Chemosensitizing interactions of clofazimine and B669 with human K562 erythroleukaemia cells with varying levels of expression of P-glycoprotein.

Differential expression of a permeability glycoprotein (P-gp) in human myeloleukaemia K562 cells grown in the presence of the anti-cancer drug, doxorubicin, gave rise to subclones with varying degrees of resistance to other anti-tumour drugs such as vinblastine and daunorubicin. Subclones K562/MMB, MMG and MMF were produced from the parental (K562/P) cell line via limiting dilution and their MDR nature confirmed with flow cytometry using an MRK 16 monoclonal antibody directed at a surface epitope of the P-gp pump. The pattern of increasing P-gp expression in the series K562/P, MMF, MMG and MMB was paralleled by increasing resistance to vinblastine and daunorubicin. When the subclones were pre-incubated with the chemosensitizing drugs clofazimine and B669, a pattern of increasing reversal of resistance to vinblastine and daunorubicin was seen in the series K562/P, MMF, MMG and MMB.

The riminophenazine agents clofazimine and B669 reverse acquired multidrug resistance in a human lung cancer cell line.

The potential of the riminophenazine agents clofazimine and B669, at therapeutically relevant concentrations, to reverse P-glycoprotein-mediated multidrug-resistance (MDR) in a human lung cancer cell line (H69/LX4) has been investigated in vitro. Cyclosporin A, a well-documented MDR-modifying agent, was included for comparison. Clofazimine, B669 and cyclosporin A at minimally cytotoxic concentrations of 1, 0.5 and 5 micrograms/ml, respectively, were equally effective in restoring sensitivity to vinblastine, doxorubicin, daunorubicin and mitomycin C in the H69/LX4 cell line. All three chemosensitizing agents also increased the accumulation of [14C]vinblastine by H69/LX4 cells. Riminophenazines, which are relatively non-toxic, non-carcinogenic and non-myelosuppressive agents, are promising contenders for evaluation in experimental and clinical oncology as modulators of acquired MDR.

The chemosensitizing potential of GF120918 is independent of the magnitude of P-glycoprotein-mediated resistance to conventional chemotherapeutic agents in a small cell lung cancer line.

GF120918, at 250 ng/ml, increased the sensitivity of a P-glycoprotein (P-gp)-mediated multidrug resistant (MDR) small cell lung cancer cell line (H69/LX4) to the P-gp substrates, paclitaxel, taxotere, vinblastine, vinorelbine, daunorubicin and etoposide to levels which were either greater (in the case of etoposide) or close to that of the parent cell line (H69/P). This was achieved in spite of the great variation in the levels of resistance of the MDR cell line for the various anti-cancer drugs tested. These data suggest that GF120918 is a potent antagonist of P-gp mediated multidrug resistance, even in the case of high levels of resistance, as was the case with paclitaxel and taxotere (2560 and 2215 fold more than the sensitive parent cell line respectively).

Demonstration of growth-inhibitory as well as growth-stimulatory factors in medium conditioned by lung lavage cells stimulated with a chemotactic factor secreted by jaagsiekte tumour cells.

Both growth-inhibitory and growth-stimulatory factors were detected in vitro in medium from chemotactically stimulated cultures of lung lavage cells. The macrophage component of the lavage cells was found to produce a growth stimulatory factor that was replaced by a growth inhibitory factor following chemotactic factor stimulation.

Production of a macrophage chemotactic factor by cultured jaagsiekte tumour cells.

The increase of alveolar macrophages in jaagsiekte sheep lungs is not caused by excessive surfactant production but is due to a chemotactic factor secreted by the tumor cells. This factor has a molecular mass in the region of 13 kilodaltons, is stable at 56 degrees C but labile at 100 degrees C and, being sensitive to proteases, indicates that it is a small protein molecule.

Isolation and preliminary characterization of the jaagsiekte retrovirus (JSRV).

Jaagsiekte, or ovine pulmonary adenomatosis, is caused by a recently discovered retrovirus. The virus cannot be cultivated in vitro at present, but a procedure is described for the isolation and purification of small amounts in the form of immune complexes with IgA from affected lungs. The virion was shown to possess a 70S RNA genome which can be transcribed by an endogenous reverse transcriptase. Nine size from 94 000 to 25 000 daltons, were found in purified preparations. Using neutralization of the viral reverse transcriptase and an enzyme immunoassay as criteria, no serological relationship could be demonstrated to representatives of type B, C and C oncoviruses, or to bovine leukemia virus, maedi-visna virus of sheep or caprine arthritis-encephalitis virus.

The possible involvement of immunosuppression caused by a lentivirus in the aetiology of jaagsiekte and pasteurellosis in sheep.

A South African isolate of ovine lentivirus was shown to cause a mild immunosuppression in sheep, reflected by a reduced delayed hypersensitivity reaction. This effect, measured in terms of skin swelling after intradermal inoculation with tuberculin, showed a positive linear relationship with the latency period before the appearance of jaagsiekte symptoms in animals co-infected with JSRV, as well as with the activity of monocytes. In a parallel study, increased susceptibility of lentivirus-infected sheep to infection with Pasteurella haemolytica was demonstrated. It is concluded that the lentivirus may play an enhancing role in both viral and bacterial infections of sheep by compromising the host's cellular immune response.

Alpha-tocopherol prevents cyclosporin A-mediated activation of phospholipase A2 and inhibition of Na+, K(+)-adenosine triphosphatase activity in cultured hamster renal tubular cells.

At concentrations of 0.5 microM and upward, cyclosporin A (CsA) caused dose-related inhibition of the growth of a hamster renal tubular cell line (HAK ATCC; CCL15) in vitro. Inhibition of cell growth was due to the cytotoxic properties of CsA which were associated with enhancement of activity of phospholipase A2 (PLA2) according to the increased generation of arachidonic acid and lysophosphatidylcholine (LPC). Arachidonate per se, at concentrations of up to 20 microM, did not affect the growth of HAK cells, while cyclooxygenase and 5-lipoxygenase inhibitors failed to protect the cells against the antiproliferative effects of CsA. However, LPC caused dose-related inhibition of the growth of HAK cells. Moreover, coincubation with lysophospholipase or alpha-tocopherol (AT, vitamin E), a PLA2 inhibitory and lysophospholipid-complexing agent, protected the HAK cells against both CsA and LPC. The Na+, K(+)-ATPase activity of HAK cells was also inhibited by CsA, with the enzyme being protected by inclusion of AT or lysophospholipase. Increased activity of PLA2 and inhibition of Na+, K(+)-ATPase preceded cytotoxicity and cytolysis. Excessive production of lysophospholipids and consequent inhibition of Na+, K(+)-ATPase in renal tubular cells is a possible mechanism of CsA-induced nephrotoxicity. The protective effects of AT suggest that this agent may be clinically useful in preventing the renal side effects of CsA.

Measurement of anti-acetylcholine receptor auto-antibodies in myasthenia gravis.

Two different acetylcholine receptor (AChR) preparations derived from amputated human muscle (AChRAMP) and from the human rhabdomyosarcoma cell line TE671 (AChRTE671) were compared in radio-immunoprecipitation assays for the detection of AChR auto-antibodies in serum specimens from 20 patients with proven myasthenia gravis. Tests performed with the AChRTE671 and AChRAMP antigen preparations were positive in all the patients and in 19/20 respectively. A high degree of correlation (r = 0.94) was evident between the two auto-antigen preparations. Assays based on the use of TE671-derived antigen represent a useful alternative to the conventional assay using AChRAMP for the detection of AChR auto-antibodies.

Passive smoking by humans sensitizes circulating neutrophils.

The proinflammatory effects of passive inhalation of cigarette smoke were investigated by exposing a total of 16 healthy, young nonsmokers (mean age 29 +/- 1.4 yr, 11 women and five men) to actively smoking individuals in a poorly-ventilated room. Neutrophil functions were measured before and after 3 h of exposure to cigarette smoke. Passive cigarette smoking was associated with increased leukocyte counts (mean increase 33%, p less than 0.005), chemotaxis (57%, p less than 0.001), and release of reactive oxidants (71%, p less than 0.005) by stimulated neutrophils. These results were confirmed in a second study designed to eliminate the possible complicating effects of serial venepuncture. Plasma concentrations of the proinflammatory cytokines interleukin-1 (IL-1) alpha, IL-1 beta, IL-6, and tumor necrosis factor alpha (TNF alpha) were not affected by passive smoking. These results indicate that inhalation of sidestream tobacco smoke promotes systemic priming of neutrophils. These potentially proinflammatory events may induce oxidant-mediated tissue damage and carcinogenesis in the lungs of passive smokers.

Novel tetramethylpiperidine-substituted phenazines are potent inhibitors of P-glycoprotein activity in a multidrug resistant cancer cell line.

The multidrug resistance (MDR)-neutralizing and cytotoxic properties of 16 novel tetramethylpiperidine (TMP)-substituted phenazines were compared with those of clofazimine and B669 using a P-glycoprotein (P-gp)-expressing undifferentiated, human leukemia cell line (K562/MMB). Unchlorinated TMP-substituted phenazine molecules were more cytotoxic than their chlorinated counterparts, while the halogenated molecules, especially those with chlorine atoms at position 3 on the aniline and phenyl rings, were less cytotoxic but more effective as chemosensitizing, P-gp-neutralizing agents. One of the TMP-substituted phenazines, B4121, increased the sensitivity of K562/MMB cells to vinblastine by 100-fold. TMP-substituted phenazines are a novel class of pharmacologic anti-cancer agents with both direct cytotoxic, as well as MDR-neutralizing anti-tumor properties.

Peptides generated from C-reactive protein by a neutrophil membrane protease. Amino acid sequence and effects of peptides on neutrophil oxidative metabolism and chemotaxis.

We have recently provided evidence that C-reactive protein (CRP) could act as an up-regulatable substrate for membrane-associated neutrophil serine protease(s). The resultant degradation of CRP yielded small soluble bioactive peptides that inhibit many of the proinflammatory functions of activated neutrophils and could oppose the tissue destructive potential of these cells. We report on the reverse phase HPLC separation of the small TCA-soluble peptides obtained when CRP is degraded with nonstimulated or PMA-stimulated neutrophils and purified neutrophil membranes. The amino acid sequence of seven peptides isolated from the CRP digest has been ascertained and synthetic peptides homologous to these sequences have been synthesized. Three of the synthetic peptides corresponding to residues 201-206 (CRP-III), 83-90 (CRP-IV), and 77-82 (CRP-V) of the intact protein were identified to significantly inhibit superoxide production from activated neutrophils at 50 microM whereas CRP-III and CRP-V in addition inhibited neutrophil chemotaxis at this concentration. These peptides act additively and their action likely involves the signal transduction pathways for neutrophil activation.

Investigation of the relative contributions of cigarette smoking and mineral dust exposure to activation of circulating phagocytes, alterations in plasma concentrations of vitamin C, vitamin E, and beta carotene, and pulmonary dysfunction in South African gold miners.

OBJECTIVES: To determine the relative effects of cigarette smoking and mineral dust exposure on numbers and activity of circulating phagocytes, plasma nutritional antioxidant state, and pulmonary function in South African gold miners. METHODS: Pulmonary function was assessed spirometrically, whereas reactive oxidant generation by circulating phagocytes, and plasma concentrations of the nutritional antioxidative nutrients vitamin C and vitamin E and beta carotene were measured with chemiluminescence, spectrophotometry, or high performance liquid chromatography respectively. RESULTS: Cigarette smoking, but not mineral dust exposure, was associated with increased numbers and pro-oxidative activity of circulating neutrophils and monocytes, decreased plasma concentrations of vitamin C, and pulmonary dysfunction. DISCUSSION: In this study group occupational exposure to mineral dust has not been found to promote increases in the numbers or reactivity of circulating phagocytes or to be a significant cause of pulmonary dysfunction, the changes found being due primarily to cigarette smoking.

Health effects of passive smoking in adolescent children.

OBJECTIVE AND DESIGN: To study the effects of passive smoking on health in adolescent schoolchildren by questionnaire, spirometry and laboratory investigations. SETTING: Two schools in the Vanderbijlpark area. PARTICIPANTS: Seven hundred and twenty-six high-school children of average age 16 years. OUTCOME MEASURES: Lung function, serological abnormality or historical (i.e. questionnaire) evidence of ill health. RESULTS: The prevalence of respiratory illness before and after 2 years, respiratory symptoms, earache over the past year, low birth weight and learning difficulties were found to be significantly increased in the children exposed to parenteral smoke in the home, especially those exposed to maternal smoking. Spirometric and laboratory parameters, however, were not affected by passive smoking.