A solid-phase approach for the synthesis of muramyl dipeptide conjugates for detection of NOD2.

Proper recognition of invading pathogens and prompt initiation of host defense mechanisms are instrumental for the maintenance of organismal homeostasis. Nucleotide-binding oligomerization domain-containing (NOD)-like receptors (NLRs) serve as pathogen-recognition receptors that specifically recognize bacterial peptidoglycans. NOD2 detects muramyl dipeptide (MDP) through its carboxy-terminal leucine rich repeats (LRRs), which enables the activation of downstream inflammatory signaling. Synthesis of MDP conjugates based on solution phase chemistry have been previously reported. Our solid phase approach synthetically provides a facile approach for the conjugation of biological probes to MDP, with the advantage of minimal functional/protecting group manipulation, and reduction in the laborious process of intermediate purification and isolation. MDP conjugates that we generated using solid phase synthesis allow detection of NOD2 is cell lysates and NOD2 subcellular localization by immunofluorescence microscopy. MDP-PEG6-Cyanine5.5 conjugate selectively colocalized with WT NOD2 but not NOD2 variant found in Crohn's disease, which lacks carboxy-terminal end and cannot bind MDP. Overall, these data indicate that distinct solid phase-produced MDP conjugates can be used to examine biological properties of NOD2 and could potentially facilitate further development of NOD2 targeting agents.

Brief Report: Increased Cotinine Concentrations are Associated With Reduced Expression of Cathelicidin (LL-37) and NOD-2 in Alveolar Macrophages of PLWH Who Smoke.

There is a strong link between cigarette smoking and pulmonary complications among people living with HIV. However, the effects of smoking on the local lung immune environment in this population remain unclear. Bronchoalveolar lavage and saliva were collected from HIV-infected smokers involved in a prospective study investigating alveolar macrophage expression of host defense molecules. Salivary cotinine concentrations were inversely related to expression of the immune cell receptor nucleotide-binding oligomerization domain-2 and the cathelicidin antimicrobial peptide LL-37. The negative correlation between salivary cotinine and LL-37 was particularly strong. Our study provides insight into how nicotine may adversely affect lung innate immunity in HIV.

Sphingosine-1-phosphate signal transducer and activator of transcription 3 signaling pathway contributes to baicalein-mediated inhibition of dextran sulfate sodium-induced experimental colitis in mice.

BACKGROUND: Baicalein has been shown to have anti-inflammatory and anti-tumor activities. However, the mechanisms underlying its anti-inflammatory effect on colitis remain unclear. METHODS: A dextran sodium sulfate (DSS)-induced model of acute colitis was established in BALB/c mice (6-8 weeks old, weighing 18-22 g). Six groups of mice received: (1) water for 10 days (control), n = 6; (2) DSS 4% solution in the drinking water for 7 days, followed by normal water for 3 days, n = 7; (3), (4), and (5) as for group 2 plus baicalein (10, 20, 40 mg/kg) administered once daily starting on day 1, n = 6; and (6) as for (2) plus 5-aminosalicylic acid (50 mg/kg) administered once daily starting on day 1, n = 6. Body weights, stool consistency, and hematochezia were recorded, and the severity of colitis was evaluated using a disease activity index. On day 11, the mice were euthanized, and organs and blood were collected for analysis. Serum inflammatory factors were detected by enzyme-linked immunosorbent assay; CD11b-positive cells were analyzed by immunofluorescence microscopy; expression of retinoic-acid-receptor-related orphan nuclear receptor gamma, sphingosine kinase 1 (SPHK1), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) was detected by immunohistochemistry; and expression of nucleotide-binding oligomerization domain 2 (NOD2), SPHK1, sphingosine 1-phosphate receptor 1 (S1PR1), total STAT3, and p-STAT3 were detected by western blotting analysis. Inter-group differences were compared using Student's t test. RESULTS: Baicalein treatment dose-dependently reduced DSS-induced weight loss (P < 0.01 or P < 0.05), splenomegaly (P < 0.01), and colonic damage, as reflected by amelioration of diarrhea, rectal bleeding, and colonic ulceration, congestion, edema (shown as colon length, P < 0.05 or P < 0.01), and inflammatory cell infiltration. Baicalein also significantly decreased the levels of inflammatory mediators in the serum (P < 0.01) and colon, and significantly inhibited expression of NOD2 SPHK1, S1PR1, and p-STAT3 in the colon (P < 0.05). CONCLUSIONS: Baicalein treatment ameliorated colitis in mice by inhibiting S1P-STAT3 signaling, suggesting that this flavonoid might be beneficial in the treatment of colitis.

[Staphylococcus aureus promotes NOD2 expression in bovine mammary epithelial cells].

Objective To investigate the role of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in the mediate recognition of Staphylococcus aureus (S. aureus) in bovine mammary epithelial cells (BMECs). Methods We infected BMECs with living S. aureus and heat-inactivated S. aureus, respectively. When the multiplicity of infection (MOI) of living S. aureus was set for 100:1, the infection time were 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 hours; when the MOI of living S. aureus was set for 10:1, 20:1, 40:1, 100:1, the infection time was 2 hours; on the other hand, 0, 104, 105, 106, 107, 108 colony-forming units (CFU)/mL of heat-inactivated S. aureus were used to infect the cells for 2 hours separately. The expression of NOD2 was detected using real-time PCR and Western blotting. Results Levels of NOD2 mRNA and protein expression significantly increased after 0.5-4 hours of infection with living S. aureus. When the cells were infected with S. aureus at different MOI, dose-dependent accumulation of NOD2 mRNA and protein could be detected in BMECs. Levels of NOD2 mRNA and protein expression were not significantly upregulated by the stimulation with heat-inactivated S. aureus. Conclusion S. aureus infection could induce the up-regulation of NOD2 expression in BMECs.

NOD2 pathway via RIPK2 and TBK1 is involved in the aberrant catabolism induced by T-2 toxin in chondrocytes.

OBJECTIVE: This study aimed to identify the key intracellular pattern recognition receptor (PRR) and its role in the unbalanced extracellular matrix gene expressions of chondrocytes treated by T-2 toxin, a potential etiological factor for cartilage damages. DESIGN: Differential expressions of intracellular PRRs after T-2 toxin treatment were screened by RT-qPCR in chondrocytes. RNAi was used to knockdown the expression of NOD2 and its two downstream signal molecules, RIPK2, and TBK1, for observing the effects of NOD2 pathway on regulation of metabolism gene expressions by RT-qPCR. The matrix metalloproteinases (MMP) activity was determined by gelatin zymography. The inhibitor of NF-κB and ROS scavenger were exploited to analyze the mechanism of NOD2 up-regulation in chondrocytes treated with T-2 toxin. RESULTS: In chondrocytes treated with T-2 toxin, anabolism genes were down-regulated whereas catabolism genes were up-regulated, and NOD2 was identified as a significantly up-regulated gene. Intervening NOD2 expression via RNAi could ameliorate the down-regulation of anabolism genes, while inhibit the up-regulation of catablolism genes induced by T-2 toxin in chondrocytes. RNAi of RIPK2 and TBK1 in chondrocytes could obtain the similar outcome. Furthermore, up-regulation of NOD2 expression induced by T-2 toxin could be abrogated by pretreating the cells with inhibitors of NF-κB and scavenger of ROS. CONCLUSION: T-2 toxin could up-regulate NOD2 expression via ROS/NF-κB pathway and activate NOD2 signaling pathway. The up-regulated NOD2 would affect the metabolism gene expressions and MMP activity in chondrocytes via RIPK2 and TBK1. The findings add new insights into understanding NOD2 effects on chondrocytes treated with T-2 toxin.

Oral microbiota and host innate immune response in bisphosphonate-related osteonecrosis of the jaw.

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts (n=30); patients with periodontal disease without a history of BP therapy (Control, n=10), patients with periodontal disease having history of BP therapy but without ONJ (BP, n=5) and patients with BRONJ (BRONJ, n=15). Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ (71.6%), BP (70.3%) and Control (59.1%). Significant differences (P<0.05) in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay (ELISA) results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix-loop-helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.

Expression and functional characterization of NOD2 in decidual stromal cells isolated during the first trimester of pregnancy.

NOD2, one of the cytosolic proteins that contain a nuclear oligomerization domain (NOD), is a pattern recognition receptor (PRR) involved in innate immune responses to intracellular pathogens. Little is known, however, about the effect of NOD2 expression on the maternal-fetal relationship. Our aim was to elucidate the functions of NOD2 in normal decidual stromal cells (DSCs) from the first trimester. Tissues and DSCs were isolated from 26 patients with normal pregnancies that required abortion. The expression of NOD2 in deciduas/decidual stromal cells (DSCs) was examined by real-time PCR, immunohistochemistry, and In-cell western. DSCs containing NOD2 were stimulated by its ligand, muramyl dipeptide (MDP). The secretion of various cytokines and chemokines were measured by ELISA and the apoptotic rate was determined by flow cytometry. Treatment with MDP significantly elevated the expression of both NOD2 mRNA and protein levels in DSCs. In addition, MDP activation of NOD2 significantly increased IL-1ß and MCP-1 cytokine expression in a dose dependent manner but had no effect on IL-12 expression. IL-1ß and TNF-α also significantly increased the expression of NOD2 in DSCs, suggesting a positive feedback loop mechanism. Moreover, MDP stimulation augmented DSC apoptosis. In summary, the results suggest that NOD2 expression in DSCs plays an important role in protecting the embryo and preventing infection in the maternal-fetal interface.

Activation of innate immune responses by Haemophilus influenzae lipooligosaccharide.

A Gram-negative pathogen Haemophilus influenzae has a truncated endotoxin known as lipooligosaccharide (LOS). Recent studies on H. influenzae LOS highlighted its structural and compositional implications for bacterial virulence; however, the role of LOS in the activation of innate and adaptive immunity is poorly understood. THP-1 monocytes were stimulated with either lipopolysaccharide (LPS) from Escherichia coli or LOS compounds derived from H. influenzae Eagan, Rd, and Rd lic1 lpsA strains. Cell surface expression of key antigen-presenting, costimulatory, and adhesion molecules, as well as gene expression of some cytokines and pattern recognition receptors, were studied. Eagan and Rd LOS had a lower capacity to induce the expression of ICAM-1, CD40, CD58, tumor necrosis factor alpha (TNF-α), and interleukin-1ß (IL-1ß) compared to LPS. In contrast, antigen-presenting (HLA-ABC or HLA-DR) and costimulatory (CD86) molecules and NOD2 were similarly upregulated in response to LOS and LPS. LOS from a mutant Rd strain (Rd lic1 lpsA) consistently induced higher expression of innate immune molecules than the wild-type LOS, suggesting the importance of phosphorylcholine and/or oligosaccharide extension in cellular responses to LOS. An LOS compound with a strong ability to upregulate antigen-presenting and costimulatory molecules combined with a low proinflammatory activity may be considered a vaccine candidate to immunize against H. influenzae.

Expression of TRAF6 and pro-inflammatory cytokines through activation of TLR2, TLR4, NOD1, and NOD2 in human periodontal ligament fibroblasts.

OBJECTIVE: Human periodontal ligament fibroblasts (HPDLFs) play a crucial role in protecting against oral bacteria in periapical tissue. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) are two major forms of innate immune sensors that recognize microbial pathogens and initiate pro-inflammatory signalling. Tumour necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein for TLR-mediated nuclear factor-κB (NF-κB) signalling pathway activation that induces the production of pro-inflammatory cytokines. The aim of this study was to investigate the expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. We also investigated the expression of TRAF6 and pro-inflammatory cytokines induced by the activation of TLRs and NODs. METHODS: The expression of TLR2, TLR4, NOD1, and NOD2 was measured by reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and immunostaining. HPDLFs were stimulated with TLR and NOD agonists. Then, the expression of TRAF6 was measured by real-time PCR and western blot. Concentrations of IL-1ß, IL-6, and IL-8 in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Finally, by using small interfering RNA (siRNA) for TRAF6, we analysed the production of IL-1ß, IL-6, and IL-8 in HPDLFs upon stimulation with TLRs and NODs agonists. RESULTS: We found clear mRNA and protein expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. The expression levels of TRAF6 and pro-inflammatory cytokines (IL-1ß, IL-6, and IL-8) were markedly up-regulated upon the activation of TLRs and NODs. Furthermore, the co-activation of TLRs and NODs had synergistic effect on the production of TRAF6 and pro-inflammatory cytokines. We also found TRAF6 suppression resulted in reduced IL-1ß, IL-6, and IL-8 expression upon TLR and NOD agonists challenge. CONCLUSION: These findings indicated that TLR2, TLR4, NOD1, and NOD2 are functional receptors in HPDLFs during innate immune responses to invading bacteria, and a combination of signalling through TLRs and NODs leads to the synergistic enhancement of inflammatory reactions in HPDLFs. In addition, TLR and NOD signalling involving TRAF6 contribute to inflammatory responses in HPDLFs.

NOD-like receptors in the human upper airways: a potential role in nasal polyposis.

BACKGROUND: Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are newly discovered cytosolic receptors belonging to the pattern-recognition receptor family. They detect various pathogen-associated molecular patterns, triggering an immune response. The knowledge about these receptors, and their role in health and disease, is limited. The aim of the present study was to characterize the expression of NOD1, NOD2, and NALP3 in the human upper airways. METHODS: Surgical samples were obtained from patients with tonsillar disease (n = 151), hypertrophic adenoids (n = 9), and nasal polyposis (n = 24). Nasal biopsies were obtained from healthy volunteers (n = 10). The expression of NOD1, NOD2, and NALP3 was analyzed using real-time PCR and immunohistochemistry. RESULTS: Expression of NOD1, NOD2, and NALP3 mRNA and protein were seen in all tissue specimens. The NLR mRNA was found to be higher in nasal polyps than in normal nasal mucosa, and local steroid treatment reduced the NLR expression in polyps. In contrast, tonsillar infection with Streptococcus pyogenes or Haemophilus influenzae did not affect the NLR expression. CONCLUSIONS: The present study demonstrates the presence of NLRs in several upper airway tissues and highlights a potential role of NLRs in chronic rhinosinusitis with polyps.

Proinflammatory effects of muramyldipeptide on human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Because human gingival fibroblasts (HGFs) are the predominant cells in periodontal tissues, we hypothesized that HGFs are contributed to receptors for components of bacteria. In this study, we focused on expression and function of nucleotide binding oligomerization domain 2 (NOD2) in HGFs, which is a mammalian cytosolic pathogen recognition molecule. MATERIAL AND METHODS: Expression of NOD2 in HGFs was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry. Production of interleukin (IL)-6, IL-8, cc chemokine ligand2, cxc chemokine ligand10 (CXCL10) and CXCL11 from HGFs was examined by enzyme-linked immunosorbent assay (ELISA). We used RT-PCR and immunohistochemistry to detect the NOD2 expression in human gingival tissues. RESULTS: We found clear NOD2 expression in HGFs. Upon stimulation with NOD2 agonist, muramyldipeptide (MDP), production of proinflammatory cytokines was enhanced. Moreover, MDP-induced production of proinflammatory cytokines was inhibited in a different manner by mitogen-activated protein kinase inhibitors and phosphatidylinositol 3-kinase inhibitor. Furthermore, MDP enhanced CXCL10 and CXCL11 productions by tumor necrosis factor-alpha (TNF-alpha)- or interferon-gamma (IFN-gamma)-stimulated HGFs, although MDP alone did not induce these chemokines. TNF-alpha and IFN-gamma increased NOD2 expression in HGFs. In addition, we detected NOD2 expression in mononuclear cells and HGFs in periodontally diseased tissues. CONCLUSION: These findings indicate that MDP which induces production of cytokines and chemokines from HGFs is related to the pathogenesis of periodontal disease.

Nod1, Nod2 and Nalp3 receptors, new potential targets in treatment of allergic rhinitis?

BACKGROUND: Recently, a new set of pattern-recognition receptors, the nucleotide-binding oligomerization domain (Nod)-like receptors (NLRs), have emerged. Their activation, either by allergens or microbes, triggers an inflammatory response. The knowledge about NLRs in human airways is limited. AIM OF THE STUDY: To investigate presence of NLRs in the human nose of healthy individuals and patients with intermittent allergic rhinitis outside and during pollen season. METHODS: The expression of Nod1, Nod2, and Nalp3 in nasal biopsies was determined with real-time RT-PCR and immunohistochemistry. Cultured primary human nasal epithelial cells (HNECs) were analyzed using real-time RT-PCR and flow cytometry to further verify the presence of NLRs in the epithelium. RESULTS: Immunohistochemical analysis revealed presence of Nod1, Nod2, and Nalp3 in the nasal epithelium. This was corroborated in cultured HNECs. Patients suffering from symptomatic allergic rhinitis exhibited lower Nod1 and Nalp3 mRNA levels than both controls and patients during pollen season. Nod2 expression was found in all specimens tested, but no differences were seen between the three groups. CONCLUSION: Nod1, Nod2, and Nalp3 receptors were found to be present in the human nose. The expression of Nod1 and Nalp3 were down-regulated during pollen season among patients with allergic rhinitis. This opens up for new insights and novel therapeutic strategies in inflammatory airway disease.

Roles of TLR2, TLR4, NOD2, and NOD1 in pulp fibroblasts.

Pulp fibroblasts express various pro-inflammatory mediators leading to marked infiltration of inflammatory cells in the progression of pulpitis. We hypothesized that pulp fibroblasts play roles in the recognition of invaded caries-related bacteria and the subsequent innate immune responses. We found clear expressions of TLR2, NOD1, and NOD2 and a faint expression of TLR4 in human dental pulp fibroblasts (HDPF) by RT-PCR and flow cytometry. We also observed that various pro-inflammatory mediators, including cytokines, chemokines, adhesion molecules, prostaglandin E(2) and its key enzyme COX-2, not iNOS or caspase-1, were markedly up-regulated by stimulation with these TLR and NOD agonists. More over, the NOD2 agonist acted synergistically with the TLR2, not the TLR4, agonist to stimulate the production of pro-inflammatory mediators in HDPF. These findings indicate that TLR2, TLR4, NOD2, and NOD1 in HDPF are functional receptors, and NOD2 is a modulator of signals transmitted through TLR2 in pulpal immune responses, leading to progressive pulpitis.

Expression of nucleotide-binding oligomerization domain 2 in normal human dental pulp cells and dental pulp tissues.

INTRODUCTION: The nucleotide-binding oligomerization domain (NOD) proteins belong to a distinct family of proteins that are implicated in the intracellular recognition of bacterial components. NOD2 appears to be a sensor of bacterial peptidoglycans because it recognizes a minimal motif present in all peptidoglycans. The interaction of NOD2 with downstream signaling molecules ultimately results in the activation of NF-kappaB and production of inflammatory mediators in innate immunity. As such, NOD2 may play an important role in the detection of bacterial pathogens and the initiation of inflammation within the dental pulp. This study was designed to evaluate the expression of NOD2 in normal human dental pulp cells (HDPCs) and human pulp tissues. METHODS: Human pulp tissue samples were collected from freshly extracted human wisdom teeth, and HDPCs were prepared from the explants of normal human dental pulp tissues. Nested reverse-transcription polymerase chain reaction (Nested RT-PCR) and Western blotting were performed to detect the expression of NOD2 messenger RNA and protein, respectively. Immunohistochemical staining was used to determine the distribution of NOD2 in the pulp tissues. RESULTS: The NOD2 messenger RNA and protein were present in normal human dental pulp tissues, with most NOD2 protein expression being localized to odontoblasts and some pulp vascular endothelial cells. In contrast, HDPCs only showed a low level of NOD2 protein expression. CONCLUSIONS: Our results suggest that NOD2 protein expressed in HDPCs and pulp tissues may play an important role in dental immune defense.

NOD2: a potential target for regulating liver injury.

The recent discovery of bacterial receptors such as NOD2 that contribute to crosstalk between innate and adaptive immune systems in the digestive tract constitutes an important challenge in our understanding of liver injury mechanisms. The present study focuses on NOD2 functions during liver injury. NOD2, TNF-alpha and IFN-gamma mRNA were quantified using real-time PCR in liver samples from patients and mice with liver injury. We evaluated the susceptibility of concanavalin A (ConA) challenge in NOD2-deficient mice (Nod2-/-) compared to wild-type littermates. We tested the effect of muramyl dipeptide (MDP), the specific activator of NOD2, on ConA-induced liver injury in C57BL/6 mice. We studied the cellular distribution and the role of NOD2 in immune cells and hepatocytes. We demonstrated that NOD2, TNF-alpha and IFN-gamma were upregulated during liver injury in mice and humans. Nod2-/- mice were resistant to ConA-induced hepatitis compared to their wild-type littermates, through reduced IFN-gamma production by immune cells. Conversely, administration of MDP exacerbated ConA-induced liver injury. MDP was a strong inducer of IFN-gamma in freshly isolated human PBMC, splenocytes and hepatocytes. Our study supports the hypothesis that NOD2 contributes to liver injury via a regulatory mechanism affecting immune cells infiltrating the liver and hepatocytes. Taken together, our results indicate that NOD2 may represent a new therapeutic target in liver diseases.

[Parenteral symptoms and intestinal complications in children with inflammatory bowel diseases in relation to card15 mutation]. / Objawy pozajelitowe i powiklania u dzieci z nieswoistymi zapaleniami jelit w zalenznosci od mutacji genu CARD15.

THE AIM OF THE STUDY: Was evaluation of the incidence of parenteral symptoms and complications in children with inflammatory bowel disease and their analysis in relation to the examined mutations of CARD15 gene. PATIENTS AND METHODS: The study involved 38 children with Crohn's disease, aged from 5 to18 years (median14) and 40 children with ulcerative colitis, aged from 6 to18 years (median14). The control group included 23 children, aged from 4 to 18 years (median15), with functional disorders of the alimentary tract resulting from lactose intolerance. In all the examined patients as well as in the control group, mutations R702W, G908R and L1007fs of the CARD15 gene were determined, according to the protocol described by Tukel et al. RESULTS: Parenteral symptoms, in the group of children with Crohn's disease, manifested as arthritis and erythema nodosum, were observed in 7 patients (18.4%), whereas in the group with ulcerative colitis they presented - in 4 children (10%). Intestinal complications in the form of stenosis, fistula, abscess and gastrointestinal bleeding were the most frequently observed changes in children with Crohn's disease (n=15; 39,5%). Parenteral symptoms were statistically significantly more frequent in children with Crohn's disease and with at least one mutation of CARD15 gene. Intestinal complications statistically appeared more often in children with Crohn's disease and mutation L1007fs. CONCLUSIONS: 1. Parenteral symptoms and intestinal complications occurred more frequently in the group of children with Crohn's disease, in comparison with the children with ulcerative colitis. 2. We observed a relation between parenteral symptoms and at least one mutation of CARD15 gene and a relation between intestinal complications and L1007fs mutation.

A novel technique for NOD2/CARD15 genotyping using PCR-SSP.

The Nucleotide-binding Oligomerisation Domain (NOD) 2 protein is encoded by the Caspase Recruitment Domain (CARD) 15 gene and has a critical role in innate immunity. Recent studies have implicated Single Nucleotide Polymorphisms (SNPs) of the NOD2/CARD15 gene with the onset of several Inflammatory Bowel Disorders (Crohn's Disease, Blau syndrome) and the progression of several malignant diseases. The identification of SNPs in the genotypes of donor and recipient pairs prior to haematopoietic stem cell transplantation have also been shown to predict for a worse outcome, specifically causing increases in the incidence and severity of acute Graft-versus-Host disease, disease relapse and mortality. In light of these widespread areas of interest, we have developed a Polymerase Chain Reaction assay using Sequence Specific Primers (PCR-SSP) to identify the three SNPs that have been implicated, (SNPs 8, 12 and 13). The assay has proven to be a rapid and accurate method of performing NOD2/CARD15 genotyping when compared to other techniques described to date.