Ernesto Massouh

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 Peyers 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.

Effect of Melia azedarach L. leaf extracts on human complement and polymorphonuclear leukocytes

The effect of the water extract of Melia azedarach L. (Meliaceae) leaves on human complement and polymorphonuclear leukocytes was investigated. This extract showed a strong anticomplementary activity, which was more pronounced in the classical pathway assay. The extract did not affect the phagocytic activity of polymorphonuclear leukocytes, nor the respiratory burst of these cells as measured by the nitro blue tetrazolium reduction assay.

Nitric oxide and HSV vaginal infection in BALB/c mice

Here we study the role of nitric oxide in the vaginal infection of Balb/c mice with herpes simplex virus type 2. Inducible nitric oxide synthase (iNOS) mRNA was detected by RT-PCR in vaginal tissue and inguinal lymph nodes early postinfection. iNOS was also found to be activated in cells recovered from vaginal washings of infected animals. Animals treated with aminoguanidine (AG), an iNOS inhibitor, showed a dose-dependent increase in vaginal pathology after viral infection compared to controls. Viral titers in vaginal washings and vaginas were higher in AG-treated mice. Treated animals presented higher PMN counts in vaginal washings compared to controls. Histopathology studies revealed a profound inflammatory exudate in vaginal tissue of treated animals. Finally, RT-PCR analysis showed increased expression of the chemokines MIP-2 and RANTES in vaginal tissue and inguinal lymph nodes of these animals.

In vitro antiphagocytic effect of Melia azedarach leaf extracts on mouse peritoneal exudate cells

The effect of Melia azedarach L. (Meliaceae) leaf extract on the phagocytic capability and respiratory burst of mouse peritoneal exudate cells was studied. The extract inhibited the phagocytosis of opsonized sheep erythrocytes. This inhibition was both dose- and time-dependent and reverted 48 h after removing the extract from the culture medium. Furthermore, chemiluminescence in treated cells was also impaired using either receptor (opsonized zymosan) or post-receptor (PMA) stimuli.

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 Peyers 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.

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 Peyers 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.

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.

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.

Aminoguanidine administered during the induction of oral tolerance alters the systemic response of the tolerised rats

Herewith we investigated the role of nitric oxide synthase (NOS)-II in the establishment of oral tolerance induced by low antigen dose. To accomplish this, we used a rat model of oral tolerance induced by intragastric administration of low doses of ovalbumin (OVA). NOS-II was inhibited in vivo during the onset of tolerance by intraperitoneal (i.p.) treatment with aminoguanidine (AMG), a selective NOS-II inhibitor. Four experimental groups were generated: (TOL), tolerised rats, receiving OVA but no AMG; (TAG), rats tolerised with OVA and simultaneously receiving AMG i.p.; (CAG), controls treated with AMG but no oral antigen; and (CONT), controls receiving neither OVA nor AMG treatment. The state of oral tolerance was evaluated in all groups by analysing several immune parameters upon subcutaneous administration of OVA in Freunds complete adjuvant. First, we were able to determine that NOS-II inhibition altered the TH1/TH2 balance in tolerised rats, driving the TH2 anti-OVA response in TOL rats towards TH1 in TAG animals, which showed enhanced delayed hypersensitivity responses. Second, splenocyte cultures from TAG rats showed lower levels of IL-10 production compared to TOL samples as determined by ELISA analysis. Last, we detected the presence of a functional distinct Tr1 regulatory T cell population in spleen samples recovered from TAG animals. Contrary to what happened with TOL Tr1 cells, the levels of Tr1 cells in TAG samples were modified by in vitro stimulation with OVA. All together, these data indicate a preponderant role for NOS-II in the process of oral tolerance induced by low antigen dose.

Effect of undernourishment on Herpes Simplex Virus Type 1 ocular infection in the Wistar rat model

Abstract.  We have studied the susceptibility to Herpes Simplex Virus Type 1 (HSV‐1) infection in malnourished rats. Groups of 10 rats were undernourished during suckling by offspring duplication. The animals were put on commercial diet and at 1, 2, 3, 5 and 8 weeks after weaning, infected in the eye by scarification with HSV‐1, strain F. Significant differences in morbidity and mortality were observed between malnourished and control groups infected three weeks after weaning. Viral titres were higher in ocular washings and brains obtained from the malnourished group. This group showed a diminution in antigen dependent lymphocyte proliferation compared to control, and significantly lower delayed type hypersensitivity reaction against inactivated virus (malnourished = 0.16 ± 0.02 mm, control = 0.26 ± 0.03 mm, p < 0.05). Neutralizing antibodies in serum were lower in the malnourished group and lower levels of interferon were obtained in the malnourished group 24 h post‐infection. We conclude that malnutrition during suckling induces a delay in the capability to overcome HSV infection.