Morphology and sedimentology of (clustered) cold-water coral mounds at the south Rockall Trough margins,NE Atlantic Ocean

Cold-water coral mounds on both margins of the Rockall Trough (NE Atlantic Ocean) have a strongly different morphology. Single, isolated mounds occur on the SE margin and are mainly found on the upper slope between 900 and 650 m water depth, while large mound clusters are found on the SW margin in water depths between 600 and 1,000 m, in a narrow zone almost parallel to the slope. Sedimentation rates on the mounds are higher than on the surrounding seabed as a result of baffling of biogenic carbonate debris and siliciclastic particles by the coral framework covering the mounds. This is confirmed by ²¹⁰Pb measurements. The individual coral growth rate can be three times higher then the vertical growth rate of the coral cover (±10 mm year⁻¹) which in turn is more than an order of magnitude higher then the present-day overall mound growth rate (±0.25 mm year⁻¹). The presence of extensive hardgrounds and firmgrounds and the three-dimensional coral framework are considered to be responsible for the stability of the relatively steep slopes of the mounds. High current velocities in the intramound areas result in local non-sedimentation and erosion, as is shown by the presence of IRD (ice-rafted debris) lag deposits on the seabed and moats around some of the mounds. The morphology and sedimentology of cold-water coral-covered (mainly Lophelia pertusa and Madrepora oculata) mounds on the southern Rockall Trough margins (NE Atlantic Ocean) is discussed and a model describing the development of these mounds is presented.     

Thymidylate synthase expression in patients with colorectal carcinoma using a polyclonal thymidylate synthase antibody in comparison to the TS 106 monoclonal antibody.

Colorectal cancer is one of the most common human cancers, for which 5-fluorouracil (5FU) is usually part of the treatment. Thymidylate synthase (TS), the target enzyme for 5FU, can be predictive for the outcome of 5FU-based therapy. TS levels in tumor samples can be determined with radiochemical enzyme assays, RT-PCR, and immunohistochemical staining. We validated TS immunohistochemistry with a polyclonal rabbit anti-human TS antibody using the avidin-biotin method. This antibody can be used on paraffin-embedded, formalin-fixed material using an antigen retrieval method with citrate buffer and microwave treatment. The antibody shows a granular cytosolic staining pattern. The reproducibility in cross-sections from colorectal tumors from 50 patients was 90% and the interobserver variability was acceptable with a kappa of 0.45. On Western blotting it detects purified TS at 36 kD, while in 5FU-treated cells the ternary complex between FdUMP, TS, and 5, 10-methylene-tetrahydrofolate is clearly visible at 38 kD, with no other interfering bands. In a separate set of tumors, immunostaining was compared with enzyme levels; Western blots correlated with enzyme levels. Because both this polyclonal antibody and the monoclonal antibody TS-106 are being used for large-scale studies, we also determined whether they could be used interchangeably. No differences were observed. This polyclonal antibody is specific and gives reproducible results. A study on a larger scale is ongoing to determine the role of TS as a predictive parameter in patients with colorectal cancer treated either with postoperative adjuvant 5FU/levamisole or with surgery only.     

Simultaneous immunoenzyme staining of vimentin and cytokeratins with monoclonal antibodies as an aid in the differential diagnosis of malignant mesothelioma from pulmonary adenocarcinoma

The occurrence and coexpression of the cytoskeletal proteins vimentin and cytokeratins were studied in malignant mesotheliomas and pulmonary carcinomas. For this purpose a double immunoenzyme staining with monoclonal antibodies was developed which made it possible to visualize vimentin and cytokeratins simultaneously within the same cell. A clear distinction between stromal cells (vimentin only) and tumour cells was also obtained. A total of 12 mesotheliomas (six mixed type and six epithelioid type) and 13 carcinomas (eight adenocarcinomas and five large cell undifferentiated carcinomas) were studied. The results revealed a clear difference between mesotheliomas and adenocarcinomas: 11 of 12 mesotheliomas showed coexpression of vimentin and cytokeratins in at least 50% of the tumour cells, while in seven of the eight adenocarcinomas none or only a few cells could be seen with this coexpression. In the undifferentiated large cell carcinomas three of five expressed both components, but in less than 25% of the cells. It is concluded that a reliable double immunoenzyme staining of vimentin and cytokeratins can be used as an additional means to distinguish malignant mesothelioma from pulmonary adenocarcinoma.     

Reactivity against complementary proteinase-3 is not increased in patients with PR3-ANCA-associated vasculitis

The etiology of anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides (AAV) is unknown, but the association between infections and autoimmunity has been studied extensively. In 2004, a novel theory was proposed that could link infection and autoimmunity. This 'theory of autoantigen complementarity' was based on the serendipitous finding of antibodies against complementary-PR3 (cPR3) in patients with PR3-ANCA-associated vasculitis. cPR3 demonstrated homology to several bacterial proteins, and it was hypothesized that PR3-ANCA develop in response to anti-cPR3 antibodies, as a consequence of the anti-idiotypic network. These data have not been confirmed in other patient cohorts. We investigated the presence of anti-cPR3 antibodies in a Dutch cohort of PR3-ANCA-associated vasculitis patients. Anti-cPR3 reactivity was determined in serum using ELISA. Two separate batches of cPR3 were used to determine reactivity in two separate cohorts of PR3-ANCA-associated vasculitis patients. We found that anti-cPR3-reactivity was not increased in our PR3-ANCA-associated vasculitis patients, in comparison to control groups. Further research will be necessary to prove the concept of autoantigen complementarity in autoimmune diseases.     

Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences

Conditions for combination of DNA in situ hybridization, using biotinylated DNA probes, with immunohistochemistry were investigated on cryostat sections, cytological preparations, and paraffin sections. We found that cryostat sections and cytological preparations are suitable for in situ hybridization of target DNA after fixation in acetone, methanol, ethanol, or Carnoy without further proteinase pretreatment. Acetone is also very suitable for immunostaining of cell surface or cytoskeleton antigens. We therefore performed combined immunoenzyme and in situ hybridization staining using this fixative. The best results were obtained when immunoperoxidase staining with diaminobenzidine/H2O2 was followed directly by in situ hybridization. In addition to immunoperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) staining with naphthol ASBI phosphate and New Fuchsin as a substrate could be used. In most instances, detection of the biotinylated hybrid with a streptavidin-biotinylated polyalkaline phosphatase method using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as the substrate was preferable. The double stainings were studied on the following test models: (a) frozen tonsil sections: cell surface antigens (pan T) and ribosomal DNA; (b) frozen genital condyloma sections; cytokeratins and human papillomavirus type 6 + 11 (HPV-6/11) DNA; (c) CaSKi cells: cytokeratins and HPV-16 DNA; (d) infected fetal lung fibroblasts: vimentin and cytomegalovirus (CMV) DNA. An adapted procedure was followed on routinely formaldehye-fixed and paraffin-embedded condyloma tissue. Immunoperoxidase staining for papilloma virus capsid antigen could be combined with DNA in situ hybridization with HPV-6/11 DNA. In this model, however, the accessibility of the target DNA had to be improved by enzyme treatment after the immunostaining and before starting the in situ hybridization.     

A conceptual model explaining benthic foraminiferal microhabitats

We present a conceptual model which explains benthic foraminiferal microhabitat preferences in terms of differences in the downward organic flux. We argue that under oligotrophic conditions the microhabitat depth is controlled by the availability of metabolizable food particles in the sediment. Under more eutrophic conditions, the ecosystem is no longer food-controlled, but instead, a critical oxygen level determines down to what depth we find a living fauna. Under food-limited conditions, anaerobic degradation of organic matter may provide an additional food source around the redox front, which could explain deep infaunal maxima reported in the literature. In a sample transect through the Adriatic Sea, both microhabitat controls (food-limited and oxygen-limited) are present. On the shelf and the upper part of the slope, the rather shallow depth of the microhabitat is controlled by a critical oxygen level. In the 1250 m deep southern basin and on the lower part of the slope, on the contrary, the availability of metabolizable organic matter, and not a critical oxygen level, determines down to what depth living foraminifera are found.     

Increased expression of Toll-like receptors by monocytes and natural killer cells in ANCA-associated vasculitis

Introduction

Toll-like receptors (TLRs) are a family of receptors that sense pathogen associated patterns such as bacterial cell wall proteins. Bacterial infections are associated with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Here, we assessed the expression of TLRs 2, 4, and 9 by peripheral blood leukocytes from patients with AAV, and investigated TLR mediated responses ex vivo.

Methods

Expression of TLRs was determined in 38 AAV patients (32 remission, 6 active disease), and 20 healthy controls (HC). Membrane expression of TLRs 2, 4, and 9, and intracellular expression of TLR9 by B lymphocytes, T lymphocytes, NK cells, monocytes and granulocytes was assessed using 9-color flowcytometry. Whole blood from 13 patients and 7 HC was stimulated ex vivo with TLR 2, 4 and 9 ligands and production of cytokines was analyzed.

Results

In patients, we observed increased proportions of TLR expressing NK cells. Furthermore, patient monocytes expressed higher levels of TLR2 compared to HC, and in a subset of patients an increased proportion of TLR4⁺ monocytes was observed. Monocytes from nasal carriers of Staphylococcus aureus expressed increased levels of intracellular TLR9. Membrane expression of TLRs by B lymphocytes, T lymphocytes, and granulocytes was comparable between AAV patients and HC. Patients with active disease did not show differential TLR expression compared to patients in remission. Ex vivo responses to TLR ligands did not differ significantly between patients and HC.

Conclusions

In AAV, monocytes and NK cells display increased TLR expression. Increased TLR expression by these leukocytes, probably resulting from increased activation, could play a role in disease (re)activation.     

Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies

A number of fixation and decalcification procedures were evaluated to determine their suitability for immunohistochemistry on trephine samples of bone marrow after paraffin embedding. In particular, the immunoreactivity of antigens characteristic for various hematopoietic cell lines (immunoglobulin heavy and light chains for plasmacytoid cells; elastase for neutrophil myeloid cells; lysozyme, alpha-1-antitrypsin and alpha-1-antichymotrypsin for hystiocytic cells; leukocyte common antigen for lymphocytes; hemoglobin and glycophorin A for erythroid cells; Factor VIII-related antigen for thrombocytoid cells) as well as some antigens specific for epithelial tumors (CEA, 115D8, and keratin) were investigated. Fixation in a mercuric chloride-formaldehyde mixture followed by decalcification in acetic acid-formaldehyde-saline proved to be the best procedure for antigen preservation and retention of morphologic detail. Moreover, there is no need of trypsinization when using this procedure. The only exception was Factor VIII-related antigen in megakaryocytes, which was best demonstrated in trypsin-digested sections of formalin-fixed and acetic acid-decalcified biopsies.