Background and Aims. Deoxyribonuclease I (DNaseI) is an endonuclease that facilitates chromatin breakdown and promotes susceptibility to autoimmune disorders. The aim of current study was to investigate serum DNase I activity in patients with inflammatory bowel diseases (IBD). Patients and Methods. A cohort of 110 IBD patients was evaluated, aged 35 ± 12 years, 77 with Crohn's disease (CD) and 33 with ulcerative colitis (UC). 50 SLE patients and 50 healthy blood donors were examined as control groups. Results. DNase I activity in IBD patients was significantly lower than in healthy individuals, but higher than in SLE patients (P < .0001). Patients with UC showed higher DNase I activity than CD patients, P = .21. DNase I activity in female patients with IBD was significantly lower than in males, P = .024; however, no differences in DNase I activity were found in relation to gender in healthy individuals. DNase I activity has shown a strong negative correlation with the serum concentration of anti-nucleosomal antibodies in the autoimmune (SLE + IBD) cohort, as well as in the separate IBD cohort. Conclusions. Reduced serum DNase I activity probably has pathogenetic consequences in IBD. Induction of autoantibodies towards nucleosomes could be a reflection of impaired DNase I activity.

Abstract BACKGROUND: Breast cancer (BC) patients still harbor a considerable risk of metastatic relapse caused by minimal residual disease (MRD) despite of complete removal of primary tumor. The aim of this study was to identify single disseminated tumor cells (DTC) in the bone marrow and circulating tumor cells (CTC) in the peripheral blood as potential biomarkers for therapy response monitoring and metastatic relapse risk prediction. Finally, the gene expression profiles of CTCs have been analyzed. METHODS: A total of 87 patients with diagnosed BC at stage I to III and 115 metastatic patients were enrolled into a prospective study between years 2008 - 2010. Peripheral blood (5ml) for CTC-detection was collected from primary BC patients before chemotherapy (CHT), after 2 cycles of CHT and after the CHT. Metastatic BC patients have been examined for CTCs before starting a new line of the treatment. Bone marrow aspirates from 16 premenopausal BC patients (mean age 31) with primary tumor were analyzed for the presence of DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 (Epimet™, AS Diagnostik, Germany) before surgery. CTC detection in blood was performed by AdnaTest BreastCancerTM(AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. cDNA from isolated CTCs has been further pre-amplified and used for multimarker qPCR gene expression profiling using Biomark® microfluidic 48x48 GE Dynamic arrays (Fluidigm, USA). qPCR results have been analyzed by GENEX vs. 5.2 software (MultiD Analysis). RESULTS: 286 CTC samples have been analyzed in total. The analysis has shown that the expression profiles of CTCs from primary BC patients have been significantly different comparing them to the CTC-profiles from metastatic BC patients for several of tested genes (e.g., CK19, GA7332, MLFIP1, SATB1, PTEN). Interestingly, before surgery of primary tumor DTCs were found in 5/16 patients (31 %) and CTCs in 7/16 (43 %). Both DTCs and CTCs together were found in 4/16 (25%) patients. In 18% of the primary BC patients no dissemination markers were found CONCLUSIONS: Information based on the CTCs-gene expression profiles could provide an additional support for therapy management. Metastatic potential of enriched CTCs will be further evaluated on the single cell level. This work has been supported by Grant Agency of Ministry of Health, Czech Republic (IGA NS9976) and Grant Agency of Charles University in Prague, Czech Republic no. 7709 and 59410. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-03-01.

In 30–50% of sub-fertile couples the male partner has poor semen quality, either because of low sperm count, poorly motile sperm or sperm with abnormal size and shape (morphology). This may result from inherent genetic factors, poor pre-natal testicular development or adult exposure to gonadotoxic agents. There are few proven therapies to improve semen quality. Therefore, the number and quality of sperm that can be recovered from the ejaculate, or obtained by surgical sperm recovery, determines the available options for assisted conception. In recent years, the reliance upon donor sperm has reduced markedly as intracytoplasmic sperm injection can be attempted. Reproductive technologies are considered relatively safe, with healthy babies born; however, there are some concerns about potential reproductive problems in males conceived in this way. These may be related to the techniques used, or the underlying infertility of the parents, and are currently the subject of follow-up studies.

Summary Calreticulin, upon translocation to the cell surface, plays a critical role in the recognition of tumour cells and in experimentally induced cellular anti-tumour immunity. However, less is known about anti-calreticulin antibodies and their role in malignancies. Using enzyme-linked immunosorbent assay (ELISA), we found immunoglobulin (Ig)A and/or IgG anti-calreticulin antibodies in sera of approximately 63% of patients with hepatocellular carcinoma (HCC), 57% of patients with colorectal adenocarcinoma (CRA) and 47% of patients with pancreatic adenocarcinoma (PACA), while healthy controls, patients with viral hepatitis C and with chronic pancreatitis reached only 2%, 20% and 31% seropositivity, respectively. We found significantly elevated mean levels of IgA anti-calreticulin antibodies (P < 0·001) in patients with HCC (78·7 ± 52·3 AU, mean ± standard deviation), PACA (66·5 ± 30·9 AU) and CRA (61·8 ± 25·8 AU) when compared to healthy controls (41·4 ± 19·2 AU). Significantly elevated mean levels of IgG anti-calreticulin antibodies (P < 0·001) were detected in patients with HCC (121·9 ± 94·2 AU), gall bladder adenocarcinoma (118·4 ± 80·0 AU) and PACA (88·7 ± 55·6 AU) when compared to healthy controls (56·7 ± 22·9 AU). Pepscan analysis revealed a large number of antigenic epitopes of calreticulin recognized by both IgA and IgG antibodies of patients with HCC and PACA, indicating robust systemic immune response. Moreover, significantly elevated levels of antibodies against peptide KGEWKPRQIDNP (P < 0·001) in these patients, tested by ELISA, confirmed the distinct character of antibody reactivity against calreticulin. The high occurrence and specificity of serum anti-calreticulin autoantibodies in the majority of patients with some gastrointestinal malignancies provide the evidence for their possible clinical relevance.

The effect of active periodontal treatment on progression of the disease and the role of in vitro immune reaction after activation of peripheral blood mononuclear cells were studied. The genotype was analyzed and serological predisposition to disease was studied. The first group of 20 patients at the begining of the study was conservatively treated for 15 years. The second group was formed from healthy persons of similar age. Slow progression of the disease was found in 10 patients and after 10 years the progressive form of the disease was found in another 10 patients. The risk composite genotype IL-1 and risk alleles of Apo-E were present more frequently in patients with progressive periodontitis. 23,3% of patients had increased serum atherosclerosis index.

IgE against mixtures of common food or respiratory allergens were determined by ELISA in healthy ( n = 38) and allergic ( n = 62) mothers and their children. Significantly higher level of IgE against respiratory allergens was found in sera of allergic mothers and in cord blood of their children. No correlation between antibody level in maternal and newborn’s sera was found; this argues against the transfer of IgE from mother to fetus and points rather to offspring’s intrauterine sensitization. Specific IgE level in cord blood was higher in children who developed later allergy than in children who did not. Specific IgE level in colostrum was low both in healthy and allergic mothers; there was no correlation between high concentration of IgE against respiratory allergens in sera of allergic mothers and their colostrum, which does not support the idea of IgE transport from blood to mammary gland. Only slightly increased colostral IgE was detected in allergic mothers whose children manifested allergy later. Allergy of the mother and high level of anti-allergen IgE in her serum and in cord blood are the main predictive factors of future occurrence of allergy in the offspring. A combination of several predictive factors could have higher prognostic value.

BACKGROUND:Fecal calprotectin test is a simple, non-invasive, rapid and inexpensive diagnostic tool allowing differentiation between GIT functional disorders and inflammatory conditions and relapse prediction in non-specific inflammatory bowel disease. In the last year, commercially available ELISA diagnostic kits, using either monoclonal or polyclonal antibodies against a heterodimeric complex, calprotectin, for the detection of fecal calprotectin, started to be marketed.OBJECTIVE:To compare two ELISA kits for the detection of fecal calprotectin differing from each other in the used type of antibody (monoclonal versus polyclonal).MATERIAL AND METHODS:Two ELISA kits were assessed: Calprotectin ELISA (Bühlmann, Basel, Switzerland) using a monoclonal antibody against calprotectin and PhiCal Calprotectin ELISA (R-Biopharm, Darmstadt, Germany) using a polyclonal antibody against calprotectin. We analyzed fecal eluates from patients with Crohn's disease (CD, n=36) and ulcerous colitis (UC, n=29) and from healthy controls (n=98). Data were analyzed using software Statistica CZ 8.0 (Statsoft, Tulsa, U.S.A.) and measurement variability parameters (linearity, repeatability, stability) and test sensitivity and specificity were established and the methods were compared.RESULTS:The two kits showed adequate accuracy (intra- and inter-assay variation < 10%). The dilution linearity test indicated superiority of the Calprotectin ELISA Bühlmann kit, in particular for high calprotectin levels. The results of the two methods showed good correlation: Pearson's correlation coefficient r = 0.83, limit difference according to Bland-Altman plot ranged from 17% to 30%. Diagnostic sensivity rates were 79% for the Calprotectin ELISA Bühlmann kit and 74% for the Calprotectin ELISA R-Biopharm kit, the test specificity rates were 87% and 84%, respectively.CONCLUSIONS:Both of the tested kits have comparably good measurement parameters, the Bühlmann kit using monoclonal antibody against calprotectin showed higher sensitivity and specificity. In view of their availability, sensitivity and performance, the fecal calprotectin ELISA kits are helpful diagnostic tools for clinical practice.

The objective of this study was to test the diagnostic accuracy of novel anti-carbohydrate assays in patients with inflammatory bowel disease, namely in Crohn's disease. These carbohydrate assays are based on oligosaccharide chitobioside carbohydrate - anti-chitobioside carbohydrate antibodies (ACCA), laminaribioside carbohydrate anti-laminaribioside carbohydrate antibodies (ALCA), and mannobioside carbohydrate - anti-mannobioside carbohydrate antibodies (AMCA). We compared these assays with the anti-Saccharomyces cerevisiae antibodies (ASCA) assay. The results of this study suggest that ASCA are still the best serological marker for Crohn's disease. Further studies are required to explore the clinical utility of ACCA, ALCA and AMCA.