Rapid selection of cultured cells with increased expression of a membrane marker (secretory component)

This study showed how immunomagnetic monodisperse microspheres (Dynabeads) can be used for rapid selection of stable and homogeneous sublines of cancer cells. The beads were activated with antibody to a specific surface membrane marker. We used a monoclonal antibody (MAb) to human secretory component (SC), the epithelial receptor for polymeric immunoglobulins (pIg). A relatively low number of particles favored rosetting of strongly positive cells. SC-expressing cells were isolated within 30 min from a colonic carcinoma cell line (HT-29.E10). After 13 months of continuous culture the selected cells had a 15 times higher cellular content and a 10 times higher secretory rate of SC than parallel cultures of the original cell line. Immunomagnetic microspheres can be recommended in experimental cancer research, e.g., in order to minimize cellular heterogeneity and to isolate cells with particular surface membrane phenotypes in differentiation studies. The selection method is rapid and simple, and eliminates laborious test systems for conventional subclone screening.     

Eating disorder that was diagnosed before pregnancy and pregnancy outcome

OBJECTIVE: The purpose of this study was to determine the association of an eating disorder that was diagnosed before pregnancy and a preterm delivery and/or the delivery of a low-birth-weight or small-for-gestational-age infant. STUDY DESIGN: This was a register-based follow-up study. We included 302 women who were hospitalized with an eating disorder before pregnancy who were delivered of 504 children and 900 control subjects who were delivered of 1552 children. The association of eating disorders, birth weight, and gestational age was assessed by bivariate and multivariate analyses. RESULTS: The risk of a low-birth-weight infant was twice as high in women with a previous eating disorder compared with women with no such disorder (odds ratio, 2.2; 95% CI, 1.4-3.2). The risk of preterm delivery and a small-for-gestational-age infant was increased to 70% and 80% (odds ratio, 1.7 [95% CI, 1.1-2.6]; odds ratio, 1.8 [95% CI, 1.3-2.4]), respectively. CONCLUSION: Women who were hospitalized for an eating disorder that was diagnosed before pregnancy were at increased risk of impaired pregnancy outcome.     

Capnography rapidly confirmed correct endotracheal tube placement during resuscitation of extremely low birthweight babies (< 1000 g)

During neonatal resuscitation, the routine use of capnography to verify correct placement of the endotracheal tube is not an established international practice. We present four cases that illustrate the successful use of immediate capnography to verify correct tracheal tube placement even in extremely low birthweight (ELBW) prematures (< 1000 g) during resuscitation. Based on this limited experience, we reached institutional consensus among paediatricians and anaesthesiologists that capnography should become standard monitoring during all endotracheal intubations in premature babies.     

HLA-DQ relative risks for coeliac disease in European populations: a study of the European Genetics Cluster on Coeliac Disease

Coeliac disease is an enteropathy due to an intolerance to gluten. The association between HLA-DQ genes and CD is well established. The majority of patients carry the HLA-DQ heterodimer encoded by DQA1*05/DQB1*02, either in cis or in trans. The remaining patients carry either part of the DQ heterodimer or DQA1*03-DQB1*0302. The aim of the study was to estimate the risks associated with different DQ genotypes in European populations. HLA information was available for 470 trio families from four countries: France (117), Italy (128), and Norway and Sweden (225). Five DQA1-DQB1 haplotypes were considered and control haplotype frequencies were estimated from the set of parental haplotypes not transmitted to the affected child. The possible genotypes were grouped into five genotype groups, based on the hierarchy of risk reported in the literature. A north-south gradient in the genotype group frequencies is observed in probands: homogeneity is strongly rejected between all country pairs. For each country, the relative risks associated with each genotype group were computed taking into account the control haplotype frequencies. Homogeneity of relative risks between countries was tested pairwise by maximum likelihood ratio statistics. The hypothesis of homogeneity of relative risks is rejected (P is approximately 10(-6)) for all country pairs. In conclusion, the gradient in the genotype group frequencies in probands is not only due to differences in haplotype frequencies but also due to differences in genotype relative risks in the studied populations; the relative risks associated with each DQ genotype group are different between northern and southern European countries; neither are they ordered in the same way.     

Association analysis of the 1858C>T polymorphism in the PTPN22 gene in juvenile idiopathic arthritis and other autoimmune diseases

A functional single nucleotide polymorphism, 1858C>T, in the PTPN22 gene, encoding a tyrosine phosphatase, has been reported to be associated with type I diabetes and some other autoimmune diseases. To further investigate whether this polymorphism may be a general susceptibility factor for autoimmunity, we performed an association study in five different autoimmune diseases, three previously not tested. We found an association with juvenile idiopathic arthritis (OR=1.41; P=0.04), not previously reported, and a tendency for an association with coeliac disease (OR=1.35; P=0.08). In primary sclerosing cholangitis, no association was observed (OR=0.95; P=0.8). Furthermore, we confirmed the increased risk in rheumatoid arthritis (OR=1.58; P=0.001), but could not find support for an association with systemic lupus erythematosus (OR=0.94; P=0.8). Altogether, we have provided further evidence of an association between autoimmune diseases and the 1858C>T polymorphism in PTPN22.     

Genome-wide linkage analysis of Scandinavian affected sib-pairs supports presence of susceptibility loci for celiac disease on chromosomes 5 and 11

Celiac disease (CD) is a common chronic inflammatory disorder of the small intestine with a multifactorial aetiology. HLA is a well-known risk factor, but other genetic factors also influence disease susceptibility. To identify the genes involved in this disorder, we performed a genome-wide scan on 106 well-defined Swedish and Norwegian families with at least two affected siblings. We investigated familial segregation of 398 microsatellite markers, and utilised non-parametric linkage analysis. The strongest linkage with disease was found to the HLA locus (6p) (P<0.000006). There were eight regions besides HLA with a point wise P value below 0.05. Among these eight regions were 11q and 5q, both of which have been suggested in several linkage studies of independent celiac disease families. We also performed a stratification analysis of families according to their HLA genotypes. This resulted in significant differences on chromosome 2q. These results indicate that 11q, 5q and possibly also 2q are true susceptibility regions in CD.     

Identification of a Gliadin T-Cell Epitope in Coeliac Disease: General Importance of Gliadin Deamidation for Intestinal T-Cell Recognition

Coeliac disease probably results from a T-cell response to wheat gliadin and is associated to HLA-DQ2. No gliadin epitopes recognized by intestinal T cells have yet been identified, limiting our understanding of the pathogenesis. Gut-lesion-derived DQ2-restricted T cells from coeliac disease patients were used to identify an epitope within a purified γ-type gliadin. The structure of the epitope was characterized by mass spectrometry and verified by synthesis. The epitope (QPQQSFPEQQ) results from deamidation of a distinct glutamine in the native structure. This deamidation is important for binding to DQ2 and T-cell recognition. Other gut-derived T cells fail to recognize the epitope, although deamidation of unfractionated gliadin enhances the response of all gut-derived DQ2-restricted T cells isolated from several patients. Several DQ2-restricted T-cell epitopes exist, but for all of them deamidation of glutamine residues appears to be critical for creation of active epitopes. Native gliadin has few negatively charged residues but is very rich in glutamine. After deamidation gliadin becomes a rich source of DQ2 epitopes thus providing a link between DQ2, gliadin and coeliac disease. The necessity for modification may have general immunological relevance.     

T lymphocyte clones recognizing an HLA-DQw3.2-associated epitope involving residue 57 on the DQ β chain

The DQw3.2 specificity has previously been recognized using genomic RFLP analysis and certain combinations of monoclonal antibodies. Here we report three CD4+ T lymphocyte clones (TLCs) generated from a DR3,4; DQw2,w3.1 responder stimulated with cells from a DR3,4; DQw2,w3.2 donor, and using a modified cloning procedure involving enrichment of IL-2 receptor-positive T cell during priming. The resulting TLCs were strongly inhibited by some monoclonal anti-DQ, but not anti-DR or -DP antibodies. In panel studies using HLA homozygous stimulating cells, it was found that the TLCs recognize an HLA epitope encoded by a DQ gene carried only by DR4,DQw3.2 haplotypes. By comparison with published DQ chain amino acid sequences of some stimulating cells able or not to induce a response in these clones, evidence was obtained that Ala at position 57 on the DQ β chain is most probably involved in the epitope. The epitope is present on cells from 12 out of 12 DR4,DQw3 insulin dependent diabetes mellitus (IDDM) patients, but on cells only from 6 out of 12 healthy DR4,DQw3 controls. Thus, a DQ-encoded epitope involving residue 57 on the DQ β chain, and which is strongly associated to IDDM, may be recognized by T cells.