Human leukocyte antigen (HLA) and single nucleotide polymorphisms (SNPs) tumor necrosis factor (TNF)-alpha-238 and-308 as genetic markers of susceptibility to psoriasis and severity of the disease in a long-term follow-up Brazilian study

Background The strongest genetic marker for psoriasis is Cw*06. Polymorphisms in the tumor necrosis factor (TNF)-alpha promoter region, especially replacement of guanine with adenine in positions -238 and -308 are related to higher TNF-alpha production and higher risk for psoriasis in Caucasoid populations, not found in Asians. We performed a case-control study of 69 patients with psoriasis type I and 70 controls, characterized clinical progression along 10-years of follow-up in mild or severe disease and determined HLA class I, II, and TNF single nucleotide polymorphisms (SNPs) -238 and -308 polymorphisms to demonstrate whether these polymorphisms may be genetic risk for susceptibility to psoriasis or severity of the disease in Brazilians. Methods Polymorphisms were identified using PCR/SSP. Alleles, genotypes, and haplotypes frequencies were compared using Fisher`s test. Results More severe disease was found in male patients. It may be suggested that alleles B*37, Cw*06, Cw*12, and DRB1*07 were associated with severe disease course, while B*57 with mild disease. No statistical difference was found between the patients and controls regarding polymorphisms frequencies in TNF SNPs. This study pointed to a higher TNF-238 G/G genotype frequency (OR: 3.21; CI: 1.06-9.71; P = 0.04) in the group with severe disease. Conclusions Polymorphisms in the TNF-alpha SNPs do not seem to be a more important genetic risk factor for psoriasis than the already known Cw*06 in Brazilian patients, but these markers may be related to clinical manifestations.

Effects of nitric oxide on neutrophil influx depends on the tissue: role of leukotriene B(4) and adhesion molecules

Background and purpose: We investigated the effect of nitric oxide synthase (NOS) inhibition on polymorphonuclear cell (PMN) influx in zymosan or lipopolysaccharide (LPS)-induced arthritis and peritonitis. Experimental approach: Wistar rats received intra-articular (i.art.) zymosan (30-1000 mu g) or LPS (1-10 mu g). Swiss C57/Bl6 mice genetically deficient in intercellular adhesion molecule-1 (ICAM-1(-/-)) or in beta(2)-integrin (beta(2)-integrin(-/-)) received zymosan either i.art. or i.p. PMN counts, leukotriene B(4) (LTB(4)), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) levels were measured in joint and peritoneal exudates. Groups received the NOS inhibitors N(G)-nitro-L-arginine methyl ester (LN), nitro-L-arginine, N-[3-(aminomemethyl) benzyl] acetamide or aminoguanidine, prior to zymosan or LPS, given i.p. or s.c. in the arthritis and peritonitis experiments respectively. A group of rats received LN locally (i.art. or i.p.), 30 min prior to 1 mg zymosan i.art. Key results: Systemic or local NOS inhibition significantly prevented PMN migration in arthritis while increasing it in peritonitis, regardless of stimuli, concentration of NOS inhibitors and species. NOS inhibition did not alter TNF-alpha and IL-10 but decreased LTB(4) in zymosan-induced arthritis. LN administration significantly inhibited PMN influx into the joints of ICAM-1(-/-) and beta(2)-integrin(-/-) mice with zymosan-arthritis, while not altering PMN influx into the peritoneum of mice with zymosan-peritonitis. Conclusions and implications: Nitric oxide has a dual modulatory role on PMN influx into joint and peritoneal cavities that is stimulus-and species-independent. Differences in local release of LTB(4) and in expression of ICAM-1 and beta(2)-integrin account for this dual role of NO on PMN migration.

Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases

Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1 alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CK beta 8/CCL23, and osteoprotegerin, which were significantly higher than in control. Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Local profile of cytokines and nitric oxide in patients with bacterial vaginosis and cervical intraepithelial neoplasia

Objective: To evaluate the local immune response in patients with bacterial vaginosis (BV) and cervical intraepithelial neoplasia (CIN), as assessed by cytokine and nitric oxide (NO) concentrations. Study design: Patients attending for routine gynaecological examination were prospectively enrolled in groups: BV (n = 25) diagnosed by clinical criteria, CIN graded I to III (n = 35, 6 CIN 1, 8 CIN 11 and 21 CIN 111) by histological analysis, and controls (n = 15) without clinical and cytological findings. Randomly selected patients within CIN group at grades 11 or III (n = 15) were re-evaluated at 60 days after surgical treatment. Endocervical (EC) and vaginal secretion samples were collected by cytobrush and the levels of cytokines (ELISA) and NO metabolite (Griess reaction) were assayed. Results: NO was assessed in all subjects, and cytokines in all controls, 15 BV and 30 CIN patients. Interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and nitrite levels were higher in EC than in vaginal secretions in BV and CIN groups. In CIN group, IL-8, IL-10 and nitrite concentrations were greater in EC and/or vaginal secretions than in BV or controls. Surgical treatment reduced IL-8 levels in EC and vaginal secretions. Conclusion: A similar local immune profile was found in BV and CIN groups. The increased local production of IL-8, IL-10 and NO in CIN suggests a role for these mediators in the immune response against tumour or tumour development. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Detrimental role of endogenous nitric oxide in host defence against Sporothrix schenckii

We earlier demonstrated that nitric oxide (NO) is a fungicidal molecule against Sporothrix schenckii in vitro. In the present study we used mice deficient in inducible nitric oxide synthase (iNOS(-/-)) and C57BL/6 wild-type (WT) mice treated with N omega-nitro-arginine (Nitro-Arg-treated mice), an NOS inhibitor, both defective in the production of reactive nitrogen intermediates, to investigate the role of endogenous NO during systemic sporotrichosis. When inoculated with yeast cells of S. schenckii, WT mice presented T-cell suppression and high tissue fungal dissemination, succumbing to infection. Furthermore, susceptibility of mice seems to be related to apoptosis and high interleukin-10 and tumour necrosis factor-alpha production by spleen cells. In addition, fungicidal activity and NO production by interferon-gamma (IFN-gamma) and lipopolysaccharide-activated macrophages from WT mice were abolished after fungal infection. Strikingly, iNOS(-/-) and Nitro-Arg-treated mice presented fungal resistance, controlling fungal load in tissues and restoring T-cell activity, as well as producing high amounts of IFN-gamma Interestingly, macrophages from these groups of mice presented fungicidal activity after in vitro stimulation with higher doses of IFN-gamma. Herein, these results suggest that although NO was an essential mediator to the in vitro killing of S. schenckii by macrophages, the activation of NO system in vivo contributes to the immunosuppression and cytokine balance during early phases of infection with S. schenckii.

The Potential Role of Suppressors of Cytokine Signaling in the Attenuation of Inflammatory Reaction and Alveolar Bone Loss Associated with Apical Periodontitis

Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by suppressors of cytokine signaling (SOCS), which down-regulate signal transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines tumor necrosis factor alpha (TNF-alpha); interleukin (IL)-10 and RANKL; and SOCS-1, -2, and -3 by real-time polymerase chain reaction in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significantly higher SOCS-1, -2, and -3, TNF-alpha, IL-10, and RANKL messenger RNA levels when compared with healthy controls. Significant positive correlations were found between SOCS1 and IL-10 and between SOCS3 and IL-10. Significant inverse correlations were observed between SOCS1 and TNF-alpha, SOCS1 and RANKL, and SOCS3 and TNF-alpha. Increased SOCS-1, -2, and -3 messenger RNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may play a role in the dynamics of periapical granulomas development. (J Endod 2008;34:1480-1484)

CD40 and CD86 upregulation with divergent CMRF44 expression on blood dendritic cells in inflammatory bowel diseases

OBJECTIVE: Dendritic cells (DC) are the only antigen-presenting cells that can activate naive T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease. METHODS: Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c(+) and CD11c(-) DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin(-) = CD3(-), CD19(-), CD14(-), CD16(-)) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86(+) and CD40(+)lin(-) cells. RESULTS: Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c(+) and CD11c(-) DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86(+) and CD40(+) DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44(+) antigen was mainly associated with CD11c(+) DC, and in UC was inversely related to the Clinical Activity Index (r = -0.69, p = 0.0002). CONCLUSIONS: There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86(+) and CD40(+) DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation. (Am J Gastroenterol 2001;96:2946-2956. (C) 2001 by Am. Coll. of Gastroenterology).

Role of cytokine gene polymorphisms in acute rejection and renal impairment after liver transplantation

Although immunosuppressive regimens are effective, rejection occurs in up to 50% of patients after orthotopic liver transplantation (OLT), and there is concern about side effects from long-term therapy. Knowledge of clinical and immunogenetic variables may allow tailoring of immunosuppressive therapy to patients according to their potential risks. We studied the association between transforming growth factor-beta, interleukin-10, and tumor necrosis factor alpha (TNF-alpha) gene polymorphisms and graft rejection and renal impairment in 121 white liver transplant recipients. Clinical variables were collected retrospectively, and creatinine clearance was estimated using the formula of Cockcroft and Gault. Biallelic polymorphisms were detected using polymerase chain reaction-based methods. Thirty-seven of 121 patients (30.6%) developed at least 1 episode of rejection. Multivariate analysis showed that Child-Pugh score (P =.001), immune-mediated liver disease (P =.018), normal pre-OLT creatinine clearance (P =.037), and fewer HLA class 1 mismatches (P =.038) were independently associated with rejection, Renal impairment occurred in 80% of patients and was moderate or severe in 39%, Clinical variables independently associated with renal impairment were female sex (P =.001), pre-OLT renal dysfunction (P =.0001), and a diagnosis of viral hepatitis (P =.0008), There was a significant difference in the frequency of TNF-alpha -308 alleles among the primary liver diseases. After adjustment for potential confounders and a Bonferroni correction, the association between the TNF-alpha -308 polymorphism and graft rejection approached significance (P =.06). Recipient cytokine genotypes do not have a major independent role in graft rejection or renal impairment after OLT, Additional studies of immunogenetic factors require analysis of large numbers of patients with appropriate phenotypic information to avoid population stratification, which may lead to inappropriate conclusions.

Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum

Background The ability of T cells, acting independently of antibodies, to control malaria parasite growth in people has not been defined. If such cell-mediated immunity was shown to be effective, an additional vaccine strategy could be pursued. Our aim was to ascertain whether or not development of cell-mediated immunity to Plasmodium falciparum blood-stage infection could be induced in human beings by exposure to malaria parasites in very low density. Methods We enrolled five volunteers from the staff at our research institute who had never had malaria. We used a cryopreserved inoculum of red cells infected with P falciparum strain 3D7 to give them repeated subclinical infections of malaria that we then cured early with drugs, to induce cell-mediated immune responses. We tested for development of immunity by measurement of parasite concentrations in the blood of volunteers by PCR of the multicopy gene STEVOR and by following up the volunteers clinically, and by measuring antibody and cellular immune responses to the parasite. Findings After challenge and a extended period without drug cure, volunteers were protected against malaria as indicated by absence of parasites or parasite DNA in the blood, and absence of clinical symptoms. Immunity was characterised by absence of detectable antibodies that bind the parasite or infected red cells, but by the presence of a proliferative T-cell response, involving CD4+ and CD8+ T cells, a cytokine response, consisting of interferon gamma but not interleukin 4 or interleukin 10, induction of high concentrations of nitric oxide synthase activity in peripheral blood mononuclear cells, and a drop in the number of peripheral natural killer T cells. Interpretation People can be protected against the erythrocytic stage of malaria by a strong cell-mediated immune response, in the absence of detectable parasite-specific antibodies, suggesting an additional strategy for development of a malaria vaccine.

Evidence for a sub-morphological inflammatory process in the liver in haemochromatosis

Background/Aims: The role of cytokines in hepatic injury has been examined for many liver diseases however little is known of the cytokine involvement in haemochromatosis. The aim of the current study was to examine the hepatic gene expression of potential proinflammatory and profibrogenic cytokines in haemochromatosis. Methods: Interferon-gamma, interleukin-10, transforming growth factor-beta(1) and tumor necrosis factor-alpha mRNA expression was assessed in liver tissue from 20 haemochromatosis patients, eight controls and eight chronic hepatitis C patients. To assess the immunophenotype of the inflammatory infiltrate in haemochromatosis, liver sections were subjected to immunohistochemistry using markers for macrophages (CD68, HAM56, MAC387) or T cells (CD3 and CD45RO). Results: Interferon-gamma mRNA was increased in both haemochromatosis (0.29+/-0.08%, P=0.01) and hepatitis C patients (1.02+/-0.32%, P=0.03) compared to controls (0.04+/-0.01%). Interleukin-10 mRNA was significantly decreased in both haemochromatosis and hepatitis C patients (0.01+/-0.003%, P=0.008 and 0.03+/-0.015%, P=0.02, respectively) compared to controls (0.12+/-0.01%). CD3 positive T-cell number was significantly correlated with increasing hepatic iron concentration (r=0.56, P=0.03). Conclusions: This study has demonstrated a distinct pattern of cytokine gene expression in haemochromatosis, which resembles that of inflammatory conditions such as chronic hepatitis C. These factors may play a role in the development of iron-induced hepatic fibrosis in haemochromatosis. (C) 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

Oral coinfection can stress peripheral lymphocyte to inflammatory activity in leprosy

INTRODUCTION: This study evaluated the intracellular profile of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10) and interferon-γ (IFN-γ) in peripheral blood mononuclear cells (PBMCs) from leprosy patients based on oral infections presence to determine whether these coinfections could be associated with pro-inflammatory activity in leprosy. METHODS: Leprosy patients regardless of clinical form and specific leprosy treatment (n=38) were divided into two groups: Group I - leprosy patients with oral infections (n=19), and Group II - leprosy patients without oral infections (n=19). Non-leprosy patients presenting oral infections were assigned to the control Group (n=10). Intracellular IL-2, IL-4, IL-10 and IFN-γ production was evaluated by flow cytometry (FACS) before and 7 days after controlling the oral infection in the Group I, before and 7 days after dental prophylaxis in the Group II, and during oral infection process in control Group. RESULTS: Low percentages of CD3+ lymphocytes bearing IL-2, IL-10 and IFN-γ were observed in the Group I and Group II at baseline and 7 days after therapy or prophylaxis compared to controls. Group I showed reduced percentages of IL-4 at baseline and 7 days after therapy compared to controls, or at baseline of Group II, and the Group II showed reduced percentages of CD3+ cells bearing IL-4 compared to control. An increase of the percentages of CD3+cells bearing IL-4 was observed in the Group I after the oral infections treatment. CONCLUSIONS: The occurrence of oral infections favors the intracellular cytokines expression and, probably, the inflammatory reaction operating as a stimulatory signal triggering the leprosy reactions.

Severe visceral leishmaniasis in children: the relationship between cytokine patterns and clinical features

Introduction The relationship between severe clinical manifestations of visceral leishmaniasis (VL) and immune response profiles has not yet been clarified, despite numerous studies on the subject. This study aimed to investigate the relationship between cytokine profiles and the presence of immunological markers associated with clinical manifestations and, particularly, signs of severity, as defined in a protocol drafted by the Ministry of Health (Brazil). Methods We conducted a prospective, descriptive study between May 2008 and December 2009. This study was based on an assessment of all pediatric patients with VL who were observed in a reference hospital in Maranhão. Results Among 27 children, 55.5% presented with more than one sign of severity or warning sign. Patients without signs of severity or warning signs and patients with only one warning sign had the highest interferon-gamma (IFN-γ) levels, although their interleukin 10 (IL-10) levels were also elevated. In contrast, patients with the features of severe disease had the lowest IFN-γ levels. Three patients who presented with more than two signs of severe disease died; these patients had undetectable interleukin 2 (IL-2) and IFN-γ levels and low IL-10 levels, which varied between 0 and 36.8pg/mL. Conclusions Our results showed that disease severity was associated with low IFN-γ levels and elevated IL-10 levels. However, further studies with larger samples are needed to better characterize the relationship between disease severity and cytokine levels, with the aim of identifying immunological markers of active-disease severity.

An alteration in the levels of populations of CD4+ Treg is in part responsible for altered cytokine production by cells of aged mice which follows injection with a fetal liver extract.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (>20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were >4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND:: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18. METHODS:: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS:: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS:: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations. (Inflamm Bowel Dis 2010).