Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs

AIMS: To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues. METHODS: This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared. RESULTS: Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessles. CONCLUSIONS: These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.     

Chip-based mtDNA mutation screening enables fast and reliable genetic diagnosis of OXPHOS patients.

PURPOSE: Oxidative phosphorylation is under dual genetic control of the nuclear and the mitochondrial DNA (mtDNA). Oxidative phosphorylation disorders are clinically and genetically heterogeneous, which makes it difficult to determine the genetic defect, and symptom-based protocols which link clinical symptoms directly to a specific gene or mtDNA mutation are falling short. Moreover, approximately 25% of the pediatric patients with oxidative phosphorylation disorders is estimated to have mutations in the mtDNA and a standard screening approach for common mutations and deletions will only explain part of these cases. Therefore, we tested a new CHIP-based screening method for the mtDNA. METHODS: MitoChip (Affymetrix) resequencing was performed on three test samples and on 28 patient samples. RESULTS: Call rates were 94% on average and heteroplasmy detection levels varied from 5-50%. A genetic diagnosis can be made in almost one-quarter of the patients at a potential output of 8 complete mtDNA sequences every 4 days. Moreover, a number of potentially pathogenic unclassified variants (UV) were detected. CONCLUSIONS: The availability of long-range PCR protocols and the predominance of single nucleotide substitutions in the mtDNA make the resequencing CHIP a very fast and reliable method to screen the complete mtDNA for mutations.     

High frequency of BRCA1/2 germline mutations in 42 Belgian families with a small number of symptomatic subjects

AIM: The initial risk assessments for BRCA1/2 mutation carriers and estimates of carrier frequencies were based on extended pedigrees with a large number of symptomatic subjects. When counselling based on BRCA gene mutation analysis was initiated, we faced requests for counselling mostly from members of small families with only two or three affected members. We report on the likelihood of finding a BRCA mutation in such small families. METHODS: In the first 100 families that came for oncogenetic counselling since September 1994, a BRCA1/2 gene mutation screen was initiated if there were two or more symptomatic first degree relatives, if one of them had ovarian cancer, or if one breast cancer was diagnosed before the age of 50 years. RESULTS: BRCA gene mutations were found and confirmed by sequencing in 14 out of 42 families (33%); 10 mutations were in the BRCA1 gene and four in the BRCA2 gene. Our findings indicate an increased probability of detecting a BRCA gene mutation when ovarian cancer occurred in the family. There is no increased probability of detecting a mutation with increasing numbers of breast cancers. Only 22% of the eligible presymptomatic family members opted for testing. The presymptomatic female carriers currently prefer breast surveillance rather than prophylactic surgery. CONCLUSION: BRCA1/2 gene mutation testing can be done with reasonable efficiency in the Belgian population when there are two symptomatic family members. The availability of testing does not lead to a high frequency of requests for testing by presymptomatic family members.     

Egfl7 knockdown causes defects in the extension and junctional arrangements of endothelial cells during zebrafish vasculogenesis.

The endothelial cell (EC) -specific secreted protein EGFL7 is important for tubulogenesis in newly forming blood vessels. We studied its role in vascular tube formation by a quantitative ultrastructural analysis of Egfl7-knockdown zebrafish embryos. At 24 hours postfertilization, the endothelia of dorsal aorta (DA) and posterior cardinal vein (PCV) were correctly anchored to the hypochord and endoderm, respectively, but failed to expand into the vascular area. This resulted in vessels with reduced or split lumen and open sheets of ECs. Concomitantly, the organization of hematopoietic cells-identified by the presence of previously undescribed membrane tubules-between DA and PCV, and within the vessels, was severely disturbed. Strikingly, ectopic cell junctions occurred across the obstructed vessel lumen, on the luminal EC surfaces, which in control conditions never display junctions of any kind. These data suggest that Egfl7 provides ECs with a cue for their extension into the vascular area and in establishing EC cell polarity.     

Immunology of DNA. VI. The effect of mercaptans on IgG and IgM anti-dsDNA.

Reduction of IgM antibodies to DNA with mercaptans such as dithioerythrol or 2-mercaptoethanol completely destroys DNA binding in the Farr assay and in the immunofluorescence technique by Crithidia luciliae. In contrast reduction of IgG antibodies to DNA results in a six-fold increase of DNA binding in the Farr assay while no effect on titres in the immunofluorescence technique can be observed. Our results lead to the following conclusions: 1) The Farr assay is selective for high avidity interactions; only a minor part of IgG antibodies to DNA is measured; 2) 7S IgM antibodies to DNA cannot be demonstrated in the Farr assay or the immunofluorescence technique; obviously only multivalent interactions, as obtained with the intact 19S IgM molecule are stable in these assays; 3) reduction of IgG leads to a greater flexibility of this molecule; this facilitates monogamous bivalent binding to DNA; 4) THE PRESENTATION OF DNA in the kinetoplast of Crithidia luciliae favours a monogamous bivalent binding of antibodies to DNA with high avidity; this accounts for occasionally observed discrepancies between anti-DNA activity in the Farr assay and in the immunofluorescence technique.     

Early detachment of titanium particles from various different surfaces of endosseous dental implants

Titanium (Ti) endosseous dental screws with different surfaces (smooth titanium--STi, titanium plasma-sprayed-TPS, alumina oxide sandblasted and acid-etched--Al-SLA, zirconium oxide sandblasted and acid etched--Zr-SLA) were implanted in femura and tibiae of sheep to investigate the biological evolution of the peri-implant tissues and detachment of Ti debris from the implant surfaces in early healing. Implants were not loaded. Sections of the screws and the peri-implant tissues obtained by sawing and grinding were analysed by light microscopy immediately after implantation (time 0) and after 14 days. All samples showed new bone trabeculae and vascularised medullary spaces in those areas where gaps between the implants and host bone were visible. In contrast, no osteogenesis was induced in the areas where the implants were initially positioned in close contact with the host bone. Chips of the pre-existing bone inducing new peri-implant neo-osteogenesis were surrounded by new bone trabeculae. The threads of some screws appeared to be deformed where the host bone showed fractures. Ti granules of 3-60 microm were detectable only in the peri-implant tissues of TPS implants both immediately after surgery and after 14 days, thus suggesting that this phenomenon may be related to the friction of the TPS coating during surgical insertion.     

Gender-related influences on the development of chronic graft-versus-host disease-induced experimental lupus nephritis.

Autoimmune diseases are far more common in women than in men. In the incidence of systemic lupus erythematosus (SLE), the female-to-male ratio is as high as 10:1. This suggests that sex hormones may play a fundamental role in determining the susceptibility to these diseases. In order to investigate the sex-related differences in the inducibility of chronic graft-versus-host disease-related experimental lupus nephritis, lymphocytes from female DBA/2 donor mice were administered to either male or female (C57BL10 x DBA/2)F1 recipients. An additional group of male recipients received lymphocytes from male DBA/2 donors. After four cell transfers, female recipients developed a significantly higher albuminuria than both male groups. Serum concentrations of autoantibodies against glomerular basement membrane (GBM), collagen IV, and laminin were significantly higher in females 2-4 weeks after induction. Levels of circulating autoantibodies against renal tubular epithelial antigens (RTE) and nuclear antigens were not different between the sexes. In transfer studies, the necessity of the presence of anti-GBM and anti-RTE autoantibodies for the development of glomerulonephritis was confirmed. These findings indicate that: (i) in this model of lupus nephritis, susceptibility to glomerulonephritis is strongly influenced by sex-related genes; and (ii) among the variety of autoantibodies occurring in this model of SLE, both anti-GBM and anti-RTE autoantibodies play a key role in the pathogenesis of glomerulonephritis.