J. Van De Water

Detection of Autoantibodies to Recombinant Mitochondrial Proteins in Patients with Primary Biliary Cirrhosis

Primary biliary cirrhosis is characterized by the presence of autoantibodies to mitochondria with specific reactivity to proteins of 74 and 52 kilodaltons (kd). The 74-kd mitochondrial protein is the E2 component--dihydrolipoamide acetyltransferase--of the pyruvate dehydrogenase complex, and the 52-kd protein is the equivalent E2 component--dihydrolipoamide acyltransferase--of the branched-chain alpha-keto acid dehydrogenase complex. Current methods for the detection of antibodies to these proteins lack specificity or sensitivity, or they are time-consuming and not readily available. We therefore developed an enzyme-linked immunoassay to quantify specific antimitochondrial antibodies in patients with primary biliary cirrhosis. Recombinant polypeptides coding for both the 74-kd and the 52-kd mitochondrial autoantigens were used to analyze 217 coded serum samples, including samples from 93 patients with primary biliary cirrhosis and 124 controls, for reactivity by our immunoassay, immunoblotting, and immunofluorescence testing. Serum samples from 89 of the 93 patients with primary biliary cirrhosis reacted with either the pyruvate dehydrogenase-E2 or the branched-chain alpha-keto acid dehydrogenase protein. None of the 124 control samples from healthy volunteers (n = 86) or patients with primary sclerosing cholangitis (n = 38) had significant reactivity. Our results indicate that the use of recombinant, cloned autoantigens provides a simple, accurate, and rapid method of quantifying and monitoring the levels of specific mitochondrial autoantibodies in the serum of patients with primary biliary cirrhosis.

Molecular mimicry in primary biliary cirrhosis. Evidence for biliary epithelial expression of a molecule cross-reactive with pyruvate dehydrogenase complex-E2.

Sera from patients with primary biliary cirrhosis (PBC) react with enzymes of the 2-oxo dehydrogenase pathways, particularly PDC-E2. These enzymes are present in all nucleated cells, yet autoimmune damage is confined to biliary epithelial cells. Using a panel of eight mouse monoclonal antibodies and a human combinatorial antibody specific for PDC-E2, we examined by indirect immunofluorescence and confocal microscopy sections of liver from patients with PBC, progressive sclerosing cholangitis, and hepatocarcinoma. The monoclonal antibodies gave typical mitochondrial immunofluorescence on biliary epithelium and on hepatocytes from patients with either PBC, progressive sclerosing cholangitis, or hepatocarcinoma. However, one of eight mouse monoclonal antibodies (C355.1) and the human combinatorial antibody reacted with great intensity and specificity with the luminal region of biliary epithelial cells from patients with PBC. Simultaneous examination of these sections with an antiisotype reagent for human IgA revealed high IgA staining in the luminal region of biliary epithelial cells in patients with PBC. IgG and IgA antibodies to PDC-E2 were detected in the bile of patients with PBC but not normal controls. We believe that this data may be interpreted as indicating that a molecule cross-reactive with PDC-E2 is expressed at high levels in the luminal region of biliary epithelial cells in PBC.

Effect of Cocoa Flavanols and Their Related Oligomers on the Secretion of Interleukin-5 in Peripheral Blood Mononuclear Cells

We previously showed that flavanols and their related oligomers (FLO) isolated from cocoa can have immunomodulatory effects on production of the cytokines interleukin-1beta (IL-1beta), IL-2, and IL-4. In the present study, we examined whether selected FLO fractions isolated from cocoa (monomer through decamer) modulate IL-5 protein secretion from resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Although FLO fractions were unstimulatory for IL-5 secretion in resting cells, PHA-induced IL-5 release from PBMC was markedly affected by certain FLO fractions. The monomeric and small oligomeric (dimer and trimer) fractions enhanced PHA stimulation by 50%, 54%, and 43%, respectively. In contrast, the larger oligomeric fractions (hexamer through decamer) inhibited IL-5 release in the range of 18% to 39%; the tetramer and pentamer showed intermediate effects. The increment in IL-5 suggests that FLO may preferentially stimulate immunoglobulin A. We suggest that in the oral cavity this could result in reduction in the risk for dental caries and periodontal disease. This work offers additional data for consideration of the health benefits of dietary FLO from a variety of foods, including those benefits associated specifically with consumption of some cocoas and chocolates.

Effect of Cocoa Procyanidins on the Secretion of Interleukin-4 in Peripheral Blood Mononuclear Cells

Given the widespread ingestion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the antiinflammatory cytokine, interleukin-4 (IL-4). Both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) were investigated at the protein secretion level. The smaller-sized cocoa fractions (tetramer or less) were unable to induce an IL-4 response (i.e., values fell below the detection limit of 0.25 pg/ml). The larger oligomeric procyanidins (pentamer or greater) stimulated secretion of IL-4 in resting PBMC by as much as 1.42 pg/ml, as shown by the nonamer. However, only the hexameric, heptameric, and decameric fractions proved to be statistically significant. Cells coincubated with PHA showed an immense increase in secretory IL-4 (21.1 ± 1.1 pg/ml). Only the monomeric fraction was able to enhance PHA-induced secretion by 48%. The other procyanidin oligomers suppressed IL-4 production;...

Effect of spirulina on the secretion of cytokines from peripheral blood mononuclear cells.

ABSTRACT The purpose of this study was to evaluate the immunomodulatory activity of Spirulina, a bluegreen alga used as a food supplement. The effects of Spirulina on the secretion of three cytokines from unstimulated and stimulated human peripheral blood mononuclear cells (PBMC) were examined. In resting PBMC, Spirulina stimulated secretion of interleukin (IL)-1beta, IL-4, and interferon (IFN)-gamma to nearly 2.0, 3.3, and 13.6 times basal levels, respectively. Spirulina induced levels of IFN-gamma (229 +/- 104 pg/ml) that were comparable to those seen after phytohemagglutinin (PHA) stimulation (476 +/- 121 pg/ml). However, it was much less mitogenic than PHA (13.1 +/- 6.9 pg/ml) with respect to the induction of IL-4 secretion (0.34 +/- 0.1 pg/ml). In PHA-stimulated cells, Spirulina enhanced secretion of IL-1beta, IL-4, and IFN-beta by 2.9, 4.0., and 1.6 times, respectively. Although Spirulina stimulates several cytokines, it is clearly more effective in the generation of a Thl-type response. This in vitro study offers additional data for consideration of the potential therapeutic benefits of Spirulina.

Anti-brain antibodies are associated with more severe cognitive and behavioral profiles in Italian children with Autism Spectrum Disorder

Circulating 45 and 62kDa antibodies targeting the cerebellum were previously associated with Autism Spectrum Disorder (ASD), lower adaptive/cognitive function and aberrant behaviors. Moreover, 37, 39 and 73kDa maternal antibodies (mAb) targeting the fetal brain were previously correlated with broad autism spectrum, irritability, abnormal brain enlargement and impaired expressive language. The present study aims towards clinically characterizing individuals with brain-targeted IgG and/or exposed to maternal antibrain antibodies in a large sample of Italian autistic children (N=355), their unaffected siblings (N=142) and mothers (N=333). The presence of patient- and mother-produced anti-brain antibodies does not confer increased risk of autism within the same sibship. However, the 45 and 62kDa antibodies are correlated with autism severity: the 45kDa Ab is associated with cognitive impairment and lower scores at the Vineland Adaptive Behavior Scales, the 62kDa Ab with motor stereotypies, while both correlate with larger head circumference (all P<0.05). On the other hand, maternal 37, 39 and 73kDa antibrain antibodies, either alone or in combination, are correlated with impaired verbal and non-verbal language development, neurodevelopmental delay and sleep/wake cycle disturbances in their autistic children (P<0.05). Presence of the 62kDa autoAb in the child is significantly associated with presence of the 39 and/or 73kDa antibodies in his/her mother. Our results confirm and extend previous observations in an ethnically distinct sample, providing further evidence of a pathomorphic role for anti-brain antibodies in autism while demonstrating their familial clustering.

Selective abnormalities in the thymic microenvironment associated with avian scleroderma, an inherited fibrotic disease of L200 chickens.

As a prelude to deciphering the mechanisms of intrathymic T-cell maturation we produced a panel of 18 monoclonal antibodies (mAbs) against chicken thymic stromal elements. Eleven of these detected epithelial cells. They were: pan-epithelial; subcapsule and peri-vascular (pan type 1 epithelium); subcapsular, perivascular and medulla; medulla; or cortex. Of particular interest were the sub-specificities within these regions, especially the subcapsular region. Four mAbs stained both epithelial and non-epithelial cells in discrete regions. In addition, three mAbs recognized only non-epithelial cells. One identified macrophages scattered throughout the thymus, another the connective tissue and another the medullary vascular endothelium. These reagents have provided an extensive profile of the thymic stromal architecture and revealed that these cells are equally as complex as the T cells whose differentiation they induce and regulate. While the mAbs provide a valuable means for studying the mechanisms of normal thymopoiesis, their clinical significance is unknown. UCD line 200 chickens develop an autoimmune disease manifest by dermal and internal organ fibrosis, T cell infiltrates of skin and other affected organs and production of multiple autoantibodies. We have used our panel of mAbs to evaluate the thymic microenvironment in these autoimmunity-prone chickens. A comparative analysis with control chickens revealed striking deficiencies in the L200 subcapsular regions coupled with excessive expression of MHC class II antigens, particularly in the cortex. We hypothesize that these abnormalities induce altered T-cell differentiation, thereby predisposing the L200 chickens to autoimmune disease.

CD8+ T cell function in children with autism

389 Psychosocial and life style influences on the genetics of inflammaging in older humans L. Coe , G. Love , J.A. Morozink , E.M. Friedman , C.D. Ryff b a University of Wisconsin, Harlow Center for Biological Psychology, Madison, WI 53715, United States b University of Wisconsin, Institute on Aging, Madison, WI 53706, United States c University of Wisconsin, Population Health, Madison, WI 53726, United

Abstract # 1787 Unique immune profiles in children with autism who experience gastrointestinal co-morbidity

Children with an autism spectrum disorder (ASD) are 6–8 times more likely to have persistent gastrointestinal (GI) symptoms. In addition, intestinal and peripheral inflammation, altered microbiome profiles, and impaired intestinal permeability have also been observed in children with ASD who exhibit GI issues. To further assess immune dysfunction and relationship to behavior in children with ASD and GI co-morbidities, we analyzed samples from children placed into the following study groups based on responses from standardized GI questionnaires: children with ASD who experience GI symptoms (ASDGI), children with ASD without a history of GI symptoms (ASDNoGI), or typically developing (TD) children. Cytokine analysis of supernatants collected from stimulated peripheral blood mononuclear cells, revealed elevated innate immune responses from ASDNoGI including increased levels of IL-1alpha, IL-1beta and TNFalpha after stimulation with Toll-Like receptor (TLR)-4 ligands compared to TD. The ASDGI group produced increased levels of mucosa-relevant cytokines IL-5, IL-15 and IL-17 compared to ASDNoGI following TLR-4 stimulation. Interestingly, the production of regulatory cytokine, TGFbeta1, was decreased across all stimulatory conditions in ASDGI compared to either ASDNoGI or TD groups. Furthermore, ASDGI scored worse on the Aberrant Behaviors Checklist than ASDNoGI. Overall, data from this study found evidence of reduced regulatory cytokines and increased mucosa-related cytokines in ASDGI which suggest that children with ASD and GI symptoms have an imbalance between regulatory and inflammatory pathways.

The potential role of the maternal immune response in autism

496 The potential role of the maternal immune response in autism J. Van de Water, D. Braunschweig, P. Ashwood University of California, Davis, 451 Health Sciences Dr., Suite 6510 GBSF, Davis, CA 95616, United States The role of the gestational maternal immune response neurodevelopment is currently of great interest. This presentation will focus on the recent findings describing one particular area of interest, the presence of maternal antibodies to fetal brain proteins in the mothers of children with autism and their potential role in the pathophysiology of this disorder. We reported that some mothers of children with autism have autoantibodies in their blood that react to fetal brain proteins. Thus, the presence of anti-fetal brain antibodies in some mothers of children with autism raises the possibility that a subset of autism cases may be caused by a pathogenic maternal IgG antibody response directed against the fetal brain during gestation. The pathologic significance of this antibody was demonstrated in Rhesus monkeys who were prenatally exposed to passive intravenous transfer of IgG class antibodies purified from mothers of children with autism. Those offspring exposed to the autism IgG produced more whole body motor stereotypies compared to those exposed to control IgG. However, the identity of the brain antigens as well as the mechanism by which the maternal immune system breaks tolerance to produce antibodies that recognize fetal brain proteins remains to be addressed. doi:10.1016/j.bbi.2010.07.218