Mylarappa Ningappa

NOD2 gene polymorphism rs2066844 associates with need for combined liver-intestine transplantation in children with short-gut syndrome.

OBJECTIVES:The nucleotide-binding oligomerization protein 2 (NOD2) gene single nucleotide polymorphisms (SNPs) associated with Crohns disease were recently associated with severe rejection after small-bowel transplantation (SBTx). The purpose of this study was to re-test this association and explore whether deficient innate immunity suggested by the NOD2 SNPs predisposes to intestine failure requiring isolated SBTx or combined liver–intestine failure requiring combined liver–SBTx (LSBTx).METHODS:Archived DNA from 85 children with primary isolated SBTx or LSBTx was genotyped with Taqman biallelic discrimination assays. To minimize confounding effects of racial differences in minor allele frequencies (MAFs), allelic associations were tested in 60 Caucasian recipients (discovery cohort). Replication was sought in an independent cohort of 39 Caucasian pediatric and adult SBTx patients.RESULTS:MAF for rs2066845 and rs2066847 was similar to that seen in 538 healthy North American Caucasians. In the discovery cohort, MAF for rs2066844 was significantly higher in LSBTx (13.5 vs. 3.6%, P=0.0007, Fishers exact test), but not in isolated SBTx recipients (2.2 vs. 3.6%, P=NS), when compared with 538 healthy Caucasians. In addition, among LSBTx recipients who received identical immunosuppression, the minor allele of rs2066844 associated with early rejection in linear regression analysis (P=0.028) (all but one of the risk alleles were found in rejectors), decreased survival (P=0.015, log-rank, Kaplan–Meier analysis), and a 20-fold greater hazard of septic death in proportional hazard analysis (P=0.030). Steroid-resistant (severe) rejection and graft loss were associated with isolated SBTx (P=0.036 and 0.082, respectively), but not with NOD2 SNPs. The association between rs2066844 and combined liver–intestine failure requiring LSBTx was significant in the replication cohort (P=0.014), and achieved greater significance in the combined cohort (P=0.00006).CONCLUSIONS:The NOD2 SNP rs2066844 associates with combined liver and intestinal failure in subjects with short-gut syndrome, who require combined liver–intestine transplantation, and secondarily with early rejection and septic deaths.

Synthesis of novel coumarin appended bis(formylpyrazole) derivatives: Studies on their antimicrobial and antioxidant activities

A series of novel coumarin pyrazole hybrids of biological interest were synthesized from the hydrazones, carbazones and thiocarbazones via Vilsmeier Haack formylation reaction. These intermediates and formyl pyrazoles were evaluated for antimicrobial and antioxidant activities. Among the series, compounds 6g and 6h showed excellent antimicrobial activity against different bacterial and fungal strains and compounds 7g, 7h were found to be potent antioxidant agents in both DPPH and hydroxyl radical scavenging assays. Further, detailed quantitative structure-activity relationship (QSAR) analysis indicated the molecular parameters that contribute to increased potency of inhibition. The above findings would further encourage our understanding in employing coumarin pyrazole hybrids as potential antibiotic agents for treating infections caused by pathogenic microbes and fungi. Further, it also paves the way for exploration of these compounds as potential therapeutic agents to treat conditions arising because of excessive oxidative damage.

Allospecific CD154 + T-cytotoxic memory cells as potential surrogate for rejection risk in pediatric intestine transplantation

Sindhi R, Ashokkumar C, Higgs BW, Gilbert PB, Sun Q, Ranganathan S, Jaffe R, Snyder S, Ningappa M, Soltys KA, Bond GJ, Mazariegos GV, Abu‐Elmagd K, Zeevi A. Allospecific CD154 + T‐cytotoxic memory cells as potential surrogate for rejection risk in pediatric intestine transplantation. 
Pediatr Transplantation 2012: 16: 83–91.

Elevated myeloid: plasmacytoid dendritic cell ratio associates with late, but not early, liver rejection in children induced with rabbit anti-human thymocyte globulin.

Background. Dendritic cells (DC) play an important role in the induction and regulation of immune responses. Methods. Myeloid CD11c+ DC (MDC), which may have inflammatory functions, and plasmacytoid CD123+ DC (PDC), which may have tolerogenic potential, were measured by flow cytometric analysis, cross-sectionally, once, in 48 children, and longitudinally (pretransplant and at days 1–60, 61–200, and 201–400 posttransplant) in 30 children after liver transplantation (LTx). All children received cadaveric (n=53) or live donor (n=25) liver allografts with rabbit anti-human thymocyte globulin induction and steroid-free tacrolimus therapy. Rejectors in both groups were those children (n=35), who experienced biopsy-proven acute cellular rejection (ACR) within 60 days of DC monitoring. Results. Among rejectors in the longitudinal and cross-sectional cohorts, the MDC:PDC ratio was higher and was associated with decreased PDC frequencies. Logistic regression analysis, leave-one-out cross-validation, and receiver operating characteristic analysis applied to 30 cross-sectional subjects revealed that an MDC:PDC ratio more than or equal to 1.78 was associated with rejector status with sensitivity and specificity of 76.9% and 88.2%, respectively. Sensitivity and specificity were replicated in the 18 remaining cross-sectional subjects (88.8% and 78.8%, respectively), but not in longitudinally monitored subjects, during the early 60-day period after LTx (30.76% and 62.50%, respectively). A significant negative correlation was observed between tacrolimus whole blood concentrations and PDC frequencies (Spearman r=−0.370, P=0.005) in 48 cross-sectional subjects in whom DC subsets were monitored 1 to 3 years after LTx, but not during the early post-LTx period. Conclusions. We conclude that an elevated MDC:PDC ratio associates with liver graft rejection, which occurs after first year in children induced with rabbit anti-human thymocyte globulin.

Increased expression of peripheral blood leukocyte genes implicate CD14+ tissue macrophages in cellular intestine allograft rejection.

Recurrent rejection shortens graft survival after intestinal transplantation (ITx) in children, most of whom also experience early acute cellular rejection (rejectors). To elucidate mechanisms common to early and recurrent rejection, we used a test cohort of 20 recipients to test the hypothesis that candidate peripheral blood leukocyte genes that trigger rejection episodes would be evident late after ITx during quiescent periods in genome-wide gene expression analysis and would achieve quantitative real-time PCR replication pre-ITx (another quiescent period) and in the early post-ITx period during first rejection episodes. Eight genes were significantly up-regulated among rejectors in the late post-ITx and pre-ITx periods, compared with nonrejectors: TBX21, CCL5, GNLY, SLAMF7, TGFBR3, NKG7, SYNE1, and GK5. Only CCL5 was also up-regulated in the early post-ITx period. Among resting peripheral blood leukocyte subsets in randomly sampled nonrejectors, CD14(+) monocytes expressed the CCL5 protein maximally. Compared with nonrejectors, rejectors demonstrated higher counts of both circulating CCL5(+)CD14(+) monocytes and intragraft CD14(+) monocyte-derived macrophages in immunohistochemistry of postperfusion and early post-ITx biopsies from the test and an independent replication cohort. Donor-specific alloreactivity measured with CD154(+) T-cytotoxic memory cells correlated with the CCL5 gene and intragraft CD14(+) monocyte-derived macrophages at graft reperfusion and early post-ITx. CCL5 gene up-regulation and CD14(+) macrophages likely prime cellular ITx rejection. Infiltration of reperfused intestine allografts with CD14(+) macrophages may predict rejection events.

Allospecific CD154+ T-cytotoxic memory cells identify recipients experiencing acute cellular rejection after renal transplantation.

Background. The novel, recently described allo (antigen)-specific CD154+T cells were evaluated for their association with acute cellular rejection (ACR) in 43 adult renal transplant recipients receiving steroid-free tacrolimus after alemtuzumab induction. Methods. Single blood samples corresponding to “for cause” allograft biopsies were assayed for CD154+naive or memory T-helper or T-cytotoxic cells in 16-hr mixed leukocyte reaction. Results. Intra- and interassay variation was less than 10% for a variety of conditions. In logistic regression, leave-one-out cross-validation, and receiver-operating characteristic analyses, the rejection-risk threshold of allospecific CD154+T-cytotoxic memory cells (TcMs) associated best with biopsy-proven ACR with a sensitivity/specificity of 88% in 32 of 43 subjects. Sensitivity/specificity of 100%/88% was replicated in blinded prediction in the remaining 11 subjects. Allospecific CD154+TcM correlated inversely with CTLA4+TcM (Spearman r=−0.358, P=0.029) and increased significantly with increasing histological severity of ACR (P=2.99E-05, Kruskall-Wallis). Conclusions. The strong association between ACR and allospecific CD154+TcM may be useful in minimizing protocol biopsies among recipients at reduced rejection risk.

Elevated Myeloid: Plasmacytoid Dendritic Cell Ratio Associates With Early Acute Cellular Rejection in Pediatric Small Bowel Transplantation

Background. Acute cellular rejection affects more than 60% of children after small bowel transplantation (SBTx). Dendritic cells (DCs) are potent antigen-presenting cells, modulate immune responses to gut microbes, and may serve as markers of rejection-prone small bowel transplantation (SBTx). Methods. Myeloid CD11c+ DC (MDC), which may have inflammatory functions, and plasmacytoid CD123+ DC (PDC), which may have tolerogenic potential, were measured by flow cytometric analysis, longitudinally (pretransplant, and at days 1 to 60, 61 to 200 posttransplant) in 23 children after SBTx. All children received cadaveric allografts with rabbit anti-human thymocyte globulin induction and steroid-free tacrolimus maintenance therapy. Rejectors were those children (n=16), who experienced biopsy-proven acute cellular rejection within 60 days of SBTx. Results. Of 69 maximum possible observations, 62 were available for analysis. Among rejectors, a significantly higher MDC:PDC ratio (P=0.004) was associated with numerically higher MDC counts and significantly lower PDC frequencies (P=0.017) during the 1- to 60-day time period, compared with nonrejectors. Logistic regression analysis, leave-one-out cross-validation, and receiver operating characteristic analysis revealed that MDC:PDC ratio more than or equal to 1.52 was associated with rejector status with sensitivity/specificity of 86/67% during the 1- to 60-day risk period for early SBTx rejection. Repeated measures analysis showed a significantly higher MDC:PDC ratio (P=0.043, F-test) among rejectors, compared with nonrejectors in cumulative data for pre-SBTx and 1- to 60-day time points. No correlation was seen between DC subsets and tacrolimus blood concentration at any time point. Conclusions. We conclude that an elevated MDC:PDC ratio associates with early small bowel allograft rejection and may, therefore, identify at-risk recipients in the clinic.

The Role of ARF6 in Biliary Atresia

Background & Aims Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA). Methods To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6). Results Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3’ flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10-7), ERK/MAPK and CREB canonical pathways (p<1 x10-34), and functional networks for cellular development and proliferation (p<1 x10-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA. Conclusions The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.

Proliferative alloresponse of T cytotoxic cells identifies rejection-prone children with small bowel transplantation.

Background. More than 60% children with small bowel transplantation (SBTx) experience acute cellular rejection. Purpose/Methods. To identify children at risk of rejection, donor- and third-party-induced proliferation of T-helper and T cytotoxic (Tc) cells, and their naïve and memory (M) subsets was evaluated simultaneously in single blood samples from 28 children who received SBTx after induction with rabbit anti-human thymocyte globulin. Proliferation was measured by dilution of the intravital dye carboxyfluorescein succinimidyl ester (CFSE) in 3- to 4-day mixed leukocyte reaction co-culture. The ratio of donor- and third-party-induced proliferation (CFSElow) of the T cells was reported as the immunoreactivity (IR) index for each subset. Rejectors were defined as those who experienced biopsy-proven acute cellular rejection within 60 days of the assay. IR more than 1 signified increased risk of rejection and IR less than 1 implied decreased risk. Results. Rejectors (n=16) and Nonrejectors (n=12) were similar in general demographics. Significantly higher counts were observed for all proliferated CFSElow T-cell subsets among rejectors, compared with nonrejectors. Logistic regression, leave-one-out cross-validation, and receiver operating characteristic analyses showed that the IR of Tc associated best with biopsy-proven rejection (sensitivity >87.5%, specificity >83.3%). IR of CFSElow Tc correlated significantly with IR of proinflammatory, allospecific CD154+Tc-M (r=0.682, P=0.005) and inversely with IR of allospecific, antiinflammatory, CTLA4+Tc-M (r=−0.638, P=0.047). Conclusions. Proliferative alloresponses of Tc cells can identify rejection-prone children receiving SBTx.

Genome-wide association studies in biliary atresia

Biliary atresia (BA) is a model complex disease resulting from interactions between multiple susceptibility loci and environmental factors. This perception is based on a heterogeneous phenotype extending beyond an absent extrahepatic bile duct to include gut and cardiovascular anomalies, and the association of BA with viral infections. Refractory jaundice and progression to cirrhosis shortly after birth can be fatal without surgical correction, and further suggests a pathogenesis during liver and bile duct development. Conclusive proof for a developmental origin would require documentation of disease progression in the perinatal or fetal liver, an impossible task for obvious reasons. We review three different sets of genome‐wide association studies (GWAS) from three different cohorts of BA patients by three different groups of investigators, which address this knowledge gap. Knockdown of each susceptibility gene identified by GWAS in zebrafish embryos impairs excretion of bile from the liver, duplicating the characteristic diagnostic finding seen in affected children. This finding is associated with impaired intrahepatic biliary network formation in zebrafish morphants. Although distinct, these susceptibility genes share several functions including roles in mechanisms for organogenesis (glypican 1 or GPC1, and adenosine diphosphate ribosylation factor 6, or ARF6) or a greater expression in fetal liver than in adult liver (adducin 3 or ADD3). Together, these studies emphasize the importance of the human evidence, and present opportunities to map novel pathways which explain the phenotypic heterogeneity of BA. WIREs Syst Biol Med 2015, 7:267–273. doi: 10.1002/wsbm.1303