The inhibitory effects of camptothecin, a topoisomerase I inhibitor, on collagen synthesis in fibroblasts from patients with systemic sclerosis.

The main manifestation of systemic sclerosis (SSc) is the overproduction of extracellular matrix, predominantly type I collagen. This study was undertaken to evaluate the effects of noncytotoxic doses of the topoisomerase I inhibitor camptothecin (CPT) on collagen production in the activated dermal fibroblasts from patients with SSc and healthy donors. The fibroblasts were cultured in the presence or absence of CPT. Production of collagenous proteins by fibroblasts was determined in cell and matrix layers by ELISA and in conditioned media by [(3)H]proline incorporation, gel electrophoresis, and autoradiography. Expression of alpha2(I) collagen (COL1A2) mRNA was measured by northern blot, and the activity of COL1A2 promoter was determined by a chloramphenicol acetyltransferase assay. CPT (10(-7) M) decreased the deposition of type I collagen by 68%, of type III by 38%, and of type VI by 21% in SSc fibroblasts and to a lesser degree in healthy controls. Similarly, CPT (10(-8) M to 10(-6) M) significantly inhibited secretion of newly synthesized collagenous proteins into conditioned media by 50%. CPT (10(-8) M to 10(-6) M) caused a significant dose-dependent inhibition of COL1A2 mRNA levels and COL1A2 promoter activity, both by as much as 60%. The inhibitory effect of CPT on collagen production by fibroblasts from patients with SSc suggests that topoisomerase I inhibitors may be effective in limiting fibrosis in such patients.

Modulation of lymphocyte-fibroblast interactions by 2-chloro-2'-deoxyadenosine (2-CdA) in subarachnoid hemorrhage.

The adherence to monolayers of human fibroblasts of chromium-51 labelled peripheral blood mononuclear cells (PBMC) from normal subjects, and from patients after single or multiple subarachnoid hemorrhage (SAH), and the effect of 2-CdA on cell adhesion have been quantified. The fraction of cells adhering to fibroblasts were the lowest for multiple SAH patients and the highest for normal subjects, which can be explained by depletion of activated lymphocytes from peripheral blood of SAH patients. 2-CdA decreased the fraction of adhering cells isolated from normal subjects, did not change the adherence of cells from single SAH patients and increased the fraction of adhering cells isolated from multiple SAH patients. We conclude that the influence of 2-CdA on the adherence of PBMC to fibroblasts is inversely related to the degree of immune system activation.

Adhesion of peripheral blood mononuclear cells to vascular endothelium in patients with systemic sclerosis (scleroderma).

OBJECTIVE: Perivascular infiltrates in skin, subcutaneous tissue, and internal organs are a characteristic feature of early systemic sclerosis (SSc). We studied the first step of migration of peripheral blood mononuclear cells (PBMC) through the vessel wall to the extravascular space, i.e., adhesion of PBMC to endothelial cells (EC), in patients with various forms of SSc (limited scleroderma, diffuse scleroderma, and the transitional form). METHODS: Radioisotope-labeled patient PBMC were coincubated with umbilical cord EC in vitro, and the percentage adhesion was measured. RESULTS: Adhesion of PBMC to EC was markedly decreased, while adhesion of isolated active rosette-forming cells (ARFC) was significantly increased, in SSc patients compared with healthy controls. Decreased adhesion of PBMC to EC was found to correlate with a diminished percentage of ARFC in the peripheral blood. Preincubation of PBMC from healthy donors with interleukin-2 (IL-2) enhanced their adhesion to EC, while preincubation of PBMC from SSc patients with this cytokine resulted in a decrease in adhesion in 10 of 14 individuals. IL-1, interferon-gamma, and transforming growth factor beta had no significant effect on adhesion of SSc patient PBMC to EC. Differences in adhesion of PBMC to EC among the SSc subgroups were not significant. CONCLUSION: Our findings suggest that in SSc, activation of subpopulations of PBMC leads to their enhanced adhesion to vascular endothelium in vivo and to migration of these cells to the extravascular space, resulting in the elimination from the peripheral blood of those PBMC with high ability to adhere to EC.

Acitretin decreases tumor cell-induced angiogenesis.

The effects of acitretin and etretinate on angiogenesis induced in Balb/c mice by intradermal injection of keratinocyte tumor cell lines were evaluated. It was shown that both retinoids are capable of inhibiting angiogenesis evoked by a human epidermoid carcinoma cell line (A431). Acitretin, but not etretinate, inhibited also angiogenesis induced by the spontaneously transformed murine keratinocyte cell line Pam 212 and by the established tumorigenic SKv cell line harboring the HPV16 genome. We suggest that inhibition of blood vessel formation may be one of the mechanisms responsible for the anticancerogenic effect of retinoids.

Beta-carotene in prevention of cutaneous carcinogenesis.

Beta-carotene, administered orally to mice, caused a decrease in angiogenesis evoked by HPV-transformed tumorigenic cell lines (SKv-t, HeLa). It did not affect angiogenesis induced by the non-tumorigenic SKv (not-t) cell line, and increased lymphocyte-induced immune angiogenesis. We suggest that the anti-cancerogenic effect of beta-carotene may be due, at least in part, to its inhibitory effect on formation of new blood vessels within the tumour mass.

[Studies of the role of cellular immunity and angiogenetic changes in the pathogenesis of circumscribed scleroderma]. / Badania nad rola odczynowosci komórkowej i zmian naczyniowych w patogenezie twardziny ograniczonej.

The studies were performed in 48 patients with morphea and included evaluation of 1) antinuclear antibodies 2) lymphocyte induced angiogenesis 3) natural killer (NK) cell activity and 4) T cell subpopulations in peripheral blood. The presence of antinuclear antibodies was found in 44.4% (8/18) patients with scleroderma linearis and in 21% (4/19) patients with morphea disseminata. Lymphocyte induced angiogenesis was increased in 41.5% (17/41) morphea patients, mainly in cases with pronounced vascular changes. The E rosette forming test showed a decreased percentage of active rosette forming cells (ARFC) and total rosette forming cells (TRFC) in peripheral blood and the NK cell activity was lowered in patients with morphea. These results obtained in patients with morphea show some similarities and differences in comparison to cellular immunity disturbances in patients with systemic scleroderma.

[Studies of the mechanism of disorders of the natural killer cell activity in patients with systemic scleroderma]. / Badania nad mechanizmem zaburzen aktywnosci komórek NK u chorych z twardzina ukladowa.

The NK activity of peripheral blood mononuclear cells (PBMC) was evaluated in 90 patients with various subsets of systemic scleroderma (SSc). The NK activity, as performed with K-562 as target cell, was found to be significantly lowered in patients with diffuse scleroderma, but did not differ from the healthy control in patients with acrosclerosis. The lowest values in the NK activity assay were obtained in patients with most extensive skin involvement and severe internal organ changes. The NK activity of healthy donors' PBMC was significantly decreased by addition of SSc patients PBMC (50:1) to the cytotoxicity assay, but was not influenced by the patients sera.

Cytotoxic effects of sera from patients with systemic scleroderma: comparison of three different in vitro methods.

Sera from 93 patients with systemic scleroderma including incipient or prodromal acroscleroderma and from 43 healthy individuals were studied for cytotoxic effects on endothelial cells by means of three different in vitro methods. Inhibition of 3H-thymidine incorporation by endothelium was caused by about 33% of sera, almost exclusively from patients with diffuse scleroderma and the transitional form: acroscleroderma--diffuse scleroderma. An antibody-dependent cellular cytotoxicity assay revealed cytotoxicity of about 37% of sera from patients with diffuse scleroderma and the transitional form but also of a proportion of sera from patients with CREST syndrome with pronounced vascular changes. Serum cytotoxic activity, revealed by both methods, was related with more frequent involvement of muscle and kidney in the patients. A direct 51Cr release assay showed the cytotoxicity only in 4 of 68 cases in diffuse scleroderma with pronounced internal organ involvement. Thus, depending on the method used, various types of cytotoxicity could be detected in sera from patients with systemic scleroderma.