The effects of formula feeding on physiological and immunological parameters in the gut of neonatal rats.

A unique model of formula feeding in the neonatal rat was utilized to investigate the effects of an enterally delivered artificial milk formula on clinically relevant immunological and biological characteristics in the gut, compared to naturally reared pups. Hooded Wistar rat pups were randomly allocated to two treatment groups: formula-fed (FF) or naturally suckled (NS). A flexible silastic intra-gastric cannula was surgically implanted into the FF pups, through which an artificial rat milk supplement was continuously delivered from day 4 to day 10 of life. Rat pups were sacrificed at 10 days of age. Body weight, small intestinal weight, mucosal CD8(+) cell numbers, and ileal lactase activity in FF animals were significantly decreased compared to their NS counterparts (P < 0.05). Numbers of eosinophils, mucosal mast cells, CD4(+) T-cells, ileal villus height and gastric emptying times were significantly increased in FF pups (P < 0.05). We have developed a new rat model of artificial feeding which possesses important immunological and biological similarities to the premature human infant.

Maternal milk regulation of cell infiltration and interleukin 18 in the intestine of suckling rat pups.

BACKGROUND: and aims: In neonates the gastrointestinal tract is exposed to food and bacterial antigens at a time when the gut mucosal immune system has not developed the ability to induce oral tolerance. This increases the risk for an inappropriate immune response to oral antigens. Transforming growth factor beta (TGF-beta) is an immunoregulatory cytokine present in high concentration in maternal milk. Interleukin 18 (IL-18) is a cytokine that mediates early immune events, and drives T cell development. We assessed the role of TGF-beta in mediating mucosal immune development and specifically the effect on endogenous IL-18. METHODS: Rat pups were randomly assigned to the following groups, naturally suckled, maternal milk via cannula, and formula fed with and without physiological levels of TGF-beta2. A comparison of the immune response profile was then carried out. Cytokine profiles, dendritic cell, intestinal mast cell, and eosinophil numbers were assessed. RESULTS: We show that feeding formula deficient in TGF-beta2 resulted in accumulated IL-18 protein release from intestinal epithelial cells and IL-18 mRNA up regulation. A proinflammatory cytokine profile resulted in the gut, along with increased numbers of activated dendritic cells, eosinophils, and mast cells. Supplementation of the formula with TGF-beta2 down regulated the proinflammatory cytokine mRNA as well as the number of activated lymphocytes, eosinophils, mast cells, CD80, and CD86 positive dendritic cells. CONCLUSION: The data suggests an important role for maternal milk, in regulating immune responses after exposure to food antigens, which might otherwise induce deleterious immune responses in the intestine of suckling neonates. This regulation is potentially mediated by milk TGF-beta2, as well as endogenous IL-18.

Immune modulation in suckling rat pups by a growth factor extract derived from milk whey.

Oral tolerance to foreign enteral antigens is not fully developed in early neonatal life. Epidemiological evidence supports a role for maternal milk in the development of immune responses, including oral tolerance. Formula fed infants have an increased susceptibility to food allergy and the later development of autoimmune disease. This may relate to the lack in infant formula of growth factors found in maternal milk. Bovine milk contains proteins, growth factors and cytokines. Various studies have outlined the immune modulating potential of bovine milk-derived products. Fractionated whey extracts have therapeutic potential in disease states where there is an excessive inflammatory reaction, and disease preventive potential for infants who are not breast-fed. We have shown that daily oral administration of a growth factor-enriched fraction from milk whey to naturally suckling rat pups between days 4-9 postnatal can down-regulate immune activation to a specific orally administered food antigen, ovalbumin, assessed by lymphocyte proliferation. In addition, non-specific down-regulation in the intestine was observed as assessed by the expression of MHC I. Treatment of rat pups with whey extract at the time of oral sensitisation to ovalbumin also resulted in an increased secretion of TGF-beta into the culture supernatant of spleen cells incubated with specific antigen. TGF-beta is an immuno-down-regulatory cytokine involved in tolerance induction. Immune modulation by extracts derived from milk whey could be of potential benefit for formula-fed and pre-term infants in reducing susceptibility to inappropriate activation to food antigens.

Localization of transforming growth factor-beta receptor types I, II, and III in the postnatal rat small intestine.

Transforming growth factor-beta2 (TGF-beta2) levels in rat milk are high in early lactation, whereas endogenous TGF-beta1 expression in the neonatal gut increases toward midweaning. Three types of transmembrane TGF-beta receptors have been identified in mammals. The receptor III (or betaglycan) binds and presents TGF-beta1 or beta2 to receptor II. Receptor I then interacts with receptor II, forming a signaling receptor complex, and propagates the signal. To determine whether TGF-beta receptor expression in the gut is also developmentally regulated, the present study assessed ontogeny of TGF-beta receptor expression in the postnatal rat small intestine. Jejunum and ileum tissues from rat pups at d 3, 10, 14, 21, and 28 of age were collected. Cryostat sections were stained with antibodies against TGF-bea receptors I, II, and III, and various cell markers by immunofluorescence. In both regions, receptor I staining was seen on apical and basolateral membranes of the villus and crypt epithelium at all ages, and staining on the apical membrane increased with age; receptor II was predominantly expressed in the crypt, and staining on the villi appeared after d 10; receptor III was distributed throughout the mucosa at early ages but diminished from the epithelium postweaning by d 28. T cells, B cells, and dendritic cells in the lamina propria expressed TGF-beta receptor III but lacked expression of receptor I and II. The pattern of TGF-beta receptor expression changes with age in a manner that may reflect the change in ligand from TGF-beta2 (milk-derived) to TGF-beta1 (endogenously produced).

Transforming growth factor-beta levels in maternal milk and expression in postnatal rat duodenum and ileum.

After birth, the gastrointestinal tract of the neonate is exposed to food and bacterial and environmental antigens. Maternal milk components may play a role in regulation of mucosal immune activity to luminal antigens. In this study we determine the ontogeny of transforming growth factor (TGF)-beta1-producing cells in the rat pup small intestine and assess maternal milk concentrations of TGF-beta. Intestinal tissue samples of duodenum and ileum were collected, processed, and stained for TGF-beta1, and in situ hybridization for TGF-beta1 mRNA was also performed on the duodenum. TGF-beta levels in milk were assayed by ELISA. TGF-beta2 levels in milk were high at d 6, and declined thereafter at d 10 and 19. TGF-beta1 was not detected. In contrast, the cell number and intensity of staining of TGF-beta1 peptide in the small intestine was low in 3- and 10-d-old rats and increased markedly by 19 d of life. In the duodenum mRNA levels mirrored this trend. TGF-beta1 expression in the lamina propria was absent before d 19, and increased progressively over time. Maternal milk TGF-beta2 levels are high in early milk and decrease during the weaning period. In contrast, endogenous TGF-beta production in the small intestine increases during the weaning period.

Cytokine dysregulation in the post-Q-fever fatigue syndrome.

The post-Q-fever fatigue syndrome (QFS) (inappropriate fatigue, myalgia and arthralgia, night sweats, changes in mood and sleep patterns) follows about 20% of laboratory-proven, acute primary Q-fever cases. Cytokine dysregulation resulting from chronic immune stimulation and modulation by persistence of Coxiella burnetii cells or their antigens is hypothesized. We studied cytokine release patterns of peripheral blood mononuclear cells (PBMC) stimulated with various ligands in short-term culture, from 18 patients with active QFS, and 27 controls: six with resolving QFS, five who had had acute primary Q-fever without subsequent QFS, eight healthy Q-fever vaccinees and eight healthy subjects without Q-fever antibody. Conditioned media (CM) from PBMC stimulated in short-term culture with Q-fever antigens, PHA or measles antigen (as an unrelated antigen) were assayed for IL-2, IL-4, IL-5, IL-6, IL-10 and IFN gamma by AgEIA, and for IL-1 and TNF alpha/beta by bioassay. Aberrant cytokine release patterns were observed with PBMC from QFS patients when stimulated with Q-fever antigens: an accentuated release of IL-6 which was significantly [p = 0.01, non-parametric one-way analysis of variance (ANOVA)] in excess of medians for all four control groups. With IL-2, the number of responders in the active QFS group was decreased relative to control groups (Fisher's exact test, p = 0.01) whereas the number of IFN gamma responders was increased (Fisher's exact test, p = 0.0008). Significant correlations were observed between concentrations of IL-6 in CM, total symptom scores, and scores for other key symptoms.

Cell surface determinants on typhoid-specific B cells isolated from peripheral blood after oral Ty21a typhoid vaccination in humans.

Antigen specific B cells (ASC) that circulate after oral immunization with the typhoid vaccine Ty21a display cell surface determinants which are potentially involved in B cell differentiation and homing to mucosal sites. These ASC were isolated from peripheral blood after oral Ty21a, and dual labelled for binding of typhoid antigen and expression of various cell surface determinants: alpha 4 integrin (CD49d), CD45RO, CD45RA, L-selectin, CD44 and CD11a. Of particular interest was the finding of CD45RO expression on ASC. A comparison of cell surface determinants on typhoid-specific cells was also made following binding to high endothelial venules on peripheral and mesenteric lymph nodes, and venules in the lamina propria of the small intestine. Generally more typhoid ASC bound to mesenteric compared with peripheral lymph node. More ASC expressing CD45RO and alpha 4 integrin (CD49d) were bound to mesenteric lymph node and small intestine than to peripheral lymph node. When expressed as a fraction of total ASC, the difference was statistically significant only for CD45RO binding to small intestine versus peripheral lymph node. No differences in expression of other homing markers on bound ASC were seen.

Antigen-specific B cells in tissues after oral typhoid vaccination.

Six human subjects who were to receive elective bowel surgery for a variety of diseases were vaccinated with the oral typhoid vaccine, Ty21a. Intestinal tissue (ileum in two, large intestine in four) removed 7-26 days after the first dose of vaccine was examined for the presence and distribution of antigen-specific B cells. This was compared with intestinal tissue derived from two unvaccinated controls. A number of B cell differentiation antigens were also assessed on these cells by immunofluorescence using dual-labelling. Antigen-specific cells were found randomly distributed in the lamina propria of all the vaccinated subjects in low frequency (6 +/- 0.5 to 37 +/- 31 [mean +/- s.e.m.] antigen specific cells/10 mm2 of tissue). The lymphocyte differentiation antigens CD45RA, CD45RO, L-selectin, CD-11a CD-38, CD-44 and VLA-4 were all found on antigen-specific cells, but no particular pattern was recognizable in this small series of six subjects with different disease processes affecting the intestine.

Dual staining of lymphocyte membrane antigens with colloidal gold and biotinylated horseradish peroxidase.

Double immunoenzymatic labelling procedures for the localization of antigens on cells in tissue sections using horseradish peroxidase (HRP) and alkaline phosphatase have been described previously, but mainly for detecting antigens on different cells. With this type of staining when two antigens are present on the same cell, an optimal colour combination that shows a high contrast between the basic colour of each enzyme substrate product is difficult to achieve and the interpretation of their mixed colour intermediate is subjective. We present a method for the simultaneous demonstration of two antigens on the same cell. The method can be used to label either single cells in suspension, or cells in paraffin fixed tissue, using a combination of a particulate label, colloidal immunogold-silver, and an enzymatic label HRP-DAB. The method is easy to perform and utilises commercially available staining kits.

Cellular and humoral responses in coeliac disease. 2. Protein extracts from different cereals.

The humoral and cellular immune responses to grain protein extracts from coeliac-toxic and non-toxic cereals were compared by use of a number of ELISA and immunoblotting methods and the indirect leucocyte migration inhibition factor (LMIF) assay. Both adult and child coeliacs had elevated levels of serum antibody to proteins from the coeliac-toxic cereals, namely bread wheat, durum wheat, rye and barley and low levels of proteins from other cereals. Using protein blotting techniques, antibody binding was greatest to gliadins/low mol mass glutenin subunits and homologous prolamins from rye and barley, consistent with the ELISA findings. Competition ELISA and preabsorption tests indicated that antibody reaction to maize storage proteins did not simply result from cross-reaction of antigliadin antibodies. In LMIF assays, only the wheat extracts had activity in coeliac patients. This is most likely partly due to loss of some of T-cell epitopes from the extraction technique required for these proteins, as well as the relatively small effects seen for even very active fractions in the LMIF assay.

Cellular and humoral responses in coeliac disease. 1. Wheat protein fractions.

The humoral and cellular immune response of coeliac individuals to various wheat protein fractions was studied using serum antibody ELISA assays and the indirect leucocyte migration inhibition factor (LMIF) assays. Greater migration inhibition factor activity was seen in coeliacs on a gluten-free-diet having low serum antibody titres, and using purified T-cells instead of total peripheral blood mononucleocytes. Gliadin was the most active fraction in both assays. Raised antibodies to low-molecular weight and high-molecular weight glutenin polypeptides was observed, though these proteins had little migration inhibition factor activity. No cellular or humoral response was seen to albumins or globulins. Proteins associated with the granules of well-washed wheat starch are distinct from gluten proteins and had little T-cell activity, correlating with clinical observations that properly prepared wheat starch is devoid of coeliac toxicity. The greater specificity of the humoral response for individual wheat protein fractions in this study, compared with the earlier reports, likely results from cross-contamination in the earlier work of each fraction with gliadin.

Phenotype and activation of milk-derived and peripheral blood lymphocytes from normal and coeliac subjects.

The phenotype of milk-derived and peripheral blood lymphocytes from normal and coeliac subjects was assessed for CD3, alpha beta-TcR (T cell receptor), gamma delta-TcR, CD4, CD8, HML-1 (human mucosal lymphocyte) determinants, and activation was measured by interleukin-2 receptor (IL-2R) expression. Milk cells from normal and coeliac subjects were analysed by manual immunofluorescence and milk and blood cells from normal subjects were analysed by flow cytometry. Milk cells from two coeliac subjects were tested for proliferation to gluten antigen. The CD4:CD8 ratio of milk lymphocytes from both normal and coeliac subjects was similar (0.78-1.1), but lower than that present in blood (1.5-2.1). The IL-2R expression of milk lymphocytes from both coeliac and normal subjects was increased by 3-6 times compared with peripheral blood cells. For example, IL-2R was present on 27.3% of milk CD3+ lymphocytes and on 8.0% of blood CD3+ lymphocytes from normal subjects. gamma delta- and HML-1+ T cells were increased 4.2-fold and 12-fold respectively compared with blood lymphocytes. Milk cells from coeliac subjects showed specific proliferation to gluten but not to soya bean antigen. We conclude that milk cells have a 'mucosal' phenotype, with increased gamma delta-TcR, CD8+ and HML-1+ T cells, and have an increased proportion of activated cells. Milk cells from coeliac subjects have no 'toxic' phenotype, but show functional reactivity by specific proliferation to gluten antigen.

Recovery of the small intestine in coeliac disease on a gluten-free diet: changes in intestinal permeability, small bowel morphology and T-cell activity.

Intestinal permeability was assessed before and 1, 2, 4, 8 and 12 weeks after commencing a gluten-free diet (GFD) in eight coeliac subjects. Intestinal morphology was quantified in six coeliac subjects on a normal diet, six coeliac subjects on a GFD, and 21 normal subjects. T-cell activity was measured in the eight coeliac subjects by soluble interleukin-2 receptor (sIL-2R) concentration (normal less than 477 U/mL). Intestinal permeability was increased 10-fold with a geometric mean value of 0.72 on a normal diet, and decreased to 0.17 at 4 weeks (P = 0.04), to 0.07 at 8 weeks (P = 0.010), and to 0.20 at 12 weeks (P = 0.015) of a GFD. Two of the eight subjects showed a poor response to gluten withdrawal. Quantitative intestinal morphology showed no significant improvement after 3 to 6 months of a GFD. Mean +/- s.d. sIL-2R concentrations in the eight subjects were increased 5-fold higher than control values at 1400 +/- 530 U/mL on a normal diet and decreased to 750 +/- 200 U/mL after 12 weeks of a GFD (P = 0.004). We conclude that intestinal permeability improves rapidly in the majority of coeliac subjects after commencing a GFD, although some abnormal permeability and increased T-cell activity persists. This may be due to varying degrees of gluten ingestion resulting in continued immune activation.

Lymphocyte activation as measured by interleukin-2 receptor expression to gluten fraction 111 in coeliac disease.

Lymphocyte activation was examined by interleukin-2 (IL-2) receptor expression on peripheral blood mononuclear cells from coeliac and control subjects. Purified T cells were incubated with gluten fraction 111 (a known toxic peptide for coeliac subjects), soyabean hydrolysate (an unrelated hydrolysed food antigen), and Concanavalin-A (Con-A, a non-specific mitogen). After 1-5 days incubation, expression of IL-2 receptors was assessed using a cellular enzyme-linked immunosorbent assay (ELISA). Gluten fraction 111 induced expression of IL-2 receptors on T lymphocytes from coeliac but not from normal subjects (P = 0.0005), whereas soyabean hydrolysate did not induce IL-2 receptor expression. Lymphocytes from both coeliac and normal subjects had similar increased IL-2 receptor expression after incubation with Con-A. Flow cytometry was also used to confirm specific expression of IL-2 receptor expression of lymphocytes from coeliac subjects. Interleukin-2 receptor expression increased from 0 to 5.4% of cultured mononuclear cells after 7 days incubation with gluten fraction III. These cells were CD3-positive and CD4-positive. We conclude that peripheral blood lymphocytes from coeliac subjects are sensitized specifically to gluten fraction III.

Infection of mice with Nematospiroides dubius: demonstration of neutrophil-mediated immunity in vivo in the presence of antibodies.

The ability of murine neutrophils to confer resistance to mice against infection by the helminth parasite Nematospiroides dubius has been investigated. Mice whose neutrophils had been 'altered' by an immunizing infection with third-stage larvae (L3) of N. dubius exhibited resistance to a challenge dose of L3 given 4 days after the immunizing infection, provided greater than 0.1 ml of immune mouse serum was passively transferred within 0-24 hr of challenge. Normal mouse serum was ineffective, as was immune serum given at the time of challenge to naive (unimmunized) mice. Neutrophils purified from the blood of mice infected 4 days previously were able to reduce the infectivity of exsheathed L3 when incubated with the latter in vitro in the presence of fresh immune serum. In contrast, peritoneal exudate cells collected at the same time were inactive in this test, indicating that activated macrophages capable of damaging L3 were not yet present and that the immunity of 4-day infected mice given immune serum was probably attributable to the presence of 'altered' neutrophils. The capacity of neutrophils to confer resistance to infection in vivo was unambiguously demonstrated by the passive transfer of 98 +/- 4% pure neutrophils, isolated from the blood of 4-day infected (C57Bl X BALB/c)F1 mice, to uninfected F1 mice. Only those mice which received both neutrophils and immune serum exhibited resistance to a challenge infection. In contrast, mice injected with an eosinophil-enriched cell preparation and immune serum were not resistant. These results indicate for the first time that neutrophils have the capacity to damage nematode parasites in vivo and that they are active against N. dubius in mice infected with this parasite.

Reduced infectivity of Nematospiroides dubius larvae after incubation in vitro with neutrophils or eosinophils from infected mice and a lack of effect by neutrophils from normal mice.

Neutrophils and eosinophils, isolated from the blood of mice infected with Nematospiroides dubius, were tested for their capacity to damage exsheathed third stage N. dubius larvae in vitro. In the presence of fresh serum from infected mice, both types of granulocyte caused a significant reduction in larval infectivity (up to 40-50%) whereas lymphocytes/monocytes prepared from the same blood samples were inactive. Neutrophils were at least as active as eosinophils, on a cell for cell basis. None of the cells exhibited larvicidal activity in the absence of serum and serum alone had no effect. The reduction in larval infectivity caused by neutrophils in the presence of fresh normal mouse serum (NMS) was only marginally less than that obtained using immune mouse serum (IMS), suggesting that complement, which is activated by the larvae via the alternative pathway and mediates the adherence of both cell types, was able to promote the larvicidal effect of these cells in vitro. In contrast to neutrophils, eosinophils were considerably less effective in NMS than in IMS. Both NMS and IMS were ineffective if they had been heat-inactivated or incubated with methylamine at pH 8.0 to destroy complement activity. The immunoglobulin fraction of IMS was also ineffective in promoting neutrophil or eosinophil-mediated larval damage. These results indicate that in this in vitro system antibodies are incapable of directing the activity of either cell type in the absence of complement. A novel finding of this study was that neutrophils from uninfected mice were unable to reduce larval infectivity in the presence of fresh NMS or IMS. 'Altered' neutrophils possessing larvicidal activity appeared in the blood of mice within 4 days of infection with N. dubius.

Adherence of murine peripheral blood eosinophils and neutrophils to the different parasitic stages of Nematospiroides dubius.

Eosinophils and neutrophils, purified by density gradient centrifugation from the blood of infected mice resistant to reinfection, were tested for their ability to adhere to the different parasitic larval stages of the murine nematode parasite Nematospiroides dubius. Cells were tested for adherence to larvae which had been sensitised with immune mouse serum (IMS) or normal mouse serum (NMS) in the presence of CA2+ and Mg2+ ions. EDTA, or EGTA. Differences were observed in the degree of cell adherence to the different stages of the parasite. However, the adherence of the two cell types to any given stage of the parasite was similar. Adherence to the sheathed infective third-stage (L3) larvae, 96 h post-infective larvae and to adult worms depended to a large degree on conditions suitable for complement activation (viz. fresh serum and the presence of Ca2+ and Mg2+ ions). Complement was activated both via the alternative pathway by the parasite itself and via the classical pathway by parasite-bound antibodies. In these conditions, cell adherence probably occurred predominantly through the interaction of leucocyte third component of complement (C3) receptors with parasite-bound C3. In contrast, adherence of cells to exsheathed L3 and to the 48 h and 72 h post-infective larval stages appeared to involve antibody/Fc receptor as well as C3/C3 receptor interaction. The data indicate that N. dubius may undergo a series of antigenic changes during its life cycle and that antibodies capable of mediating granulocyte attachment are elicited predominantly against the early tissue developmental forms of the parasite.