RESUMO
SPROUTY-2 (SPRY2) is a modulator of tyrosine kinase receptor signaling with receptor- and cell type-dependent inhibitory or enhancing effects. Studies on the action of SPRY2 in major cancers are conflicting and its role remains unclear. Here we have dissected SPRY2 action in human colon cancer. Global transcriptomic analyses show that SPRY2 downregulates genes encoding tight junction proteins such as claudin-7 and occludin and other cell-to-cell and cell-to-matrix adhesion molecules in human SW480-ADH colon carcinoma cells. Moreover, SPRY2 represses LLGL2/HUGL2, PATJ1/INADL and ST14, main regulators of the polarized epithelial phenotype, and ESRP1, an epithelial-to-mesenchymal transition (EMT) inhibitor. A key action of SPRY2 is the upregulation of the major EMT inducer ZEB1, as these effects are reversed by ZEB1 knock-down by means of RNA interference. Consistently, we found an inverse correlation between the expression level of claudin-7 and those of SPRY2 and ZEB1 in human colon tumors. Mechanistically, ZEB1 upregulation by SPRY2 results from the combined induction of ETS1 transcription factor and the repression of microRNAs (miR-200 family, miR-150) that target ZEB1 RNA. Moreover, SPRY2 increased AKT activation by epidermal growth factor, whereas AKT and also Src inhibition reduced the induction of ZEB1. Altogether, these data suggest that AKT and Src are implicated in SPRY2 action. Collectively, these results show a tumorigenic role of SPRY2 in colon cancer that is based on the dysregulation of tight junction and epithelial polarity master genes via upregulation of ZEB1. The dissection of the mechanism of action of SPRY2 in colon cancer cells is important to understand the upregulation of this gene in a subset of patients with this neoplasia that have poor prognosis.
Assuntos
Neoplasias do Colo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Adesão Celular/genética , Linhagem Celular Tumoral , Polaridade Celular/genética , Proliferação de Células/fisiologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Células Epiteliais , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Fenótipo , Proteína Proto-Oncogênica c-ets-1/genética , Transdução de Sinais , Transfecção , Regulação para Cima , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
The presence of hypoxic regions in solid tumors is an adverse prognostic factor for patient outcome. Here, we show that hypoxia induces the expression of Ephrin-A3 through a novel hypoxia-inducible factor (HIF)-mediated mechanism. In response to hypoxia, the coding EFNA3 mRNA levels remained relatively stable, but HIFs drove the expression of previously unknown long noncoding (lnc) RNAs from EFNA3 locus and these lncRNA caused Ephrin-A3 protein accumulation. Ephrins are cell surface proteins that regulate diverse biological processes by modulating cellular adhesion and repulsion. Mounting evidence implicates deregulated ephrin function in multiple aspects of tumor biology. We demonstrate that sustained expression of both Ephrin-A3 and novel EFNA3 lncRNAs increased the metastatic potential of human breast cancer cells, possibly by increasing the ability of tumor cells to extravasate from the blood vessels into surrounding tissue. In agreement, we found a strong correlation between high EFNA3 expression and shorter metastasis-free survival in breast cancer patients. Taken together, our results suggest that hypoxia could contribute to metastatic spread of breast cancer via HIF-mediated induction of EFNA3 lncRNAs and subsequent Ephrin-A3 protein accumulation.
Assuntos
Neoplasias da Mama/metabolismo , Loci Gênicos , Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Longo não Codificante/biossíntese , RNA Neoplásico/biossíntese , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Hipóxia Celular/genética , Linhagem Celular Tumoral , Efrina-A3/genética , Efrina-A3/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteínas de Neoplasias/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Peixe-ZebraRESUMO
BACKGROUND: Wheezing is one of the most frequent causes of visit to emergency rooms among children. However, data on wheezing burden are mostly provided at healthcare setting, and particularly only for infants. AIMS: We sought to estimate the prevalence of wheezing in children under 4 years and to assess potential risk factors in the community. DESIGN: This was a cross-sectional analysis of a population-based cohort study. METHODS: The sample comprised children aged <4 years living in Salvador, Brazil. Data were collected via home visits when the parents/guardians were interviewed. Data were recorded on standardized forms. RESULTS: Of 1534 children, mean age was 21 ± 14 months (minimum 3 days; maximum 47 months; 6% <2 months); 780 (51%) were males and 501 [33%; 95% confidence interval (95% CI): 30-35%] reported wheezing in the last 12 months. Among wheezers, 321 (64%) had occasional wheezing. Overall, 180 (12%; 95% CI: 10-14%) had recurrent wheezing and 157 (10%; 95% CI: 9-12%) had asthma. For children in the first, second, third and fourth year of life wheezing was reported in 23, 41, 34 and 37%, respectively. Mother atopic-related disease was independently associated with recurrent wheezing (AdjPR[95% CI]: 1.54 [1.12-2.11]) and asthma (AdjPR[95% CI]: 1.54 [1.10-2.16]). Smoker at home (AdjPR[95% CI]: 1.34 [1.07-1.67]) and low birth weight (AdjPR[95%CI]: 1.38 [1.05-1.81]) were independently associated with occasional wheezing. CONCLUSIONS: One-third of under 4 years reported wheezing; history of mother's atopic-related disease was an independent risk factor for recurrent wheezing and asthma; smoker at home and low birth weight were independent risk factors for occasional wheezing.
Assuntos
Sons Respiratórios/etiologia , Distribuição por Idade , Asma/epidemiologia , Brasil/epidemiologia , Pré-Escolar , Estudos Transversais , Saúde da Família , Feminino , Humanos , Lactente , Recém-Nascido de Baixo Peso/fisiologia , Recém-Nascido , Masculino , Prevalência , Recidiva , Fatores de Risco , Poluição por Fumaça de Tabaco/estatística & dados numéricosRESUMO
Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that displays broad anti-tumor activity based on dual targeting of the tumor microenvironment (anti-angiogenic action) and the tumor cells (direct anti-tumor action). Here, we show that PEDF expression is high in melanocytes, but it is lost during malignant progression of human melanoma. Using a high-throughput analysis of the data from microarray studies of molecular profiling of human melanoma, we found that PEDF expression is lost in highly invasive melanomas. In paired cell lines established from the same lesion but representing the high and low extremes of malignant potential, abundant PEDF expression was restricted to the poorly aggressive counterparts. We used RNA interference to directly address the functional consequences of PEDF silencing. PEDF knockdown in poorly aggressive melanoma cell lines augmented migration, invasion and vasculogenic mimicry, which translated into an increased in vivo metastatic potential. PEDF interference also significantly enhanced the migratory and invasive capability of normal melanocytes and moderately increased their proliferative potential. Our results show that loss of PEDF enables melanoma cells to acquire an invasive phenotype and, therefore, modulation of this multifunctional factor could be critical for the malignant progression of human melanoma.
Assuntos
Movimento Celular , Proteínas do Olho/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Linhagem Celular Tumoral , Proteínas do Olho/genética , Perfilação da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/patologia , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Fatores de Crescimento Neural/genética , Análise de Sequência com Séries de Oligonucleotídeos , Serpinas/genéticaRESUMO
Saliva plays important roles in facilitation of a bloodmeal, lubrication of mouthparts, and parasite transmission for some vector insects. Salivary composition changes during the lifetime of an insect, and differences in the salivary profile may influence its functions. In this report, the amount and profile of salivary gland protein of the American visceral leishmaniasis vector Lutzomyia longipalpis (Lutz & Neiva, 1912) were analyzed at different times of insect development and diet. Protein content from unfed female sand flies increased significantly with age, and a significant difference was observed in sugar-fed females during the first 10 d of adult life. Salivary protein content sharply decreased 1 d after blood feeding, with gradual increase in concentration the following days. SDS-polyacrylamide gel electrophoresis analysis revealed that most polypeptides present in the saliva of sugar-fed also were present in the saliva of blood-fed females. Understanding changes in sand fly's saliva contents at distinct days after emergence and the influence of a bloodmeal in this aspect may reveal the role played by saliva during leishmaniasis transmission.
Assuntos
Envelhecimento/fisiologia , Dieta , Proteínas de Insetos/metabolismo , Psychodidae/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Animais , Feminino , Regulação da Expressão GênicaRESUMO
Experimental animal models have been used for the study of the physiopathogenesis of leishmaniasis, on some occasions with success, while in other situations such as bone alterations that accompany tegumentary leishmaniasis, especially in diffuse cutaneous form (DCL), the mechanisms are still unknown. In the present study, we determined these alterations in an animal model susceptible to Leishmania (L) amazonensis. Amastigotes of L. (L) amazonensis isolated from patients with diffuse cutaneous leishmaniasis (DCL) were inoculated into the hind paws of eight BALB/c mice, macroscopic and histopathological aspects were analyzed. After 90 and 120 days of evolution, histopathological analysis demonstrated a mononuclear cell infiltrate rich in plasma cells and intense parasitism of intra- and extra-medullary macrophages, with areas of bone necrosis and discrete involvement of cartilaginous tissue. The results show that the inflammatory process developed during L. (L) amazonensis infection might cause bone tissue destruction and secondarily affect the joints.
Assuntos
Leishmania/crescimento & desenvolvimento , Leishmaniose Tegumentar Difusa/patologia , Osteomielite/parasitologia , Animais , Modelos Animais de Doenças , Membro Posterior/parasitologia , Membro Posterior/patologia , Histocitoquímica , Humanos , Leishmaniose Tegumentar Difusa/imunologia , Leishmaniose Tegumentar Difusa/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Osteomielite/imunologia , Osteomielite/patologiaRESUMO
The first steps in leishmaniasis are critical in determining the evolution of the disease. Major advances have recently been done in understanding this crucial moment. Fundamental research in parasite-vector interaction, parasite biology, insect saliva, and vertebrate host response have shed new light and uncovered a most fascinating and complex moment in leishmaniasis. We review here some of these aspects and we try to connect them in a logical framework.
Assuntos
Animais , Humanos , Camundongos , Insetos Vetores , Leishmania , Leishmaniose , Psychodidae , Interações Hospedeiro-ParasitaRESUMO
Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-g, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ
Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Linfócitos B , Citocinas , Leishmaniose Cutânea , Citocinas , Imuno-Histoquímica , Interferon-alfa , Interferon gama , Leishmaniose Cutânea , Óxido Nítrico Sintase , Estatísticas não ParamétricasRESUMO
The initial encounter of Leishmania cells and cells from the immune system is fundamentally important in the outcome of infection and determines disease development or resistance. We evaluated the anti-Leishmania amazonensis response of naive volunteers by using an in vitro priming (IVP) system and comparing the responses following in vivo vaccination against the same parasite. In vitro stimulation allowed us to distinguish two groups of individuals, those who produced small amounts of gamma interferon (IFN-gamma) (n = 16) (low producers) and those who produced large amounts of this cytokine (n = 16) (high producers). IFN-gamma production was proportional to tumor necrosis factor alpha and interleukin 10 (IL-10) levels but did not correlate with IL-5 production. Volunteers who produced small amounts of IFN-gamma in vitro remained low producers 40 days after vaccination, whereas high producers exhibited increased IFN-gamma production. However, 6 months after vaccination, all individuals tested produced similarly high levels of IFN-gamma upon stimulation of their peripheral blood mononuclear cells with Leishmania promastigotes, indicating that low in vitro producers respond slowly in vivo to vaccination. In high IFN-gamma producers there was an increased frequency of activated CD8(+) T cells both in vitro and in vivo compared to the frequency in low producers, and such cells were positive for IFN-gamma as determined by intracellular staining. Such findings suggest that IVP responses can be used to predict the pace of postvaccination responses of test volunteers. Although all vaccinated individuals eventually have a potent anti-Leishmania cell-mediated immunity (CMI) response, a delay in mounting the CMI response may influence resistance against leishmaniasis.
Assuntos
Interferon gama/biossíntese , Leishmaniose/imunologia , Vacinas Protozoárias/imunologia , Adolescente , Adulto , Linfócitos T CD8-Positivos/imunologia , Previsões , Humanos , Imunidade Inata , Ativação Linfocitária , Masculino , Receptores de Interleucina-2/isolamento & purificação , Subpopulações de Linfócitos T/imunologia , VacinaçãoRESUMO
Sickle cell disease has a worldwide distribution and is a public health problem in Brazil. Although vaso-occlusive crisis (VOC) is one of the most important clinical features of the disease, there are still several steps of its pathogenesis which are unknown. The increase of the chemotactic factor interleukin 8 (IL-8) has been reported to be involved in sickle cell disease crisis, but this has not been demonstrated conclusively. In the present study we analyzed serum IL-8 levels by ELISA and hematological parameters and hemoglobin patterns by standard techniques in 23 (21 SS and 2 SC) Brazilian patients with sickle cell syndromes during VOC caused by different inducing factors, 22 (21 SS and 1 SC) sickle cell patients out of crisis, and 11 healthy controls. Increased IL-8 levels were observed in 19 of 23 VOC patients (79.2 percent), 3 of them with more than 1,000 pg/ml. Seventeen of 22 (77.3 percent) non-crisis patients showed low IL-8 levels (less than 15 pg/ml). Healthy controls had low IL-8 levels. A significant difference in serum IL-8 levels was observed between crisis and non-crisis sickle cell patients (P<0.0001). There was no correlation between IL-8 levels and hematological data or hemoglobin patterns. High serum IL-8 levels were observed in VOC patients independently of the crisis-inducing factor. We conclude that in the studied population, IL-8 concentration may be a useful VOC marker, although the mechanism of the pathogenic process of sickle cell VOC syndromes remains unclear
Assuntos
Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Anemia Falciforme , Arteriopatias Oclusivas , Interleucina-8 , Anemia Falciforme , Arteriopatias Oclusivas , Biomarcadores , Brasil , Hemoglobina Falciforme , Hemoglobinas , Fatores de Risco , SíndromeRESUMO
Leishmaniasis, caused by infection with the protozoan parasite Leishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-gamma) production by several different sources, listed in order of contribution: CD4(+) T lymphocytes, CD4(-), CD8(-) lymphocytes, and CD8(+) T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4(+) T lymphocytes (IFN-gamma(+) and IL-10(-)/IL-4(-)), Tc1 CD8(+) T cells (IFN-gamma(+), and IL-10(-)/IL-4(-)), and a high frequency of TNF-alpha-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.
Assuntos
Antígenos de Protozoários/imunologia , Citocinas/biossíntese , Citometria de Fluxo , Leishmaniose Cutânea/imunologia , Proteínas de Protozoários/imunologia , Células Th1/imunologia , Humanos , Interferon gama/biossíntese , Interleucina-10/biossíntese , Proteínas Recombinantes/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Although malignant melanomas are often associated with cytotoxic lymphocyte infiltration, these cells are largely ineffective in inducing tumour cell kill, indicating that the melanoma cells have protective mechanisms. These mechanisms are not fully understood, but cytokines and redox-active antioxidant proteins such as catalase, superoxide dismutase, thioredoxin (Trx) and Trx reductase (TrxR) present in the tumour cells constitute part of this protection. In this study firstly we investigated the constitutive intracellular expression of Trx, TrxR, the cytokines interleukin (IL)-1alpha, IL1beta, IL2, IL4, IL6, IL8, IL10, tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) in normal melanocytes and ten primary and metastatic malignant melanoma cell lines. Secondly, we analysed whether redox stimulation by Trx alone or in combination with the phorbol ester PMA affected the expression and release of TNFalpha. Thirdly, we explored the possible correlation between Trx/TrxR expression and resistance to exogenous TNFalpha. All the cultured cells showed intracellular overexpression of Trx and TrxR, which was not always the case for melanoma cells in vivo (tissue sections). The predominant intracellular cytokines found were TNFalpha, IL1alpha and IL1beta. In spite of its presence in the Golgi apparatus, none of the cell lines secreted TNFalpha constitutively, and only one melanoma, FM3, released detectable amounts after stimulation. In contrast, U-937 monocyte control cells released high amounts of TNFalpha on identical stimulation. All the melanoma cell lines were relatively resistant against exogenous TNFalpha, and there was a significant correlation (P < 0.01) between intracellular Trx/TrxR expression and TNFalpha resistance (IC50). In conclusion, Trx and TrxR, as well as TNFalpha, IL1alpha and IL1beta, were highly expressed in cultured normal skin melanocytes and malignant melanoma cell lines. In contrast to U-937 monocytic cells, TNFalpha showed a secretory block in these cells, suggesting a cytoprotective and possible autocrine role for TNFalpha. The intracellular expression of Trx and TrxR together with endogenous TNFalpha was correlated with the resistance to TNFalpha-induced cytotoxicity.
Assuntos
Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Tiorredoxina Dissulfeto Redutase/biossíntese , Tiorredoxinas/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Sobrevivência Celular/efeitos dos fármacos , Combinação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Técnicas Imunoenzimáticas , Interferon gama/metabolismo , Interleucinas/metabolismo , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanoma/secundário , Microscopia Confocal , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/farmacologia , Tiorredoxinas/farmacologia , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células U937/metabolismoRESUMO
This study examined the T cell responses in the early phase of Leishmania braziliensis infection. Cytokine profiles, lymphoproliferative responses, and skin test results in 25 patients with early cutaneous leishmaniasis (ECL; illness duration <60 days) were compared with those in persons with late cutaneous leishmaniasis (LCL; illness duration >2 months). Absent or low lymphoproliferative responses were observed in 8 (32%) of 25 patients and an absence of interferon (IFN)-gamma production in 9 (41%) of 22 patients prior to therapy. IFN-gamma production in ECL (mean +/- SD) was lower than in LCL (293+/-346 vs. 747+/-377 pg/mL, respectively; P<.01). In contrast, interleukin (IL)-10 production in ECL (mean +/- SD) was higher than in LCL (246+/-56 vs. 50+/-41 pg/mL, respectively; P<.01). Restoration of lymphoproliferative responses and IFN-gamma production was achieved when monoclonal antibody to IL-10 or IL-12 was added to the cultures. These results show that T cell responses during early-phase infection are down-regulated by IL-10 and may facilitate parasite multiplication.
Assuntos
Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Células Th1/imunologia , Animais , Regulação para Baixo , Humanos , Interferon gama/biossíntese , Interleucina-10/biossíntese , Leishmaniose Cutânea/parasitologia , Ativação Linfocitária , Linfócitos T/imunologia , Fatores de TempoRESUMO
E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65 percent smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75 percent in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57 percent by SC and 49 percent by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization
Assuntos
Camundongos , Animais , Antígenos de Protozoários/administração & dosagem , Leishmania major/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/imunologia , Timidilato Sintase/imunologia , Leishmaniose Cutânea/imunologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos MutantesRESUMO
Treatment for multibacillary leprosy is presently performed with a multidrug therapy (MDT) scheme maintained for 2 years. Leprosy treatment however can benefit from the reduction of length. The lack of interferon-gamma (IFN-gamma) production by lepromatous leprosy (LL) patients' lymphocytes lead us to use this cytokine in the treatment of multibacillary leprosy associated with MDT in the treatment of multibacillary leprosy, and monitor several clinical and immunological parameters during the course of treatment. A total of 20 multibacillary leprosy patients were evaluated, 10 treated with MDT alone, and 10 treated with MDT + 10 daily doses of 2 x 10(6) international units (IU) of recombinant human IFN-gamma/m2 followed by 10 daily doses of 10(7) IU IFN-gamma/m2, intramuscularly, during the first 20 days of MDT. IFN-gamma was well tolerated and did not cause any increase in the rate of leprosy reactions development during treatment. Decrease of bacillary load, fall of anti-Mycobacterium leprae IgG serum antibodies, changes of histological pattern, as well as changes in lymphocyte proliferation assay in response to mitogens (PHA or PWM), M. leprae antigen or PPD was similar in both groups of patients. Among several soluble immunological markers measured before and 30 days after beginning of treatment, levels of soluble IL-2R receptor increased in patients treated with MDT plus IFN-gamma whereas decreased in patients treated with MDT alone. Soluble ICAM-1 levels decreased in the MDT group but did not change in the MDT + IFN-gamma treated patients. Soluble CD4 and soluble CD8 markers did not change significantly in either group of patients. Neopterin, a marker of macrophage activation, increased in all but one patient treated with MDT + IFN-gamma but in none treated with MDT alone, indicating that IFN-gamma was active in vivo. Our findings indicate that despite being able to promote macrophage activation in multibacillary leprosy patients a short course of systemically administered IFN-gamma is not able to change the clinical course of a long standing disease such as leprosy.
Assuntos
Interferon gama/uso terapêutico , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Criança , Clofazimina/uso terapêutico , Dapsona/uso terapêutico , Quimioterapia Combinada , Feminino , Humanos , Interferon gama/biossíntese , Hanseníase/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Neopterina/análise , Proteínas Recombinantes , Rifampina/uso terapêutico , Pele/microbiologiaRESUMO
The cell-mediated immune response is critical in the resistance to and recovery from leishmaniasis. Cytokines are central elements in mounting an immune response and have received a great deal of attention in both human and experimental leishmaniasis. IFN-gamma is responsible for macrophage activation leading to leishmanicidal mechanisms. Understanding the balance of cytokines that lead to enhanced production of or synergize with IFN-gamma, and those cytokines that counterbalance its effects is fundamental for developing rational immunotherapeutic or immunoprophylactic approaches to leishmaniasis. Here we focus on the cytokine balance in human leishmaniasis, particularly IL-10 as an IFN-gamma opposing cytokine, and IL-12 as an IFN-gamma inducer. The effects of these cytokines were evaluated in terms of several parameters of the human immune response. IL-10 reduced lymphocyte proliferation, IFN-gamma production and cytotoxic activity of responsive human peripheral blood mononuclear cells. Neutralization of IL-10 led to partial restoration of lymphoproliferation, IFN-gamma production and cytotoxic activity in unresponsive visceral leishmaniasis patients. IL-12 also restored the responses of peripheral blood mononuclear cells from visceral leishmaniasis patients. The responses obtained with IL-12 are higher than those obtained with anti-IL-10, even when anti-IL-10 is combined with anti-IL-4.
Assuntos
Citocinas/fisiologia , Leishmaniose/imunologia , Brasil , Humanos , Interleucina-10 , Interleucina-12RESUMO
The production of IFNgamma, IL-10, IL-13 and TNFalpha was determined using PBMC from 7 tuberculoid (TT) and 7 lepromatous leprosy (LL) patients, after stimulation with several mycobacterial antigens, in an attempt to characterize the cytokine responses to these antigens. The results showed that TT patients displayed higher IFNgamma levels than LL patients with the mycobacterial antigens tested, but no differences in IL-10 production were observed between the two groups. MLSC antigen was associated with the lowest IFNgamma production in TT and LL groups. Only BCG could be identified with stimulation of IFNgamma production in some LL patients. The mycobacterial antigens SP+, SP- and BCG were associated with higher TNFalpha production in patients and controls, suggesting that these antigens could be involved in immunopathological effects. Our findings showed that the antigens tested were associated with a heterogeneous cytokine production in leprosy patients. Further studies are required to establish if an individual antigen can be identified as inducing a protective immune response in leprosy.
Assuntos
Antígenos de Bactérias/imunologia , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-13/imunologia , Hanseníase Virchowiana/imunologia , Hanseníase Tuberculoide/imunologia , Leucócitos Mononucleares/imunologia , Mycobacterium bovis/imunologia , Mycobacterium leprae/imunologia , Fator de Necrose Tumoral alfa/imunologia , Estudos de Casos e Controles , Células Cultivadas , Humanos , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-13/sangue , Hanseníase Virchowiana/sangue , Hanseníase Tuberculoide/sangue , Ativação Linfocitária/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The cell-mediated immune response is critical in the resistance to and recovery from leishmaniasis. Cytokines are central elements in mounting an immune response and have received a great deal of attention in both human and experimental leishmaniasis. IFN-gamma is responsible for macrophage activation leading to leishmanicidal mechanisms. Understanding the balance of cytokines that lead to enhanced production of or synergize with IFN-gamma, and those cytokines that counterbalance its effects is fundamental for developing rational immunotherapeutic or immunoprophylactic approaches to leishmaniasis. Here we focus on the cytokine balance in human leishmaniasis, particularly IL-10 as an IFN-gamma opposing cytokine, and IL-12 as an IFN-gama inducer. The effects of these cytokines were evaluated in terms of several parameters of the human immune response. IL-10 reduced lymphocyte proliferation, IFN-gamma production and cytotoxic activity of responsive human peripheral blood mononuclear cells. Neutralization of IL-10 led to partial restoration of lymphoproliferation, IFN-gamma production and cytotoxic activity in unresponsive visceral leishmaniasis patients. IL-12 also restored the responses of peripheral blood mononuclear cells from visceral leishmaniasis patients. The responses obtained with IL-12 are higher than those obtained with anti-IL-10, even when anti-IL-10 is combined with anti-IL-4.
Assuntos
Humanos , Citocinas/fisiologia , Leishmaniose/imunologia , Leishmaniose/fisiopatologia , Brasil , Interleucina-10 , Interleucina-12RESUMO
Thioredoxin (Trx) is an intracellular multifunctional 12-kDa protein with a reduction/oxidation (redox) active disulfide constitutively expressed by most cells of the human body. Trx can also be released by cells such as lymphocytes upon activation or oxidative stress exposure and exert a cocytokine and cytoprotective activity. In addition, a truncated 10-kDa form of Trx has been reported. In order to better understand the function of full-length and truncated Trx, we have produced, for the first time, specific monoclonal antibodies, which can discriminate between the two forms. Using these novel antibodies, designated alpha Trx1 to alpha Trx4, a panel of cell lines derived from human B and T lymphocytes, monocytes, granulocytes, and melanomas was analyzed by immunochemical techniques. The cellular distribution differed between the two forms. All lines contained full-length Trx, also located to a minor extent on the cell surface. One exception was the melanoma cell line FM28.4, which did not show any Trx expression. Truncated Trx was present in most cells in minimal amounts only, whereas the monocytic cell lines THP-1 and U-937 expressed high amounts on the cell surface, as shown by flow cytometric analysis of living cells and confocal laser-scanning microscopy. The biological importance and function of the short versus long forms of Trx as detected by the antibodies are discussed.
Assuntos
Tiorredoxinas/química , Tiorredoxinas/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Antígenos de Superfície/análise , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Western Blotting , Membrana Celular/química , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Expressão Gênica/fisiologia , Humanos , Membranas Intracelulares/química , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Dados de Sequência Molecular , Peso Molecular , Tiorredoxinas/imunologia , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/fisiologiaRESUMO
Parasite-specific cytotoxicity in human leishmaniasis was evaluated in an autologous system. PBL from cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML) patients were exposed to Leishmania amazonensis-infected autologous macrophages for 7 days and then used as effector cells in a cytotoxic assay using 51Cr-labeled autologous infected macrophages as targets. Results are reported as LU per 10(7) PBMC. Cytotoxic activity is present in ML (9.7 +/- 2.1 LU/10(7) PBMC) but not in CL (1.5 +/- 2.4 LU/10(7) PBMC) patients' lymphocytes, and the differences were highly significant (p < 0.0001). Both CD8+ T cells and NK cells exhibited cytotoxic activity. Addition of rIL-12, but not of IFN-gamma, during the generation of effector cells increased cytotoxic responses against infected macrophages. On the other hand, addition of mAb against human IL-12 or IFN-gamma during the stimulation of PBL significantly decreased the cytotoxic responses. Addition of IL-10 led to diminished cytotoxic responses, whereas the addition of anti-IL-10 did not significantly increase the cytotoxic responses. The observation of parasite-driven autologous cytotoxic responses in patients with ML, the destructive form of leishmaniasis, but not in CL, suggests that this phenomenon is involved in tissue pathology rather than in protection. Understanding the regulation of cytotoxic responses in leishmaniasis may be relevant to strategies aimed at limiting pathologic tissue destruction.