Partially hydrolyzed whey proteins prevent clinical symptoms in a cow's milk allergy mouse model and enhance regulatory T and B cell frequencies.

SCOPE: Partially hydrolyzed cow's milk proteins are used to prevent cow's milk allergy in children. Here we studied the immunomodulatory mechanisms of partial cow's milk hydrolysates in vivo. METHODS AND RESULTS: Mice were sensitized with whey or partially hydrolyzed whey using cholera toxin. Whey-specific IgE levels were measured to determine sensitization and immune cell populations from spleen, mesenteric lymph nodes and Peyer's patches after oral whey administration were measured by flowcytometry. Whey-specific IgE and IgG1 levels in partial whey hydrolysate sensitized animals were enhanced, but challenge did not induce clinical symptoms. This immunomodulatory effect of partial whey hydrolysate was associated with increased regulatory B and T cells in the spleen, together with a prevention of IgM-IgA class switching in the mesenteric lymph nodes and an increased Th1 and activated Th17 in the Peyer's patches. CONCLUSION: Partial hydrolysate sensitization did not induce whey-induced clinical symptoms, even though sensitization was established. Increased regulatory cell populations in the systemic immune system and a prevention of increased total Th1 and activated Th17 in the intestinal immune organs could contribute to the suppression of allergic symptoms. This knowledge is important for a better understanding of the beneficial effects of hydrolysates.

Immunohistochemical detection of disease-associated prion protein in the intestine of cattle naturally affected with bovine spongiform encephalopathy by using an alkaline-based chemical antigen retrieval method.

An alkaline-based chemical antigen retrieval pretreatment step was used to enhance immunolabeling of disease-associated prion protein (PrP(Sc)) in formalin-fixed and paraffin-embedded tissue sections from cattle naturally affected with bovine spongiform encephalopathy (BSE). The modified chemical method used in this study amplified the PrP(Sc) signal by unmasking PrP(Sc) compared with the normal cellular prion protein. In addition, this method reduced nonspecific background immunolabeling that resulted from the destruction of the residual normal cellular form of prion protein, and reduced the treatment time compared with the usual autoclave pretreatment step. Immunolabeled PrP(Sc) was thereby clearly detected in the myenteric plexus of the ileum in naturally occurring BSE cattle.

The antiallergic effects and acute toxicity of Lactobacillus crispatus KT-11 cultured in food grade medium.

We synthesized a medium consisting of commercial food supplements (food grade medium) that could be used to cultivate Lactobacillus crispatus KT-11 (KT-11), and investigated the antiallergic effects and acute toxicity of KT-11 cultured in this medium. We found that the growth of KT-11 in the food grade medium was comparable to that in DeMan-Rogosa-Sharpe (MRS) medium. Sneezing event was reduced in ovalbumin (OVA)-sensitized BALB/c mice given a diet supplemented with KT-11 grown in the food grade medium (FG-KT-11 group) when compared to mice given a diet supplemented with KT-11 grown in MRS medium (MRS-KT-11 group). The number of CD80(+)CD11b(+) Peyer's patch cells was significantly lower in the FG-KT-11 group than in the MRS-KT-11 group, while IL-12(+)CD11b(+) Peyer's patch cells were higher in the FG-KT-11 group. Only minimal acute toxicity was observed in ICR mice given 1000 or 2000 mg of FG-KT-11/kg body weight. These results suggest that FG-KT-11 represents a safe antiallergic food material.

Effects of glutamine added to enteral nutrition on Peyer's patch apoptosis in severely burned mice.

BACKGROUND AND AIMS: This study aimed to investigate the influence of enteral glutamine (GLN) supplementation on Peyer's patch apoptosis in severely burned mice. METHODS: Thirty-four mice were randomly assigned to a normal group (n=10), an EN group (n=12) and an EN supplemented with GLN (EN+GLN) group (n=12) and the mice in the EN and EN+GLN groups received a full-thickness scald burn over 20% total body surface area (TBSA) on the back. The burned mice then were fed orally with a common EN or an isonitrogenous and isocaloric EN supplemented with GLN for 7 days. On day 7 after injury, all surviving mice were euthanised and the entire intestine was collected. The percentage of apoptotic cells and cell percentage of phenotype in Peyer's patches were determined by flow cytometry (FCM). The FasL expression in Peyer's patches was analysed by reverse transcription polymer chain reaction (RT-PCR) and FCM. Both TNF-alpha levels and caspase-3 activity in Peyer's patches were also assessed. RESULTS: The results revealed that the percentage of lymphocyte subsets in Peyer's patches after burn injury significantly altered: the percentage of CD4 and CD19 cells declined and the percentage of CD8 cells correspondingly increased, when compared with the normal control mice (p<0.05). On the other hand, the total apoptotic ratio and all lymphocytes subset apoptosis in Peyer's patches were markedly increased (p<0.05), which were consistent with up-regulation in the FasL expression at the levels of both mRNA and protein, TNF-alpha levels and caspase-3 activity in Peyer's patches. Enteral GLN supplementation partially reversed these changes: the total apoptotic ratio and all lymphocytes subpopulation apoptosis in Peyer's patches were markedly decreased when compared with the EN group (p<0.05). The percentage of lymphocyte subsets within Peyer's patches also restored the condition prior to injury. However, no significant differences in the FasL expression, including mRNA and protein, were observed between the EN and EN+GLN groups. Although, both TNF-alpha levels and caspase-3 activity in Peyer's patches were lower in the EN+GLN group than in the EN group (p<0.05). CONCLUSIONS: This study suggests that enteral GLN supplementation is superior to a common enteral nutrition with respect to attenuating apoptosis in Peyer's patches, which might be more effective in decreasing TNF-alpha levels and down-regulating caspase-3 activity in Peyer's patches.

Morphological changes and virus distribution in the ileum of colostrum-deprived calves inoculated with non-cytopathic bovine viral diarrhoea virus genotype-1.

Eight colostrum-deprived calves were inoculated intranasally with a non-cytopathic strain of bovine viral diarrhoea virus (BVDV) genotype-1 and killed in batches of two at 3, 6, 9 and 14 days post-inoculation (dpi). Two non-inoculated animals with similar background served as controls. All infected calves developed mild pyrexia and transient leucopenia due primarily to lymphopenia. Viraemia was correlated with body temperature and inversely related to leucocyte count. Ileal Peyer's patches developed mild follicular lymphoid depletion from 3dpi. This change was accompanied by cellular fragmentation and pyknosis, characteristic of apoptosis, which was most prominent from 6dpi. Lymphocyte apoptosis was confirmed by ultrastructural examination. Stellate cells and macrophages located in the lymphoid follicles were identified as infected by virus from 3dpi and the number of these infected cells increased until 9dpi. Fewer lymphocytes expressed BVDV antigen. Macrophages had morphological features consistent with activation of secretory and phagocytic function from 3dpi. These findings suggest that BVDV is only directly responsible for the destruction of a small number of lymphocytes. Although lymphocyte infection coincided with the onset of apoptosis, the intensity of infection was disproportionate to the marked depletion of gut-associated lymphoid tissue, particularly during the early stages of this process. Characterization of the indirect pathogenic mechanisms involved in the lymphoid depletion associated with BVDV infection will require additional study.

Bioadhesive capacity and immunoadjuvant properties of thiamine-coated nanoparticles.

The general aim of this work was to develop polymeric nanoparticle carriers with bioadhesive properties, and to evaluate its adjuvant potential for oral vaccination. Thiamine was used as specific ligand-nanoparticle conjugate (TNP) to target specific sites within the gastrointestinal tract, enterocytes and Peyer's patches. The affinity of nanoparticles to the gut mucosa was studied in orally inoculated rats. In contrast to conventional non-coated nanoparticles (NP), higher levels of TNP were found in the ileum tissue, showing a strong capacity to be captured by Peyer's patches. TNP were characterized by an AUCadh which was found to be three times higher than for control NP. To investigate the adjuvant capacity of TNP, ovalbumin (OVA) was used as standard antigen. Oral immunization of BALB/c mice with OVA-TNP induced stronger serum titers of specific IgG2a and IgG1 and mucosal IgA compared to OVA-NP. This mucosal immune response (IgA) was about 4-titers higher than that elicited by OVA-NP. These results suggest the use of thiamine-coated nanoparticles as particle vectors for oral vaccine and immunotherapy delivery strategies.

[Effects of Huoxiang Zhengqi liquid on enteric mucosal immune responses in mice with Bacillus dysenteriae and Salmonella typhimurium induced diarrhea].

OBJECTIVE: To explore the effects of Huoxiang Zhengqi liquid (HXZQ) on enteric mucosal immune responses in mice with Bacillus dysenteriae and Salmonella typhimurium induced diarrhea (BSD). METHOD: Mice were randomly divided into four groups with 10 mice in each group: control group (control), BSD group, Huoxiang Zhengqi liquid treated BSD groups at high dosage and low dosage (HXZQ high, HXZQ low). HXZQ was administrated from the day of diarrhea induction at dosage of 5.21 g kg(-1) and 0.52 g kg (-1) respectively. Peyer's patch and periphery lymphocytes were prepared for flow cytometry, and level of TNF-alpha in periphery and enteric tissue homogenate were determined with ELISA. Student's t-test was used for statistics. RESULT: Mice in BSD group started showing continuous diarrhea at the day of induction till the fourth day when the mice were sacrificed. Diarrhea in the mice of HXZQ high and low groups lasted for 36 and 54 h respectively. There were more CD4+ and CD8+ cells in periphery, less CD4+ cells in peyer's patch in BSD mice comparing to normal mice. In peyer's patch, there were more CD8+ cells in mice in HXZQ high and low groups and more CD4+ in mice in HXZQ high group. Higher level TNF-alpha in periphery and intestinal tissue homogenate in BSD group were observed. Mice in HXZQ high group showed the decreased level TNF-alpha in periphery and enteric tissue homogenate. CONCLUSION: The immune regulation on peyer's patch CD4+ and CD8+ cells and suppression on TNF-alpha level in enteric homogenate might partially explain the effect of HXZQ on improvement of BSD.

Effects of dexamethasone on lymphoid tissue in the gut and thymus of neonatal calves fed with colostrum or milk replacer.

An increased susceptibility to disease in neonatal calves may be attributable to high glucocorticoid levels that influence immune reactions. We tested whether dexamethasone (DEXA) administration influences the proliferation, apoptosis, and number of B- and T-lymphocytes in Peyer's patches (PP) and thymus in calves fed colostrum (C) or a milk-derived formula. All calves were subcutaneously administered bovine colostrum-derived immunoglobulin G and fed chicken-egg derived immunoglobulins that protected against rotavirus and pathogenic Escherichia coli. The DEXA (30 microg/kg of BW daily) was injected for 4 d into groups fed colostrum on the first 3 d (CD+) and those fed the formula that contained nutrients in amounts as in colostrum but no immunoglubulin G (FD+). Groups CD- and FD were fed the same as the other two groups, but did not receive DEXA. Immunohistochemical methods were used to evaluate cell proliferation rates (by labeling of 5-bromo-2'-deoxyuridine), apoptosis rates (by terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling). Numbers of T- and B-lymphocytes were determined with antibodies specific for CD3 and CD79 surface proteins. There were significant effects (P < 0.05) of DEXA treatment (decrease of cell proliferation rates in follicles of PP and thymus, increase of apoptotic rate in follicles of PP and thymus, decrease of B-lymphocyte numbers in follicles of PP, increase of B-lymphocyte numbers in domes of PP, increase of T-lymphocyte numbers in follicles of PP, and a decrease of intraepithelial T-lymphocyte numbers). There were significant effects (P < 0.05) of C feeding (decrease of cell proliferation rates in follicles of PP and of B-lymphocyte numbers in interfollicular areas, domes, and follicular-associated epithelium of PP, and an increase of cell proliferation rate in the thymus). A DEXA x feeding interaction (P < 0.001) was found for cell proliferation rate in the thymus. In conclusion, DEXA treatment decreased cell proliferation rates in follicles of PP and thymus and enhanced apoptotic rates in follicles of PP. Colostrum feeding decreased cell proliferation rates, likely of B-lymphocytes, in follicles of PP and numbers of B-lymphocytes in domes, follicular-associated epithelium, and interfollicular areas of PP and enhanced cell proliferation rates and selectively modified DEXA effects in the thymus.

Salmonella serotype Typhimurium infection of bovine Peyer's patches down-regulates plasma membrane calcium-transporting ATPase expression.

To identify host genes differentially expressed during Salmonella enterica serotype Typhimurium infection, an RNA differential display was made with total RNA extracted from ileal loops that were infected with Salmonella Typhimurium 2.5 h after infection. Down-regulated cDNA was identified in bovine Peyer's patches after infection that was highly homologous to a human plasma membrane calcium-transporting ATPase (PMCA). Differential expression of PMCA, evaluated by Northern analysis, was found to have more than a 4.6-fold decrease in expression of mRNA (size, approximately 5.1 kb). PMCA mRNA was detected by in situ hybridization exclusively within epithelial cells in the Peyer's patches. cDNA (4.4 kb) was amplified by rapid amplification of cDNA ends, cloned, and sequenced and showed a high homology to hPMCA. Bovine PMCA is down-regulated in epithelial cells of Peyer's patches after infection with Salmonella Typhimurium and, subsequently, may influence cellular calcium levels that contribute to the inflammatory processes in the pathogenesis of diarrhea.

Effect of black tea on azoxymethane-induced colon cancer.

Two sets of experiments on the role of tea in azoxymethane (AOM) induced colon cancer were performed. The first test involved male F344 rats given 1.25% solutions of black tea beginning at 5 weeks of age and ending at 51 days of age. At 6 and 7 weeks of age, they received 15 mg/kg AOM and were held for 50 weeks. Another group received the AOM dosage at 6 and 7 weeks and were placed on the tea solutions 2 days after the last AOM dosage, at 51 days of age, and held for the 50-week period. The end point was the occurrence and multiplicity of colon cancer, classified as in situ, exophytic, invasive and Peyer's patch carcinomas. Tea failed to affect the incidence and multiplicity of colon cancers when given during or after the AOM administration, but tea after AOM increased the multiplicity of exophytic carcinomas. In a second series of tests, solutions of 0.6, 1.25, 1.75 or 2.5% tea were given, beginning 1 week prior to the two AOM doses and extending for 42 weeks. Also, one group received 1.25% tea and 1.85% whole milk. The incidence of exophytic or invasive colon cancer and tumor multiplicity were similar in all treatment groups, although the incidence of exophytic neoplasms was higher with 2.5% tea. Thus, chronic administration failed to significantly change the incidence and multiplicity of the AOM-induced colon cancers. These findings are accounted for by the underlying mechanism, namely the fact that tea solutions do not alter the amount of cytochrome P-4502E1 required for the metabolic activation of AOM.

Glycyl-L-glutamine-enriched total parenteral nutrition maintains small intestine gut-associated lymphoid tissue and upper respiratory tract immunity.

BACKGROUND: i.v. administration of a total parenteral nutrition (TPN) solution results in small intestinal gut-associated lymphoid tissue (GALT) atrophy, lowers small intestinal immunoglobulin A (IgA) levels, and impairs upper respiratory tract secretory IgA-mediated mucosal immunity; isonitrogenous supplementation of TPN with 2% glutamine attenuates these changes. This experiment examines whether a 2% glycyl-L-glutamine-enriched TPN solution reverses i.v. TPN-induced changes as effectively as L-glutamine. METHODS: Male Institute of Cancer Research (ICR) mice underwent intranasal inoculation with H1N1 influenza virus to establish immunity. After 3 weeks, mice were randomized to chow, i.v. feeding of a TPN solution, glutamine-enriched TPN, or glycyl-L-glutamine-enriched TPN. After 4 days of feeding, mice were challenged intranasally with influenza virus and killed at 40 hours to determine viral shedding from the respiratory tract; normal convalescent mice do not shed virus because they possess intact IgA-mediated mechanisms Lymphocytes were isolated from Peyer's patches, the intraepithelial layer, and lamina propria to determine cell yields. RESULTS: Total lymphocyte yield in the Peyer's patches, the intraepithelial layer, and lamina propria decreased with TPN but remained normal with glutamine and glycyl-L-glutamine. Upon challenge, 70% of the mice in the TPN group shed virus in nasal secretions, whereas only 20% of the glutamine-treated group, 18% of glycyl-L-glutamine group and none of the Chow group were virus positive. CONCLUSIONS: L-Glutamine and glycyl-L-glutamine have similar effects on i.v. administered TPN-associated (GALT) atrophy and decreased upper respiratory tract immunity.

[Intestinal invagination in childhood: our experience]. / Invaginazione intestinale in età pediatrica: esperienza personale.

The Authors report their experience in the treatment of twenty-one patients with intussusception operated in the Paediatric Surgery Division ASL FG/3 between January 1988 and December 1994. Eighty-nine percent of the patients were between 2 and 12 months of age, with a peak between 2 and 6 months (60%). Ultrasound allowed to identify the pathological picture in forty-two percent of the cases and diagnosis was confirmed by barium enema. In 8 patients a pathology more or less responsible of the intussusception or associated with it was detected. The time between the onset of symptoms and admission as well as the relation between intestinal resection and duration of symptomatology were analyzed. The resection rate was maximal (55%) in those patients presenting symptoms beyond 25 hours. Intestinal resection was performed in 9 patients (47%): 6 ileo-colic; 1 ileo-colic resection extended to the splenic flexure; and 2 ileo-ileal ones. In this series no postoperative complications or recurrences or deaths were registered. Long term results proved that ileo-colic resection, even in few-months old infants, is well tolerated.

Intussusception revisited: clinicopathologic analysis of 261 cases, with emphasis on pathogenesis.

In the ten-year period from 1978 through 1987, 261 patients with intussusception were admitted to Chang Gung Memorial Hospital. The diagnosis was established by barium enema or at laparotomy. The patients were divided into two groups; there were 228 children ranging in age from 1 month to 14 years, and 33 adults. Among the children, 134 (59%) were male and 94 (41%) were female, a ratio of 1.4:1. There was no clear seasonal incidence. The age group most commonly affected was between 3 and 11 months of age (72.4%). The classic triad of abdominal pain, vomiting, and rectal bleeding was encountered in 187 cases (82%). Two hundred one cases (88%) were idiopathic, without any definite leading point. In these cases, the ileocecal area was the site most commonly involved (82%), hypertrophic Peyer's patches of the terminal ileum being responsible for 39% of the idiopathic intussusceptions in the ileocolic area. Enlargement of the mesenteric lymph nodes occurred in 67 of the idiopathic cases (33%). Local pathology or the leading point precipitating intussusception was found in 27 cases (12%); there were eight benign tumors, six malignant tumors, and 13 tumor-like lesions. In 32 of the 33 cases in adults, there was a definite contributing pathologic entity, including 18 benign tumors, 11 malignant tumors, and three tumor-like lesions. In infants and young children, there is usually no apparent predisposing disease, and a contributing or causative local pathologic lesion is seldom found. In contrast, intussusception in adults is almost invariably caused by some preexisting lesion involving the bowel wall. Furthermore, trauma, lymphoid hyperplasia, pregnancy, and viral infection may be possible predisposing factors in the production of intussusception.