Publications by authors named "Vânia Sammartino Mariano"

18 Publications

  • Page 1 of 1

Detection of Dysbiosis and Increased Intestinal Permeability in Brazilian Patients with Relapsing-Remitting Multiple Sclerosis.

Int J Environ Res Public Health 2021 04 27;18(9). Epub 2021 Apr 27.

Microbiology Program, Institute of Biosciences, Humanities and Exact Sciences, São Paulo State University, Sao Jose do Rio Preto 15054-000, Brazil.

Dysbiosis, associated with barrier disruption and altered gut-brain communications, has been associated with multiple sclerosis (MS). In this study, we evaluated the gut microbiota in relapsing-remitting patients (RRMS) receiving disease-modifying therapies (DMTs) and correlated these data with diet, cytokines levels, and zonulin concentrations. Stool samples were used for 16S sequencing and real-time PCR. Serum was used for cytokine determination by flow cytometry, and zonulin quantification by ELISA. Pearson's chi-square, Mann-Whitney, and Spearman's correlation were used for statistical analyses. We detected differences in dietary habits, as well as in the gut microbiota in RRMS patients, with predominance of and and decreased . Interleukin-6 concentrations were decreased in treated patients, and we detected an increased intestinal permeability in RRMS patients when compared with controls. We conclude that diet plays an important role in the composition of the gut microbiota, and intestinal dysbiosis, detected in RRMS patients could be involved in increased intestinal permeability and affect the clinical response to DTMs. The future goal is to predict therapeutic responses based on individual microbiome analyses (personalized medicine) and propose dietary interventions and the use of probiotics or other microbiota modulators as adjuvant therapy to enhance the therapeutic efficacy of DMTs.
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http://dx.doi.org/10.3390/ijerph18094621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123689PMC
April 2021

Human papillomavirus is not associated to non-small cell lung cancer: data from a prospective cross-sectional study.

Infect Agent Cancer 2019 2;14:18. Epub 2019 Aug 2.

1Teaching and Research Institute, Molecular Oncology Research Center, Barretos Cancer Hospital - Pio XII Foundation, Barretos, Brazil.

Background: The pathogenesis of lung cancer is triggered by a combination of genetic and environmental factors, being the tobacco smoke the most important risk factor. Nevertheless, the incidence of lung cancer in non-smokers is gradually increasing, which demands the search for different other etiological factors such as occupational exposure, previous lung disease, diet among others. In the early 80's a theory linked specific types of human papillomavirus (HPV) to lung cancer due to morphological similarities of a subset of bronchial squamous cell carcinomas with other HPV-induced cancers. Since then, several studies revealed variable rates of HPV DNA detection. The current study aimed to provide accurate information on the prevalence of HPV DNA in lung cancer.

Methods: Biopsies were collected from 77 newly diagnosed non-small cell lung cancer (NSCLC) patients treated at the Thoracic Oncology Department at Barretos Cancer Hospital. The samples were formalin fixed and paraffin embedded (FFPE), histologic analysis was performed by an experienced pathologist. DNA was extracted from FFPE material using a commercial extraction kit and HPV DNA detection was evaluated by multiplex PCR and HPV16 specific real-time PCR.

Results: HPV was not identified in any of the samples analysed (69).

Conclusions: Our data demonstrated a lack of HPV DNA in a series of NSCL cancers.
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http://dx.doi.org/10.1186/s13027-019-0235-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679449PMC
August 2019

Intestinal Dysbiosis in Autoimmune Diabetes Is Correlated With Poor Glycemic Control and Increased Interleukin-6: A Pilot Study.

Front Immunol 2018 25;9:1689. Epub 2018 Jul 25.

Microbiome Study Group, School of Health Sciences Dr. Paulo Prata (FACISB), Barretos, Brazil.

Intestinal dysbiosis associated with immunological deregulation, leaky gut, bacterial translocation, and systemic inflammation has been associated with autoimmune diseases, such as type 1 diabetes (T1D). The aim of this study was to investigate the intestinal dysbiosis in T1D patients and correlate these results with clinical parameters and cytokines. The present study was approved by the Barretos Cancer Hospital (Process number 903/2014), and all participants have signed the informed consent in accordance with the Declaration of Helsinki, and answered a questionnaire about dietary habits. Stool samples were used for bacterial 16S sequencing by MiSeq Illumina platform. IL-2, IL-4, IL-6, IL-10, IL-17A, TNF, and IFN-γ plasma concentrations were determined by cytometric bead arrays. The Pearson's chi-square, Mann-Whitney and Spearman correlation were used for statistical analyses. Alpha and beta diversities were conducted by using an annotated observed taxonomic units table. This study included 20 patients and 28 controls, and we found significant differences ( < 0.05) among consumption of vegetables, proteins, milk and derivatives, spicy food, and canned food when we compare patients and controls. We detected intestinal dysbiosis in T1D patients when we performed the beta diversity analysis ( = 0.01). The prevalent species found in patients' stool were the Gram-negatives , and . The inflammatory interleukin-6 was significantly increased ( = 0.017) in patients' plasma. Furthermore, we showed correlation among patients with poor glycemic control, represented by high levels of HbA1 percentages and Bacteroidetes, Lactobacillales, and relative abundances. We concluded that there are different gut microbiota profiles between T1D patients and healthy controls. The prevalent Gram-negative species in T1D patients could be involved in the leaky gut, bacterial translocation, and poor glycemic control. However, additional studies, with larger cohorts, are required to determine a "signature" of the intestinal microbiota in T1D patients in the Brazilian population.
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http://dx.doi.org/10.3389/fimmu.2018.01689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068285PMC
July 2018

The relationship between esophageal cancer, chagasic megaesophagus and HPV: myths, tales or reality?

Histol Histopathol 2018 Nov 24;33(11):1135-1149. Epub 2018 Apr 24.

Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil.

A supposed role for persistent high-risk human papillomavirus (HPV) infection in esophageal squamous cell carcinoma (ESCC) etiology has been suggested by a number of studies. Concomitantly, megaesophagus induced by the Trypanosoma cruzi cell-cycle activity also shows a potential association with ESCC. This review discusses esophageal cancer and the potential association between chagasic megaesophagus and HPV as risk factors for ESCC development.
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http://dx.doi.org/10.14670/HH-11-993DOI Listing
November 2018

HPV infection and p53 and p16 expression in esophageal cancer: are they prognostic factors?

Infect Agent Cancer 2017 13;12:54. Epub 2017 Oct 13.

Teaching and Research Institute, Barretos Cancer Hospital - Pius XII Foundation, Rua Antenor Duarte Vilela, 1331, Dr. Paulo Prata, Barretos, São Paulo 14784-400 Brazil.

Background: Esophageal squamous cell carcinoma (ESCC) is a highly lethal malignant tumor. Currently, Human papillomavirus (HPV) is suggested as a potential risk factor for esophageal cancer (EC) in addition to the classic risk factors, alcohol and tobacco, but this hypothesis still remains contradictory. We sought to investigate wether HPV and well-known biomarkers (p16 and p53) and patient-related factors that may have impact on survival of ESCC.

Methods: We conducted a prospective cohort study. By using multiplex PCR, we determined the prevalence of high risk HPV in ESCC, and evaluated the immunohistochemical expression of p16 and p53, molecular markers related to esophageal carcinogenesis in order to verify the potential influence of these variables in patients's survival. Survival rates were estimated using Kaplan-Meier methods. A multivariate confirmatory model was performed using Cox proportional hazards regression.

Results: Twelve (13.8%) of 87 patients were HPV-DNA positive. Positive reactions of p16 and p53 were 10.7% and 68.6%, respectively. Kaplan-Meier analysis indicated that men ( = 0.025) had poor specific-cancer survival and a shorter progression-free survival ( = 0.050) as compared to women; III or IV clinical stage ( < 0.019) had poor specific-cancer survival and a shorter progression-free survival ( < 0.001) compared to I and II clinical stage; not submitted to surgery (<0.001) and not submitted to chemoradiotherapy ( = 0.039) had a poor specific-cancer survival, as well. The multivariate analysis showed that HPV, p16 and p53 status are not predictive parameters of progression-free and specific-cancer survival.

Conclusion: HPV infection and p53 and p16 expression are not prognostic factors in ESCC.
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http://dx.doi.org/10.1186/s13027-017-0163-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640908PMC
October 2017

Detection of Increased Plasma Interleukin-6 Levels and Prevalence of and in the Feces of Type 2 Diabetes Patients.

Front Immunol 2017 15;8:1107. Epub 2017 Sep 15.

Microbiome Study Group, School of Health Sciences Dr. Paulo Prata (FACISB), Barretos, Brazil.

Intestinal dysbiosis and metabolic endotoxemia have been associated with metabolic disorders, such as obesity, insulin resistance, and type 2 diabetes (T2D). The main goal of the present study was to evaluate the intestinal dysbiosis in Brazilian T2D patients and correlate these data with inflammatory cytokines and lipopolysaccharides (LPS) plasma concentrations. This study was approved by the Ethics Committees from Barretos Cancer Hospital and all individuals signed the informed consent form. Stool samples were required for DNA extraction, and the V3/V4 regions of bacterial 16S were sequenced using an Illumina platform. Peripheral blood was used to quantify inflammatory cytokines and plasma LPS concentrations, by CBA flex and ELISA, respectively. Statistical analyses were performed using Mann-Whitney and Spearman's tests. Analysis of variance, diversity indexes, and analysis of alpha- and beta-diversity were conducted using an annotated Operational Taxonomic Unit table. This study included 20 patients and 22 controls. We observed significant differences ( < 0.01) in the microbiota composition (beta-diversity) between patients and controls, suggesting intestinal dysbiosis in Brazilian T2D patients. The prevalent species found in patients' feces were the Gram-negatives , and . The proinflammatory interleukin-6 (IL-6) was significantly increased ( < 0.05) in patients' plasma and LPS levels were decreased. We find correlations between the proinflammatory interferon-gamma with Gram-negatives and species, and a positive correlation between the LPS levels and reads. The and species were associated with insulin resistance in previous studies. In this study, we suggested that the prevalence of Gram-negative species in the gut and the increased plasma IL-6 in patients could be linked to low-grade inflammation and insulin resistance. In conclusion, the and species could represent an intestinal microbiota signature, associated with T2D development. Furthermore, the identification of these Gram-negative bacteria, and the detection of inflammatory markers, such as increased IL-6, could be used as diabetes predictive markers in overweight, obese and in genetically predisposed individuals to develop T2D.
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http://dx.doi.org/10.3389/fimmu.2017.01107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605568PMC
September 2017

Prevalence of high risk HPV DNA in esophagus is high in Brazil but not related to esophageal squamous cell carcinoma.

Histol Histopathol 2018 Apr 6;33(4):357-363. Epub 2017 Sep 6.

Teaching and Research Institute, Barretos Cancer Hospital - Pius XII Foundation, Brazil.

Backgrounds: The first publication that associated Human Papillomavirus (HPV) infection and esophageal cancer was published in 1982. However, data are still contradictory and require further investigation. The aim of this study was to identify high risk HPV DNA in esophageal tissue of patients with and without esophageal squamous cell carcinoma (ESCC) and correlate HPV presence with classical risk factors.

Methods: Invited patients signed the informed consent form, and interviews were conducted in order to obtain information about sociodemographic and lifestyle behavior. During endoscopy, esophageal biopsies were collected from case and controls. Multiplex polymerase chain reaction genotyping was conducted on endoscopic biopsies to identify HPV types and HPV-16 was further evaluated by specific PCR real time.

Results: Among 87 cases, 12 (13.8%) had tumors harboring high risk HPV DNA and among 87 controls, 12 (13.8%) had high risk HPV DNA (OR:1.025 [CI:0.405:2.592]). Variables regarding consumption of alcohol and use of tobacco continued to characterize risk factors even after adjustments by presence or absence of high risk HPV.

Conclusion: HPV was demonstrated to be frequently and similarly associated to normal and malignant esophageal tissues, but not as an independent risk factor to esophageal cancer.

Impact: To contribute to the Brazilian population data on this subject, which is still contradictory.
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http://dx.doi.org/10.14670/HH-11-929DOI Listing
April 2018

Paracoccin distribution supports its role in Paracoccidioides brasiliensis growth and dimorphic transformation.

PLoS One 2017 28;12(8):e0184010. Epub 2017 Aug 28.

Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil.

Paracoccidioides brasiliensis yeast was reported to express paracoccin, a GlcNAc-binding protein that displays N-acetyl-β-d-glucosaminidase (NAGase) activity. Highly specific anti-paracoccin antibodies have been previously used to examine the localization of paracoccin in yeast and inhibit its growth in vitro. In the present study, anti-paracoccin antibodies were used to characterize, by scanning confocal microscopy, the distribution of paracoccin in P. brasiliensis hyphae, transition forms from hyphae to yeast, and mature yeast. In the mycelial phase, paracoccin was detected mainly in the hyphae tips, where it demonstrated a punctate distribution, and was associated with the cell wall. During the first 48 hours after a temperature shift from 26°C to 37°C, paracoccin expression in the differentiating hyphae was mainly detected in the budding regions, i.e. lateral protrusions, and inside the new daughter cells. There was an increased number of chlamydoconidia that expressed a high concentration of paracoccin on their surfaces and/or in their interiors 72-96 hours after the temperature shift. After 120 hours, yeast cells were the predominant form and their cytoplasm stained extensively for paracoccin, whereas Wheat Germ Agglutinin (WGA) staining was predominant on their exterior walls. After 10 days at 37°C, the interior of both mother and daughter yeast cells, as well as the budding regions, stained intensely for paracoccin. The comparison of mRNA-expression in the different fungal forms showed that PCN transcripts, although detected in all evaluated morphological forms, were higher in hypha and yeast-to-hypha transition forms. In conclusion, the pattern of paracoccin distribution in all P. brasiliensis morphotypes supports prevalent beliefs that it plays important roles in fungal growth and dimorphic transformation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184010PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573292PMC
October 2017

High systemic IL-6 is associated with worse prognosis in patients with non-small cell lung cancer.

PLoS One 2017 17;12(7):e0181125. Epub 2017 Jul 17.

Teaching and Research Institute, Barretos Cancer Hospital-Pio XII Foundation, Barretos, Sao Paulo, Brazil.

Characteristic cytokine patterns have been described in different cancer patients and they are related to their diagnosis, prognosis, prediction of treatment responses and survival. A panel of cytokines was evaluated in the plasma of non-small cell lung cancer (NSCLC) patients and healthy controls to investigate their profile and relationship with clinical characteristics and overall survival. The case-controlled cross-sectional study design recruited 77 patients with confirmed diagnosis of NSCLC (cases) and 91 healthy subjects (controls) aimed to examine peripheral pro-inflammatory and anti-inflammatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNF and IFN-γ) by Cytometry Beads Arrays (CBA Flex) in. The cytokine IL-6 showed a statistically significant difference among groups with increased expression in the case group (p < 0.001). The correlation between the cytokines expression with patient's clinical characteristics variables revealed the cytokine IL-6 was found to be associated with gender, showing higher levels in male (p = 0.036), whereas IL-17A levels were associated with TNM stage, being higher in III-IV stages (p = 0.044). We observed worse overall survival for individuals with high levels of IL-6 when compared to those with low levels of this cytokine in 6, 12 and 24 months. Further studies of IL-6 levels in independent cohort could clarify the real role of IL-6 as an independent marker of prognostic of NSCLC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181125PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513446PMC
September 2017

Lung cancer samples preserved in liquid medium: One step beyond cytology.

Diagn Cytopathol 2017 Oct 6;45(10):915-921. Epub 2017 Jun 6.

Barretos Cancer Hospital/Pio XII Foundation, Molecular Oncology Research Center, Brazil.

Lung cancer is one of the most common cancer types in men and women worldwide with a high mortality rate. World Health Organization (WHO) classification has accepted biopsy as the primary sample for lung cancer diagnosis, pathological classification and molecular testing for management of patients, yet, the use of alternative sampling procedures is highly encouraged. Bronchial cytological samples require a less invasive collection technique and may be suitable for pathological and molecular analysis and storage in liquid medium. Furthermore, the molecular analysis of bronchial cytological samples allows the detection of molecular biomarkers, which may be useful for the selection of molecular targeted therapies. Thus, the purpose of this review is to describe the usefulness of bronchial cytological samples preserved in liquid medium from lung cancer patients for pathological diagnosis and molecular investigation.
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http://dx.doi.org/10.1002/dc.23743DOI Listing
October 2017

Intestinal dysbiosis and probiotic applications in autoimmune diseases.

Immunology 2017 09 29;152(1):1-12. Epub 2017 Jun 29.

Barretos Cancer Hospital, Barretos, São Paulo, Brazil.

In humans, a complex interaction between the host immune system and commensal microbiota is required to maintain gut homeostasis. In this symbiotic relationship, the microbiota provides carbohydrate fermentation and digestion, vitamin synthesis and gut-associated lymphoid tissue development, as well as preventing colonization by pathobionts, whereas the host offers a niche and nutrients for the survival of the microbiota. However, when this mutualistic relationship is compromised and an altered interaction between immune cells and microorganisms occurs, the gut microbiota may cause or contribute to the establishment of infectious diseases and trigger autoimmune diseases. Researchers have made efforts to clarify the role of the microbiota in autoimmune disease development and find new therapeutic approaches to treat immune-mediated diseases. However, the exact mechanisms involved in the dysbiosis and breakdown of the gut epithelial barrier are currently unknown. Here, we provide a general overview of studies describing gut microbiota perturbations in animal models of autoimmune diseases, such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. Moreover, we include the main studies concerning dysbiosis in humans and a critical discussion of the existing data on the use of probiotics in these autoimmune diseases.
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http://dx.doi.org/10.1111/imm.12765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543467PMC
September 2017

The Relation of HPV Infection and Expression of p53 and p16 Proteins in Esophageal Squamous Cells Carcinoma.

J Cancer 2017 9;8(6):1062-1070. Epub 2017 Apr 9.

Teaching and Research Institute, Molecular Oncology Research Center, Barretos Cancer Hospital - Pio XII Foundation, Brazil.

To investigate the HPV prevalence and characterize the expression of potential molecular surrogate markers of HPV infection in esophageal squamous cell carcinoma. The prevalence of HPV in individuals with and without esophageal cancer (EC) was determined by using multiplex PCR; p16 and p53 protein levels were assessed by immunohistochemistry (IHC). High-risk HPV (hr-HPV) was found in the same frequency (13.8%) in esophageal squamous cell carcinoma (ESCC) and in healthy individuals. The p53 expression was positive in 67.5% of tumor tissue, 20.0% of adjacent non-tumoral tissue and 1.8% of normal esophageal tissue. p16 was positive in 11.6% of esophageal cancer cases and 4.7% of adjacent non-tumoral tissue. p16 was undetectable among control group samples. p53 and p16 levels were not significantly associated with the HPV status. These results suggest that hr-HPV types are not associated with the development of ESCC and that p53 and p16 protein expression have no relationship with HPV infection in normal or cancerous esophagus.
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http://dx.doi.org/10.7150/jca.17080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436260PMC
April 2017

A Low-Cost HPV Immunochromatographic Assay to Detect High-Grade Cervical Intraepithelial Neoplasia.

PLoS One 2016 20;11(10):e0164892. Epub 2016 Oct 20.

Molecular Oncology Research Center, Barretos Cancer Hospital/Pio XII Foundation, Barretos, Sao Paulo, Brazil.

Objective: To evaluate the reproducibility and accuracy of the HPV16/18-E6 test.

Methods: The study population was comprised of 448 women with a previously abnormal Pap who were referred to the Barretos Cancer Hospital (Brazil) for diagnosis and treatment. Two cervical samples were collected immediately before colposcopy, one for the hr-HPV-DNA test and cytology and the other for the HPV16/18-E6 test using high-affinity monoclonal antibodies (mAb). Women with a histologic diagnosis of cervical intraepithelial neoplasia grade 2 or 3 were considered to be positive cases. Different strategies using a combination of screening methods (HPV-DNA) and triage tests (cytology and HPV16/18-E6) were also examined and compared.

Results: The HPV16/18-E6 test exhibited a lower positivity rate compared with the HPV-DNA test (19.0% vs. 29.3%, p<0.001) and a moderate/high agreement (kappa = 0.68, 95%CI: 0.60-0.75). It also exhibited a significantly lower sensitivity for CIN2+ and CIN3+ detection compared to the HPV-DNA test and a significantly higher specificity. The HPV16/18-E6 test was no different from cytology in terms of sensitivity, but it exhibited a significantly higher specificity in comparison to ASCH+. A triage test after HPV-DNA detection using the HPV16/18-E6 test exhibited a significantly higher specificity compared with a triage test of ASCH+ to CIN2+ (91.8% vs. 87.4%, p = 0.04) and CIN3+ (88.6% vs. 84.0%, p = 0.05).

Conclusion: The HPV16/18-E6 test exhibited moderate/high agreement with the HPV-DNA test but lower sensitivity and higher specificity for the detection of CIN2+ and CIN3+. In addition, its performance was quite similar to cytology, but because of the structural design addressed for the detection of HPV16/18-E6 protein, the test can miss some CIN2/3+ lesions caused by other high-risk HPV types.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164892PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072685PMC
June 2017

IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells.

PLoS One 2016 22;11(2):e0149721. Epub 2016 Feb 22.

Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4+ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4+ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149721PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767177PMC
July 2016

Recognition of TLR2 N-glycans: critical role in ArtinM immunomodulatory activity.

PLoS One 2014 3;9(6):e98512. Epub 2014 Jun 3.

Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, USP, São Paulo, Brasil.

TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098512PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4043963PMC
January 2015

Lysophosphatidylcholine triggers TLR2- and TLR4-mediated signaling pathways but counteracts LPS-induced NO synthesis in peritoneal macrophages by inhibiting NF-κB translocation and MAPK/ERK phosphorylation.

PLoS One 2013 30;8(9):e76233. Epub 2013 Sep 30.

Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil ; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular- INCT-EM, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.

Background: Lysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway.

Methodology/principal Findings: HEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells.

Conclusions/significance: The above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076233PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848743PMC
June 2014

Monocyte Migration Driven by Galectin-3 Occurs through Distinct Mechanisms Involving Selective Interactions with the Extracellular Matrix.

ISRN Inflamm 2013 25;2013:259256. Epub 2013 Feb 25.

Programa de pós-graduação em Imunologia Básica e Aplicada, FMRP/USP, Ribeirão Preto, SP, Brazil.

Monocyte migration into tissues, an important event in inflammation, requires an intricate interplay between determinants on cell surfaces and extracellular matrix (ECM). Galectin-3 is able to modulate cell-ECM interactions and is an important mediator of inflammation. In this study, we sought to investigate whether interactions established between galectin-3 and ECM glycoproteins are involved in monocyte migration, given that the mechanisms by which monocytes move across the endothelium and through the extravascular tissue are poorly understood. Using the in vitro transwell system, we demonstrated that monocyte migration was potentiated in the presence of galectin-3 plus laminin or fibronectin, but not vitronectin, and was dependent on the carbohydrate recognition domain of the lectin. Only galectin-3-fibronectin combinations potentiated the migration of monocyte-derived macrophages. In binding assays, galectin-3 did not bind to fibronectin, whereas both the full-length and the truncated forms of the lectin, which retains carbohydrate binding ability, were able to bind to laminin. Our results show that monocytes migrate through distinct mechanisms and selective interactions with the extracellular matrix driven by galectin-3. We suggest that the lectin may bridge monocytes to laminin and may also activate these cells, resulting in the positive regulation of other adhesion molecules and cell adhesion to fibronectin.
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http://dx.doi.org/10.1155/2013/259256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3767352PMC
September 2013

Influence of N-glycosylation on the morphogenesis and growth of Paracoccidioides brasiliensis and on the biological activities of yeast proteins.

PLoS One 2011 21;6(12):e29216. Epub 2011 Dec 21.

Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.

The fungus Paracoccidioides brasiliensis is a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The cell wall of P. brasiliensis is a network of glycoproteins and polysaccharides, such as chitin, that perform several functions. N-linked glycans are involved in glycoprotein folding, intracellular transport, secretion, and protection from proteolytic degradation. Here, we report the effects of tunicamycin (TM)-mediated inhibition of N-linked glycosylation on P. brasiliensis yeast cells. The underglycosylated yeasts were smaller than their fully glycosylated counterparts and exhibited a drastic reduction of cell budding, reflecting impairment of growth and morphogenesis by TM treatment. The intracellular distribution in TM-treated yeasts of the P. brasiliensis glycoprotein paracoccin was investigated using highly specific antibodies. Paracoccin was observed to accumulate at intracellular locations, far from the yeast wall. Paracoccin derived from TM-treated yeasts retained the ability to bind to laminin despite their underglycosylation. As paracoccin has N-acetyl-β-d-glucosaminidase (NAGase) activity and induces the production of TNF-α and nitric oxide (NO) by macrophages, we compared these properties between glycosylated and underglycosylated yeast proteins. Paracoccin demonstrated lower NAGase activity when underglycosylated, although no difference was detected between the pH and temperature optimums of the two forms. Murine macrophages stimulated with underglycosylated yeast proteins produced significantly lower levels of TNF-α and NO. Taken together, the impaired growth and morphogenesis of tunicamycin-treated yeasts and the decreased biological activities of underglycosylated fungal components suggest that N-glycans play important roles in P. brasiliensis yeast biology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029216PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3244461PMC
May 2012
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