Inhibition of Cross-Reactive Carbohydrate Determinants in Allergy Diagnostics.

Despite being clinically largely irrelevant, antibodies against cross-reactive carbohydrate determinants (CCD) are an important issue in the in vitro diagnostics, as they may produce false positive or falsely elevated results of the immunoglobulin E class (asIgE) in relation to the actually present level of asIgE. The present chapter demonstrates an effective resolution of this diagnostic issue by the use of a CCD inhibitor in in vitro tests. A synthetic CCD inhibitor, Polycheck® CCD inhibitor, was used in the laboratory diagnostics of 24 children diagnosed with allergic diseases. The anti-CCD antibody content was measured in the serum using a Polycheck® Atopic 30-I panel (Biocheck GmbH; Münster, Germany), a screening assay for the quantitative determination of multiple allergen-specific IgE. We found that the baseline anti-CCD antibody content, without the CCD inhibitor, ranged from 0.7 to 3.5 kU/L in the sera of the majority of 16 out of 24 children. When the CCD inhibitor was applied, the anti-CCD antibody content decreased in 16, remained unchanged in 3, and increased in 5 samples. In samples positive for plant allergens, the asIgE content dropped by an average of 72% when the CCD inhibitor was used in the assay, except the antibodies to tree and grass pollen allergens, for which the asIgE content remained above 100 kU/L. We conclude that the use of a CCD inhibitor in in vitro assays is a viable option to mitigate the influence of anti-CCD antibodies on the measured level of asIgE immunoglobulin, which increase the reliability of testing particularly in cases displaying multiple allergies.

Clinical utility of quantitative multi-antibody Polycheck immunoassays in the diagnosis of coeliac disease.

AIM: To evaluate the clinical utility of multi-antibody strategies in the diagnosis of coeliac disease (CD), the new quantitative Polycheck immunoassays were analysed. METHODS: Polycheck Celiac Panels (PCPs) are immunoenzyme screening assays for the quantitative measurement of coeliac-specific immunoglobulin class G (IgG) or class A (IgA) in serum. Lines of relevant antigens are coated together with five IgG or IgA standard lines used for the standard curve as positive control. PCP IgA consists of human recombinant human tissue transglutaminase (tTG) and deamidated gliadin peptides (DGP) as targets to detect IgA antibodies. PCP IgG consists of tTG, DGP and IF (intrinsic factor) antigens to detect antibodies in IgG class. PCPs were performed on 50 CD patients, including 6 cases with selective IgA deficiency, and 50 non-coeliac controls. CD diagnosis was performed according to the ESPGHAN recommendations: The presence of specific anti-tTG-IgA or anti-DGP-IgG (in the case of IgA deficiency) antibodies, typical histopathological changes in duodenal mucosa described in Marsh-Oberhüber classification as at least grade 2. The diagnosis of the majority of the control subjects was functional gastrointestinal disorders. The PCP results were compared with reference EliA Celikey. RESULTS: The usage of PCPs led to the correct identification of all CD patients. In our study, PCPs showed 100% agreement with the histopathological results. PCP IgA test showed a 98% concordance and correlated positively (R = 0.651, P = 0.0014) with EliA Celikey test. The highest specificity and positive predictive value (both 100%) were observed for the detection of Polycheck anti-tTG-IgA antibodies. The highest sensitivity and negative predictive value (both 100%) were achieved by Polycheck anti-DGP-IgG antibody detection. The best performance (98% sensitivity and negative predictive value, 100% specificity and positive predictive value, diagnostic accuracy - AU ROC 99%) was observed for the strategy of using both PCP IgA and IgG and determining positive outcomes of the test with two or more coeliac-specific antibodies detected. The majority of coeliac patients had multiple antibodies. All four antibodies were detected in 7 (14%) cases, 19 children (38%) were positive for three antibodies and 23 (46%) were positive for two antibodies. CONCLUSION: The present study showed that detection of coeliac-specific antibodies with multi-antibody PCPs is effective and efficacious in the diagnosis of CD.

Expression of Programmed Death 1 Ligand in Different Compartments of Chronic Lymphocytic Leukemia.

BACKGROUND: The programmed death 1 (PD-1) receptor pathway is responsible for the negative regulation of both T and B lymphocytes upon activation of these cells. There is growing evidence that chronic lymphocytic leukemia (CLL) cells exploit the PD-1 ligand (PD-L1) to resist antitumor immune reactions and maintain their survival by shaping their own microenvironment. METHODS: We used a quantitative RT-PCR method to analyze PD-L1 gene expression in bone marrow and peripheral blood mononuclear cells, representing the proliferation and accumulation compartments of CLL. RESULTS: PD-L1 expression was found to be significantly higher in 112 CLL patients than in controls. Levels of PD-L1 expression in bone marrow and peripheral blood were comparable and showed a positive correlation. Furthermore, expression of PDL1 strongly correlated with expression of PD-1 receptor in mononuclear cells from the same compartment, and was not affected by incubation with immunomodulatory drug thalidomide. CONCLUSION: PD-L1 expression is shared between CLL cells localized in distinct disease compartments, demonstrating that PD-1/PD-L1 a universal target for therapy.

The function of a novel immunophenotype candidate molecule PD-1 in chronic lymphocytic leukemia.

Programmed death-1 (PD-1) is a negative receptor expressed on lymphocytes including malignant B cells in chronic lymphocytic leukemia (CLL). In this work, we found that patients with CLL had a higher expression of PD-1 transcript (PDCD1) than healthy volunteers (p < 0.0001). PDCD1 expression was comparable between CLL cells from accumulation (peripheral blood) and proliferation (bone marrow) disease compartments. In blood samples of patients with mutated IGHV genes PDCD1 expression was higher than with unmutated IGHV (p = 0.0299). We demonstrated that phosphorylation of SYK and LYN, key B-cell receptor signaling kinases, was independent of PD-1 expression in patients with CLL, while ZAP-70 phosphorylation in negative tyrosine residue 292 showed strong inverse correlation (r = - 0.8, p = 0.0019). No associations between five single nucleotide polymorphisms of PDCD1, their expressions and susceptibility to CLL were found. In conclusion, PD-1 might be an independent, universal marker of CLL cells and a part of their activated phenotype, and subsequently might modulate the function of ZAP-70.

[Evaluation of usefulness of Polycheck method in the detection autoantibodies in patients with systemic lupus erythematosus, Sjögren's syndrome and systemic sclerosis]. / Ocena przydatnosci metody Polycheck w wykrywaniu autoprzeciwcial u chorych na toczen rumieniowaty ukladowy, zespól Sjögrena i twardzine ukladowa.

INTRODUCTION: To recognize the connective tissue diseases (CTD), which include lupus erythematosus (SLE), Sjögren's syndrome (SS) and Systemic sclerosis (SSc) it is necessary to determine the presence of autoantibodies (Ab). There is still work going on to find effective and equivocal detection methods. THE AIM OF STUDY: To evaluate the usefulness, clinical value and innovativeness of the Polycheck method in the detection of autoantibodies in patients with connective tissue diseases. MATERIALS AND METHODS: The study involved 178 people: 153 patients of the Department of Rheumatology and 25 healthy people. According to the main diagnosis the patients were divided into 3 groups: SLE-59, ZS-45, SSc-49. The presence and concentrations of Ab were determined by using multiparametric enzyme-linked immunosorbent assay Polycheck Rheuma (Biocheck, GmbH, Münster, Germany). Statistical data analysis was performed using Statistica v10.0. RESULTS: In our study we found thatthe frequency of antibodies characteristic of the SS: anti-SS-A/Ro 52, anti-SS-A Ro 60 and anti-SS-B/La was significantly higher in patients with SS group compared to TRU, TU, and GK (p <0.05); concentrations of Ab were lower in TRU and in SSc group. Marker Ab for SLE anti-dsDNA were only present in patients with SLE. Anti-Sm antibodies were more common in this patient population too. Antibodies associated with diagnosis of SSc anti-SCL-70 and anti-CENP B were significantly more often observed in patients with SSc in higher concentrations compared to the other groups examined (anti-SCL-70 p<0,0005). The Ab concentration analysis performed with use of this analysis help us to confirm diagnosis in particular patients. CONCLUSIONS: The multiparametric enzyme-linked immunosorbent assay Polycheck Rheuma is helpful in making a quick and comprehensive identification of autoantibodies in pts with suspected CTD. The concentration analysis performed with use of this method enables an accurate diagnosis despite the ambiguous clinical picture.

Programmed death-1 and its ligand are novel immunotolerant molecules expressed on leukemic B cells in chronic lymphocytic leukemia.

Programmed death-1 (PD-1) is an immunoreceptor predominantly expressed on exhausted T cells, which through an interaction with its ligand (PD-L1), controls peripheral tolerance by limiting effector functions of T lymphocytes. qRT-PCR for PD-1, PD-L1 and their splicing forms as well as flow cytometric assessment of surface expression was performed in a cohort of 58 chronic lymphocytic leukemia (CLL) patients. In functional studies, we assessed the influence of the proliferative response of leukemic B-cells induced by IL-4 and CD40L on PD-1 transcripts and expression on the protein level. The median level of PD-1, but not PD-L1, transcripts in CLL patients was higher in comparison to healthy volunteers (HVs, n = 43, p = 0.0057). We confirmed the presence of PD-1 and PD-L1 on the CLL cell surface, and found the expression of PD-1, but not PD-L1, to be higher among CLL patients in comparison to HVs (47.2% vs. 14.8%, p<0.0001). The Kaplan-Meier curves for the time to progression and overall survival in groups with high and low surface expression of PD-1 and PD-L1 revealed no prognostic value in CLL patients. After stimulation with IL-4 and CD40L, protein expression of PD-1 was significantly increased in samples that responded and up-regulated CD38. PD-1, which is aberrantly expressed both at mRNA and cell surface levels in CLL cells might represent a novel immunotolerant molecule involved in the pathomechanism of the disease, and could provide a novel target for future therapies.