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Biomedical subjects

C L Silva

Publications and source records attributed to C L Silva.

At least 73 records · Page 4Linked to original sources
PubMed1994

Towards a DNA vaccine against tuberculosis.

Expression of the gene for a single mycobacterial antigen (Mycobacterium leprae hsp65) in adult Balb/c mice resulted in substantial cell-mediated protection against challenge with M. tuberculosis. CD4 and CD8 T cells cloned from spleens of such immunized mice passively transferred protection to non-immunized mice, and CD8 cells selectively lysed macrophages infected with M. tuberculosis. Three modes of expressing the gene have been tested: (1) expression from a retroviral vector (pZIPNeoSV) in implanted J774 tumour cells, (2) expression from the same vector via bone marrow cells transfected in vitro and used to reconstitute irradiated mice, and (3) in a preliminary experiment, from CMV immediate-early and hydroxymethylglutaryl Co-A reductase promoters injected as plasmid DNA into muscle.

Animals↗
PubMed1994

Involvement of cell wall glucans in the genesis and persistence of the inflammatory reaction caused by the fungus Paracoccidioides brasiliensis.

The role of cell wall polysaccharides in leucocyte recruitment and granuloma formation in paracoccidioidomycosis was investigated. The inflammatory cells recruitment to the peritoneal cavity in rats inoculated with cell wall fraction (CW-265 or F1-265) from an avirulent strain of Paracoccidioides brasiliensis (Pb265), was greater than that observed for the cell wall fraction (CW-HC or F1-HC) recovered from the virulent strain (PbHC). Moreover, the inoculation of F1-HC and F1-265 into the subcutaneous layer of mice resulted in the formation of nodular and not progressive granulomatous lesions. The size and mean time of evolution of these lesions was proportional to the degree of virulence of the sample from which they were derived. Analyses showed that both F1 fractions contained beta-glucan and chitin. Only beta-glucan was able to trigger attraction and concentric organization of polymorphonuclear neutrophils and macrophages at the inflammatory foci, and the difference in the concentration of this compound in the cell walls of PbHC and Pb265 could explain the inflammatory capacity exhibited by the two strains of P. brasiliensis.

Animals↗
PubMed1994

Protection against tuberculosis by passive transfer with T-cell clones recognizing mycobacterial heat-shock protein 65.

We have previously shown that mice vaccinated by injection with J774 macrophage-like tumour cells that expressed Mycobacterium leprae heat-shock protein (hsp) 65 as a transgene had acquired a remarkably high degree of protection against subsequent challenge with virulent M. tuberculosis. We show here that antigen-specific T cells cloned from spleens of such vaccinated animals can transfer a high level of protection to non-vaccinated recipients. The most efficient cells were of T-cell receptor (TCR) alpha beta+ and CD4- CD8+ type and specifically lysed mycobacteria-infected macrophages. These findings are consistent with the importance for protective immunity of engaging the endogenous antigen-presenting pathway to bias the immune response towards a cytolytic action against a mycobacterial antigen that is expressed at the surface of infected macrophages. TCR gamma delta+ and TCR alpha beta+ cells interacted synergistically.

Animals↗
PubMed1994

A single mycobacterial protein (hsp 65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis.

We used a retroviral shuttle vector [pZIPNeoSV(X)] to transfect a monocyte-like murine tumour cell line (J774.G8) with the Mycobacterium leprae gene encoding heat-shock protein (hsp) 65. The antigen was expressed and presented on the surface of the transfected cell in association with major histocompatibility complex (MHC) class I and class II for recognition by T cells from specifically sensitized mice. We show here that when these transfected cells were used as a vaccine and introduced parentally into syngeneic (BALB/c) mice they conferred a remarkably high degree of protective immunity against subsequent challenge with either M. bovis bacillus Calmette-Guérin (BCG) or M. tuberculosis H37Rv.

Animals↗
PubMed1993

Cell wall fractions from Paracoccidioides brasiliensis induce hypergammaglobulinemia.

The antibody response against the antigen sheep red blood cells (SRBC) was investigated in mice pre-treated with formalin-killed Paracoccidioides brasiliensis or with cell wall fractions of the fungus. Pre-treatment with P. brasiliensis, as well as with the Fl fraction and beta-glucan significantly increased the anti-SRBC antibody response in the experimental groups as compared to the control group that received only SRBC. This immunomodulatory effect varied with the different doses employed and with pre-treatment time. We conclude that the cell wall fractions of P. brasiliensis might play an important role in the hypergammaglobulinemia associated with Paracoccidioidomycosis.

Animals↗
PubMed1993

Tumour necrosis factor production in vivo and in vitro in response to Paracoccidioides brasiliensis and the cell wall fractions thereof.

Tumour necrosis factor (TNF) was detected in serum from mice challenged with Paracoccidioides brasiliensis. The serum TNF levels of mice challenged with an avirulent strain were significantly higher than those of mice challenged with a virulent strain, and the same was observed for the TNF levels of mice challenged with a cell wall fraction (F1) from the two fungal strains. Fraction F1 consisted of chitin and beta-glucan; but although the chitin contents were similar for the two strains, the avirulent strain allowed a greater content of beta-glucan. The beta-glucan, purified from both strains, increased serum TNF levels in an identical dose-dependent manner, whereas purified chitin did not induce serum TNF levels. P. brasiliensis, the F1 fractions and beta-glucan induced macrophages to secrete TNF in vitro. The differences in TNF levels, induced by the different fungal strains, were correlated with the beta-glucan concentrations in the cell walls of both the avirulent and virulent strains of P. brasiliensis. These findings support a role for TNF in the pathogenicity of P. brasiliensis.

Animals↗
PubMed1993

Major histocompatibility complex non-restricted presentation to CD4+ T lymphocytes of Mycobacterium leprae heat-shock protein 65 antigen by macrophages transfected with the mycobacterial gene.

When the immunodominant 65,000 MW heat-shock protein of Mycobacterium leprae (ML65hsp) was expressed from the transfected mycobacterial gene in the mouse macrophage cell line J774.G8, the antigen was recognized by specifically sensitized CD4+ splenocytes in association with major histocompatibility complex (MHC) class II and CD4. Inhibition by monensin, leupeptin and chloroquine but not brefeldin A indicated dependence of presentation upon endosomal antigen processing. Although direct access of the endogenously synthesized antigen to the endosomal pathway of presentation, without extracellular release followed by endocytosis, could not be discounted, antigen was present in supernatants of the transfected cells in a form that could be presented by fixed macrophages and a form that required further processing for presentation. Each of three monoclonal antibodies (mAb) specific for widely separated linear amino acid epitopes of the antigen strongly inhibited recognition, suggesting steric interference with antigen-presenting cell (APC)-T cell interaction. Tests with splenocytes from vaccinated congenic mice indicated that recognition was not restricted by MHC haplotype. The significance and mechanism of this apparent MHC context-independent interaction of the presented antigen with specific T-cell receptor (TcR) remain to be explored.

Animals↗
PubMed1993

Correlation between TNF production, increase of plasma C-reactive protein level and suppression of T lymphocyte response to concanavalin A during erythema nodosum leprosum.

The complex symptoms observed in lepromatous leprosy patients with reactive episodes of the erythema nodosum leprosum (ENL) type are associated with different serum components actively participating in the acute inflammatory reaction. Among them are the tumor necrosis factor (TNF) and the acute-phase protein C-reactive protein (CRP). TNF and CRP were found at significantly more elevated concentrations in the serum of patients with ENL, with a positive correlation of about 95% when compared with patients with nonreactive lepromatous leprosy (L) or tuberculoid leprosy (T) or with control individuals. Furthermore, in another series of experiments CRP had a specific and significant suppressive action on concanavalin A (ConA)-induced lymphoproliferation in cultures from patients and controls, the reduction being more marked (75%) in patients with ENL. By extrapolation from its known actions, production of TNF may have a number of potential consequences for the immunobiology of ENL. Thus, TNF may cause direct injury to compromised cells, facilitating mononuclear cell activation and production of cytokines such as interleukin-1 and interleukin-6, and upregulating hepatocyte expression of CRP. Both CRP and TNF in high serum concentrations have the ability to enhance the acute inflammatory process in ENL, favoring increased macrophage activation and phagoctyosis, and contributing to the elimination of damaged cells and bacilli, as well as in the reduction of T-suppressor cells, with a consequent improvement in the immunologic response of ENL patients.

Adolescent↗
PubMed1993

The role of the complement system in the neutrophil functions stimulated in vitro by an alkali-insoluble cell wall fraction of Paracoccidioides brasiliensis.

We investigated the capacity of an alkali-insoluble cell wall polysaccharide fraction (F1) of Paracoccidioides brasiliensis to induce rat polymorphonuclear neutrophil (PMN) migratory and chemiluminescence (CL) responses. Normal rat serum pre-incubated with F1 induced a chemotactic neutrophil response which was fully abolished by heat-inactivation. The participation of the alternative complement pathway was more effective than that of the classical pathway since depletion of factor B by heating at 50 degrees C reduced PMN migration, whereas blockade of the classical pathway with EGTA left the migratory response practically unchanged. Opsonized serum F1 induced a significant release of oxygen radicals from PMN as measured by CL. The complement system was also found to be involved in this activity since serum inactivation at 56 degrees C altered the CL response. In addition to complement-derived fragments, other serum opsonins, probably cross-reacting antibodies, were required for optimal interaction between PMN and opsonized particles. These results contribute to the understanding of the role of fungal components and of the complement system in the inflammatory response observed in paracoccidioidomycosis.

Animals↗
PubMed1992

Detection of cellular immunity with the soluble antigen of the fungus Sporothrix schenckii in the systemic form of the disease.

Sporothrix schenckii is the etiologic agent of sporotrichosis, a mycosis of world-wide distribution more commonly occurring in tropical regions. The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully understood but apparently include both the humoral and cellular responses. In the present investigation, cellular immunity was evaluated by in vivo and in vitro tests in mice infected with yeast-like forms of S. schenckii. The disease developed systemically and cellular immunity was evaluated for a period of 10 weeks. The soluble antigen utilized in the tests was prepared from yeast form of the fungus through the sonication (20 min: 10 sonications at 50 W at 2-min intervals). Delayed hypersensitivity and lymphocyte transformation tests showed that the cellular immune response was depressed between the 4th and 6th week of infection when the animals were challenged with the soluble fungal antigen. This depression frequently indicates worsening of the disease, with greater involvement of the host. This is a promising field of research for a better understanding of the pathogeny of this mycosis.

Animals↗
PubMed1992

Mycobacterium leprae 65hsp antigen expressed from a retroviral vector in a macrophage cell line is presented to T cells in association with MHC class II in addition to MHC class I.

Mycobacterium leprae lives free in the cytoplasm in infected macrophages. To test if an M. leprae antigen released into the cytoplasm would associate with major histocompatibility complex (MHC) class II we introduced the gene encoding the 65 kDa heat-shock protein (ML65hsp) into a retroviral shuttle vector (pZIPNeoSV(X)) and transfected the murine macrophage cell line J774G8. S1 nuclease mapping and Western blot analysis of the transfected cell line (CJ11) showed that specific messenger RNA and ML65hsp antigen were stably expressed. Presence of antigen at the cell surface was demonstrated by flow cytometric analysis with specific monoclonal antibodies (mAb). Antigen-specific T lymphocytes were stimulated by CJ11 cells to proliferate and release interleukins (IL-2 and IL-3). These responses were blocked by mAbs specific for either MHC class II or for the mycobacterial antigen. The endogenous antigen was also recognised by MHC class I-dependent cytotoxic T cells; cytotoxicity was inhibited by mAbs against either MHC class I molecules or ML65hsp. Thus, production of ML65hsp within the host cytoplasm resulted in association of the antigen with both MHC class I and MHC class II antigen-presenting structures and evoked both lymphocyte proliferation and cytotoxicity towards the antigen-presenting cell. These findings may be relevant to the development of recombinant subunit vaccines against intracellular pathogens.

3T3 Cells↗
PubMed1992

Tumor necrosis factor and macrophage activation are important in clearance of Nocardia brasiliensis from the livers and spleens of mice.

The roles of tumor necrosis factor (TNF) and macrophage activation in clearance of Nocardia brasiliensis from BALB/c mouse livers and spleens were evaluated. TNF activity was detectable in sera from animals at all stages of infection. Treatment of infected mice with an antiserum against TNF significantly enhanced the experimental infection as judged by enumeration of CFU in the spleens and livers of infected mice. In another set of experiments, a population of activated macrophages from the peritoneal cavities of N. brasiliensis-infected mice was studied by using a cytostatic assay. The observed cytotoxic activity of these activated macrophages against L929 cells was mediated by TNF, since this activity was inhibited by anti-TNF antiserum treatment. The level of TNF activity generated in vitro in the presence of lipopolysaccharide (LPS) by peritoneal macrophages from infected mice was higher than that of adherent peritoneal cells obtained from normal mice after challenge with LPS. When the nocardiacidal activity of peritoneal cells from N. brasiliensis-infected mice was estimated in vitro, a significant decrease in the number of CFU recovered was observed. Moreover, nocardiacidal activity of peritoneal cells obtained from N. brasiliensis-infected mice previously treated with anti-TNF antiserum was significantly reduced compared with the activity of cells obtained from infected mice previously treated with normal rabbit serum and that of cells from uninfected mice. These data suggest a role for TNF in resistance to N. brasiliensis infection.

Animals↗
PubMed1992

Serum levels of tumor necrosis factor in insulin-dependent diabetic patients.

We determined the serum concentrations of tumor necrosis factor (TNF) in 15 nondiabetic healthy subjects and in 36 insulin-dependent (type I) diabetic outpatients. The mean (+/- SD) annual fasting plasma glucose, urine glucose and HbA1 levels of the diabetic group were 179 +/- 71 mg/dl, 13.0 +/- 13.2 g/day and 12.3 +/- 1.3%, respectively. The mean serum TNF concentration measured by immunoradiometric assay of the diabetic group (8.6 +/- 1.9 pg/ml) was significantly higher than healthy controls (6.9 +/- 0.9 pg/ml). Within the diabetic group, there was no correlation between serum TNF levels and either duration of diabetes or indices of metabolic control. However, serum TNF levels progressively increased from the well to the poorly controlled diabetic groups: 8.1 +/- 1.5 (G), 8.2 +/- 1.4 (F) and 9.4 +/- 2.4 (P) pg/ml, respectively, which parallel levels of HbA1 (%): 8.4 +/- 2.4, 11.7 +/- 1.8 and 14.6 +/- 1.2, respectively. Serum TNF levels of the diabetic patients with chronic complications (N = 7, 9.5 +/- 2.3 pg/ml) and without complications (N = 29, 8.4 +/- 1.7 pg/ml) were statistically higher than control subjects. The progressive increase of the serum TNF levels from the well to the poorly controlled diabetic groups suggests a relationship between levels of this cytokine and protein glycosylation.

Adolescent↗
PubMed1989

Inflammation induced by a glycolipid fraction from Mycobacterium leprae.

1. The inflammatory properties of a glycolipid fraction isolated from human recovered Mycobacterium leprae were investigated. The inflammatory reaction induced in mouse lung by the inoculation of the glycolipid fraction adsorbed to charcoal particles was characterized by a large influx of macrophages at various stages of maturation and of epithelioid cells around the particles. 2. When injected as an aqueous emulsion into the footpad of mice, the same fraction evoked a dose-dependent massive influx of mononuclear (MN) cells. The inflammatory reaction reached a peak at 6 days. The minimal effective dose of glycolipid was 0.1 micrograms. 3. The kinetics of inflammatory cell migration was studied by total and differential counts of leucocytes that migrated to the peritoneal cavity of mice inoculated intraperitoneally with the glycolipid fraction. This fraction initially induced intense polymorphonuclear (PMN) migration, which was later reduced, with a simultaneous increase in MN cells. 4. Adherent peritoneal cells (APC) incubated with glycolipid released one or more soluble factor(s) which induce active PMN and MN cell chemotaxis in vivo as well as in vitro. Thus, the MN cells may be attracted to the site of glycolipid inoculation by factor(s) released through the interaction of macrophages with the glycolipid fraction. 5. The present results demonstrate that a glycolipid containing trehalose and mycolic acid isolated from M. leprae reproduces some aspects of the fundamental lesion of leprosy.

Animals↗