Neutral detergent-soluble fiber improves gut barrier function in twenty-five-day-old weaned rabbits.

The effect of neutral detergent-soluble fiber level on gut barrier function and intestinal microbiota was examined in weaned rabbits. A control diet (AH) containing 103 g of neutral detergent-soluble fiber/ kg of DM included alfalfa hay as main source of fiber. Another diet (B-AP) was formulated by replacing half of the alfalfa hay with a mixture of beet and apple pulp resulting in 131 g of soluble fiber/kg of DM. A third diet (OH) was obtained by substituting half of the alfalfa hay with a mix of oat hulls and a soybean protein concentrate and contained 79 g of soluble fiber/kg of DM. Rabbits weaned at 25 d and slaughtered at 35 d were used to determine ileal digestibility, jejunal morphology, sucrase activity, lamina propria lymphocytes, and intestinal microbiota. Suckling 35-d-old rabbits were used to assess mucosa morphology. Mortality (from weaning to 63 d of age) was also determined. Villous height of the jejunal mucosa increased with soluble fiber (P = 0.001). Rabbits fed with the greatest level of soluble fiber (BA-P diet) showed the highest villous height/ crypt depth ratio (8.14; P = 0.001), sucrase specific activity (8,671 mumol of glucose/g of protein; P = 0.019), and the greatest ileal starch digestibility (96.8%; P = 0.002). The opposite effects were observed in rabbits fed decreased levels of soluble fiber (AH and OH diets; 4.70, 5,848 mumol of glucose/g of protein, as average, respectively). The lowest ileal starch digestibility was detected for animals fed OH diet (93.2%). Suckling rabbits of the same age showed a lower villous height/crypt depth ratio (6.70) compared with the B-AP diet group, but this ratio was higher than the AH or OH diet groups. Lower levels of soluble fiber tended (P = 0.074) to increase the cellular immune response (CD8+ lymphocytes). Diet affected IL-2 production (CD25+, P = 0.029; CD5+CD25+, P = 0.057), with no clear relationship between soluble fiber and IL-2. The intestinal microbiota biodiversity was not affected by diets (P >/= 0.38). Rabbits fed the B-AP and AH diets had a reduced cecal frequency of detection compatible with Campylobacter spp. (20.3 vs. 37.8, P = 0.074), and Clostridium perfringens (4.3 vs. 17.6%, P = 0.047), compared with the OH diet group. Moreover, the mortality rates decreased from 14.4 (OH diet) to 5.1% (B-AP diet) with the increased presence of soluble fiber in the diet. In conclusion, increased levels of dietary soluble fiber improve mucosal integrity and functionality.

[Antineutrophil cytoplasmic antibodies in nonvasculitis diseases: clinical correlates and target antigens]. / Anticuerpos anticitoplasma de neutrófilo en enfermedades distintas a las vasculitis idiopáticas: correlaciones clínicas y especificidades antigénicas.

The value of antineutrophil cytoplasmic antibodies (ANCA) in the diagnosis of several types of idiopathic vasculitis has been well-documented: In these diseases the ANCA show two classical immunofluorescence patterns, C-ANCA and P-ANCA, which have antigen specificity for the myeloperoxidase and proteinase 3, respectively. However, the appearance of ANCA in very different diseases other than the mentioned vasculitis, has been documented in recent years. In these diseases, the ANCA generally have atypical immunofluorescence patterns and are directed against neutrophil antigens that are different from the previous two, their clinical value still being under debate.

Anticuerpos anticitoplasma de neutrófilo en enfermedades distintas a las vasculitis idiopáticas: correlaciones clínicas y especificidades antigénicas / Antineutrophil cytoplasmic antibodies in nonvasculitis diseases: clinical correlates and target antigens

Es bien conocido el valor de los anticuerpos anticitoplasma de neutrófilo (ANCA) en el diagnóstico de varios tipos de vasculitis idiopáticas. En estas enfermedades estos autoanticuerpos muestran dos patrones clásicos de inmunofluorescencia, C-ANCA y P-ANCA, que poseen, respectivamente, especificidad antigénica por la proteinasa 3 y la mieloperoxidasa. Sin embargo, en los últimos años se ha documentado la aparición de ANCA en muy diversas patologías distintas a las mencionadas vasculitis. En estas enfermedades los ANCA suelen mostrar patrones atípicos por inmunofluorescencia e ir dirigidos a antígenos neutrofílicos distintos de los dos anteriores, estando su valor clínico aún sujeto a debate (AU)

The value of antineutrophil cytoplasmic antibodies (ANCA) in the diagnosis of several types of idiopathic vasculitis has been well-documented: In these diseases the ANCA show two classical immunofluorescence patterns, C-ANCA and P-ANCA, which have antigen specificity for the myeloperoxidase and proteinase 3, respectively. However, the appearance of ANCA in very different diseases other than the mentioned vasculitis, has been documented in recent years. In these diseases, the ANCA generally have atypical immunofluorescence patterns and are directed against neutrophil antigens that are different from the previous two, their clinical value still being under debate (AU)

Beneficial effects of HLA class II incompatibility in living donor liver transplantation.

OBJECTIVE: To analyze the impact of HLA matching in both patient and graft evolution after LDLT. MATERIAL AND METHODS: Twenty recipients underwent LDLT with follow-up of 3 to 30 months. HLA typing was performed on all donor-recipient pairs; class I antigens were typed using serological methods and class II loci (DRB1 and DQB1) using low-resolution molecular typing. Recipient sera were cross-matched with donor lymphocytes. Antigen mismatches were analyzed for each locus individually, for each class as a whole and for HLA class I immunogenic triplets according to HLA Matchmaker software. RESULTS: Eighteen of 20 donor-recipient pairs were HLA haploidentical. All but one of the recipients had a negative cross-match before transplantation. While there was not a statistically significant correlation between HLA class I mismatches and the incidence of acute rejection episodes, HLA class II matching in DRB1 and DQB1 loci appeared to be associated with a higher incidence of acute rejection episodes after LDLT. Both host-versus-graft (HvG) and graft-versus-host (GvH) HLA class II compatibilities correlated with rejection episodes, especially for the GvH direction. CONCLUSIONS: HLA class II matching for DRB1 and DQB1 loci appears to be associated with a higher incidence of acute rejection episodes after LDLT. In this study, mismatches in class I HLA antigens are not related to an higher incidence of acute rejection episodes nor other complications after LDLT. Further studies are needed to unveil the role of HLA matching in LDLT.

Los linfocitos intraepiteliales en el diagnóstico de la enfermedad celíaca latente-potencial / Intraepithelial lymphocytes in the diagnosis of latent-potential celiac disease

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Anti-transglutaminase IgA ELISA: clinical potential and drawbacks in celiac disease diagnosis.

BACKGROUND: Since the identification of tissue transglutaminase (tTG) as the antigen for the anti-endomysial antibodies (EMA), several antigen-specific immunoassays have been reported for celiac disease (CD) screening. A first objective was to evaluate the suitability for CD screening of three different IgA tTG ELISAs, two of them based on guinea pig liver tTG (gp-tTG) (an in-house ELISA with a partially purified extract and a commercial ELISA with purified gp-tTG antigen) and a third recombinant human tTG (rh-tTG) ELISA. The results are compared with EMA and with the final clinical diagnosis. A second objective was to analyze antibody reactivities in those patients with anti-tTG and EMA discrepancies. METHODS: ELISA and EMA tests were used to measure IgA anti-tTG levels in sera from 259 patients (107 had CD and 72 had Type I diabetes mellitus). RESULTS: The purified gp-tTG ELISA was highly sensitive (97.7%) and specific (98.8%) in the detection of CD, almost equaling EMA. Rh-tTG ELISA did not improved the sensitivity of EMA, but its specificity was slightly superior. Immunoblot analysis with partially purified gp-tTG extract, the antigen most frequently used for anti-tTG detection, showed that the majority of false positives were due to IgA reactivities to contaminant proteins present in the liver antigenic extract. This low specificity was particularly problematic in diabetics. CONCLUSION: Purified tTG ELISAs, either with purified guinea pig liver or recombinant human antigens, can be used as quantitative and observer-independent alternatives to the traditional and time-consuming EMA in the screening of CD.

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99 percent with rBoIFN-alphaC and by 84 percent with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity.

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

Intestinal intraepithelial lymphocytes contain a CD3- CD7+ subset expressing natural killer markers and a singular pattern of adhesion molecules.

Intestinal intraepithelial lymphocytes (i-IEL) represent one of the largest, non-organized lymphoid population in the body. They are located outside the epithelial basement membrane among the mucosal epithelial cells. We, and previously other groups, have reported the presence of a CD7+CD3-IEL subset in the epithelium of human small intestine. This subset is drastically reduced in coeliac disease (CD) patients. In the present work we accomplish a better phenotypic characterization of this CD3-IEL subset and demonstrate the expression of typical natural killer (NK) cell markers. Most, if not all, CD3-CD7+ cells express NKPR1 (CD161)[98% +/- 2] and CD122[92% +/- 6]. In addition, a variable percentage express CD2[55% +/- 16], CD94[24% +/- 18], CD56[44% +/- 21] and CD16[12% +/- 4], however, no CD57 expression was observed. Moreover, these cells contain perforin granules[75% +/- 5], supporting a potential cytolytic ability. Regarding adhesion molecules, CD18 and CD44 expression is absent, which is consistent with a limited capacity of migration. Altogether, these data suggest the presence of intraepithelial NK cells in human intestinal epithelium, a compartment where cytotoxic effectors have not been clearly defined.

Intraepithelial lymphocytes and coeliac disease: permanent changes in CD3-/CD7+ and T cell receptor gammadelta subsets studied by flow cytometry.

UNLABELLED: Permanent changes in intestinal intraepithelial lymphocytes have been observed in coeliac patients. The aim of this investigation was to study small intestinal intraepithelial lymphocytes by using flow cytometry and to evaluate its diagnostic value in coeliac disease. Three-colour flow cytometry analyses were performed on isolated epithelial cells of 117 intestinal biopsies obtained from 113 children (54 coeliac disease, 4 other enteropathies, 18 Helicobacter pylori associated gastritis and 37 normal controls). A multiple logistic regression model was developed to select the best intraepithelial lymphocytes subset predictor of coeliac disease. Coeliac patients had significant higher levels of T cell receptor gammadelta intraepithelial lymphocytes than control patients (p < 0.01), H. pylori patients (p < 0.01) and other enteropathies (p < 0.05). The density of CD3-/CD7+ intraepithelial lymphocytes, a intraepithelial lymphocyte subset poorly characterized by immunohistochemical methods, was significantly lower in coeliac patients than in the control group (p < 0.01). H. pylori group (p < 0.01) and other enteropathies (p < 0.01). Both changes remained altered independent of the coeliac patient's diet. The data were used on a logistic regression analysis in order to calculate sensitivity [94.4%; 95% confidence interval (CI) 83.7-98.6%], specificity (94.9%; 95% CI 84.9-98.7%) and likelihood ratio for a positive test 18.5 (95% CI 6.1-55.8) in the diagnosis of coeliac disease. CONCLUSION: Changes in T cell receptor gammadelta and CD3-/CD7+ intraepithelial lymphocytes subsets are permanently observed in paediatric coeliac disease. Their assessment, by three-colour flow cytometry on routine diagnostic biopsies, permits a better characterization of coeliac enteropathy and represents a valuable procedure to identify coeliac patients with different clinical presentations.

Mechanistic aspects of the induction of apoptosis by lauryl gallate in the murine B-cell lymphoma line Wehi 231.

The effect of lauryl gallate (antioxidant E-312) has been studied on the mouse B-cell lymphoma line Wehi 231. This compound is able to inhibit protein tyrosine kinases (PTKs) in whole cells and in crude extracts with a better efficiency than other well-known PTK inhibitors such as herbimycin or genistein. Initial events triggered upon the incubation of cells with lauryl gallate in phosphate-buffered saline (up to 1 h) include the inhibition of tyrosine phosphorylation, discharge of the mitochondrial transmembrane potential, and induction of mRNA for Bcl-2. Long-term cultures in complete medium supplemented with fetal calf serum (up to 24 h) in the presence of this compound exhibit clear apoptotic features such as increase in phosphatidylserine in the cell surface, decrease in the functionality of mitochondria, cytochrome c release to the cytosol, activation of caspases, hypodiploidy, and oligonucleosomal breakdown of DNA. Comparison between Wehi cells overexpressing Bcl-2 (Wehi-bcl-2) with Wehi-neo cells shows a delay in the manifestations of the apoptotic signs, indicating that Bcl-2 has a partial protective effect on the apoptosis induced by lauryl gallate. The proapoptotic effect of lauryl gallate is not dependent on DNA or protein synthesis, is not blocked by the chelation of calcium, and is not reverted by N-acetylcysteine.

Inhalant allergy to egg yolk and egg white proteins.

BACKGROUND: Several egg white and egg yolk and avian proteins have been described as a cause of inhalant allergy. Sometimes inhalational type I hypersensitivity to these proteins is associated with food allergy to egg. OBJECTIVE: We studied two patients who experienced respiratory and food allergic symptoms upon exposure to egg or avian antigens through the inhalative or digestive routes. Clinical and immunological studies were carried out in order to identify individual allergens from these sources that could be responsible for crossreactivity reactions. RESULTS: Patient 1 showed IgE sensitization to egg yolk livetins, feathers, and chicken serum. Specific bronchial challenge with chicken albumin and livetin extracts elicited a positive early asthmatic response and an increase in serum eosinophil cationic protein. Immunoblot and CAP-inhibition studies in this patient supported that chicken albumin (alpha-livetin) was the crossreactive antigen present in egg yolk and chicken serum and feathers. Patient 2 showed sensitization to egg white, ovomucoid and lysozyme. However, SDS-PAGE and immunoblot studies demonstrated contaminating lysozyme in the ovomucoid extract and identified lysozyme as the main allergen causing egg sensitization in this patient. Conjunctival challenge test confirmed allergy to lysozyme. CONCLUSION: Egg yolk and egg white proteins may act not only as ingested allergens but also as aeroallergens. Immunological studies using highly purified preparations of egg proteins are useful for the accurate diagnosis of allergic reactions to egg proteins and to identify individual allergens that may be responsible for crossreactivity reactions.

Flow cytometry description of a novel CD3-/CD7+ intraepithelial lymphocyte subset in human duodenal biopsies: potential diagnostic value in coeliac disease.

Intraepithelial lymphocytes (IEL) represent a heterogeneous cellular compartment of unknown functions and controversial ontogeny. Previous observations in humans indicate that the majority of IEL subsets express the CD3 complex associated with either the alphabeta or the gammadelta T-cell receptor components, and describe the characteristic increase of CD3+TCRgammadelta+ IEL in coeliac disease. In the present work, we analyze the surface antigen expression of intraepithelial lymphocytes isolated from duodenal biopsies of control subjects and coeliac disease patients. We describe a CD3-CD7 + IEL subset frequently found in control subjects (41.41+/-21.8), with the following features: 1) most of these cells are CD45R0+ CD103+ and CD44- CD28- CD5-; 2) a significant percentage express CD56 (44.7%+/-21.3), CD2 (55.1%+/-16.2), and CD94 (16.2%+/-7.3). Furthermore, they are CD122+ and CD25-; 3) this CD3- IEL subset exhibit an activated phenotype expressing higher levels of CD69, CD103, and CD38 than the CD3+ subset. Interestingly, this CD3- subset is drastically reduced in CD patients (2.2+/-2.9 in active disease, 6.3+/-4.6 in treated patients versus 41.4+/-21.8 in control subjects). The imbalanced ratio "increased TCRgammadelta versus decreased CD3- CD7+" is a permanent finding in CD patients following clinical and histological remission. This parameter might provide helpful diagnostic information (easily obtained by 3-color FCM from diagnostic biopsies), and suggest a potential implication in the pathogenesis of coeliac disease.