Incidental Diagnosis of Rheumatic Myocarditis during Cardiac Surgery-Impact on Late Prognosis.

Rheumatic fever (RF) and rheumatic heart disease (RHD) are still highly prevalent, particularly in low- and middle-income countries. RHD is a neglected and underdiagnosed disease for which no specific laboratory diagnostic test is completely reliable. This is a retrospective observational study, which included 118 patients with RHD who underwent cardiac surgery from 1985 to 2018. The aim of this investigation was to evaluate the clinical, epidemiological, echocardiographic and pathological characteristics in two cohorts of RHD patients: one cohort with Aschoff bodies present in their pathological results and the other without such histopathological characteristics. No conventional clinical and laboratory tests for RHD myocarditis were able to identify active carditis during the preoperative phase of valve repair or replacement. Patients who had Aschoff bodies in their pathological results were younger (median age of 13 years (11-24 years) vs. 27 years (17-37 years), p = 0.001) and had higher rate of late mortality (22.9% vs. 5.4%, p = 0.043). In conclusion, the presence of Aschoff bodies in pathological findings may predict increased long-term mortality, emphasizing the importance of comprehensive pathology analysis for suspected myocarditis during heart surgery.

StreptInCor, a Group A Streptococcal Adsorbed Vaccine: Evaluation of Repeated Intramuscular Dose Toxicity Testing in Rats.

Streptococcus pyogenes infections continue to be a worldwide public health problem, causing various diseases in humans, with rheumatic fever and rheumatic heart disease being the most harmful manifestations. Impetigo and post-streptococcal glomerulonephritis are also important sequelae of skin infections. We have developed a candidate vaccine epitope (StreptInCor) that presents promising results in diverse animal models. To assess whether the StreptInCor alum-adsorbed vaccine could induce undesirable effects, a certified independent company conducted a repeated intramuscular dose toxicity evaluation in Wistar rats, a choice model for toxicity studies. We did not observe significant alterations in clinical, hematological, biochemical, anatomical, or histopathological parameters due to vaccine administration, even when the animals received the highest dose. In conclusion, repeated intramuscular doses did not show signs of macroscopic or other significant changes in the clinical or histopathological parameters, indicating that StreptInCor can be considered a safe candidate vaccine.

Ficolin-3 in rheumatic fever and rheumatic heart disease.

Rheumatic fever (RF) and chronic rheumatic heart disease (RHD) are complications of oropharyngeal infection caused by Streptococcus pyogenes. Despite the importance of the complement system against infections and autoimmunity diseases, studies on the role of the lectin pathway in RF and RHD are scarce. Thus, our aim was to evaluate the association of ficolin-3 serum levels, FCN3 polymorphisms and haplotypes with the susceptibility to RF and RHD. We investigated 179 patients with a history of RF (126 RHD and 53 RF only) and 170 healthy blood donors as control group. Ficolin-3 serum concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Three FCN3 single nucleotide polymorphisms (SNPs rs532781899, rs28362807 and rs4494157) were genotyped through the sequence-specific PCR method. Lower ficolin-3 serum levels were observed in RF patients when compared to controls (12.81 µg/mL vs. 18.14 µg/mL respectively, p < 0.0001, OR 1.22 [1.12-1.34]), and in RHD in comparison to RF only (RFo) (12.72 µg/mL vs. 14.29 µg/mL respectively, p = 0.016, OR 1.38 [1.06-1.80]). Low ficolin-3 levels (<10.7 µg/mL) were more common in patients (39.5 %, 30/76) than controls (20.6 %, 13/63, p = 0.018, OR = 2.51 [1.14-5.31]), and in RHD (44.4 %, 28/63) than RFo (15.4 %, 2/13, p = 0.007, OR = 3.08 [1.43-6.79]). On the other hand, FCN3 polymorphism/haplotypes were not associated with ficolin-3 serum levels or the disease. Low ficolin-3 levels might be associated with RF, being a potential marker of disease progression.

Rheumatic Fever: A Model of Autoimmune Disease due to Molecular Mimicry between Human and Pathogen Proteins.

Acute rheumatic fever (ARF) is caused by an autoimmune response to throat infection with Streptococcus pyogenes in individuals who present some susceptibility genes. Rheumatic heart disease (RHD) is the major sequela and can cause heart failure and premature mortality. The disease is mediated by humoral and cellular immune responses. In this review, we present the major events that can trigger heart lesions.

Group A Streptococcus Adsorbed Vaccine: Repeated Intramuscular Dose Toxicity Test in Minipigs.

Streptococcus pyogenes infection continues to be a worldwide public health problem causing various diseases in humans and plays an important role in the pathogenesis of rheumatic fever and rheumatic heart disease. We developed a vaccine candidate to prevent S. pyogenes infections, identified as StreptInCor, that presented promising results in mouse models. A certified and independent laboratory conducted two repeated intramuscular dose toxicity tests (28 days, four weekly injections). The first test, composed of four experimental groups treated with 0 (vehicle), 50, 100 or 200 µg/500 µL StreptInCor, did not show significant alterations in clinical, hematological, biochemical or anatomopathological parameters related to the administration of StreptInCor. In addition to the parameters mentioned above, we evaluated the cardiac function and valves of animals by echocardiography before and after administration of 200 µg/500 µL StreptInCor versus placebo. We did not observe any changes related to StreptInCor administration, including changes in cardiac function and valves in animals, after receiving the highest dose of this vaccine candidate. The results obtained in the two repeated intramuscular dose toxicity tests showed that this vaccine formulation did not induce harmful effects to the tissues and organs studied, indicating that the candidate vaccine is well tolerated in minipigs.

Thymopoiesis in Pre- and Post-Hematopoietic Stem Cell Transplantation.

Hematopoietic stem cell transplantation (HSCT) is an important therapeutic option for some hematological diseases. However, patients who undergo HSCT acquire a state of immunodeficiency that causes significant mortality. Reconstitution of thymic function is needed to support the immune system. One way to measure thymic function is through T-cell receptor excision circle (TREC) quantification. TRECs are generated by T-cell receptor gene rearrangements during T-cell maturation in the thymus and represent a reliable marker for thymic output. In this study, we aimed to assess aging and malignant hematological diseases as two important factors that may influence thymic output before HSCT. We observed that patients before HSCT presented signal joint TREC (sjTREC) numbers lower than 606.55 copies/µg DNA (low values) compared with healthy individuals, with an odds ratio (OR) of 12.88 [95% confidence interval (CI): 5.26-31.53; p < 0.001]. Our results showed that a group of older individuals (≥50 years old), comprising both healthy individuals and patients, had an OR of 10.07 (95% CI: 2.80-36.20) for low sjTREC values compared with younger individuals (≤24 years old; p < 0.001). Multiple logistic regression analysis confirmed that both older age (≥50 years old) and malignant hematological diseases and their treatments were important and independent risk factors related to thymic function impairment (p < 0.001). The median sjTREC value for patients of all ages was significantly lower than the sjTREC median for the subgroup of older healthy individuals (≥50 years old; p < 0.001). These data suggested that patients before HSCT and healthy individuals exhibited age-dependent thymic impairment, and that prior treatment for hematological diseases may exacerbate aging-related deterioration of natural thymic function. Furthermore, we analyzed these patients 9 months post-HSCT and compared patients who underwent autologous HSCT with those who underwent allogeneic HSCT. Both groups of patients achieved sjTREC copy numbers similar to those of healthy individuals. We did not find a close relationship between impaired thymic function prior to HSCT and worse thymic recovery after HSCT.

Acute rheumatic fever.

Acute rheumatic fever is caused by an autoimmune response to throat infection with Streptococcus pyogenes. Cardiac involvement during acute rheumatic fever can result in rheumatic heart disease, which can cause heart failure and premature mortality. Poverty and household overcrowding are associated with an increased prevalence of acute rheumatic fever and rheumatic heart disease, both of which remain a public health problem in many low-income countries. Control efforts are hampered by the scarcity of accurate data on disease burden, and effective approaches to diagnosis, prevention, and treatment. The diagnosis of acute rheumatic fever is entirely clinical, without any laboratory gold standard, and no treatments have been shown to reduce progression to rheumatic heart disease. Prevention mainly relies on the prompt recognition and treatment of streptococcal pharyngitis, and avoidance of recurrent infection using long-term antibiotics. But evidence for the effectiveness of either approach is not strong. High-quality research is urgently needed to guide efforts to reduce acute rheumatic fever incidence and prevent progression to rheumatic heart disease.

Sickening or Healing the Heart? The Association of Ficolin-1 and Rheumatic Fever.

Rheumatic fever (RF) and its subsequent progression to rheumatic heart disease (RHD) are chronic inflammatory disorders prevalent in children and adolescents in underdeveloped countries, and a contributing factor for high morbidity and mortality rates worldwide. Their primary cause is oropharynx infection by Streptococcus pyogenes, whose acetylated residues are recognized by ficolin-1. This is the only membrane-bound, as well as soluble activator molecule of the complement lectin pathway (LP). Although LP genetic polymorphisms are associated with RF, FCN1 gene's role remains unknown. To understand this role, we haplotyped five FCN1 promoter polymorphisms by sequence-specific amplification in 193 patients (138 with RHD and 55, RF only) and 193 controls, measuring ficolin-1 serum concentrations in 78 patients and 86 controls, using enzyme-linked immunosorbent assay (ELISA). Patients presented lower ficolin-1 serum levels (p < 0.0001), but did not differ according to cardiac commitment. Control's genotype distribution was in the Hardy-Weinberg equilibrium. Four alleles (rs2989727: c.-1981A, rs10120023: c.-542A, rs10117466: c.-144A, and rs10858293: c.33T), all associated with increased FCN1 gene expression in whole blood or adipose subcutaneous tissue (p = 0.000001), were also associated with increased protection against the disease. They occur within the *3C2 haplotype, associated with an increased protection against RF (OR = 0.41, p < 0.0001) and with higher ficolin-1 levels in patient serum (p = 0.03). In addition, major alleles of these same polymorphisms comprehend the most primitive *1 haplotype, associated with increased susceptibility to RF (OR = 1.76, p < 0.0001). Nevertheless, instead of having a clear-cut protective role, the minor c.-1981A and c.-144A alleles were also associated with additive susceptibility to valvar stenosis and mitral insufficiency (OR = 3.75, p = 0.009 and OR = 3.37, p = 0.027, respectively). All associations were independent of age, sex or ethnicity. Thus, minor FCN1 promoter variants may play a protective role against RF, by encouraging bacteria elimination as well as increasing gene expression and protein levels. On the other hand, they may also predispose the patients to RHD symptoms, by probably contributing to chronic inflammation and tissue injury, thus emphasizing the dual importance of ficolin-1 in both conditions.

Immunological Balance Is Associated with Clinical Outcome after Autologous Hematopoietic Stem Cell Transplantation in Type 1 Diabetes.

Autologous hematopoietic stem cell transplantation (AHSCT) increases C-peptide levels and induces insulin independence in patients with type 1 diabetes. This study aimed to investigate how clinical outcomes may associate with the immunological status, especially concerning the balance between immunoregulation and autoreactivity. Twenty-one type 1 diabetes patients were monitored after AHSCT and assessed every 6 months for duration of insulin independence, C-peptide levels, frequencies of islet-specific autoreactive CD8+ T cells (CTL), regulatory lymphocyte subsets, thymic function, and T-cell repertoire diversity. In median follow-up of 78 (range 15-106) months, all patients became insulin-independent, resuming insulin after median of 43 (range 6-100) months. Patients were retrospectively divided into short- or prolonged-remission groups, according to duration of insulin independence. For the entire follow-up, CD3+CD4+ T-cell numbers remained lower than baseline in both groups, whereas CD3+CD8+ T-cell levels did not change, resulting in a CD4/CD8 ratio inversion. Memory CTL comprehended most of T cells detected on long-term follow-up of patients after AHSCT. B cells reconstituted to baseline levels at 2-3 months post-AHSCT in both patient groups. In the prolonged-remission-group, baseline islet-specific T-cell autoreactivity persisted after transplantation, but regulatory T cell counts increased. Patients with lower frequencies of autoreactive islet-specific T cells remained insulin-free longer and presented greater C-peptide levels than those with lower frequencies of these cells. Therefore, immune monitoring identified a subgroup of patients with superior clinical outcome of AHSCT. Our study shows that improved immunoregulation may balance autoreactivity endorsing better metabolic outcomes in patients with lower frequencies of islet-specific T cells. Development of new strategies of AHSCT is necessary to increase frequency and function of T and B regulatory cells and decrease efficiently autoreactive islet-specific T and B memory cells in type 1 diabetes patients undergoing transplantation.

Rheumatic Heart Disease and Myxomatous Degeneration: Differences and Similarities of Valve Damage Resulting from Autoimmune Reactions and Matrix Disorganization.

Autoimmune inflammatory reactions leading to rheumatic fever (RF) and rheumatic heart disease (RHD) result from untreated Streptococcus pyogenes throat infections in individuals who exhibit genetic susceptibility. Immune effector mechanisms have been described that lead to heart tissue damage culminating in mitral and aortic valve dysfunctions. In myxomatous valve degeneration (MXD), the mitral valve is also damaged due to non-inflammatory mechanisms. Both diseases are characterized by structural valve disarray and a previous proteomic analysis of them has disclosed a distinct profile of matrix/structural proteins differentially expressed. Given their relevance in organizing valve tissue, we quantitatively evaluated the expression of vimentin, collagen VI, lumican, and vitronectin as well as performed immunohistochemical analysis of their distribution in valve tissue lesions of patients in both diseases. We identified abundant expression of two isoforms of vimentin (45 kDa, 42 kDa) with reduced expression of the full-size protein (54 kDa) in RHD valves. We also found increased vitronectin expression, reduced collagen VI expression and similar lumican expression between RHD and MXD valves. Immunohistochemical analysis indicated disrupted patterns of these proteins in myxomatous degeneration valves and disorganized distribution in rheumatic heart disease valves that correlated with clinical manifestations such as valve regurgitation or stenosis. Confocal microscopy analysis revealed a diverse pattern of distribution of collagen VI and lumican into RHD and MXD valves. Altogether, these results demonstrated distinct patterns of altered valve expression and tissue distribution/organization of structural/matrix proteins that play important pathophysiological roles in both valve diseases.

Acute rheumatic fever and rheumatic heart disease.

Acute rheumatic fever (ARF) is the result of an autoimmune response to pharyngitis caused by infection with group A Streptococcus. The long-term damage to cardiac valves caused by ARF, which can result from a single severe episode or from multiple recurrent episodes of the illness, is known as rheumatic heart disease (RHD) and is a notable cause of morbidity and mortality in resource-poor settings around the world. Although our understanding of disease pathogenesis has advanced in recent years, this has not led to dramatic improvements in diagnostic approaches, which are still reliant on clinical features using the Jones Criteria, or treatment practices. Indeed, penicillin has been the mainstay of treatment for decades and there is no other treatment that has been proven to alter the likelihood or the severity of RHD after an episode of ARF. Recent advances - including the use of echocardiographic diagnosis in those with ARF and in screening for early detection of RHD, progress in developing group A streptococcal vaccines and an increased focus on the lived experience of those with RHD and the need to improve quality of life - give cause for optimism that progress will be made in coming years against this neglected disease that affects populations around the world, but is a particular issue for those living in poverty.

Status of research and development of vaccines for Streptococcus pyogenes.

Streptococcus pyogenes is an important global pathogen, causing considerable morbidity and mortality, especially in low and middle income countries where rheumatic heart disease and invasive infections are common. There is a number of promising vaccine candidates, most notably those based on the M protein, the key virulence factor for the bacterium. Vaccines against Streptococcus pyogenes are considered as impeded vaccines because of a number of crucial barriers to development. Considerable effort is needed by key players to bring current vaccine candidates through phase III clinical trials and there is a clear need to develop a roadmap for future development of current and new candidates.

Streptococcus pyogenes strains in Sao Paulo, Brazil: molecular characterization as a basis for StreptInCor coverage capacity analysis.

BACKGROUND: Several human diseases are caused by Streptococcus pyogenes, ranging from common infections to autoimmunity. Characterization of the most prevalent strains worldwide is a useful tool for evaluating the coverage capacity of vaccines under development. In this study, a collection of S. pyogenes strains from Sao Paulo, Brazil, was analyzed to describe the diversity of strains and assess the vaccine coverage capacity of StreptInCor. METHODS: Molecular epidemiology of S. pyogenes strains was performed by emm-genotyping the 229 isolates from different clinical sites, and PCR was used for superantigen profile analysis. The emm-pattern and tissue tropism for these M types were also predicted and compared based on the emm-cluster classification. RESULTS: The strains were fit into 12 different emm-clusters, revealing a diverse phylogenetic origin and, consequently, different mechanisms of infection and escape of the host immune system. Forty-eight emm-types were distinguished in 229 samples, and the 10 most frequently observed types accounted for 69 % of all isolates, indicating a diverse profile of circulating strains comparable to other countries under development. A similar proportion of E and A-C emm-patterns were observed, whereas pattern D was less frequent, indicating that the strains of this collection primarily had a tissue tropism for the throat. In silico analysis of the coverage capacity of StreptInCor, an M protein-conserved regionally based vaccine candidate developed by our group, had a range of 94.5 % to 59.7 %, with a mean of 71.0 % identity between the vaccine antigen and the predicted amino acid sequence of the emm-types included here. CONCLUSIONS: This is the first report of S. pyogenes strain characterization in Sao Paulo, one of the largest cities in the world; thus, the strain panel described here is a representative sample for vaccine coverage capacity analysis. Our results enabled evaluation of StreptInCor candidate vaccine coverage capacity against diverse M-types, indicating that the vaccine candidate likely would induce protection against the diverse strains worldwide.

Distinct mitral valve proteomic profiles in rheumatic heart disease and myxomatous degeneration.

Rheumatic heart disease (RHD) affects heart-valve tissue and is the most serious consequence of group A Streptococcus infection. Myxomatous degeneration (MXD) is the most frequent valvopathy in the western world. In the present work, key protein expression alterations in the heart-valve tissue of RHD and MXD patients were identified and characterized, with controls from cadaveric organ donors. Proteins were separated by two-dimensional (2D)-electrophoresis and identified by mass spectrometry. We found 17 differentially expressed protein spots, as compared to control samples. We observed an increased expression of ASAP-2 in the RHD patients' valves, while collagen-VI, haptoglobin-related protein, prolargin, and cartilage oligomeric protein showed reduced expression. Valve tissue of MXD patients, on the other hand, presented lower expression of annexin-A1 and A2, septin-2, SOD (Cu/Zn), and transgelin. Tissue samples from both valvopathies displayed higher expression of apolipoprotein-A1. Biglycan was downexpressed in both diseases. Vimentin and lumican showed higher expression in RHD and lower in MXD. These results suggest that key pathogenetic mechanisms are intrinsically distinct in RHD and MXD.

Analysis of the coverage capacity of the StreptInCor candidate vaccine against Streptococcus pyogenes.

Streptococcus pyogenes is responsible for infections as pharyngitis, sepsis, necrotizing fasciitis and streptococcal toxic shock syndrome. The M protein is the major bacterial antigen and consists of both polymorphic N-terminal portion and a conserved region. In the present study, we analyzed the in vitro ability of StreptInCor a C-terminal candidate vaccine against S. pyogenes to induce antibodies to neutralize/opsonize the most common S. pyogenes strains in Sao Paulo by examining the recognition by sera from StreptInCor immunized mice. We also evaluated the presence of cross-reactive antibodies against human heart valve tissue. Anti-StreptInCor antibodies were able to neutralize/opsonize at least 5 strains, showing that immunization with StreptInCor is effective against several S. pyogenes strains and can prevent infection and subsequent sequelae without causing autoimmune reactions.

Rheumatic Heart Disease: Molecules Involved in Valve Tissue Inflammation Leading to the Autoimmune Process and Anti-S. pyogenes Vaccine.

The major events leading to both rheumatic fever (RF) and rheumatic heart disease (RHD) are reviewed. Several genes are involved in the development of RF and RHD. The inflammatory process that results from S. pyogenes infection involves the activation of several molecules such as VCAM and ICAM, which play a role in the migration of leukocytes to the heart, particularly to the valves. Specific chemokines, such as CXCL3/MIP1α as well as CCL1/I-309 and CXCL9/Mig, attract T cells to the myocardium and valves, respectively. The autoimmune reactions are mediated by both the B- and T-cell responses that begin at the periphery, followed by the migration of T cell clones to the heart and the infiltration of heart lesions in RHD patients. These cells recognize streptococcal antigens and human-tissue proteins. Molecular mimicry between streptococcal M protein and human proteins has been proposed as the triggering factor leading to autoimmunity in RF and RHD. The production of cytokines from peripheral and heart-infiltrating mononuclear cells suggests that T helper 1 and Th17 cytokines are the mediators of RHD heart lesions. The low numbers of IL-4 producing cells in the valvular tissue might contribute to the maintenance and progression of the valve lesions. The identification of a vaccine epitope opens a perspective of development of an effective and safe vaccine to prevent S. pyogenes infections, consequently RF and RHD.

StreptInCor: a candidate vaccine epitope against S. pyogenes infections induces protection in outbred mice.

Infection with Streptococcus pyogenes (S. pyogenes) can result in several diseases, particularly in children. S. pyogenes M protein is the major virulence factor, and certain regions of its N-terminus can trigger autoimmune sequelae such as rheumatic fever in susceptible individuals with untreated group A streptococcal pharyngitis. In a previous study, we utilized a large panel of human peripheral blood cells to define the C-terminal protective epitope StreptInCor (medical identity), which does not induce autoimmune reactions. We recently confirmed the results in HLA-transgenic mice. In the present study, we extended the experimental assays to outbred animals (Swiss mice). Herein, we demonstrate high titers of StreptInCor-specific antibodies, as well as appropriate T-cell immune responses. No cross-reaction to cardiac myosin was detected. Additionally, immunized Swiss mice exhibited 87% survival one month after challenge with S. pyogenes. In conclusion, the data presented herein reinforce previous results in humans and animals and further emphasize that StreptInCor could be an effective and safe vaccine for the prevention of S. pyogenes infections.