Tissue engineering of bone for mandibular augmentation in immunocompetent minipigs: preliminary study.

Large mandibular defects caused by trauma, infection or resection of a tumour are still a major problem for plastic and maxillofacial surgeons. The modern concept of tissue engineering combines the osteoinductive effects of osteogenic cells with a suitable scaffold structure to promote differentiation of osteoblasts and optimal matrix production. Critical size mandibular bone defects were therefore made to investigate the osteogenic potential of periosteal cells and a bioabsorbable polymer fleece (Ethisorb 510) in minipigs. Periosteal cells were isolated from four minipigs, expanded in vitro and seeded with fibrin glue into Ethisorb 510 fleeces. Tissue constructs were used to repair critical size mandibular defects and compared with two minipigs with untreated bone defects. Bone healing was evaluated after 90 and 180 days by radiographs and a histological scoring system. The radiographs showed increased radiodensity of defects filled with the cell-fibrin-fleece-constructs compared with the untreated control group after 90 and 180 days in vivo. The defects repaired by the cell-fibrin-scaffolds (180 days in vivo) obtained the highest histological mean score 2.9 (range 2-3), while defects filled by cell-fibrin-scaffolds (90 days in vivo) achieved a mean score of 2.1 (range 2-3). In contrast, the control group (n = 2) scored 1 and 2. The results show that a combination of periosteal cells and polymer fleeces may be a promising approach for clinical mandibular augmentation.     

Konstitutionelle Hypermobilität

Benign hypermobility is a constitutional condition and is linked to somatic type. The spinal column and the extremity joints are more flexible from childhood and throughout adult life in the average person affected. This body build is believed to be due to thin, hypotonic and even weak muscles in most cases. Symptoms, clinical examination, the development of pain and patient management, including possible methods of prevention, are presented.     

Clozapine-induced agranulocytosis and hereditary polymorphisms of clozapine metabolizing enzymes: no association with myeloperoxidase and cytochrome P4502D6

The pathomechanisms of most drug-induced agranulocytoses are unclear; however, there are some studies pointing to genetic determinants. Some drug-induced agranulocytoses such as clozapine-induced agranulocytosis (CA) may be regarded as an idiosyncratic drug reaction because of its preclinical and clinical characteristics. To study some aspects of the genetic background of CA further, polymorphisms of specific metabolizing enzyme systems of clozapine were examined. Thirty-one schizophrenic patients with CA and 77 schizophrenic comparison subjects without this adverse effect underwent genotyping of a recently discovered G(-463)A polymorphism of myeloperoxidase (MPO) gene and cytochrome P4502D6. Neither the MPO mutation nor specific genotypes of cytochrome P4502D6 were associated with CA. Both were equally distributed among CA patients and controls. Thus, our data suggest lack of evidence of an association of CA and genetically variable activity of these specific drug metabolizing enzymes; however, this may be due to statistical reasons only. Thus, further studies with greater CA samples are necessary to draw final conclusions about these genetically based hypotheses.     

First clinical evaluation of the CELL-DYN® 3200 haematology analyser

A prototype of the CELL-DYN 3200® haematology analyser was evaluated in a tertiary care hospital laboratory. Precision, effects of sample ageing, linearity, carry-over, and com-parability of cellular blood counts and five-part leucocyte differentiation were determined in accordance with the ICSH guidelines for the evaluation of blood cell analysers; the results were satisfactory for all parameters tested: haemoglobin concentration, RBC, MCV, WBC, platelet count, and counts of neutrophils, lymphocytes, monocytes, and eosinophils. Two-hundred and forty-seven routine blood samples were used for the comparability studies. The cellular blood count results from the CELL-DYN 3200® and the Bayer Diagnostic H-1 systems corresponded closely (correlation coefficient r > 0.96 for all parameters). For 201 samples without an instrument-generated suspect flag the same was true with regard to the dif-ferential parameters, although somewhat lower correlation was observed for monocyte counts (r = 0.88). Comparisons to 400-cell microscopic differentials gave similar results (r > 0.93 for neutrophil, lymphocyte and eosinophil counts). Our results suggest that the CELL-DYN 3200® analyser will serve the needs for automated blood cell counting and differential leucocyte counting in a tertiary care hospital laboratory.     

Manufacture and in vitro Characterization of a Solvent/Detergent-Treated Human Plasma

We have developed a modified solvent/detergent (S/D) treatment to inactivate viruses in human plasma using 1% w/w final concentration of tri(n-butyl) phosphate (TNBP) and Triton X-100 and an incubation period of 4 h at 30 degrees C. The procedure inactivates > or = 10(6) chimpanzee-infectious doses (CID50) of HBV, > or = 10(5) CID50 of HCV, and > or = 10(6.2) tissue culture infectious doses (TCID50) of HIV. After virus inactivation, eleven plasma batches were lyophilized and 12 batches were deep-frozen until further use. The batches were characterized by extensive laboratory tests including measurement of clotting factors I-XIII, von Willebrand factor, plasminogen, inhibitors of blood coagulation and fibrinolysis, and other clinically important plasma proteins. All parameters were determined before and after S/D treatment. Twelve conventional single donor plasma units served as control. There were no marked losses of activities of clotting factors, antithrombin III, protein C, plasminogen, and C1-esterase inhibitor due to treatment. After the S/D step, the levels of these parameters were within the normal range in all batches. The same holds true for total protein, immunoglobulins, albumin, complement factors C3 and C4, haptoglobin, hemopexin, caeruloplasmin, alpha 1-antitrypsin, and pH. Protein S and alpha 2-antiplasmin activities decreased by about 50% and were frequently found to be slightly below the lower limit of the respective normal range after treatment. The interindividual variations of all proteins analysed were significantly lower than in the single donor plasma units. The S/D procedure did not lead to increases of markers indicating activation of hemostasis.(ABSTRACT TRUNCATED AT 250 WORDS)     

IgM anti-dsDNA antibodies in systemic lupus erythematosus: negative association with nephritis

Antibodies against dsDNA of the IgM class were measured in sera of 352 patients with systemic lupus erythematosus, 81 blood donors and 189 patients with rheumatoid arthritis using a new ELISA based on human recombinant dsDNA as antigen. IgM anti-dsDNA antibodies were found in 52.3% of the sera from patients with systemic lupus erythematosus, but in none of the sera from 81 normal controls and 189 patients with rheumatoid arthritis. The association of these autoantibodies with 31 clinical and 37 laboratory parameters was calculated. There was a highly significant negative correlation between IgM anti-dsDNA antibodies and nephritis as well as all the laboratory parameters indicating renal disease (elevated serum creatinine concentration, proteinuria, erythrocyte casts in the urine). IgM anti-dsDNA antibodies indicate protection of lupus patients against the development of lupus nephritis. Further experiments will show whether application of IgM anti-dsDNA antibodies is effective in treating lupus nephritis. Received: 10 August 1998 / Accepted: 11 September 1998     

Antigen stored in dendritic cells after macropinocytosis is released unprocessed from late endosomes to target B cells

B lymphocytes can be triggered in lymph nodes by nonopsonized antigens (Ag), potentially in their native form. However, the mechanisms that promote encounter of B lymphocytes with unprocessed antigens in lymph nodes are still elusive. We show here that antigens are detected in B cells in the draining lymph nodes of mice injected with live, but not fixed, dendritic cells (DCs) loaded with antigens. This highlights active processes in DCs to promote Ag transfer to B lymphocytes. In addition, antigen-loaded DCs found in the draining lymph node were CD103+. Using 3 different model Ag, we then show that immature DCs efficiently take up Ag by macropinocytosis and store the internalized material in late endocytic compartments. We find that DCs have a unique ability to release antigens from these compartments in the extracellular medium, which is controlled by Rab27. B cells take up the regurgitated Ag and the chemokine CXCL13, essential to attract B cells in lymph nodes, enhances this transfer. Our results reveal a unique property of DCs to regurgitate unprocessed Ag that could play an important role in B-cell activation.     

Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids

Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.Release of virus particles from infected cells is the last essential step of the viral replication cycle. In the course of this process, virions face the challenging task of crossing the cell envelope. Viruses have developed an arsenal of diverse strategies to overcome this problem. Most bacterial viruses are lytic and induce lysis of the infected cell with help of the holin-endolysin system (1), whereas others disrupt the host cell envelope via inhibition of the murein biosynthesis pathway (2). The morphological and genomic properties of archaeal viruses (3) suggested that their egress from host cells may have unusual traits that are different from those of bacterial viruses. Indeed, although most archaeal viruses exit cells without lysis, some, in particular the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) and Sulfolobus turreted icosahedral virus (STIV), are lytic and exploit a special mechanism of virion egress (4–8). During the infection cycle of these viruses, pyramidal protrusions with sevenfold rotational symmetry form in the host cell membrane. As the final step of the infection cycle the virus-associated pyramids (VAPs) open outwards along the seams of their seven facets, creating ∼100-nm apertures through which the newly formed virions escape from the host cell (4, 7). VAPs consist of multiple copies of an ∼10-kDa virus-encoded protein, which we term “PVAP” (Protein forming Virus-Associated Pyramids/SIRV2_P98) (7–9). Surprisingly, PVAP assembles into membrane pyramids even when expressed heterologously in archaeal and bacterial expression systems, demonstrating that no other viral proteins are required for VAP formation (7). The mechanism by which VAPs self-assembles in the membrane remains unknown.In the present study we used electron cryotomography to investigate morphological features of SIRV2 replication and the formation of VAPs at different time points after infection. By subtomogram averaging, we determined a 3D map of the VAP. This map, in combination with secondary structure predictions of PVAP and the expression of wild-type (WT) PVAP or a variety of truncation mutants in archaeal, bacterial and eukaryotic cells allows us to propose a model showing how PVAP arranges to form the sevenfold pyramids. These insights are fundamental for understanding how this mechanism can be exploited as a universal tool to engineer the formation and controlled opening of large pores in biological or artificial lipid bilayers.