Patrícia Sammarco Rosa

Instituto Lauro de Souza Lima, Бауру, São Paulo, Brazil

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Publications (32)71.68 Total impact

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    ABSTRACT: Jorge Lobo's disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.
    Preview · Article · Dec 2015 · PLoS ONE
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    ABSTRACT: Leprosy, an infectious disease caused by Mycobacterium leprae, affects millions of people worldwide. However, little is known regarding its molecular pathophysiological mechanisms. In this study, a comprehensive assessment of human mRNA was performed on leprosy skin lesions by using DNA chip microarrays, which included the entire spectrum of the disease along with its reactional states. Sixty-six samples from leprotic lesions (10TT, 10BT, 10BB, 10BL, 4LL, 14R1, and 10R2) and nine skin biopsies from healthy individuals were used as controls (CC) (ages ranged from 06 to 83 years, 48 were male and 29 female). The evaluation identified 1580 differentially expressed mRNAs [Fold Change (FC) ≥ 2.0, p ≤ 0.05] in diseased lesions vs. healthy controls. Some of these genes were observed in all forms of the disease (CD2, CD27, chit1, FA2H, FAM26F, GZMB, MMP9, SLAMF7, UBD) and others were exclusive to reactional forms (Type “1” reaction: GPNMB, IL1B, MICAL2, FOXQ1; Type “2” reaction: AKR1B10, FAM180B, FOXQ1, NNMT, NR1D1, PTX3, TNFRSF25). In literature, these mRNAs have been associated with numerous pathophysiological processes and signaling pathways and are present in a large number of diseases. The role of these mRNAs maybe studied in the context of developing new diagnostic markers and therapeutic targets for leprosy.
    Full-text · Article · Nov 2015 · Frontiers in Genetics
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    ABSTRACT: Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1404-5) contains supplementary material, which is available to authorized users.
    Full-text · Article · Mar 2015 · Acta Neuropathologica
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    ABSTRACT: Mycobacterium leprae infects macrophages and Schwann cells inducing a gene expression program to facilitate its replication and progression to disease. MicroRNAs (miRNAs) are key regulators of gene expression and could be involved during the infection. To address the genetic influence of miRNAs in leprosy, we enrolled 1,098 individuals and conducted a case-control analysis in order to study four miRNAs genes containing single nucleotide polymorphism (miRSNP). We tested miRSNP-125a (rs12975333 G.T), miRSNP-223 (rs34952329 *.T), miRSNP-196a-2 (rs11614913 C.T) and miRSNP-146a (rs2910164 G.C). Amongst them, miRSNP-146a was the unique gene associated with risk to leprosy per se (GC OR = 1.44, p = 0.04; CC OR = 2.18, p = 0.0091). We replicated this finding showing that the C-allele was over-transmitted (p = 0.003) using a transmission-disequilibrium test. A functional analysis revealed that live M. leprae (MOI 100:1) was able to induce miR-146a expression in THP-1 (p,0.05). Furthermore, pure neural leprosy biopsies expressed augmented levels of that miRNA as compared to biopsy samples from neuropathies not related with leprosy (p = 0.001). Interestingly, carriers of the risk variant (C-allele) produce higher levels of mature miR-146a in nerves (p = 0.04). From skin biopsies, although we observed augmented levels of miR-146a, we were not able to correlate it with a particular clinical form or neither host genotype. MiR-146a is known to modulate TNF levels, thus we assessed TNF expression (nerve biopsies) and released by peripheral blood mononuclear cells infected with BCG Moreau. In both cases lower TNF levels correlates with subjects carrying the risk C-allele, (p = 0.0453 and p = 0.0352; respectively), which is consistent with an immunomodulatory role of this miRNA in leprosy.
    Full-text · Article · Sep 2014 · PLoS neglected tropical diseases
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    ABSTRACT: Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs able to control leprosy and tuberculosis, mainly due to extensively drug-resistant tuberculosis (XDR-TB), is pressing. Our group recently showed that M. leprae is able to induce lipid body biogenesis and cholesterol accumulation in macrophages and Schwann cells, facilitating its viability and replication. Considering these previous results, we investigated the efficacies of two statins on the intracellular viability of mycobacteria within the macrophage, as well as the effect of atorvastatin on M. leprae infections in BALB/c mice. We observed that intracellular mycobacteria viability decreased markedly after incubation with both statins, but atorvastatin showed the best inhibitory effect when combined with rifampin. Using Shepard's model, we observed with atorvastatin an efficacy in controlling M. leprae and inflammatory infiltrate in the BALB/c footpad, in a serum cholesterol level-dependent way. We conclude that statins contribute to macrophage-bactericidal activity against Mycobacterium bovis, M. leprae, and M. tuberculosis. It is likely that the association of statins with the actual multidrug therapy effectively reduces mycobacterial viability and tissue lesion in leprosy and tuberculosis patients, although epidemiological studies are still needed for confirmation.
    Full-text · Article · Jul 2014 · Antimicrobial Agents and Chemotherapy

  • No preview · Article · Jul 2014
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    ABSTRACT: We synthesized a series of novel dapsone-thalidomide hybrids (3a-i) by molecular hybridization and evaluated their potential for the treatment of type 2 leprosy reactions. All of the compounds had analgesic properties. Compounds 3c and 3h were the most active antinociceptive compounds and reduced acetic acid-induced abdominal constrictions by 49.8% and 39.1%, respectively. The hybrid compounds also reduced tumor necrosis factor-α levels in lipopolysaccharide-stimulated L929 cells. Compound 3i was the most active compound; at concentrations of 15.62 and 125μM, compound 3i decreased tumor necrosis factor-α levels by 86.33% and 87.80%, respectively. In nude mice infected with Mycobacterium leprae in vivo, compound 3i did not reduce the number of bacilli compared with controls. Compound 3i did not have mutagenic effects in Salmonella typhimurium strains TA100 and TA102, with or without metabolic activation (S9 mixture). Our results indicate that compound 3i is a novel lead compound for the treatment of type 2 leprosy reactions.
    No preview · Article · May 2014 · Bioorganic & Medicinal Chemistry Letters
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    ABSTRACT: Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.
    Preview · Article · Mar 2014 · Journal of Visualized Experiments
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    ABSTRACT: We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulates in ML-infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL-R, CD36, SRA-1, SR-B1, and LRP-1) and enzymes involved in Cho biosynthesis were investigated by qRT-PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular cholesterol, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML-infected macrophages to synthesize Cho and sequester exogenous LDL-Cho. Notably, Cho co-localized to ML-containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.
    Full-text · Article · Feb 2014 · Cellular Microbiology
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    ABSTRACT: Overpopulation of urban areas results from constant migrations that cause disordered urban growth, constituting clusters defined as sets of people or activities concentrated in relatively small physical spaces that often involve precarious conditions. Aim. Using residential grouping, the aim was to identify possible clusters of individuals in São José do Rio Preto, Sao Paulo, Brazil, who have or have had leprosy. Methods. A population-based, descriptive, ecological study using the MapInfo and CrimeStat techniques, geoprocessing, and space-time analysis evaluated the location of 425 people treated for leprosy between 1998 and 2010. Clusters were defined as concentrations of at least 8 people with leprosy; a distance of up to 300 meters between residences was adopted. Additionally, the year of starting treatment and the clinical forms of the disease were analyzed. Results. Ninety-eight (23.1%) of 425 geocoded cases were located within one of ten clusters identified in this study, and 129 cases (30.3%) were in the region of a second-order cluster, an area considered of high risk for the disease. Conclusion. This study identified ten clusters of leprosy cases in the city and identified an area of high risk for the appearance of new cases of the disease.
    Full-text · Article · Oct 2013 · The Scientific World Journal
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    ABSTRACT: Angiogenesis and lymphangiogenesis are the processes of neovascularization that evolve from preexisting blood and lymphatic vessels. There are few studies on angiogenesis and none on lymphangiogenesis in leprosy. Thus, the role of neovascularization in the pathophysiological mechanisms of the disease was studied across the spectrum of leprosy, its reactional states and its residual lesions. Seventy-six biopsies of leprosy skin lesions and seven healthy controls were selected. Fifty-five serum samples were used for the detection of CD105 by ELISA. Histological sections were stained with antibodies against CD31 (blood and lymphatic vessels), D2-40/podoplanin (lymphatic vessels), and CD105/endoglin (neovessels). Microvessels were counted in 100 high-power fields (400x) and the number of vessels was evaluated in relation to the extension of the inflammatory infiltrate (0-3), to the bacillary index (0-6) and to the clinical forms. Angiogenesis, as marked by CD31 and CD105, was observed across the leprosy spectrum, compared with the controls. Additionally, there was a positive correlation between these markers with extension of the infiltrate (p <0.0001). For D2/40, lymphangiogenesis was observed in the tuberculoid form (p <0.0001). There was no statistical significance for values of CD105 detected in plasma by ELISA. Angiogenesis is present across the spectrum of leprosy and in its reactional forms. The increase in the number of vessels, as detected by CD31 and CD105 staining, is related to the extension of the inflammatory infiltrate. Samples from reactional lesions have a higher number of CD31+ and CD105+ stained vessels, which indicates their involvement in the pathophysiological mechanisms of the reactional states. The regression of lesions is accompanied by the regression of neovascularization. Drugs inhibiting angiogenesis may be relevant in the treatment of leprosy, in addition to multidrugtherapy, and in the prevention of the development of reactions.
    Full-text · Article · Sep 2013 · PLoS ONE
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    ABSTRACT: Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.
    Full-text · Article · Jun 2013 · PLoS ONE
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    ABSTRACT: Leprosy in children is correlated with community-level factors, including the recent presence of disease and active foci of transmission in the community. We performed clinical and serological examinations of 1,592 randomly selected school children (SC) in a cross-sectional study of eight hyperendemic municipalities in the Brazilian Amazon Region. Sixty-three (4%) SC, with a mean age of 13.3 years (standard deviation = 2.6), were diagnosed with leprosy and 777 (48.8%) were seropositive for anti-phenolic glycolipid-I (PGL-I). Additionally, we evaluated 256 house-hold contacts (HHCs) of the students diagnosed with leprosy; 24 (9.4%) HHC were also diagnosed with leprosy and 107 (41.8%) were seropositive. The seroprevalence of anti-PGL-I was significantly higher amongst girls, students from urban areas and students from public schools (p < 0.0001). Forty-five (71.4%) new cases detected amongst SC were classified as paucibacillary and 59 (93.6%) patients did not demonstrate any degree of physical disability at diagnosis. The results of this study suggest that there is a high rate of undiagnosed leprosy and subclinical infection amongst children in the Amazon Region. The advantages of school surveys in hyperendemic areas include identifying leprosy patients at an early stage when they show no physical disabilities, preventing the spread of the infection in the community and breaking the chain of transmission.
    Full-text · Article · Dec 2012 · Memórias do Instituto Oswaldo Cruz

  • No preview · Article · Nov 2012 · Immunobiology
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    Full-text · Article · Mar 2011 · Indian journal of dermatology, venereology and leprology
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    ABSTRACT: Jorge Lobo's disease is a cutaneous and subcutaneous mycosis that affects patients in the Amazon region. The number of patients is relatively small, but the real situation of the disease as public health problem is not known, because Jorge Lobo's disease is not a notifiable disease. This study aims to report the clinical evolution in patients affected and to determine the prevalence and areas of occurrence of the disease. A retrospective study was carried out based on the analysis of the clinical records, which included a collection of photographs of patients in the Department of Sanitary Dermatology, in Rio Branco, and patients seen in the interior of the state. In a decade, in Rio Branco, 249 cases of the disease were reported, 30 were females and 219 males. Of these patients, 153 had localized lesions, 94 of them were on one ear, 55 had multifocal lesions and 41 had disseminated lesions. The average time between the onset of symptoms and diagnosis was 19 years. The average age at the time of diagnosis was 53 years, and ages ranged from 14 to 96 years.
    Preview · Article · Oct 2010 · Revista do Instituto de Medicina Tropical de São Paulo
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    ABSTRACT: Leprosy is still a worldwide public health problem. Brazil and India show the highest prevalence rates of the disease. Natural infection of armadillos Dasypus novemcinctus with Mycobacterium leprae has been reported in some regions of the United States. Identification of bacilli is difficult, particularly due to its inability to grow in vitro. The use of molecular tools represents a fast and sensitive alternative method for diagnosis of mycobacteriosis. In the present study, the diagnostic methods used were bacilloscopy, histopathology, microbiology, and PCR using specific primers for M. leprae repetitive sequences. PCR were performed using genomic DNA extracted from 138 samples of liver, spleen, lymph nodes, and skin of 44 D. novemcinctus, Euphractus sexcinctus, Cabassous unicinctus, and C. tatouay armadillos from the Middle Western region of the state of São Paulo and from the experimental station of Embrapa Pantanal, located in Pantanal da Nhecolândia of Mato Grosso do Sul state. Also, the molecular analysis of 19 samples from internal organs of other road killed species of wild animals, such as Nasua nasua (ring-tailed coati), Procyon cancrivoros (hand-skinned), Cerdocyon thous (dog-pity-bush), Cavia aperea (restless cavy), Didelphis albiventris (skunk), Sphigurrus spinosus (hedgehog), and Gallictis vittata (ferret) showed PCR negative data. None of the 157 analyzed samples had shown natural mycobacterial infection. Only the armadillo inoculated with material collected from untreated multibacillary leprosy patient presented PCR positive and its genomic sequencing revealed 100% identity with M. leprae. According to these preliminary studies, based on the used methodology, it is possible to conclude that wild mammals seem not to play an important role in the epidemiology of leprosy in the Middle Western region of the São Paulo state and in the Pantanal of Mato Grosso do Sul state.
    Full-text · Article · Feb 2010 · The Brazilian journal of infectious diseases: an official publication of the Brazilian Society of Infectious Diseases
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    ABSTRACT: Procedures involving the use of Mycobacterium leprae and Lacazia loboi, uncultivated organisms, depend on the collection of material from the lesions of patients or experimental animals. This study compared fine-needle aspiration (FNA) and skin biopsy methods for obtaining bacilli and fungal cells to experimentally infect animals. Lepromas from one armadillo and one enlarged footpad of a mouse previously inoculated with L. loboi were submitted to FNA and biopsy. Materials collected were processed for inoculation in mice. Acid-fast bacilli (AFB) collected by two FNA procedures yielded 7.2×10(7) and 5.3×10(6) AFB/ml and biopsies yielded 1.58×10(8) and 3.5×10(8) AFB/ml from each leproma. Yeast-like cells of L. loboi collected by FNA yielded 1.0×10(6) fungal cells/ml and biopsy 1.0×10(7) fungal cells/ml. After 8 months, inoculated animals were sacrificed and the inoculated footpads submitted to histopathological examination and counting of AFB and fungal cells. The results obtained by the two methods were comparable for both microorganisms. Biopsy may be replaced by FNA during harvesting of material for different purposes, especially for experimental inoculation of mice in leprosy and Jorge Lobo's disease, with the advantage of FNA being a simpler, less invasive, and less costly method.
    Full-text · Article · Feb 2010 · International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases
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    ABSTRACT: Several pathogens that cause important zoonotic diseases have been frequently associated with armadillos and other xenarthrans. This mammal group typically has evolved on the South American continent and many of its extant species are seriously threatened with extinction. Natural infection of armadillos with Paracoccidioides brasiliensis in hyperendemic areas has provided a valuable opportunity for understanding the role of this mammal in the eco-epidemiology of Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Latin America. This study aimed to detect P. brasiliensis in different xenarthran species (Dasypus novemcinctus, Cabassous spp., Euphractus sexcinctus, Tamandua tetradactyla and Myrmecophaga tridactyla), by molecular and mycological approaches, in samples obtained by one of the following strategies: i) from road-killed animals (n = 6); ii) from naturally dead animals (n = 8); iii) from animals that died in captivity (n = 9); and iv) from living animals captured from the wild (n = 2). Specific P. brasiliensis DNA was detected in several organs among 7/20 nine-banded armadillos (D. novemcinctus) and in 2/2 anteaters (M. tridactyla). The fungus was also cultured in tissue samples from one of two armadillos captured from the wild. Members of the Xenarthra Order, especially armadillos, have some characteristics, including a weak cellular immune response and low body temperature, which make them suitable models for studying host-pathogen interaction. P. brasiliensis infection in wild animals, from PCM endemic areas, may be more common than initially postulated and reinforces the use of these animals as sentinels for the pathogen in the environment.
    Full-text · Article · Nov 2009 · BMC Research Notes
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    ABSTRACT: Lacazia loboi is a geographically restricted, uncultivated fungal pathogen of humans and dolphins. Previous investigations using 18S small unit rDNA, chitin synthase 2 and gp43 DNA sequences positioned L. loboi as a close relative of Paracoccidioides brasiliensis. However, given the few individuals of L. loboi studied and the high degree of genetic variation observed in P. brasiliensis, the existence of L. loboi as an independent species has been questioned. To investigate the phylogenetic position of this species, we conducted a phylogenetic analysis using 20 L. loboi collections (L. loboi was obtained from proven cases of lacaziosis and 14 collections were maintained in mice, the others were analyzed from DNA taken directly from infected human tissue.). L. loboi DNA sequence was compared to that from 17 P. brasiliensis strains that represented the known variation in this species, and outgroup taxa in the Onygenales (Ajellomyces and Coccidioides species). Our analyses used DNA sequence from ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-ribosylation factor, and gp43. Nucleotide variation among strains of L. loboi was minor but numerous nucleotide mismatches and multiple gaps were found for these gene regions among members in the Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred using neighbor-joining, maximum parsimony and Bayesian analyses showed no significant conflict and depicted L. loboi as a well-supported, monophyletic group that was sister to the Paracoccidioides clade. These results argue for maintaining L. loboi as a taxon independent from Paracoccidioides within the Ajellomycetaceae.
    Full-text · Article · Jun 2009 · Mycological Research

Publication Stats

198 Citations
71.68 Total Impact Points

Institutions

  • 2002-2015
    • Instituto Lauro de Souza Lima
      Бауру, São Paulo, Brazil
  • 2009
    • Michigan State University
      • Department of Microbiology and Molecular Genetics
      East Lansing, MI, United States
  • 2005
    • Federal University of Minas Gerais
      Cidade de Minas, Minas Gerais, Brazil