p8 Deficiency causes siderosis in spleens and lymphocyte apoptosis in acute pancreatitis.

OBJECTIVES: The gene p8 was initially described in pancreatic tissue during acute experimental pancreatitis, a disease that is characterized by a systemic immune response. Although early reports suggested that p8 affects leukocyte migration during acute pancreatitis (AP), no studies revealing its immune-modulatory effects have been performed. METHODS: We investigated the composition of the cellular immune system in naive p8 knockout (p8(−/−)) mice and compared with matched wild-type mice during pancreatitis. RESULTS: In young mice, there were no relevant differences in the composition of peripheral and splenic CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD11b(+)Gr-1(-), and Gr-1 cells. In mature p8(−/−) mice, increased splenic CD4CD25FoxP3 cells, spleen siderosis, and increased marginal zones in the splenic white pulp were found. During AP, peripheral and splenic CD3(+) and CD3CD4 declined stronger in the p8(−/−) mice. The spleen of the p8(−/−) mice showed severe hypoplasia of the white pulp and mild hyperplasia of the red pulp. This was associated with a significantly increased rate of apoptosis. CONCLUSIONS: We conclude that p8 has no impact on the cellular composition of the adaptive and innate immune systems in noninflammatory conditions. However, it may limit apoptosis and maintain homeostasis of the immune reaction during AP.

Eight-color immunophenotyping of T-, B-, and NK-cell subpopulations for characterization of chronic immunodeficiencies.

BACKGROUND: The heterogeneity of primary and secondary immunodeficiencies demands for the development of a comprehensive flow cytometric screening system, based on reference values that support a standardized immunophenotypic characterization of most lymphocyte subpopulations. METHODS: Peripheral blood samples from healthy adult volunteers (n = 25) were collected and split into eight panel fractions (100 µl each). Subsequently, premixed eight-color antibody cocktails were incubated per specific panel of whole blood to detect and differentiate cell subsets of: (i) a general lymphocyte overviews, (ii) B-cell subpopulations, (iii) CD4+ subpopulations, (iv) CD8+ subpopulations, (v) regulatory T-cells, (vi) recent thymic emigrants (RTE), (vii) NK-cell subpopulations, and (viii) NK-cell activation markers. All samples were lysed, washed, and measured by flow cytometry. FACS DIVA software was used for data analysis and calculation of quadrant statistics (mean values, standard error of mean, and percentile ranges). RESULTS: Whole blood staining of lymphocytes provided the analysis of: (i) CD3+, 4+, 8+, 19+, 16/56+, and activated CD4/8 cells; (ii) immature, naïve, nonswitched/switched, memory, (activated) CD21(low) , transitional B-cells, plasmablasts/plasmacells; (iii and iv) naïve, central memory, effector, effector memory, TH1/TH2/TH17-like, and CCR5+CD8-cells; (v) CD25+, regulatory T-cells (naïve/memory, HLA-DR+); (vi) α/ß- and γ/δ-T-cells, RTE in CD4/CD8 cells; (vii) immature/mature CD56(bright) , CD94/NKG2D+ NK-cells; and (viii) Nkp30, 44, 46, and CD57+NK-cells. Clinical examples and quadrant statistics are provided. CONCLUSION: The present study represents a practical approach to standardize the immunophenotyping of most T-, B-, and NK-cell subpopulations. That allows differentiating whether abnormalities or developmental shifts observed in lymphocyte subpopulations originates either from primary or secondary immunological disturbance.

Serologic assay based on gliadin-related nonapeptides as a highly sensitive and specific diagnostic aid in celiac disease.

BACKGROUND: Celiac disease (CD) is induced by wheat gliadins and related cereal proteins. Anti-gliadin antibodies (AGAs) are present in the serum of CD patients, but these antibodies have lower diagnostic specificity and sensitivity than autoantibodies [anti-endomysium antibodies (AEmAs) and anti-tissue transglutaminase antibodies (AtTGAs)]. Recently, AGAs from CD patients were found to recognize deamidated gliadin peptides, probably formed by the action of tissue transglutaminase. METHODS: We synthesized several gliadin peptides and their glutamine-glutamic acid-substituted counterparts on cellulose membranes and tested their recognition by IgA in sera of 52 AEmA-positive CD patients and 76 AEmA-negative controls in a luminescence assay. For comparison, we assayed IgA concentrations of AGAs, AtTGAs, and AEmAs. For measurement of AtTGAs, we used the human recombinant antigen. RESULTS: We identified several nonapeptides that were detected with high specificity by IgA in CD patients. Diagnostic accuracy of the peptide antibody assay was highest when peptide PLQPEQPFP was used in combination with peptide PEQLPQFEE within one assay. AGAs were above the cutoff in 14 of the controls, but only 5 of the controls were positive for peptide antibodies. For comparison, 82% and 94% of samples were correctly classified by AGAs and the combination nonapeptide assay, respectively (P = 0.007), and the AtTGAs correctly classified 98%. CONCLUSION: The peptide antibody assay has higher diagnostic accuracy than AGAs for distinguishing patients with CD from controls, and has diagnostic accuracy similar to that of AtTGAs.

Intrathecal synthesis of autoantibodies against tissue transglutaminase.

Anti-tissue transglutaminase (tTG) antibodies (AtTGA) are typically found in serum of patients with untreated coeliac disease (CD). tTG catalyses crosslinking of peptides an activity supposed to be important in neurological disorders. tTG occurs in cerebrospinal fluid (CSF) and its assay in CSF was suggested to be diagnostically useful. However, nothing is known about AtTGA in CSF. Therefore, in 129 unselected CSF-serum pairs IgA- and IgG-AtTGA were assayed by ELISA using human recombinant tTG. For comparison, IgA- and IgG-anti-gliadin antibodies (AGA), typically coexisting with AtTGA were measured. Albumin, total IgA and IgG and further parameters were determined according to routine programme recommended by the European CSF consensus group. AtTGA were detected in 27 (IgA) and in 63 (IgG) CSF samples. Antibody indices (AI) could be calculated for AtTGA from 21 (IgA) and from 61 (IgG) sample pairs. AI for AtTGA was >2 in 11 (IgA) and in 22 (IgG) sample pairs, hinting to intrathecal antibody synthesis. AI for AGA was >2 only for 1 (IgA) and 2 (IgG) sample pairs. Patients with normal routine findings had significantly higher AI for IgA-AtTGA than patients with abnormal findings. This is the first demonstration of AtTGA in CSF and their intrathecal synthesis. The pathogenetic relevance of this new autoantibody species remains to be clarified.