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.     

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.     

Immunohistochemical study on tissue transglutaminase and copper-zinc superoxide dismutase in human myocardium: Its relevance to apoptosis detected by the nick end labelling method

The influence of free radicals on apoptosis was studied in the human heart; 45 autopsy cases were examined by the nick end labelling method (NELM) that detects DNA fragmentation. Immunostaining for copper-zinc superoxide dismutase (CuZn-SOD) and tissue transglutaminase (tTG) induced frequently during apoptosis were also studied. Positive immunoreaction for tTG was detected in mucinous degeneration of myocardial cells; these same cells were also positive for CuZn-SOD but negative for NELM. Myocardial cells showing basophilic alterations after haematoxylin and eosin staining were also positive for CuZn-SOD but negative for the other markers examined. Positive nuclear reaction by NELM was only observed in myocardial cells showing contraction band necrosis or irregularly shaped nuclei surrounding recent or long-standing infarcted foci. In these the other two markers were negative. Since mucinous degeneration lacks the distinguishing morphological features of apoptosis, immunoreactive tTG in this lesion may not imply that the cells are undergoing apoptosis. tTG can be induced in non-apoptotic conditions and may not be involved in apoptosis induced by infarction. Histological disassociation between CuZn-SOD expression and apoptosis suggests the possibility of a cytoprotective role played by endogenous CuZn-SOD against free radical generation in the human heart.     

Cloning of TPA-inducible early (TIE) genes by differential hybridization using TPA-Nonresponsive variant of mouse 3T3-L1 cells

The tumor promoter 12-O -tetradecanoylphorbol-13-acetate (TPA) induces DNA synthesis in quiescent 3T3-L1 cells but not in its variant VT-1 cells. A gt10 cDNA library was constructed using poly(A)⁺ RNA from 3T3-L1 cells that were stimulated by TPA for 20 min. Radioactive cDNA probes were prepared from mRNAs of TPA-treated 3 T3-L1 and VT-1 cells and used for screening of the 3T3-L1 cDNA library by differential hybridization. Nine of 6000 phage plaques hybridized only to the 3T3-L1 cDNA probe. Analysis of the nucleotide sequence of five of these clones indicated a high degree of homology with human or mouse type I and type III collagen genes. Three other independent clones showed no homology with any known DNA sequences. These isolated clones of TPA-inducible early (TIE) genes may be useful to study the signal transduction pathway of phorbol esters.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.