The mechanisms of coronary restenosis: insights from experimental models

Since its introduction into clinical practice, more than 20 years ago, percutaneous transluminal coronary angioplasty (PTCA) has proven to be an effective, minimally invasive alternative to coronary artery bypass grafting (CABG). During this time there have been great improvements in the design of balloon catheters, operative procedures and adjuvant drug therapy, and this has resulted in low rates of primary failure and short-term complications. However, the potential benefits of angioplasty are diminished by the high rate of recurrent disease. Up to 40% of patients undergoing angioplasty develop clinically significant restenosis within a year of the procedure. Although the deployment of endovascular stents at the time of angioplasty improves the short-term outcome, 'in-stent' stenosis remains an enduring problem. In order to gain an insight into the mechanisms of restenosis, several experimental models of angioplasty have been developed. These have been used together with the tools provided by recent advances in molecular biology and catheter design to investigate restenosis in detail. It is now possible to deliver highly specific molecular antagonists, such as antisense gene sequences, to the site of injury. The knowledge provided by these studies may ultimately lead to novel forms of intervention. The present review is a synopsis of our current understanding of the pathological mechanisms of restenosis.     

Defects of natural killer cell activity in children with untreated acute lymphocytic leukemia

Summary Natural killer (NK) activity against cells of the K-562 line was significantly depressed in 12 of 18 children (66%) with untreated acute lymphocytic leukemia (ALL). No suppression of allogeneic NK activity was observed with sera of the patients, regardless of the level of NK depression. The ability of peripheral blood lymphocytes (PBLs) to suppress allogeneic NK activity was tested in two ALL patients — one with no detectable NK activity, and one with high NK activity. No NK-suppressive activity was found with PBLs of the areactive patient; PBLs of the reactive patients exhibited some suppressive activity, but only at a particular suppressor-to-effector cell ratio. Leukemic blasts were resistant to killing by autologous NK cells stimulated by IFN, as well as to killing by allogeneic, IFN-stimulated PBLs. Leukemic blasts of an ALL patient inhibited lysis of K-562 cells in an 18-h, but not in a 4-h NK assay. The inhibition could partly be reversed by pretreatment of ALL cells with alpha interferon, suggesting that the blasts might inhibit the lysis of K-562 targets in a competitive manner. Disturbed function and/or regulation of NK cells may influence attempts at NK cell activation by lymphokines.Abbreviations ALL acute lymphocytic leukemia - AML acute myeloid leukemia - CLL chronic lymphocytic leukemia - CML chronic myeloid leukemia - IFN interferon - IL-2 interleukin-2 - LGL large granular lymphocyte - NK cell natural killer cell - PBL peripheral blood lymphocyte - Poly I:C polyinosinic-polycytidylic acid     

Analogies and differences between ω-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells

 The characteristics of the binding sites for the Conus magus toxins ω-conotoxin MVIIC and ω-conotoxin MVIID, as well as their effects on K⁺-evoked ⁴⁵Ca²⁺ entry and whole-cell Ba²⁺ currents (I _Ba), and K⁺-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [¹²⁵I] ω-conotoxin GVIA to bovine adrenal medullary membranes was displaced by ω-conotoxins GVIA, MVIIC and MVIID with IC₅₀ values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [¹²⁵I] ω-conotoxin MVIIC, which was displaced by ω-conotoxins MVIIC, MVIID and GVIA with IC₅₀ values of around 30, 80 and 1.200 nM, respectively. The sites recognized by ω-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC₅₀ values of around 1 nM). Both ω-conotoxin MVIIC and MVIID blocked I _Ba by 70–80%; the higher the [Ba²⁺]_o of the extracellular solution the lower the blockade induced by ω-conotoxin MVIIC. This was not the case for ω-conotoxin MVIID; high Ba²⁺ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba²⁺. A further difference between the two toxins concerns their reversibility; washout of ω-conotoxin MVIIC did not reverse the blockade of I _Ba while in the case of ω-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by ω-conotoxin GVIA, suggesting that it is associated with N-type Ca²⁺ channels. Blockade of K⁺-evoked ⁴⁵Ca²⁺ entry produced results which paralleled those obtained by measuring I _Ba. Thus, 1 μM of each of ω-conotoxin GVIA and MVIIA inhibited Ca²⁺ uptake by 25%, while 1 μM of each of ω-conotoxin MVIIC and MVIID caused a 70% blockade. K⁺-evoked catecholamine secretory responses were not reduced by ω-conotoxin GVIA (1 μM). In contrast, at 1 μM both ω-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca²⁺ channels that contribute to the regulation of secretion and are sensitive to ω-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for ω-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca²⁺ channels or an entirely new subtype of voltage-dependent high-threshold Ca²⁺ channel. Received: 16 April 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

Distribution of cytoskeletal and contractile proteins in normal and tumour bearing salivary and lacrimal glands

Summary We have evaluated by means of immunocytochemistry the distribution of various cytoskeletal and contractile proteins (cytokeratins, vimentin, desmin and -smooth muscle actin) in 23 salivary or lacrimal gland primary tumours (15 pleomorphic adenomas and 8 carcinomas in pleomorphic adenoma), one third of which contained areas of normal gland. Normal epithelial luminal cells were stained by cytokeratin antibodies with a general specificity, while myoepithelial cells were selectively stained by a monoclonal antibody (SK2-27) reacting in immunoblots with cytokeratin polypeptides 14, 16 and 17, according to the classification of Moll et al. (1982) and by an antibody directed against -smooth muscle actin (Skalli et al. 1986). In pleomorphic adenomas, both epithelial and myoepithelial cells displayed typical topographic distributions; moreover, myoepithelial cells showed two distinct cytoskeletal phenotypes. These findings could account in part for the heterogeneity of aspects observed in this tumour. In carcinomas, malignant cells were always positive to cytokeratin antibodies with general specificity and myoepithelial cells were absent as judged by anticytokeratin SK2-27 and anti--smooth muscle actin immunostainings. However, interestingly, there was in all cases a strong positivity for -smooth muscle actin in stromal cells, similarly to what has previously been described for mammary carcinoma (Skalli et al. 1986). Our findings may be useful for the interpretation of the histogenesis of salivary and lacrimal tumour and stromal cells.     

Peripheral nerve grafts lacking viable Schwann cells fail to support central nervous system axonal regeneration

Summary Peripheral nerve grafts were implanted bilaterally into the diencephalon of adult hamsters. One graft segment contained both viable Schwann cells and their basal lamina tubes. The Schwann cell population in the second graft segment was killed by freezing prior to implantation. Seven weeks after graft implantations, the extracranial end of each graft segment was exposed, transected and labelled with a fluorescent tracer substance. One week after the labelling procedure each animal was perfused and the diencephalon and midbrain were examined. Ultrastructural analyses of both types of graft demonstrated the persistence of the Schwann cell-derived basal lamina tubes. Retrogradely labelled neurons were found in all cases in which an intact graft remained in place for two months, but were seen in only one case with a frozen graft. Large numbers of myelinated and unmyelinated axons were seen within the intact grafts, but no axons were found in the previously frozen grafts. These results indicate that lesioned CNS axons are able to regenerate vigorously when provided with an environment which includes viable Schwann cells. But, CNS axons regenerate less well, if at all, when Schwann cells are absent. Further, it appears that Schwann cell-derived basal lamina tubes, when isolated from their parent cells, are insufficient to initiate or sustain CNS axonal regeneration.This material is based upon work supported by the National Science Foundation under grant BNS-8416911     

The relationship between epithelial Ia expression and the inflammatory cell infiltrate during experimental oral carcinogenesis

Summary The development of oral epithelial expression of Ia antigens and its relationship to the presence of IL-2r⁺ (CD25⁺) cells was investigated in rats treated with the water soluble carcinogen 4-nitroquinoline-N-oxide (4NQO). Acetone fixed frozen sections of the palate and tongue were stained using an indirect immunoperoxidase technique and monoclonal antibodies to rat Ia (I-A & I-E) and IL-2 receptor. After 4 weeks 4NQO treatment all rats expressed oral epithelial Ia but thereafter (2–9 months) expression was present in only 20–40% of animals. Epithelial expression of Ia by histologically normal, dysplastic and neoplastic epithelium was always associated with the presence of an underlying inflammatory cell infiltrate containing CD25⁺ cells. Overall there were significantly more CD25⁺ cells in tissue specimens containing Ia⁺ epithelium compared with Ia^– epithelium. Furthermore, during the first 4 weeks of carcinogen treatment, a significant positive correlation was found between the CD25⁺ cell density and occurrence of focal epithelial Ia expression. These results, together with analysis of the T cell, NK cell, macrophage and B cell content of the infiltrates induced by 4NQO, suggest that the CD25⁺ cells represent activated T cells. Thus, our results in this experimental model are consistent with the idea that epithelial expression of Ia is the result of production of IFN- by locally activated T cells.     

A model for antigen-induced T cell unresponsiveness based on autophosphorylative protein tyrosine kinase activity

Helper T cell signaling is initiated by the aggregation of TCRwith the induction of tyrosine kinase activity as one of theearliest consequences. Here, a theoretical model for antigen-inducedunresponsiveness is presented that relies on a cascade of tyrosinephosphorylation- dephoshorylation cycles. A mechanism is describedfor both desensitization in the presence of antigen and persistentlowering of cell responsiveness after stimulus removal. An importantcomponent of the model, leading to bistability, is the presenceof autophosphorylating protein tyrosine kinases in the earlysteps of TCR signaling. One of its predictions is that, followingstimulation, the net phosphorylative activity of these receptor-associatedtyrosine kinases will remain above background level after removalof the antigen. It is proposed that this residual tyrosine kinaseactivity is linked to a deficient signal transduction capacityof the TCR system that leads to a state of prolonged unresponsiveness.In addition, the present analysis defines the notion of a signalingthreshold for hyporesponsiveness induction, associated witha durable switch and amplification of the net tyrosine kinaseactivity. This approach emphasizes the role of tyrosine kinasesin the down-regulation of cellular competence.     

Myoblasts fail to stimulate T cells but induce tolerance

Recent interest in myoblast transfer and in the use of myoblastsas vehicles in gene therapy has made it important to understandthe potential immunogenicity of allogeneic or neoantlgen-expresslngmyoblasts. Given the problems of producing a pure populationof myoblasts, In this study we used a tumour-derived musclecell line (TE671), with phenotyplc features of myoblasts, whichwe transfected to express HLA-DR1. However, this cell line wasunable to stimulate either established HLA-DR1-specific alloreactlveT cell clones or a primary alloresponse. Nor could it presenthaemagglutlnln peptide HA 306–324 to DR1-restricted, HA306–324-speciflc T cell clones or lines. Indeed, prelncubatlonwith DR1-expressing TE671 and HA 306–324 rendered suchT cells tolerant as Judged by their subsequent inability toproliferate in response to a DR1⁺ B cell line plus peptide HA306–324. These results imply that myoblasts do not providecostlmulatory signals, and are therefore unlikely to stimulateallospeclfic T cells following myoblasts transplantation orto initiate neoantlgen-speclfic immune responses following Invivo transfection.     

Ganglion cell density and oil droplet distribution in the retina of brown-eared bulbul (Hysipetes amaurotis)

This study was intended to determine the number and density of both retinal ganglion cells and the oil droplets of cone photoreceptor cells in brown-eared bulbul (Hysipetes amaurotis). For this study birds were killed with proper dose of anesthetic (pentobarbital, 30 mg/kg), and the eyes were removed from the orbital cavity to isolate the retina. For the ganglion cell study retinal whole-mount specimens were prepared and stained with 0.1% cresyl violet. The different types of oil droplets were counted from color microphotographs of freshly prepared retinal samples. The mean total number of ganglion cells was estimated at approximately 2.5×10⁶; with an average density of 16 523 cells/mm². Two high-density areas, namely the central area (CA) and the dorso-temporal area (DTA), are located in the central and dorso-temporal retinas, respectively, in bulbuls (24 032 cells/mm² in the CA; 23 113 cells/mm² in the DTA). Small ganglion cells persisted in the highest density areas, whereas the largest soma sizes were found in the lowest density areas of the retina. Four types of different colored oil droplets — red, orange, green and clear — were identified with an average density of 29 062/mm². Among the different colors, the green oil droplets had a significantly higher population (13 083/mm²) than the others across the retina. The central retina had a significantly higher number of all types of oil droplets, at a density of 60 552/mm². The density and size of the different colored oil droplets were inversely related across the regions of the retina. Taken together, it is concluded that the CA of the retina is an excellent quality area for visual perception due to peak density of ganglion cells and oil droplets. Moreover, each specific oil droplet makes a distinct contribution to visual perception, thereby ensuring that the bird has a retina that best matches its natural environment and feeding behavior.