Isolation of a coronavirus from kidney biopsies of endemic Balkan nephropathy patients.

Endemic Balkan nephropathy (EBN) is a kidney disease of unknown etiology limited to Bulgaria, Rumania and former Yugoslavia. Primary kidney tissue cultures were established as explants from tissue obtained at operations from 5 EBN patients with urinary tract tumors. Four out of the five biopsy specimens on extended culture incubation at 33 degrees C yielded a coronavirus virus (EBNV) which was cytopathogenic for human fibroblast and Vero cells. In cells inoculated with EBNV, cytoplasmic immunofluorescence was found using antisera for human coronaviruses OC43 and 229E as well as the porcine transmissible gastroenteric virus and avian (chicken) bronchitis virus. In neutralization tests, EBNV failed to react with antisera to these viruses. Using hyperimmune serum raised with EBNV, positive cytoplasmic immunofluorescence was seen with cells infected with OC43, 229E, TGV and significantly with the kidney tissue of the biopsy specimens from the EBN patients. A screen for neutralizing antibody using the EBN virus revealed that 87.2% of EBN patients on dialysis were positive, also 74% of people from an endemic area were also positive, while only 13.5% from outside were positive. It is suggested that a coronavirus is involved in the etiology of the disease and that humans are an incidental host of a coronavirus zoonosis.

Temperature enhancement of syncytium formation by HIV and Sendai virus.

Syncytium formation, the characteristic cytopathic effect (CPE) of the human immunodeficiency virus (HIV) and cell fusion by Sendai virus, is accelerated by increasing the ambient temperature to values at which normal metabolic activity is inhibited. Uninfected C8166, CEM, and H9 cells were absorbed at 4 degrees C onto monolayers of H9 cells chronically infected with HIV and incubated subsequently at either 37 degrees C or 45 degrees C. Similarly chick and human erythrocytes and Hela cells were agglutinated with Sendai virus at 4 degrees C before incubation at temperatures of up to 50 degrees C. With both viruses the rate of cell fusion was directly related to temperature. Since membrane fluidity is dependent on the phase-transition temperature points of the membrane lipids it is proposed that sufficient membrane fluidity is essential for cell fusion to occur. The implication of these observations on the cytopathology of HIV is discussed.

Fatty acid composition of normal and malignant cells and cytotoxicity of stearic, oleic and sterculic acids in vitro.

The aim of this study was to investigate the hypothesis that saturated fatty acids are differentially cytotoxic to cancer cells. Three studies were undertaken to: (1) measure the toxicities of stearic and oleic acids to normal and malignant cells in vitro, (2) assess if there is any relationship between toxicity and relative fatty acid composition and (3) determine whether the relative fatty acid composition of a cancer cell line could be modified by sterculic acid, an inhibitor of delta-9-desaturase. Stearic (18:0) and oleic (18:1) acids inhibited the colony-forming abilities of five human cancer cell lines and two non-neoplastic cell lines in a dose-dependent fashion. The concentration of oleic acid required to reduce colony formation ability by 50% was 2.5-6.0-fold greater than that of stearic acid. Addition of sterculic acid to a cancer cell line resulted in steady-state levels of stearic acid and increasing percentage of oleic acid.

Manipulation of body fat composition with sterculic acid can inhibit mammary carcinomas in vivo.

Sterculic acid, a delta-9-desaturase inhibitor, administered to rats caused a rise in the stearic:oleic acid ratio of total lipids in peripheral red cells, serum and liver (P less than 0.001). As a reduction in the stearic:oleic acid ratio has been described in cancer cells, we investigated the effect of sterculic acid on tumour growth. Female F344 rats were injected subcutaneously with two different doses of sterculic acid for 4 weeks prior to, and 4 weeks following, implantation of a nitrosomethylurea-induced mammary tumour. Tumour growth was inhibited equally by the two doses of sterculic acid (P less than 0.001). A rise in the stearic:oleic acid ratio of tumours was observed in rats treated for only 16 days with sterculic acid. Manipulation of the tissue stearic:oleic acid ratio inhibits transplanted mammary tumour growth in rats.

Syncytia formation in HIV-1 infected cells is associated with an increase in cellular oleic acid.

Infection of cells with the human immunodeficiency virus type-1 (HIV-1) usually results in the formation of giant multinuclear cells (syncytia) [(1986) Nature 322, 470-474; (1986) Nature 322, 725-728; (1985) Hum. Pathol. 18, 760-765; (1987) Ann. Neurol. 21, 490-496]. The appearance of syncytia is associated with an increase in the monounsaturated oleic acid content. This report describes experiments which compare the activity of known antiviral agents with that of saturated fatty acid derivatives in inhibiting oleic acid and syncytia formation. A concept is introduced which proposes that infection of cells with the human immunodeficiency virus causes a rise in cellular oleic acid which leads to increased membrane fluidity.

Alpha 2 interferon in the prevention of N-nitroso-methyl urea induced breast cancer in rats.

Human interferons have been shown to be effective treatment for hairy cell leukaemia and are now commercially available. Their role in treatment of solid tumours has yet to be established. This study assessed the value of alpha 2 interferon (IFN) in an experimental breast cancer model. Four groups of female Sprague-Dawley rats were studied. The first received three intravenous injections (7 mg/kg) of N-nitroso-methyl urea (NMU) at weeks 0, 3 and 7. The second received the same NMU dosage regime plus IFN (100,000 IU, twice weekly for 3 weeks). A third received IFN alone and the fourth was a control group receiving three intravenous injections of normal saline. At week 16, 19 of 20 rats in the NMU alone group had developed tumours significantly more than four of 15 rats with tumour in the NMU plus IFN group (P less than 0.001). Both the mean tumour number/rat and the mean tumour weight/rat was significantly more in the NMU group than the NMU plus IFN group P less than 0.05). No rats in the IFN alone or control group developed tumour. These data suggest that IFN prevents carcinogen induced breast cancer in rats. It may have a role in the prevention and treatment of human breast cancer.

Stearic acid and carcinogenesis.

Decreased membrane rigidity is one of the characteristics of malignant cells, resulting in part from the desaturation of stearic acid into oleic acid. In this study we investigated the influence of stearic acid on tumour cell inhibition in vitro and tumour development in vivo. Stearic acid inhibited the colony-forming ability of 4 out of 5 rat and two human tumour continuous cell lines in vitro. In contrast, the colony-forming ability of rat fibroblasts was not inhibited and that of human foetal lung fibroblasts was inhibited at a higher dose than that required to inhibit human tumour cell lines. Using a model of rat mammary carcinoma induced by nitroso-methyl urea (NMU) the subcutaneous injection of stearic acid at weekly intervals prevented tumour development in 5 to 10 rats. Using iodostearic acid twice weekly, 11 of 19 rats were alive and tumour free at week 22 whilst all of 14 animals injected with NMU alone had died of tumour by the 16th week. The ratio of stearic to oleic acids in erythrocyte membranes was significantly reduced in the tumour-bearing rats, but was normal in tumour-free animals treated with stearic or iodostearic acid. These preliminary data indicate that stearic acid inhibits tumour development in rats.

Natural IFN-alpha therapy in hairy-cell leukaemia (namalva-type IFN--Wellferon).

More than 50 patients with hairy-cell leukaemia have now been entered into the British Wellferon (IFN) study, at different centres under the coordination of the Wellcome Research Laboratories. While the treatment duration and dosage varied between patients, the initial dose was usually 3 megaunits daily by intramuscular or subcutaneous injection. Although flu-like symptoms and mild somnolence were commonly experienced side-effects; cessation of IFN treatment as a result of such effects was necessary in only three patients. All patients irrespective of previous treatment showed some response and a complete (less than 5% hairy cells, HCs) bone marrow response was observed in 17. The degree of response was related to the duration of therapy. Immunological markers showed that there was no apparent increase in natural killer (NK) cells and no return of normal B lymphocytes. Light-chain-restricted B cells became reduced in parallel with the disappearance of morphological HCs. Absolute numbers of T cells were reduced, Leu2a+ preferentially, resulting in an increase in helper/suppressor ratios. The ratios (saturation index, SI) of saturated to unsaturated 18 carbon fatty acids (C18FA) of erythrocyte or leukocyte membranes, while abnormal in untreated patients, approached normal levels during IFN therapy. It is concluded that prolonged alpha IFN therapy is highly effective in HCL. The mechanism of action of IFN remains unknown, but indirect surface-marker data favours a direct effect on HCs.

Saturation index of blood cell membrane fatty acids before and after IFN treatment in hairy cell leukemia.

Cells from the peripheral blood of 32 patients with hairy cell leukemia (HCL) were analyzed for cell membrane fatty acid composition. A consistent and significant change was found in the relative amounts of stearic and oleic acid components, which is expressed as the saturation index (SI), the ratio of stearic to oleic acid content. In all patients, SI was reduced in the erythrocytes, leukocytes, and platelets. On extended treatment with interferon, the hematological improvement was accompanied by an increase in SI. In two patients, one deteriorating after cessation of treatment and the other on a low maintenance dose of interferon, concurrent influenza infection induced temporary improvements in the blood cell counts, as well as increase in SI values. These results indicate that endogenous interferon can also be effective in inducing hematological and SI improvement in this disease. The relevance of low SI values and concomitant increase in membrane fluidity to the malignant transformation is discussed. It is proposed that one effect of interferon is to reduce the production of growth factors by reducing the malignant cell mass.

Increased erythrocyte stearic acid desaturation in rats with chemically induced colorectal carcinomas.

Our previous studies have demonstrated a desaturation of stearic acid related to oleic acid in the lipid layer of erythrocytes in patients with malignancies. This study investigated the stearic acid desaturation in red blood cell membranes of rats during the induction of colorectal tumours. Male Sprague-Dawley rats were injected weekly with dimethylhydrazine (DMH) and sacrificed at 4-week intervals. Blood was withdrawn via heart puncture, collected in EDTA bottle and erythrocytes separated by centrifugation. Total lipid extraction was carried out and analysed with gas liquid chromatography. In the control rats (injected with normal saline) the mean of the stearic to oleic acid ratio in erythrocyte membranes was 2.0 +/- 0.3 (n = 28, range 1.51-2.62) compared to a mean of 0.94 +/- 0.16 (n = 0.5-1.23) in tumour bearing rats (p less than 0.001). The increased desaturation occurred in parallel with appearance of tumours. These data suggest the regulation of stearic acid desaturation is an important adaptive mechanism of membrane fluidity and could be a useful chemical marker for malignancy.

Desaturation-producing factor present in the tissue, blood, and urine of cancer patients.

Patients with liver neoplasms have a significantly lower stearic to oleic ratio (SI) in tumour (x = 0.36) vs non-tumour liver tissues (x = 0.75; P less than 0.001). The SI of circulating erythrocytes is lower in cancer patients (n = 100; X = 0.69) than noncancer patients (n = 50; x = 1.45) and normal subjects (n = 50; x = 1.5) (p less than 0.001). It was also found that urine protein extracts from cancer patients reduce the SI of cultured cell membrane (n = 10; x = 0.74) as compared to urine from noncancer patients (n = 10; x = 1.02; p less than 0.001). This study suggests that patients with cancer release a desaturation-producing factor from the malignant cells into the systemic circulation and subsequently into the urine. The observed increased unsaturation (oleic acid) in the circulating erythrocytes can be useful in the diagnosis and postoperative monitoring of cancer patients.

Reduction in the stearic to oleic acid ratio in human malignant liver neoplasms.

Total lipid extracts of liver tissue from 14 patients with primary and secondary liver tumours were analysed for relative values of saturation of 18 carbon chain length fatty acids (C18FA). The saturation indices (ratio C18S:C18u) of the tumour areas were significantly and consistently lower than the corresponding values in the non-tumour areas (P less than 0.001). It is proposed that the relative increase in unsaturated C18FA (oleic acid) could prove to be a chemical marker reflecting deficient cellular control of the desaturation of stearic acid.

Increase of oleic acid in erythrocytes associated with malignancies.

Total lipid extracts of erythrocyte cell membranes from 60 patients with documented malignancies, 41 patients with various acute and chronic diseases, and 40 healthy subjects were analysed. The results were expressed as ratios of stearic to oleic acid, reflecting the degree of desaturation of stearic acid. The mean ratios for the healthy subjects and controls without cancer were 1.5 (SD 0.27) and 1.45 (0.28), respectively, whereas the ratios for patients with malignancies were consistently lower than the cut off point of 1.0, with a mean of 0.69 (0.15) (p less than 0.001). The desaturation ratio was also significantly lower (p less than 0.001) in the group with recurrent tumours (mean 0.75 (0.04)) compared with those with no evidence of recurrent tumours (mean 1.55 (0.27)). It is suggested that the increased unsaturation (oleic acid) in the circulating erythrocytes may be useful in the diagnosis and postoperative monitoring of patients with cancer.

Reduction in the stearic to oleic acid ratio in leukaemic cells--a possible chemical marker of malignancy.

Total lipid extracts of peripheral blood cells from patients with chronic leukaemias were analysed for relative values of saturation of the eighteen carbon chain length fatty acids (C 18 FA). The results are expressed as saturation index (C 18 S:C 18 U) of the saturated C 18 FA (stearic acid) over the unsaturated C 18 FA (oleic, linoleic and linolenic acids). The saturation indices of the white blood cells (WBC) and the red blood cells (RBC) in specimens from 14 patients with chronic granulocytic leukaemia (CGL) and 17 patients with chronic lymphocytic leukaemias (CLL) were significantly and consistently lower than control specimens. It is proposed that the relative increase in the unsaturated oleic acid could prove to be a chemical marker of malignancy reflecting a deficient cellular control of the process of stearic acid desaturation. The theoretical implications of the implied increase in membrane fluidity for the cells are discussed.

Reduction in the stearic to oleic acid ratio in the circulating red blood cells: a possible tumour marker in solid human neoplasms.

Gas liquid chromatography study of the 18 carbon chain length fatty acids (C18FA) of the human red blood cells (RBCs) was performed on 65 patients with various clinical disorders. It was found that the stearic to oleic acid ratio (SI) of the RBCs was significantly lower (P less than 0.001) in patients with malignant conditions (n = 20, SI = 0.62 +/- 0.16) compared with pathological non-malignant diseases (n = 10, SI = 1.19 +/- 0.2) and the normal control group (n = 35, SI = 1.57 +/- 0.5). Our early results suggest that the increased unsaturation (oleic acid) in the circulating RBCs could be used as a chemical marker in various solid neoplasms.