Cytokine mRNA repertoire of articular chondrocytes from arthritic patients, infants, and neonatal mice.

Articular chondrocytes from nine arthritic patients, five infants, and Balb/c neonatal mice were analyzed for the presence of various cytokine mRNAs by a reverse transcriptase polymerase chain reaction (RT-PCR). Four cytokine mRNAs, interleukin (IL)-6, IL-8, IL-11, and macrophage colony stimulating factor (M-CSF), were detected in all human chondrocytes, regardless of source. IL-10, IL-12p35, and tumor necrosis factor alpha (TNF-alpha) transcripts were found in at least 12 of the 14 human samples. IL-13, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and TNF-beta mRNAs were found more predominantly in infant samples and in samples from patients with rheumatoid arthritis (RA) compared with samples from patients with osteoarthritis (OA). Another group of cytokine mRNAs, IL-1 (alpha, beta), IL-4, IL-5, and IL-7, were only weakly expressed in some human samples. The cytokine transcripts that were not found were IL-2, IL3, and interferon gamma (IFN-gamma). Because of the large array of cytokine transcripts detected, human chondrocyte preparations were further purified by reacting them with a monoclonal antibody specific to chondrocyte differentiation antigen and subjecting them to fluorescent-activated cell sorting. A similar array of cytokines was found between the sorted and unsorted chondrocytes, although TNF-alpha, G-CSF and GM-CSF transcripts appeared to be upregulated during the sorting process. Human chondrocytes that dedifferentiated into fibroblasts (a 40-day and a 77-day culture) no longer expressed mRNAs for IL-1, G-CSF, GM-CSF, and TNF-alpha, but all other cytokine mRNAs remained detectable. Although certain phenotypic characteristics were lost, including reactivity to chondrocyte-specific monoclonal antibodies and morphological features, chondrocytes in long-term culture still expressed cytokine mRNAs. As expected, more consistent results were obtained when seven preparations of chondrocytes from neonatal Balb/c mice were examined using available cytokine primers. They contained IL-1, IL-5, IL-6, IL-7, IL-12, GM-CSF, M-CSF, transforming growth factor beta (TGF-beta), TNF-alpha, and TNF-beta mRNAs but lacked IL-2, IL-3, IL-4, IL-10, and IFN-gamma mRNAs. Future experiments to define conditions by which these cytokine protein products are expressed are needed to help assess their roles in chondrocyte biology and in disease states.

Differentiation antigens of human articular chondrocytes and their tissue distribution as assessed by monoclonal antibodies.

Five monoclonal antibodies (HuMC1, HuMC2, HuMC3, HuMC4 and HuMC5) raised against intact human articular chondrocytes were tested with chondrocyte samples from arthritic and non-arthritic patients by surface immunofluorescence and by immunoperoxidase labeling of fixed cells. All acetone-fixed samples reacted strongly with the monoclonal antibodies but some variation in the percentages of intact chondrocytes positive by immunofluorescence was noted. Under culturing conditions which induced de-differentiation, epitopes recognized by HuMC1, HuMC3 and HuMC4 disappeared with time in culture. In contrast, reactivities to HuMC2 and HuMC5 either persisted or increased as the culture became more fibroblastic and these antibodies bound to antigens on human fibroblast cell lines. HuMC1, HuMC3 and HuMC4 reacted with purified adult and fetal proteoglycan. HuMC2 and HuMC5 exhibited only slight or no reactivity to either proteoglycan. All five monoclonal antibodies reacted with chondrocytes in frozen articular cartilage but HuMC2 and HuMC5 failed to react to chondrocytes in paraffin-embedded cartilage sections. Only HuMC1, HuMC3 and HuMC4 recognized matrix components in both frozen and paraffin-embedded cartilage. When tested against 29 different non-cartilaginous tissues, each of the monoclonal antibodies had distinctive reactivity patterns, suggesting that each reacted to different epitopes. HuMC3 reacted with neurons in the cerebral cortex and cerebellum, indicating that it may recognize epitopes shared on the S-100 protein. HuMC1 showed the greatest specificity for chondrosarcomas. These antibodies are useful for identifying differentiated chondrocytes, providing information on the distribution of chondrocyte antigens shared by other human tissue, assessing the extent of chondrocyte heterogeneity in a population and aiding in the classification of chondrosarcomas.

Proliferative activity as an adjunct in the diagnosis of prostatic adenocarcinoma.

Sixty-nine men underwent transrectal ultrasound-directed biopsy of the prostate. One biopsy core from each side of each gland was sent for DNA flow cytometric testing (138 total specimens). Results were correlated with findings from standard hematoxylin and eosin staining of other cores. Twelve patients (17.4%) had biopsies with histopathologic evidence of prostatic carcinoma. Of 57 patients (82.6%) with benign biopsies, two had stage A prostate adenocarcinoma noted on subsequent transurethral resection. Proliferative activity was calculated from DNA histograms by adding the percentage of nuclei in the proliferative (S and G2/M) phases of the cell-division cycle. Mean proliferative activity for the malignant group (19.08) was significantly higher (p < 0.001) than that of the benign group (13.43). Inflammation was associated with elevated proliferative activity scores among benign glands. Proliferative activity is an objective, easily obtainable indicator of the biological activity of a population of cells which, when elevated, may suggest a need for repeat biopsy in patients with otherwise normal prostate biopsies. Flow cytometry may have value as a complement to standard histologic analysis of transrectal core biopsies in the diagnosis of prostate cancer.

The expression of major histocompatibility antigens on human articular chondrocytes.

Human articular chondrocytes were analyzed for major histocompatibility (MHC) antigens. The cells were obtained by enzymatic digestion of surgical specimens from patients with osteoarthrosis and rheumatoid arthritis having joint replacement. The analyses of Class I and Class II antigens were performed using microcytotoxicity techniques, as with conventional human leukocyte antigen (HLA) typing, and by immunofluorescence on a flow cytometer with monoclonal antibodies. Eighty-five percent of the chondrocytes had strong representation of Class I molecules, and individual specificities for HLA-A, HLA-B, and HLA-C could be detected. Class II antigens generally were not expressed on chondrocytes from noninflammatory states. In osteoarthrosis, some cells expressed HLA-DP and HLA-DQ, whereas in rheumatoid arthritis, there was an increase in the expression of all Class II antigens. Incubation of chondrocytes with gamma-interferon caused the strong induction of HLA-DR and HLA-DP, whereas HLA-DQ expression was largely unaffected. These results have broad implications for tissue transplantation and autoimmune disease.

Action of tolytoxin on cell morphology, cytoskeletal organization, and actin polymerization.

Tolytoxin, a cytostatic, antifungal macrolide produced by blue-green algae of the genus Scytonema, is a potent, reversible inhibitor of cytokinesis in cultured mammalian cells. Treatment of KB cells with 2-16 nM tolytoxin results in profound morphological changes, beginning with the formation of zeiotic processes and culminating in nuclear protrusion. In L1210 cells, cytokinesis is inhibited by as little as 2 nM tolytoxin, while karyokinesis proceeds normally, resulting in polynucleation. Tolytoxin specifically disrupts microfilament organization in A10 cells, while having no apparent effect on microtubules or intermediate filaments. Tolytoxin inhibited actin polymerization in vitro and also caused the depolymerization or fragmentation of F-actin in vitro. Tolytoxin exhibits effects that closely resemble those of cytochalasin B but is effective at concentrations 1/50-1/1,000 that of cytochalasin B.

A potent naturally occurring low-molecular weight blastogenic inhibitory factor from edible black tree fungus.

Crude dialyzable extracts of Auricularia polytricha (black tree fungus) when added to lymphoprep-isolated blood mononuclear cells (PBL) stimulated with mitogens (PHA and PWM) showed significant inhibition of 3H-thymidine (3H-TdR) incorporation in vitro. Similar extracts of Agaricus biporus and Cortinellus shiitake demonstrated essentially no suppression of 3H-TdR uptake by PBL cells stimulated with PHA or PWM. A 50 micrograms/well (200 microliter) concentration of A. polytricha reduced 3H-TdR uptake of PHA stimulated PBL cells from different donors by 65.4 to 99.8% and similarly reduced 3H-TdR uptake of PWM stimulated PBL cells by 89.6 to 99.9%. Viability examination of PBL cells in RPMI medium with A. polytricha extract alone, mitogens alone, and mixtures of mitogens and extract for 72 hr at 37 degrees C showed per cent survivals as follows: medium alone, 93%; PWM, 91%; PHA, 77%; BTF extract (200 micrograms/ml), 57%; PWM + BTF extract, 47%, and PHA + BTF extract, 39%. Data presented show that Auricularia polytricha contained a low dalton potent blastogenic inhibitory factor(s).

A rapid enzyme-immunoassay for the detection of ciguatoxin in contaminated fish tissues.

An enzyme-immunoassay procedure for the detection of ciguatoxin has been developed using the sheep anti-ciguatoxin serum described earlier in the radioimmunoassay method for ciguatoxin. In the enzyme-immunoassay procedure, the sheep anti-ciguatoxin was coupled to horseradish peroxidase. Analyses of fish tissues showed that the enzyme-immunoassay procedure distinguished between clinically documented toxic and nontoxic tissues (P less than 0.001). Comparisons of the enzyme-immunoassay method with the radioimmunoassay for ciguatoxin and with a mouse bioassay demonstrated significant associations (P less than 0.001 and P less than 0.01, respectively). Preliminary studies showed that purified ciguatoxin inhibited the binding of sheep anti-ciguatoxin horseradish peroxidase to toxic fish tissues. Results suggest that the enzyme-immunoassay procedure may be valuable for routine direct assessment of ciguatoxin in fish tissues because of its practicality, sensitivity and specificity.

Presence of prostaglandins (PGs) in Tetrahymena pyriformis, GL and the effect of aspirin.

Tetrahymena pyriformis GL appears to require prostaglandins, either B, E or F series for growth, as demonstrated by the deleterious effect of aspirin. The latter inhibits prostaglandin synthetase (cyclooxygenase). Aspirin inhibited 50% growth of a 24 hr culture of T. pyriformis at a dose of approximately 200 micrograms/ml and completely inhibited at 600 micrograms/ml. Extraction with acid ethyl acetate: isopropanol solvent of 2.8 x 10(8) cells yielded 41.3 mg of lipid of which 62.8% and 34.5% were PGE2 and PGB, respectively. It is suggested that PGs are important for the growth of T. pyriformis and that the organism may be a useful source of natural PGs. Additionally, T. pyriformis may be useful in studies of the PGs pathway.

Comparison of three different assays for the assessment of ciguatoxin in fish tissues: radioimmunoassay, mouse bioassay and in vitro guinea pig atrium assay.

Moderate correlation was shown between the following tests: RIA: mouse bioassay (r = 0.58); RIA: guinea pig atrium assay (r = 0.62); guinea pig atrium assay: mouse bioassay (r = 0.62). All three assay procedures showed good correlation when ciguatoxin was present in tissues in high concentrations. The results also suggest that more than one structurally related toxin may be present in the fish tissues.

Effects of specific antibodies on the interaction between the fungus Candida albicans and human oral mucosa.

Host-parasite interactions were studied in four groups: non-infected controls; infected carriers of Candida albicans without evidence of candidiasis; subjects with acute candidiasis; and subjects with chronic candidiasis. Specific anti-candida antibodies were demonstrated in saliva from subjects of all four groups; the titres reflected the degree of antigenic stimulation, being significantly higher in candidiasis than in controls or infected carriers. The adherence of C. albicans to buccal epithelial cells was not significantly different in a given saliva, regardless of whether the assay was carried out with autologous C. albicans and epithelial cells or with a stock strain in a standardized assay. Therefore, the standard assay was used to study the effects of specific salivary antibodies on adherence. A significant inverse correlation was found between salivary IgA anti-candida antibodies and the adherence of C. albicans to buccal epithelial cells, suggesting that IgA antibodies can inhibit adherence of candida to the oral mucosa. In some instances, removal of antibodies led to a significant increase in adherence; however, often this was not the case, indicating that some but not all of the antibodies present were capable of inhibiting the adherence of C. albicans to epithelial cells.

Significant increase of plasma prostaglandins in cancer patients.

Plasma prostaglandin (PGs) levels of normal individuals, cardiac and autoimmune disease patients were similar with the following mean and S.E.M. in ng/ml values: Normal, 45.4 +/- 6.4; cardiac, 48.3 +/- 4.2; and autoimmune, 52.4 +/- 4.8. On the other hand, cancer patients showed a significant increase in plasma PGS with a mean +/- S.E.M. of 102.0 +/- ng/ml as compared to the values of normal individuals, cardiac and autoimmune patients. It is suggested that, in part, the increase in PGs may be associated with the depressed state of the cell-mediated immunity in cancer patients.

Distribution of prostaglandins B, E and F series in plasma of cancer patients.

Plasma prostaglandins (PGs) of normal individuals and cancer patients have been examined. With the exception of a breast carcinoma patient, all patients demonstrated significant elevations in PGE levels. Lung cancer patients showed a significant decrease in PGF, while stomach cancer patients showed a marked decrease in PGB. It is suggested that the changes in PGs may play a significant role in host-tumor metabolism.

The effect of purified ciguatoxin on mitogen responses of mouse spleen lymphoid cells.

Ciguatoxin at 10 micrograms per tube showed a slight suppressive effect on mouse unstimulated spleen lymphoid cells. The same CTX concentration demonstrated a significant effect on mouse lymphoid cells in blastogenesis stimulated by mitogens. The lower levels of CTX (1 and 0.1 microgram) had essentially no effect on the spleen cells with the exception of one experiment. Since the high CTX concentration used was sufficient to kill two twenty gm mice, it is suggested that spleen cells are relatively resistant to CTX. Since the primary effects of CTX in vivo appear to be neurological in nature, to achieve the dose levels to affect the immune system would be extremely lethal for the whole animal.

Relationship between germination of Candida albicans and increased adherence to human buccal epithelial cells.

A strong correlation was shown between germination and increased adherence of Candida albicans to human buccal epithelial cells, indicating that germination or other changes in the fungi accompanying germination were responsible for enhanced adherence. Partial inhibition of germination by cysteine resulted in a comparably lower adherence. Preferential adherence of germinated fungi occurred in competition assays with nongerminated and germinated fungi. The enhanced adherence to human mucosal cells of germinated C albicans could represent one mechanism contributing to the pathogenicity of the organism.

Adherence of Candida albicans to human buccal epithelial cells.

The adherence of Candida albicans to human buccal epithelial cells after 2 h at 37 degrees C was significantly greater in human saliva than in phosphate-buffered saline. in saliva, viable fungi adhered much better than did nonviable fungi, and this adherence was greater at 37 than at 25 degrees C. Viable yeasts, preincubated in saliva for 90 min at 37 degrees C before being washed and mixed with epithelial cells in phosphate-buffered saline, adhered better than nonviable yeasts or yeasts preincubated in phosphate-buffered saline. Enhanced adherence in saliva appeared to be associated with germination of the yeast cells. Conditions permitting germination (growth in tissue culture medium 199 at 37 degrees C but not at 25 degrees C) also supported enhanced adherence. After germination had occurred, the fungi could be killed with Formalin without interfering with their rapid and efficient adherence to epithelial cells. These data indicate that the enhanced adherence of C. albicans observed after incubation in saliva is related to changes in the fungi, rather than to a requirement for prolonged interaction between fungi and epithelial cells.

Effect of phospholipase C on lymphocyte responses to mitogen.

Peripheral lymphocytes treated with phospholipase C (phosphatidylcholine cholin phosphohydrolase E [3.1.4.3]) were examined for their response to mitogen and rosette formations. High levels of phospholipase C (greater than 0.01 unit) showed significant toxic effects on the peripheral lymphocytes as examined by the trypan blue exclusion test. This was attributable to "impurities" in the Cl. perfringens phospholipase C preparation since recovery of the mitogen responses were incomplete after heat inactivation of the enzyme. Active phospholipase C at 0.005 unit significantly (50%) suppressed the PHA response with little or no effect on the PWM stimulation. Similarly, a significant suppression of E rosette formation occurred with phospholipase C (0.005 u) treated lymphocytes. Suppression of similarly treated lymphocytes to EAC rosette was slight. It is suggested that the removal of phosphorylated amines from membrane surfaces affects T-cells more than B-cells and that the use of phospholipase C is a useful means of examining membrane functions of the lymphoid system.