Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis

Abstract: A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.     

Inter- and intramolecular interactions in polyelectrolyte complex formation with polyampholytes

Employing polyampholytes (inclusively polybetaines) of different chemical structure containing carboxylic groups and various basic nitrogen functions, homosymplex formation, as well as the competition between homo- and heterosymplex formation on addition of an appropriate polyelectrolyte, was investigated in dependence of pH and ionic strength by means of viscometry and turbidimetry. With most, but not with all, of the polyampholytes, the expected viscosity minimum at the isoelectric point, with its steepness depending on polyampholyte structure, was observed. Competition of homo- and heterosymplex formation at and near the isoelectric point is mainly governed by the pK values of the species involved, the level of zwitter-ion formation of the polyampholyte and the effect of non-Coulombic interactions, for example, via hydrogen bonds.     

Down-modulation of CD4+ T helper type 2 and type 0 cells by T helper type 1 cells via Fas/Fasligand interaction

Fas was recently demonstrated to be the major target molecule engaged by CD4⁺ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4⁺ or CD8⁺ CTL. A reciprocal relationship in Fas and Fas-ligand expression was observed in CD4⁺ T helper (Th)1- and Th2-type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas-ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas-ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4-mediated lysis. A functional consequence of the inverse Fas-Fas-ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4⁺ CTL and a CD8⁺ CTL clone in a Fas-dependent manner. These results suggest that cytolytic CD4⁺ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas-mediated lysis.     

C-peptide immunoreactive neurons in human brain

C-peptide/C-peptide-like immunoreactivity was shown to be present in the cytoplasm of the soma and the proximal part of apical dendrites of some pyramidal cells in the Neocortex (Gyrus precentralis) and Hippocampus of man. C-peptide (connecting peptide) is a metabolic product in insulin biosynthesis and its localization in neurons is a proof for extrapancreatic insulin production.     

Clinical and pathological characterization of an osteoma in a barred owl

Osteomas are rare in birds. An osteoma of the left proximal radius was diagnosed in an adult barred owl based on gross, radiographic, and pathologic findings.     

Mikrokallusformationen der Spongiosa Ein bisher unterschätzter reparativer Mechanismus des Skelettsystems

Zusammenfassung Mikrokallusformationen lassen sich in nahezu allen Skelettabschnitten der Spongiosa nachweisen. Mikrokallus besteht aus Geflechtknochen, der sich an lokal überbelasteten Stellen in der Spongiosa bildet. Mit Hilfe einer speziellen Pr?parationstechnik wurden 26 skelettgesunde und 11 Wirbels?ulen von F?llen mit Osteoporose untersucht. Mikrokallusformationen finden sich bevorzugt bei Frauen ?lter als 45 Jahre in den unteren Wirbels?ulenabschnitten. Dabei hat die Mikroarchitektur der Spongiosa (TBPf) einen st?rkeren Einflu? auf die Anzahl der Mikrokalli, als individuelle Trabekelparameter (Tb.N, BV/TV und Tb.Th). Nur in 33 % der Formationen lassen sich Frakturspalten nachweisen. Mikrokallusformationen k?nnen nichtinvasive Knochenmassemessungen verf?lschen. Auch wenn Mikrokallusformationen Indikatoren für eine Instabilit?t der Spongiosa sind, tragen sie zur Knochenregeneration bei, und die Entstehung neuer Trabekel ist durch sie m?glich. Die Vorstellung, da? Osteoporose das Resultat einer verminderten Mikrokallusbildung ist, trifft nicht zu.      

Mitogenicity of Entamoeba histolytica extracts for murine lymphocytes.

Aqueous extracts or aqueous extracts of delipidated Entamoeba histolytica (E.h.e.) contain a mitogenic principle for murine lymphocytes. As detected by [3H]-thymidine incorporation and blast transformation, E.h.e. acted predominantly on T cells of splenic origin, but not on thymocytes or bone marrow cells. Furthermore, E.h.e. induced proliferation of a subset of non-T-cells which is present in the spleen of athymic nude mice, adhered to nylon wool, but could not be activated to produce antibody. It seems possible that polyclonal activation of lymphocytes by E. histolytica might play a role in the disturbance of the immune system as manifested in the impaired cell mediated immune response of E. histolytica infected hosts.     

Immunohistochemistry (S 100, KL 1) and human papillomavirus DNA hybridization on Morbus Bowen and bowenoid papulosis

Summary In this study 55 paraffin embedded samples defined as Bowen's disease or bowenoid papulosis were investigated with antibodies against S 100 protein and keratins (KL 1). S 100-positive cells were quantified and related to defined section area of the epidermal compartment by computer-assisted image analysis. The density of S 100-positive cells was compared with normal skin and was particularly related to growth patterns and keratinization of the different lesions under study. S 100-positive dendritic cells were found to be reduced overall in bowenoid lesions when compared with normal skin. Lesions with high counts of S 100-positive dendritic cells most frequentty showed a solitary growth pattern with highly conserved architecture and differentiation and no tendency to stromal invasion. In contrast, cases with low counts of S 100-positive cells very often showed multifocal development, a high degree of architectural disturbance and dedifferentiation. In this group, stromal invasion (cases of invasive carcinoma associated with Bowen's disease) was seen more often. Interestingly, this latter group of cases also revealed a peculiar keratin pattern. Frequently, the basal cell layer was decorated with KL 1 antibody, which usually recognizes only suprabasaly located keratinocytes. No differences between Bowen's disease and bowenoid papulosis were found in terms of densities of S 100-positive dendritic cells and keratin pattern. In our experience, extragenital Bowen's disease and genital Bowen's disease can not be distinguished on purely morphological grounds or with the immunocytochemical approach presented here. Interestingly, when employing in situ hybridization with HPV 16 probes three of seven samples of genital Bowen's disease harboured HPV 16 DNA, whereas six cases of extragenital disease were negative.Supported by the Deutsche Forschungsgemeinschaft (Lo 285/2-4)