Tissue factor pathway inhibitor can interact with platelets

Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. However, there is no report on interaction between TFPI and platelets other than that by Tsuji, who found that whole blood anticoagulated with TFPI exhibited remarkable decrease in platelet count. Our study revealed that washed platelets suspended in modified Tyrode's buffer (8 mM CaCl2) containing TFPI exhibit platelet aggregation. However, platelets aggregation was observed without TFPI, but its increase and intensity were slow and weak, compared to that in the presence of TFPI. This aggregation was inhibited by anti-CD41 (anti-GPIIb) antibody. This finding suggested that TFPI promotes platelet aggregation.     

Evidence of allosteric conformational changes in the antibody constant region upon antigen binding

We have addressed the question of whether antigen binding induces a conformational change in the heavy chain constant (C(H)) domain of antibodies using staphylococcal protein A or streptococcal protein G as probes, since these proteins are known to bind to IgG domains such as C(H)1 and C(H)2-C(H)3 domains. Biosensor assays on interactions between these proteins and mouse IgG specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) or their enzymatic fragments conducted in the presence or absence of the hapten, NP-epsilon-aminocaproic acid (NP-Cap), showed that the binding of IgG to these proteins was inhibited by the binding of NP-Cap. The results of isothermal titration calorimetry also revealed that the association constant for the interaction of protein A with IgG2b decreased by the addition of NP-Cap. These results suggested that antigen binding induced conformational changes in binding sites for protein G or protein A located at C(H)1 and C(H)2-C(H)3 domains, respectively.     

Chitosan hydrogel as a drug delivery carrier to control angiogenesis

An aqueous solution of photocrosslinkable chitosan containing azide groups and lactose moieties (Az-CH-LA) incorporating paclitaxel formed an insoluble hydrogel within 30 s of ultraviolet light (UV) irradiation. The chitosan hydrogel showed strong potential for use as a new tissue adhesive in surgical applications and wound dressing. The fibroblast growth factor (FGF)-2 molecules retained in the chitosan hydrogel and in an injectable chitosan/IO₄-heparin hydrogel remain biologically active, and were gradually released from the hydrogels as they biodegraded in vivo. The controlled release of biologically active FGF-2 molecules from the hydrogels caused induction of angiogenesis and collateral circulation occurred in healing-impaired diabetic (db/db) mice and in the ischemic limbs of rats. Paclitaxel, which is an antitumor reagent, was also retained in the chitosan hydrogel and remained biologically active as it was released on degradation of the hydrogel in vivo. The chitosan hydrogels incorporating paclitaxel effectively inhibited tumor growth and angiogenesis in mice. The purpose of this review is to describe the effectiveness of chitosan hydrogel as a local drug delivery carrier for agents (e.g., FGF-2 and paclitaxel) to control angiogenesis. It is thus proposed that chitosan hydrogel may be a promising new local carrier for drugs such as FGF-2 and paclitaxel to control vascularization.     

Genetic approaches for the identification of apoptotic components

Jun amino-terminal kinase (JNK) mediates a physiological stress signal that leads to cell death. However, the role of the JNK pathway in intrinsic cell death execution mechanisms is largely unknown. In a genetic screen for dominant suppressors of Reaper (Rpr)-induced cell death, we identified Drosophila chromosomal regions that contain genes which are homologous to apoptosis signal-regulating kinase (ASK1) and Drosophila tumor necrosis factor receptor-associated factor 1 (DTRAF1). We present evidence that the killer signal initiates the JNK pathway via proteasome-mediated degradation of Drosophila inhibitor of apoptosis protein 1 (DIAP1) to promote cell death.     

Microsatellite instability in primary and metastatic lung carcinomas

Fifty-seven primary lung carcinomas and 35 metastatic lung carcinomas were analyzed for microsatellite instability at 11 different chromosomal loci. Although no instability was detected in 37 small cell lung carcinomas (SCLC), it was frequently detected in non-small cell lung carcinomas (NSCLC) (16/55, 29%). In NSCLC, the incidence of replication errors (RERs) in metastatic tumors (12/22, 55%) was significantly higher than that in primary tumors (4/33, 12%) (P = 0.0021). Among 10 pairs of primary tumors and corresponding metastases, there were 4 cases which manifested the identical RER phenotypes in both primary and metastatic tumors. In two cases, RER phenotypes were detected in metastatic but not in primary tumors. Never was an RER phenotype found only in a primary tumor but not in the metastases. RERs were detected more frequently in stage III or IV tumors (3/8, 38%) than stage I or II tumors (1/25, 4%) (P = 0.0359). Tumor cells with allelic losses on chromosome arm 3p or 18q tended to have RER phenotypes (P = 0.0432 and P = 0.0187, respectively). The data suggest that microsatellite instability is common in NSCLC but not in SCLC, and that genomic instability appears late in tumor progression and plays an important role in the acquisition of more malignant phenotypes in NSCLC.     

PROLIFERATIVE MYOSITIS AND FASCIITIS: Report of Five Cases with an Ultrastructural and Immunohistochemical Study

Cases of proliferative myositis and fasciitis were studied immunohisto-chemically and ultra structurally for further understanding of the nature of ganglion cell-like giant cells. Blood coagulation factor XIIIa, fibronectin, myoglobin, myosin, CPK MM, and alpha-1-antichymotrypsin were detected in three cases of proliferative myositis and two cases of proliferative fasciitis by the avid in-biotin-peroxidase complex method. Factor XIIIa (a fibrin-stabilizing factor) and flbronectin were strongly positive in the giant cells, but not in striated muscle fibers. A small quantity of myosin was demonstrated in the giant cells, but myoglobin and CPK MM were never demonstrated in these cells. No alpha-1-antichymotrypsin was demonstrated in the giant cells. One case of proliferative myositis showed ultrastructural features suggestive of fibroblast rather than muscle cell or histiocytic origin. Strongly positive factor XIIIa in the giant cells is suggestive of the fact that they are active fibroblasts.     

Evaluation of an automated hematologyanalyzer (CELL-DYN 4000) for counting CD4+ T helper cells at low concentrations

In human immunodeficiency virus (HIV) infection, CD4 cell counts are useful in defining the disease state, monitoring antiviral treatment, and identifying patients at risk for opportunistic infections. Counting CD4 cells typically relies on traditional immuno-flow cytometric analyzers that require opening the tube for manipulation of the blood sample. In addition to automated blood cell counting, the CELL-DYN 4000 hematology analyzer performs a completely enclosed and automated analysis of the T lymphocyte subsets. We studied the performance characteristics of this method in blood samples containing low levels of CD4+ T cells. In one set of experiments, we emulated low level CD4 counts by use of a CD4 Positive Isolation Kit to deplete the CD4+ cells from blood samples. We used the FACScan analyzer for reference counts. Measurements were made exactly 12 hr after venepuncture in samples that were stored at room temperature. In normal samples and those with low CD4+ cell counts, there was excellent correlation between the results of the CELL-DYN and FACScan methods. Using the CELL-DYN 4000 analyzer, the precision of CD4+ and CD8+ T-cell counts was high (CV = 2 to 8%). The CD4+ T-cell count was linear over a wide range (35 to 1640 cells/microl). This study shows that CD4 and CD8 T cell counts using the CELL-DYN 4000 analyzer is suitable for normal samples and also for those with low CD4+ T cell counts. The method is rapid and automated, and blood specimens remain enclosed, minimizing the biohazard of exposure to blood of HIV patients.