Functional immunomics: Microarray analysis of IgG autoantibody repertoires predicts the future response of NOD mice to an inducer of accelerated diabetes

One's present repertoire of antibodies encodes the history of one's past immunological experience. Can the present autoantibody repertoire be consulted to predict resistance or susceptibility to the future development of an autoimmune disease? Here we developed an antigen microarray chip and used bioinformatic analysis to study a model of type 1 diabetes developing in non-obese diabetic (NOD) male mice in which the disease was accelerated and synchronized by exposing the mice to cyclophosphamide at 4 weeks of age. We obtained sera from 19 individual mice, treated the mice to induce cyclophosphamide-accelerated diabetes (CAD), and found, as expected, that 9 mice became severely diabetic while 10 mice permanently resisted diabetes. We again obtained serum from each mouse afterCAD induction. We then analyzed the patterns of antibodies in the individualmice to 266 different antigens spotted on the antigen chip. We identified a select panel of 27 different antigens (10% of the array) that revealed a pattern of IgG antibody reactivity in the pre-CAD serathat discriminated between the mice resistant or susceptible to CAD with 100% sensitivity and 82% specificity (p=0.017). Surprisingly, the set of IgG antibodies that was informative before CAD induction did not separate the resistant and susceptible groups after the onset of CAD; new antigens became criticalfor post-CAD repertoire discrimination. Thus, at least for a model disease, present antibody repertoires can predict future disease; predictive and diagnostic repertoires can differ; and decisive information about immune system behavior can be mined by bioinformatic technology. Repertoires matter.Comment: See Advanced Publication on the PNAS website for final versio

Characteristics of the Early Immune Response Following Transplantation of Mouse ES Cell Derived Insulin-Producing Cell Clusters

Background The fully differentiated progeny of ES cells (ESC) may eventually be used for cell replacement therapy (CRT). However, elements of the innate immune system may contribute to damage or destruction of these tissues when transplanted. Methodology/Principal Findings Herein, we assessed the hitherto ill-defined contribution of the early innate immune response in CRT after transplantation of either ESC derived insulin producing cell clusters (IPCCs) or adult pancreatic islets. Ingress of neutrophil or macrophage cells was noted immediately at the site of IPCC transplantation, but this infiltration was attenuated by day three. Gene profiling identified specific inflammatory cytokines and chemokines that were either absent or sharply reduced by three days after IPCC transplantation. Thus, IPCC transplantation provoked less of an early immune response than pancreatic islet transplantation. Conclusions/Significance Our study offers insights into the characteristics of the immune response of an ESC derived tissue in the incipient stages following transplantation and suggests potential strategies to inhibit cell damage to ensure their long-term perpetuation and functionality in CRT

A Practical Guide to Rodent Islet Isolation and Assessment

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes

The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells

Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases

Cigarette smoking, genetic polymorphisms and colorectal cancer risk: the Fukuoka Colorectal Cancer Study

Background: It is uncertain whether smoking is related to colorectal cancer risk. Cytochrome P-450 CYP1A1, glutathione-S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1) are important enzymes in the metabolism of tobacco carcinogens, and functional genetic polymorphisms are known for these enzymes. We investigated the relation of cigarette smoking and related genetic polymorphisms to colorectal cancer risk, with special reference to the interaction between smoking and genetic polymorphism. Methods: We used data from the Fukuoka Colorectal Cancer Study, a population-based case-control study, including 685 cases and 778 controls who gave informed consent to genetic analysis. Interview was conducted to assess lifestyle factors, and DNA was extracted from buffy coat. Results: In comparison with lifelong nonsmokers, the odds ratios (OR) of colorectal cancer for <400, 400-799 and ≥800 cigarette-years were 0.65 (95 % confidence interval [CI], 0.45-0.89), 1.16 (0.83-1.62) and 1.14 (0.73-1.77), respectively. A decreased risk associated with light smoking was observed only for colon cancer, and rectal cancer showed an increased risk among those with ≥400 cigarette-years (OR 1.60, 95 % CI 1.04-2.45). None of the polymorphisms under study was singly associated with colorectal cancer risk. Of the gene-gene interactions studied, the composite genotype of CYP1A1*2A or CYP1A1*2C and GSTT1 polymorphisms was associated with a decreased risk of colorecta

Serum Amyloid A Induces NLRP-3-Mediated IL-1β Secretion in Neutrophils

Background/Aims: Serum amyloid A (SAA) is an acute phase reactant with significant immunological activities, including effects on cytokine synthesis and neutrophil chemotaxis. Neutrophils can also release cytokines with proinflammatory properties. IL-1β is a key proinflammatory cytokine, the secretion of which is controlled by inflammasome. We investigated the proinflammatory effects of SAA in vitro in relation to the NLRP3 inflammasome in neutrophils. Methodology/Principal Findings: Human neutrophils isolated form healthy subjects were stimulated with serum amyloid A (SAA). The cellular supernatants were analyzed by western blot using anti-IL-1β or anti-caspase-1 antibodies. IL-1β or Nod-like receptor family, pyrin domain containing 3 (NLRP3) mRNA expressions were analyzed by real-time PCR or reverse transcription-PCR (RT-PCR) method. SAA stimulation induced pro-IL-1β mRNA expression in neutrophils. Furthermore, SAA engaged the caspase-1-activating inflammasome, resulting in the production of active IL-1β. SAA-induced pro-IL-1β expression was marginally suppressed by the Syk specific inhibitor, R406, and SAA-induced pro-IL-1β processing in neutrophils was prevented by R406. Furthermore, SAA-induced NLRP3 mRNA expression was completely blocked by R406. Analysis of intracellular signaling revealed that SAA stimulation activated the tyrosine kinase Syk and mitogen-activated protein kinase (MAPK). Conclusions/Significance: These results demonstrate that the innate neutrophil immune response against SAA involves a two-step activation process: an initial signal promoting expression of pro-IL-1β and a second signal involving Syk-dependent activation of the NLRP3 inflammasome and caspase-1, allowing processing of pro-IL-1β and secretion of mature IL-1β

Bacterial Load of Pneumococcal Serotypes Correlates with Their Prevalence and Multiple Serotypes Is Associated with Acute Respiratory Infections among Children Less Than 5 Years of Age

Background: Among pneumococcal serotypes, some serotypes are more prevalent in the nasopharynx than others; determining factors for higher prevalence remain to be fully explored. As non-vaccine serotypes have emerged after the introduction of 7-valent conjugate vaccines, study of serotype specific epidemiology is in need. When two or more serotypes co-colonize, they evolve rapidly to defend host\u27s immune responses; however, a clear association of cocolonization with a clinical outcome is lacking. Methods: Children less than 5 years old who were admitted to hospital due to acute respiratory infections (ARI) (n = 595) and healthy children (n = 350) were recruited. Carriage of pneumococcus was determined by culture and lytA PCR in the nasopharyngeal samples. Serotype/serogroup detection and its quantification were done by the nanofluidic real time PCR system. Spearman\u27s correlation and logistic regression were used to examine a correlation of serotype/serogroup specific bacterial load with its prevalence and an association of co-colonization with ARI respectively. Results: Serotype/serogroup specific bacterial load was correlated with its prevalence, both in ARI cases (Spearman\u27s rho = 0.44, n = 186; P<0.0001) and healthy children (Spearman\u27s rho = 0.41, n = 115; P<0.0001). The prevalence of multiple serotypes was more common in ARI cases than in healthy children (18.5% vs 7.1%; aOR 2.92, 95% CI: 1.27-6.71; P = 0.01). The dominant serotype in the co-colonization had a 2 log10 higher bacterial load than the subdominant serotype, both in ARI cases (P<0.001) and healthy children (P<0.05). Conclusions: High bacterial load in the nasopharynx may help transmit pneumococci among hosts, and increase the chance of successful acquisition and colonization. Co-colonization of multiple serotypes of pneumococci is linked with ARI, which infers the interactions of multiple serotypes may increase their pathogenicity; however, they may compete for growth in number

Identification of Disease-Promoting HLA Class I and Protective Class II Modifiers in Japanese Patients with Familial Mediterranean Fever

Objectives: The genotype-phenotype correlation of MEFV remains unclear for the familial Mediterranean fever (FMF) patients, especially without canonical MEFV mutations in exon 10. The risk of FMF appeared to be under the influence of other factors in this case. The contribution of HLA polymorphisms to the risk of FMF was examined as strong candidates of modifier genes. Methods: Genotypes of HLA-B and -DRB1 loci were determined for 258 mutually unrelated Japanese FMF patients, who satisfied modified Tel-Hashomer criteria, and 299 healthy controls. The effects of carrier status were evaluated for the risk of FMF by odds ratio (OR). The HLA effects were also assessed for clinical forms of FMF, subsets of FMF with certain MEFV genotypes and responsiveness to colchicine treatment. Results: The carriers of B?39:01 were increased in the patients (OR = 3.25, p = 0.0012), whereas those of DRB1?15:02 were decreased (OR = 0.45, p = 0.00050), satisfying Bonferroni\u27s correction for multiple statistical tests (n = 28, p<0.00179). The protective effect of DRB1?15:02 was completely disappeared in the co-existence of B?40:01. The HLA effects were generally augmented in the patients without a canonical MEFV variant allele M694I, in accordance with the notion that the lower penetrance of the mutations is owing to the larger contribution of modifier genes in the pathogenesis, with a few exceptions. Further, 42.9% of 14 colchicine-resistant patients and 13.5% of 156 colchicine-responders possessed B?35:01 allele, giving OR of 4.82 (p = 0.0041). Conclusions: The differential effects of HLA class I and class II polymorphisms were identified for Japanese FMF even in those with high-penetrance MEFV mutations