LUIZA GUILHERME GUGLIELMI
Projetos de Pesquisa
Unidades Organizacionais
Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina
LIM/19 - Laboratório de Histocompatibilidade e Imunidade Celular, Hospital das Clínicas, Faculdade de Medicina - Líder
LIM/19 - Laboratório de Histocompatibilidade e Imunidade Celular, Hospital das Clínicas, Faculdade de Medicina - Líder
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- Sickening or Healing the Heart? The Association of Ficolin-1 and Rheumatic Fever(2018) CATARINO, Sandra Jeremias; ANDRADE, Fabiana Antunes; BOLDT, Angelica Beate Winter; GUILHERME, Luiza; MESSIAS-REASON, Iara JoseRheumatic 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 highmorbidity andmortality rates worldwide. Their primary cause is oropharynx infection by Streptococcus pyogenes, whose acetylated residues are recognized by ficolin-1. This is the onlymembrane-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, theminor 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, theymay 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.
- Rheumatic heart disease: molecules involved in valve tissue inflammation leading to the autoimmune process and anti-S. pyogenes vaccine(2013) GUILHERME, Luiza; KALIL, JorgeThe major events leading to both rheumatic fever (RE) and rheumatic heart disease (RHD) are reviewed. Several genes are involved in the development of RE 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/MIP1a as well as CCL1/I-309 and CXCL9/Mig, attractT 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 humantissue proteins. Molecular mimicry between streptococcal M protein and human proteins has been proposed as the triggering factor leading to autoimmunity in RE 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 1154 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 RE and RHD.
- Thymopoiesis in Pre- and Post-Hematopoietic Stem Cell Transplantation(2018) ROCHA, Luis Klaus A. da; BARROS, Samar Freschi de; BANDEIRA, Francine; BOLLINI, Alexia; TESTA, Lucia Helena de A.; SIMIONE, Anderson Joao; SOUZA, Marina de O. e; ZANETTI, Lilian P.; OLIVEIRA, Leila Cibele S. de; SANTOS, Ana Claudia F. dos; SOUZA, Mair Pedro de; COLTURADO, Vergilio Antonio R.; KALIL, Jorge; MACHADO, Clarisse M.; GUILHERME, LuizaHematopoietic 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/mu g 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.
- CXCL9/Mig Mediates T cells Recruitment to Valvular Tissue Lesions of Chronic Rheumatic Heart Disease Patients(2013) FAE, Kellen C.; PALACIOS, Selma A.; NOGUEIRA, Luciana G.; OSHIRO, Sandra E.; DEMARCHI, Lea M. F.; BILATE, Angelina M. B.; POMERANTZEFF, Pablo M. A.; BRANDAO, Carlos; THOMAZ, Petronio G.; REIS, Maxwell dos; SAMPAIO, Roney; TANAKA, Ana C.; CUNHA-NETO, Edecio; KALIL, Jorge; GUILHERME, LuizaRheumatic fever (RF) is an autoimmune disease triggered by Streptococcus pyogenes infection frequently observed in infants from developing countries. Rheumatic heart disease (RHD), the major sequel of RF, leads to chronic inflammation of the myocardium and valvular tissue. T cells are the main population infiltrating cardiac lesions; however, the chemokines that orchestrate their recruitment are not clearly defined. Here, we investigated the expression of chemokines and chemokine receptors in cardiac tissue biopsies obtained from chronic RHD patients. Our results showed that CCL3/MIP1 alpha gene expression was upregulated in myocardium while CCL1/I-309 and CXCL9/Mig were highly expressed in valvular tissue. Auto-reactive T cells that infiltrate valvular lesions presented a memory phenotype (CD4(+)CD45RO(+)) and migrate mainly toward CXCL9/Mig gradient. Collectively, our results show that a diverse milieu of chemokines is expressed in myocardium and valvular tissue lesions and emphasize the role of CXCL9/Mig in mediating T cell recruitment to the site of inflammation in the heart.
- Pilot scale production of the vaccine adjuvant Proteoliposome derived Cochleates (AFCo1) from Neisseria meningitidis serogroup B(2013) ZAYAS, Caridad; GONZALEZ, Domingo; ACEVEDO, Reinaldo; CAMPO, Judith del; LASTRE, Miriam; GONZALEZ, Elizabeth; ROMEU, Belkis; CUELLO, Maribel; BALBOA, Julio; CABRERA, Osmir; GUILHERME, Luisa; PEREZ, OliverThe use of new adjuvants in vaccine formulations is a subject of current research. Only few parenteral adjuvants have been licensed. We have developed a mucosal and parenteral adjuvant known as AFCo1 (Adjuvant Finlay Cochleate 1, derived from proteoliposomes of N. meningitidis B) using a dialysis procedure to produce them on lab scale. The immunogenicity of the AFCo1 produced by dialysis has been already evaluated, but it was necessary to demonstrate the feasibility of a larger-scale manufacturing process. Therefore, we used a crossflow diafiltration system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The aim of this work was to produce AFCo1 on pilot scale, while conserving the adjuvant properties. The proteoliposomes (raw material) were resuspended in a buffer containing sodium deoxycholate and were transformed into AFCo1 under the action of a calcium forming buffer. The detergent was removed from the protein solution by diafiltration to a constant volume. In this CFS, we used a hollow fiber cartridge from Amicon (polysulfona cartridge of 10 kDa porosity, 1mm channel diameter of fiber and 0.45 m(2) area of filtration), allowing production of a batch of up to 20 L. AFCo1 were successfully produced by tangential filtration to pilot scale. The batch passed preliminary stability tests. Nasal immunization of BALB/c mice, induced specific saliva IgA and serum IgG. The induction of Th1 responses were demonstrated by the induction of IgG2a, IFN gamma and not IL-5. The adjuvant action over Neisseria (self) antigens and with co-administered (heterologous) antigens such as ovalbumin and a synthetic peptide from haemolytic Streptococcus B was also demonstrated.
conferenceObject RESTRICTED AND SKEWED TCR VB REPERTOIRE IN CHROMOSOME 22Q11.2 DELETION(2012) ARANTES, J. M.; GRASSI, M. S.; SANTOS, N. M.; GUILHERME, L.; KULIKOWSKI, L. D.; DUTRA, R. L.; WATANABE, L. A.; JACOB, C. M. A.; ZAGO, C. A.; CARNEIRO-SAMPAIO, M.Introduction: Chromosome 22q11 deletion is the most common human deletion and is found in the majority of patients with DiGeorge and velo-cardio-facial syndromes. Many patients have a mild to moderate immunodeficiency, and most have cardiac anomaly. Objective: To evaluate TCR repertoire diversity in infants with 22q11.2 deletion identified at FMUSP ward for congenital heart diseases. Methods: TCR Vβ variable chain repertoire was analyzed by the TCRBV CDR3 lenght spectratyping technique, and repertoire diversity was quantified utilizing the complexity score (CS), that represents the sum of the number of peaks for each one of the 24 BV families. 22q11.2 deletion was detected utilizing multiplex ligation-dependent probe amplification. First case report: A 9-month-old boy was identified in a survey among infants with complex congenital heart anomalies. He was born from non-consanguineous parents, weighing 2845g and presenting microcephaly, micrognathia, ocular hypertelorism and low set left ear, renal involvement, left atrial isomerism and pulmonary atresia. He also had hypocalcemia and hypoplasticthymus. He has lymphopenia=3,800 cells/mm3 (CD3=1,454 cells/mm3, CD4=888cells/mm3, CD8=537cells/mm3), thrombocytopenia=55,000, IgG+=1,285mg/dL, IgM=123mg/dL, IgA=132mg/dL. Results: The patient presented CS=49, in contrast with 2 healthy age-matched male infants with 127 and 135. Four young healthy adults presented CS between 165 and 178. The patient presented mostly olygoclonal distribution and even absence of TCRBV families, while healthy donors exhibited mainly polyclonal non-Gaussian distributions. Conclusions: The evaluation of new cases as well as the follow-up the patients will demonstrate if the repertoire diversity correlates with clinical severity.- HLA class II transgenic mice develop a safe and long lasting immune response against StreptInCor, an anti-group A streptococcus vaccine candidate(2011) GUERINO, Milton T.; POSTOL, Edilberto; DEMARCHI, Lea M. F.; MARTINS, Carlo O.; MUNDEL, Luiz R.; KALIL, Jorge; GUILHERME, LuizaStreptococcus pyogenes infections remain a health problem in several countries because of post-streptococcal sequelae, such as rheumatic fever and rheumatic heart disease. We developed a vaccine epitope (StreptInCor) composed of 55 amino acid residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. Recently, by using human blood samples, we showed that the StreptInCor epitope is able to bind to different HLA class 11 molecules and that it could be considered a universal vaccine epitope. In the present work, we evaluated the immune response of HLA class II transgenic mice against aluminum hydroxide-absorbed StreptInCor. After a period of one year, several organs were analyzed histologically to verify the safety of the candidate vaccine epitope. Our results showed that StreptInCor is able to induce robust and safe and long lasting immune response without deleterious reactions in several organs. In conclusion, the results presented here indicate that StreptInCor could be considered a safe vaccine against severe streptococcus-induced diseases.
- Analysis of the coverage capacity of the StreptInCor candidate vaccine against Streptococcus pyogenes(2014) AMICIS, Karine M. De; BARROS, Samar Freschi de; ALENCAR, Raquel E.; POSTOL, Edilberto; MARTINS, Carlo de Oliveira; ARCURI, Helen Andrade; GOULART, Cibelly; KALIL, Jorge; GUILHERME, LuizaStreptococcus 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 Fever: A Model of Autoimmune Disease due to Molecular Mimicry between Human and Pathogen Proteins(2020) KALIL, Jorge; GUILHERME, LuizaAcute 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.
- Ficolin-3 in rheumatic fever and rheumatic heart disease(2021) CATARINO, Sandra Jeremias; ANDRADE, Fabiana Antunes; BAVIA, Lorena; GUILHERME, Luiza; MESSIAS-REASON, Iara JoseRheumatic 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 mu g/mL vs. 18.14 mu g/mL respectively, p < 0.0001, OR 1.22 [1.12-1.34]), and in RHD in comparison to RF only (RFo) (12.72 mu g/mL vs. 14.29 mu g/mL respectively, p = 0.016, OR 1.38 [1.06-1.80]). Low ficolin-3 levels (<10.7 mu g/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.