Nod2 influences microbial resilience and susceptibility to colitis following antibiotic exposure.

Inflammatory bowel disease (IBD) etiology involves genetic susceptibility, environmental triggers, and the gut microbiome. Antibiotic exposure is associated with IBD, both in early life and adulthood. Here, we investigated whether Nod2-deficiency influenced response of the gut microbiota to antibiotics and subsequent colitis susceptibility. Wild-type and Nod2-/- littermate mice were treated with amoxicillin as adults or neonates, and fecal samples were collected for 16S rRNA sequencing. Five weeks after antibiotic exposure, dextran sulfate sodium (DSS) colitis was induced. Antibiotic treatment altered the microbiota of adult WT and Nod2-/- mice, but recovery was delayed in Nod2-/- mice. Neonatal antibiotic treatment significantly changed the microbiota at weaning in WT and Nod2-/- littermates; however, Nod2-/- mice maintained reduced microbial diversity 14 days after cessation of antibiotics. Although treatment of adult mice did not influence susceptibility to colitis, neonatally treated Nod2-/- mice developed a more severe colitis. Moreover, the colitis phenotype was transferable through fecal transplantation into germ-free Nod2-/- recipients, and was associated with changes in intestinal T cells and the cytokine milieu following inflammation. These data demonstrate that neonatal antibiotic exposure has long-lasting influence on the microbiota and mucosal immunity, and may explain how NOD2 contributes to the risk of intestinal inflammation.

Higher Adalimumab Drug Levels are Associated with Mucosal Healing in Patients with Crohn's Disease.

BACKGROUND AND AIMS: The current approach to managing the loss of response to anti-tumour necrosis factor (TNF) agents is generally empirical. Prior studies have suggested that adalimumab levels of >4.9 µg/mL are required to achieve clinical remission. Our aim was to identify an optimal adalimumab level to achieve endoscopic healing in Crohn's disease (CD). METHODS: A cohort of 60 CD patients treated with adalimumab between 2005 and 2013 were reviewed for the study. Demographic and clinical information was obtained from chart review and patient interview. Disease activity was determined using the Harvey-Bradshaw index (HBI), ileocolonoscopy reports and C-reactive protein (CRP) levels. Clinical remission was defined as HBI <5. Endoscopic remission/mucosal healing (MH) was defined as the absence of any ulceration in all ileocolonic segments. Trough adalimumab levels and adalimumab antibody levels were tested using a liquid-phase mobility shift assay. RESULTS: Lower median CRP was significantly associated with MH 1.2mg/dl vs no MH 14.4mg/dl (p = 6.93×10(-6)). Higher adalimumab trough level was significantly associated with MH (median 14.7 µg/mL in those with MH vs 3.4 µg/mL in those without, p = 6.25×10(-5)). Higher adalimumab trough level was also significantly associated with the combined outcome of clinical and endoscopic remission (median 13.0 vs 4.8 µg/mL, p = 5.36×10(-3)). A cut-off of 8.14 µg/ml best discriminated subjects with MH from those without MH, with sensitivity and specificity of 91.4 and 76.0%, respectively (positive and negative predictive values 84.2 and 86.4%, respectively). CONCLUSIONS: Higher adalimumab levels were significantly associated with MH. This study suggests that attaining MH alone or a combined outcome of clinical and endoscopic remission is more likely to occur in those patients who achieve an adalimumab trough level of at least 8.14 µg/mL.

Management of diarrhea induced by epidermal growth factor receptor tyrosine kinase inhibitors.

Treatment for non-small-cell lung cancer (nsclc) is moving away from traditional chemotherapy toward personalized medicine. The reversible tyrosine kinase inhibitors (tkis) erlotinib and gefitinib were developed to target the epidermal growth factor receptor (egfr). Afatinib, an irreversible ErbB family blocker, was developed to block egfr (ErbB1), human epidermal growth factor receptor 2 (ErbB2), and ErbB4 signalling, and transphosphorylation of ErbB3. All of the foregoing agents are efficacious in treating nsclc, and their adverse event profile is different from that of chemotherapy. Two of the most common adverse events with egfr tkis are rash and diarrhea. Here, we focus on diarrhea. The key to successful management of diarrhea is to treat early and aggressively using patient education, diet, and antidiarrheal medications such as loperamide. We also present strategies for the effective assessment and management of egfr tki-induced diarrhea.

Characterisation of complementary and alternative medicine use and its impact on medication adherence in inflammatory bowel disease.

BACKGROUND: Complementary and alternative medicine (CAM) use among inflammatory bowel disease (IBD) patients is common. We characterised CAM utilisation and assessed its impact on medical adherence in the IBD population. AIM: To characterise CAM utilisation and assess its impact on medical adherence in the IBD population. METHODS: Inflammatory bowel disease patients recruited from an out-patient clinic at a tertiary centre were asked to complete a questionnaire on CAM utilisation, conventional IBD therapy, demographics and communication with their gastroenterologist. Adherence was measured using the self-reported Morisky scale. Demographics, clinical characteristics and self-reported adherence among CAM and non-CAM users were compared. RESULTS: We recruited prospectively 380 IBD subjects (57% Crohn's disease; 35% ulcerative colitis, and 8% indeterminate colitis). The prevalence of CAM use was 56% and did not significantly vary by type of IBD. The most common reason cited for using CAM was ineffectiveness of conventional IBD therapy (40%). The most popular form of CAM was probiotics (53%). CAM users were younger than non-CAM users at diagnosis (21.2 vs. 26.2, P < 0.0001) and more likely than non-CAM users to have a University-level education or higher (75% vs. 62% P = 0.006). There was no overall difference in adherence between CAM and non-CAM users (Morisky score: 1.0 vs. 0.9, P = 0.26). CONCLUSIONS: The use of complementary and alternative medicine is widely prevalent among IBD patients, and is more frequent among those with experience of adverse effects of conventional medications. From this cross-sectional analysis, complementary and alternative medicine use does not appear to be associated with reduced overall adherence to medical therapy.

Canadian Association of Gastroenterology Clinical Practice Guidelines: The use of tumour necrosis factor-alpha antagonist therapy in Crohn's disease.

BACKGROUND: Guidelines regarding the use of infliximab in Crohn's disease were previously published by the Canadian Association of Gastroenterology in 2004. However, recent clinical findings and drug developments warrant a review and update of these guidelines. OBJECTIVE: To review and update Canadian guidelines regarding the use of tumour necrosis factor-alpha antibody therapy in both luminal and fistulizing Crohn's disease. METHODS: A consensus group of 25 voting participants developed a series of recommendation statements that addressed pertinent clinical questions and gaps in existing knowledge. An iterative voting and feedback process was used in advance of the consensus meeting in conjunction with a systematic literature review to refine the voting statements. These statements were brought to a formal consensus meeting held in Montreal, Quebec (March 2008), wherein each statement underwent discussion, reformulation, voting and subsequent revision until group consensus was obtained (at least 80% agreement). OUTCOME: The 47 voting statements addressed three themes: induction therapy, maintenance therapy and safety issues. As a result of the iterative process, 23 statements achieved consensus and were submitted for publication. CONCLUSION: In the past five years, tumour necrosis factor-alpha antagonist therapy has become a cornerstone in the management of moderate-to-severe Crohn's disease refractory to conventional treatment algorithms. The evidentiary base supporting the use of these drugs in Crohn's disease is substantial and strengthened by results from longterm clinical and molecular studies. However, significant gaps in knowledge exist, particularly with regard to treatment failure. Confidence in the safety of these drugs is increasing, provided that therapy is administered in a clinical setting in which potential complications can be readily recognized and treated.

Review and clinical perspectives for the use of infliximab in ulcerative colitis.

Infliximab is a chimeric, monoclonal anti-tumour necrosis factor-alpha antibody. It has been previously demonstrated to be an effective treatment for patients with Crohn's disease who do not achieve the desired response with conventional treatments. Although the etiology of ulcerative colitis (UC) differs from that of Crohn's disease, randomized controlled trials have demonstrated that infliximab is also beneficial for the treatment of moderate to severe UC in patients who are either intolerant of or refractory to immunosuppressant agents or steroids, or those who are steroid-dependent. A review of the literature is followed by practical recommendations regarding infliximab that address the needs of clinicians and UC patients. Where there is a lack of evidence-based information, the expert panel provides its combined opinion derived from the members' clinical experiences.

Induction versus noninduction antiviral therapy for chronic hepatitis C virus in patients with congenital coagulation disorders: a Canadian multicentre trial.

BACKGROUND: Patients with congenital coagulation disorders and chronic hepatitis C virus (HCV) infection have multiple risk factors (ie, infection predominantly with genotype-1 HCV, long duration of the disease, HIV coinfection and male sex) for poor response to antiviral therapy. The present study compared induction therapy with interferon-alpha (IFN-alpha)-2b with standard IFN-alpha2b therapy. Pegylated IFN was not available at the time that the study was initiated. PATIENTS AND METHODS: A randomized study was performed comparing the efficacy of traditional IFN-alpha2b therapy (group A -- three million units, three times weekly for 24 to 48 weeks) and daily ribavirin (1.0 g to 1.2 g according to weight for 24 to 48 weeks), with induction IFN-alpha2b therapy (group B -- three million units, daily for eight weeks followed by the same dose administered three times a week for a further 16 to 40 weeks) and daily ribavirin (same dose as above) in IFN-naive patients with congenital coagulation disorders and chronic HCV infection. RESULTS: Between 2000 and 2003, 54 HIV-negative patients were recruited and randomly assigned to group A or B (n=27 each). Both groups were comparable in terms of age, sex, ethnicity, body mass index, baseline HCV RNA titre, viral genotype, liver fibrosis stage and type of coagulation disorder. Induction therapy did not significantly alter sustained virological response rates (group A 50%, group B 50%; P=1.0). Multiple logistic regression analysis indicated that induction therapy did not benefit individuals with difficult-to-treat infection (ie, those infected with genotypes 1 and 4, or those with high baseline viral loads). CONCLUSIONS: There was no benefit with induction antiviral therapy for HCV infection in individuals with congenital coagulation disorders.

Increased epithelial uptake of protein antigens in the ileum of Crohn's disease mediated by tumour necrosis factor alpha.

BACKGROUND AND AIMS: The exact nature of the epithelial barrier defect in Crohn's disease remains to be elucidated. Previously we showed increased permeability to proteins in ileal Crohn's disease. Our aims were to study if this barrier defect (a) involves endocytotic uptake of antigens and (b) is related to low grade inflammation not detectable by histology. METHODS: Macroscopically normal segments of distal ileum of Crohn's disease patients (n = 10) were subgrouped into non-inflamed (histologically unaffected) and slightly inflamed tissues and studied in Ussing chambers, with normal ileal specimens from colon cancer patients (n = 9) as controls. Endocytotic uptake into enterocytes of the protein antigen horseradish peroxidase was assessed by measuring the area of horseradish peroxidase containing endosomes in electron photomicrographs. Mucosal tumour necrosis factor alpha (TNF-alpha) mRNA was quantified using real time polymerase chain reaction. For comparison, the effects of low doses of TNF-alpha on endosomal uptake of horseradish peroxidase were studied in cultured T84 cells grown on filter supports. RESULTS: The area of horseradish peroxidase containing endosomes was increased (p<0.001) in enterocytes of non-inflamed ileum of Crohn's disease (2.8 (0.7) mum(2)/300 mum(2)) compared with control ileum (0.6 (0.06)). In non-inflamed mucosa, a significant association between endosomal uptake and mucosal expression of TNF-alpha mRNA (p = 0.03) was found. Low concentrations of TNF-alpha (0.25-1.0 ng/ml) enhanced the endosomal uptake of horseradish peroxidase in polarised T84 cells, without affecting transepithelial electrical resistance. CONCLUSIONS: Our findings suggest increased endosomal uptake of antigens in ileal Crohn's disease that may be mediated by TNF-alpha. These data highlight the transcellular route of antigen uptake in barrier dysfunction and implicate the interaction between epithelial cells and the innate immune system in the development of mucosal inflammation.

Detection of species-specific helicobacter ribosomal DNA in intestinal biopsy samples from a population-based cohort of patients with ulcerative colitis.

The inflammatory bowel diseases are considered an abnormal host immune response to an environmental stimulus. Evidence suggests a role for intestinal bacteria in initiating and/or providing an ongoing stimulus for inflammation in inflammatory bowel disease. Helicobacter pylori is the major cause of active chronic gastritis and peptic ulcers in humans and has been linked to gastric carcinoma and lymphoma. Studies in various animal models, particularly mice, have identified enterohepatic Helicobacter species that are capable of causing hepatitis and enterocolitis. We hypothesize that Helicobacter species may have a role in maintaining inflammation in humans with inflammatory bowel disease. In order to investigate this, biopsy specimens were obtained from patients with and without inflammatory bowel disease. DNA was extracted from the tissues and subjected to PCR with primers designed to detect the ribosomal DNA of members of the Helicobacter species. DNA from six biopsy samples from 60 inflammatory bowel disease patients tested positive. This included 5 of 33 ulcerative colitis patients that were positive compared to 0 of 29 age-matched controls (P < 0.04). Sequencing of the bands produced by PCR amplification revealed >or=99% homology with H. pylori. These results indicate that a member of the Helicobacter species may be involved in some cases of ulcerative colitis.

Defining the roles of perforin, Fas/FasL, and tumour necrosis factor alpha in T cell induced mucosal damage in the mouse intestine.

BACKGROUND AND AIMS: Mucosal flattening and epithelial cell apoptosis are typical features of T cell induced inflammatory diseases of the bowel, such as coeliac disease and graft versus host disease. Mice injected with a T cell activating anti-CD3 antibody develop a severe diarrhoeal illness. We describe the histological features of this enteropathy and define the effector mechanisms involved in T cell induced mucosal injury in this in vivo model. METHODS: Wild-type and genetically modified mice were injected with the anti-CD3 antibody 3C11 (50 microg). Changes in the murine intestine were characterised by light microscopy analysis and terminal uridine nick-end labelling (TUNEL) assay. The role of perforin, Fas/Fas ligand (FasL), tumour necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) in T cell induced mucosal damage was assessed using selected immunodeficient mouse strains. RESULTS: T cell activation caused severe damage, including small intestinal mucosal flattening and apoptosis of crypt epithelial cells. Mucosal damage was unaltered in anti-CD3 treated mice lacking IFN-gamma, Fas, or TNF-alpha receptors. In mice lacking TNF-alpha receptors and Fas (TNF-R1xR2 lpr/lpr strain), enterocyte apoptosis was diminished but there was no significant reduction in tissue damage. Apoptosis and mucosal injury were significantly reduced in perforin knockout mice. Abrogation of both FasL and perforin (perforin KOxgld mice) further significantly reduced tissue damage and apoptotic bodies. CONCLUSIONS: T cell induced mucosal injury is mediated by the combined effect of multiple pathways but predominantly by perforin. The redundancy of the mechanisms of tissue damage will have significant impact on therapeutic strategies aimed at specific and targeted inhibition of inflammatory processes.

TH1/TH2,3 imbalance due to cytokine-producing NK, gammadelta T and NK-gammadelta T cells in murine pregnancy decidua in success or failure of pregnancy.

PROBLEM: Recurrent spontaneous abortion in DBA/2-mated CBA/J mice has been attributed to the production of Th1 cytokines (tumor necrosis factor [TNF]-alpha and interferon [IFN]-gamma) by asialoGM1+ natural killer (NK) cells and Vgamma1.1delta6.3+ T cells that infiltrate decidua by day 6.5, during the peri-implantation period. Abortions can be prevented by a second population of Vgamma1.1delta6.3 cells, which infiltrate on day 8.5 of gestation, and produce the Th2 cytokine interleukin (IL)-10 and Th3 cytokine transforming growth factor (TGF)-beta2. In low abortion rate immunocompetent mice, most of the TGF-beta2 is derived from gammadelta T cells. However, TGF-beta2-producing cells are present in the decidua of pregnant severe combined immune deficient (SCID) mice, which lack gammadelta T cells. METHODS: The cells in day 13.5 decidua of CBA x DBA/2 matings and SCID x SCID matings were identified using flow cytometry and combined surface staining for gammadelta and/or asialoGM1, and intracellular cytokine staining for TNF-alpha, IFN-gamma, and TGF-beta2,3. RESULTS: TGF-beta2 and TNF-alpha were found in asialoGM1+ NK cells in SCID mouse decidua. In CBA x DBA/2 mated mice, two major and one minor subsets of cytokine-positive cells were identified: -gammadelta-only T cells, double positive asialoGM1+ gammadelta+ (NK-gammadelta T) cells, and a small number of asialoGM1 +gammadelta- NK-only cells. The NK-only and NK-gammadelta T subsets showed a greater Th1/Th2,3 pattern of intracellular staining compared with the gammadelta-only subset. In the CBA x DBA/2 and SCID x SCID systems, Th1/Th2,3 ratios could not predict actual observed abortion rates but did correlate with susceptibility to abortions (if exposed to an additional stimulus such as stress). The known effect of in vivo administration of anti-asialoGM1 antibody on abortion rates within groups of mice exposed to such stresses could also be predicted. CONCLUSION: gammadelta+ cells in decidua (e.g. Vgamma1+ cells which can recognize trophoblasts) differ based on the presence or absence of the NK marker-asialo-GM1. NK-gammadelta T cells may be quite important in the Th1 response in early pregnancy that predisposes to abortions in CBA x DBA/2 matings, whereas gammadelta T-only cells appear to be protective. In pregnant SCID mice, the TNF-alpha+/TGF-beta2+ NK population is greatly expanded. An activating stimulus (such as stress or endotoxin) appears to be as important in triggering abortions, as is the Th1/Th2,3 ratio at the feto maternal interface.

Murine T cell determination of pregnancy outcome.

At the fetomaternal interface, maternal effector cells come in intimate contact with fetal trophoblast cells which express paternal antigens. Failure of fetal trophoblast cells to activate maternal Th1 immune responses has been attributed in part to the absence of classical Class I and Class II major histocompatibilty complex (MHC) antigen expression and elaboration of factors which reduce TcR expression and shift any immune responses which may occur to Th2. Classical TcR alphabeta(+) T cells have not been found to be able to respond to trophoblasts. Recently, TcR gammadelta(+) T cells have been characterized in the low-abortion-rate pregnant C57Bl/10 mouse decidua, and the Vgamma1(+) subset may be able to respond to trophoblasts in a non-MHC-dependent manner. Trophoblast-recognizing T cells with Vgamma1 receptors are also present in the decidua of CBA/J mice pregnant by DBA/2, an abortion-prone mating combination. To test the role of the Vgamma1 subset of decidual gammadelta T cells in abortion-prone pregnancies, we altered this subset by injecting monoclonal anti-Vgamma1.1 antibody on gestation day 5.5, 1 day after implantation. This reduced detectability of a Vgammadelta subset producing TNF-alpha and reduced the abortion rate. Anti-Vgamma2, which reacts with a similar proportion of decidual gammadelta T cells as anti-Vgamma1.1, failed to prevent abortions. Vdelta6.3(+) cells are prominent at the fetomaternal interface, and anti-Vdelta6 antibody injected on day 5.5 prevented abortions. TGF-beta2(+) gammadelta cells first appear on day 8.5 of pregnancy; anti-Vgamma1.1 antibody injection on day 8.5 depleted these cells and boosted abortions; anti-Vdelta6.3 given on day 8.5 boosted abortions to the same level. These results suggest that two populations of Vgamma1.1(+)delta6.3(+) T cells may arise in the decidua: an early population that is Th1, abortogenic, and present during the time of implantation, and a Th2/3 cell subset that is present in the decidua later during pregnancy and which is pregnancy-protective.

Replication-defective adenovirus infection reduces Helicobacter felis colonization in the mouse in a gamma interferon- and interleukin-12-dependent manner.

Helicobacter infection leads to chronic inflammation of the stomach. Although the infection persists in spite of an immune response, animal studies have shown that adjuvant-based oral vaccines can protect against infection and even eliminate established infection. These vaccines are thought to induce a Th2 immune response, counterbalancing the Th1 response seen with natural infections. As a prelude to using adenovirus vectors carrying cytokine genes to modulate the immune response to established Helicobacter felis infection, we first examined the effect of the replication-defective adenovirus (RDA) vector itself. C57BL/6 mice chronically infected with H. felis (8 to 10 weeks) received intramuscular injections of RDA. The effect of RDA on the severity of H. felis colonization and the degree of gastric inflammation was assessed 2 weeks later. RDA caused a significant decrease in H. felis colonization without significantly altering the associated inflammation. RDA did not alter the H. felis-specific immunoglobulin G1 (IgG1), IgG2a, and IgA responses in the serum but was associated with an increase in gamma interferon (IFN-gamma)-producing CD8(+) spleen cells. To determine if IFN-gamma or Th1 cytokines were involved in the response to RDA, we examined RDA treatment of H. felis infection in mice lacking either IFN-gamma or interleukin-12 (IL-12). RDA failed to alter H. felis colonization in either of these two mouse strains. Thus, viral infection of mice chronically infected with H. felis led to a significant decrease in H. felis colonization in an IFN-gamma- and IL-12-dependent manner. These results demonstrate that Th1 responses associated with systemic viral infection can influence an established H. felis infection.

Immunomodulation of helicobacter infection.

Helicobacter pylori leads to a chronic infection in humans that is associated with gastric inflammation and a vigorous immune response. Despite the humoral and cellular responses that can be detected in both human and animal models of helicobacter infection, the immune response fails to eliminate the organism. Eradication failure may be due to the niche in which H pylori confines itself, well away from direct contact with elements of the immune system. Alternatively, the general tendency of the intestinal immune response to down- regulate reactivity to noninvasive luminal bacteria also may contribute to the failure to eliminate helicobacter infection. Results of vaccine studies in mouse models indicate that modulating the helper T cell response from a T helper cell type 1 to a T helper cell type 2 response likely is required for the prevention and elimination of helicobacter infection. Understanding the mechanisms by which the immune response controls bacterial infections will allow for the design of novel strategies of immune modulation and the development of vaccines for both the treatment and prevention of H pylori.

Characterization of enteric functional changes evoked by in vivo anti-CD3 T cell activation.

Specific in vivo T cell activation initiated by treatment with anti-CD3 antibodies leads to diarrhea and structural damage of the intestinal mucosa. In this study, the effect of T cell-induced mucosal damage on jejunal epithelial ion transport, muscle contractility, and neuronal ACh release was assessed in Ussing chambers, organ baths, and a specialized perfusion apparatus, respectively. Time-matched control mice received hamster serum containing irrelevant antibodies. Jejunal segments from anti-CD3-treated mice displayed a significantly elevated epithelial baseline short-circuit current (which indicates increased ion transport) and a concomitant reduction in responsiveness to prosecretory stimuli (nerve stimulation, carbachol, and forskolin). Longitudinal smooth muscle displayed altered spontaneous contractile activity, length-tension relationships, and carbachol-stimulated contraction in tissues excised from mice 20 and 40 h posttreatment. Anti-CD3 treatment did not affect stimulated ACh release from myenteric plexus neurons. We conclude that specific T cell activation via anti-CD3 antibody results in dramatic alterations in jejunal epithelial and smooth muscle function. Such T cell-induced changes in intestinal function may contribute to the symptomatology of T cell-mediated enteropathies, including graft-versus-host disease, celiac disease, and idiopathic inflammatory bowel disease.

T lymphocyte-dependent and -independent intestinal smooth muscle dysfunction in the T. spiralis-infected mouse.

We examined the profile of increased intestinal muscle contractility after primary infection with Trichinella spiralis in the mouse, correlating it with parasite expulsion. We also examined the extent to which the changes in muscle contraction were T lymphocyte dependent, by infecting athymic and SCID mice. Infection was accompanied by increased tension development by intestinal muscle. Two components of this response were identified, a rapid peak increase in tension generation observed on day 6 postinfection, and a smaller but sustained increase in tension evident thereafter in euthymic BALB/c mice. The peak muscle response was significantly delayed in infected athymic and SCID mice, along with a corresponding reduction in the magnitude of the sustained component. These changes were accompanied by reduced parasite expulsion in athymic and SCID mice, compared with euthymic mice. Reconstitution of T cell function in athymic mice restored both the acute and sustained profile of muscle contraction seen in euthymic mice, and this was accompanied by faster expulsion of the worms. These results identify T cell-dependent and -independent components of the muscle response to nematode infection in the mouse and suggest that the onset of the peak contractile response, as well as the magnitude of the sustained muscle response, contributes to parasite eviction from the gut.

Protection by CD4 or CD8 T cells against pulmonary Mycobacterium bovis bacillus Calmette-Guérin infection.

Mice deficient in CD8 T cells demonstrated levels of Th1 cytokines and granulomatous responses in the lungs very similar to those demonstrated by normal control mice and were fully capable of controlling pulmonary mycobacterial infection by Mycobacterium bovis BCG as assessed at day 37 postinfection. In comparison, mice deficient in CD4 T cells had similar levels of interleukin-12 (IL-12) and tumor necrosis factor alpha but lower levels of gamma interferon in the lungs and were still able to mount tissue granulomatous responses and control pulmonary mycobacterial infection. In contrast, IL-12(-/-) mice with impaired CD4 and CD8 T-cell responses had a markedly weakened control of infection, whereas SCID mice deficient in all T cells succumbed to such pulmonary mycobacterial infections.

Compartmentalized transgene expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mouse lung enhances allergic airways inflammation.

To investigate the role of GM-CSF in asthmatic airways inflammation, we have targeted GM-CSF transgene to the airway cells in a mouse model of ovalbumin (OVA)-induced allergic airways inflammation, a model in which there is marked induction of endogenous IL-5 and IL-4 but not GM-CSF. Following intranasal delivery of a replication-deficient adenoviral gene transfer vector (Ad), transgene expression was found localized primarily to the respiratory epithelial cells. Intranasal delivery of 0.03 x 10(9) plaque-forming units (PFU) of AdGM-CSF into naive BALB/c mice resulted in prolonged and compartmentalized release of GM-CSF transgene protein with a peak concentration of approximately 80 pg/ml detected in bronchoalveolar lavage fluid (BALF) at day 7, but little in serum. These levels of local GM-CSF expression per se resulted in no eosinophilia and only a minimum of tissue inflammatory responses in the lung of naive mice, similar to those induced by the control vector. However, such GM-CSF expression in the airways of OVA-sensitized mice resulted in a much greater and sustained accumulation of various inflammatory cell types, most noticeably eosinophils, both in BALF and airway tissues for 15-21 days post-OVA aerosol challenge, at which times airways inflammation had largely resolved in control mice. While the levels of IL-5 and IL-4 in BALF and the rate of eosinophil apoptosis were found similar between different treatments, there was an increased number of proliferative leucocytes in the lung receiving GM-CSF gene transfer. Our results thus provide direct experimental evidence that GM-CSF can significantly contribute to the development of allergic airways inflammation through potentiating and prolonging inflammatory infiltration induced by cytokines such as IL-5 and IL-4.

Lack of both types 1 and 2 cytokines, tissue inflammatory responses, and immune protection during pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin in IL-12-deficient mice.

Understanding of key cytokines and the nature of protective immune responses in pulmonary mycobacterial diseases remains a task of paramount importance. In this study, both wild-type (wt) and IL-12-deficient (IL-12(-/-)) mice were infected by airways inoculation of live Mycobacterium bovis bacille Calmette-Guérin (BCG). The type 1 cytokines IL-12, IFN-gamma, and TNF-alpha, but not the type 2 cytokines IL-4 and granulocyte macrophage (GM)-CSF, markedly increased in the lung and peripheral blood of wt mice postinfection, which resulted in the development of intense granulomatous responses and the effective control of mycobacterial infection in the lung. In contrast, IL-12(-/-) mice demonstrated a lack of both types 1 and 2 cytokines in the lung and blood and a severely impaired tissue immune-inflammatory response lacking not only macrophages and neutrophils but CD4 and CD8 T cells and NK cells in the lung throughout the entire course of study. Total lung mononuclear cells isolated from these mice, in contrast to wt mice, had an impaired recall immune response to Ag challenge in vitro. These impaired responses resulted in an uncontrolled local growth and systemic spread of bacilli. Our findings reveal that IL-12 plays an irreplaceable role in the initiation of Th1 responses, and the loss of its function cannot be compensated for by alternative mechanisms in the lung. This cytokine, together with IFN-gamma and TNF-alpha, and granulomatous inflammation are critically required for the effective control of pulmonary mycobacterial infection. Our results also indicate that the absence of type 1 cytokines does not necessarily favor a Th2 response.