A peptide sequence from platelet factor 4 (CT-112) is effective in the treatment of type II collagen induced arthritis in mice.

OBJECTIVE: Platelet factor 4 (PF-4) is a critical alpha chemokine in inflammation and injury responses, with multiple effects upon cellular activities. Discrete peptide sequences of the PF-4 molecule have been shown to retain biological activity. Our aim was to examine the influence of the PF-4 derived octapeptide (CT-112; TTSQVRPR) on type II collagen induced arthritis in mice, to determine if this peptide exhibited antiinflammatory properties. METHODS: DBA/1 mice were treated with CT-112 from either the time of immunization with type II collagen or from the initial onset of arthritis. RESULTS: CT-112 both prevented the development of arthritis in mice treated prophylactically and reduced progression of disease in animals treated therapeutically, and was active when delivered by either subcutaneous injection or oral gavage. No marked immunosuppressive effects were observed during CT-112 treatment, with only moderate decrease in antibody levels and mitogen responses. A significant reduction of the circulating levels of IL-1 was a consistent finding in mice treated therapeutically with CT-112. CONCLUSION: These data suggest PF-4 derived octapeptide exerts antiinflammatory effects of experimental arthritis in mice.

Raloxifene (LY156758) produces antimetastatic responses and extends survival in the PAIII rat prostatic adenocarcinoma model.

The benzothiophene antiestrogen, raloxifene (LY156758), has selective estrogen pharmacological antagonist activity in rats. The PAIII rat prostatic adenocarcinoma model was used to evaluate the effects of this agent on the lymphatic and pulmonary metastasis and survival in tumor-bearing male Lobund-Wistar (LW) rats. Raloxifene was inactive against colony formation of PAIII cells in vitro. Similarly, following subcutaneous (s.c.) implantation of 10(6) PAIII cells in the tail, s.c. administration of raloxifene (2.0, 10.0, or 20.0 mg/kg/day) for 30 days failed to demonstrate cytoreductive activity against primary tumor growth in the tail. However, in these same animals, raloxifene administration produced significant (P < 0.05) inhibition of PAIII metastasis from the primary tumor in the tail to the gluteal and iliac lymph nodes (maximal responses = 89% and 81% from control values, respectively). PAIII metastasis to the lungs was significantly inhibited by raloxifene treatment. Numbers of pulmonary foci in PAIII-bearing rats were significantly (P < 0.05) reduced by raloxifene administration in a dose-related manner (maximal reduction = 97% from control values). In these animals, maximal regression of 20% for ventral prostate and 21% for seminal vesicle were also seen after raloxifene administration (P < 0.05 for both). Coadministration of E2B and raloxifene had no consistent antagonistic effect upon the antitumor responses produced by raloxifene. Raloxifene (40.0 mg/kg/day for 28 days) produced marked decreases in PAIII metastasis in the lymphatic and pulmonary components. Continued administration of the compound produced significant (P < 0.05) extension of survival of PAIII-bearing rats. Further studies are needed to define the maximal antitumor efficacy and the mechanism of action of raloxifene in urogenital solid tumor animal models. These data support the contention that raloxifene represents a class of active antimetastatic agents with potential efficacy in the treatment of hormone-insensitive human prostatic cancer.

Anti-inflammatory prednisolone derivatives inhibit collagen synthesis and pro-alpha(1) (I) collagen promoter activity in rat skin fibroblasts.

Inhibition of wound healing by anti-inflammatory steroids is associated with decreased collagen synthesis. Methyl-20-dihydroprednisolonate has been previously reported to be anti-inflammatory without inhibiting collagen synthesis in skin when given either subcutaneously or intraperitoneally. The data we now report show that this prednisolone derivative, as well as two 9alpha fluorinated derivatives inhibit both collagen synthesis and pro-alpha(1) (I) collagen gene promoter activity in rat skin fibroblasts. Our data suggest that metabolism, absorption, or distribution of these corticosteroids results in their inability to inhibit collagen synthesis in vivo. In addition our data indicate that fluorination in the 9alpha position of the adrenal steroid nucleus is not required for the inhibition of collagen synthesis by methyl-20-dihydroprednisolonate.

Acute hemolytic anemia after oral administration of L-tryptophan in ponies.

The hematologic and pathologic effects of orally administered L-tryptophan and indoleactic acid and of L-tryptophan administered IV were studied in ponies. Sixteen adult Shetland ponies were allotted into 4 experimental groups. Group 1 consisted of 5 ponies (1-5) given 0.6 g of tryptophan/kg of body weight in a water slurry via stomach tube. Group 2 included 4 ponies (6-9) given 0.35 g of tryptophan/kg orally. Group-3 ponies (10-13) were given 0.35 g of indoleacetic acid/kg orally. Group 4 consisted of 3 ponies (14-16) given a single 4-hour IV infusion of 0.1 g of tryptophan/kg. Restlessness, increased respiratory rate, hemolysis, and hemoglobinuria were detected in 4 of the 5 group-1 ponies. Only pony 7 in group 2 developed hemolysis, hemoglobinuria, and a significant increase in respiratory rate. Renal pathologic lesions, consistent with hemoglobinuric nephrosis, were seen in ponies 2, 4, 5, and 7. Bronchiolar degeneration was evident in 4 of 9 ponies given tryptophan orally. The importance of these respiratory lesions was unknown. Clinical or pathologic abnormalities were not noticed in the ponies of groups 3 and 4. Mean plasma tryptophan values increased significantly in groups 1 and 2 at 6 hours after dosing. A second peak of tryptophan was detected in both groups at 12 hours. Values returned to predose values by 48 hours. Plasma indole and 3-methylindole concentrations were detectable in only 2 ponies (4 and 7). In vitro incubations of cecal fluid from ponies 6, 8, and 9 yielded a percentage conversion of tryptophan to indole of 16.75%, 5.84%, and 7.96%, respectively. 3-Methylindole was not produced. These results suggested that indole was the major metabolite of orally administered tryptophan in these ponies.

Acute hemolytic anemia induced by oral administration of indole in ponies.

Eight ponies were allotted to 2 groups of 4. Group-1 ponies (1-4) were given 0.2 g of indole/kg of body weight orally and group-2 ponies (5 to 8) were given 0.1 g of indole/kg. Various physical, hematologic, and physiologic measurements were obtained after administration of indole. Intravascular hemolysis and hemoglobinuria were detected in both groups within 24 hours of dosing. Hemolysis was reflected by decreases in PCV, hemoglobin concentration, and RBC count, and an increase in indirect bilirubin. Erythrocyte fragility appeared to increase in both groups at 8 hours after dosing and peaked at 16 hours after dosing. At 72 hours after dosing, the RBC fragility value was less than predose measurements. Heinz body formation was noticed in group-2 ponies, but not in group 1. Plasma indole concentrations increased in both groups from the nondetectable predose concentrations. Group-1 values were 203% of group-2 values. In group 2, plasma indole was nondetectable by 12 hours, whereas low concentrations could still be measured in the group-1 ponies at 24 hours. Ponies in group 1 died or were euthanatized between 24 and 72 hours after dosing, whereas group-2 ponies were euthanatized between 48 and 120 hours. At necropsy, all body fat, mucous membranes, and elastic tissue were stained yellow. Hemoglobinuric nephrosis was the most prominent microscopic lesion. Results of this study indicated that indole, a metabolite of the amino acid tryptophan, causes acute intravascular hemolysis in ponies.

Effect of carbohydrate structure and concentration on the non-enzymatic glycosylation and subsequent cross-linking of collagen.

It has been previously demonstrated that non-enzymatic glycosylation and subsequent cross-linking of proteins can occur at high or greater than physiological concentrations of glucose. Soluble collagen was incubated in the presence of increasing glucose concentrations. The amount of cross-linked collagen was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Our findings reveal that cross-linking due to non-enzymatic glycosylation occurs at or near physiological concentrations of glucose (3.11-4.22 mM). In addition, this glucose induced cross-linking is a time dependent reaction. When collagen was incubated with a variety of different carbohydrates it was found that ketoses are more active cross-linking agents than aldoses. The addition of a reactive group (such as an amine) alpha to the aldehyde group on the carbohydrate increases the cross-linking activity of glucose 2.8 fold. Blockage of the reactive group alpha to the aldehyde (such as N-acetyl glucosamine or 2-deoxy-D-glucose) totally abolishes glycosylation activity. Both 5-C and 7-C carbohydrates are more active than 6-C carbohydrates. Thus, although glucose may be the most abundant carbohydrate capable of non-enzymatic glycosylation and subsequent cross-linking, it is not the most chemically reactive. However, the significance of these findings to the pathogenesis of diabetes needs to be defined.

Experimental instability in the rabbit lumbar spine.

The authors performed mechanical, biochemical, and histologic analyses of changes in the rabbit lumbar spine occurring after instability had been induced by facet removal to find whether this intervention produced an experimental model for intervertebral disc degeneration. Sham operated animals and an unoperated control group were used for comparison. Half of the operated animals were housed under conditions to promote higher physical activity than the other animals housed individually in small cages. Acutely, the removal of facet joints increased the flexibility of intervertebral joints. Over the following year, this increase in flexibility was reduced to close to control levels in all groups of animals. Within the intervertebral discs, there was no significant change in proportions or solubility of collagen or proteoglycans after surgery, nor was there microscopic or macroscopic evidence of disc degeneration. The surgical procedure produced hypermobility of the spine, but there was a subsequent restabilization, and the intended disc degeneration was not produced. These findings indicate that some as yet unidentified soft tissue repair process, facilitated by activity, overcame the hypermobility created at surgery, so degenerative changes in the intervertebral discs did not result. We suggest that other animal models of disc degeneration may represent a failure of reparative response to acute injury.

Proliferation of human peripheral blood lymphocytes induced by recombinant human interleukin 2: contribution of large granular lymphocytes and T lymphocytes.

Recombinant human interleukin 2 (rH IL-2) in the presence or absence of additional stimuli, was found to be able to induce and support the proliferation of human peripheral blood lymphocytes (PBLs). These proliferative effects were observed at low doses (less than or equal to 10 U/ml) of interleukin 2 (IL-2) only when additional signals (antigen, mitogen) were provided. However, higher doses (greater than or equal to 100 U/ml) of rH IL-2 significantly stimulated the proliferation of PBL even in the absence of exogenous lectin, antigen, or allogeneic serum. The subpopulation of lymphocytes most responsive to these higher doses of rH IL-2 was the large granular lymphocyte (LGL), the morphologic homologue of natural killer activity. After the separation of human PBLs on discontinuous Percoll gradients, cells from fraction 2 (greater than 90% LGLs) responded in a dose-dependent manner to rH IL-2 alone, whereas cells from fraction 6 (greater than 90% T cells) were only slightly responsive to rH IL-2 alone. A portion of the proliferation of cells from fraction 2 was dependent on the expression of the TAC receptor, because the prior removal of TAC-positive cells significantly reduced IL-2-induced lymphocyte proliferation. These results demonstrate that human LGL that have not been exogenously stimulated can proliferate in direct response to IL-2, and suggest that LGL are the major cellular phenotype in the proliferative response that has been observed clinically.

Skin lysyl oxidase activity is not rate limiting for collagen crosslinking in the glucocorticoid-treated rat.

Lysyl oxidase activity in the skin of rats receiving triamcinolone diacetate (12 mg/kg) for three consecutive days was decreased by sixty-four percent as compared to control values. A decrease of lysyl oxidase activity was observed twelve hours after the initial glucocorticoid injection. The decreased lysyl oxidase activity was accompanied by a forty-nine percent decrease of acetic acid extractable collagen. There was also a forty-two percent decrease in the alpha/beta ratio of the acetic acid soluble skin collagen of glucocorticoid-treated animals. These data indicate that although skin lysyl oxidase activity is decreased by glucocorticoid treatment, the crosslinking of acid extracted collagen as measured by the alpha/beta ratio and collagen solubility is increased. Accordingly lysyl oxidase activity is not rate limiting for collagen crosslink formation in the skins of rats treated with glucocorticoids.

Collagen accumulation in the neonatal rat skin: absence of fibrillar collagen degradation during normal growth.

Collagen-bound collagenase activity was assayed in the entire skins of growing neonatal rats. The levels of fibrillar collagen degradation were found to be extremely low throughout the first six days of life. Less than one percent of the collagen accumulated during one day's growth could be degraded by the collagenase bound to the extracellular fibrils of the skin. Control experiments, which included the addition of purified rat uterine collagenase to the skins both before and after homogenization, showed that collagenase activity is easily detectable in the tissue when it is present. It therefore appears that the vast majority of skin collagen, once deposited as fibrils in the skin, fails to turn over during growth. Support for this concept was provided by experiments in which neonatal animals were injected with the potent synthetic glucocorticoid, triamcinolone. Very low levels of collagen-bound collagenase, comparable to those observed in control animals, were found in the steroid-treated animals. Furthermore, the inhibition of collagen synthesis in these animals resulted in a constant chemical content of collagen as well as a constant amount of proteinaceous [14C]-hydroxyproline after injection of [14C]-proline over a 72-hour period. Our results strongly suggest that the bulk of fibrillar collagen does not participate in a dynamic equilibrium between synthesis and degradation during normal neonatal growth. In addition, the results in steroid-treated animals suggest that the rate of collagen accumulation during this period appears to be essentially a function only of collagen synthesis.

Auricular chondritis in fawn-hooded rats. A spontaneous disorder resembling that induced by immunization with type II collagen.

A spontaneous apparently unique auricular chondritis in the pinna of fawn-hooded rats is described. The chondritis was bilateral, with adult onset, and resulted in a marked thickening of the auricular cartilage. Microscopically, islands of proliferative cartilage were present, and at the margins between the normal cartilage and the thickened abnormal cartilage a marked cellular inflammatory response was present. The condition was familial in rats of the fawn-hooded strain and appeared to be unrelated to the platelet storage pool deficiency in this strain of rats. Biochemically no increased synthesis of pinna cartilage was detected. No histopathologic lesions were detected in other cartilaginous tissues of affected rats. The lesions in the pinna bore a striking resemblance to those induced in rats by immunization with Type II collagen. The spontaneous condition described herein may, therefore, represent a unique model of relapsing polychondritis of human beings, a disease with auricular chondritis associated with antibodies to Type II collagen.

Secretion of mucin by explants of rabbit and human cervix in organ culture.

Small explants (2-3 mm3) of endocervix from virgin, estrous rabbits, and from hospitalized patients undergoing hysterectomy for nonneoplastic disease, were placed in organ culture and maintained in serum-free media for 4 days at 35 degrees C in a humid environment of 95% air/5% CO2. Waymouth's MB 752/1 with 10-5 M hydrocortisone succinate, 10-7 M retinyl acetate, and 1 microgram/ml insulin proved to be an excellent medium for maintaining these tissues, as judged by examination with light and scanning electron microscopy after incubation for 5 days. The explants incorporated the radiolabeled glycoprotein precursor, tritiated glucosamine, and secreted labeled mucin glycoproteins in vitro. Mucin released into the culture medium contained sialic acid and hexosamine in a molar ratio of approximately 0.5-0.8:1.0. Although some alterations occur in the morphology of secretory cells and their products after maintenance in culture for several days, the system can be utilized for studying various aspects of the cell biology of cervical mucin secretion.

Isolation and some properties of equine alpha 1-antitrypsin.

1. Equine alpha 1-antitrypsin was isolated from horse plasma by a combination of ammonium sulfate and acidification precipitation followed by ion-exchange chromatography on DEAE-cellulose, molecular sieve chromatography on Sephadex G-200 and affinity chromatography on Cibacron Blue-agarose. 2. The purified protein showed a single precipitin arc on immunoelectrophoresis in agarose but gave two bands on discontinuous polyacrylamide gel electrophoresis (PAGE). 3. Both bands appeared to interact equally with trypsin and were thought to represent two isoinhibitors of equine alpha 1-AT.

Collagen lysyl oxidase activity in the lung increases during bleomycin-induced lung fibrosis.

Intratracheal administration of bleomycin caused pulmonary fibrosis in rats. Bleomycin sulfate (640 micro grams/165 g b.wt. in 0.5 ml of sterile saline) was instilled Intratracheally into male Fisher 344 rats (169 +/- 5 g), whereas control animals received 0.5 ml of sterile saline by the same route. One, 3, 7, 14, 28 and 322 days after instillation, the animals were killed, the lungs were homogenized in 6 M urea, 0.01 M NaCl, 0.001 M potassium phosphate (pH 8.3) and the homogenates were subjected to ultracentrifugation. The 106,000 x g supernate was assayed for lysyl oxidase activity. Total lung hydroxyproline and desmosine content was determined at each time point. Lysyl oxidase specific activity in the lung was elevated significantly 3 days after bleomycin treatment, peaked 14 days after bleomycin treatment at 230% above the control value and was returned toward normal 28 days after treatment. The increase of lysyl oxidase activity preceded the maximal increase of total lung hydroxyproline and desmosine which occurred 28 days after bleomycin instillation.

Isolation of collagen from the skins of Ehlers-Danlos syndrome-affected dogs by acetic acid extraction and pepsin digestion.

Collagen was isolated by acetic acid extraction in the presence of protease inhibitors and also by pepsin digestion from the skins of dogs affected with the Ehlers-Danlos syndrome and the skins on non-affected dogs. The collagen preparations isolated by acetic acid extraction from the Ehlers-Danlos syndrome-affected dog skin contained a greater proportion of alpha-chains than the collagen preparations from the normal dog skin. When the collagen from the Ehlers-Danlos syndrome-affected dog skin was reduced with NaBH4 before heat denaturation, and electrophoresis, there was a greater proportion of beta-chains present. The collagen isolated from the normal dog skin was not affected by the NaBH4 reduction. Collagen preparations isolated by pepsin digestion from both the Ehlers-Danlos syndrome-affected dog skin and the non-affected dog skin contained the same quantity of alpha- and beta-chains. In addition, collagen from both affected and non-affected dog skins isolated by pepsin digestion contained 10-11% type III collagen as determined by the interrupted sodium dodecyl sulfate polyacrylamide gel electrophoresis method. Pepsin digestion of the collagens isolated by acetic acid extraction in the presence of protease inhibitors from the skins of affected and non-affected dogs eliminated the differences between the alpha:beta ratios of the affected and non-affected collagen preparations.

Dermatosparaxis in a Himalayan cat: II. Ultrastructural studies of dermal collagen.

Dermatosparaxis is a connective tissue disease, primarily of sheep and cattle, that results from deficient activity of the NH2-terminal procollagen peptidase. It is characterized by fragile, loose skin that is easily torn with minor trauma. We have identified a cat twith a defect in this procollagen peptidase which affects only a small proportion of the collagen molecules; the majority of the collagen is processed normally. Nonetheless, as seen by transmission and scanning electron microscopy, this population of aberrant collagen molecules significantly alters the structure of individual collagen fibrils, the assembly of fibrils into fiber bundles and the integration of fiber bundles into a normal, woven network in the reticular dermis of skin. Although the clinical findings are less severe than those in sheep and cattle where the enzymatic defect is more complete, the ultrastructural abnormalities are marked and demonstrate that a minority of abnormal collagen molecules cn have a major effect on the structure and function of connective tissues.