Haematological side effects of antiepileptic drug treatment in patients with epilepsy.

OBJECTIVES: Little is known about the haematological side effects of the newer antiepileptic drugs (AEDs), but recent case reports have raised concerns regarding the possibility of altered thrombocyte counts or function in some patients during levetiracetam (LEV) treatment. The aim of our study was to investigate haematological changes in patients treated with the newer AEDs, LEV and lamotrigine (LTG), compared with the older AEDs, valproate (VPA) and carbamazepine (CBZ). METHODS: This cross-sectional study included 251 patients with epilepsy of both genders, aged 18-45 years, using AED monotherapy: 52 patients on LEV (31 men, 21 women), 80 on LTG (37 men, 43 women), 90 on CBZ (61 men, 29 women), 29 on VPA (15 men, 14 women), and 79 healthy controls (36 men, 43 women). Haemoglobin (Hb), white blood cells (WBC) and platelet (thrombocyte) counts were estimated. The subjects were recruited from hospitals in south-eastern Norway and Innsbruck, Austria. RESULTS: Significantly lower platelet counts were recorded in both men and women on LEV monotherapy. In the LEV group, platelets were 14% lower (40.68 × 10(9) /l lower) than in the control group. There was no difference according to sex or age of the patients. Only minor changes in haematological parameters were observed for the other drugs investigated. CONCLUSIONS: Both men and women treated with LEV monotherapy have lower blood platelet counts than healthy controls, with no difference in Hb or WBC. Haematological changes observed with the other AEDs were minor.

Enzymatic treatment of spermatozoa with a trypsin solution, SpermSolute: improved motility and enhanced ATP concentration.

We have developed a solution, fully described in this report, that can be used in a pretreatment procedure to promote liquefaction and to enhance motility during preparation of spermatozoa. It was applied to native ejaculates prior to swim-up and, in parallel investigations, motility and adenosine triphosphate concentration were compared in treated and untreated samples, which revealed that the solution significantly improved both parameters. The solution, named SpermSolute, is based on a proteinase (trypsin), which previously has been shown to stimulate the activity of a glycolytic key-enzyme. We speculate that our findings reflect intracellular enzyme activation and we suggest that our formula can be used in sperm preparation to prevent cell aggregates and to promote liquefaction, in addition to promotion of motility.

Improvement of a cytokine (TNF-α) bioassay by serum-free target cell (WEHI 164) cultivation.

The elaboration of a sensitive bioassay for assessment of tumour necrosis factor alpha (TNF-α) in a defined medium is described. The assay is based on the cytotoxic effect of TNF-α on a target cell line, the murine fibrosarcoma WEHI 164 clone 13. Cytotoxicity was assessed by detecting the rate of tetrazolium salt reduction employing a spectrophotometer (ELISA-reader). A similar bioassay was used previously to assess TNF-α, though this was dependent on cell growth in a medium containing serum. By employing a synthetic serum replacement, the WEHI cells were adapted to growth in a defined medium which allowed both the propagation of the cell line and the assay to be performed under completely defined conditions. Thus, factors in serum that may influence the TNF-α assessment, such as growth factors, cytokines, soluble cytokine-receptors and macroglobulin, were avoided. The only protein required in this bioassay was insulin, while albumin was added as a carrier protein and to protect the cytokine against loss of biological activity during multiple freeze and thaw cycles. The present assay was optimised to achieve a high sensitivity and, by testing endogenous TNF-α originating from the macrophage-like cell line RAW in both the serum-free and serum-based assay, we found the highest sensitivity in the assay based on defined medium. The LC50 of recombinant mouse and human TNF-α were in the serum-free and serum-based assays considered to be 25 and 50 pg mL-1, respectively. The demonstration of a culture condition that enables long-term cultivation of target cells and a bioassay in a completely defined medium is in our opinion a substantial contribution to more reliable cytokine assessment.

[Anterior interosseus nerve syndrome]. / Nervus interosseus anterior-syndrom.

A prolonged period of illness in a patient showing symptoms of reflexdystrophy is described. After going through several examinations and finally being referred to a pain clinic the patient demanded a second opinion from an orthopaedic surgeon. Her symptoms turned out to have been caused by a compression syndrome of the anterior interosseus nerve.

MR imaging of the wrist in carpal tunnel syndrome.

PURPOSE: To determine whether specific parameters measured on MR images correlated to electrophysiological changes in carpal tunnel syndrome (CTS). MATERIAL AND METHODS: Prospective clinical examinations were made of 20 patients with suspected CTS. We performed bilateral electrophysiological examinations of the median nerve and bilateral MR imaging of the wrists. RESULTS: The electrophysiological examination suggested median nerve entrapment in 18 wrists. These wrists were compared to the remaining 22 electrophysiologically normal wrists. In addition, we compared both wrists in 12 patients with unilateral symptoms of CTS without reference to the electrophysiological findings. We found no difference in specific MR parameters between the 2 groups. CONCLUSION: Neither symptoms nor electrophysiological findings in CTS were related to specific MR parameters.

A sensitive serum-free colorimetric assay for the detection of cytotoxic effects of pesticides.

A new method of cell quantification in the Hybritest bioassay is described. This test is based on culturing the hybridoma cell line 1E6 in the serum-free medium RPMI-SR3 and determination of cytotoxicity by growth inhibition. By employing a tetrazolium salt (MTT), the cell number were quantified colorimetrically using an ELISA reader. This assay carried out in microtiter plates saved time, labour and expenses. It was demonstrated that the sensitivity of 1E6 to pesticide toxicity (glyphosate and cypermethrine) was considerably higher in serum-free medium than in serum-containing medium. Furthermore, it is suggested that this assay may represent an alternative to the use of living animals in toxicological experiments.

A new sensitive cell culture test for the assessment of pesticide toxicity.

A new, simple and sensitive cell culture test for the determination of cytotoxicity of pesticides is described. This test is based on assessment of the growth inhibitory effect of pesticides on the rapidly growing mouse hybridoma cell line 1E6 cultivated in a defined serum-free medium (RPMI-SR3). In addition the cytotoxicity in serum-containing medium was investigated. The results showed that the sensitivity of 1E6 cell towards the toxicity of the pesticides; carbofuran, cypermethrin, lindane, glyphosate and 2,4-D was considerably higher in the serum-free medium than in serum-containing medium. Also IC50 values of these pesticides were compared with LD50 values obtained from the literature. The ratio between the in vitro IC50 and the in vivo LD50 showed that the present cell culture test determine the toxicity of low levels of pesticides with much higher sensitivity than tests based on using animals.

A simplified serum-free method for preparation and cultivation of human granulosa-luteal cells.

A simplified method for the preparation and long-term cultivation of granulosa-luteal cells in serum-free medium is described. The cells were harvested from women undergoing in-vitro fertilization, enriched by sedimentation and dissociated by enzymatic treatment. We demonstrated, by introducing a synthetic serum replacement (SSR2), that these primary cell cultures cultivated in monolayers on an extracellular matrix may be used in experiments exceeding 7 days with low cell loss and cell death. No adverse effect on progesterone production was found. There was a high diversity in progesterone production between cells from individual patients. After several days in culture, the cells were challenged with human chorionic gonadotrophin which revived the rapidly decreasing progesterone production. We were unable to demonstrate an increase in cell number after 7 days of cultivation when the cells were grown in medium supplemented with either serum or SSR2. The mitogens epidermal growth factor and basic fibroblast growth factor had no influence on proliferation. We also found that the present method prevents leukocyte contamination in the granulosa-luteal cell cultures. Compared with the common method based on the enrichment of granulosa-luteal cells on a density gradient (Ficoll/Percoll), this method saves time, labour and expense, in addition to augmenting purity.

A sensitive sperm-motility test for the assessment of cytotoxic effect of pesticides.

A sensitive sperm-motility test for the evaluation of cytotoxic effects of carbofuran and glyphosate in a defined protein-free culture medium is described. The sperm motility was compared to that obtained with a protein-containing medium. The use of protein-free medium considerably increased the sensitivity of sperm cells from rabbit and human to the toxic effects of the pesticide. The respective IC50 values (the concentration needed to cause 50% inhibition of sperm motility) in protein-free medium of carbofuran and glyphosate were 321 and 48.2 microM with human sperm, and 116 and 23.5 microM with rabbit sperm. Whereas, the corresponding values in protein-containing medium were 920 and 740 microM, and 910 and 500 microM with human and rabbit sperm, respectively. Our results show that testing human and rabbit sperm in protein-free medium proves to be a more sensitive method than that in protein-containing medium. Additionally, the use of rabbit sperm is a more sensitive test system than human sperm. This study suggests that the rabbit sperm test appears to have a potential for the assessment of toxicity on human reproduction.

Toxic effects of carbofuran and glyphosate on semen characteristics in rabbits.

The present study was undertaken to investigate the effect of chronic treatment with two sublethal doses of Carbofuran (carbamate insecticide) and Glyphosate (organophosphorus herbicide) on body weight and semen characteristics in mature male New Zealand white rabbits. Pesticide treatment resulted in a decline in body weight, libido, ejaculate volume, sperm concentration, semen initial fructose and semen osmolality. This was accompanied with increases in the abnormal and dead sperm and semen methylene blue reduction time. The hazardous effect of these pesticides on semen quality continued during the recovery period, and was dose-dependent. These effects on sperm quality may be due to the direct cytotoxic effects of these pesticides on spermatogenesis and/or indirectly via hypothalami-pituitary-testis axis which control the reproductive efficiency.

Mitochondrial disease and reduced sperm motility.

Mitochondrial dysfunction reduces aerobic energy production and results in symptoms from various tissues, depending on metabolic demands. Mitochondrial adenosine triphosphate (ATP) is essential for sperm motility. Sperm motility was investigated in a patient with a mitochondrial disease caused by reduced activity of the mitochondrial enzyme complexes I and IV, and in two control subjects. Spermatozoa were cultured in media containing various energy substrates. Motility was judged by light microscopy, and ultrastructure by transmission electron microscopy. In the patient with mitochondrial disease, 12% of the spermatozoa were motile in the medium containing only glucose. There was a three-fold increase in motile spermatozoa when pyruvate and succinate were present together with glucose. In contrast, the spermatozoa of both control subjects had best motility in the presence of substrates for complex I, and no further increase was observed when succinate was added. Glucose and pyruvate enter the respiratory chain at complex I, and succinate at complex II. Electron microscopy of spermatozoa from the patient with mitochondrial disease revealed mitochondria with increased matrix, thickening of membranes, parallelization of cristae and lipid inclusions, which are characteristic findings in mitochondrial disorders. Abnormal mitochondria were also found in a spermatid, suggesting that the ultrastructural changes of mitochondria are primary rather than secondary to degeneration of the spermatozoa. The results indicate that mitochondrial dysfunction causes reduced sperm motility in some men.

Endocrine composition of follicular fluid comparing human chorionic gonadotrophin to a gonadotrophin-releasing hormone agonist for ovulation induction.

Concentrations of inhibin, oestradiol and progesterone were determined in pre-ovulatory follicular fluid from 16 women undergoing in-vitro fertilization and embryo transfer treatment. A prospective randomized design was used such that ovulation was induced in eight women with human chorionic gonadotrophin (HCG) (9000 IU), and in eight women with an endogenous surge of luteinizing hormone (LH) and follicle stimulating hormone (FSH) caused by a single injection of gonadotrophin-releasing hormone agonist (GnRHa). Inhibin was measured by an enzyme-linked immunosorbent assay, and oestradiol and progesterone were measured by radio-immunoassay. Concentrations of inhibin and progesterone are significantly higher in follicular fluids collected after ovulation induction with HCG compared with ovulation induction with GnRHa (P < 0.001, P < 0.02, respectively). Concentrations of oestradiol were similar in the two groups. This study shows that the method by which ovulation is triggered significantly affects the micro-environment of the oocyte just prior to ovulation. The results indicate that HCG causes a prolonged luteotrophic effect well before ovulation, compared to an endogenous surge of gonadotrophins caused by GnRHa, and suggest that follicular maturation with an endogenous surge of gonadotrophins may be closer to the natural cycle than those cycles in which HCG is administered for ovulation induction. In addition, this study shows that the concentrations of inhibin and progesterone in follicular fluid may be valuable parameters in assessing the midcycle LH surge requirements for induction of ovulation.

Growth of cells in a new defined protein-free medium.

The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum. Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR.

A new protective solution for hypothermic storage of free vein grafts in cardiovascular surgery.

In order to reduce the operative injury of the endothelium in free reversed vein grafts, cultured human endothelial cells were used to test the optimal concentration of the constituents of a flushing solution for improved protection of the endothelium. The following solution proved to be the most suitable when tested at 20 degrees C; mannitol 160 mmol l-1, glucose 15 mmol l-1, NaCl 30 mmol l-1, KHCO3 5 mmol l-1, K2SO4 10 mmol l-1, KH2PO4 4 mmol l-1, MgSO4 20 mmol l-1, CaCl2 1.5 mmol l-1, potassium citrate 1.0 mmol l-1, Pluronic F-68 20 mg l-1, HEPES 4 mmol l-1, HEPES-Na 6 mmol l-1, pH 7.25, osmolality 325 mosmol kg-1 H2O. When endothelial cell injury was measured by a 51Cr-release assay, the new solution protected human endothelial cells in culture during hypothermic incubation better than isotonic NaCl, St Thomas' cardioplegic solution or Krebs-Henseleit's buffer. Transmission and scanning electron microscopy showed that the endothelium of human saphenous vein grafts was well preserved following 6 h of incubation at 20 degrees C with the new solution. As determined by morphometry using scanning electron microscopy, the endothelium of free porcine vein grafts was better preserved after incubation for 2 h at 20 degrees C with the new solution than with either isotonic NaCl (p = 0.02) or diluted, heparinized blood (p = 0.02) as the incubation medium, all cases observed following 2 h of subsequent arterial flow. The present study indicates that the endothelium of free vein grafts can be well protected against hypothermia when the flushing and irrigation fluid has a composition favouring endothelial protection. It appears likely that such treatment of vein grafts will reduce the frequency of vein graft narrowing and occlusion, post-operatively.

Medium-associated luteinization expressed as progesterone release in granulosa-luteal cells isolated from patients undergoing in-vitro fertilization.

The present study describes the effect of culture medium components on progesterone release from human granulosa-luteal cells isolated from patients undergoing in-vitro fertilization (IVF). Progesterone release was selectively measured as a central parameter of in-vitro luteinization, a process believed to decrease the success rate of IVF treatments. Ten different media of relevance to embryo culture were investigated for their effect on progesterone release in unstimulated granulosa cell cultures and in cultures stimulated with human chorionic gonadotrophin (HCG) (1 IU/ml) during 4 days in vitro. Culture media supplemented with human serum yielded the greatest secretion of progesterone. Supplementation with fetal calf serum caused an intermediate pattern of progesterone release. Substitution of serum with a synthetic replacement (Medi-CultR SSR 1 and 2), lacking hormones, cholesterol and growth factors, led to a minimal output of progesterone from granulosa-luteal cells. Complex media (RPMI 1640 and Ham's F10) generally caused a greater progesterone release than simple salt solution (EBSS). No effect of insulin was detected when added to serum-free media.

Experience with GnRH agonists in an in vitro fertilization (IVF) program.

The experience using pre-treatment with GnRH-agonists in an IVF-program are reported. 55 patients having a 'poor response', premature LH surge, or follicular luteinization in previous treatment cycles were treated for a total of 68 cycles. Therapy with GnRH agonists was initiated in the midluteal phase, and given by self-administration subcutaneously. Stimulation started with exogenous gonadotropins on a fixed day (Saturday) after pituitary desensitization had first been obtained, and resulted in all follicular punctures being performed on weekdays. Five treatment cycles (7.4%) were cancelled because of 'poor response'. Where the indication for GnRH-agonist therapy was previous 'poor response', a cancellation rate of 36.4% was observed, whereas a cancellation rate of only 1.8% was found in the other indication groups (p less than 0.001). Altogether 26 clinical pregnancies were achieved, five of these ending in a spontaneous abortion. The rate of deliveries was 33% per oocyte retrieval, and 40% per pre-embryo replacement.

Optimization and simplification of culture conditions in human in vitro fertilization (IVF) and preembryo replacement by serum-free media.

The results of 220 consecutive IVF treatments are presented, comparing the use of culture media supplemented with either patient serum (Group 1; n = 110), or Medi-Cult SSR 2 synthetic serum replacement with pyruvate, and human serum albumin (HSA) (GEA BioTech, Hvidovre, Denmark) (Group 2; n = 110). In both groups the Medi-Cult Hybritest was used for routine quality testing. A significantly (P less than 0.05) increased rate of deliveries/ongoing pregnancies was observed with the Group 2 medium. However, no significant differences in fertilization rate, cleavage rate, or implantation rate were observed. It is concluded that the serum-free culture medium described and the testing for absence of cytotoxicity in a sensitive bioassay (Hybritest) have yielded culture conditions capable of sustaining the development in vitro of human preembryos without impairing the fertilization process or the implantation rate, ultimately resulting in a significantly increased rate of deliveries/ongoing pregnancies and an apparently decreased abortion rate. The potential harmful effects of serum and the need for blood sampling and preparation further increase the advantages of replacing serum with the synthetic serum replacement SSR 2 in an IVF program.

A new cell culture assay for quality control in IVF.

A new, simple and sensitive bioassay for quality testing in IVF is described. This bioassay (Medi-Cult Hybritest, GEA Ltd, Biotech Division, Hvidovre, Denmark) is based on the culture of the rapidly growing mouse hybridoma cell line 1E6 in a defined serum-free culture medium. The use of serum-free conditions greatly increases the sensitivity to toxic substances, due to the absence of binding proteins. The testing of known toxic agents showed that this assay disclosed cytotoxicity with a high sensitivity. The Hybritest thus provides a simple yet sensitive and reproducible bioassay for quality control of culture media, water, chemical compounds and equipment in an IVF programme. Tests of different batches of culture media showed that media for IVF should be processed from powder and high-quality sterile water. It is important not only to test the single components of the culture media, but also the final product in a sensitive test system.

Increased relative frequency of suppressor monocytes in peripheral blood in early pregnancy.

The adherent cell fraction (AdC) of human peripheral blood mononuclear cells (PBM) contains two cell types of opposing function in vitro. Dendritic cells (DC) act as antigen presenting cells (APC) in vitro, while monocytes (Mo) have a suppressive effect on antigen activation of T cells. In this report we show that pregnant women (PW) during the first trimester have a significantly increased relative frequency of suppressor Mo compared with nonpregnant healthy controls. The T cell response to PPD (purified protein derivative of tuberculin) was significantly lower in the PW, but after removal of Mo by adherence the T cell response was about the same in the two groups. These observations indicate the PW during the first trimester have the same number of T cells reactive to PPD and normal functioning DC. The relative suppression expressed on a per Mo basis was the same in both groups, which indicates that the increased suppression in the PW was caused by an increased number of Mo, and not by changes in their activation state.