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Biomedical subjects

P Panayiotidis

Publications and source records attributed to P Panayiotidis.

At least 37 records · Page 2Linked to original sources
PubMed1996

Downregulation of interleukin-2 and apha-chain interleukin-2 receptor biosynthesis by cisplatin in human peripheral lymphocytes.

To further investigate the mechanisms by which the antineoplastic agent cisplatin interferes with immune function, we studied its effect on the biosynthesis of interleukin-2 (IL-2) and its alpha-chain receptor (IL-2Ralpha). Normal human peripheral blood lymphocytes (PBL) were activated in vitro with phytohemagglutinin (PHA), and anti-CD3 antibody in the presence of various concentrations of cisplatin. Purified T cells were also cultured with anti-CD3 antibody and costimulated by CD80 (B7-1, B7/BB1)-transfected P815 mastocytoma cells in the presence of cisplatin. Tritiated thymidine incorporation assays, an enzyme-linked immunosorbent assay for soluble IL-2Ralpha determination, and RNA dot-blot analysis and hybridization with IL-2- and IL-2Ralpha-specific probes were used. PHA-induced and anti-CD3 antibody-induced proliferation of PBL were significantly inhibited by cisplatin at concentrations attainable in vivo. This inhibition was not due to direct cell death as shown by the absence of trypan blue uptake in the presence of high concentrations of cisplatin. Therapeutic concentrations of cisplatin (1 microgram/ml) also inhibited the IL-2-dependent proliferation of purified T cells, mediated via the CD28-CD80 costimulatory pathway. In addition, the amount of soluble IL-2Ralpha released in the T cell culture supernatants was decreased by cisplatin in a dose-dependent fashion, suggesting that inhibition of cell proliferation was associated with a parallel decrease in IL-2Ralpha production. These effects correlated with a specific cisplatin-induced downregulation of both IL-2 and IL-2Ralpha messenger RNA accumulation in PHA-stimulated PBL that was dependent on the concentration of the drug. These findings suggest that the immunomodulatory effects of cisplatin may result in part from its capacity to directly downregulate the IL-2/IL-2R system in activated lymphocytes.

Antineoplastic Agents↗
PubMed1996

Regulation of bcl-2 and fas expression in primary activation of human peripheral lymphocytes is not sensitive to dexamethasone or cyclosporin-A.

The regulation of bcl-2 and fas (Apo-1/CD95) gene product expression plays a significant role in lymphocytes proliferation, survival, and apoptosis. Dexamethasone (Dex) and the immunosuppressive agent cyclosporin-A (CsA) inhibit primary activation of lymphocytes by distinct, though overlapping mechanisms that trigger undefined signals and can induce or prevent apoptosis in lymphoid cells in vitro. Here we demonstrate that Dex and CsA, at concentrations that markedly inhibit phytohemagglutinin (PHA)-induced proliferation of normal human peripheral blood lymphocytes, suppress the activation-dependent expression of interleukin 2 (IL-2) and the alpha-chain IL-2 receptor in a dose-dependent fashion without affecting the inducible accumulation and kinetics of either bcl-2 or fas mRNAs. Similar results were obtained when PHA-stimulated lymphocytes were cultured in the presence of the CsA analogue FK-506 or rapamycin. Moreover, the inducible maximal expression of either bcl-2 or fas protein levels on 48-h PHA-activated lymphocytes was not changed in the presence of either Dex or CsA. These findings show that the cell activation-induced biosynthesis of bcl-2 and fas proteins is not affected by immunosuppressive agents, suggesting that the expression of IL-2 and both bcl-2 and fas genes is regulated through independent mechanisms.

Cells, Cultured↗
PubMed1996

Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro.

Leukaemic cells from most cases of B-chronic lymphocytic leukaemia die rapidly by apoptosis in vitro unless they are cultured in the presence of interleukin-4 or interferon alpha or gamma. We now report prolonged survival of purified CLL cells cultured on bone marrow (BM) derived stromal cells in the absence of exogenous growth factors. In 10 cases of CLL examined 0-61% (mean 14.7%) of the cells were viable after 10 d culture in medium alone, whereas in the presence of BM stromal cells 10-102% (mean 47.0%) of cells were recovered alive (P < 0.005) in 7/10 cases of CLL, cells remained viable after 30 d of culture in BM stromal cells with cell recovery of 12-65%. These long-term cultured CLL cells were Epstein Barr virus negative, shown by the failure to detect the ENBA-2 and BZLF1 genes of EBV by PCR analysis. Identity between day 0 and day 30 CLL cells was demonstrated by sequence analysis of their clonal IgH CDR3 region. Adherence of CLL cells to BM stromal cell layers was critical for their protection from apoptosis. Separation of CLL cells from stroma by 0.45 micron culture filters resulted in loss of the protective effect of the stromal cells. Stromal cells were also able to protect CLL cells from hydrocortisone-induced apoptotic cell death. Our findings provide an in vitro system that can be used to analyse the growth requirements of CLL cells and their chemosensitivity in an in vitro environment that mimics the in vivo milieu.

Aged↗
PubMed1996

Interleukin-6 inhibits apoptosis and tumour necrosis factor induced proliferation of B-chronic lymphocytic leukaemia.

There is now good evidence that tumour necrosis factor [TNF] stimulates DNA synthesis of B-chronic lymphocytic leukaemia (B-CLL) cells. The malignant clone produces TNF, and addition of exogenous TNF up-regulates the TNF mRNA in B-CLL cells. Interleukin-6 (rIL-6) may also be important in this growth loop. We studied the interaction of TNF and IL-6 in the regulation of DNA synthesis (3H-TdR uptake), cytokine release and cell survival in CLL cells in vitro. Addition of TNF (100 U/ml over 5 days) enhanced DNA synthesis from 718 +/- 284 (mean cpm +/- SE) to 2730 +/- 545 compared to cells cultured in medium alone (n = 16, p < 0.01). TNF-alpha induced DNA synthesis was inhibited in all cases studied by the addition of anti-TNF monoclonal antibody (5 micrograms/ml) to cell cultures. Spontaneous IL-6 protein release was enhanced in the presence of TNF (100 U/ml and 250 U/ml) by CLL cells at 48 hours of culture 143.6% and 172% (p < 0.05, n = 6). At 120 hours of culture, the increase was 323% and 412.5% (4 of 7 cases) of the control respectively. IL-6 (100 U/ml or greater) increased spontaneous DNA synthesis (3H-TdR uptake) but, in the presence of high concentrations of TNF-alpha, inhibited TNF induced DNA synthesis in a dose dependent manner. Cell survival was reduced in the presence of anti-IL-6 mAb, while IL-6 was able to protect CLL cells from spontaneous apoptosis. These results suggest that IL-6 in an autocrine manner may inhibit DNA synthesis but prolongs survival in CLL cells. Increased serum IL-6 levels were detected in 27 of 50 cases of CLL, the mean level being significantly higher in Rai Stage III and IV cases compared to Rai Stage O-II cases.

Adult↗
PubMed1996

Factors that influence activated CD8+ T-cell apoptosis in patients with acute herpesvirus infections: loss of costimulatory molecules CD28, CD5 and CD6 but relative maintenance of Bax and Bcl-X expression.

The expanded CD8+ T-lymphocyte population arising in response to viral infection controls the virus but could also prove damaging to the host unless safely removed at the end of the immune response. Apoptosis provides a mechanism whereby this can be achieved, as apoptotic cells are recognized and engulfed by macrophages. Peripheral blood CD8+ T lymphocytes from individuals with acute viral infections were highly susceptible to apoptosis after short-term culture in vitro. This spontaneous cell death could be prevented by interleukin-2 (IL-2) and was related to a decreased expression of Bcl-2 but not Bax or Bcl-XL, additional molecules that promote or prevent apoptosis, respectively, as well as an increase in CD95. After stimulation with anti-CD3 antibody, T cells from these patients also underwent an activation-induced cell death (AICD) that could not be prevented by IL-2. Interestingly, CD8+ T cells from this patient group expressed lower than normal levels of three costimulatory molecules, CD28, CD5 and CD6, suggesting that stimulation in the absence of a second signal is a possible mechanism for the defective reactivation of these cells. Thus multiple mechanisms, including loss of Bcl-2, increased CD95 and loss of costimulatory molecules, place constraints on the survival and reactivation of activated CD8+ T cells after viral infections. This enables immune activation to be controlled and cellular homeostasis to be re-established during resolution of viral diseases in vivo.

Acute Disease↗
PubMed1995

A trinucleotide deletion together with a base duplication event at codon 439 in the human tyrosinase gene identifies a mutational hotspot.

Molecular analysis of the human tyrosinase gene in two patients suffering from a temperature-sensitive form of albinism has identified a thymine triplet deletion at codon 439 which is accompanied by a duplication of the immediately preceding cytosine residue. This results in a two base pair frame shift leading to premature termination at codon 448, giving a truncated protein. Its relationship to other mutations in tyrosinase and the possible cause are discussed. The temperature-sensitive phenotype is due to the guanine to adenine mutation at codon 422, known to generate a temperature-sensitive enzyme. The CTTT at F439 in tyrosinase is also present at F508 in CFTR, the main mutation causing cystic fibrosis.

Adolescent↗
PubMed1995

Serum soluble interleukin-2 receptors as an index of the biological activity of thyroid hormones in hyperthyroidism.

In order to examine whether serum soluble Interleukin-2 Receptors (sIL-2R) could be used as a marker of the biological effects of the thyroid hormones, we measured the sIL-2R, sex hormone binding globulin and beta-2 microglobulin levels in thirty-three hyperthyroid patients (14 with Graves' disease, 17 with Toxic Nodular Goiter and 2 with toxic adenoma) before and during treatment with antithyroid drugs. We found that serum sIL-2R concentrations of the patients, at diagnosis, were significantly higher compared with normal controls (2424 +/- 1447 vs 459 +/- 184 U/ml). All hyperthyroid patients had sIL-2R levels > mean + 2SD of normal controls, with 28 of the 33 patients having sIL-2R concentrations higher than 1011 U/ml (mean + 3SD of normal controls). Only 15 patients had SHBG levels higher than 3SD above the mean for the normal controls and 28 had SHBG levels 2SD above the mean for the normal controls. Three of the 5 hyperthyroid patients with normal SHBG levels at presentation had abnormally high sIL-2R levels. In all patients sIL-2R levels decreased gradually during therapy down to normal levels when euthyroidism was achieved. A strong positive correlation was found between sIL-2R, SHBG and T3 and T4 concentrations. Serum B2-microglobulin (B2-m) levels were higher than the upper normal limit only in 9 patients, but a significant decrement was observed in all patients when euthyroidism was achieved. The above results indicate that serum sIL-2R levels could be a useful marker of the in vivo biological effects of the thyroid hormones on lymphocytes in hyperthyroid patients.

Adult↗
PubMed1995

Effective treatment of disease-related anaemia in B-chronic lymphocytic leukaemia patients with recombinant human erythropoietin.

Nine B-chronic lymphocytic leukaemia (B-CLL) patients suffering from anaemia, due to no obvious cause except their disease, were treated with recombinant human erythropoietin (r-HuEPO). The treatment protocol provided a closed label phase of 3 months duration, during which the patients received r-HuEPO or placebo in a ratio of 2:1, followed by an open label phase, also of 3 months duration, during which r-HuEPO was administered to all patients three times a week s.c. r-HuEPO was given a dose of 150 U/kg of body weight with an escalation of 50 U/kg up to a maximum of 300 U/kg three times a week. Complete response was achieved in 5/9 (55%) patients and partial response in 3/9 (33%). The response obtained was independent of the pretreatment serum EPO levels, the duration of anaemia, the concomitant administration of chemotherapy, the presence of splenomegaly, or the degree of bone marrow infiltration by lymphocytes. It appears that r-HuEPO is very effective in reversing the disease-related anaemia of B-CLL patients.

Aged↗
PubMed1995

Iron chelators induce apoptosis in proliferating cells.

The iron chelators 1,2-dimethyl-3-hydroxypyrid-4-one (L1) and desferrioxamine (DFO) were found to induce apoptosis of proliferating activated T-lymphocytes and of the promyelocytic cell line HL60, but not of resting peripheral blood lymphocytes or granulocytes. The induction of apoptosis was quantified by propidium iodide staining of apoptotic/dead cells and flow cytometry. In activated T-lymphocytes incubated with the chelators at equivalent iron-binding concentrations (300 microM L1 or 100 microM DFO) for 24 h, L1 caused a 54% increase in cell death and DFO a 57% increase. In HL60 cells L1 caused a 50% increase in cell death and DFO a 40% increase. DNA cytofluorometry of HL60 cells treated with either chelator showed an increase in the percentage of cells with hypodiploid DNA content. Presaturation of the chelators with ferric chloride abrogated these effects. L1 and DFO did not induce apoptosis in resting peripheral blood lymphocytes or granulocytes, even after 48 h of incubation.

Apoptosis↗
PubMed1995

Expression and function of the FAS antigen in B chronic lymphocytic leukemia and hairy cell leukemia.

The expression and function of the FAS antigen was analyzed in 21 patients with B chronic lymphocytic leukemia (CLL) and four with hairy cell leukemia (HCL) using a specific IgM monoclonal antibody and FACS analysis. The FAS antigen was expressed in a minority (5-41%, mean 15.6%) of the CLL cells in 10 of 21 CLL patients and this expression was not modified during spontaneous or hydrocortisone-induced apoptosis of CLL cells. In contrast, culture with gamma-interferon (gamma-IFN) upregulated the expression of FAS in all CLL patients, with 65-100% (mean 84.8%) of the cells being positive after 2 days in vitro culture. Culture with alpha-IFN induced FAS expression in 15 of 19 CLL patients tested, with 15-74% (mean 34%) of the cells being FAS+ after 2 days culture. IL-4 and IL-10, lymphokines that inhibit and promote CLL apoptosis respectively, did not modify the expression of FAS. These results from FACS analysis were consistent with FAS mRNA analysis of fresh and cultured CLL cells, using a semi-quantitative reverse transcriptase (RT)-PCR technique. Although IL-4 and IFNs prevent apoptotic cell death of CLL cells in vitro, the present results show that IFNs induce the expression of the apoptosis-inducing protein FAS. However, FAS+ CLL cells were not killed in the presence of anti-FAS monoclonal antibody (while the FAS+ Jurkat and four lymphoblastoid cell lines were killed). This resistance is not due to a mutated FAS protein, since only wild-type FAS cDNA was demonstrated in the leukemic cells of three CLL patients. In four HCL patients 34-53% (mean 44.5%) of the leukemic cells were FAS+ and they were also resistant to the anti-FAS mediated cytotoxicity. The combination of high bcl-2 protein levels and resistance to anti-FAS mediated cytotoxicity may contribute to the extended in vivo survival of CLL and HCL cells.

Aged↗
PubMed1994

Alpha-interferon (alpha-IFN) protects B-chronic lymphocytic leukaemia cells from apoptotic cell death in vitro.

B-chronic lymphocytic leukaemia cells (CLL) are prone to apoptotic cell death when cultured in vitro. Apoptosis and loss of the bcl-2 protein is prevented in CLL cells cultured in the presence of interleukin-4. In this study we analysed the effects of alpha-IFN on the DNA fragmentation, bcl-2 protein levels and cell survival in purified B-cells from 16 CLL patients. Alpha-IFN (10(3) U/ml) reduced the degree of spontaneous DNA fragmentation of CLL cells after a 30 h culture period (from a mean of 22.2% in control cultures to 10.5%, P < 0.01). This inhibition was accompanied by preservation of bcl-2 protein and an increased survival of CLL cells compared to control cultures. In parallel, alpha-IFN inhibited hydrocortisone induced DNA fragmentation in CLL cells. The effects of alpha-IFN on DNA synthesis of CLL cells were variable (in two patients a decrease and in seven an increase in 3H-thymidine uptake) and did not correlate with the effect on DNA fragmentation. In conclusion, our data suggest that alpha-IFN, like IL-4 and gamma-IFN, inhibits apoptosis of CLL cells. These in vitro data indicate that the clinical responses of some CLL patients to alpha-IFN cannot be explained by a direct cytotoxic effect of alpha-IFN on circulating CLL cells. Alternatively, alpha-IFN may inhibit the proliferation of the small fraction of clonogenic CLL progenitors, or interfere with cellular interactions necessary for the survival and growth of CLL cells.

Aged↗
PubMed1994

The association of systemic lupus erythematosus and myasthenia gravis.

Two women with the rare association of systemic lupus erythematosus (SLE) and myasthenia gravis (MG) are reported. The first patient developed SLE (arthritis, severe thymectomy for MG. The second patient developed SLE (oral ulcers, arthritis, serositis, leukopenia, high titres of anti-DNA and anti-nuclear antibodies) 4 years prior to the clinical and serological onset of MG. Lymphocyte subsets and in vitro proliferative responses of peripheral blood mononuclear cells to mitogens were normal in both patients. A review of the literature revealed 26 additional patients with definite SLE coexisting with MG. Besides the theoretical interest of this association, the differential diagnosis of fatigue in patients with SLE should always include the possibility of MG.

Adult↗
PubMed1994

Influenza virus vaccine in B-cell chronic lymphocytic leukaemia patients.

The clinical reaction and the immunological response to influenza virus vaccine were studied in 43 B-cell chronic lymphocytic leukaemia patients. The Vaxigrip vaccine was administered containing the antigens A/Ghizhou/54/89, A/Singapore/6/86, and B/Yamagata/16/88. The side-effects observed were minimal and well tolerated. Antibody production with titres > 1:20 on day 15 was observed at least for one antigen in 35 patients (81%). In 23 of them (63%) this response was retained on days 30 and 60. Patients with IgG levels (< 700 mg/dl) responded less well as compared to those having normal IgG levels (> 700 mg/dl).

Adult↗
PubMed1993

Interleukin-4 inhibits apoptotic cell death and loss of the bcl-2 protein in B-chronic lymphocytic leukaemia cells in vitro.

When monoclonal B cells from B-chronic lymphocytic leukaemia (B-CLL) patients are cultured in vitro, they die by apoptosis. Apoptotic cell death occurred in the B cells from 20/24 B-CLL patients after 26-30 h in in vitro culture, with 14.3-59.0% (mean 33.6%) of their DNA being fragmented in approximately 180 base pair multimers. After 8-10 d culture, 90-100% of the B-CLL cells were dead. Cell death and DNA fragmentation were inhibited in the presence of 0.5-5 ng/ml human recombinant interleukin-4 (IL-4) and viable monoclonal B cells could be maintained in culture up to 3 weeks. At 5 ng/ml, IL-4 reduced DNA fragmentation after a 26-30 h culture to 2.2-33.3% (mean 14.9%). IL-4 inhibited apoptosis without stimulating cell proliferation. In four patients the cells were resistant to apoptosis in vitro and they could be maintained for up to 4 weeks in culture medium alone. DNA fragmentation in all patients was increased in the presence of the RNA synthesis inhibitor actinomycin-D. Western blot analysis of cell lysates showed expression of the bcl-2 protein in all 11 B-CLL patients studied. However, during culture, bcl-2 protein levels were preserved only in patients resistant to apoptosis and were reduced in those susceptible to apoptosis. Reduction of bcl-2 protein levels was inhibited in cells cultured in the presence of IL-4. These data offer an explanation for the difference between the long life in vivo and rapid death in vitro of B-CLL cells and indicate that IL-4 may participate in the extended survival of these non-dividing cells in vivo.

Adult↗
PubMed1991

Effects of various metals on DNA synthesis and lymphokines production by human peripheral blood lymphocytes in vitro.

1. The presence of Cd2+, Ni2+, Cu2+, Mn2+ and Co2+ (10(-4)-10(-6) M) caused a dose-dependent inhibition of DNA synthesis in phytohemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) from normal human donors. No inhibition of DNA synthesis was observed in the presence of Mg2+ and Al3+. 2. Spontaneous and PHA induced interleukin-1 (IL-1) was enhanced in the presence of Mn2+. 3. The release of interleukin-2 (IL-2) and soluble interleukin-2 receptors (sIL-2R) was decreased by Cd2+, Cu2+ and Mn2+. 4. Spontaneous tumor necrosis factor-alpha (TNF-alpha) production was enhanced by Co2+ and Al3+, but decreased by Cd2+. 5. gamma-Interferon (gamma-IFN) production was decreased in the presence of Cd2+ and Cu2+ and increased in the presence of Al3+.

Aluminum↗
PubMed1991

High levels of serum soluble interleukin-2 receptors in hyperthyroid patients: correlation with serum thyroid hormones and independence from the etiology of the hyperthyroidism.

The serum levels of soluble interleukin-2 receptors (s-IL-2R) and soluble CD8 antigens (s-CD8) were measured in 33 patients with Graves' disease (GD), 29 with toxic nodular goiter (TNG), 6 with toxic adenoma (TA), and 12 with hypothyroidism, as well as in 11 patients with infectious mononucleosis (known to have high s-IL-2R and s-CD8 levels) and 34 normal controls. Serum levels of T3 and T4, both total and free, and of TSH were simultaneously determined. s-IL-2R levels were significantly higher in all patients with hyperthyroidism (mean +/- SD, 3276 +/- 1273 U/mL for GD, 4183 +/- 1832 for TNG, and 1671 +/- 648 for TA) compared to normal control values (P less than 0.001 for GD and TNG and P less than 0.01 for TA), while in the euthyroid state they were within the normal range (535 +/- 240 U/mL). Hypothyroid patients had significantly lower s-IL-2R levels compared to normal controls (P less than 0.05). A positive correlation (P less than 0.001) between serum s-IL-2R levels and total/free T3-T4 levels was found in these groups of patients, while no correlation between s-CD8 levels and s-IL-2R/T3/T4 was found. These findings suggest an association between hyperthyroxinaemia and activation of human lymphocytes in vivo.

Adenoma↗