The major T cell epitope on type II collagen is glycosylated in normal cartilage but modified by arthritis in both rats and humans

Type II collagen (CII) is a target for autoreactive T cells in both rheumatoid arthritis and the murine model collagen-induced arthritis. The determinant core of CII has been identified as CII260-270, and the alteration of this T cell epitope by posttranslational modifications is known to be critical for development of arthritis in mice. Using CII-specific T cell hybridomas we have now shown that the immunodominant T cell epitope in the normal (healthy) human and rat joint cartilage is O-glycosylated at the critical T cell receptor recognition position 264 with a mono- or di-saccharide attached to a hydroxylysine. In contrast, in the arthritic human and rat joint cartilage there are both glycosylated and non-glycosylated CII forms. Glycosylated CII from normal cartilage could not be recognized by T cells reactive to peptides having only lysine or hydroxylysine at position 264, showing that antigen-presenting cells could not degrade the O-linked carbohydrate. Thus, the variable forms of the glycosylated epitope are determined by the structures present in cartilage, and these vary during the disease course. We conclude that the chondrocyte determines the structures presented to the immune system and that these structures are different in normal versus arthritic states.     

Growth control of human mammary cancer cells (MCF-7 cells) in culture: effect of estradiol and growth factors in serum-containing medium

The growth control of estrogen-dependent mammary cancer is very complex and only partly understood. The present study was undertaken in order to establish conditions for growth control of MCF-7 cells in monolayer culture with focus on the effect of estradiol-17 beta, fetal calf serum, and growth factors. The effect of charcoal-stripped fetal calf serum (CSFCS) on cell growth was dependent upon the presence of hormones or growth factors in the medium. In the presence of insulin (or insulin-like growth factor 1) and in the absence of estradiol-17 beta, increasing concentrations of CSFCS, 0.625-20%, produced a bell-shaped growth response curve. Serum concentrations greater than 2.5% inhibited cell growth in the absence of estradiol-17 beta, whereas CSFCS in a dose-dependent way up to 10% stimulated growth in the presence of estradiol-17 beta (5 x 10(-10) mol/liter). The growth inhibitory effect of CSFCS could not be demonstrated in the absence of insulin (or insulin-like growth factor 1) and estradiol-17 beta. CSFCS stimulated growth in a dose-dependent way in the presence of estradiol-17 beta and also in the absence of insulin. Both the putative growth inhibitor and stimulator were found to be heat stable and not dialyzable. Epidermal growth factor stimulated growth but was unable to eliminate the growth inhibitory effect of 5-10% CSFCS. Interleukin-1 alpha inhibited MCF-7 cell growth in a dose-dependent way and produced a 75% reduction in cell number at a concentration of 5 x 10(-10) mol/liter. This inhibition was almost totally overcome by estradiol-17 beta. It is concluded that serum appears to contain factors with both stimulatory and inhibitory effects on the growth of MCF-7 cells. The inhibitory effect can be eliminated by estradiol (5 x 10(-10) mol/liter). In the presence of estradiol cell growth is stimulated by CSFCS in a dose-related way up to 5-10%. Taken together these data seem to indicate that estradiol stimulates cell growth in two principal ways: partly by eliminating the effect of an inhibitor, in support of a "negative hypothesis," and partly by an effect whereby estradiol permits a growth stimulator in CSFCS to be expressed, in support of the "indirect positive hypothesis."     

Pressure Transfer Between Intracranial and Cochlear Fluids in Patients With Meniere's Disease

Objectives: To elucidate the pressure transfer between intracranial and labyrinthine fluids in patients with well‐defined unilateral Meniere's disease. Study Design: Eleven patients previously exposed to hypobaric pressure agreed to be investigated further with the tympanic membrane displacement (TMD) technique. TMD was used to indirectly analyze perilymph pressure changes as the result of changes in body position. Methods: Repeated measurements for both the diseased and the healthy ears were made with the patients supine and then in a sitting position. The TMD parameters for the maximum inward displacement, the Vi, and the mean volume displacement, the Vm, were calculated and compared. Results: The paired comparison showed statistically significant larger Vi values for both ears in the supine position. A similar tendency was observed for the Vm value. This difference of the Vi was significantly larger for the diseased ear compared with the currently healthy ear. The results were compared to the audiometric and electrocochleographic results previously obtained on the same patients when they were subjected to hypobaric pressure. Patients who experienced the largest differences in hearing level thresholds in the lower frequencies also showed the greatest differences in TMD values as the result of postural changes. Conclusions: Despite the limited number, the statistically supported results suggest a relation between the efficiency of the routes of pressure transfer and the observed effect of hypobaric exposure. The results also indicate that for the patients tested, the routes of communication are more effective in the diseased ear than in the healthy ear—a condition that may relate to the pathogeneses of Meniere's disease.     

Formaldehyde and cancer morbidity among male employees in Denmark

Formaldehyde, a genotoxic and potent animal carcinogen, is widespread in the working environment as well as in private homes. The risk for cancer morbidity in Denmark during 1970–84 was estimated from standardized proportionate incidence ratios (SPIR) among men whose longest employment had been held since 1964, at least 10 years before diagnosis, in 265 companies in which exposure to formaldehyde was identified. The results do not support the hypothesis that formaldehyde is associated with lung cancer (SPIR=1.0,410 cases). Significantly elevated risks were found for cancers of the colon (SPIR=1.2,166 cases), kidney (SPIR=1.3,60 cases), and sino-nasal cavities (SPIR=2.3,13 cases). For sino-nasal cancer, a relative risk of 3.0 (95 percent confidence interval=1.4–5.7) was found among blue-collar workers with no probable exposure to wood dust, the major confounder. This study provides further evidence that occupational exposure to formaldehyde increases the risk for sino-nasal cancer.     

Radiosensitizing effect of estramustine in malignant gliomain vitro andin vivo

Summary Estramustine-phosphate (EMP), a combination of nornitrogen mustard and 17-estradiol, has been demonstrated to exert specific antiproliferative effects on human glioma cellsin vitro. The cytotoxic effect is, at least partially, mediated by inhibiting microtubule function. In this study the combined effect of EMP and radiation was evaluated in the human glioma cell-lines, 251-MG and 105-MG,in vitro, and in the rat glioma BT4Cin vitro andin vivo. In all cell-lines an additive effect of EMP and radiation was obtainedin vitro. Assuming equal effect of EMP is obtained in subsequent radiation fractions, the cell kill will be increased from 2–3 to 5–10 logs if delivering 30 fractions of 2 Gy combined with EMP. In the BT4C rat model the combined effect was found to be synergistic. Flow cytometry demonstrated an arrest in G2/M phase in all cell-lines after EMP treatment. This block in G2/M phase in addition to the previously demonstrated induction of free oxygen radicals, and the increase of blood flow with an assumed subsequent increase of oxygenation, might provide an explanation for the observed radiosensitizing effect of estramustine.     

In vitro effect of r-verapamil on acute myelogenous leukemia blast cells: studies of cytokine secretion and cytokine-dependent blast proliferation

The in vitro effect of the dextroisomer r-verapamil on blast cells derived from patients with acute myelogenous leukemia (AML) was studied. R-verapamil caused a dose-dependent inhibition of AML blast proliferation in the presence of stem-cell factor, leukemia inhibitory factor, interleukin 4, interleukin 6, and interleukin 10 when these cytokines were tested both alone and in different combinations. R-verapamil also inhibited the growth of clonogenic AML blast cells. The antiproliferative effect was not specific for AML blast cells, because r-verapamil also inhibited cytokine-dependent proliferation of blast cells derived from patients with acute lymphoblastic leukemia. The inhibitory effects of r-verapamil and anti-IL1 serum were additive, suggesting that the antiproliferative effect of r-verapamil does not depend solely on inhibition of IL1-mediated effects. Although r-verapamil inhibited spontaneous AML blast proliferation, for a majority of patients it caused only minimal, if any, inhibition of spontaneous cytokine secretion (IL1, IL1, TNF, IL6) by AML blast cells. Thus, although inhibition of IL1 effects may contribute in certain patients to the antiproliferative effect of r-verapamil, mechanisms other than IL1 inhibition seem to be more important in mediating the effects of r-verapamil.Abbreviations ALL Acute lymphocytic leukemia - AML acute myelogenous leukemia - cpm counts per minute - ELISA enzyme-linked immunosorbent assay - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor - IL interleukin - IF leukemia inhibitory factor - PBMC peripheral blood mononuclear cells - RR relative response - SCF stem cell factor - TNF tumor necrosis factor      

Plasma concentrations and renal clearance of orotic acid in argininosuccinic acid synthetase deficiency

Argininosuccinic acid synthetase deficiency (ASD) is a rare disorder of urea cycle metabolism, with pronounced citrullinemia and orotic aciduria being characteristic biochemical features. To further investigate the role of plasma orotic acid and its possible use for monitoring the metabolic status in ASD, we determined plasma orotic acid, amino acid, and ammonium levels in plasma samples collected over a period of 3 years from a patient who is now 8 years of age. Orotic acid plasma concentrations varied widely from less than 1 μmol/l to more than 60 μmol/l. The renal clearance of orotic acid was eightfold the glomerular filtration rate, thus supporting an active mechanism underlying the excretion of this pyrimidine. Data obtained during a metabolic crisis yielded a statistically significant linear correlation of orotic acid plasma levels with those of glutamine and ammonium, which are generally accepted for assessment of the successful treatment of this disorder. Our data revealed no advantage of plasma orotic acid concentrations over the established amino acids (glutamine and arginine) and ammonium for determining acute treatment responses. Since several effects of high levels of orotic acid have been described in mammals, further research is necessary to assess a possible contribution of orotic acid to the pathogenesis of ASD and the use of plasma orotic acid levels in the long-term monitoring of these patients. Received: 3 November 1998 / Revised: 3 May 1999 / Accepted: 3 May 1999     

Detection of polycyclic aromatic hydrocarbon metabolites by high-pressure liquid chromatography after purification on immunoaffinity columns in urine from occupationally exposed workers

Objective: The objective in our study was to quantitate benzo[a]pyrene (B[a]P) metabolites by a combination of immunoaffinity chromatography and high-pressure liquid chromatography (HPLC) with fluorescence detection in urine from workers exposed to high levels of polycyclic aromatic hydrocarbons (PAH). Furthermore, by the simultaneous quantitation of 1-hydroxypyrene, the correlation between the B[a]P-tetrol and 1-hydroxypyrene would provide a means of evaluating the validity of 1-hydroxypyrene as a surrogate biomarker for occupational exposure to the potent carcinogen B[a]P in an electrode paste plant. Methods: The study was carried out at an electrode paste plant that produces electrode paste for Söderberg electrodes. A total of 34 pre- and post-shift urine samples and 17 personal air samples were collected from 17 workers during a normal work week. The concentration of 1-hydroxypyrene was measured in all urine samples. A recent method of quantitating B[a]P-r-7, t-8, t-9, c-10-tetrol in urine of humans exposed to low levels of PAH has been described. A modified version of this method involving purification of urine samples on immunoaffinity columns and HPLC analysis with fluorescence detection was used on urine samples from workers exposed to high levels of PAH. A monoclonal antibody (8E11) with binding affinity to B[a]P-tetrols was used. This antibody also binds several PAH-DNA adducts and metabolites, including 1-hydroxypyrene. Gas chromatography/mass spectroscopy (GC/MS) was also used for identification of metabolites isolated by HPLC fractionation. Results: From personal air sampling the mean exposure to particulate PAHs was 38?μg/m³. The mean concentration of urinary 1-hydroxypyrene was 3.9?μmol/mol creatinine in preshift samples and 10.2?μmol/mol creatinine in postshift samples. We could not identify detectable amounts of urinary B[a]P-tetrol by HPLC or fluorescence spectroscopy after purification on immunoaffinity columns. However, in the HPLC analysis we identified several hydroxyphenantrene metabolites that were detected at relatively high concentrations in all of the workers' urine samples. We could not separate 2- and 3-hydroxyphenanthrene (2?+?3-OH-Phe) in peak 1, and peak 2 contained both 1- and 9-hydroxyphenanthrene (1?+?9-OH-Phe). The phenanthrene metabolites were mainly conjugated to glucuronic acid and sulfate. There was a significant correlation between the 1-hydroxypyrene concentration and 2?+?3-OH-Phe (r?=?0.73) and 1?+?9-OH-Phe (r?=?0.64) in the urine samples. 1-Hydroxypyrene was measured in all post-shift urine samples but was not significantly correlated with workplace pyrene exposure, indicating that skin exposure is an important route of pyrene exposure in this factory. As with 1-hydroxypyrene, dermal PAH uptake may also account for the poor correlation between 2?+?3- and 1?+?9-OH-Phe and ambient phenanthrene. Discussion: Since dermal uptake is likely to be important in occupational PAH exposure in addition to inhalation, estimation of total PAH exposure is best achieved by quantitation of PAHs excreted into body fluids. However, it remains unclear whether there might be a difference in uptake and urinary excretion of 3-ring, 4-ring, or 5-ring PAHs and in the correlation between these metabolites and ambient-air PAH measurements. In summary, using immunaffinity chromatography, we did not find detectable amounts of B[a]P-tetrol in urine from workers occupationally exposed to PAH. However, by an HPLC/immunoaffinity method, relatively high amounts of 1-hydroxypyrene as well as 2?+?3- and 1?+?9-OH-Phe were quantitated in the urine samples, both of which are relevant as biomarkers of PAH exposure.     

Chronic treatment with epidermal growth factor stimulates growth of the urinary tract in the rat

Twenty-four male Wistar rats, 8 weeks old, were allocated into three groups and treated with human recombinant epidermal growth factor (EGF) administered subcutancously in doses of 0, 30, and 150 g/kg per day for 4 weeks. Blood sampling was done every 2nd week and urine sampling was done for 2 consecutive days every week. The most striking finding was that the ureters were dose dependently enlarged, due to growth of all layers of the ureteric wall. The urothelium of the bladder showed considerable hyperplasticity with a widening of the basal proliferative compartment and a normal differentiation pattern as observed by the expression of carbohydrate epitopes, characterized with lectinohistochemistry. Blood examination revealed a decrease in blood haemoglobin concentration and a slight increase in serum creatinine concentration in the high-dose group. There were no effects of EGF on the urinary excretion of electrolytes, proteins, and endogenous EGF.