RESUMO
Background:Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17.Methods:Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18.Results:The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 μg), goat (HU50 = 13.2 ± 0.3 μg), rabbit (HU50 = 34.7 ± 0.5 μg), and human (HU50 = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction...(AU)
Assuntos
Animais , Anêmonas-do-Mar , Venenos de Cnidários/análise , Venenos de Cnidários/química , Perforina/análise , Perforina/uso terapêutico , Espectrometria de Massas , Neoplasias Pulmonares/terapiaRESUMO
American tegumentary leishmaniasis displays two main clinical forms: cutaneous (CL) and mucosal (ML). ML is more resistant to treatment and displays a more severe and longer evolution. Since both forms are caused by the same Leishmania species, the immunological response of the host may be an important factor determining the evolution of the disease. Herein, we analyzed the differentiation and memory profile of peripheral CD4(+) and CD8(+) T lymphocytes of patients with CL and ML and their Leishmania-T. cruzi co-infected counterparts. We measured the expression of CD27, CD28, CD45RO, CD127, PD-1 and CD57, together with interferon-γ and perforin. A highly differentiated phenotype was reflected on both T subsets in ML and preferentially on CD8(+) T cells in CL. A positive trend toward a higher T differentiation profile was found in T. cruzi-infected CL and ML patients as compared with Leishmania single infections. Association between CD8(+) T-cell differentiation and illness duration was found within the first year of infection, with progressive increase of highly differentiated markers over time. Follow-up of patients with good response to therapy showed predominance of early differentiated CD8(+) T cells and decrease of highly differentiated cells, while patients with frequent relapses presented the opposite pattern. CD8(+) T cells showed the most striking changes in their phenotype during leishmaniasis. Patients with long-term infections showed the highest differentiated degree implying a relation between T differentiation and parasite persistence. Distinct patterns of CD8(+) T differentiation during follow-up of different clinical outcomes suggest the usefulness of this analysis in the characterization of Leishmania-infected patients.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/patologia , Coinfecção/patologia , Leishmaniose Mucocutânea/patologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/análise , Diferenciação Celular , Criança , Feminino , Seguimentos , Humanos , Imunofenotipagem , Interferon gama/análise , Masculino , Pessoa de Meia-Idade , Perforina/análise , Adulto JovemRESUMO
BACKGROUND: CD4+/CD8+ double positive (DP) T cells have been described in healthy individuals as well as in patients with autoimmune and chronic infectious diseases. In chronic viral infections, this cell subset has effector memory phenotype and displays antigen specificity. No previous studies of double positive T cells in parasite infections have been carried out. METHODOLOGY/PRINCIPAL FINDINGS: Seventeen chronic chagasic patients (7 asymptomatic and 10 symptomatic) and 24 non-infected donors, including 12 healthy and 12 with non-chagasic cardiomyopathy donors were analyzed. Peripheral blood was stained for CD3, CD4, CD8, HLA-DR and CD38, and lymphocytes for intracellular perforin. Antigen specificity was assessed using HLA*A2 tetramers loaded with T. cruzi K1 or influenza virus epitopes. Surface expression of CD107 and intracellular IFN-γ production were determined in K1-specific DP T cells from 11 chagasic donors. Heart tissue from a chronic chagasic patient was stained for both CD8 and CD4 by immunochemistry. Chagasic patients showed higher frequencies of DP T cells (2.1% ± 0.9) compared with healthy (1.1% ± 0.5) and non-chagasic cardiomyopathy (1.2% ± 0.4) donors. DP T cells from Chagasic patients also expressed more HLA-DR, CD38 and perforin and had higher frequencies of T. cruzi K1-specific cells. IFN-γ production in K1-specific cells was higher in asymptomatic patients after polyclonal stimulation, while these cells tended to degranulate more in symptomatic donors. Immunochemistry revealed that double positive T cells infiltrate the cardiac tissue of a chagasic donor. CONCLUSIONS: Chagasic patients have higher percentages of circulating double positive T cells expressing activation markers, potential effector molecules and greater class I antigenic specificity against T. cruzi. Although K1 tetramer positive DP T cell produced little IFN-γ, they displayed degranulation activity that was increased in symptomatic patients. Moreover, K1-specific DP T cells can migrate to the heart tissue.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Cardiomiopatia Chagásica/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/análise , Linfócitos T CD4-Positivos/química , Linfócitos T CD8-Positivos/química , Cardiomiopatia Chagásica/patologia , Feminino , Antígenos HLA/análise , Humanos , Interferon gama/biossíntese , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Perforina/análise , Subpopulações de Linfócitos T/químicaRESUMO
Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAE-Sepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE.(AU)
Assuntos
Animais , Perforina/análise , Anêmonas-do-Mar/classificação , Porosidade , Sulfato de Amônio/efeitos adversos , Sódio/análiseRESUMO
Tuberculous pleurisy is a naturally occurring site of Mycobacterium tuberculosis (Mtb) infection. Herein, we describe the expression of activation, natural killer (NK) and cell migration markers, as well as effector functions from gammadeltaT cells in peripheral blood (PB) and pleural effusion (PE) from tuberculosis patients (TB). We observed a decreased percentage of circulating gammadeltaT from TB patients and differential expression of NK as well as of chemokine receptors on PB and PE. Two subsets of gammadeltaT cells were differentiated by the CD3/gammadeltaT cell receptor (gammadeltaTCR) complex. The gammadeltaTCR(low) subset had a higher CD3 to TCR ratio and was enriched in Vdelta2(+) cells, whereas most Vdelta1(+) cells belonged to the gammadeltaTCR(high) subset. In PB from TB, most gammadeltaTCR(high) were CD45RA(+)CCR7(-) and gammadeltaTCR(low) were CD45RA(+/-)CCR7(+)CXCR3(+). In the pleural space the proportion of CD45RA(-)CCR7(+)CXCR3(+) cells was higher. Neither spontaneous nor Mtb-induced interferon (IFN)-gamma production was observed in PB-gammadeltaT cells from TB; however, PE-gammadeltaT cells showed a strong response. Both PB- and PE-gammadelta T cells expressed surface CD107a upon stimulation with Mtb. Notably, PE-gammadeltaTCR(low) cells were the most potent effector cells. Thus, gammadeltaT cells from PB would acquire a further activated phenotype within the site of Mtb infection and exert full effector functions. As gammadeltaT cells produce IFN-gamma within the pleural space, they would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a T helper type 1 profile.
Assuntos
Complexo CD3/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T/imunologia , Tuberculose Pleural/imunologia , Adolescente , Adulto , Biomarcadores/análise , Estudos de Casos e Controles , Feminino , Imunofluorescência/métodos , Humanos , Memória Imunológica , Interferon gama/análise , Proteína 1 de Membrana Associada ao Lisossomo/análise , Proteínas de Membrana Lisossomal/análise , Masculino , Pessoa de Meia-Idade , Perforina/análiseRESUMO
Some cases of T-cell acute lymphoblastic leukaemia (ALL) express markers found in natural-killer (NK) cells, such as CD56 and CD16. Out of 84 T-cell ALL cases diagnosed at our Institution, CD56 and/or CD16 was detected in 24 (28.5%), which we designated T/NK-ALL group. Clinical features, laboratory characteristics, survival and expression of cytotoxic molecules were compared in T/NK-ALL and T-ALL patients. Significant differences were observed regarding age (24.9 vs. 16.4 years in T/NK-ALL and T-ALL, respectively, P = 0.006) and platelet counts (177 x 10(9)/l vs. 75 x 10(9)/l in T/NK-ALL and T-ALL, respectively, P = 0.03). Immunophenotypic analysis demonstrated that CD34, CD45RA and CD33 were more expressed in T/NK-ALL patients, whereas CD8 and terminal deoxynucleotidyl transferase were more expressed in T-ALL patients (P < 0.05). The mean overall survival (863 vs. 1869 d, P = 0.02) and disease-free survival (855 vs. 2095 d, P = 0.002) were shorter in patients expressing CD56/CD16. However, multivariate analysis identified CD56/CD16 as an independent prognostic factor only for DFS. Cytotoxic molecules were highly expressed in T/NK-ALL compared to T-ALL. Perforin, granzyme B and TIA-1 were detected in 12/17, 4/17 and 7/24 T/NK-ALL patients and in 1/20, 0/20 and 1/20 T-ALL respectively (P < 0.001, P = 0.036 and P = 0.054). Therefore, the presence of CD56/CD16 was associated with distinct clinical features and expression of cytotoxic molecules in the blasts.