated by induction of a ROS-inducible protein, apurinic/apyrimidinic endonuclease 1 (APE1), and an enhanced interaction of APE1 with the transcriptional coactivator p300. Surprisingly, with accumulation of HIF1α, further transcriptional activation of mcl1 was not observed. We identified a HIF-binding site (HBS) in the hif1α promoter and showed that increased HIF1α expression, whether H. pylori -induced or hypoxia-mimetic agent, CoCl2-induced, resulted in enhanced HIF1α binding to its own promoter. This resulted in a transcriptionally inactive hif1α promoter since hif1α HBS lacks HIF ancillary sequence (HAS) required for HIF1 transcriptional activity. We conclude that enhanced binding of “nonfunctional” HIF1α to hif1α promoter and limiting availability of p300 in the cell serves as checkpoints for uncontrolled HIF1α activity.

Leptin-stimulated Stat3 activation in proopiomelanocortin (POMC)-expressing neurons of the hypothalamus plays an important role in maintenance of energy homeostasis. While Stat3 activation in POMC neurons is required for POMC expression, the role of elevated basal Stat3 activation as present in the development of obesity has not been directly addressed. Here, we have generated and characterized mice expressing a constitutively active version of Stat3 (Stat3-C) in POMC neurons (Stat3-CPOMCmice). On normal chow diet, these animals develop obesity as a result of hyperphagia and decreased POMC expression accompanied by central leptin and insulin resistance. This unexpected finding coincides with POMC-cell-specific, Stat3-mediated upregulation of SOCS3 expression inhibiting both leptin and insulin signaling as insulin-stimulated PIP3(phosphatidylinositol-3,4,5 triphosphate) formation and protein kinase B (AKT) activation in POMC neurons as well as with the fact that insulin's ability to hyperpolarize POMC neurons is largely reduced in POMC cells of Stat3-CPOMCmice. These data indicate that constitutive Stat3 activation is not sufficient to promote POMC expression but requires simultaneous PI3K (phosphoinositide 3-kinase)-dependent release of FOXO1 repression. In contrast, upon exposure to a high-fat diet, food intake and body weight were unaltered in Stat3-CPOMCmice compared with control mice. Taken together, these experiments directly demonstrate that enhanced basal Stat3 activation in POMC neurons as present in control mice upon high-fat feeding contributes to the development of hypothalamic leptin and insulin resistance.

The endogenous purine nucleoside adenosine is an important antiinflammatory mediator that contributes to the control of CD4+ T cell responses. While adenosine clearly has direct effects on CD4+ T cells, it remains to be determined whether actions on APC such as dendritic cells (DC) are also important. In this report we characterize DC maturation and function in BMDC stimulated with LPS in the presence or absence of the nonselective adenosine receptor agonist NECA (5′-N-ethylcarboxamidoadenosine). We found that NECA inhibited TNF-α and IL-12 in a concentration-dependent manner, whereas IL-10 production was increased. NECA-treated BMDC also expressed reduced levels of MHC class II and CD86 and were less effective at stimulating CD4+ T cell proliferation and IL-2 production compared with BMDC exposed to vehicle control. Based on real-time RT-PCR, the A2A adenosine receptor (A2AAR) and A2BAR were the predominant adenosine receptors expressed in BMDC. Using adenosine receptor subtype selective antagonists and BMDC derived from A2AAR−/− and A2BAR−/−mice, it was shown that NECA modulates TNF-α, IL-12, IL-10, and CD86 responses predominantly via A2BAR. These data indicate that engagement of A2BAR modifies murine BMDC maturation and suggest that adenosine regulates CD4+ T cell responses by selecting for DC with impaired immunogencity.

Regulatory T cells (known as "Treg") express apyrases (CD39) and ecto-5'-nucleotidase (CD73) and contribute to their inhibitory function by generating adenosine. We investigated the expression of CD39 and CD73 on human T helper (Th) cells and the role of CD73 in regulating Helicobacter felis-induced gastritis and colonization.Human CD4+ Th cells, gastric T cells, or Treg subsets were stimulated and assayed for the expression of CD39 and CD73 by means of reverse-transcriptase polymerase chain reaction and flow cytometry. The effect of CD73 on proliferation and cytokine production was assessed, and the presence of gastritis, proinflammatory cytokine expression, or colonization of H. felis was evaluated in CD73-deficient (CD73-/-) mice or recipient mice given control or CD73-/- Treg.CD4+ T cells expressed CD39 and CD73, particularly in CD25+Foxp3+ Treg from peripheral blood or gastric mucosa. Activation significantly increased CD73 expression on all Th cells. Inhibition of CD73 enhanced production of interferon-gamma. Gastritis in H. felis-infected CD73-/- mice was significantly worse than that in wild-type mice and was accompanied by increased levels of proinflammatory cytokines and reduced bacterial colonization, whereas Treg from CD73-/- mice did not inhibit gastritis.CD39 and CD73 expressed by Th cells contribute to local accumulation of adenosine and attenuation of gastritis, which may favor persistent infection.

Insulin- and leptin-stimulated phosphatidylinositol-3 kinase (PI3K) activation has been demonstrated to play a critical role in central control of energy homeostasis. To delineate the importance of pathways downstream of PI3K specifically in pro-opiomelanocortin (POMC) cell regulation, we have generated mice with selective inactivation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in POMC-expressing cells (PDK1(DeltaPOMC) mice). PDK1(DeltaPOMC) mice initially display hyperphagia, increased body weight, and impaired glucose metabolism caused by reduced hypothalamic POMC expression. On the other hand, PDK1(DeltaPOMC) mice exhibit progressive, severe hypocortisolism caused by loss of POMC-expressing corticotrophs in the pituitary. Expression of a dominant-negative mutant of FOXO1 specifically in POMC cells is sufficient to ameliorate positive energy balance in PDK1(DeltaPOMC) mice but cannot restore regular pituitary function. These results reveal important but differential roles for PDK1 signaling in hypothalamic and pituitary POMC cells in the control of energy homeostasis and stress response.

Recruitment of lymphocytes to sites of inflammation requires the sequential engagement of adhesion molecules and chemokine receptors. Of these, the lectin-like molecule CD44 has been particularly implicated in inflammatory trafficking. Using a TNF-driven model of chronic ileitis (i.e., B6.129P-TnfΔARE mice) that recapitulates many features of Crohn’s disease, we demonstrate dynamic changes in the expression and functional state of CD44 on CD8+ T cells. These cells coexpress CD44 and L-selectin, giving them a surface phenotype similar to that of central memory T cells. Yet functionally they exhibit the phenotype of effector T cells, because they produce IFN-γ. Unexpectedly, depletion of the CD8+ population had no effect on the severity of ileitis. Further analyses showed a second CD8+ population that lacked CD44, but expressed CD103, produced TGF-β, inhibited the proliferation of CD4+ in vitro, and attenuated adoptively transferred ileitis in vivo, most likely counteracting the proinflammatory role of the CD44high subset. Collectively, these data suggest that the presence or absence of CD44 and CD103 on the CD8+ lymphocyte surface defines functionally distinct subsets of CD8+ T cells in vivo. These inflammation-driven populations exert distinct roles during the development of chronic ileitis, and influence the balance of effector and regulatory functions in the chronically inflamed small intestine.

Colonization of the gastric mucosa by Helicobacter pylori gives rise to chronic gastritis, which may be complicated by peptic ulcer disease [1] or gastric adenocarcinoma [2] and mucosa-associated lymphoid tissue lymphoma [3]. Ulcers and gastric cancer may represent divergent results of the repair process arising from disruption of the balance between cellular proliferation and apoptosis in the gastric epithelium, resulting in part from binding of bacteria to epithelial cells [4]. The genotype of the infecting strain, specifically expression of the cag pathogenicity island (cag PAI), is one of many factors that has been found to impact gastric epithelial apoptosis. Expression of the 40 kb cag pathogenicity island (cag PAI), present within 50–70% of H. pylori strains [5], has been linked to increased overall disease severity [6] and a greater likelihood of progression to gastric cancer [7,8]. cag PAI-positive strains have been shown to enhance apoptosis in T lymphocytes [9], but the specific impact of the cag PAI on the induction of apoptosis in gastric epithelial cells remains in contention [10–14]. Expression of cytotoxin-associated gene A (cagA), located within the cag PAI, plays a key role in the apparent enhanced virulence of cag PAI-positive strains of H. pylori . Several genes encoded by the cag PAI are devoted to the development of a type four (IV) secretion system [15]. Once bacteria have bound to the cell surface, the type IV secretion system enables delivery of gene products, such as CagA, directly into gastric epithelial cells, dramatically altering cell behavior [16]. Some consequences of the delivery of CagA into epithelial cells are the secretion of pro-inflammatory cytokines, changes in cell structure, and altered cellular proliferation [17,18]. Whether these intracellular events resulting from infection with cag PAIbearing strains are directly related to programmed cell death and whether the resultant apoptosis is affected by bacterial binding and bacterial viability are not clear. To examine this further we evaluated the effect of cag PAI expression in H. pylori on binding of bacteria to gastric epithelial cells and also apoptosis using cultured human gastric epithelial cell lines.

Much of the gene content of the human gastric pathogen Helicobacter pylori (∼1.7-Mb genome) is considered essential. This view is based on the completeness of metabolic pathways, infrequency of nutritional auxotrophies, and paucity of pathway redundancies typically found in bacteria with larger genomes. Thus, genetic analysis of gene function is often hampered by lethality. In the absence of controllable promoters, often used to titrate gene function, we investigated the feasibility of an antisense RNA interference strategy. To test the antisense approach, we targeted alkyl hydroperoxide reductase (AhpC), one of the most abundant proteins expressed by H. pylori and one whose function is essential for both in vitro growth and gastric colonization. Here, we show that antisense ahpC (as- ahpC ) RNA expression from shuttle vector pDH37::as- ahpC achieved an ∼72% knockdown of AhpC protein levels, which correlated with increased susceptibilities to hydrogen peroxide, cumene, and tert -butyl hydroperoxides but not with growth efficiency. Compensatory increases in catalase levels were not observed in the knockdowns. Expression of single-copy antisense constructs (expressed under the urease promoter and containing an fd phage terminator) from the rdxA locus of mouse-colonizing strain X47 achieved a 32% knockdown of AhpC protein levels (relative to wild-type X47 levels), which correlated with increased susceptibility to organic peroxides but not with mouse colonization efficiency. Our studies indicate that high levels of AhpC are not required for in vitro growth or for primary gastric colonization. Perhaps AhpC, like catalase, assumes a greater role in combating exogenous peroxides arising from lifelong chronic inflammation. These studies also demonstrate the utility of antisense RNA interference in the evaluation of gene function in H. pylori .

BackgroundThe ability to measure the expression of proinflammatory cytokines from intestinal biopsies in patients with Crohn's disease in an accurate and reproducible way is critical for proof-of-concept and mechanism-of-action trials; however, the number of biopsies from a segment of the ileum or colon required to yield reproducible results has not been rigorously evaluated. We examined intestinal biopsies from patients with Crohn's disease to validate methods for detecting changes in inflammatory gene expression.MethodsTo evaluate the reproducibility of gene expression measurements, intestinal biopsies were obtained from designated segments from 6 healthy controls, 6 patients with active Crohn's disease, and 6 patients with inactive Crohn's disease. Disease activity was based on the simple endoscopic score for Crohn's disease. Expression of 7 proinflammatory genes was measured from each biopsy using quantitative polymerase chain reaction. Using a linear mixed effects model, the power to detect transcriptional changes corresponding to active and inactive Crohn's disease was calculated.ResultsTotal simple endoscopic score for Crohn's disease score corresponds with expression of most inflammatory biomarkers. For most genes, 2 to 5 biopsies are needed to reduce sampling error to

The initiating etiologic factor in Crohn's disease (CD) remains unclear. SAMP1/YitFc (SAMP) mice develop chronic ileitis similar to human CD. We used bone marrow chimeras to determine if SAMP ileitis results from a primary immunological defect or from dysregulated mucosal immunity secondary to intrinsic, nonhematopoietic (e.g., epithelial) dysfunction. SAMP mice receiving wild-type (AKR) BM developed severe ileitis, whereas SAMP BM did not confer ileitis to WT recipients. WT lymphocytes from reconstituted SAMP mice resembled native SAMP populations in regard to surface phenotype and cytokine production. Ilea from native SAMP mice and SAMP recipients of wild-type BM displayed decreased epithelial barrier resistance ex vivo and increased epithelial permeability in vivo compared to native WT mice and AKR recipients of SAMP BM. This permeability defect preceded the development of ileal inflammation, was present in the absence of commensal bacteria, and was accompanied by altered ileal mRNA expression of the tight junction proteins claudin-2 and occludin. Our results provide evidence that the primary defect conferring ileitis in SAMP mice originates from a nonhematopoietic source. Generation of pathogenic lymphocytes is a consequence of this defect and does not reflect intrinsic proinflammatory leukocyte properties. Decreased barrier function suggests that defects in the epithelium may represent the primary source of SAMP ileitis susceptibility.

In 1984, Barry Marshall and Robin Warren proposed a role for bacterial infections in the pathogenesis of gastroduodenal disease, which triggered an avalanche of research intended to prove or disprove their theory. The result has been a series of advances that have enhanced our understanding of these diseases and completely modernized the clinical approach to their management. In just over 20 years, many aspects of the immunopathogenesis of these diseases have been dissected at the molecular level, with key pathogenic mechanisms being validated by the identification of genes that are associated with the development of gastric cancer. There has been particular emphasis on understanding the molecular structures associated with Helicobacter pylori and their role in modifying the host responses. Gastric immune and inflammatory responses have emerged as key elements in the pathogenesis of gastritis and epithelial cell damage. This review summarizes important findings emanating from basic research primarily related to the immunopathogenesis of H pylori that have advanced the practice of medicine or our understanding of gastroduodenal disease.

CD74 is known as the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) that regulates the cell biology and functions of MHC class II molecules. Class II MHC and Ii expression was believed to be restricted to classical antigen-presenting cells (APC); however, during inflammation, other cell types, including mucosal epithelial cells, have also been reported to express class II MHC molecules. Given the importance of Ii in the biology of class II MHC, we sought to examine the expression of Ii by gastric epithelial cells (GEC) to determine whether class II MHC molecules in these nonconventional APC cells were under the control of Ii and to further support the role that these cells may play in local immune and inflammatory responses during Helicobacter pylori infection. Thus we examined the expression of Ii on GEC from human biopsy samples and then confirmed this observation using independent methods on several GEC lines. The mRNA for Ii was detected by RT-PCR, and the various protein isoforms were also detected. Interestingly, these cells have a high level expression of surface Ii, which is polarized to the apical surface. These studies are the first to demonstrate the constitutive expression of Ii by human GEC.

Respiratory syncytial virus (RSV) is the major etiologic agent of severe epidemic lower respiratory tract infections in infancy. Airway mucosal inflammation plays a critical role in the pathogenesis of RSV disease in both natural and experimental infections. RSV is among the most potent biological stimuli that induce the expression of inflammatory genes, including those encoding chemokines, but the mechanism(s) that controls virus-mediated airway inflammation in vivo has not been fully elucidated. Herein we show that the inoculation of BALB/c mice with RSV results in rapid activation of the multisubunit IκB kinase (IKK) in lung tissue. IKK transduces upstream activating signals into the rate-limiting phosphorylation (and proteolytic degradation) of IκBα, the inhibitory subunit that under normal conditions binds to the nuclear factor (NF)-κB complex and keeps it in an inactive cytoplasmic form. Mice treated intranasally with interleukin-10 or with a specific cell-permeable peptide that blocks the association of the catalytic subunit IKKβ with the regulatory protein NEMO showed a striking reduction of lung NF-κB DNA binding activity, chemokine gene expression, and airway inflammation in response to RSV infection. These findings suggest that IKKβ may be a potential target for the treatment of acute or chronic inflammatory diseases of the lung.

Helicobacter pylori is the primary cause of peptic ulcer disease and an etiologic agent in the development of gastric cancer. H. pylori infection is curable with regimens of multiple antimicrobial agents, and antimicrobial resistance is a leading cause of treatment failure. The Helicobacter pylori Antimicrobial Resistance Monitoring Program (HARP) is a prospective, multicenter U.S. network that tracks national prevalence rates of H. pylori antimicrobial resistance. Of 347 clinical H. pylori isolates collected from December 1998 through 2002, 101 (29.1%) were resistant to one antimicrobial agent, and 17 (4.8%) were resistant to two or more antimicrobial agents. Eighty-seven (25.1%) isolates were resistant to metronidazole, 45 (12.9%) to clarithromycin, and 3 (0.9%) to amoxicillin. On multivariate analysis, black race was the only significant risk factor (p < 0.01, hazard ratio 2.04) for infection with a resistant H. pylori strain. Formulating pretreatment screening strategies or providing alternative therapeutic regimens for high-risk populations may be important for future clinical practice.

Introduction: The cytogenetic abnormalities and the response to induction therapy have been regarded as the most important prognostic parameters in acute myelogenous leukemia (AML) patients. Recent studies have demonstrated that internal tandem duplications and specific D-835 point mutations of the Flt3 gene, as well as the angioregulatory phenotype represent additional adverse prognostic factors. The aim of the study was to investigate possible associations between genetic abnormalities, differentiation status and angioregulatory phenotype in native human AML blasts. Method: Native AML blasts derived from consecutive patients were cultured in vitro and concentrations of angioregulatory molecules determined in the supernatants. Results: Most patients released at least two different angioregulatory mediators. Pro-angiogenic interleukin 8 (IL8) was released at relatively high levels for most patients, many of these patients showed additional release of pro-angiogenic vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). High release of anti-angiogenic IL12 was associated with high release of pro-angiogenic IL8 and VEGF. Furthermore, patients with D-835 mutations showed increased IL12 release, whereas patients with normal karyotype had decreased HGF release. Myelomonocytic differentiation was associated with IL18 release and CD34 expression with low IL12 release. Conclusion: Our results suggest that native human AML blasts have a pro-angiogenic phenotype. Although the investigated genetic abnormalities are associated with variation in the in vitro release of angioregulators, these differences are relatively small and do not quantitatively involve the most important IL8 release. It therefore seems unlikely that this phenotypic variation can explain the prognostic impact of the genetic abnormalities.

Intestinal epithelial cell function is compromised by local immune and inflammatory responses. In this study, we examined the possibility that intestinal epithelial cell injury occurs in the presence of activated inflammatory cells, such as neutrophils and macrophages, via production of reactive oxygen species (ROS). Following exposure to 50-150 microM H2O2 levels of mRNA and protein for Fas and, to a lesser degree, Fas-L were increased and intestinal epithelial cells underwent apoptosis. Treatment of H2O2-exposed cells with agonistic anti-Fas antibody, but not isotype control antibody, significantly enhanced apoptosis. Apoptosis was associated with the activation of caspase 8, while Z-IETD, an inhibitor of caspase 8, blocked apoptosis of H2O2-exposed intestinal epithelial cells. Thus, ROS induced Fas and Fas-L expression in association with intestinal epithelial cell apoptosis. These data support the hypothesis that, following exposure to oxidative stress, enterocytes are primed for cell death via Fas-mediated pathways.

Antigens which penetrate the mucosal epithelium of the intestine stimulate B lymphocytes to produce local IgA that minimizes antigen uptake across the epithelium 1 . Yet in the intestinal epithelium there are as many lymphocyte-like cells as in the spleen. Some are lymphocytes 2 but because the cells are heterogeneous a more precise term for them would be intraepithelial leukocytes (IEL). They are directly in line with macromolecules in transit across the epithelium 2,3 and increase in number in many enteric inflammatory processes 3,4 . Here Peter Ernst and his colleagues review the diverse properties of IEL that have been uncovered by isolation procedures which separate IEL from lamina proprial cells, their potential role as effectors of cell-mediated immunity and their contribution to the prevention and pathogenesis of enteric disease.

This chapter talks about some of the challenges from the various layers of the host response to which Helicobacter pylori must adapt in order to survive in the gastric niche. The outcome of a persistent infection interacting with a chronic inflammatory response is discussed in the context of the survival of the organism and the impact this may have on the host. An infection introduced into the digestive tract must immediately deal with antimicrobial factors that exist in the mucous secretions of the oral cavity and stomach. The mucus overlaying the gastric epithelium provides an "antiseptic paint" containing antibodies and antimicrobial factors such as lactoferrin and lysozyme that protect mucosal tissues. Gastric antibodies may be ineffective at limiting the infection for several reasons that are more obvious when one considers how antibodies usually confer protection. Helper T-cell (Th) responses have been viewed as primarily belonging to one of two major subsets, Th1 or Th2, on the basis of their cytokine profile. The presence of an ineffective T-cell response suggests that the selection of a very limited T-cell subset facilitates persistent infection by H. pylori. Th1 cells and the associated cell-mediated immunity may be tolerable when kept under control, but an overwhelming Th1 response, in combination with host genetics, specific strains of bacteria, and other environmental factors, could contribute to epithelial cell damage and ulcerogenesis. Since humans are believed to be a major reservoir for H. pylori, the continued survival of the bacterial species depends largely on human-to-human transmission.

Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

A highly increased risk of myelodysplasia (MDS) and acute myeloid leukaemia (AML) is well established in patients previously treated for other malignancies with alkylating agents or topoisomerase II inhibitors. More recently, single cases of acute lymphoblastic leukaemia (ALL), often presenting balanced translocations involving chromosome band 11q23, have been observed. We present two such cases with t(4;11)(q21;q23), one of whom had previously received only single-agent chemotherapy with 4-epi-doxorubicin. A review of the literature since 1992 including these two patients reveals a total of 23 cases of ALL or lymphoblastic lymphoma after chemotherapy presenting balanced translocations to 11q23. All 23 patients had previously received at least one topoisomerase II inhibitor, and in two patients 4-epi-doxorubicin had been administered as single-agent chemotherapy for breast cancer. The latency period to development of t-ALL was 24 months or less in 20 out of 22 cases. The MLL gene was found to be rearranged in 14 out of 14 cases, and in three out of six cases the breakpoint was at the telomeric part of the gene, as observed in most cases of AML following therapy with topoisomerase II inhibitors. These results indicate that patients with ALL and balanced translocations to chromosome band 11q23 following chemotherapy with topoisomerase II inhibitors in the future should be included with cases of MDS or AML in calculations of risk of leukaemia.

Approximately 50% of humanity is infected with Helicobacter pylori. This lifelong infection elicits a marked host response, including a robust gastric IgA response. However, natural infection fails to yield protective immunity. Rather than providing protection, the chronic inflammatory response associated with natural infection can contribute to tissue damage and the pathogenesis of gastroduodenal disease, including atrophic gastritis, peptic ulcer, and gastric cancer. These immune responses are attributed to a subset of helper T cells, so-called Th1 cells, that enhance cell-mediated immunity and induce damage to the gastric epithelium. Thus, it is desirable to have effective vaccines that could prevent and cure infection and that may modify the host response in a manner that prevents immune-mediated disease. Using animal models as a tool to understand the immunobiology of Helicobacter infections, several investigators have shown that effective vaccines can be developed. Thus, prophylactic and even therapeutic vaccines have been described in various animal models. The basis for the effectiveness of these vaccines appears related to their ability to alter the gastric immune response, from a homogeneous Th1 response to a mixed Th1 and Th2 response. Interestingly, immunity can occur in the absence of B cells, suggesting that novel IgA-independent mechanisms exist that confer protection against a luminal infection. Thus, H. pylori infection provides a model with which new mechanisms of immunological protection can be identified and applied to other mucosal infections.

Approximately 50% of humanity is infected with Helicobacter pylori. It is a life-long infection that elicits a marked host inflammatory response; however, natural infection fails to yield protective immunity. Rather than providing protection, the chronic inflammatory response associated with natural infection contributes to tissue damage and the pathogenesis of gastroduodenal disease, including atrophic gastritis, peptic ulcer, and gastric cancer. While bacterial factors are important triggers of inflammation, many subjects infected with strains bearing putative virulence factors remain free from disease. Recent genetic studies have implicated the host’s immune and inflammatory responses, suggesting that disease results from an interaction between bacterial and environmental factors in genetically susceptible hosts. Other digestive diseases, including celiac disease and inflammatory bowel disease, mimic this paradigm, where it appears that luminal triggers only manifest disease in subjects with the right combination of host and environmental factors. Since infection with H. pylori is relatively common, it is possible to study the impact of a specific etiologic agent on the pathogenesis of disease in humans. This approach has illuminated the complexity of the pathogenic mechanisms, but the advances achieved to date may provide some hints regarding the pathogenesis of chronic inflammatory diseases elsewhere in the digestive tract.

▪ Abstract Helicobacter pylori is a gram-negative bacterium that resides under microaerobic conditions in a neutral microenvironment between the mucus and the superficial epithelium of the stomach. From this site, it stimulates cytokine production by epithelial cells that recruit and activate immune and inflammatory cells in the underlying lamina propria, causing chronic, active gastritis. Although epidemiological evidence shows that infection generally occurs in children, the inflammatory changes progress throughout life. H. pylori has also been recognized as a pathogen that causes gastroduodenal ulcers and gastric cancer. These more severe manifestations of the infection usually occur later in life and in a minority of infected subjects. To intervene and protect those who might be at greatest risk of the more severe disease outcomes, it is of great interest to determine whether bacterial, host, or environmental factors can be used to predict these events. To date, several epidemiological studies have attempted to define the factors affecting the transmission of H. pylori and the expression of gastroduodenal disease caused by this infection. Many other laboratories have focused on identifying bacterial factors that explain the variable expression of clinical disease associated with this infection. An alternative hypothesis is that microorganisms that cause lifelong infections can ill afford to express virulence factors that directly cause disease, because the risk of losing the host is too great. Rather, we propose that gastroduodenal disease associated with H. pylori infection is predominantly a result of inappropriately regulated gastric immune responses to the infection. In this model, the interactions between the immune/inflammatory response, gastric physiology, and host repair mechanisms would dictate the disease outcome in response to infection.

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC− cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.