María Estela Roux

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.

Nutrition disorders and immunologic parameters: study of the thymus in growing rats

OBJECTIVES We studied the effect of a low-quality dietary protein on cellular proliferation and maturation in the thymus of growing rats over time. METHODS After weaning Wistar rats were fed a diet containing 6.5 g/100 g of corn flour for 6, 10, 18, and 45 d (M groups). For comparison, other rats were fed a diet containing 6.5 g/100 g of casein (Cas groups), and well-nourished age-matched control rats were fed a commercial laboratory diet (C groups). Food intake, body weight, thymus weight, total number of thymocytes, and the percentages of CD43(+) and Thy1(+) thymocyte phenotypic antigen determinants were measured. RESULTS M versus Cas and C groups showed significant differences (P < 0.01) in body and thymus weights after 6 d of feeding, and the total number of thymocytes and the percentages of CD43(+) and Thy1(+) were significantly lower after 10 d of feeding. The results indicated that consuming a cereal diet for short or long periods causes thymus atrophy in growing rats, with significant reductions in the total number of T-cells concomitant with increases in the number of immature thymocytes. CONCLUSIONS The data showed that, in addition to low-protein concentration, low-quality dietary protein is a limiting factor in certain steps of cellular intrathymic pathways, probably related to the requirement of specific amino acids for optimal immune response.

Comparative immunohistochemical study of M-CSF and G-CSF in feto-maternal interface in a multiparity mouse model.

Multiparity status has been found to bring beneficial effects both to the maintenance of pregnancy and to the offspring; however, these effects have not been fully explained. We have previously reported that placentae obtained from multiparous females belonging to a syngeneic mouse crossbreeding showed an important increase in the number of placental macrophages, suggesting that they might constitute a protective subpopulation. Taking into account that macrophage‐colony stimulating factor (M‐CSF) and granulocyte‐colony stimulating factor (G‐CSF) have proved to modulate macrophage activity and that both factors and/or their receptors have been found at feto–maternal interface, in this paper we analyzed the presence of M‐CSF and G‐CSF in placental tissue employing the same multiparity mouse model in order to investigate the influence of parity status on local immunoregulation factors of macrophage activity.

Effect of short term refeeding on the thymus of growing rats after marginal and severe protein deprivation

Abstract The recovery of thymus of growing rats after marginal and severe protein deprivation at weaning was studied. Diets containing 15% or 20% casein were fed during a five day experimental period; age-matched control groups (36 and 45 days of age) received stock diet from weaning. At the end of the refeeding period body weight (bw) was determined and animals were killed. Thymus was removed; weight and cell number were determined. The mature T cell population was characterized by indirect immunofluorescence using the monoclonal antibody W3/13. Body weight (g) and thymus weight (mg) increased significantly in all the experimental groups after 5 days of refeeding; in spite of this none of them reached the values of their age-matched control (p 0.75 (g) were observed in the marginal protein deprived rats refed with 15% or 20% protein diet when compared to their respective age-matched control; thymus weight of severe depleted animals remained significantly lower. All experimental groups showed higher number of thymocytes tran the respective protein deprived group; after refeeding with both diets only the marginal deprived group reached the values of the age-matched control. None of the refed groups reached the percentage and absolute number of mature W3/13 + T cell population found in the respective well nourished controls; nevertheless, the percentage of W3/13 + cells was significantly higher in the groups fed the 20% protein diet. These results confirm the interaction between dielary protein concentration and the degree of wasting; they also point to the time of refeeding as all important factor since a short period is enough to reverse the damage produced by marginal protein malnutrition on cellular proliferation, but not on the appearance of specific antigenic determinants.

Mucosal immunity induced by orally administered transgenic plants

Oral immunization is an efficient means to induce protection at the portal entrance for many pathogens. Therefore, the design of efficient edible vaccines through transgenic plants represents a challenging alternative to the traditional injectable ones. We have previously reported the construction of transgenic potato plants expressing the genes coding for the immunogenic proteins of Newcastle Disease Virus (NDV) and their immunogenicity in mice. All mice receiving transgenic plant extracts in incomplete Freunds adjuvant produced specific antibodies. Animals fed with transgenic leaves also showed a specific response against NDV. The aim of the present study was to continue the evaluation of the mucosal immune response. Adult Balb/c mice were fed with potato leaves for a month and on day 36 mucosal samples were collected. ELISAs performed on intestinal washes showed that transformed plants elicited the synthesis of NDV-specific IgG and IgA antibodies. In addition, anti-NDV IgA antibodies were detected in supernatants of cultured small intestine fragments of mice fed with the recombinant immunogens, suggesting the presence of NDV-specific IgA secreting plasma cells in the intestinal tissue. Moreover, we detected specific anti-NDV antibodies in intestinal fluids after oral immunization with F and HN transgenic plants. Also, indirect immunofluorescence on intestinal tissue was performed. The present results suggest that these immunogens, F and HN glycoproteins of NDV, when orally administered, would enhance the number of IgA(+) B cells, and the cytotoxic cellular immune response via CD8(+) T cells, found in the gut lamina propria that is in accordance with our first findings.

Developmental study of immunocompetent cells in the bronchus-associated lymphoid tissue (BALT) from Wistar rats

The aim of the present report was to study in growing Wistar rats the development of immunocompetent cells in the bronchus-associated lymphoid tissue (BALT). We found at day 4 postpartum, a high number of TCRgamma/delta+ T cells and very few CD8alpha+, CD8beta+, CD5+, TCRalpha/beta+ T cells in BALT. The latter cells and CD4+ T cells increase with age. Even though T cells expressing TCRgamma/delta outnumber those expressing TCRalpha/beta early in development, until 45 days of age, alpha/beta+ predominate over gamma/delta+ T cells only in adult rats (60 days of age). Moreover, a predominance of suppressor/cytotoxic T cells over T-helper cells was found in 60 days old rats. Surprisingly, more CD8alpha+ than CD8beta+ T cells in BALT are observed. The number of IgA+ B and CD4+ T cells found in the BALT increases with age. The early appearance - 4 days of age - of all T-cell phenotypes in BALT especially of gamma/delta+ T cells may imply a benefit to respond to inhaled antigen soon after birth.

Development of T lymphocytes in the nasal-associated lymphoid tissue (NALT) from growing Wistar rats.

The aim of the present report was to study the development of several T-lymphocyte subsets in the nasal-associated lymphoid tissue (NALT) of growing Wistar rats. CD5+ and CD4+ lymphocytes gradually increased with age. A predominance of CD8α+ over CD4+ T cells was found from 7 to 45 days but from 45 to 60 days of age T helper cells outnumbered the cytotoxic subpopulation. The majority of CD8+ T lymphocytes expressed the heterodimeric isoform. The most relevant findings by immunohistochemistry are: (1) the predominance of TCRγδ+ and CD8α+ cells at 7 days postpartum over all the other T-cell subpopulations; and (2) that TCRγβ+ outnumbered TCRαβ+ T cells from 7 to 45 days postpartum whereas αβ T cells predominated in 45- and 60-day-old rats. Besides, cytometric studies have shown that the percentages of TCRγ+, CD8+, as well as the population coexpressing both phenotypes (TCRγδ+CD8α+), were significantly higher in rats at 7 days postpartum when compared to 60 day-old rats. In the present study, the finding of a high number of γδ+ and CD8+ T cells early in NALT development may indicate the importance of these subpopulations in the protection of the nasal mucosa in suckling and weaning Wistar rats.

Compartmentalisation between gut and lung mucosae in a model of secondary immunodeficiency: effect of thymomodulin.

Compartmentalisation of mucosal immune response seems to be the result mainly of the preferential migration of activated cells back to their inductive sites. The aim of this report was to demonstrate, in a model of secondary immunodeficiency in Wistar rats (severely protein deprived at weaning and refed with casein 20 %; group R21), that the oral administration of Thymomodulin (group: R21TmB) has different effects on gut and BALT (Bronchus-associated lymphoid tissue). Tissue sections (5μ) were studied by immunohistochemistry 1). The oral administration of Thymomodulin restores only in gut Lamina propria (LP) the IgA B and CD4 T cell populations to control levels. The CD8a and CD25 subpopulations do not vary in gut as they return to control levels when refed with 20% casein diet. All the populations mentioned above remained decreased even after receiving Thymomodulin by the oral route. However, the same behaviour was observed for the TCRδ T cells that were decreased and return to normal levels in both mucosae by the effect of the immunomodulator; 2) when studying the iIEL (intestinal intraepithelial lymphocytes) CD8α, CD25 and TCRγδ T cells, that were increased in R21, return to control levels in R21TmB. In BALT intraepithelium CD8α and CD25 T cells remained decreased, while only TCRγδ T cells (increased in R21) return to control values. Conclusions: 1) there exists a compartmentalisation between both mucosae, as T CD4+ and IgA B+ cells are restored by TmB only in gut; 2) only those iIEL involved in inflammation (CD8α+/CD25+ and TCRγδ+/CD25+) are normalised by means of the Thymomodulin 3) however, in BALT, only TCRγδ+ T cells are restored 4) the oral administration of the present immunomodulator may be useful as a therapeutic agent, although the preferential survival in the tissue of initial stimulation is the major factor in the preferential distribution of activated cells.

Multiparity increases trophoblast invasion and vascular endothelial growth factor expression at the maternal-fetal interface in mice

To analyze immunomodulating effects related to parity status, we studied trophoblast invasion grade, placental expression and systemic concentration of VEGF and its receptor Flt-1 in normal fertile (CBA/JxBALB/c) mice and abortion-prone (CBA/JxDBA/2) H-2(d)xH-2(k) mice. BALB/c or DBA/2 mated CBA/J females were, respectively, divided into the following groups: primiparous young (3.0+/-0.5 months old); primiparous old (8.5+/-0.5 months old) and multiparous old (8.5+/-0.5 months old, with 4 pregnancies). Immunohistochemical analysis of term placentae from both multiparous groups revealed various layers of invasive trophoblast tissue, identified as cytokeratin+/vimentin- cells, in contrast to the single layer detected in the placentae of primiparous animals, indicating that multiparity increases trophoblast invasion regardless of the success of the pregnancy outcome. Invasive trophoblast tissue from primiparous CBA/JxDBA/2 placentae showed diminished VEGF expression in comparison with the normal fertile group, while both multiparous groups demonstrated high expression of VEGF in the invasive trophoblast tissue. Placental expression of Flt-1 was similar in all groups. However, the primiparous CBA/JxBALB/c group showed the highest plasma concentration of sFlt-1 at term, while both multiparous groups demonstrated low circulating levels. No differences in circulating VEGF levels were observed among the groups. These results demonstrate an increase in trophoblast invasion tissue and expression of VEGF in the maternal-fetal interface in multiparous mice compared to primiparous mice. Moreover, the placenta appears to be able to regulate the circulating levels of VEGF by releasing sFlt-1.

Impairment of IgA Expression and Cell Mediated Immunity Observed on Peyer’s Patches of Protein-Depleted Rats at Weaning and Then Fed on 20% Casein

The gastrointestinal immune system has many unique structural and functional features, that include the Peyer’s patch and the secretory IgA system (1). Precursors of the IgA plasma cells are first recognizable as bone marrow-derived B cells in the Peyer’s patches (1–3). IgA committed Peyer’s patch cells, that bear surface IgA, leave Peyer’s patches to mesenteric lymph nodes, where they further maturate and acquire the capacity to seed the intestinal lamina propria as IgA plasma cells (2,4).