PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD

Lipid Metabolism in Subclinical Hypothyroidism: Plasma Kinetics of Triglyceride-Rich Lipoproteins and Lipid Transfers to High-Density Lipoprotein Before and After Levothyroxine Treatment

Background: Subclinical hypothyroidism (SCH) has been associated with atherosclerosis, but the abnormalities in plasma lipids that can contribute to atherogenesis are not prominent. The aim of this study was to test the hypothesis that patients with normocholesterolemic, normotriglyceridemic SCH display abnormalities in plasma lipid metabolism not detected in routine laboratory tests including abnormalities in the intravascular metabolism of triglyceride-rich lipoproteins, lipid transfers to high-density lipoprotein (HDL), and paraoxonase 1 activity. The impact of levothyroxine (LT4) treatment and euthyroidism in these parameters was also tested. Methods: The study included 12 SCH women and 10 matched controls. Plasma kinetics of an artificial triglyceride-rich emulsion labeled with radioactive triglycerides and cholesteryl esters as well as in vitro transfer of four lipids from an artificial donor nanoemulsion to HDL were determined at baseline in both groups and after 4 months of euthyroidism in the SCH group. Results: Fractional clearance rates of triglycerides (SCH 0.035 +/- 0.016 min(-1), controls 0.029 +/- 0.013 min(-1), p=0.336) and cholesteryl esters (SCH 0.009 +/- 0.007 min(-1), controls 0.009 +/- 0.009 min(-1), p=0.906) were equal in SCH and controls and were unchanged by LT4 treatment and euthyroidism in patients with SCH, suggesting that lipolysis and remnant removal of triglyceride-rich lipoproteins were normal. Transfer of triglycerides to HDL (SCH 3.6 +/- 0.48%, controls 4.7 +/- 0.63%, p=0.001) and phospholipids (SCH 16.2 +/- 3.58%, controls 21.2 +/- 3.32%, p=0.004) was reduced when compared with controls. After LT4 treatment, transfers increased and achieved normal values. Transfer of free and esterified cholesterol to HDL, HDL particle size, and paraoxonase 1 activity were similar to controls and were unchanged by treatment. Conclusions: Although intravascular metabolism of triglyceride-rich lipoproteins was normal, patients with SCH showed abnormalities in HDL metabolism that were reversed by LT4 treatment and achievement of euthyroidism.

Abnormal collagen deposition in synovia after collagen type V immunization in rabbits

Sinovitis in Scleroderma (SSc) is rare, usually aggressive and fully resembles rheumatoid arthritis. Experimental models of SSc have been used in an attempt to understand its pathogenesis. Previous studies done in our laboratory had already revealed the presence of a synovial remodeling process in rabbits immunized with collagen V. To validate the importance of collagen type V and to explore the quantitative relationship between this factor and synovia remodeling as well as the relationship between collagen type V and other collagens, we studied the synovial tissue in immunized rabbits. Rabbits (N= 10) were immunized with collagen V plus Freund's adjuvant and compared with animals inoculated with adjuvant only (N= 10). Synovial tissues were submitted to histological analysis, immunolocalization to collagen I, III and V and biochemical analysis by eletrophoresis, immunoblot and densitometric method. The synovial tissue presented an intense remodeling process with deposits of collagen types I, III and V after 75 and 120 days of immunization, mainly distributed around the vessels and interstitium of synovial extracellular matrix. Densitometric analysis confirmed the increased synthesis of collagen I, III and V chains (407.69 +/- 80.31; 24.46 +/- 2.58; 70.51 +/- 7.66, respectively) in immunized rabbits when compared with animals from control group (164.91 +/- 15.67; 12.89 +/- 1.05; 32 +/- 3.57) (p<0.0001). We conclude that synovial remodeling observed in the experimental model can reflect the articular compromise present in patients with scleroderma. Certainly, this experimental model induced by collagen V immunization will bring new insights in to pathogenic mechanisms and allow the testing of new therapeutic strategies to ameliorate the prognosis for scleroderma patients.

Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

Background: The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGFbeta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods: Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V ( 25 mu g/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05. Results: IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 +/- 0.118 vs. 0.874 +/- 0.282, p < 0.001), bronchioles (0.294 +/- 0.139 vs. 0.646 +/- 0.172, p < 0.001) and in the septal interstitium (0.027 +/- 0.014 vs. 0.067 +/- 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 +/- 0.07 vs. 1.0 +/- 0.528, p = 0.002) and V (1.12 +/- 0.42 vs. 4.74 +/- 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression ( p < 0.0001). Conclusions: Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

Relatedness and HLA-DRB1 typing may discriminate the magnitude of the genetic susceptibility to tuberculosis using a household contact model

Background Tuberculosis clusters in families may be due to increased household exposure, shared genetic factors, or both. Household contact studies are useful to control exposure because socioeconomic and environmental conditions are similar to all subjects, allowing the evaluation of the contribution of relatedness to disease development. Methods In this study, the familial aggregation of tuberculosis using relatedness and a specific inherited marker (HLA-DRB1) was evaluated. Fifty families, which had at least two cases of tuberculosis diagnosed within the past 5 years, were selected from a cohort of tuberculosis carried out in Recife, Brazil. The first case diagnosed was considered to be a primary case. The secondary attack rate of tuberculosis in household contacts was estimated according to the degree of relatedness. The relative risk of having tuberculosis based on the degree of relatedness household and the population attributable fraction to relatedness were also estimated. HLA-DRB1 typing and attributable etiologic/preventive fractions were calculated among sick and healthy household contacts. Results Compared to unrelated contacts, the relative risk for tuberculosis adjusted for age was 1.38 (95% CI 0.86 to 2.21). Relatedness contributed 23% to the development of tuberculosis at the population levels. The HLA-DRB1*04 allele group (OR = 2.44; p =0.0324; etiologic fraction =0.15) was overrepresented and the DRB1*15 allele group (OR=0.48; p=0.0488; protective fraction=0.19) was underrepresented among household contacts exhibiting tuberculosis. The presence of DRB1 shared alleles between primary cases and their contacts was a risk factor for tuberculosis (p=0.0281). Conclusion This household contact model together with the utilisation of two genetic variables permitted the evaluation of genetic factors contributing towards tuberculosis development.

Administration of M. leprae Hsp65 Interferes with the Murine Lupus Progression

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K(409)A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F(1) mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F1 serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K(409)A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K(409)A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process.

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10. RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+)IL-17(+), CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+)IFN-gamma(+) and CD4(+)IL-17(+) T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Continuous monitoring of ascorbate transport through neuroblastoma cells with a ruthenium oxide hexacyanoferrate modified microelectrode

The uptake of ascorbate by neuroblastoma cells using a ruthenium oxide hexacyanoferrate (RuOHCF)-modified carbon fiber disc (CFD) microelectrode (r = 14.5 mu m) was investigated. By use of the proposed electrochemical sensor the amperometric determination of ascorbate was performed at 0.0 V in minimum essential medium (MEM, pH = 7.2) with a limit of detection of 25 mu mol L(-1). Under the optimum experimental conditions, no interference from MEM constituents and reduced glutathione (used to prevent the oxidation of ascorbate during the experiments) was noticed. The stability of the RuOHCF-modified electrode response was studied by measuring the sensitivity over an extended period of time (120 h), a decrease of around 10% being noticed at the end of the experiment. The rate of ascorbate uptake by control human neuroblastoma SH-SY5Y cells, and cells transfected with wild-type Cu,Zn-superoxide dismutase (SOD WT) or with a mutant typical of familial amyotrophic lateral sclerosis (SOD G93A), was in agreement with the level of oxidative stress in these cells. The usefulness of the RuOHCF-modified microelectrode for in vivo monitoring of ascorbate inside neuroblastoma cells was also demonstrated.

Suppressive action of melatonin on the TH-2 immune response in rats infected with Trypanosoma cruzi

Control of the acute phase of Trypanosoma cruzi infection is critically dependent on cytokine-mediated macrophage activation to intracellular killing, natural killer (NK) cells, CD4(+) T cells, CD8(+) T cells and B cells. Cell-mediated immunity in T. cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Here we studied the role of cytokines in the regulation of innate and adaptive immunity during the acute phase of T. cruzi infection in Wistar rats. Melatonin is an effective regulator of the immune system. Macrophages and T lymphocytes, which have melatonin receptors, are target cells for the immunomodulatory function of melatonin. In this paper melatonin was orally given via two protocols: prior to and concomitant with infection. Both treatments were highly effective against T. cruzi with enhanced action for the concomitant treatment. The data suggest an up-regulation of the TH-1 immune response as all analyzed parameters, interleukin (IL)-4, IL-10, transforming growth factor-beta 1 and splenocyte proliferation, displayed reduced levels as compared with the untreated counterparts. However, the direct effects of melatonin on immune cells have not been fully investigated during T. cruzi infection. We conclude that in light of the current results, melatonin exerted important therapeutic benefits through its immune regulatory effects.

Influence of melatonin therapy and orchiectomy on T cell subsets in male Wistar rats infected with Trypanosoma cruzi

Gonadal steroids exert an important influence on the host immune response during infection. Changes resulting from the absence or replacement of gonadal hormones may represent a distinct evolution of a particular parasite. Taking into account the greater susceptibility of males to parasites, the magnitude of the immune response seems to depend on the interaction of many hormones that will act synergistically with other immune cells. The aims of this research were to evaluate the effects of the luck of male sex hormones due to orchiectomy, and the influence of oral administration of melatonin on the immune response of male Wistar rats infected with the Y strain of Trypanosoma cruzi. The percentage of CD3(+) CD4(+) and CD3(+) CD8(+) lymphocyte T cell subsets were evaluated using flow cytometry and the measurement of IL-2 and IL-12. For all parameters examined, a synergistic action of melatonin and orchiectomy on the host`s immune response was observed, promoting an effective response against the parasite during the acute phase of infection. These results offer insight into other possibilities for possibly controlling T. cruzi proliferation through melatonin therapy and also the stimulatory effects on host`s immune response triggered by the absence of male gonadal steroids during the acute phase of infection.

Allelic Variation Investigation of the Estrogen Receptor Within an Australian Multiple Sclerosis Population

Multiple Sclerosis (MS) is a central nervous system (CNS) chronic inflammatory demyelinating disease leading to various neurological disabilities. The disorder is more prevalent for women with a ratio of 3:2 female to male. Objectives: To investigate variation within the estrogen receptor 1 (ESR1) polymorphism gene in an Australian MS case-control population using two intragenic restriction fragment length polymorphisms; the G594A located in exon 8 detected with the BtgI restriction enzyme and T938C located in intron 1, detected with PvuII. One hundred and ten Australian MS patients were studied, with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 110 age, sex and ethnicity matched controls were investigated as a comparative group. No significant difference in the allelic distribution frequency was found between the case and control groups for the ESR1 PvuII (P = 0.50) and Btg1 (P = 0.45) marker. Our results do not support a role for these two ESR1 markers in multiple sclerosis susceptibility, however other markers within ESR1 should not be excluded for potential involvement in the disorder.

Multiple Sclerosis with Idiopathic Dilated Cardiomyopathy: A Case Report

Multiple sclerosis and idiopathic dilated cardiomyopathy are two conditions in which an autoimmune process is implicated in the pathogenesis. There is evidence to support clustering of autoimmune diseases in patients with multiple sclerosis and their families. To our knowledge, this is the first report of idiopathic dilated cardiomyopathy occurring in a patient with multiple sclerosis.

Autoimmune Neurological Disease

This book provides a comprehensive and critical overview of the immunological aspects of autoimmune neurological disease. These diseases include common conditions such as multiple sclerosis, the Guillain–Barré syndrome and myasthenia gravis. The introductory chapters on antigen recognition and self–non-self discrimination, and on neuroimmunology, are followed by chapters on specific diseases. These are presented in a standardized format with sections on clinical features, genetics, neuropathology, pathophysiology, immunology and therapy. Each chapter has a concluding section which summarizes key points and suggests directions for future research. Animal models of autoimmune neurological disease are also covered in detail because of their importance in understanding the human diseases. The book is suitable for clinicians and neurologists managing patients with these diseases, and for immunologists, neuroscientists and neurologists investigating the pathogenesis and pathophysiology of these disorders.

Immunology of multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) leading to demyelination, axonal damage, and progressive neurologic disability. The development of MS is influenced by environmental factors, particularly the Epstein-Barr virus (EBV), and genetic factors, which include specific HLA types, particularly DRB1*1501-DQA1*0102-DQB1*0602, and a predisposition to autoimmunity in general. MS patients have increased circulating T-cell and antibody reactivity to myelin proteins and gangliosides. It is proposed that the role of EBV is to infect autoreactive B cells that then seed the CNS and promote the survival of autoreactive T cells there. It is also proposed that the clinical attacks of relapsing-remitting MS are orchestrated by myelin-reactive T cells entering the white matter of the CNS from the blood, and that the progressive disability in primary and secondary progressive MS is caused by the action of autoantibodies produced in the CNS by ­meningeal lymphoid follicles with germinal centers.

Immunopathology and Pathophysiology of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS), and is widely studied as an animal model of the human CNS demyelinating diseases, including multiple sclerosis (Raine, 1984). EAE can be induced by inoculation with whole CNS tissue, purified myelin basic protein (MBP) or myelin proteolipid protein (PLP), together with adjuvants. It may also be induced by the passive transfer of T cells specifically reactive to these myelin antigens. EAE may have either an acute or a chronic relapsing course. Acute EAE closely resembles the human disease acute disseminated encephalomyelitis, while chronic relapsing EAE resembles multiple sclerosis. EAE is also the prototype for T-cell-mediated autoimmune disease in general. This chapter will focus on the immunopathology and pathophysiology of EAE, which are the subjects of investigation in my laboratory.