Group-specific amplification of HLA-DQA1 revealed a number of genomic full-length sequences including the novel HLA alleles DQA1*01:10 and DQA1*01:11.

In this article, we describe a subgroup-specific amplification assay for HLA-DQA1 that encompasses the whole coding region and allows us to sequence full-length HLA-DQA1 genes. We introduce the novel alleles HLA-DQA1*01:10 and HLA-DQA1*01:11. Moreover, we were able to confirm the full-length genomic sequence data of the alleles HLA-DQA1*01:07, HLA-DQA1*03:01:01, HLA-DQA1*03:02, HLA-DQA1*04:01:02, HLA-DQA1*04:02, HLA-DQA1*05:03, HLA-DQA1*05:05:01:02 and HLA-DQA1*06:01:01. A complete genomic overview of all six HLA-DQA1 allele groups is now available from the submission of our data to the IMGT/HLA database. Because our approach facilitates the analysis of all HLA-DQA1 allele sequences, HLA-DQA1 may become the first HLA locus from which all subgroup members will be known in detail in the near future.

Immune response to Cystoisospora suis in piglets: local and systemic changes in T-cell subsets and selected mRNA transcripts in the small intestine.

Infections of neonatal piglets with Cystoisospora suis are responsible for substantial economic losses in pig production. To investigate kinetics of T-cell populations, which are possibly involved in this infection, lymphocytes from blood, spleen, mesenteric lymph nodes and the jejunal mucosa of infected and noninfected piglets were investigated by flow cytometry and immunohistochemistry at five time points during the acute phase of primary infection. Additionally, mRNA expression levels of pattern recognition receptors and immunomodulatory cytokines in the jejunum were investigated. T-cell receptor-γδ(+) T cells were found to be increased in the gut mucosa 4 days after infection and were most likely involved in the primary local immune response. Five to eleven days later, cytotoxic T cells peaked in this location, which was preceded by an expansion of this lymphocyte population in the mesenteric lymph nodes. In intestines of infected piglets, mRNA expressions of TLR-2, NOD2 and TNF-α were significantly upregulated, suggesting an involvement in parasite recognition, immune response and possibly also in immunopathology. Taken together, this study identifies cellular and molecular players involved in the early immune responses against C. suis, but their precise role in the pathogenesis and control of this neonatal disease requires further investigation.

A novel HLA-A allele, A*66:17 was revealed in a family of a leukemia patient during high-resolution HLA typing.

HLA -A*66:17, presented here, shows a single-nucleotide polymorphism in exon 4 in comparison to A*66:01.

The novel allele DRB1*07:23 was revealed by LABType HD DRB1 typing test and confirmed by sequence-based HLA typing.

The novel allele HLA-DRB1*07:23 shows a single nucleotide change in comparison to DRB1*07:01:01:01.

B*07:156, revealed by sequence-based typing of a leukemia patient and confirmed by allele-specific amplification.

We present the full-length sequence of the novel allele HLA-B*07:156, closely related to B*07:02:01.

Sequence-based typing confirmed a new HLA-A allele, A*31:53, in a liver recipient patient.

In this report, we present the full-length coding sequence of A*31:53, a novel allele with a single-nucleotide difference in exon 3 with respect to A*31:01:02.

HLA-DQB1*06:41 shown by sequence-based typing may be associated with DRB1*13:02.

In this report we present DQB1*06:41, a novel allele probably associated with DRB1*13:02.

Sequence-based typing confirmed a new HLA-A allele, A*68:71.

In this report, we present the full length coding sequence of A*68:71, a novel allele with a single nucleotide difference to A*68:45.

[Effect of biological glues on vascular wall in an experimental model of aortic dissection]. / Vliv biologických lepidel na cévní stenu v modelu disekce aorty v experimentu.

INTRODUCTION: The use of biological glues and their application between the two dissection layers and into the anastomosis region is a common integral part of surgical management of thoracic aortic dissections. AIM: The aim of the experimental study was to assess and evaluate histopathological changes of vascular wall following deposition of the following three types of glue--GRF, Tissucol, Bioglue, based on qualitative and quantitative parameters. The secondary aim of the study was to assess dynamics of these changes depending on the glue effect duration and to formulate expected behaviour of the vascular wall during the time beyond the experimental period. METHODOLOGY: The dissection model was performed with pigs of the same gender and age, assigned to four groups. Different glues were used to close artificial infrarenal aortic dissections in Group 1-3, while direct suturing and no glue was used to close false lumini in Group 4. Samples of the dissected aorta were then collected at Month 1, 6 and 12 and then histologically examined. RESULTS: Upon assessment of the whole group of qualitative and quantitative parameters, the most significant changes in the smooth muscle histological picture were observed with the use GRF glue. The smooth muscle changes following the Bioglue application and, in particular, Tissucol glue application, are similar to changes observed in Group 4, where no glue was used. CONCLUSION: Based on the results, the authors present a hypothesis that, in a long-time horizont, vascular wall destructions, eventually redissections, are likely to occur more frequently in patients, in whom GRF glue is used.

HLA-DQB1*0404, a novel DQB1-allele detected in a volunteer blood platelet donor typed for HLA.

Introduction of a novel human leukocyte antigen-DQB1 allele, DQB1*0404, which featured one nucleotide mismatch in comparison with DQB1*0402.

A new polymorphic position in exon 2 shows the novel allele HLA-DPB1*123:01.

Introduction of a novel human leukocyte antigen-DPB1 allele, DPB1*123:01, which featured one nucleotide mismatch in comparison with DPB1*02:01:02.

Changes in lymphocyte populations in suckling piglets during primary infections with Isospora suis.

Isospora suis, a common intestinal parasite of piglets, causes neonatal porcine coccidiosis, which results in reduced and uneven weaning weights and economic losses in pig production. Nevertheless, there are no detailed studies available on the immune response to I. suis. The aim of this study was to carry out phenotypical characterization of lymphocytes during primary infections on day 3 after birth. Infected and noninfected piglets were investigated between days 7 and 16 after birth. Lymphocytes from the blood, spleen and mesenteric lymph nodes (flow cytometry) and of the jejunal mucosa (immunohistochemistry) were analysed. A decrease in T cells, especially with the phenotype of resting T-helper cells, T-cell receptor-gammadelta-T cells, and regulatory T cells in the blood, spleen and mesenteric lymph nodes was noticeable. An increase in cells with the phenotype of natural killer cells in the spleen of infected animals was found, and the subset of TcR-gammadelta-T cells was strongly increased in the gut mucosa. Our findings suggest an accelerated migration of those cells into the gut. This study provides a strong indication for the involvement of adaptive and innate immune response mechanisms in the primary immune response to I. suis, especially of TcR-gammadelta-T cells as a linkage between innate and adaptive immunity.

HLA-B*1832, a novel B allele was found through high-resolution HLA typing of a Spanish blood donor.

In this paper, we describe the novel human leukocyte antigen (HLA)-B*1832 allele that we found in a female Spanish volunteer blood donor for clinical investigation during her high-resolution HLA typing. The HLA-B typing is B*1801, 1832, and the DNA sequence is homozygous with the exception characterized by a nucleotide exchange 'C' to 'A' at position 505, which, in consequence, replaced arginine at codon 169 (CGC) by serine in the new allele B*1832.

In a study for acne vulgaris, sequence-based HLA typing showed a novel DPB1 allele, DPB1*2402.

In this paper, we characterize the novel human leucocyte antigen (HLA)-DPB1*2402 allele that we found in a patient suffering from acne vulgaris. In comparison to the closest related allele DPB1*0401, HLA-DPB1*2402 has a single nucleotide exchange at position 115 (202), T replaces G. In consequence, codon 39 (68) TAC encodes for tyrosine in the novel allele instead of aspartic acid 39 (68) GAC in DPB1*0401.

Sequence-based HLA high-resolution retyping of a bone marrow donor/recipient pair reveals the novel HLA allele DQB1*0322.

In this paper, we characterize the novel human leukocyte antigen (HLA)-DQB1*0322. We found this novel allele in a hematopoietic stem cell donor. The donor and the recipient were high-resolution HLA retyped using sequence-based typing. Both, the female patient and her donor were previously typed HLA identical, which was confirmed with the exception of the novel DQB1 allele. The novel allele is characterized by a nucleotide exchange 'G' to 'A' at position 485 in exon 3. This affected codon 130-arginine (CGG), which is replaced by glutamine (CAG) in the new allele DQB1*0322. The transplant was performed because of an acute myeloid leukemia at first remission.

Distribution of Connective Tissue in the Male and Female Porcine Liver: Histological Mapping and Recommendations for Sampling.

The pig is a large animal model that is often used in experimental medicine. The aim of this study was to assess, in normal pig livers, sexual dimorphism in the normal fraction of hepatic interlobular and intralobular connective tissue (CT) in six hepatic lobes and in three macroscopical regions of interest (ROIs) with different positions relative to the liver vasculature. Using stereological point grids, the fractions of CT were quantified in histological sections stained with aniline blue and nuclear fast red. Samples (415 tissue blocks) were collected from healthy piglets, representing paracaval, paraportal and peripheral ROIs. There was considerable variability in the CT fraction at all sampling levels. In males the mean fraction of interlobular CT was 4.7 ± 2.4% (mean ± SD) and ranged from 0% to 11.4%. In females the mean fraction of the interlobular CT was 3.6 ± 2.2% and ranged from 0% to 12.3%. The mean fraction of intralobular (perisinusoidal summed with pericentral) CT was <0.2% in both sexes. The interlobular CT represented >99.8% of the total hepatic CT and the fractions were highly correlated (Spearman r = 0.998, P <0.05). The smallest CT fraction was observed in the left medial lobe and in the paracaval ROI and the largest CT fraction was detected in the quadrate lobe and in the peripheral ROI. For planning experiments involving the histological quantification of liver fibrosis and requiring comparison between the liver lobes, these data facilitate the power analysis for sample size needed to detect the expected relative increase or decrease in the fraction of CT.

Differences in intestinal size, structure, and function contributing to feed efficiency in broiler chickens reared at geographically distant locations.

The contribution of the intestinal tract to differences in residual feed intake (RFI) has been inconclusively studied in chickens so far. It is also not clear if RFI-related differences in intestinal function are similar in chickens raised in different environments. The objective was to investigate differences in nutrient retention, visceral organ size, intestinal morphology, jejunal permeability and expression of genes related to barrier function, and innate immune response in chickens of diverging RFI raised at 2 locations (L1: Austria; L2: UK). The experimental protocol was similar, and the same dietary formulation was fed at the 2 locations. Individual BW and feed intake (FI) of chickens (Cobb 500FF) were recorded from d 7 of life. At 5 wk of life, chickens (L1, n = 157; L2 = 192) were ranked according to their RFI, and low, medium, and high RFI chickens were selected (n = 9/RFI group, sex, and location). RFI values were similar between locations within the same RFI group and increased by 446 and 464 g from low to high RFI in females and males, respectively. Location, but not RFI rank, affected growth, nutrient retention, size of the intestine, and jejunal disaccharidase activity. Chickens from L2 had lower total body weight gain and mucosal enzyme activity but higher nutrient retention and longer intestines than chickens at L1. Parameters determined only at L1 showed increased crypt depth in the duodenum and jejunum and enhanced paracellular permeability in low vs. high RFI females. Jejunal expression of IL1B was lower in low vs. high RFI females at L2, whereas that of TLR4 at L1 and MCT1 at both locations was higher in low vs. high RFI males. Correlation analysis between intestinal parameters and feed efficiency metrics indicated that feed conversion ratio was more correlated to intestinal size and function than was RFI. In conclusion, the rearing environment greatly affected intestinal size and function, thereby contributing to the variation in chicken RFI observed across locations.

How many Layers has the Adventitia? - Structure of the Arterial Tunica Externa Revisited.

Tunica adventitia or tunica externa is the outer layer of the blood vessel wall. It consists of connective tissue with vasa and nervi vasorum and plays a key role in vascular health. The aim of our study was to compare the wall layers beyond tunica media in arteries of different type and location. The following arteries of pig, dog and cat were processed histologically and analysed by light microscopy: aorta ascendens, arcus aortae, aorta thoracica, aorta abdominalis, arteria (a.) femoralis, a. tibialis cranialis, a. carotis communis, a. lingualis, a. basilaris, a. cerebralis media, a. testicularis and aa. jejunales. We found two layers of connective tissue outside the media: (1) a compact layer with many elastic fibres in muscular and few in elastic arteries and (2) an outer layer of loose connective tissue. The compact layer was missing in aorta ascendens, arcus aortae and intracranial vessels. Adventitial stripping removed only the loose connective tissue layer. In spite of the still present compact layer, stripped arteries were very flimsy. We suggest using the term 'tunica externa' for the compact connective tissue layer and 'tunica adventitia' for the outermost loose connective tissue layer as in other organs. The presence of the tunica externa differs between species, arteries and arterial side, as well as the removability of tunica adventitia and tunica externa by anatomical dissection.

Malignant potential and cytogenetic characteristics of occult disseminated tumor cells in esophageal cancer.

Although micrometastatic cancer cells in lymph nodes can be detected by monoclonal antibodies against epithelial or tumor-associated antigens, it remains unclear whether these cells are precursors of overt metastases or shedded tumor cells with a limited life span. Here we used esophageal cancer as a model to evaluate the prognostic significance and biological characteristics of such micrometastases. In lymph nodes classified as tumor free by conventional histopathological staging, tumor cells were identified with monoclonal antibody Ber-EP4 in 89 of 126 patients (71%) with completely resected (R0) esophageal carcinomas. Multivariate survival analysis underlined the strong and independent prognostic significance of Ber-EP4-positive cells in "node-negative" (pN0) patients. To assess the biology of Ber-EP4-positive cells, we established tumor cell lines from an immunohistochemically positive lymph node and the autologous primary tumor. p53 mutational analysis and multiplex-fluorescence in situ hybridization revealed common aberrations shared between both cell lines, whereas an insertion of chromosome 13 material in the short arm of chromosome 1 was only observed in micrometastatic cells. The tumorigenicity and metastatic potential of both cell lines were demonstrated in severe combined immunodeficient mice. In conclusion, our data provide first direct evidence for the malignant potential of micrometastatic cancer cells.

Phenotypic characteristics of cell lines derived from disseminated cancer cells in bone marrow of patients with solid epithelial tumors: establishment of working models for human micrometastases.

Bone marrow (BM) is a clinically relevant site of micrometastatic disease in patients with solid epithelial tumors. It is, therefore, important to establish suitable models that allow the in-depth characterization of disseminated tumor cells present at low frequencies of 10(-5)-10(-6) nucleated BM cells. The aim of this study was to assess common phenotypic features of nine tumor cell lines established from BM of patients with cancer of the prostate (four cell lines), breast (two cell lines), lung (two cell lines), and colon (one cell line) using immunocytochemistry, flow cytometry, and reverse transcription-PCR. All cell lines stained positive for both cytokeratins, the epithelial intermediate filaments, and the epithelial cell adhesion molecule E-cadherin, and they lacked markers of BM-derived cells. The tumor origin of the cell lines was supported by the expression of the ErbB2 oncogene (seven of nine) and MAGE mRNA (eight of eight). All cell lines coexpressed cytokeratin and vimentin, the mesenchymal intermediate filament, indicating an epithelial-mesenchymal transition of micrometastatic cells. The invasive phenotype of the immortalized cells was also reflected by the consistent expression of several metastasis-associated adhesion molecules, including alpha5 (eight of nine), alpha6 (five of nine), alphaV (nine of nine), beta1 (nine of nine), and beta3 (nine of nine) integrin subunits and the Mr 67,000 laminin receptor (seven of nine). Contrary to our expectations, metastasis-promoting CD44 variant isoforms were only detected on two lines, whereas all cell lines expressed MUC18/melanoma cell adhesion molecule and intercellular adhesion molecule-1, two members of the immunoglobulin superfamily of adhesion molecules that are not frequently found on primary carcinoma cells. The consistent expression of various epithelial and tumor-associated antigens provides evidence that the established cell lines are derived from disseminated cancer cells present in the BM. The invasive phenotype of the immortalized cells was mirrored by their epithelial-mesenchymal transition and the expression of several metastasis-associated molecules, which might be potential candidates for novel therapeutic targets.