HLA-DR2 in two sibships with insulin-dependent diabetes mellitus.

We report two families selected from 124 genotyped Caucasian insulin-dependent diabetes mellitus (IDDM) families because of unusual features. In both families, all offspring are affected and four out of six bear the allele HLA-DR2 which is an uncommon phenotype among diabetic patients. Onset before the age of 1 year in all the patients of one family, association with optic atrophy in the other, and the existence of pairs of affected sibs of different HLA types in both, are infrequent findings and support the evidence of heterogeneity in IDDM.     

Alveolar bone loss in rats after immunization with Actinomyces viscosus.

We investigated a possible cause-and-effect relationship between sensitization against Actinomyces viscosus Nyl and destructive periodontal disease in RIC-Sprague-Dawley rats. Germfree rats (66) were either immunized with A. viscosus Nyl (day 20) or orally infected with A. viscosus Nyl (days 38 and 39) or both. We measured alveolar bone loss in maxillary and mandibular molars, in vitro T-lymphocyte responsiveness, and serum antibody titers. In immunized and monoassociated rats bone loss in both jaws progressed rapidly between days 37 and 72, whereas the rate of further resorption decreased until day 100. In monoassociated rats, development of bone loss was much slower, and the maximum resorption measured was, at best, half of the bone loss compared with the former group. However, no amplification of bone loss by immunization was observed in a second experiment using 63 conventional rats kept in relative gnotobiosis. Antibody titers to A. viscosus Nyl in gnotobiotic monoassociated rats were higher in immunized animals, whereas no difference was found in the respective groups of the relative gnotobiotic experiment. The fact that immunization more than doubled alveolar bone loss in gnotobiotic monoassociated rats confirms the allergic nature of the disease. The lack of such an effect under conventional conditions points to suppressor mechanisms whose decrease might convert stable periodontal lesions into progressive ones.     

Immunocytochemical characterization of lymphocytes in benign and malignant lymphocyte - rich serous effusions

Summary The cytological diagnosis of malignant Lymphoma in serous effusions can be difficult because reactive lymphocytes may be morphologically indistinguishable from malignant cells in lymphocytic and other low grade Non-Hodgkin's lymphomas. As a result of the present study, diagnostic accuracy can be improved by means of B- and T-cell enumeration using an immunoalkaline-phosphatase method (IAP). 30 cytological specimens, including 28 pleural, 1 pericardial and 1 ascitic fluids, were studied with a panel of monoclonal anti B- and anti T-cell antibodies (PAN B, kappa, lambda, T1, T2, OKT4, T8). Reactive lymphocytic effusions were characterized by a predominance of T cells constituting 80% of all lymphocytes with an excess of helper/inducer cells (mean helper to suppressor ratio 3.0) and by a surface kappa to surface lambda ratio of 1.6 on B-cells. Tuberculous effusions showed a similar distribution of lymphocyte-subpopulations whilst most of the carcinomatous fluids showed a lower percentage of T cells (lowest value 67%) and lower Th:Ts ratio (mean 2.0). Lymphoid cells in samples of five B-cell lymphomas were characterized by T-cell depression ( 70%). B-cells in three cases expressed clear cut light chain monoclonality which was at least suggested in the other two cases.Lymphoid cells from two cases of Hodgkin's disease expressed an indistinct immunological pattern. Labelling of cytoplasmic immunoglobulins (heavy and light chains) using the peroxidase antiperoxidase method (PAP) may be important to characterize neoplasms of the plasma cell series.It is concluded that the chosen panel of antibodies in combination with IAP labelling method may be of great value in identifying B-cell lymphomas. The technique can be used in the routine laboratory and storage of unlabelled and labelled slides over long periods is possible.Dedicated to Professor K. Lennert, Kiel, on the occasion of his 65th birthdayThis study was supported by the Krebsliga St. Gallen/Appenzell     

Setting out the frame conditions for feasible use of FFPE derived RNA

Introduction

The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.

Assessment of phagocytic and antimicrobial activity of human granulocytes

A microassay for assessing two functions of polymorphonuclear leukocytes in the presence or absence of complement is presented. Requiring only minute amounts of blood (0.1 ml) and a minimum of laboratory equipment (microscope, incubator, and centrifuge), it allows the quantitation of phagocytosis and intracellular killing of microorganisms by leukocytes. To demonstrate the value of this assay, the phagocytic and microbicidal activity of leukocytes from healthy subjects and patients against Candida albicans was investigated. Apart from individual cases, no differences in the phagocytic activity between groups of healthy subjects and patients with candida vaginitis or different types of cancer could be found. However, the killing capacity of the leukocytes from women with recurrent candida vaginitis was reduced. The leukocytes of one patient showing a very low killing capacity lacked myeloperoxidase. Also, low values of killing were seen with leukocytes from three patients suffering from osteosarcoma, chronic lymphatic leukemia, or Hodgkins disease.     

Defensin deficiency, intestinal microbes, and the clinical phenotypes of Crohn's disease

Crohn's disease is a chronic, inflammatory disease of the intestinal mucosa. Although intestinal bacteria are implicated in disease pathogenesis, the etiology is still unclear. The main location of disease is the small intestine (ileum) and the colon. Ileal disease has been linked to a mutation in the NOD2 gene. Defensins are antimicrobial peptides and in the ileum, are mainly expressed in Paneth cells, epithelial cells that also express NOD2. In the colon, defensins are expressed by enterocytes or metaplastic Paneth cells. Crohn's disease patients with ileal involvement, compared with controls or Crohn's patients without ileal involvement, have diminished expression of ileal Paneth cell defensins. This decrease is even more pronounced in Crohn's patients displaying a NOD2 mutation. In contrast, Crohn's disease of the colon is characterized by an impaired induction of beta-defensins in enterocytes. The colonic expression of the constitutive beta-defensin 1 is also decreased in the inflamed colonic mucosa, but this decrease is less specific to Crohn's disease, as it can also be found in ulcerative colitis patients. In conclusion, the regional localizations of Crohn's disease, ileal or colonic disease, can be linked to different defensin profiles. Crohn's disease of the ileum is associated with diminished defensin expression in Paneth cells. Crohn's disease of the colon is associated with diminished beta-defensin expression in enterocytes. Thus, it can be speculated that decreased defensin levels lead to a weakened intestinal barrier function to intestinal microbes and might be crucial in the pathophysiology of Crohn's disease.     

Enhanced expression of transforming growth factor-beta type I and type II receptors in wound granulation tissue and hypertrophic scar.

In the present study we have analyzed and compared, by immunohistochemistry and in situ hybridization, the expression pattern of the R4/ALK5 transforming growth factor (TGF)-beta type I receptor (RI) and the TGF-beta type II receptor (RII) in normal human skin, in wounded skin at various stages during the transition of wound granulation tissue to scar, and in long-persisting post-burn hypertrophic scars. In normal human skin, expression of RI and RII was clearly visible in the epidermis, in epidermal appendages, and in vascular cells, although only a small number of dermal fibroblasts revealed detectable levels of TGF-beta receptor expression. In contrast, granulation tissue fibroblasts showed strong expression of both TGF-beta receptor types, although in normal-healing excisional wounds their density decreased during granulation tissue remodeling. However, in post-burn hypertrophic scars, RI- and RII-overexpressing fibroblasts were found in high densities up to 20 months after injury. From these findings we suggest that the repair process of deep wounds involves the transformation of a subset of fibroblastic cells toward an increased TGF-beta responsiveness and a transient accumulation of these cells at the wound site. In addition, our study provides evidence that excessive scarring is associated with a failure to eliminate TGF-beta receptor-overexpressing fibroblasts during granulation tissue remodeling, which leads to a persistent autocrine, positive feedback loop that results in over-production of matrix proteins and subsequent fibrosis.     

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Three novel human VMD2-like genes are members of the evolutionary highly conserved RFP-TM family

The RFP-TM protein family was first described in Caenorhabditis elegans as hypothetical transmembrane proteins containing a conserved 350-400 amino acid domain including the invariant peptide motif RFP. The VMD2 gene underlying Best disease was shown to represent the first human member of the RFP-TM protein family. More than 97% of the disease-causing mutations are located in the N-terminal RFP-TM domain implying important functional properties. Here, we have identified three novel VMD2-related human genes (VMD2L1, VMD2L2 and VMD2L3) demonstrating a high degree of conservation in their respective RFP-TM domains. Each of the VMD2-like proteins has a unique C-terminus that lack similarity to other proteins or motifs. By FISH analysis, VMD2L1 was localised to chromosome 19p13.2-p13.12, VMD2L2 to 1p32.3-p33 and VMD2L3 to 12q14.2-q15. RT-PCR analyses revealed tissue-restricted expression of the three genes with both VMD2L1 and VMD2L2 abundantly transcribed in colon. VMD2L1 is present in the retinal pigment epithelium while VMD2L3 shows predominant expression in skeletal muscle.     

Immunohistochemical distribution of chromogranins A and B and secretogranin II in neuroendocrine tumours of the gastrointestinal tract

The aim of the present study was to investigate immunohistochemically the distribution of chromogranin A, chromogranin B, and secretogranin II in a series of 152 neuroendocrine tumours of the gastrointestinal tract. Tumour tissues from 25 argyrophil gastric carcinoids, 18 gastrin and 5 somatostatin-producing tumours, 4 gangliocytic paragangliomas, 49 classical argentaffin and 2 L cell appendiceal carcinoids, 27 classical ileal carcinoids, 17 rectal carcinoids, and 5 poorly differentiated neuroendocrine tumours of the stomach and rectum were immunostained with antibodies against chromogranin A, chromogranin B, and secretogranin II. Chromogranin A was the major granin expressed in gastric carcinoids and in serotonin-producing carcinoids of the appendix and the ileum. In contrast, strong chromogranin B and secretogranin II immunoreactivity was found in rectal carcinoids, in which chromogranin A was rarely expressed. Since chromogranin A is a widely used marker for neuroendocrine differentiation, it is of diagnostic importance that some gastrin-producing tumours, gangliocytic paragangliomas, poorly differentiated neuroendocrine carcinomas, and appendiceal L cell carcinoids completely lacked chromogranin A positivity. It is concluded that the various neuroendocrine tumours of the gastrointestinal tract show distinctly different patterns of granin expression, probably reflecting their histogenetical origin.