Imbalanced IL10/TGF-ß production by regulatory T-lymphocytes in patients with HTLV-1-associated myelopathy/ tropical spastic paraparesis.

BACKGROUND: Human T-cell lymphotropic virus type 1 (HTLV-1), also denominated Human T-cell leukemia virus-1, induces immune activation and secretion of proinflammatory cytokines, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Regulatory T lymphocytes (Tregs) may control of inflammation through the production of regulatory cytokines, including IL10 and TGF-ß. In this study we determined the frequencies of CD4 + and CD8 + Tregs in a HAM/TSP population, compared to asymptomatic carriers and uninfected individuals, as well as investigated the profiles of regulatory and inflammatory cytokines. METHODS: Asymptomatic HTLV-1 carriers and HAM/TSP patients were matched by sex and age. The frequencies of IL10- and/or TGF-ß-producing Tregs were quantified by flow cytometry. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify HTLV-1 proviral load and the mRNA expression of cytokines and cellular receptors in peripheral blood mononuclear cells. RESULTS: Total frequencies of CD4 + Tregs, as well as the IL10-producing CD4 + and CD8 + Treg subsets, were statistically higher in patients with HAM/TSP compared to asymptomatic HTLV-1-infected individuals. In addition, a positive correlation was found between the frequency of CD4 + IL10 + Tregs and proviral load in the HAM/TSP patients evaluated. A positive correlation was also observed between gene expression of proinflammatory versus regulatory cytokines only in HAM / TSP group. CONCLUSIONS: A higher frequencies of IL10-producing Tregs were identified in patients with HAM/TSP. Imbalanced production of IL10 in relation to TGF-ß may contribute to the increased inflammatory response characteristically seen in HAM/TSP patients.

Leishmania braziliensis exosomes activate human macrophages to produce proinflammatory mediators.

Exosomes, organelles measuring 30-200nm, are secreted by various cell types. Leishmania exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein 63, proteins exerting signaling activity and those containing mRNA and miRNA. Studies have demonstrated that Leishmania donovani exosomes downregulate IFN-γ and inhibit the expression of microbicidal molecules, such as TNF and nitric oxide, thus creating a microenvironment favoring parasite proliferation. Despite lacking immunological memory, data in the literature suggest that, following initial stimulation, mononuclear phagocytes may become "trained" to respond more effectively to subsequent stimuli. Here we characterized the effects of macrophage sensitization using L. braziliensis exosomes prior to infection by the same pathogen. Human macrophages were stimulated with L. braziliensis exosomes and then infected with L. braziliensis. Higher levels of IL-1ß and IL-6 were detected in cultures sensitized prior to infection compared to unstimulated infected cells. Moreover, stimulation with L. braziliensis exosomes induced macrophage production of IL-1ß, IL-6, IL-10 and TNF. Inhibition of exosome secretion by L. braziliensis prior to macrophage infection reduced cytokine production and produced lower infection rates than untreated infected cells. Exosome stimulation also induced the consumption/regulation of NLRP3 inflammasome components in macrophages, while the blockade of NLRP3 resulted in lower levels of IL-6 and IL-1ß. Our results suggest that L. braziliensis exosomes stimulate macrophages, leading to an exacerbated inflammatory state that may be NLRP3-dependent.

Differential gene expression analysis of sickle cell anemia in steady and crisis state.

Sickle cell anemia is one of the most prevalent genetic diseases worldwide, showing great clinical heterogeneity. This study compared the gene expression patterns between sickle cell anemia pediatric patients in steady state and in crisis state, as compared to age-paired, healthy individuals. RNA sequencing was performed from these groups of patients/controls using Illumina HiSeq 2500 equipment. The resulting differentially expressed genes were loaded into QIAGEN's ingenuity pathway analysis. The results showed that EIF2 pathway and NRF2-mediated oxidative stress-response pathways were more highly activated both in steady state and in crisis patients, as compared to healthy individuals. In addition, we found increased activation of eIF4 and p70S6K signaling pathways in crisis state compared to healthy individuals. The transcription factor GATA-1 was found exclusively in steady state while SPI was found exclusively in crisis state. IL6 and VEGFA were found only in crisis state, while IL-1B was found exclusively in steady state. The regulator effects analysis revealed IgG1 as an upstream regulator in steady state compared to healthy individuals, resulting in invasion of prostate cancer cell lines as the disease/function outcome. For crisis-state patients versus healthy individuals, two networks of regulator effects revealed STAT1, CD40LG, TGM2, IRF7, IRF4, and IRF1 acting as upstream regulators, resulting in disease/function outcomes, including engulfment of cells and aggregation of blood cells and inflammation of joints. Our results indicated genes and pathways that can provide clues on the molecular events involved in the severity of sickle cell disease.

The miRNA 361-3p, a Regulator of GZMB and TNF Is Associated With Therapeutic Failure and Longer Time Healing of Cutaneous Leishmaniasis Caused by L. (viannia) braziliensis.

L. (viannia) braziliensis infection causes American Tegumentary Leishmaniasis (ATL), with prolonged time to healing lesions. The potent inflammatory response developed by the host is important to control the parasite burden and infection however an unbalanced immunity may cooperate to the tissue damage observed. The range of mechanisms underlying the pathological responses associated with ATL still needs to be better understood. That includes epigenetic regulation by non-coding MicroRNAs (miRNAs), non-coding sequences around 22 nucleotides that act as post-transcriptional regulators of RNAs encoding proteins. The miRNAs have been associated with diverse parasitic diseases, including leishmaniasis. Here we evaluated miRNAs that targeted genes expressed in cutaneous leishmaniasis lesions (CL) by comparing its expression in both CL and normal skin obtained from the same individual. In addition, we evaluated if the miRNAs expression would be correlated with clinical parameters such as therapeutic failure, healing time as well as lesion size. The miR-361-3p and miR-140-3p were significantly more expressed in CL lesions compared to normal skin samples (p = 0.0001 and p < 0.0001, respectively). In addition, the miR-361-3p was correlated with both, therapeutic failure and healing time of disease (r = 0.6, p = 0.003 and r = 0.5, p = 0.007, respectively). In addition, complementary analysis shown that miR-361-3p is able to identify with good sensitivity (81.2%) and specificity (100%) patients who tend to fail initial treatment with pentavalent antimonial (Sbv). Finally, the survival analysis considering "cure" as the endpoint showed that the higher the expression of miR-361-3p, the longer the healing time of CL. Overall, our data suggest the potential of miR-361-3p as a prognostic biomarker in CL caused by L. braziliensis.

Resolvin D1 drives establishment of Leishmania amazonensis infection.

Previous studies have indicated that the balance between different eicosanoids reflect the intensity of the inflammatory profile in patients with tegumentary leishmaniasis. More recently, pro-resolution lipid mediators have been shown to play critical roles in dampening pathological inflammatory processes to reestablish homeostasis in a diverse range of experimental settings. Among these lipid mediator, resolvins from D series have been described as potent anti-inflammatory and immunomodulatory mediators, and its activities include inhibition of leukocyte chemotaxis and blockage production of proinflammatory cytokines, while increasing the expression of regulatory mediators. Whether resolvins play significant roles in establishment and persistence of Leishmania infection is currently unknown. We addressed this question in the current study by assessing circulating levels of D-series resolvins in tegumentary leishmaniasis patients presenting with localized or diffuse disease. We found heightened expression of resolvin D1 in diffuse cutaneous leishmaniasis which was correlated with expression profile of biomarkers associated with disease pathogenesis. Additional in vitro experiments using primary human macrophages indicated that resolvin D1 may promote intracellular Leishmania amazonensis replication through a mechanism associated with induction of heme oxygenase-1. These results suggest that targeting resolvin D1 could serve as potential strategy for host directed therapy in diffuse cutaneous leishmaniasis.

Mitochondrial genome instability in colorectal adenoma and adenocarcinoma.

Mitochondrial dysfunction is regarded as a hallmark of cancer progression. In the current study, we evaluated mitochondrial genome instability and copy number in colorectal cancer using Next Generation Sequencing approach and qPCR, respectively. The results revealed higher levels of heteroplasmy and depletion of the relative mtDNA copy number in colorectal adenocarcinoma. Adenocarcinoma samples also presented an increased number of mutations in nuclear genes encoding proteins which functions are related with mitochondria fusion, fission and localization. Moreover, we found a set of mitochondrial and nuclear genes, which cooperate in the same mitochondrial function simultaneously mutated in adenocarcinoma. In summary, these results support an important role for mitochondrial function and genomic instability in colorectal tumorigenesis.

COL1A1 and miR-29b show lower expression levels during osteoblast differentiation of bone marrow stromal cells from Osteogenesis Imperfecta patients.

BACKGROUND: The majority of Osteogenesis Imperfecta (OI) cases are caused by mutations in one of the two genes, COL1A1 and COL1A2 encoding for the two chains that trimerize to form the procollagen 1 molecule. However, alterations in gene expression and microRNAs (miRNAs) are responsible for the regulation of cell fate determination and may be evolved in OI phenotype. METHODS: In this work, we analyzed the coding region and intron/exon boundaries of COL1A1 and COL1A2 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. COL1A1 and miR-29b expression were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System. RESULTS: We have identified eight novel mutations, where of four may be responsible for OI phenotype. COL1A1 and miR-29b showed lower expression values in OI type I and type III samples. Interestingly, one type III OI sample from a patient with Bruck Syndrome showed COL1A1 and miR-29b expressions alike those from normal samples. CONCLUSIONS: Results suggest that the miR-29b mechanism directed to regulate collagen protein accumulation during mineralization is dependent upon the amount of COL1A1 mRNA. Taken together, results indicate that the lower levels observed in OI samples were not sufficient for the induction of miR-29b.

Simvastatin reduces CCL2 expression in monocyte-derived cells by induction of a repressive CCL2 chromatin state.

Statins exert anti-inflammatory characteristics, besides their lipid lowering properties, and may display beneficial effects for the treatment of inflammatory diseases. One possible explanation is that statins interfere in the deregulated gene transcription patterns associated with immune-mediated diseases, although the precise mechanism is not fully understood. Besides gene regulatory proteins, epigenetic mechanisms play an important role in the orchestration of gene expression. Disturbances in the tightly controlled epigenetic mechanisms influence the cellular portrait of expressed genes resulting in the protein dysfunctions found in many inflammatory diseases. In this study, we found that simvastatin reduces secretion and gene expression of CCL2 in monocyte-derived immature dendritic cells and in type 1 macrophages, which is accompanied by increased levels of the 3meK27H3 and 3meK9H3 repressive histone marks and decreased levels of the permissive histone marks AcH3 and 3meK4H3 in CCL2 promoter chromatin. The repressive chromatin status of the CCL2 promoter region affected recruitment of the NF-κB p65 subunit, which controls CCL2 transcription. The down-regulation of CCL2 in these immune cells may therefore impact their chemotactic activity and reduce their recruitment to sites of tissue injury.

Colon preneoplasia after carcinogen exposure is enhanced and colonic serotonergic system is suppressed by food deprivation.

Calorie restriction regimens usually promote health and extend life-span in mammals. This is partially related to their preventive effects against malignancies. However, certain types of nutritional restriction failed to induce beneficial effects. The American Institute of Nutrition defines calorie restriction as diets which have only 40% fewer calories, but provide normal amounts of necessary food components such as protein, vitamins and minerals; whereas, food restriction means 40% less of all dietary ingredients plus 40% less calories. Our study aimed to test the hypothesis that the latter type of food deprivation (40% less food than consumed by standard fed rats) might increase cancer risk instead of reducing it, as is generally assumed for all dietary restrictive regimens. Since the endogenous modulation of the colon serotonergic system has been observed to play a role during the early steps of carcinogenesis we also investigated whether the serotoninergic system could be involved in the food intake modulation of cancer risk. For this, rats were exposed to a carcinogen and subjected to food deprivation for 56 days. Triglyceride levels and visceral adipose tissue were reduced while hepatic and colonic lipid peroxidation was increased. This dietary restriction also decreased serotonin levels in colon, and gene expression of its intestinal transporter and receptors. Finally, the numbers of preneoplastic lesions in the colon tissue of carcinogen-exposed rats were increased. Our data suggest that food deprivation enhances formation of early tumorigenic lesions by suppressing serotonergic activity in colon tissue.

Increased expression of miR-221 is associated with shorter overall survival in T-cell acute lymphoid leukemia.

BACKGROUND: CD56 expression has been associated with a poor prognosis in lymphoid neoplasms, including T-cell acute lymphoblastic leukemia (T-ALL). MicroRNAs (miRNAs) play an important role in lymphoid differentiation, and aberrant miRNA expression has been associated with treatment outcome in lymphoid malignancies. Here, we evaluated miRNA expression profiles in normal thymocytes, mature T-cells, and T-ALL samples with and without CD56 expression and correlated microRNA expression with treatment outcome. METHODS: The gene expression profile of 164 miRNAs were compared for T-ALL/CD56+ (n=12) and T-ALL/CD56- (n=36) patients by Real-Time Quantitative PCR. Based on this analysis, we decided to evaluate miR-221 and miR-374 expression in individual leukemic and normal samples. RESULTS: miR-221 and miR-374 were expressed at significantly higher levels in T-ALL/CD56+ than in T-ALL/CD56- cells and in leukemic blasts compared with normal thymocytes and peripheral blood (PB) T-cells. Age at diagnosis (15 or less vs grater than 15 years; HR: 2.19, 95% CI: 0.98-4.85; P=0.05), miR-221 expression level (median value as cut off in leukemic samples; HR: 3.17, 95% CI: 1.45-6.92; P=0.004), and the expression of CD56 (CD56-vs CD56+; HR: 2.99, 95% CI: 1.37-6.51; P=0.006) were predictive factors for shorter overall survival; whereas, only CD56 expression (HR: 2.73, 95% CI: 1.03-7.18; P=0.041) was associated with a shorter disease-free survival rate. CONCLUSIONS: miR-221 is highly expressed in T-ALL and its expression level may be associated with a poorer prognosis.

Antiproliferative effects of fluoxetine on colon cancer cells and in a colonic carcinogen mouse model.

The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G(0)/G(1) phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.

High-fat diet causes an imbalance in the colonic serotonergic system promoting adipose tissue enlargement and dysplasia in rats.

A high-fat (HF) diet, the serotonergic system and stromal elements have all been implicated in colon carcinogenesis. We investigated whether the colonic serotonergic system could play a main role in the development of colonic dysplasia and stromal reactivity in carcinogen-treated rats under HF diet. For this, dimethylhydrazine-treated rats were fed with standard diet and a HF diet. Fat distribution was quantified by computerized tomography exam, serotonergic activity was analyzed by high-performance liquid chromatography, gene expression, and immunohistochemistry, which along with histopathological technique enabled us to enumerate dysplasia, microvessels density, cell proliferation and COX-2 expression. We found that the HF diet induced an increase in the amount of visceral adipose tissue, even without expressive changes in the average body weight. This was correlated with a loss of serotonergic balance in colon tissue. Moreover, the HF diet promoted dysplasia and microvessel density in association with increased proliferation and COX-2 expression within pericryptal colonic stroma. Our current findings suggest that a HF diet promotes the enlargement of adipose tissue via loss of control in colon serotonergic activity, which enhances colonic dysplasia by supporting microvessel development.

miR-15a and 16-1 are downregulated in CD4+ T cells of multiple sclerosis relapsing patients.

The pathology of relapsing-remitting multiple sclerosis (RR-MS) is largely attributed to activated autoreactive effector T lymphocytes. The influence of microRNAs on the immune response has been shown to occur in different pathways of lymphocyte differentiation and function. Here, the expression of the miRNAs miR-15a/16-1 in PBMC, CD4(+), and CD8(+) from RR-MS patients has been investigated. BCL2, a known miR-15a/16-1 target, has also been analyzed. The results have shown that miR-15a/16-1 is downregulated in CD4(+) T cells, whereas BCL2 is highly expressed in RR-MS patients only. Our data suggest that miR-15a/16-1 can also modulate the BCL2 gene expression in CD4(+) T cells from RR-MS patients, thereby affecting apoptosis processes.

Fluoxetine induces preventive and complex effects against colon cancer development in epithelial and stromal areas in rats.

Fluoxetine (FLX) is a drug commonly used as antidepressant. However, its effects on tumorigenesis remain controversial. Aiming to evaluate the effects of FLX treatment on early malignant changes, we analyzed serotonin (5-HT) metabolism and recognition, aberrant crypt foci (ACF), proliferative process, microvessels, vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2) expression in colon tissue. Male Wistar rats received a daily FLX-gavage (30mgkg(-1)) and, a single dose of 1,2 dimethylhydrazine (DMH; i.p., 125mgkg(-1)). After 6 weeks of FLX-treatment, our results revealed that FLX and nor-fluoxetine (N-FLX) are present in colon tissue, which was related to significant increase in serotonin (5-HT) levels (P<0.05) possibly through a blockade in SERT mRNA (serotonin reuptake transporter; P<0.05) resulting in lower 5-hydroxyindoleacetic acid (5-HIAA) levels (P<0.01) and, 5-HT2C receptor mRNA expressions. FLX-treatment decreased dysplastic ACF development (P<0.01) and proliferative process (P<0.001) in epithelia. We observed a significant decrease in the development of malignant microvessels (P<0.05), VEGF (P<0.001), and COX-2 expression (P<0.01). These findings suggest that FLX may have oncostatic effects on carcinogenic colon tissue, probably due to its modulatory activity on 5-HT metabolism and/or its ability to reduce colonic malignant events.

The melatonin action on stromal stem cells within pericryptal area in colon cancer model under constant light.

Constant light (LL) is associated with high incidence of colon cancer. MLT supplementation was related to the significant control of preneoplastic patterns. We sought to analyze preneoplastic patterns in colon tissue from animals exposed to LL environment (14 days; 300 lx), MLT-supplementation (10mg/kg/day) and DMH-treatment (1,2 dimethylhydrazine; 125 mg/kg). Rodents were sacrificed and MLT serum levels were measured by radioimmunoassay. Our results indicated that LL induced ACF development (p < 0.001) with a great potential to increase the number of CD133(+) and CD68(+) cells (p < 0.05 and p < 0.001). LL also increased the proliferative process (PCNA-Li; p < 0.001) as well as decreased caspase-3 protein (p < 0.001), related to higher COX-2 protein expression (p < 0.001) within pericryptal colonic stroma (PCCS). However, MLT-supplementation controlled the development of dysplastic ACF (p < 0.001) diminishing preneoplastic patterns into PCCS as CD133 and CD68 (p < 0.05 and p < 0.001). These events were relative to decreased PCNA-Li index and higher expression of caspase-3 protein. Thus, MLT showed a great potential to control the preneoplastic patterns induced by LL.

Ultra-deep sequencing reveals the microRNA expression pattern of the human stomach.

BACKGROUND: While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia. METHODOLOGY/PRINCIPAL FINDINGS: A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue. CONCLUSIONS/SIGNIFICANCE: This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.

The profile of gene expression of human marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent precursors present in adult bone marrow, that differentiate into osteoblasts, adipocytes and myoblasts, and play important roles in hematopoiesis. We examined gene expression of these cells by serial analysis of gene expression, and found that collagen I, secreted protein acidic and rich in cysteine (osteonectin), transforming growth factor beta- (TGF-beta) induced, cofilin, galectin-1, laminin-receptor 1, cyclophilin A, and matrix metalloproteinase-2 are among the most abundantly expressed genes. Comparison with a library of CD34(+) cells revealed that MSCs had a larger number of expressed genes in the categories of cell adhesion molecule, extracellular and development. The two types of cells share abundant transcripts of many genes, some of which are highly expressed in myeloid progenitors (thymosin-beta 4 and beta 10, fos and jun). Interleukin-11 (IL-11), IL-15, IL-27 and IL-10R, IL-13R and IL-17R were the most expressed genes among the cytokines and their receptors in MSCs, and various interactions can be predicted with the CD34(+) cells. MSCs express several transcripts for various growth factors and genes suggested to be enriched in stem cells. This study reports the profile of gene expression in MSCs and identifies the important contribution of extracellular protein products, adhesion molecules, cell motility, TGF-beta signaling, growth factor receptors, DNA repair, protein folding, and ubiquination as part of their transcriptome.

Gravidez na adolescência: falta de informaçäo? / Pregnancy in adolescence: no information?

A gravidez na adolescência cosntitui uma das preocupaçöes mais importantes relacionadas à conduta sexual dos adolescentes frente ao percentual cada vez mais elevado da sua ocorrência, em consequência a uma iniciaçäo cada vez mais precoce, sem o uso adequado de métodos anticincepcionais. Além disso, é considerada um importante problema de Saúde Pública por estar intimamente relacionada às altas taxas de mortalidade infantil, perinatal e materna. O presente trabalho foi desenvolvido na Unidade Básica de Saúde do Conjunto Habitacional Maria Cecília e sua área de abrangência (Zona Norte de Londrina), onde dados fornecidos pela 17ª Regional de Saúde mostram ser esta uma regiäo com alto percentual de adolescentes grávidas. Com o objetivo de traçar o perfil dos adolescentes dessa regiäo, foram feitas entrevistas com 25 adolescentes (sexo feminino) na faixa atária entre 13 e 19 anos. Constatamos que das 25 adolescentes (grávidas e/ou já tiveram filhos), 52 por cento casadas atualmente e 44 por cento solteiras, sendo 64 por cento com 1§ grau incompleto e 20 por cento com 1§ grau concluído. As respostas mostram que 68 por cento näo conversam com os pais sobre sexo, as informaçöes säo obtidas principalmente com os amigos (60 por cento) e a prática sexual é iniciada em 64 por cento entre 15 e 16 anos e 24 por cento entre 13 e 14 anos. Os motivos dessa prática precoce incluem sexo como parte do namoro (60 por cento), atraçäo física (16 por cento), rebeldia e desestrutura familiar (8 por cento). Concluímos que a falta de informaçäo näo é o principal fator responsável pelo elevado índice de gravidez na adolescência. Assim, propomos a implantaçäo de programas profissionais tendo adolescentes como populaçäo alvo, que orientem quanto ao uso de métodos contraceptivos e prevençäo contínua, com atendimento integral, multiprofissional e intersetorial