Immunosuppressive activity and tissue polypeptide antigen content of human ascitic fluids.

Ascitic fluids from patients with cancer, cirrhosis, and congestive heart failure and from a patient with noninfectious tuberculosis contain measurable levels of tissue polypeptide antigen (TPA). Only the cancer patients had levels higher than 2.0 microgram TPA per ml. The average TPA levels of 29 cancer patients was 6.4 microgram/ml compared to 0.9 microgram/ml for the controls. Seventeen of 22 cancer ascitic fluids and 7 of 9 fluids from patients with liver disease were immunosuppressive as measured by the inhibition of [3H]thymidine incorporation into phytohemagglutinin-stimulated lymphocytes. Fluids from a patient with congestive heart failure and a patient with noninfectious tuberculosis were not suppressive. We were unable to obtain a significant correlation coefficient between immunosuppression and TPA levels in these fluids. In addition, TPA levels remained constant over a period of 18 months of testing, whereas the in vitro immunosuppressive activity was lost in 9 to 10 months. Sephadex G-200 fractionation of the ascitic fluid resulted in the TPA and immunosuppressive activity eluting in the first large molecular weight peak from the column. Although the 2 activities eluted together in this fractionation, the data suggest that TPA is not responsible for the immunosuppression.

Differential effects of an immunosuppressive fraction from ascites fluid of patients with ovarian cancer on spontaneous and antibody-dependent cytotoxicity.

The ascites fluids from humans with cancer that has metastasized to the peritoneum is suppressive of the immune response both in vitro and in vivo. The active moiety is present in a high-molecular-weight fraction which elutes in the void volume of a Sephadex G-200 column. This fraction (designated Peak I) has been shown to inhibit a number of in vitro responses and will inhibit the antibody response to sheep red blood cells in vivo. In this report, the Peak I protein fraction from the ascites of patients with ovarian cancer is shown to inhibit spontaneous cytotoxicity of human mononuclear cells against the myeloid cell line K562 and against the T-lymphoid cell line Molt-4F. This fraction was active at concentrations of 1 to 3 mg/ml. In contrast to this finding, it was not possible to demonstrate an inhibition of antibody-dependent cell cytotoxicity against chicken red blood cells. Preincubation of the effector cells with Peak I prior to addition to the chicken red blood cell targets or modification of the antibody concentration in the assay did not result in suppression; in fact, in many experiments, the Peak I potentiated cytotoxicity against chicken red blood cells. The Peak I proteins were also tested in an antibody-dependent cell cytotoxicity assay against a transformed cell line (HeLa cells), and there was no significant suppression of cytotoxicity. Peak I protein fractions prepared as controls from normal human serum and a congestive heart failure fluid were not suppressive, whereas the Peak I fraction from a cirrhotic fluid was suppressive of natural killing activity.

Immunosuppressive activity of ascitic fluid from patients with cancer metastatic to the peritoneum.

We have examined the immunosuppressive effects of ascitic fluids from patients with advanced cancer metastatic to the peritoneum and compared them with noncancerous abdominal or pleural effusions, serum from cancer patients, or the proteins human serum albumin and bovine gamma-globulin. The ascitic fluids from cancer patients produce a nontoxic, dose-dependent suppression of DNA and protein synthesis of phytohemagglutinin-stimulated human peripheral blood lymphocytes. This suppression reached 100% with an ascitic protein concentration of 4 to 6 mg/ml, whereas control effusions, serum from cancer patients, or added extraneous proteins were not suppressive. Ascitic proteins also suppress the mixed lymphocyte reaction and the response of human peripheral blood lymphocytes to the antigen keyhold limpet hemocyanin, in vivo, the primary plaque-forming response to sheep red blood cells in mice could be suppressed, and this suppression was not due to antigenic competition.

Suppression of tumor-specific cell-mediated cytotoxicity by immunoregulatory alpha-globulin and by immunoregulatory alpha-globulin-like peptides from cancer patients.

The suppression of tumor-specific cell-mediated cytotoxicity by human immunoregulatory alpha-globulin (IRA), by a peptide fraction derived from IRA, and by IRA-like peptides from the serum of cancer patients was studied in a syngeneic murine tumor-host system. Splenic lymphocytes from tumor-immunized mice were cytotoxic specific tumor cells in vitro as measured by the [125]-iododeoxyuridine release microcytotoxicity assay. However, this effect was significantly depressed if 1.25 to 5 mg of IRA per ml were added to the cultures. Pooled lyophilized normal human serum protein was inactive. IRA peptide and IRA-like peptide fractions from cancer patients were also highly suppressive of cell-mediated cytotoxicity at much lower concentrations (0.05 to 0.5 mg/ml). Control human serum peptide, which failed to inhibit the induction of hemolytic plaque-forming cells in sheep erythrocyte-injected mice, had no effect on cell-mediated cytotoxicity. IRA and IRA-like peptide fractions were not cytotoxic to the effector lymphocytes or to the target cells at the concentrations used.

Isolation of a bone-resorptive factor from human cancer ascites fluid.

A protein fraction that induces the resorption of bone explants in organ culture was isolated from the ascitic fluid of patients with advanced cancer metastatic to the peritoneal cavity. Partial purification was achieved by means of gel filtration, affinity chromatography, and ion-exchange chromatography. The isolated fraction, the components of which have an apparent molecular weight of 60,000, was found to be heterogeneous by disc gel electrophoresis and to be composed primarily of proteins with relatively acidic electrophoretic properties. The specific bone-resorptive activity of this protein fraction was greatly increased over that of the unfractionated starting material, and the activity could be completely destroyed upon incubation with pronase and on heating. As determined by immunoassay and extraction procedures with various solvents, the bone-resorptive action of the isolated fraction was not attributable to the presence of parathyroid hormone, prostaglandin E2 or vitamin D-like sterols. In parallel experiments the supernatants of phytohemagglutinin-stimulated normal human peripheral leukocytes were subjected to identical chromatographic techniques, and a proten fraction with a molecular weight of 60,000, which resembled the resorptive fraction isolated from cancer ascites fluid and which contained significant bone-resorptive activity, was also partially purified.

p38 mitogen-activated protein kinase inhibitors--mechanisms and therapeutic potentials.

The pyridinylimidazole compounds, exemplified by SB 203580, originally were prepared as inflammatory cytokine synthesis inhibitors. Subsequently, the compounds were found to be selective inhibitors for p38 mitogen-activated protein kinase (MAPK), a member of the MAPK family. SB 203580 inhibits the catalytic activity of p38 MAPK by competitive binding in the ATP pocket. Four homologues of p38 MAPK have been identified to date, and interestingly, their biochemical properties and their respective sensitivities to the inhibitors are distinct. X-ray crystallographic analysis of p38-inhibitor complexes reinforces the observations made from site-directed mutagenesis studies, thereby providing a molecular basis for understanding the kinase selectivity of these inhibitors. The p38 MAPK inhibitors are efficacious in several disease models, including inflammation, arthritis and other joint diseases, septic shock, and myocardial injury.

Enhanced superoxide production by rat alveolar macrophages stimulated in vitro with biological response modifiers.

The kinetics of superoxide release and the effects of several biological response modifiers (BRM) on superoxide release from rat pulmonary alveolar macrophages (AM) have been studied. These cells produced superoxide anion both spontaneously and in response to phorbol myristate acetate (PMA) in a dose-related manner. The response to PMA peaked in approximately 2 hr and maintained plateau levels for an additional 2-3 hr before subsiding. Pretreatment of the macrophages in vitro with a number of immunostimulants enhanced the production of superoxide above that of controls. The release of superoxide in response to the immunostimulants was a slow phenomenon that took place over a 3-5 hr time period. Lymphokine-containing supernatants from concanavalin A (con A)-stimulated rat spleen cells (LK-Sup), murine recombinant gamma interferon (rMuIFN-gamma), nigeran, and muramyl dipeptide (MDP) enhanced this response in a dose-related manner. Poly I:C and Salmonella typhosa lipopolysaccharide (LPS) stimulated rat alveolar macrophages at low but not high concentrations. In contrast to the alveolar macrophages, rat peritoneal exudate cells were not activated by immunostimulants to produce increased amounts of superoxide.

Activation of rat alveolar macrophages by gamma interferon to inhibit Toxoplasma gondii in vitro.

We have investigated the effects of murine interferons on the ability of rat alveolar macrophages (AM) to inhibit the proliferation of the intracellular protozoan Toxoplasma gondii. This activity was determined by measuring suppression of 3H-uracil uptake into the Toxoplasma and by microscopic enumeration of the intracellular organisms. Recombinant gamma interferon (rMulFN-gamma), but not alpha/beta interferon (IFN-alpha/beta) was able to activate AM for antimicrobial activity in vitro. Maximum activation was achieved by incubation with 50-200 units/ml rMulFN-gamma and the activity was lost at one unit/ml. The highest levels of activation were obtained when macrophages were incubated with interferon for 48-72 h prior to the challenge with Toxoplasma organisms. Activation could still be obtained, however, when the interferon was added to the cultures as late as 2 h after the phagocytosis of Toxoplasma. Neither MDP nor low concentrations (1-1-ng/ml) of S. typhosa lipopolysaccharide (LPS) were able to activate these cells to inhibit the growth of Toxoplasma. Phagocytosis of Toxoplasma by AM did not result in the release of O2-, in fact the spontaneous release of O2- by these cells was inhibited by Toxoplasma. This inhibition was reversed by preincubation of the cells with rMulFN-gamma.

Administration of an immunomodulatory azaspirane, SK&F 105685, or human recombinant interleukin 1 stimulates myelopoiesis and enhances survival from lethal irradiation in C57Bl/6 mice.

The immunomodulatory azaspirane SK&F 105685 has immunosuppressive activity in animal models of autoimmune disease such as adjuvant-induced arthritis and experimental autoimmune encephalomyelitis. The mechanism of SK&F 105685 appears to be the induction of nonspecific suppressor cell (SC) activity. SC appear to be "null cells," that is, cells that lack specific cell surface markers of mature B cells, T cells, natural killer (NK) cells, or macrophages. Because we hypothesized that the induction of SC was associated with enhanced hematopoiesis, we sought to determine the hematopoietic potential of SK&F 105685. Recombinant interleukin 1 alpha (rIL-1) was included as a positive control for hematopoietic stimulation in our studies. We demonstrate here that administration of SK&F 105685 increases the number of granulocyte-macrophage colony-forming units (CFU-GM) within the bone marrow 24 h after injection in a dose-dependent manner. In addition, the percentage of CFU-GM in S-phase of the cell cycle was significantly increased, as was colony-stimulating activity (CSA) present in the serum of treated animals. In our experiments IL-1 did not increase marrow CFU-GM; however, splenic CFU-GM, the proportion of CFU-GM in S-phase of the cell cycle, and serum CSA were all increased 24 h after a single treatment. Administration of SK&F 105685 24 h prior to lethal irradiation resulted in a dose-related increase in the number of surviving mice. These results demonstrate that SK&F 105685 and rIL-1 stimulate myelopoiesis in vivo and suggest a mechanism by which prophylactic treatment with these agents protects mice from otherwise lethal irradiation.

Regulation of colony-stimulating activity production from bone marrow stromal cells by the hematoregulatory peptide, HP-5.

It has been reported that the granulocyte-derived hematoregulatory pentapeptide, HP-5, and its dimer (HP-5b) have potent hematoregulatory properties. The proposed mechanism of action for HP-5b is synergy with colony-stimulating activity (CSA) resulting in enhanced myeloid colony formation in vitro. We now demonstrate that the effects of HP-5b on enhanced colony formation are indirect and mediated by an effect on CSA production by bone marrow stromal cells. Bone marrow stromal cell culture systems from mice, rats, and humans were used as target cells for the action of HP-5 monomer and dimer. Cell-free supernatants from these cultures were assayed for CSA in a murine granulocyte-macrophage colony-forming unit (CFU-GM) assay. Supernatants from stromal cell cultures pulsed for 1 h with HP-5b resulted in increased murine CFU-GM colony proliferation with an estimated half-maximal effective concentration (EC50) of 1-5 ng/ml. This increase in CFU-GM proliferation was neutralized by anti-macrophage colony-stimulating factor (anti-M-CSF) antibodies. The HP-5 monomer was without effect on constitutive CSA production by stromal cells, but it antagonized HP-5b-induced CSA production in a dose-responsive manner with an estimated half-maximal inhibitory concentration (IC50) of 0.2-0.4 ng/ml. The ability of HP-5 monomer to antagonize HP-5b induction of CSA appears specific in that HP-5 monomer failed to alter interleukin 1 (IL-1) or lipopolysaccharide (LPS)-induced stromal cell CSA production.

Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo.

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

Cyclosporin analogs modified in the 3,7,8-positions: substituent effects on peptidylprolyl isomerase inhibition and immunosuppressive activity are nonadditive.

Four analogs of cyclosporin A (CsA) were synthesized to determine if the biological activities of CsA analogs generated by multiple amino acid replacements are predictable from the effects on biological activity of analogs with single residue changes. CsA analogs [Phe7]CsA (8a), [D-MeAla3,Phe7]CsA (8b), [D-Ser8,Phe7]CsA (8c), and [D-MeAla3,Phe7,D-Ser8]CsA (8d) were designed by modification of positions 3, 7, and 8, which are adjacent to one effector region of the cyclophilin-bound CsA complex. The syntheses of CsA analogs 8a-d were carried out by suitable modifications of the reported strategy. Each analog was characterized by NMR in deuterated chloroform and DMSO solutions, and their biological activities as inhibitors of cis-trans-peptidyl prolyl isomerase (PPIase), inhibitors of proliferation in BDF1 mouse spleen cells stimulated with concanavalin A (Con A), and inhibitors of IL-2 release stimulated with PMA/ionomycin by Jurkat cells were determined. Incorporation of the phenylalanine residue in position 7 diminished activities 5-8-fold. Substitution at position 3 decreased activity nearly 2-fold, and substitution at position 8 did not lower activities. However, when all three modifications (D-MeAla3,Phe7, and D-Ser8) were incorporated into one molecule, the resulting analog, 8d, was found to bind more tightly to cyclophilin than CsA (Ki = 3 +/- 1.5 vs 6 +/- 2 nM) and to produce the full immunosuppressive effect in the other assay systems. Our structure-activity results show that combinations of substitutions that individually lower PPIase or immunosuppressive activity produce fully active analogs when combined in a single compound. These results suggest that other, multimodified CsA derivatives may be discovered that possess excellent or improved immunosuppressive activities even though they contain a substitution that otherwise reduces immunosuppressive activity.

Antiarthritic and suppressor cell inducing activity of azaspiranes: structure-function relationships of a novel class of immunomodulatory agents.

Spirogermanium (1; 8,8-diethyl-N,N-dimethyl-2-aza-8- germaspiro[4.5]decane-2-propanamine dihydrochloride) is a potent cytotoxic agent in vitro which has demonstrated limited activity in experimental animal tumor models. Subsequently, it has been reported that spirogermanium has antiarthritic and suppressor cell-inducing activity. We have synthesized a series of substituted 8-hetero-2-azaspiro[4.5]decane and 9-hetero-3-azaspiro[5.5]undecane analogues of spirogermanium to identify the heteroatom requirements for in vivo antiarthritic and suppressor cell-inducing activity. This structure-activity relationship study has identified that appropriately substituted silicon and carbon analogues of spirogermanium retain both antiarthritic and immunosuppressive activity, with the 8,8-dipropyl (carbon) analogue being among the most active. Following the identification of N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine++ + dihydrochloride (9) as a more active analogue than spirogermanium, a series of 8,8-dipropyl analogues with various amine substituents were synthesized. A number of these analogues had activity similar to that of 9. A correlation between activity in the adjuvant arthritic rat and the ability to induce suppressor cells (r = 0.894, p less than 0.001) suggests an association between the two pharmacologic effects. While the precise biochemical mechanism(s) for the pharmacological activity is unclear, these data suggest that compounds within this series, e.g., N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine++ + dihydrochloride, may provide effective therapy in diseases of autoimmune origin and/or the prevention of rejection in tissue transplantation.

1-substituted 4-aryl-5-pyridinylimidazoles: a new class of cytokine suppressive drugs with low 5-lipoxygenase and cyclooxygenase inhibitory potency.

A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production. The members of the earlier series exhibited varying degrees of potency as inhibitors of the enzymes of arachidonic acid metabolism, PGHS-1 and 5-LO. Several of the more potent CSBP ligands and TNF biosynthesis inhibitors among the present series of N-1-alkylated imidazoles (A) were tested as inhibitors of PGHS-1 and 5-LO and were found to be weak to inactive as inhibitors of these enzymes. One of the compounds, 9 (SB 210313) which lacked measureable activity as an inhibitor of the enzymes of arachidonate metabolism, and had good potency in the binding and in vivo TNF inhibition assays, was tested for antiarthritic activity in the AA rat model of arthritis. Compound 9 significantly reduced edema and increased bone mineral density in this model.

Abrogation by rapamycin of accelerated rejection in sensitized rats by inhibition of alloantibody responses and selective suppression of intragraft mononuclear and endothelial cell activation, cytokine production, and cell adhesion.

This study evaluated the efficacy and mode of action of rapamycin (RPM) in a model of accelerated (24-hr) rejection of LBNF1 cardiac allografts in specifically sensitized LEW rats. RPM treatment (0.25 mg/kg/day i.p.) between the day of sensitizing skin grafts (day -7) and subsequent heart (day 0) transplantation (Tx), abrogated fulminant rejection and prolonged cardiac allograft survival to 46 +/- 22 days (mean +/- SD, P < 0.0001). The delayed introduction of RPM until day -2 or day -1 was equally effective, whereas treatment initiated after cardiac Tx was ineffectual. Untreated accelerated rejection was associated with strong production of circulating IgM, whereas an IgG alloantibody response was not detected until after rejection was complete. RPM therapy (day -7 to -1) diminished this systemic IgM response and prevented the switch from IgM to IgG alloantibody production. Immunohistologic evaluation at 24 hr after cardiac Tx showed that compared with untreated hosts RPM treatment largely abolished intragraft cellularity, and was associated with decreased mononuclear and endothelial cell activation. Specifically, Ia and ICAM-1 upregulation was abolished, and no cells elaborating IL-2 or IFN-gamma were detected. In addition, RPM treatment prevented intragraft production of the proinflammatory cytokines IL-1 beta, IL-6, and IL-8. The effects of RPM therapy on recipient cellular responses were evaluated in vitro by mixed lymphocyte reaction. Surprisingly, the donor-specific proliferative response of cells from RPM-treated hosts at 1 or 7 days after Tx was markedly increased, compared with cells from rejecting, untreated controls, and bioassay of IL-2 within supernatants of MLR cultures showed comparable levels of IL-2 in both groups. The effects of RPM upon adhesion properties of lymph node lymphocytes were also tested in an in vitro binding assay. The binding of naive cells to sections of cardiac allografts collected from RPM-treated hosts at 24 hr post-Tx was decreased compared with that in untreated recipients. Interestingly, the binding of mononuclear cells to high endothelial venules of peripheral lymph nodes in RPM-treated hosts remained relatively high. Thus, treatment with RPM prevents and/or erases the sensitization, which otherwise leads to accelerated allograft rejection. Abrogation of allograft injury by RPM was associated with profound and long-lasting depression of host IgM and IgG alloantibody responses in the circulation, and selective downregulation of host cellular immunity and endothelial activation at the graft site. In contrast, antigen alloreactivity and endothelial adhesivity in peripheral lymphoid tissues were spared, indicating novel and potent selective effects of RPM therapy in allograft recipients.

Induction of nonspecific x-irradiation-resistant suppressor cell activity in vivo and prolongation of vascularized allograft survival by SK&F 105685, a novel immunomodulatory azaspirane.

SK&F 105685 is a novel azaspirane with immunosuppressive activity in animal models of autoimmune disease. This study evaluates the efficacy and mechanism of action of the compound in rat recipients of cardiac allografts. Short-term SK&F 105685 therapy (20 mg/kg/day by gavage) proved effective both in the pretreatment (days -14 to -8 or -7 to -1; allograft at day 0) and treatment (days 0 to 6) protocols, with cardiac allograft survival prolonged to 14-17 days (acute rejection = 7 days; P < 0.001). SK&F 105685 pretreatment exerted at least additive effects with subtherapeutic CsA (1.5 mg/kg/day x 7 days i.m.) given after transplantation, with 50% of allografts surviving > 50 days. SK&F 105685 therapy diminished the immunohistological features of acute rejection, with the cellular infiltrate suppressed and the induction of IL-2/transferrin receptors, and elaboration of IL-2/IFN-gamma essentially abolished, as compared with the grafts in untreated hosts. These correlated with normal frequency of CD4, CD5, CD8 phenotype subsets and B cells in recipient lymphoid organs, as shown by flow microfluorimetry. Adoptive transfer of untreated or x-irradiated (2000 rads) spleen cells from SK&F 105685-modulated hosts significantly prolonged the survival of donor-specific or third-party test cardiac allografts to 10-15 days, suggesting the presence of nonspecific x-irradiation-resistant suppressor cells in the transferred inoculum. Their activity could be enriched by Percoll density centrifugation and screened by the ability to inhibit Con A-driven proliferation of normal cells in the coculture assay. The light-density x-irradiation-resistant spleen cell fraction (1.07 g/ml) was consistently and significantly more suppressive than the heavy-density (1.09 g/ml) interface, or the corresponding unseparated cells. Thus SK&F 105685 therapy abrogates rejection response and significantly prolongs the survival of vascularized cardiac allografts in rats. This effect is associated with selective depression of host alloreactivity/immune activation at the graft site, and simultaneous induction of suppressor cells in recipient spleen, comparable to natural or nonspecific suppressor cells generated by TLI. This unique activity profile is consistent with the concept that SK&F 105685 should be considered as a critical chemical adjunct in novel therapeutic strategies representing TLI-equivalent.

Down-regulation of chondrocyte aggrecan and type-II collagen gene expression correlates with increases in static compression magnitude and duration.

The goal of this study was to examine the simultaneous effects of mechanical compression of chondrocytes on mRNA expression and macromolecular synthesis of aggrecan and type-II collagen. Bovine cartilage explants were exposed to different magnitudes and durations of applied mechanical compression, and levels of aggrecan and type-IIa collagen mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase were measured and quantified by Northern blot analysis. Synthesis of aggrecan and type-II collagen protein was measured by radiolabel incorporation of [35S]sulfate and [3H]proline into macromolecules. The results showed a dose-dependent decrease in mRNA levels for aggrecan and type-II collagen, with increasing compression relative to physiological cut thickness applied for 24 hours. Radiolabel incorporation into glycosaminoglycans and collagen also decreased with increasing compression in a dose-related manner similar to the changes seen in mRNA expression. The modulation of aggrecan and type-II collagen mRNA and protein synthesis were dependent on the duration of the compression. Aggrecan and type-II collagen mRNA expression increased during the initial 0.5 hours of static compression; however, 4-24 hours after compression was applied total mRNA levels had significantly decreased. The synthesis of aggrecan and collagen protein decreased more rapidly than did mRNA levels after the application of a step compression. Together, these results suggest that mechanical compression rapidly alters chondrocyte aggrecan and type-II collagen gene expression on application of load. However, our results indicate that the observed decreases in biosynthesis may not be related solely to changes in mRNA expression. The mechanisms by which mechanical forces affect different segments of the biosynthetic pathways remain to be determined.

Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro.

Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.

Therapeutic activity of SK&F 105685, a novel azaspirane with suppressor-cell inducing activity.

SK&F 105685, methotrexate (MTX) and chloroquine (CHL) were examined for their ability to inhibit paw inflammation in the adjuvant arthritic rat (AA) and for their ability to induce non-specific suppressor cells (SC) in the spleens of these animals. Compared to SK&F 105685 (84-90%) inhibition and MTX (91% inhibition) CHL, at the maximally tolerated dose, only inhibited paw lesions by 29%. MTX did not induce splenic SC activity and, despite the structural similarities between CHL and SK&F 105685, only the latter induced suppressor cells.

Human non-interferon macrophage activating factor: biological and biochemical characterization.

A HTLV-1 transformed T cell line has been demonstrated to constitutively produce and secrete a lymphokine with macrophage activating properties. This lymphokine was biologically and biochemically distinct from interferon gamma, the interleukins, the colony stimulating factors and lymphotoxin. In vitro treatment of human monocytes with the partially purified lymphokine resulted in the acquisition of microbicidal and tumoricidal activities. The activity was associated with a protein with an apparent molecular weight of 55,000 daltons and an isoelectric point of 5.5. The potential for the efficacy of such a lymphokine in activating macrophages in vivo provides aspirations for its use in the therapy/prevention of opportunistic infections in immunocompromised hosts.