Anti-Group A Streptococcal Vaccine Epitope STRUCTURE, STABILITY, AND ITS ABILITY TO INTERACT WITH HLA CLASS II MOLECULES

Streptococcus pyogenes infections remain a health problem in several countries due to poststreptococcal sequelae. We developed a vaccine epitope (StreptInCor) composed of 55 amino acids residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. The nuclear magnetic resonance (NMR) structure of the StreptInCor peptide showed that the structure was composed of two microdomains linked by an 18-residue alpha-helix. A chemical stability study of the StreptInCor folding/unfolding process using far-UV circular dichroism showed that the structure was chemically stable with respect to pH and the concentration of urea. The T cell epitope is located in the first microdomain and encompasses 11 out of the 18 alpha-helix residues, whereas the B cell epitope is in the second microdomain and showed no alpha-helical structure. The prediction of StreptInCor epitope binding to different HLA class II molecules was evaluated based on an analysis of the 55 residues and the theoretical possibilities for the processed peptides to fit into the P1, P4, P6, and P9 pockets in the groove of several HLA class II molecules. We observed 7 potential sites along the amino acid sequence of StreptInCor that were capable of recognizing HLA class II molecules (DRB1*, DRB3*, DRB4*, and DRB5*). StreptInCoroverlapping peptides induced cellular and humoral immune responses of individuals bearing different HLA class II molecules and could be considered as a universal vaccine epitope.

Cutaneous vasculitis in systemic lupus erythematosus: association with anti-ribosomal P protein antibody and Raynaud phenomenon

Ninety-one consecutive systemic lupus erythematosus (SLE) patients (American College of Rheumatology criteria) with a history of cutaneous vasculitis were compared to 163 SLE controls without this clinical manifestation from July to December 2007 in order to determine the possible clinical and serological association of this manifestation. Data were obtained in an ongoing electronic database protocol and autoantibodies to anti-double-stranded DNA, anti-Sm, anti-RNP, anti-Ro/SS-A, anti-La/SS-B, and anticardiolipin and ribosomal P protein antibody (anti-P) were detected by standard techniques. Exclusion criteria were the presence of anti-phospholipid syndrome or antibodies, Sjogren syndrome, and a history of thrombosis. The mean age (38.5 +/- 11.5 vs. 37.8 +/- 11.6 years, p = 0.635), disease duration (12.5 +/- 7.8 vs. 11.8 +/- 7.9 years, p = 0.501), and frequency of white race (71.4% vs. 70.5%, p = 0.872) and female sex (96.8% vs. 93.7%, p = 0.272) were comparable in both groups. The vasculitis group had a higher frequency of malar rash (97.9% vs. 87.4%, p = 0.004), photosensitivity (91.4% vs. 81.6%, p = 0.030), and Raynaud phenomenon (RP; 27.7% vs. 7.5%, p < 0.001), whereas all other clinical manifestation including renal and central nervous system involvements were similar to the control group. Laboratorial data revealed that only anti-P (35.1% vs. 12.1%, p < 0.001) was more frequent in patients with vasculitis. In a multivariate logistic regression model, cutaneous vasculitis was associated to the presence of RP (OR = 3.70; 95% confidence interval [CI] = 1.73-8.00) and anti-P (OR = 3.42; 95% CI = 1.76-6.66). In summary, SLE cutaneous vasculitis characterizes a subgroup of patients with more RP and anti-P antibodies but not accompanied by a higher frequency of renal and central nervous system involvements.

Late response to anti-TNF-alpha therapy in refractory mucocutaneous lesions of Beh double dagger et`s disease

Beh double dagger et`s disease (BD) is a multisystem chronic inflammatory disorder characterized by oral and genital ulceration and ocular involvement. Recurrent oral and genital ulcers are the most common symptoms of BD and occur in more than 80% of patients. The treatments of these disease manifestations include colchicine, corticosteroids and immunosuppressive drugs in severe cases. Anti-TNF-alpha therapy may be useful in refractory severe BD, particularly for ocular, central nervous system, gastrointestinal and refractory mucocutaneous lesions. During a 2-year period, 280 patients suffering from rheumatic diseases received anti-TNF-alpha agents at the infusion center of our University Hospital. Two patients (0.7%) presented BD; one of them had celiac disease as well, with recalcitrant mucocutaneous lesions that were not responsive to immunosuppressive drugs. We reported those patients who were successfully treated with infliximab and adalimumab, despite their late response.

Anti-inflammatory effect of bee venom on antigen-induced arthritis in rabbits: Influence of endogenous glucocorticoids

Aim of the study: This study assessed the involvement of endogenous glucocorticoids (GCs) in the anti-arthritic properties of bee venom (BV) on antigen-induced arthritis (AIA) in rabbits. Materials and methods: BV (1.5-6 mu g/kg/day) was injected for 7 days before AIA induction, whereas the control group received sterile saline. The total and differential leukocyte count. PGE(2) levels in synovial fluid and synovial membrane cell infiltrate were evaluated. The contribution of GCs to BV action was assessed in rabbits treated with BV plus metyrapone, an inhibitor of GC synthesis, or RU-38 486, a steroid antagonist. Results: Treatment with BV (1.5 mu g/kg/day) reduced the leukocyte count and PGE2 level (18571 +/- 1909 cells/mm(3) and 0.49 +/- 0.05 ng/mL, respectively) as well as the cellular infiltrate compared with the control group (40968 +/- 5248 cells/mm(3) and 2.92 +/- 0.68 ng/mL, p < 0.05). The addition of metyrapone to BV treatment completely reversed the inhibition of AIA, whereas RU-38 486 was ineffective. Conclusion: Our data show that bee venom treatment prevents the development of antigen-induced arthritis in rabbits through the action of GCs. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Pentoxifylline reduces pro-inflammatory and increases anti-inflammatory activity in patients with coronary artery disease - A randomized placebo-controlled study

The balance between different immunological stimuli is essential in the progression and stabilization of atherosclerotic plaques. Immune regulation has been suggested as potential target for the treatment of atherosclerotic disease. We sought to determine whether treatment with pentoxifylline, a phosphodiesterase inhibitor with immunomodulating properties, could reduce the pro-inflammatory response observed in patients with acute coronary syndromes (ACS) and increase anti-inflammatory activity. In a double-blind, prospective, placebo-controlled study, 64 patients with ACS were randomized to receive pentoxifylline 400 mg TID or placebo for 6 months. Analysis of the pro-inflammatory markers, Greactive protein (CRP), interleukin (IL)-6, IL-12, interferon-gamma and tumor necrosis factor (TNF)-alpha and the anti-inflammatory cytokines, transforming growth factor (TGF)-beta 1 and IL-10 were done at baseline, 1 and 6 months. Pentoxifylline treatment significantly reduced the adjusted levels of CRP and TNF-alpha compared to placebo after 6 months (P=0.04 and P < 0.01, respectively). IL-12 increase was significantly less pronounced with pentoxifylline (P=0.04). The levels of the anti-inflammatory cytokine, IL-10, also declined significantly less in the pentoxifylline group compared to placebo (P < 0.01) with a trend towards a higher increase of TGF-beta 1 in the former group (P=0.16). Pentoxifylline reduces pro-inflammatory and increases anti-inflammatory response in patients with ACS and may have beneficial clinical effects on cardiovascular events. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Clinicopathological evaluation of in vivo epidermal nuclear fluorescence

Nuclear fluorescence in keratinocytes is an occasional phenomenon, often present in autoimmune diseases, especially in connective-tissue disease (CTD); however, its clinical significance remains unclear. To investigate the profile of patients with positive nuclear staining on direct immunofluorescence (DIF) of skin samples. A retrospective analysis of 28 patient records from our immunodermatology laboratory was performed between May 2003 and June 2006. Inclusion criteria were the presence of autoantibodies (IgG, IgA or IgM) or complement (C3) binding keratinocyte nuclei on DIF. The most prevalent diseases related to the nuclear keratinocyte DIF staining were systemic lupus erythematosus (n = 9), mixed CTD (n = 3), overlap syndrome (n = 3), Sjogren`s syndrome (n = 1), and CREST (calcinosis, Raynaud`s phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia) syndrome (n = 1). Serum antinuclear antibody (ANA) was positive in 20 of 28 patients, with titres varying from 1 : 160 to 1 : 1280. Of the 20 patients with positive anti-nuclear antibodies (ANA), 17 were positive for anti-extractable nuclear antigen antibodies, 12 had anti-SSA/Ro, 11 had anti-SSB/La and 8 had anti-ribonucleoprotein. Eight patients were negative for ANA. Positive predictive value of in vivo ANA for systemic CTDs was 75%. The present data suggest that in vivo ANA evaluation is an additional and feasible auxiliary tool for diagnosing CTDs.

Aerobic training reverses airway inflammation and remodelling in an asthma murine model

Aerobic training (AT) decreases dyspnoea and exercise-induced bronchospasm, and improves aerobic capacity and quality of life; however, the mechanisms for such benefits remain poorly understood. The aim of the present study was to evaluate the AT effects in a chronic model of allergic lung inflammation in mice after the establishment of airway inflammation and remodelling. Mice were divided into the control group, AT group, ovalbumin (OVA) group or OVA+AT group and exposed to saline or OVA. AT was started on day 28 for 60 min five times per week for 4 weeks. Respiratory mechanics, specific immunoglobulin (Ig)E and IgG(1), collagen and elastic fibres deposition, smooth muscle thickness, epithelial mucus, and peribronchial density of eosinophils, CD3+ and CD4+, IL-4, IL-5, IL-13, interferon-gamma, IL-2, IL-1ra, IL-10, nuclear factor (NF)-kappa B and Foxp3 were evaluated. The OVA group showed an increase in IgE and IgG1, eosinophils, CD3+, CD4+, IL-4, IL-5, IL-13, NF-kappa B, collagen and elastic, mucus synthesis, smooth muscle thickness and lung tissue resistance and elastance. The OVA+AT group demonstrated an increase of IgE and IgG(1), and reduction of eosinophils, CD3+, CD4+, IL-4, IL-5, IL-13, NF-kappa B, airway remodelling, mucus synthesis, smooth muscle thickness and tissue resistance and elastance compared with the OVA roup (p < 0.05). The OVA+AT group also showed an increase in IL-10 and IL-1ra (p < 0.05), independently of Foxp3. AT reversed airway inflammation and remodelling and T-helper cell 2 response, and improved respiratory mechanics. These results seem to occur due to an increase in the expression of IL-10 and IL-1ra and a decrease of NF-kappa B.

Pro- and Anti-inflammatory Cytokines in Steatosis and Steatohepatitis

Fatty liver disease is a problem in both bariatric patients and in patients with moderate obesity. Tumor necrosis factor (TNF)-alpha has been frequently measured in nonalcoholic steatohepatitis (NASH) with or without diabetes, but less is known about interleukin (IL)-6 and IL-10. Moderately obese patients (n = 80) with histologically proven steatosis (n = 29) and NASH (n = 51) were recruited. Serum levels of cytokines were documented along with clinical information. The aim was to identify the correlates of such biomolecules in a stable population. Diabetes tended to be more associated with NASH (52.5% instead of 41.4%, P = 0.015), with no difference of age, gender, or body mass index regarding steatosis. For the entire population, cytokine changes were not significant, including TNF-alpha and IL-6. In diabetics only, all markers tended to diminish with NASH, especially IL-10 (P = 0.000). IL-10 correlated with homeostatic model assessment index (P = 0.000) and other variables of glucose homeostasis in diabetes, thus representing a major marker of the disease. (1) Generally inconsistent changes in pro- and anti-inflammatory cytokines occurred when NASH was globally compared to steatosis. (2) In contrast, downregulation of IL-6 and IL-10 was perceived in diabetics with NASH. (3) Arterial hypertension did not play a role in these circumstances. (4) IL-10 maintained strong correlations with glucose metabolism indices. (5) TNF-alpha could not be incriminated for progressive liver damage, as values failed to increase in NASH. (6) Investigations of IL-10 and other counterregulatory cytokines are lacking in this context and deserve further studies.

Performance of Two Commercial ELISAs for Detecting IgA Anti-Human and Anti-Guinea Pig Tissue Transglutaminase Antibodies

Background: Sensitivity and specificity of anti-human tissue transglutaminase antibodies (anti-htTGA) seem to be superior to those of anti-tissue transglutaminase of guinea pig (anti-gptTGA) for screening patients with celiac disease (CD), but there are still controversies. The aim of this study was to evaluate the performance of two INOVA ELISA kits to detect IgA anti-htTGA and anti-gptTGA in patients with and without CD. Methods: The study groups were comprised of 49 anti-endomysial antibody (EMA)-positive untreated-CD, and 123 controls (EMA-negative treated CD, EMA-negative chronic diarrhea, autoimmune hepatitis, inflammatory bowel disease and healthy people). Results: The agreement between the two ELISAs was statistically significant in all study groups and there was no significant difference between them (92.7% agreement; kappa=0.70; kappa p=0.001; McNemar p=1). All patients with serum reactivity of more than 100 units had histologic diagnosis of CD. In seven of 10 patients with treated-CD who had control biopsies, villous atrophy was still present in four who tested positive by both kits. Two of three celiacs with histologic remission tested positive for both anti-tTGA. Conclusions: the anti-gptTGA and anti-htTGA determination were equally efficient in identifying patients with untreated-CD with high titers of EMA. Whatever the anti-tTGA ELISA used, the reactivity above 100 units was always related to active CD diagnosed by histologic alterations in intestinal biopsies. The anti-tTGA reactivity by both kits was not only similar in determining histologic activity in the follow-up of CD after a gluten free diet, but also in identifying positive sera from the control groups, regardless if CD has been confirmed by duodenal biopsies. (Clin. Lab. 2010;56:29-35)

Profile of Autoantibodies Against Phosphorylcholine and Cross-reactivity to Oxidation-Specific Neoantigens in Selective IgA Deficiency With or Without Autoimmune Diseases

Immunoglobulin A deficiency (IgAD) is considered the most common form of primary immunodeficiency. The majority of IgA-deficient individuals are considered asymptomatic, even though IgAD has been associated with an increased frequency of recurrent infections, allergy, and autoimmune diseases. In this study we evaluate the Natural autoantibodies (NatAbs) reactivity to phosphorylcholine (PC) and to some pro-inflammatory molecules in IgAD with or without autoimmune disorders. We observed that in the absence of IgA there is an enhancement of IgG subclasses functioning as NatAbs against PC. Immunoglobulin G (IgG) against lipopolysaccharide, C-reactive protein, and IgA was found in IgAD, regardless of the autoimmune manifestations. Nonetheless, IgAD patients with autoimmune disease showed significantly higher IgG reactivity against pro-inflammatory molecules, such as cardiolipin, oxidized low-density lipoproteins, and phosphatidylserine, with positive correlation between them. In conclusion, the IgG NatAbs against PC may represent a compensatory defense mechanism against infections and control excess of inflammation, explaining the asymptomatic status in the IgA deficiency.

Passive Acquisition of Protective Antibodies Reactive with Bordetella pertussis in Newborns via Placental Transfer and Breast-feeding

Although acquisition of anti-pertussis antibodies by the newborn via placental transfer has been demonstrated, a subsequent recrudescence of pertussis infection is often observed, particularly in infants. The present study investigated the passive transfer of anti-pertussis IgG and IgA antibodies to term newborns and their ability to neutralize bacterial pathogenicity in an in vivo experimental model using mice intracerebrally challenged with viable Bordetella pertussis. Forty paired samples of maternal/umbilical cord sera and colostrum were obtained. Anti-pertussis antibodies were analysed by immunoenzymatic assay and by Immunoblotting. Antibody neutralizing ability was assessed through intracerebral B. pertussis challenges in mice. Anti-pertussis IgG titres were equivalent in both maternal and newborn sera (medians = 1:225 and 1:265), with a transfer rate of 118%. The colostrum samples had variable specific IgA titres (median = 1:74). The immunoblotting assays demonstrated identical recognition profiles of paired maternal and newborn serum pools but different bacterial recognition intensities by colostrum pools. In the animal model, significant differences were always observed when the serum and colostrum samples and pools were compared with the positive control (P < 0.05). Unlike samples with lower anti-pertussis titres, samples with high titres showed protective capacities above 50%. Pertussis-absorbed serum and colostrum pools protected 30% of mice and purified IgG antibodies protected 65%. Both pooled and single-sample protective abilities were correlated with antibody titres (P < 0.01). Our data demonstrated the effectiveness of anti-pertussis antibodies in bacterial pathogenesis neutralization, emphasizing the importance of placental transfer and breast-feeding in protecting infants against respiratory infections caused by Bordetella pertussis.

Acquisition of Serum Antibodies Reactive With Enterohemorrhagic Escherichia coli Virulence-Associated Factors by Healthy Brazilian Children and Adults

Background: Patients with hemorrhagic colitis or hemolytic uremic syndrome due to enterohemorrhagic Escherichia coli (EHEC) develop serum IgM and IgG response to lipopolysaccharide (LPS) and to virulence factors such as intimin. The small numbers of cases of diarrhea associated with EHEC strains in Brazil suggests a pre-existing immunity probably due to previous contact with diarrheagenic E. coli. Our aim was to evaluate the development of the serum antibody repertoire to EHEC virulence factors in Brazilian children and adults. Methods: Serum IgM and IgG antibodies were determined by enzyme-linked immunosorbent assay with LPS O111, LPS O26, and LPS O157 in 101 children between 2 months and 10 years of age and in 100 adult sera, by immunoblotting with protein membrane extracts and purified beta intimin; the ability of adult sera to neutralize Shiga toxin2 was also investigated. Results: Children older than 24 months had IgM concentrations reactive with the 3 LPS equivalent to those seen in the adult group, and significantly higher than the group of younger children (P < 0.05). Anti-O26 and anti-O157 LPS IgG concentrations were equivalent between the 2 groups of children and were significantly different from the adult group (P < 0.05). The anti-O111 LPS IgG levels in older children were intermediate between the younger group, and adults (P < 0.05). Immunoblotting revealed strong protein reactivity, including the conserved and variable regions of beta intimin and more than 50% of the adult samples neutralized Shiga toxin 2. Conclusions: Our results demonstrate an increasing anti-LPS and antiprotein antibody response with age, which could provide protection against EHEC infections.

Anti-C1q Antibodies in Juvenile-Onset Systemic Lupus Erythematosus

The objective of this study was to evaluate the presence of anti-C1q antibodies Hospital Israelita Albert Einstein Research Institute, Sao Paulo, Brazil in 67 juvenile Systemic lupus erythematosus (JSLE) patients and 26 healthy controls and to assess the association of these antibodies with disease activity, nephritis, and presence of anti-double-stranded (ds)DNA. Anti-C1q antibodies were detected by ELISA. A higher frequency of anti-C1q antibodies was observed in JSLE patients compared to controls (20% vs. 0%, P = 0.016). Specificity of these antibodies was 100% [95% confidence interval (CI) 86.7-100%] and sensitivity was 19.4% (95% CI 10.7-30.8%) for a lupus diagnosis. The median anti-C1q antibodies was higher in JSLE patients compared to controls [median (range) 9.4 (5.5-127) vs. 7.3 (5-20) units, P = 0.004]. Remarkably, a positive Spearman`s coefficient was found between anti-dsDNA and anti-C1q units (r = 0.42, P = 0.0004, 95% CI 0.19-0.60). Our results confirm a low frequency of anti-C1q antibody in our lupus populations, but the presence of anti-C1q antibodies appears to be a good marker for JSLE diagnosis.

The spectrum of autoantibodies in IPEX syndrome is broad and includes anti-mitochondrial autoantibodies

IPEX syndrome is a congenital disorder of immune regulation caused by mutations in the FOXP3 gene, which is required for the suppressive function of naturally arising CD4 + CD25 + regulatory T cells. In this case series we evaluated serum samples from 12 patients with IPEX syndrome for the presence of common autoantibodies associated with a broad range of autoimmune disorders. We note that 75% of patients (9/12) had 1 or more autoantibodies, an incidence far above the cumulative rate observed in the general population. The range of autoantibodies differed between patients and there was no predominant autoantibody or pattern of autoantibodies present in this cohort. Surprisingly, one patient had high-titer anti-mitochondrial antibodies (AMA) typically associated with primary biliary cirrhosis (PBC) although the patient had no signs of cholestasis. PBC is a well-characterized autoimmune disease that occurs primarily in women and includes the serological hallmarks of serum AMA and elevated IgM which were both present in this patient. PBC is virtually absent in children with the exception of one reported child with interleukin 2 receptor a (CD25) deficiency which is associated with an IPEX-like regulatory T cell dysfunction. Based on the present data and the available literature we suggest a direct role for CD4 + CD25 + regulatory T cells in restraining B cell autoantibody production and that defects in regulatory T cells may be crucial to the development of PBC. (C) 2010 Elsevier Ltd. All rights reserved.

Quality control of Toxoplasma gondii in meat packages: Standardization of an ELISA test and its use for detection in rabbit meat cuts

Toxoplasma gondii causes severe disease both to man and livestock and its detection in meat after slaughtering requires PCR or biological tests. Meat packages contain retained exudate that could be used for serology due to its blood content. Similar studies reported false negative assays in those tests. We standardized an anti-T. gondii IgG ELISA in muscle juices from experimentally infected rabbits, with blood content determination by cyanhemoglobin spectrophotometry. IgG titers and immunoblotting profiles were similar in blood, serum or meat juice, after blood content correction. These assays were adequate regardless of the storage time up to 120 days or freeze-thaw cycles, without false negative results. We also found 1.35% (1/74) positive sample in commercial Brazilian rabbit meat cuts, by this assay. The blood content determination shows ELISA of meat juice may be useful for quality control for toxoplasmosis monitoring. (C) 2011 Elsevier Ltd. All rights reserved.