Visceral leishmaniasis in the crab-eating fox (Cerdocyon thous) in south-east Brazil.
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Biomedical subjects
Publications and source records attributed to C M Gontijo.
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Skin biopsies from 53 patients with American cutaneous leishmaniasis (ACL) from the State of Minas Gerais, Brazil, were used for a characterization of the Leishmania parasites. A pair of primers flanking the conserved region of the Leishmania minicircle kDNA was used to obtain amplified DNA via the polymerase chain reaction. The amplified products were subsequently hybridized with Leishmania subgenus-specific radiolabeled probes. Parasites from 49 out of 53 samples (92.5%) were characterized as belonging to the subgenus Viannia and four (7.5%) as belonging to the subgenus Leishmania. Clinical, epidemiological and molecular evidence allow us to conclude that Leishmania (V.) braziliensis and Leishmania (L.) amazonensis are the species present in the patients studied and that L. (V.) braziliensis is the predominant species in the State of Minas Gerais, Brazil.
A new protocol is described for immunization of outbred Swiss mice. The procedure is based on subcutaneous implantation of antigen-coupled polyester-polyurethane sponges cut into disks of 10 mm in diameter vs 2 mm in thickness. Antigen coupling was performed by overnight incubation of the sponge with a solution of ovalbumin (Ova) (2 mg/ml) diluted in sodium carbonate buffer, pH 9.6. The amount of ovalbumin that was taken up by the sponge was between 71.4 to 82.5 micrograms. This was estimated by comparing the Ova absorbance at 280 nm in coating buffer solutions before and after incubation. To compare the efficiency of the proposed method, experimental groups immunized with the antigen in the presence of adjuvants (10 micrograms in Al(OH)3 or 100 micrograms in complete Freund's adjuvant (CFA)) were run in parallel. The data obtained after the 3rd week of immunization indicate that both cellular and humoral immune responses were achieved. These were assayed by antigen-induced footpad swelling and ELISA (specific antibodies), respectively. The levels of both immune responses elicited were similar to the responses observed in mice immunized with ovalbumin in the presence of Al(OH)3. The method might represent an advantage when immunizing with pathogenic antigens. Preliminary experiments have suggested that the antigen remains immobilized or bound to the sponge for a long period of time, since there is an increment on the cell population inside the sponges after boosting the animals. If so, the undesirable effects of immunization would be reduced.
Oral tolerance is a phenomenon that may occur in animals exposed to protein antigens for the first time by the oral route. They become unable to produce immune responses at the levels normally observed when they are immunized parenterally with antigen in the presence of adjuvants. Lipids have been used as adjuvants for both parenteral and oral immunization. In the present study we coupled ovalbumin with palmitate residues by incubating the protein with the N-hydroxysuccinimide palmitate ester and tested the preparation for its ability to induce oral tolerance. This was performed by giving 20 mg of antigen to mice by the oral route 7 days prior to parenteral immunization in the presence of A1(OH)3. Mice were bled one week after receiving a booster that was given 2 weeks after primary immunization. Specific antibodies were detected by ELISA. Despite the fact that the conjugates are as immunogenic as the unmodified protein when parenterally injected in mice, they failed to induce oral tolerance. This discrepancy could be explained by differences in the intestinal absorption of the two forms of the antigen. In fact, when compared to the non-conjugated ovalbumin, a fast and high absorption of the lipid-conjugated form of ovalbumin was observed by "sandwich" ELISA.
Palmitate-conjugated monoclonal antibodies specific to ovalbumin were inserted into the cell membrane of normal resting B cells and LPS-activated blasts. These two decorated B cells were tested for their ability to act as antigen-presenting cells for ovalbumin-specific I-Ad-restricted T-cell hybridomas. It was found that the antibody-decorated resting B cells presented antigen more efficiently than non-decorated controls. However, no increment was observed when decorated LPS blasts were compared with non-decorated blasts. This is explained by the fact that the inserted antibodies quickly disappeared from the cell membrane of LPS blasts, while they were retained for a long period in the membrane of resting B cells.
A palmitate-conjugate derivative of ovalbumin which can be inserted into the membrane of B cells has been prepared. The ability of these cells to act as antigen-presenting cells for specific T lymphocytes obtained from immunized mice was tested. It was found that the conjugates were more efficiently processed and presented than the naive form of the antigen. Palmitate-conjugated antibodies specific to ovalbumin were also inserted into the cell membrane of normal B lymphocytes. These cells were pulsed with the antigen and tested as antigen-presenting cells for T cells obtained from immunized mice. The antibody-decorated B cells presented ovalbumin more efficiently than non-decorated controls. Whether antibody-decorated, antigen-pulsed B cells could prime T cells in vivo was investigated. Some priming activity was found.
We have introduced some modifications in the technique called "cell decoration" in order to increase the amount of lipid-conjugated antibodies which can be incorporated into the membrane of B cells. As shown by FACS analysis, we have obtained an approximately 4-fold increment in the amount of specific antibodies incorporated into the cell membrane. The procedure, which consists of successive changes of the medium that contains the lipid-conjugated antibodies, avoided changes on parameters that interfere with cell viability. The proposed modification resulted in an approximately 2-fold enhancement of the ability of decorated B cells to act as antigen presenting cells for specific T hybridomas.
We have investigated the ability of different cells from non-immunized mice of the BALB/c strain to present antigen to two ovalbumin-specific I-Ad-restricted T hybridomas. Lipopolysaccharide-activated B-cell blasts were found to be the most efficient antigen-presenting cells. Purified small and dense splenic B cells also stimulated the hybridomas, although not to the same extent as the activated blasts, but comparable to non-fractionated spleen cells. Glutaraldehyde-treated B cells failed to present antigen, whereas F(ab')2 anti-mouse IgM-treated B cells exhibited markedly increased ability to present antigen. Using flow cytometry, we further purified the resting B cells by sorting the small lymphocytes to ensure that the ability of these cells to activate the hybridomas was not due to contamination with large non-resting B cells. The sorted small B cells retained the ability of antigen presentation. Their resting state was confirmed by the fact that they did not incorporate [3H]-thymidine as shown by autoradiographic analysis.
Monoclonal antibodies specific for ovalbumin were conjugated to palmitate and inserted into the membrane of normal spleen B cells. Their presence in the membrane, as well as their ability to bind ovalbumin, was established by immunofluorescence. The so called anti-ovalbumin-'decorated' B cells were tested for their ability to act as antigen-presenting cells for ovalbumin-specific I-Ad-restricted T-cell hybridomas. It was found that the antibody-decorated B cells presented antigen more efficiently than non-decorated B cells.
We do not agree with the analysis of Langman and Cohn on the function of Ig receptors. We have reviewed the available literature regarding anti-Ig activation of B cells and found it contradictory and unconvincing. We have presented experimental evidence on the inability of Ig receptors on B cells to mediate activation or tolerogenic signals. We suggest that the Ig receptors serve to focus antigen to specific B cells so the B cells can be activated by TI antigens or helper T cells. The Ig molecules also bind foreign antigen and thereby initiate internalization and antigen processing. The processed peptides are exported to the membrane, where they associate with MHC class II antigens, thus transforming B cells into efficient antigen-presenting cells.
1. We show that mouse strains differ widely in susceptibility to tolerance induction and/or immunization (priming) following contact of protein antigens (ovalbumin, human or bovine gamma globulins) with different mucosal surfaces. 2. When compared to a control group pretreated with saline, mice pretreated by the oral (intragastric) route with antigen became significantly less responsive to subsequent parenteral immunization (i.e., tolerant). This was observed in most, but not all, antigen/strain combinations. 3. Similar, although less prominent changes were induced by pretreatments with antigen by the ocular (conjunctival) route. 4. No significant effects were observed following pretreatments by the nasal, vaginal or rectal routes. 5. Genes present in strains selected for multispecific "high" or "low" responsiveness are included among those involved in tolerance induction following mucosal contacts with protein antigens.
Inbred mouse strains vary widely in their susceptibility to the induction of tolerance following oral (intragastric) administration of ovalbumin. Marked differences were found between strains that form a congenic pair differing at the H-2 complex: C3H/HeJ (H-2k) and C3H.SW (H-2b)-which were very susceptible and resistant to tolerance induction, respectively. In contrast, no significant differences were found between A/J (H-2a) and A.BY (H-2b) congenics, which were both susceptible, nor among C57BL/10J congenics, which were uniformly resistant to tolerance induction. We conclude that H-2-linked genes determine tolerance susceptibility in conjunction with background genes.
Granulocytes and mononuclear cells from normal subjects are able to kill Schistosoma mansoni schistosomula in the presence of human complement in vitro. In contrast, mononuclear cells from chronic schistosomiasis patients failed to kill the parasites. The relative efficiencies expressed in terms of the mean percentage killing of schistosomula for 15 experiments were 50.5 +/- 3.2 and 49.7 +/- 3.5 for granulocytes from normal and infected patients, and 50.8 +/- 2.8 against 23.0 +/- 3.2 for mononuclear cells from normal and infected patients, respectively. The killing effect of granulocytes dropped from 48.7 +/- 2.8 to 22.1 +/- 2.2 when autologous mononuclear cells from chronically infected patients were added to the system. Similar inhibitory effect of granulocyte function was obtained when these cells were incubated with normal mononuclear cells precultured with concanavalin A. Extracts prepared from mononuclear cells obtained from infected patients had the same inhibitory effect of intact cells on the complement-dependent granulocyte cytotoxicity.
By centrifuging heparinised human blood through a discontinuous Ficoll-Hypaque density gradient, it is possible to separate simultaneously mononuclear cells, neutrophils, and eosinophils.
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The effect of deltamethrin of the sandfly population in a focus of American Cutaneous Leishmaniasis in Viana, Espírito Santo State is described. The phlebotomine population density was determined inside and outside houses during a one year period in a treated and untreated area. The results showed a significant reduction in the number of sandflies inside houses in the treated area compared with both the untreated area and the same area before spraying. Despite having a residual action for 12 months after spraying the insecticide was ineffective outside houses. It is concluded therefore that deltamethrin is an efficient insecticide for reducing the number of phlebotomine sandflies inside houses.
In order to characterize the epidemiology of American Cutaneous Leishmaniasis (ACL) in a periurban area of the municipality of Sabará in the metropolitan region of Belo Horizonte (MRBH), an area until then considered free of the disease, a cross sectional survey was undertaken in 1990. The survey of the population consisted of 1119 interviews and 881 clinical examinations using Montenegro's skin test (MST). A low prevalence (3.7%) of positive MST was encountered. The disease had been occurring in the area for about 20 years in the form of sporadic cases. The predominant species of sandfly both in domestic areas and nearby areas of secondary vegetation was Lutzomyia whitmani. A canine survey of delayed hypersensitivity to the antigen P10,000 identified only one dog with a positive reaction out of 113 examined. The transmission of ACL in MRBH was confirmed. The occurrence of the disease in women, children and individuals with no contact with forest areas as well as the presence of potential vector species in the domiciliar environment, suggests the transmission of the disease in this environment.