Brandon W. Higgs

Development of Potential Pharmacodynamic and Diagnostic Markers for Anti-IFN-α Monoclonal Antibody Trials in Systemic Lupus Erythematosus.

To identify potential pharmacodynamic biomarkers to guide dose selection in clinical trials using anti-interferon-alpha (IFN-α) monoclonal antibody (mAb) therapy for systemic lupus erythematosus (SLE), we used an Affymetrix human genome array platform and identified 110 IFN-α/β-inducible transcripts significantly upregulated in whole blood (WB) of 41 SLE patients. The overexpression of these genes was confirmed prospectively in 54 additional SLE patients and allowed for the categorization of the SLE patients into groups of high, moderate, and weak overexpressers of IFN-α/β-inducible genes. This approach could potentially allow for an accurate assessment of drug target neutralization in early trials of anti-IFN-α mAb therapy for SLE. Furthermore, ex vivo stimulation of healthy donor peripheral blood mononuclear cells with SLE patient serum and subsequent neutralization with anti-IFN-α mAb or anti-IFN-α receptor mAb showed that anti-IFN-α mAb has comparable effects of neutralizing the overexpression of type I IFN-inducible genes as that of anti-IFNAR mAb. These results suggest that IFN-α, and not other members of type I IFN family in SLE patients, is mainly responsible for the induction of type I IFN-inducible genes in WB of SLE patients. Taken together, these data strengthen the view of IFN-α as a therapeutic target for SLE.

Allospecific CD154+ T Cells Associate with Rejection Risk After Pediatric Liver Transplantation

Antigen‐specific T cells, which express CD154 rapidly, but remain untested in alloimmunity, were measured with flow cytometry in 16‐h MLR of 58 identically‐immunosuppressed children with liver transplantation (LTx), to identify Rejectors (who had experienced biopsy‐proven rejection within 60 days posttransplantation). Thirty‐one children were sampled once, cross‐sectionally. Twenty‐seven children were sampled longitudinally, pre‐LTx, and at 1–60 and 61–200 days after LTx. Results were correlated with proliferative alloresponses measured by CFSE‐dye dilution (n = 23), and CTLA4, a negative T‐cell costimulator, which antagonizes CD154‐mediated effects (n = 31). In cross‐sectional observations, logistic regression and leave‐one‐out cross‐validation identified donor‐specific, CD154 + T‐cytotoxic (Tc)‐memory cells as best associated with rejection outcomes. In the longitudinal cohort, (1) the association between CD154 + Tc‐memory cells and rejection outcomes was replicated with sensitivity/specificity 92.3%/84.6% for observations at 1–60 days, and (2) elevated pre‐LTx CD154 + Tc‐memory cell responses were associated with significantly increased incidence (p = 0.02) and hazard (HR = 7.355) of rejection in survival/proportional hazard analysis. CD154 expression correlated with proliferative alloresponses (r = 0.835, p = 7.1e‐07), and inversely with CTLA4 expression of allospecific CD154 + Tc‐memory cells (r =−0.706, p = 3.0e‐05). Allospecific CD154 + T‐helper‐memory cells, not CD154 + Tc‐memory, were inhibited by increasing Tacrolimus concentrations (p = 0.026). Collectively, allospecific CD154 + T cells provide an estimate of rejection risk in children with LTx.

Putative psychosis genes in the prefrontal cortex: combined analysis of gene expression microarrays

BackgroundRecent studies have shown similarities between schizophrenia and bipolar disorder in phenotypes and in genotypes, and those studies have contributed to an ongoing re-evaluation of the traditional dichotomy between schizophrenia and bipolar disorder. Bipolar disorder with psychotic features may be closely related to schizophrenia and therefore, psychosis may be an alternative phenotype compared to the traditional diagnosis categories.MethodsWe performed a cross-study analysis of 7 gene expression microarrays that include both psychosis and non-psychosis subjects. These studies include over 400 microarray samples (163 individual subjects) on 3 different Affymetrix microarray platforms.ResultsWe found that 110 transcripts are differentially regulated (p < 0.001) in psychosis after adjusting for confounding variables with a multiple regression model. Using a quantitative PCR, we validated a set of genes such as up-regulated metallothioneins (MT1E, MT1F, MT1H, MT1K, MT1X, MT2A and MT3) and down-regulated neuropeptides (SST, TAC1 and NPY) in the dorsolateral prefrontal cortex of psychosis patients.ConclusionThis study demonstrates the advantages of cross-study analysis in detecting consensus changes in gene expression across multiple microarray studies. Differential gene expression between individuals with and without psychosis suggests that psychosis may be a useful phenotypic variable to complement the traditional diagnosis categories.

Gene Expression and Genetic Variation Data Implicate PCLO in Bipolar Disorder

BACKGROUND Genetic variation may contribute to differential gene expression in the brain of individuals with psychiatric disorders. To test this hypothesis, we identified genes that were differentially expressed in individuals with bipolar disorder, along with nearby single nucleotide polymorphisms (SNPs) that were associated with expression of the same genes. We then tested these SNPs for association with bipolar disorder in large case-control samples. METHODS We used the Stanley Genomics Database to extract gene expression and SNP microarray data from individuals with bipolar disorder (n = 40) and unaffected controls (n = 43). We identified 367 genes that were differentially expressed in the prefrontal cortex of cases vs. controls (fold change > 1.3 and FDR q-value < .05) and 45 nearby SNPs that were associated with expression of those same genes (FDR q-value < .05). We tested these SNPs for association with bipolar disorder in a meta-analysis of genome-wide association studies (GWAS) including 4,936 cases and 6,654 healthy controls. RESULTS We identified 45 SNPs that were associated with expression of differentially expressed genes, including HBS1L (15 SNPs), HLA-DPB1 (15 SNPs), AMFR (8 SNPs), PCLO (2 SNPs) and WDR41 (2 SNPs). Of these, one SNP (rs13438494), in an intron of the piccolo (PCLO) gene, was significantly associated with bipolar disorder (FDR adjusted p < .05) in the meta-analysis of GWAS. CONCLUSIONS These results support the previous findings implicating PCLO in mood disorders and demonstrate the utility of combining gene expression and genetic variation data to improve our understanding of the genetic contribution to bipolar disorder.

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.

Allospecific CD154+ T cells identify rejection-prone recipients after pediatric small-bowel transplantation.

BACKGROUND Up to 70% of children with small bowel transplantation (SBTx) experience acute cellular rejection (ACR). Allospecific CD154+ T cells predict liver ACR in children in a novel, 16-hour mixed leukocyte response (MLR) assay, but remain untested in SBTx. METHODS The expression of CD154 was measured in 4 subsets-naive (N) and memory (M) CD154+ T-helper (Th) and T-cytotoxic (Tc) cells (ie, CD154+ ThN, CD154+ ThM, CD154+ TcN, and CD154+ TcM, respectively)-in the MLR of single blood samples obtained from 32 children with SBTx within 60 days of SBTx biopsy. Children showing ACR in these biopsies were termed Rejectors. The ratio of donor-induced to third-party-induced CD154+ T cells was called the immunoreactivity index (IR). We hypothesized that IR >1 denoted increased donor-specific alloreactivity and increased risk of rejection; in contrast, IR <1 implied decreased risk. CD154 expression was correlated with the expression of CTLA4, a negative T-cell costimulator that antagonizes and is inversely related to CD154 (n = 18). RESULTS Rejectors showed significantly greater numbers of donor-specific CD154+ T-cell subsets. Logistic regression analysis and leave-one-out cross validation followed by receiver operating characteristic analysis showed that, among the 4 subsets, IR > or =1.23 for CD154+ TcM identified Rejectors with a sensitivity and specificity of 93% and 88%. Also, a significant negative correlation was observed between CD154 expression and CTLA4 expression in allospecific Tc (Spearmans rho = -0.616, P = .006) but not in Th. CONCLUSION Allospecific CD154+ TcM identify rejection-prone children with SBTx.

Effects of typical and atypical antipsychotic drugs on gene expression profiles in the liver of schizophrenia subjects

BackgroundAlthough much progress has been made on antipsychotic drug development, precise mechanisms behind the action of typical and atypical antipsychotics are poorly understood.MethodsWe performed genome-wide expression profiling to study effects of typical antipsychotics and atypical antipsychotics in the postmortem liver of schizophrenia patients using microarrays (Affymetrix U133 plus2.0). We classified the subjects into typical antipsychotics (n = 24) or atypical antipsychotics (n = 26) based on their medication history, and compared gene expression profiles with unaffected controls (n = 34). We further analyzed individual antipsychotic effects on gene expression by sub-classifying the subjects into four major antipsychotic groups including haloperidol, phenothiazines, olanzapine and risperidone.ResultsTypical antipsychotics affected genes associated with nuclear protein, stress responses and phosphorylation, whereas atypical antipsychotics affected genes associated with golgi/endoplasmic reticulum and cytoplasm transport. Comparison between typical antipsychotics and atypical antipsychotics further identified genes associated with lipid metabolism and mitochondrial function. Analyses on individual antipsychotics revealed a set of genes (151 transcripts, FDR adjusted p < 0.05) that are differentially regulated by four antipsychotics, particularly by phenothiazines, in the liver of schizophrenia patients.ConclusionTypical antipsychotics and atypical antipsychotics affect different genes and biological function in the liver. Typical antipsychotic phenothiazines exert robust effects on gene expression in the liver that may lead to liver toxicity. The genes found in the current study may benefit antipsychotic drug development with better therapeutic and side effect profiles.

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.

Allospecific CD154+ B cells associate with intestine allograft rejection in children.

Background. As a significant determinant of T- and B-cell cooperation, CD154 has been used to identify allospecific T-cytotoxic memory cells (TcM) for rejection risk assessment with high sensitivity or specificity but not for alloreactive B-cells, especially among recipients predisposed to acute cellular and humoral rejection, that is, children with intestinal transplantation (ITx). Methods. Single blood samples from 32 pediatric ITx after lymphocyte depleting induction therapy were obtained within 30 days of protocol biopsies. Samples were assayed for allospecific CD154+CD19+ B cells and allospecific CD154+ TcM in 16-hr live-cell mixed leukocyte reaction using multiparametric flow cytometry. Results were expressed as the immunoreactivity index (IR) or the ratio of donor- to third-party-induced CD154+ B cells or TcM. The rejection threshold IR of B cells was determined by logistic regression, leave-one-out cross-validation, and receiver operating characteristic analyses. Results. Biopsy-proven acute cellular rejection was present in 15 subjects (rejectors) and absent in 17 (nonrejectors). In archived serum samples from 16 of 32 subjects donor-specific anti-HLA antibodies (DSA) were assayed by Luminex bead array. DSA were absent in all 7 nonrejectors but present in 7 of 9 rejectors. The IR of allospecific CD154+CD19+ B cells more than or equal to 1.351 was associated with rejector status and was present in 13 of 15 rejectors (sensitivity 87%) and absent in 15 of 17 nonrejectors (specificity 88%). Excellent correlations were seen between CD154+CD19+ B cells and CD154+ TcM (Spearman &rgr;=0.647, P=0.0001) but could not be tested independently for DSA, which was highly correlated with rejector status and with CD154+ TcM. Conclusions. Allospecific CD154+CD19+ B cells identify rejection-prone children with ITx and can likely substitute for T-cell alloreactivity in estimating rejection risk in this rare subject population.