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Biomedical subjects

J Fló

Publications and source records attributed to J Fló.

15 recordsLinked to original sources
PubMed2001

Codelivery of DNA coding for the soluble form of CD86 results in the down-regulation of the immune response to DNA vaccines.

The costimulatory pathway that includes CD80, CD86, CD28, and CTLA-4 plays a key role in regulating T cell activation and tolerance and is a promising therapeutic target. We have studied the possibility of down-regulating the immune response to DNA vaccine by codelivery of a plasmid coding for the extracellular domains of CD86 (pDelta86). We found that DeltaCD86 was able to inhibit the engagement of FcCTLA-4 but not of FcCD28 to CD80 and CD86 expressed on COS cells. Coadministration of plasmid pDelta86 encoding for the extracellular domains of CD86 along with a plasmid encoding for the glycoprotein D (pgD) of herpes simplex virus-2 (a membrane-bound protein) by the im route in mice resulted in a strong inhibition of the cell-mediated immune response in the spleen and in draining lymph nodes. In addition, when pDelta86 was coadministered together with a plasmid encoding for the ovalbumin (pOVA) (a soluble protein), a strong inhibition of the cell-mediated immune response was observed in draining lymph nodes and only a partial inhibition was found in the spleen. Furthermore, only a partial down-regulation of the humoral immune response was observed. The mechanism involved could be a preferential engagement of DeltaCD86 to CTLA-4 leading to the transmission of a negative signal to T lymphocytes.

Abatacept↗
PubMed2001

Oral transgene vaccination mediated by attenuated Salmonellae is an effective method to prevent Herpes simplex virus-2 induced disease in mice.

An attenuated strain of Salmonella typhimurium has been used as a carrier for oral genetic immunization. The eukaryotic expression vector pCMV containing the gene of the glycoprotein D (gD) of the herpes simplex virus 2 was used to transform Salmonellae. The oral immunization with the transformed salmonellae elicited a strong cellular immune response in both, the mucosal and systemic compartments (spleen, ileal lymph nodes and Peyer patches). The immune response mainly consisted in a dramatic activation of IFN-gamma-secreting cells. Twenty hours following the challenge with five lethal doses of virus, mRNA for IFN-gamma was observed in vaginal tissues from mice immunized with salmonella harboring the plasmid pgD but not in tissues from mice immunized by the intramuscular route with pgD. After an intravaginal challenge all immunized mice survived without developing symptoms. Furthermore, the immunization with Salmonella resulted in a more effective control of viral shedding than intramuscular immunization. We have unequivocally demonstrated by the introduction of an intron in the green fluorescent protein that the expression of the plasmid was due to the transcription of the protein by an eukaryotic nuclear process and not as a result of expression of the protein by the bacteria. Macrophages and dendritic cells were found expressing the protein in systemic and mucosal compartments of the immune system.

Administration, Oral↗
PubMed2000

Superiority of intramuscular route and full length glycoprotein D for DNA vaccination against herpes simplex 2. Enhancement of protection by the co-delivery of the GM-CSF gene.

Immunization with naked DNA has been analyzed in two critical variables: the site of injection and the cellular compartment to which the coded protein is directed. The gene for the full length of the glycoprotein D (gD) of HSV-2 under the control of the citomegalovirus (CMV) promoter was injected via the intradermal (i.d.) or the intramuscular (i.m.) routes in mice. Immunization in the quadricep muscle was superior to the intradermal immunization in the footpads. A stronger activation of IFN-gamma-secreting cells in the spleen and draining lymph nodes (DLN) was induced, resulting in a more efficient protection against an intravaginal challenge. In order to analyze the effect of the cellular localizations of the coded protein, the DNA for the truncated form of the gD (DeltagD) was injected via the i.m. route. Immunization with a vector encoding for DeltagD resulted in higher antibody levels in serum and vaginal washes than immunization with the gene for the full length gD. However, immunization with the DeltagD DNA elicited a much weaker cell-mediated immune response and was inferior to gD DNA in providing protection against a lethal intravaginal challenge with HSV. Co-injection of an expression cassette for the granulocyte-macrophage colony-stimulating factor (GM-CSF) increased both the humoral and cell-mediated immune response with both gD and DeltagD. A strong activation of IL-4-secreting cells was observed in the spleen and DLN together with an increase in the number of IFN-gamma-secreting cells. In addition, a reduction in the vaginal virus titers after an intravaginal challenge was observed in mice co-injected with the GM-CSF gene as compared to those immunized with pCDNAgD only.

Administration, Cutaneous↗
PubMed2000

Modulation of the immune response to DNA vaccine by co-delivery of costimulatory molecules.

We have investigated methods for modulating immune responses, against herpes simplex virus (HSV), generated from DNA vaccination by co-delivery of genes encoding costimulatory molecules. A strong delayed-type hypersensitivity (DTH) reaction was induced in mice co-injected via the intradermal (i.d.) route with a eukaryotic expression plasmid encoding the CD80 molecule (pCD80) and a plasmid encoding the glycoprotein D of the HSV-2 (pgD). Furthermore, when spleen cells from these mice were cultured in the presence of inactivated HSV, a significant increase in the expression of interleukin-2 receptor (IL-2R) was observed in the CD4 subset compared with mice immunized only with pgD. Analysis of cytokine synthesis at the single-cell level indicated that CD80 genes induce a significant increase in the number of interferon-gamma (IFN-gamma)-, IL-2- and IL-4-secreting cells in the spleen. On the other hand, co-administration of the CD80 gene via the intramuscular (i.m.) route did not induce an increase in the cell-mediated immune response. When a plasmid carrying the CD86 gene (pCD86) was co-injected via the i.m. route with the pgD plasmid, a small decrease in the number of IFN-gamma-secreting cells was observed. This down-regulation of the immune response was also observed when eukaryotic expression cassettes for CD80 and for CD86 were co-administered with the pgD plasmid via the i.d. route. However, co-injection of pCD86 via the i.m. route produced a small increase in the number of IL-4-secreting cells. When immunized mice were challenged intravaginally with 100 plaque-forming units of virus, only co-injection of the CD80 gene by the i.d. route provoked an adjuvant effect compared with mice immunized with pgD alone. A reduction in the titres of HSV in vaginal washings was observed together with a decrease in the lesion score.

Adjuvants, Immunologic↗
PubMed1998

Priming or tolerization of the B- and Th2-dependent immune response by the oral administration of OVA-DNP is determined by the antigen dosage.

In the present report we established antigen dosages that induce oral tolerance of Th1 and Th2 lymphocytes or instead prime B- and Th2-dependent immune response and induce the tolerance of Th1 lymphocytes. Using different hapten-carrier systems, we found that low doses of OVA-DNP administered orally primed B and Th2 cells. On the other hand, no priming of B or Th2 cells was found in high-dose-OVA-DNP-fed rats. Low-dose-OVA-DNP-fed rats showed a strong mucosal immune response, with a high number of IgA anti-DNP antibody-forming cells in the lamina propria, while no mucosal immune response was observed in high-dose-OVA-DNP-fed rats. Thirty days after the immunization, tolerization of Th1 lymphocytes was confirmed in low- and high-dose-OVA-DNP-fed rats by diminished antigen-specific proliferation in vitro, reduced titers of anti-DNP IgG2a in serum, reduced expression of CD25 and CD134 molecules in cultured cells exposed to the antigen, reduced DTH reaction, and reduced IL-2 synthesis in culture. On the other hand, a high dose of OVA-DNP led to Th1 and Th2 tolerance, with an inhibition of specific IgG1 and IgG2a anti-DNP antibodies in serum after a parenteral challenge with OVA in CFA. This functional evidence was supported by the direct examination of IL-2 and IL-4 production. Furthermore, whereas in vitro assays seem to indicate that active suppression could be the responsible for Th1 tolerization in low-dose-OVA-DNP-fed rats, the results obtained after the transference of spleen or MLN cells to naive recipients support the idea that a subtractive mechanism is behind the tolerization of Th1 lymphocytes.

Administration, Oral↗
PubMed1998

Oral administration of one dose of cholera toxin induces a systemic immune response prior to a mucosal immune response by a direct presentation in the spleen.

In the present report the results indicate that the oral administration of one dose of CT in rats results in an antibody immune response in the spleen 48 h later, whereas no antitoxin antibody forming cells were found in the Peyer patches (PP), mesenteric lymph node (MLN) and lamina propria (LP) of the small intestine. At this time the main isotype of the antitoxin antibodies in the spleen were IgG and IgM, 5 days after the priming, few antitoxin AFC were observed in the MLN, IgG being the main isotype, whereas no IgM antitoxin AFC were found. At 1 week after priming the number of antitoxin AFC in the MLN reached similar values to those observed in the spleen. When cells from the spleen of rats primed orally with one dose of CT were cultured during 4 days in the presence of inhibitory doses of anti-Ia MAb (OX6), the number of antitoxin AFC was diminished when compared with that observed when cells were cultured in the absence of anti-Ia. The main isotype of antitoxin AFC observed when cells were analyzed after culture was IgM and it was the isotype most affected by the treatment with MAb anti-Ia. These results strongly suggest that an in situ presentation of the antigen did occur in the spleen. On the other hand, when the secondary immune response was studied 48 h after boosting, antitoxin AFC were found in the PP, MLN, SP and LP and 5 days after the booster a 20-30-fold increase was observed in all lymphoid tissues studied, indicating that the secondary immune response found in the spleen was mainly due to the recruitment of memory cells from Peyer's patches. However, when spleen cells were cultured 48 h after the immunization in the presence of inhibitory doses of anti-Ia a little decrease in the number of AFC was observed when compared with the controls (in absence of anti-Ia). The analysis of the antitoxin antibodies in sera and intestinal fluids were in line with the results presented above. The results shown in this report indicate that the systemic immune response observed after the oral administration of CT could be due in part to an in situ presentation of the antigen in the systemic compartments, especially in the spleen.

Administration, Oral↗
PubMed1998

Long-term antibodies after an oral immunization with cholera toxin are synthesized in the bone marrow and may play a role in the regulation of memory B-cell maintenance at systemic and mucosal sites.

To study the importance of the bone marrow in the long-term antibody response, IgG and IgA antitoxin antibody-forming cells were evaluated by ELISPOT in Peyer's patches, mesenteric lymph nodes, spleen, lamina propria of the small intestine and bone marrow at several times after oral immunization with cholera toxin. The mesenteric lymph node was the site having the major frequency of IgG antitoxin during the first two weeks after priming, whereas lamina propria was the site with a major number of IgA antitoxin antibody-forming cells. However, from 3 weeks until 10 months after priming, bone marrow became the site with the major frequency of IgG, and especially IgA antitoxin antibody-forming cells (without taking into account the lamina propria). This result indicates that bone marrow was responsible for the long-term antibody response and raises questions concerning the mechanisms involved in the maintenance of antibody production. The importance of bone marrow as a site of antibody production was great when we analysed results as the true contribution of the total number of antitoxin antibody-forming cells, taking into account the number of cells recovered from each organ. When we analysed the anatomical location of memory B and T cells by adoptive transference, we found that cells from mesenteric lymph nodes and spleen were able to transfer a strong antibody response to naive syngeneic recipients, whereas bone marrow cells transferred a weak antibody response.

Administration, Oral↗
PubMed1996

Reversible effects on B and T cells of the gut-associated lymphoid tissues in rats malnourished during suckling: impaired induction of the immune response to intra-Peyer patches immunization with cholera toxin.

To define the alterations provoked by malnutrition during suckling (20 pups/dam) in the gut-associated lymphoid tissues of rats, Peyer patch (PP) and mesenteric lymph node (MLN) cells were studied by flow cytometry. After weaning (21 days of age), rats malnourished during suckling (MNR) showed an increase in the CD4+ CD45RC+ subset together with a decrease in the CD4+ CD45RC- subset (P < 0.01). These alterations remained even after 3 weeks of refeeding with stock diet. The CD4+CD8+ subset was not increased in the MNR, indicating that a release of cortical thymocytes did not occur. At weaning the percentage of CD4+Thy1+ cells was decreased in the MNR, indicating a low number of cells released from the thymus. When the B cell lineage was studied, we found a decreased percentage of precursors in the bone marrow and a decreased percentage of mature B cells in the periphery. When the MNR were immunized intra-PP with cholera toxin (CT) after 1 week of refeeding, the specific IgG and IgA and IgM antibody-forming cells (measured by ELISPOT) were diminished in the PP, MLN, and spleen when compared to the age-matched controls (P < 0.001). These results were coincident with the ELISA titers obtained in the sera and in the intestinal fluids. When CT was administered after 2 weeks of refeeding, the number of IgM anti-toxin AFC approached control values, but the number of IgA and IgG AFC continued to be low. When 3 weeks of refeeding was allowed before the CT delivery, the immune response in the MNR approached control values. These results indicate that malnutrition during suckling provokes alterations in B and T lymphocytes and produces a lack in the induction of the primary and secondary immune responses in the GALT which reversed after 3 weeks of refeeding.

Animals↗
PubMed1996

Oral administration of a bacterial immunomodulator enhances the immune response to cholera toxin.

Attempts to achieve IgA responses in the intestine by oral immunization with non replicating antigens have been characterized by ineffective responses of short duration unless long term dosages are administered. Cholera toxin (CT) is an exception in that it is able to produce a high secretory and systemic immune response. We study the effects of a bacterial immunomodulator [3 x 10(10) Propionibacterium granulosum ml-1 and lipopolysaccharide (LPS) 5 mg ml-1] on the immune response to CT orally administered to Wistar rats. The immunomodulator was orally administered as follows: in schedule 1 during 7 days prior to the first dose of CT; and in schedule 2, 2 days before, together, and 3 days after the first dose of CT. Schedules 1 and 2 were effective in increasing the specific IgA in the intestinal fluid and specific IgG in serum (P < 0.001) when compared to controls. Besides, schedule 2 was more effective than schedule 1 when the levels of specific IgG in serum or specific IgA in intestinal fluid was measured (P < 0.05). Total IgA in the intestinal fluid was increased in rats receiving the immunomodulator (P < 0.01). However, the ratio of specific IgA per total IgA was higher in rats receiving treatment 1 or 2 when compared to controls (P < 0.01). The number of antitoxin antibody producing cells was not increased in the Peyer patches, but a significant increase was observed in the mesenteric lymph nodes and spleen when compared to controls (P < 0.05). The administration of LPS alone produced an increase in the antitoxin immune response when compared to controls, but it was lower than those produced by the administration of the immunomodulator. These results indicate that this immunomodulator is an effective adjuvant of the mucosal and systemic immune response to CT. The mechanisms of action possibly involve nonespecific and specific modulations of the immune response.

Adjuvants, Immunologic↗
PubMed1995

The bone marrow as a site of antibody production after a mucosal immunization.

To study the importance of the bone marrow in the production of specific antibodies after a mucosal immunization with cholera toxin, the IgG, IgA and IgM specific antibody forming cells were evaluated by ELISPOT in Peyer patches, mesenteric lymph node (MLN), spleen, blood and bone marrow (BM). When 50-day-old rats were immunized intra-Peyer patches, a similar number of IgG and IgA antitoxin antibody forming cells (AFC) were found in the BM, whereas in the other lymphoid tissues a higher number of IgG antitoxin AFC were found. In all sites the peak of AFC was obtained 2 weeks after immunization. The administration of CT to 35-week-old rats resulted in a stronger immune response in all lymphoid tissues studied, but the proportion of antitoxin AFC contributed by the BM had not changed. One oral dose of cholera toxin resulted in a low number of antitoxin AFC, whereas when two or three doses of CT were administered orally an increase in the number of AFC was observed in the BM, reaching similar or higher numbers of IgG and IgA AFC than in the spleen. In all cases the highest number of AFC/10(6) cells was observed in the MLN, whereas antitoxin AFC were not found in the blood. The total number of AFC recovered from each organ was calculated taken into account that the BM of one femur represents 9% of the total BM. So, it was found that the BM is an important site in the production of IgG antitoxin antibodies, being the main site in the IgA antitoxin antibody production.

Administration, Oral↗
PubMed1994

Deficient induction of the immune response to oral immunization with cholera toxin in malnourished rats during suckling.

Malnourished rats during suckling were orally immunized with cholera toxin (CT) after different periods of refeeding. Intestinal fluids, sera, and supernatant fluids from cultured mesenteric lymph node (MLN) cells were obtained after rats were given three doses of CT and analyzed by enzyme-linked immunosorbent assay (ELISA) to evaluate the specific antibody response. Serum-specific immunoglobulin G (IgG), IgA, and IgM were severely diminished in malnourished rats immunized with three doses of CT after 1 week of refeeding when compared with those of controls. Also, a decreased IgA ELISA titer of the intestinal fluids and abrogation of the capacity to neutralize the CT in the intestinal ligated loop test were found. When a booster was given at 113 days of age, the immune response continued to be affected in the serum and the intestinal fluid. The results from the analysis of the supernatant fluids from cultured MLN cells were coincident with those mentioned above. When one dose of CT was administered into Peyer's patches (PP) after 1 week of refeeding, an impaired immune response was found in the intestinal fluid of malnourished rats during suckling compared with that of controls. This result together with the analysis of supernatant from MLN and PP cell cultures suggests that antigen triggering in the PP was affected. When the refeeding period was extended to 30 days and then the first dose of CT was administered, the antibody immune responses in intestinal fluid serum and supernatant fluid approached control values. These observations reinforce the fact that the gut-associated lymphoid tissue immaturity of the rats when they received the first CT dose (at 28 days old) was the main reason for the decreased immune response observed in the experimental group.

Administration, Oral↗
PubMedDate not supplied

Impairment of B and T cell maturation in gut associated lymphoid tissues due to malnutrition during lactation.

Previously we found that malnutrition during lactation in rats produces an impairment in the immune response to cholera toxin. In this report we found that malnutrition during lactation provokes in 28-day-old rats an increase of Thy1+ c mu+ cells in gut associated lymphoid tissues concomitantly with a decrease of sIgA+ B cells. No differences were found in the percentages of the IgM+ B cell populations. Furthermore, no differences were found in the Peyer's patch (PP) and mesenteric lymph node (MLN) T cell subsets in weaning rats when compared to controls. However, after 1 week of refeeding a higher percentage of the Thy1+ c mu- subset together with a lower percentage of CD5+, CD4+, and CD8+ T cells, were found in malnourished rats when compared to controls. The above results may indicate that B-cell maturation is delayed in malnourished rats at two stages of differentiation: (a) in the passage of pre-B cells (Thy1+ c mu+) to immature B cells (s mu+), and (b) in the switch from s mu+ B cells to s alpha+ B cells. The decrease of CD5+, CD4+, and CD8+ T cells together with an increase of the Thy1+ c mu- subset in gut-associated lymphoid tissues (GALT) may indicate that T-cell maturation is also delayed. Results obtained at weaning may be due to an engraftment by maternal milk-derived lymphocytes in the pups.

Animals↗
PubMedDate not supplied

Age-related changes of naive and memory CD4 rat lymphocyte subsets in mucosal and systemic lymphoid organs.

The purpose of this study was to investigate in rats, by double-label immunofluorescence and flow cytometric analysis, the age related changes in the CD4 subset of gut-associated lymphoid tissues and spleen. We found that the percentage of CD4+ T cells in Peyer's patches (PP) and spleen (SP) increased during the first 6 weeks after weaning. An age-related decrease of the CD4 subset was observed in SP of aged rats, but not in their PP. In all lymphoid tissues studied, an age-related decrease of the Thy-1+ subset was observed from weaning to 2 years of age. Analysis of the naive CD4 subset (CD45RC+) showed that in SP this subset increased during the first 9 weeks of age, and declined in aged rats. However, in PP this subset presented a slow decrease from weaning until 2 years of age. Together with the decrease of the naive subset, a sharp increase of the memory/activated CD4+ cells (CD45RC- Thy-1-) was observed in PP, and to a lesser extent in SP. When the maturation of the CD4 T cells in PP was followed during the first week after weaning, we found that an important proportion of this subset changes its phenotype at this time, from recent thymic emigrant (CD45RC- Thy-1+) to naive T cell (CD45RC+ Thy-1-) and then to activated/memory cell (CD45RC- Thy-1-). Therefore it appeared that CD4 T cells from PP mature faster than SP CD4 T cells, and they are not subject to the deleterious effect of aging. One surprising point was the different kinetics of the CD4 T cells observed in mesenteric lymph nodes (MLN). No age-related changes were observed in the CD4 subset at this site. Furthermore, the percentage of the CD45RC+ cells did not decrease in aged rats, and in the first 9 weeks of life an increase of this subset was observed.

Aging↗
PubMedDate not supplied

Long term effects of malnutrition during lactation on GALT.

In this report we present data that help to define the impact of malnutrition during the suckling period on the gut associated lymphoid tissues (GALT). Fifty-day old rats malnourished during lactation presented a diminished percentage of s alpha +B cells and IgA level in the intestinal fluid. Also, a decrease in the CD4+ and CD8+ T cell subsets was found. At 120 days of age the percentage of s alpha +B cells and IgA level in the intestinal fluid was similar to the control. However, the percentage of T cells remained altered. When three doses of chorea toxin were administered orally since day 28, the IgA anti CT antibodies were diminished in the intestinal fluid, while the immunization schedule started after seven days of refeeding (28 days of age). This impairment of the immune response remained even after a CT booster was given to animals 113 days old. This diminishing in the CD4+ T cells may be the major cause of the nonresponsiveness described herewith.

Animals↗