Antibasement membrane antibody-mediated experimental conjunctivitis.

In ocular cicatricial pemphigoid, the binding of circulating antibodies to conjunctiva is believed to initiate an antibody-mediated cytotoxic response that results in inflammation and tissue damage. To develop a model of antibody-mediated conjunctival inflammation, we examined the effect on conjunctiva of local or systemic administration of a murine monoclonal antibody against basement membrane of stratified squamous epithelium. Neonatal rabbits were given either a single subconjunctival or intraperitoneal injection of the antibody. Eyes were graded clinically for inflammation and conjunctival biopsies were performed. After subconjunctival injection, clinical and histologic inflammation as well as murine antibody and rabbit complement binding to conjunctival basement membrane were detected. With systemic administration there was post-injection clinical inflammation, and conjunctival basement membrane-bound murine antibody was detected. There was no difference observed in conjunctival mitotic rate or goblet cell frequency between treatment groups and controls, following either route of administration. We have created, therefore, a model for antibody-mediated conjunctivitis in rabbits by local or systemic administration of a monoclonal antibody against a component of stratified squamous epithelial basement membrane.

Ulcerated edematous limbs: effect of edema removal on transcutaneous oxygen measurements.

We studied the effect of edema removal on transcutaneous oxygen tension (TcPO2) in eight patients with leg ulcers. An external pneumatic compression device was effective in removing edema from the ulcerated limbs over a 5-day course of treatment, at which time all treated limbs were clinically free of edema. TcPO2 levels at room air at the ulcer site were markedly reduced when compared with the control chest site, both before and after external pneumatic compression device therapy (p less than 0.001). Edema removal, however, did not significantly alter TcPO2 values (p greater than 0.9). No significant positional effects on TcPO2 values were noted with patients supine, sitting, or with the affected limbs elevated. Five patients (63%) had increased TcPO2 values adjacent to the ulcer site when receiving supplemental oxygen. For these patients, TcPO2 values increased after oxygen inhalation, both before and after edema removal (p less than 0.05). We conclude that TcPO2 values are markedly reduced in ulcerated edematous limbs. Edema, however, may not constitute a barrier to oxygen diffusion through the skin and does not account for the low TcPO2 values in the ulcerated edematous limb. Therefore the positive therapeutic effect of removing edema is unlikely to be related to better oxygenation of the tissues.

Effect of a platelet release fraction on glycosaminoglycan synthesis by cultured dermal fibroblasts from patients with progressive systemic sclerosis.

Confluent cultures of dermal fibroblasts from patients with progressive systemic sclerosis (PSS) and healthy controls were investigated for the synthesis of glycosaminoglycans (GAG) in response to a platelet release fraction (PRF) obtained by treatment of pooled platelets from normal individuals with adenosine 5'-diphosphate (ADP). PRF, at concentrations of 1 to 50 micrograms protein/ml, produced a linear increase in GAG synthesis that was always greater in cultures of PSS fibroblasts than in cultures of normal fibroblasts (P less than 0.001). A partial inhibition of GAG synthesis was observed with 100 micrograms/ml of PRF. The increased GAG synthesis in cultures incubated with PRF was not due to ADP. These findings demonstrate a difference in response to PRF between PSS and normal fibroblasts and may reflect an increased responsiveness of PSS fibroblasts to platelet factors.

Transforming growth factor-beta: selective increase in glycosaminoglycan synthesis by cultures of fibroblasts from patients with progressive systemic sclerosis.

Transforming growth factor-beta (TGF-beta) has been found in all cells examined thus far, and has been shown to play an important role in inflammation and connective tissue formation. We now report that TGF-beta, alone or in combination with epidermal growth factor (EGF), led to a preferential increase in glycosaminoglycan synthesis by cultures of dermal fibroblasts from patients with progressive systemic sclerosis (PSS) when compared with normal fibroblasts (p less than 0.001). Transforming growth factor-beta increased collagen synthesis to the same extent in both PSS and normal fibroblasts, whereas EGF had no stimulatory activity on collagen synthesis. The addition of EGF to cultures incubated with TGF-beta led to a decrease in collagen synthesis compared with the effect seen with TGF-beta alone (p less than 0.02). These studies suggest that TGF-beta may play an important role in the accumulation of connective tissue seen in PSS and that the combined action of multiple growth factors may modulate the synthetic activity of human dermal fibroblasts.

Dermal pericapillary fibrin in venous disease and venous ulceration.

Pericapillary fibrin deposition is thought to contribute to the pathogenesis of venous ulceration. To our knowledge, however, there is no previous evidence that pericapillary fibrin is deposited in the tissue adjacent to venous ulcers. We prospectively studied patients with ulcers of the lower extremities for the presence of dermal pericapillary fibrin in the skin adjacent tot he ulcers. On direct immunofluorescence, pericapillary fibrin was found in 14 (93%) of the 15 patients with venous ulceration but in only one (7%) of the 14 subjects with ulcers due to other causes. We also confirmed the presence of dermal pericapillary fibrin in legs with venous disease without ulcerations. We conclude that the pericapillary fibrin is easily demonstrable in the dermis adjacent to venous ulcers. In the evaluation of ulcers due to uncertain causes, the presence of dermal pericapillary fibrin may provide additional diagnostic help.

Effect of basement membrane entactin on epidermal cell attachment and growth.

A preparation of laminin and entactin (Matrix), extracted from the basement membrane of the murine cell line M1536-B3 was used to evaluate the effect of entactin on epidermal cell attachment and growth in culture. Cell growth on Matrix was significantly higher than on laminin or plastic, in the presence or absence of serum. In attachment assays, the attachment of cells to laminin (137% of control) or to Matrix (158% of control) was significantly higher when compared with plastic (p less than 0.01), and the attachment to Matrix was higher than to laminin (p less than 0.05). Varying the amount of laminin or Matrix used as a substratum showed each enhanced cell attachment to the same maximum value (approximately 50% attached cells or 160% of control), but maximal attachment was achieved with lower amounts of Matrix (15 micrograms Matrix vs 15-30 micrograms laminin, p less than 0.05). Inhibition studies with anti-entactin and anti-laminin antibodies were used to assay the specific contribution of entactin to cell attachment to Matrix. Pretreatment of the substratum with increasing amounts of anti-entactin antibody decreased cell attachment to Matrix in a concentration-dependent manner, with cell attachment to Matrix eventually falling to the same level as that obtained with laminin. There was no effect with anti-entactin on cell attachment to laminin or to plastic controls, and nonspecific rabbit IgG had no effect on any group. Similar experiments were performed using 2 different concentrations of anti-laminin antibody. At a low concentration, anti-laminin antibody decreased attachment to laminin (to a level equivalent to the plastic control). At a higher concentration anti-laminin decreased attachment to Matrix, but to a level that was still greater than the plastic control. The anti-laminin antibody had no effect on attachment to the plastic control and nonspecific rat IgG in equivalent amounts had no effect on attachment to any of the substrata. These results indicate that Matrix, containing laminin and entactin, enhanced cell attachment above the level seen with laminin alone, and that this effect was probably due to the presence of entactin in the Matrix.

Support and stimulation of epidermal cell outgrowth from porcine skin explants by platelet factors.

The effect of a platelet homogenate fraction (PHF) on epidermal cell outgrowth from porcine skin explants was examined in culture medium with and without 5% foetal bovine serum (FBS). Explants were sustained and outgrowth was initiated, supported and stimulated by PHF, in the absence of FBS. PHF-supported outgrowth was concentration-dependent up to 130 micrograms protein/ml. PHF activity was not removed by heat treatment at 100 degrees C for 2 min but was destroyed by heating at 100 degrees C for 5 min. The factor responsible for epidermal cell outgrowth in PHF may be epibolin or serum spreading factor, or may be a platelet-derived factor as yet undescribed. Viability and outgrowth were stimulated by the 'high molecular weight' fraction of PHF (greater than 30 kd). PHF plus 5% FBS gave inhibition of outgrowth, which was not PHF concentration-dependent; however, this inhibitory effect was variable with different PHF preparations. This study is the first to demonstrate an effect of platelet-derived factors on epidermal cell viability and outgrowth.