Specific and sensitive quantitation of 2,2'-dichlorodiethyl sulphide (sulphur mustard) in water, plasma and blood: application to toxicokinetic study in the rat after intravenous intoxication. A sensitive and specific capillary gas chromatographic method has been developed to measure trace amounts of 2,2'-dichlorodiethyl sulphide (sulphur mustard) in environmental or biological samples. Sulphur mustard was isolated from water or plasma by a solid-phase extraction procedure and from blood by liquid-liquid extraction. The accuracy and precision of the methods were demonstrated using replicate analyses of spiked water, plasma or blood: within-run and between-run variabilities were less than 20%. These analytical methods were used to evaluate the rate of sulphur mustard degradation in water or plasma. Good linear calibration curves, with a detection limit of 45 ng/ml, were obtained for quantitation and determination of sulphur mustard in blood following its intravenous administration to rats. Initial toxicokinetic data were obtained.


Identification of a benzhydrolic metabolite of ketoprofen in horses by gas chromatography-mass spectrometry and high-performance liquid chromatography. A benzhydrolic metabolite of ketoprofen, formed by reduction of the keto group of the drug, has been identified by gas chromatography-mass spectrometry in equine plasma and urine. After partial synthesis, its structure has been confirmed by UV, IR and 1H NMR spectroscopy. The kinetics of ketoprofen and this metabolite have been monitored in plasma by high-performance liquid chromatography. The two products were quantified in plasma up to 4 and 3 h, respectively, and were detected in urine up to 72 and 24 h, respectively, after a single intravenous administration to horses at the dose of 2.2 mg/kg. Simultaneous detection of both compounds increases the reliability of antidoping control analysis.


Gas chromatographic determination of 2- and 3-dechloroethylifosfamide in plasma and urine. The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 micrograms/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.


Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection. An improved method for the determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid (alendronate) in human urine and an assay in human plasma are described. The methods are based on co-precipitation of the bisphosphonate with calcium phosphates, automated pre-column derivatization of the primary amino group of the bisphosphonic acid with 2,3-naphthalene dicarboxyaldehyde (NDA)-N-acetyl-D-penicillamine (NAP) or cyanide (CN-) reagents, and high-performance liquid chromatography (HPLC) with electrochemical (ED) or fluorescence detection (FD). The feasibility of ED of the NDA-CN- derivative of aldendronate has been demonstrated, and a HPLC-ED assay in human urine has been validated in the concentration range 2.5-50.0 ng/ml. In order to eliminate the cyanide ion from the assay procedure, several other nucleophiles in the NDA derivatization reaction were evaluated. An NDA-NAP reagent was found to produce highly fluorescent derivatives of alendronate. The assay in urine based on NDA-NAP derivatization and HPLC-FD has been developed and fully validated in the concentration range 1-25 ng/ml. Based on the same NDA-NAP derivatization, an assay in human plasma with a limit of quantification of 5 ng/ml has also been developed. Both HPLC-FD assays were utilized to support various human pharmacokinetic studies with alendronate.


Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography. An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane-isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound alpha 1-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.


Simultaneous determination of sulphadiazine and trimethoprim in plasma and tissues of cultured fish for residual and pharmacokinetic studies. Clean-up and high-performance liquid chromatographic methods for the simultaneous determination of sulphadiazine and trimethoprim in fish plasma and tissues have been developed. The average recovery of sulphadiazine varied from 74% in liver to 92% in plasma, whereas that of trimethoprim varied from 60% in liver to 97% in plasma. The sample pretreatment procedures were simple, selective and robust, having a limit of quantification of 250 ng/ml for trimethoprim and 50 ng/ml for sulphadiazine in plasma, 15 ng/g for sulphadiazine and 80 ng/g for trimethoprim in muscle, and 30 ng/g for sulphadiazine and 160 ng/g for trimethoprim in liver. The assay was tested on plasma from Atlantic salmon treated with Tribrissen.


Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection. An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C18 reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C18 ODS Hypersil analytical column (5 microns particle size; 250 mm x 4.6 mm I.D.) using an acetonitrile-water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N',N'-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75-400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.


Sensitive micromethod for column liquid chromatographic determination of fluoxetine and norfluoxetine in human plasma. A rapid, selective and sensitive micromethod has been developed for the determination of fluoxetine (FLU) and its demethylated metabolite norfluoxetine (N-FLU) using a 250-microliters plasma sample and column liquid chromatography with ultraviolet detection at 226 nm. The limit of detection is 2.0 ng/ml for both FLU and N-FLU. Peak-height ratios are linear over a concentration range of 10-800 and 10-1000 ng/ml for FLU and N-FLU, respectively. Acceptable coefficients of variation are demonstrated for both within-run and day-to-day assays. Selected drugs were checked for interference. The method, which requires a very small volume of plasma, is sensitive enough for pharmacokinetic studies in animals, clinical pharmacology studies and drug monitoring in children or adult patients.


Enantioselective separation of some polyunsaturated epoxy fatty acids by high-performance liquid chromatography on a cellulose phenylcarbamate (Chiralcel OC) stationary phase. cis-Epoxides of linoleic acid, alpha-linolenic acid, arachidonic acid and the omega 3-epoxide of eicosapentaenoic acid were chromatographed on a cellulose trisphenylcarbamate (Chiralcel OC) stationary phase in the normal-phase mode. The R,S and S,R enantiomers of methyl-14(15)epoxyeicosatrienoate, methyl-9(10)epoxyoctadecadienoate and methyl-9(10)epoxyoctadecenoate could be partly resolved. The R,S enantiomer of methyl-14(15)epoxyeicosatrienoate eluted before the S,R enantiomer. [14C]14(15)Epoxyeicosatrienoic acid was isolated from an incubation of [14C]20:4n-6 with microsomes of rabbit kidney cortex and the S,R enantiomer was found to predominate (about 2:1).


Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection. We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid-liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3-0.8 pg injected) and reproducible (coefficients of variation 1-10%), and shows good accuracy (93-98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.


Separation and characterization of the precursor and activated forms of porcine and human pancreatic colipase by reversed-phase liquid chromatography. Reversed-phase liquid chromatography was used as an alternative method for the characterization of the precursor and activated forms of porcine and human pancreatic colipase. Using a Beckman Ultrasphere column with an increasing acetonitrile gradient in 0.1% trifluoroacetic acid, it was possible to obtain well-resolved separation of the precursor form of colipase (procolipase) from its trypsin-activated derivative. This protocol was used (1) to study the activation of porcine procolipase by trypsin or thrombin in vitro, (2) to assess the homogeneity of porcine colipase preparations used in tridimensional structure studies and in combination with immunoaffinity chromatography, (3) to identify the form of colipase present in samples of human pancreatic juice.


Gas chromatographic-mass spectrometric determination of plasma selegiline using a deuterated internal standard. A gas chromatographic-mass spectrometric method is described for the determination of plasma selegiline. Tetradeuteroselegiline was synthesized and served as the internal standard. Human plasma samples (1 ml) containing 1-6 ng of selegiline were acidified, washed with diethyl ether-hexane, then alkalinized and extracted with heptane-isoamyl alcohol. Analytical separations were performed on a dimethylsilicone capillary column. Detection was by selected ion monitoring of the electron impact generated m/z 96 and 100 alpha-cleavage fragments of drug and internal standard, respectively.


Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen. A rapid, inexpensive and sensitive high-performance liquid chromatographic method for the quantitation of ibuprofen enantiomers from a variety of biological fluids is reported. This method uses a commercially available internal standard and has significantly less interference from endogenous co-extracted solutes than do previously reported methods. The method involves the acid extraction of drug and internal standard [(+/-)-fenoprofen] from the biological fluid with isooctane-isopropanol (95:5) followed by evaporation and derivatization with ethylchloroformate and R-(+)-alpha-phenylethylamine. Excellent linearity was observed between the peak-area ratio and enantiomer concentration (r > 0.99) over a concentration range of 0.25-50 micrograms/ml. This method is suitable for the quantitation of ibuprofen from single-dose pharmacokinetic studies involving either rats or humans.


Determination of gliclazide in serum by high-performance liquid chromatography using solid-phase extraction. A simple and sensitive high-performance liquid chromatographic method for a routine assay of gliclazide in serum is described. Serum samples spiked with glibenclamide (internal standard) were applied to Bond Elut C18 cartridges. After washing with phosphate buffer (pH 7.5) and water, the cartridge was eluted with 60% methanol. The eluate was evaporated to dryness. The residue was dissolved in methanol and injected onto an octadecyl silica column (5 microns, 150 mm x 4.6 mm I.D.). The mobile phase was 0.04 M potassium dihydrogenphosphate (pH 4.6)-acetonitrile-isopropyl alcohol (5:4:1, v/v). Ultraviolet detection at 227 nm was used. The minimum detectable level of gliclazide was 20 ng/ml.


Determination of metformin in plasma by high-performance liquid chromatography. A high-performance liquid chromatographic method for the determination of metformin, an oral antidiabetic agent, in plasma is described. Plasma samples containing the internal standard, phenformin, are eluted through Amprep extraction columns before injection into the chromatographic column, packed with microBondapak phenyl. The eluent is monitored at 236 nm. At a mobile phase flow-rate of 1.35 ml/min, the retention times of metformin and phenformin are 2.8 and 5.6 min, respectively. The intra-day coefficients of variation are 1.5 and 4.3% at metformin concentrations of 0.05 and 1 mg/l, respectively.


High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine. A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.


Serum inhibin levels in gonadotrophin stimulated in-vitro fertilization/gamete intra-fallopian transfer cycles. Serum inhibin concentrations of 64 cycles of in-vitro fertilization--embryo transfer (IVF-ET) or gamete intra-Fallopian transfer (GIFT) have been analysed retrospectively. No significant difference was observed in serum inhibin levels of cycles stimulated with buserelin and human menopausal gonadotrophin (HMG) or HMG alone. During the late follicular phase, serum inhibin was higher in cycles resulting in pregnancy than in cycles without a pregnancy (peak values on day +1: 8.3 versus 6.4 IU/ml, respectively). The same difference was found between stimulation cycles resulting in a viable or a non-viable pregnancy (peak values on day +1: 8.3 versus 7.5 IU/ml). However, these differences were not significant. During the early luteal phase, serum inhibin values were similar in these groups of patients. Our results indicate that the use of the gonadotrophin-releasing hormone (GnRH) analogue buserelin, in combination with HMG, for ovarian stimulation does not affect inhibin production by granulosa cells in vivo. The late follicular and early luteal concentrations of serum inhibin have to be considered unsuitable as predictors in IVF/GIFT cycles with respect to pregnancy and pregnancy outcome.


Endocrinological environment with regard to the number of microcysts in patients with polycystic ovary syndrome. Androgen, oestrogen and gonadotrophin levels in patients with the polycystic ovary syndrome (PCOS) were compared with those in normal ovulatory women (controls), and were correlated with the number of microcysts in the polycystic ovaries studied. The androgen levels (testosterone, delta 4-androstenedione and dehydroepiandrosterone-sulphate), oestrogen levels (oestrone and oestrone/oestradiol-17 ratio) and gonadotrophin levels [luteinizing hormone (LH) and LH/follicle stimulating hormone (FSH) ratio] of PCOS patients were significantly higher than those of controls. The number of microcysts in polycystic ovaries was significantly correlated with androstenedione, which in turn was also significantly correlated with LH and LH/FSH ratio. These correlations may play a part in the maintenance of chronic anovulation in PCOS.


Expression of functional growth hormone receptors in human granulosa cells. Both clinical and experimental evidence suggest that growth hormone may be of importance for ovarian function. The present study investigated whether growth hormone receptors are expressed in human granulosa cells. Granulosa cells were isolated either from natural cycles or from stimulated cycles in the course of in-vitro fertilization. Total RNA hybridized with a 32P-labelled rat growth hormone receptor cRNA probe revealed one major transcript with an estimated size of 4.5 kb and one minor transcript with an estimated size of 1.3 kb. Biotinylated growth hormone was used to analyse growth hormone binding. Competitive growth hormone binding was detected in freshly isolated granulosa cells, as well as in cultured cells. Growth hormone augmented basal and/or follicle stimulating hormone-stimulated steroidogenesis in granulosa cells obtained from patients with natural cycles, but the response to growth hormone stimulation showed considerable variation. We conclude that functional growth hormone receptors are present in human granulosa cells and that growth hormone, therefore, may have an important role in ovarian function.


Progesterone supplementation increases luteal phase endometrial thickness and oestradiol levels in in-vitro fertilization. Luteal phase supplementation with natural progesterone appears to increase the pregnancy rate in in-vitro fertilization (IVF). The objective of this investigation was to examine the effect of intravaginal progesterone on endometrial thickness and hormonal parameters 7-9 days after embryo transfer. IVF patients receiving progesterone supplementation (Prog +, n = 64), who did not conceive, were compared to patients not receiving progesterone (Prog -, n = 23) because of failed fertilization. These two groups were also compared to 20 women (Preg) who conceived and to 16 women (control) in the mid-luteal phase of natural cycles. Endometrial thickness was greater (P < 0.01) in the Prog + (0.88 +/- 0.04 cm) and Preg (0.92 +/- 0.02 cm) groups compared to the Prog - (0.71 +/- 0.05 cm) and control (0.65 +/- 0.05 cm) groups. Mean luteal phase serum oestradiol levels were also higher (P < 0.05) in the Prog + (1118 +/- 112 pmol/l) and Preg (2267 +/- 757 pmol/l) groups than in the Prog - (574 +/- 70 pmol/l) and control (468 +/- 38 pmol/l) groups. These findings suggest that progesterone supplementation may affect pregnancy rates in IVF by increasing endometrial thickness, thereby enhancing receptivity for implantation. The mechanism through which this effect occurs is unclear but may involve serum oestradiol elevation.


Cytokine expression in human endometrium throughout the menstrual cycle. Recent evidence suggests that diverse endometrial functions may be regulated by cytokines. In this report, the presence of protein and mRNA of cytokines were studied in human endometrium throughout the menstrual cycle. The presence of the interleukin-1 (IL-1) alpha, interleukin-1 (IL-1) beta, interleukin receptor antagonist (IRAP), interleukin-6 (IL-6) and transforming growth factor (TGF)-alpha proteins were demonstrated by immunohistochemical staining. The IL-1 alpha and TGF-alpha proteins were strongly expressed and IL-1 beta protein was weakly expressed in all the cells in the stroma as well as epithelial cells. IRAP was markedly expressed in the cells with morphological features of macrophages scattered in the stroma, and the expression of IL-6 protein was predominant in the endometrial epithelium. Diffuse cytoplasmic expression of IL-1 alpha in endometrial epithelium during the proliferative phase contrasted markedly with its enhanced luminal expression during the secretory phase of the menstrual cycle. In addition, the presence of the mRNA of these cytokines in endometrium was established throughout the entire menstrual cycle by reverse transcription-polymerase chain reaction (RT-PCR). Abundant expression of cytokines in human endometrium emphasizes the significant roles that cytokines play in cell-cell interactions and in regulating endometrial functions.


Exposure to LHRH agonists in early pregnancy following the commencement of mid-luteal buserelin for IVF stimulation. Exposure to buserelin in early pregnancy has been reported following its use in in-vitro fertilization treatment cycles. The number of reported cases is small. There are still no answers regarding embryotoxicity, and the need for luteal support is still unclear. A further six cases are reported here, five receiving luteal support and resulting in the delivery of healthy children, and one, without luteal support ending in a first trimester miscarriage. We propose that further data should be collected to allow adequate monitoring and follow-up of these pregnancies.


Pituitary gonadotrophin secretory capacity during the luteal phase in superovulation using GnRH-agonists and HMG in a desensitization or flare-up protocol. Pituitary gonadotrophin reserve and basal gonadotrophin secretion were tested during the luteal phase in women superovulated with buserelin/human menopausal gonadotrophin (HMG) in a desensitization (n = 17) or flare-up protocol (n = 7). In the desensitization protocol the luteinizing hormone-releasing hormone (LHRH) stimulated serum LH and follicle stimulating hormone (FSH) concentrations remained impaired at least until day 14 after arrest of the agonist. In the flare-up protocol basal and stimulated LH secretion was still abnormal on days 14 and 15 after human chorionic gonadotrophin (HCG) injection. Normal basal serum FSH concentrations were measured at the end of the luteal phase in the flare-up protocol, but the response of FSH to LHRH injection was still subnormal. We conclude that gonadotrophin function remained impaired until the end of the luteal phase after desensitization and flare-up GnRH-agonist and HMG stimulation protocols. Corpus luteum stimulation with exogenous HCG or substitution therapy using natural progesterone are required to prevent the possible negative effects resulting from pituitary dysfunction after GnRH-agonist treatment.


Pituitary-ovarian suppression by the standard and half-doses of D-Trp-6-luteinizing hormone-releasing hormone depot. A total of 20 women with regular menstrual cycles normal ovulatory function were randomized to receive 3.75 mg or 1.87 mg of D-Trp-6-luteinizing hormone-releasing hormone (LHRH) in microcapsules either in the early follicular phase (day 2) or during the mid-luteal phase (day 21) of the menstrual cycle. Pituitary--ovarian function was studied by sequential plasma measurements of oestradiol, follicle stimulating hormone, luteinizing hormone and progesterone. Our results show that the full, standard dose (3.75 mg) of D-Trp-6-LHRH depot and the half-dose (1.87 mg) were equally effective in their desensitizing action, and that desensitization occurs more rapidly with early follicular phase administration of the analogue.


A randomized trial between GIFT and ovarian stimulation for the treatment of unexplained infertility and failed artificial insemination by donor. This study was designed to investigate the surplus effect of gamete intra-Fallopian transfer (GIFT) over ovarian stimulation alone, in patients with unexplained infertility. A total of 50 patients with unexplained infertility of at least 3 years duration, or unexplained failure of artificial insemination by donor (AID) for at least 12 cycles, meeting strict inclusion criteria, were randomly selected for either two GIFT cycles or two ovarian stimulation cycles. Ovarian stimulation was combined with timed intercourse, or timed cervical donor insemination. In 38 completed GIFT cycles, five clinical pregnancies (13.2% per cycle) occurred and in 44 ovarian stimulation cycles four clinical pregnancies occurred (9.1% per cycle). Five remaining GIFT cycles were converted into in-vitro fertilization leading to two pregnancies. Of the 50 patients suffering from unexplained infertility, the 23 who did not have AID gave rise to four pregnancies out of 39 cycles (10.3%); from the remaining 27 patients who underwent AID, seven pregnancies were achieved out of 48 cycles (14.6%). No statistical differences between GIFT and ovarian stimulation treatment were found. Therefore, the GIFT success rates can be explained at least in part, if not fully, by the effect of ovarian stimulation alone. Consequently, ovarian stimulation should be considered in unexplained infertility before more elaborate forms of assisted reproduction are used.


Prognostic value of two putative sperm function tests: hypo-osmotic swelling and bovine sperm mucus penetration test (Penetrak). The clinical value of two inexpensive and easy to perform sperm function tests (hypo-osmotic swelling and bovine sperm mucus penetration tests) were examined in a prospective study of in-vivo conception rates in 325 couples. Both tests were of no significant value alone or in combination with traditional semen characteristics in predicting pregnancy outcome. The two most predictive factors of conception were the length of infertility and percentage of progressively motile spermatozoa. Our data support the further investigation of sperm function using more sophisticated quantitative tests, specifically the examination of sperm motility.


Changes in carnitine and acetylcarnitine in human semen during cryopreservation. L-Carnitine and acetylcarnitine concentrations were determined in spermatozoa and seminal plasma from 15 men, in both fresh ejaculate and frozen-thawed semen with cryoprotective medium. Sperm motility was also evaluated. In fresh samples, the levels of carnitine and acetylcarnitine in seminal plasma were comparable whereas in spermatozoa, acetylcarnitine predominated. Cryopreservation did not change the carnitine and acetylcarnitine levels in seminal plasma nor the carnitine concentration in spermatozoa; by contrast, the acetylcarnitine level in spermatozoa was decreased in 14 cases (110 +/- 8 versus 210 +/- 20 nmol/10(8) cells). This decrease in acetylcarnitine content was greater during semen dilution in cryoprotectant than after the freezing/thawing process. Motility was also decreased in all cases after the freezing/thawing process. These results suggest that acetylcarnitine recovery in spermatozoa is further evidence of the deleterious effect of the cryoprotective medium in the cryopreservation of semen.


Unexplained in-vitro fertilization failure: implication of acrosomes with a small reacting region, as revealed by a monoclonal antibody. To determine the acrosomal characteristics related to in-vitro fertilization (IVF) outcome, spermatozoa from 50 men whose wives had resorted to IVF have been studied by indirect immunofluorescence microscopy with anti-human pro-acrosin monoclonal antibody 4D4 (mAb 4D4), prior to and after incubation in a capacitating medium. The antibody labelled only the acrosomal principal region (APR), revealing its shape (i.e. normal, small or amorphous) and its status (i.e. unreacted, partially or totally reacted). The IVF outcome distinguished: (i) spermatozoa which were able to fertilize at least one oocyte in vitro (group I; n = 25) and (ii) spermatozoa which failed to fertilize any oocyte in vitro (group II; n = 25). The semen characteristics of the two sperm groups, including the acrosome morphology, were similar according to conventional analysis. The mAb 4D4 detected in both the whole and the swim-up sperm cell fractions a lower percentage of normal APR in group II (< 50% for 10 patients in group II versus one patient from group I), which was related to a higher percentage of small APR. Moreover, after 21 h incubation, group II had a lower acrosomal loss index. The spermatozoa of five patients of this infertile group II did not undergo acrosomal modification whereas spermatozoa of all group I patients underwent the acrosomal reaction. The data showed that the relationship between acrosomal anomalies and IVF failure is mainly due to an increased incidence of acrosomes with a reduced size of the region involved in the acrosome reaction. Immunodiagnosis of this acrosomal region by means of mAb 4D4 is informative for IVF outcome.


Effect of pentoxifylline on sperm movement characteristics in normozoospermic and asthenozoospermic specimens. Preliminary evidence has suggested that the phosphodiesterase inhibitor pentoxifylline augments the fertilizing potential of asthenozoospermic sperm samples, presumably by improving sperm movement. Here, we used computer-assisted sperm movement analysis to compare the effects of pentoxifylline in normozoospermic and asthenozoospermic specimens. The study focused on the following issues: the changes in individual movement characteristics in response to pentoxifylline, the rapidity of the response, the effect of sperm capacitation on the response, the persistence of the response after drug removal and the variability of responses among asthenozoospermic individuals. Data obtained show that (i) pentoxifylline increases the curvilinear velocity, path velocity, straight-line velocity, lateral head displacement, beat cross frequency and sperm hyperactivation in both normozoospermic and asthenozoospermic specimens, (ii) pentoxifylline does not modify the percentage of motile spermatozoa, (iii) the pentoxifylline effect reaches a maximum within 10 min of treatment in fresh semen as well as in capacitated sperm suspensions and persists for at least 2 h after drug removal and (iv) pentoxifylline improves the movement characteristics in most asthenozoospermic individuals. Results are discussed with regard to methods of therapeutic application of pentoxifylline as an enhancer of sperm movement in assisted reproductive technology.


Usefulness of the hypo-osmotic swelling test for evaluation of human sperm fertilization. We examined spermatozoa from 135 male patients consulting for infertility to evaluate the usefulness of the hypo-osmotic swelling (HOS) test of sperm fertility for the swim-up washing method. HOS test values significantly improved following the treatment of spermatozoa using the swim-up washing method. There were no differences in the rate of sperm motility between the normal group and the oligozoospermic group following treatment, but the HOS test identified significant differences in swelling rates in both groups before and after treatment. The results of the HOS test following swim-up washing were higher during fertilization attempts for 11 cases of successful intra-uterine insemination (IUI) than during unsuccessful IUI attempts; there were no significant differences between the control group and the successful IUI cases. Moreover, using the lower-normal limits for overall sperm swelling (52%) and g-type sperm swelling (30%) obtained from a normal control group, we found that swelling rates were higher when IUI was successful. These findings indicate that the HOS test is an effective measure of sperm fertilizing function when the swim-up washing method is used for sperm treatment. The score of g-type sperm swelling can be used as a substitute for overall sperm swelling.


Pregnancy after zygote intra-fallopian transfer using spermatozoa from a patient with Kallmann's syndrome. A successful zygote intra-Fallopian transfer has been performed using spermatozoa from a patient with Kallmann's syndrome. Spermatogenesis was induced by hormonal treatment but even so, semen quality remained slightly impaired and no pregnancy occurred over 4 years, despite ovulation induction in the patient's spouse. Finally she conceived after assisted procreation by in-vitro fertilization and delivered a healthy baby.


Sonographic transcervical balloon tuboplasty. The purpose of this study was to evaluate sonographic guidance for transcervical tubal catheterization and transcervical balloon tuboplasty of patients with bilateral proximal tubal occlusion. Cornual catheterization and transcervical balloon tuboplasty were performed under sonographic guidance. Injection of micro-bubble emulsion confirmed tubal recanalization. Tubal patency demonstrated by sonography was confirmed by injection of contrast material under fluoroscopy. Four women with bilateral proximal tubal occlusion confirmed by previous hysterosalpingogram and laparoscopy underwent sonographically guided transcervical balloon tuboplasty. Patients with distal or peritubal damage were excluded from this study. Bilateral tubal patency confirmed by sonography and subsequent fluoroscopy was achieved in all four patients. One patient conceived spontaneously, a month following the procedure, and delivered at term. Sonographically guided transcervical balloon tuboplasty can be performed successfully on patients with proximal tubal occlusion. Identification of the catheter tip and successful cannulation of the internal tubal ostia were easier to perform under fluoroscopy. Further improvements in sonographic equipment and catheter technology will hopefully eliminate radiation and replace fluoroscopy during the performance of transcervical balloon tuboplasty. Sonographic transcervical tubal catheterization may, therefore, become a simple and cost-effective procedure for the diagnosis and treatment of patients with proximal tubal occlusion.


Fibrin glue for reanastomosis of the fallopian tube in the rabbit: adhesions and fertility. Fertility and adhesions were examined in 41 rabbits in three treatment groups following the construction of microsurgical anastomoses with fibrin glue and by conventional suturing techniques. The three types of treatment were: isthmic anastomoses without resection, ampullary anastomoses, isthmic anastomoses following resection of oviduct segments. There were no significant differences between the two methods of performing the anastomoses in any of the three groups with respect to number of ovulations, number of implantations, nidation index, and pregnancy rate. The extent of the formation of adhesions was also no different between the two types of anastomosis construction. From the functional point of view, fibrin glueing of the tubes can be considered as good as microsurgical suturing. Another study is planned to examine whether this is also true from the morphological viewpoint. It must be remarked that fibrin glueing does not appear to be suitable for anastomoses in which a lumen-adjusting technique is needed or for deep tubocornual or intramural anastomoses.


Beta-N-acetylhexosaminidase activity in human oocytes and preimplantation embryos. beta-N-acetylhexosaminidase is a lysosomal enzyme, which has two isoenzymes: beta-Hex A, a trimer consisting of one alpha-chain and two beta-chains (alpha beta 2) and beta-Hex B, a tetramer formed of four beta-chains (beta 2 beta 2). Genetic defects in the alpha-chain lead to Tay-Sachs disease, whereas mutations in the beta-chain gene lead to Sandhoff disease. In a previous study we developed a microassay for total beta-N-acetylhexosaminidase and used this for measuring activities in mouse oocytes and preimplantation embryos. In this study, to assess the feasibility of transferring this technique to the human for the purposes of preimplantation diagnosis for Tay-Sachs and Sandhoff disease, beta-Hex activity was assayed in human oocytes and embryos and in the medium in which they had been cultured. We showed that although the activity of beta-N-acetylhexosaminidase in human oocytes and embryos was > 500 times higher than in the mouse, it was not detectable in the culture medium and the activity in oocytes and embryos remained virtually constant throughout human preimplantation development, making it difficult to distinguish embryonic from maternal enzyme activity. In the absence of this distinction it would be inappropriate to use beta-N-acetylhexosaminidase activity for the purposes of preimplantation diagnosis of Sandhoff or Tay-Sachs disease. These experiments demonstrate that measuring the beta-N-acetylhexosaminidase activity in human embryos cannot be used at present for preimplantation diagnosis.


Quantitative analysis of cellular glutathione in early preimplantation mouse embryos developing in vivo and in vitro. The relative levels of reduced glutathione (GSH) have been measured fluorimetrically in individual eggs and early embryos from two mouse strains, one of which shows developmental arrest in vitro. GSH levels fell by approximately 20-25% at fertilization and by approximately 45% by the late 2-cell and early 4-cell stages. No differences were observed between strains or between embryos cultured in vitro or in vivo. Addition of exogenous H2O2 or diethylmaleate depleted GSH. GSH levels were not affected significantly after inhibition of GSH-peroxidase by mercaptosuccinate nor of catalase by aminotriazole. Mercaptosuccinate did not inhibit development but catalase inhibition caused arrest at the 2-cell stage. Addition of exogenous GSH or thioredoxin did not promote development of 'blocking' embryos through the 2-cell block. It is concluded that early embryos lack a mercaptosuccinate sensitive peroxidase activity for removing H2O2, which may be removed by catalase or the glutathione-S-transferase system. It is suggested that GSH may have a role in detoxifying peroxidated lipids. The results are consistent with a role for reactive oxygen species in the 2-cell block.


Can the mouse embryo provide a good model for the study of abnormal cellular development seen in human embryos? Mouse 2-cell embryos arrested in development, either due to the effect of in vitro culture conditions ('2-cell block') or after exposure to the protein synthesis inhibitor anisomycin, were examined to determine the effect of the level of protein synthesis on development. The rate of protein synthesis was found directly to reflect the developmental potential of the embryos. Embryos cultured in the highest dose of the drug failed to divide and had the lowest rate of protein synthesis over the period of investigation, whereas untreated viable 2-cell embryos in the control group had the highest rate of protein synthesis and developed normally. Measurement of the nuclear DNA showed that both arrested and non-arrested embryonic cells completed DNA replication. Furthermore, drug-arrested embryos, like embryos which 'block' in culture, remained morphologically intact when left in culture. Disruption of the nuclear integrity and formation of micronuclei, as is frequently observed in arrested human embryos, was not seen in mouse embryos. These results indicate that developmentally arrested mouse embryos may not be a good model for studying cellular dysfunction in early human development. Experimentation using human material is required to address directly the problem of abnormal human development.


Reliable gender screening for human preimplantation embryos, using multiple DNA target-sequences. Dependable methods were developed for preimplantation sexing of human IVF embryos, for use in clinical settings where prospective parents are at high risk for transmission of X-linked diseases. Using single cultured cells and blastomeres from human embryos as model systems, a multiplex protocol was developed for rapid analysis via nested polymerase chain reaction (PCR). Reliability was enhanced by co-amplification of conserved amelogenin gene segments from both X and Y chromosomes, as well as Y-linked DYZ1 repetitive elements. Each cell was manually isolated and individually washed to avoid potential contaminants. Multiplex amplification allowed recognition of spurious amplification failures specific to particular amelogenin single-copy targets. The X-linked internal control and multiple Y-linked markers allowed recognition and exclusion of most aberrant samples, thus averting potential misdiagnosis. The optimized single-cell protocol reduced experimental sexing errors to < 2% (1/60), but also revealed potential pitfalls of single-cell analysis. With human triploid embryos, separate sampling of individual blastomeres provided concordant female or male signals. Slight modification adapted the procedure for diagnosis of biopsy material from blastocyst stage embryos, allowing separate analysis of multiple tubes containing multiple cells for improved reliability.


CA 125 concentrations in ovarian 'chocolate' cyst fluid can differentiate an endometriotic cyst from a cystic corpus luteum. In a prospective study, the concentrations of CA 125, 17 beta-oestradiol and progesterone were assayed in 52 consecutive ovarian cysts, laparoscopically suspected to be endometriomas. Cysts with dark brown 'chocolate' fluid (n = 42) were excised by CO2-laser endoscopy. Cysts with clear fluid were diagnosed by pathology as follicular cysts (n = 5) or pseudoperitoneal cysts (n = 5). Fluids (n = 53) aspirated during echo-guided puncture for in-vitro fertilization (IVF) were assayed simultaneously. Of the 42 women undergoing a cystectomy, the clinical diagnosis of an endometrioma was confirmed by pathology in only 68%, the other cases being corpora lutea (27%) or follicular cysts (5%). Cyst fluids from corpora lutea had lower CA 125 concentrations (< 1000 IU/ml) together with high 17 beta-oestradiol concentrations (> 2000 pg/ml) and/or high progesterone concentrations (> 100 ng/ml). Endometriotic cysts had either very high CA 125 concentrations (> 10,000 IU/ml) as occurred in 78% or lower CA 125 concentrations (< 1000 IU/ml) together with low 17 beta-oestradiol and/or progesterone concentrations. 'Chocolate' fluid-containing cysts aspirated during IVF had similar concentration profiles of CA 125, 17 beta-oestradiol and progesterone and the diagnoses derived from these concentrations were not contradicted in 19/27 women undergoing a laparoscopy within 4 months. In eight women, however, with high CA 125 concentrations in their cyst fluid, no endometriotic cysts were found at laparoscopy. Only 68% of cysts containing 'chocolate' material were endometriotic cysts and CA 125 could be useful in making this diagnosis. This method is recommended when dark brown fluid is aspirated in IVF.


Ovarian steroid receptor expression in endometriosis and in two potential parent epithelia: endometrium and peritoneal mesothelium. Endometriosis is an oestrogen dependent condition and it is expected that the tissue of origin of endometriosis will express receptors for the ovarian steroids. Two epithelia, endometrium and peritoneal mesothelium, are the potential parent epithelium. Oestrogen and progesterone receptor expression has been studied immunohistochemically in (i) timed endometrial biopsies from 25 normal subjects and 27 patients with endometriosis, (ii) 25 endometriotic biopsies and (iii) 42 peritoneal biopsies. Endometrium but not peritoneal mesothelium expresses both oestrogen and progesterone receptors. No difference in the intensity of staining between endometria of normal subjects compared with the endometria of patients with endometriosis was noted. In paired endometrial and endometriotic biopsies, the intensity of staining for the oestrogen receptor in stromal cells and for the progesterone receptor in both glandular and stromal cells was less in the endometriotic biopsies. These data provide circumstantial evidence for an endometrial origin for endometriosis although quantitative differences exist in receptor expression between endometrium and endometriosis.


The effect of injecting endometriotic 'chocolate' cyst fluid into the peritoneal cavity of mice. 'Chocolate' fluid aspirated from endometriotic cysts was injected into the peritoneal cavity of mice. No peritoneal adhesions or endometriosis were noted when the animals were killed 7-37 days later.


Enzyme immunoassay (EIA) for equine chorionic gonadotropin/pregnant mare serum gonadotropin (eCG/PMSG). A simple, accurate, sensitive enzyme immunoassay (EIA) has been developed that permits the measurement of equine Chorionic Gonadotropin activity in pregnant mare plasmas or serums as well as in commercial and highly-purified preparations. This assay is specific for eCG and eLH which share the same polypeptide structure but differ in their oligosaccharidic chains. The more important result is that this EIA has been found to be give data in very close agreement with the in vivo assay. Therefore this very rapid and convenient assay can be used to measure the activity of eCG/PMSG in pregnant mares serums in in-field conditions as well as in crude or highly-purified preparations.


Enzyme-linked immunosorbent assay of CS-518, a thromboxane synthetase inhibitor, in rabbit plasma and platelets. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of CS-518, a novel thromboxane synthetase inhibitor. Antisera against CS-518 were obtained from rabbits immunized with bovine serum albumin linked to CS-518 via carboxylic acid introduced into the imidazolyl ring (for ELISA-1) or via 6-carboxylic acid directly (for ELISA-2). Each of two CS-518 derivatives was conjugated to horseradish peroxidase by a N-succinimidyl ester method, and it was used as a labeled-antigen in homogeneous combination with antisera. In ELISA-1, CS-518 was detectable in a range of 5pg-1ng, and all cross-reactivities with main metabolites were less than 5%, in contrast to high affinity to the taurine and glucuronic acid conjugates of CS-518 in ELISA-2. Validity of ELISA-1 was confirmed by a high-performance liquid chromatography and ELISA-1 enabled specific determination of CS-518 in plasma samples deproteinized by methanol. When ELISA-1 was applied to determine CS-518 in platelets after oral administration to rabbits, CS-518 uptake up to maximum capacity in platelets (4.2-5.4 x 10(6) M) and slow elimination of CS-518 from platelets (T1/2 = 36-41 hr) were observed independent of CS-518 doses. These results confirm that CS-518 binds to thromboxane synthetase in platelets with high affinity.


A sensitive enzyme immunoassay for the detection of a synthetic affinity ligand, the reactive yellow 13 dye. Dye-affinity chromatography is widely and increasingly used for the isolation of various proteins. In particular, the purification of transthyretin can be efficiently achieved by affinity chromatography on immobilized Reactive Yellow 13. Measurement of trace-amounts of dye leaching from affinity columns is important because of possible toxicity or side effects linked with the presence of dye in therapeutic transthyretin preparations. A competitive enzyme immunoassay was developed to monitor yellow-dye column leaching. Biotinylated rabbit anti-Reactive Yellow 13 antibodies (immunoglobulin G fraction) were used as principal reagent. The assay is specific sensitive to 1 ng/ml of Reactive Yellow 13, has a good reproducibility and allows the accurate detection of the dye in the presence of transthyretin.


Immunocapture assay for quantification of human IgA antibodies to parasite antigenic enzymes. Application with the alkaline phosphatase of Schistosoma mansoni. Conditions are described for using solid phase adsorbed jacalins in an immunocapture assay for IgA antibodies to the alkaline phosphatase of Schistosoma mansoni. Microtiter plates were activated with polylysine and jacalins were covalently adsorbed by means of glutaraldehyde. From three different jacalins, the one purified from seeds of Artocarpus tonkinensis showed the lowest non-specific adsorption and was used for further studies. Comparing solutions of bovine serum albumin, ovalbumin and Tween 20, it was shown that the latter was most successful in blocking non-specific adsorption. Low serum dilutions resulted in a less efficient IgA capture by the adsorbed jacalin than higher dilutions. Under optimal working conditions, a high correlation could be shown between the presence of specific anti-alkaline phosphatase antibodies of IgA isotype and IgG isotype.


Beyond simple pooling for HIV screening. We discuss some theoretical features underlying the successful uses of pooling in testing HIV seroprevalence. In particular it is shown that there is a scaling relation for the distribution of positive sera among the pools. A multi-stage pooling method consisting of repeatedly halving the positive pools is proposed. Concentrating on the number of tests required for screening all positive individuals the method is shown to be highly efficient in low prevalence situations.


Search for retrovirus in the chronic fatigue syndrome. To examine peripheral blood and skeletal muscle from patients with chronic fatigue syndrome for exogenous retrovirus. Blood samples from 30 patients and muscle biopsy specimens of 15 patients were examined for retroviral sequences by DNA extraction, polymerase chain reaction (PCR), and Southern blotting hybridisation. Sera were examined for human foamy virus by western immunoblotting and indirect immunofluorescence techniques. No differences between the patient and control populations was found for any of the PCR primer sets used (gag, pol, env, and tax regions of HTLV I/II). An endogenous gag band was observed in both the patient and control groups. All sera were negative for antibody to human foamy virus. The results indicate that there is no evidence of retroviral involvement in the chronic fatigue syndrome.


Quantification of pancreatic secretory trypsin inhibitor in colonic carcinoma and normal adjacent colonic mucosa. To measure the content of immunoreactive human pancreatic secretory trypsin inhibitor (irPSTI) in colonic carcinoma and adjacent normal colonic mucosa. From a stable hybridoma cell line producing monoclonal antibodies specific for human PSTI, a specific enzyme linked immunosorbent assay (ELISA) for human PSTI was developed. In a precipitation assay system these antibodies bound human PSTI in a dose-dependent manner. The specimens were obtained from resectional surgery. The content of irPSTI was 19.9 micrograms/g protein (0.55 micrograms/g tissue wet weight) in colonic carcinoma. In adjacent normal colonic mucosa 43.6 micrograms/g protein (1.12 micrograms/g tissue wet weight) was shown. The enzymatic degradation of surrounding tissue necessary for tumour cell invasion could be facilitated by this relative deficit of the inhibitor in infiltrative carcinoma.


Helicobacter pylori, gastritis, and peptic ulceration in the elderly. To determine the histopathological types of gastritis, presence of H pylori, and of peptic ulceration in patients aged 70 and over, compared with younger adults. Gastric antral and corpus biopsy specimens from 112 elderly patients were classified and graded histologically according to the Sydney system. Details of recent antibiotic and non-steroidal anti-inflammatory drug use were recorded. Eighty four of the patients were positive for H pylori IgG antibodies and parietal cell antibodies. The results were compared with those from a series of 124 adult patients aged under 60. H pylori were visible at histological examination in only 57 of 87 (65.5%) elderly patients with chronic gastritis (excluding "special forms") compared with 72 of 79 (91.1%) of the younger patients with gastritis (p < 0.0002). Severe atrophy of the corpus mucosa was significantly associated with absence of H pylori (p < 0.002), and was present in eight of 30 elderly patients with helicobacter negative gastritis. Other explanations for absence of H pylori include recent antibiotic intake, more intestinal metaplasia, and lower bacterial load in elderly patients (p < 0.05). Autoimmune gastritis and NSAID use did not seem to be relevant. Serodiagnosis showed reduced sensitivity (81%) in patients who were helicobacter positive histologically, but was positive in 14 of 23 (61%) with H pylori negative gastritis histologically, suggesting either current infection that had been missed or previous infection. Peptic ulceration was significantly associated with NSAID use, but not with H pylori in the elderly. The spectrum of gastritis is different in the elderly, compared with younger adults, due to a significant group with chronic gastritis who are H pylori negative on histological examination. NSAID use, but not demonstration of H pylori (at histological examination) is associated with peptic ulceration in the elderly.


Strains of Escherichia coli O157:H8 from human diarrhoea belong to attaching and effacing class of E coli. To determine whether 17 Escherichia coli O157:H8 strains isolated from patients with diarrhoea in the United Kingdom were putative pathogens. The strains had been isolated by the use of O157 antiserum, available for the detection of Vero cytotoxin (VT) producing strains of E coli O157 that are usually of flagellar (H) type 7, but may also be non-motile. The strains were examined for VT production, for their ability to adhere to HEp-2 cells, and for hybridisation with several DNA probes that recognise pathogenic properties of E coli. Their ability to ferment sorbitol and to produce beta-glucuronidase was also investigated, as these tests are used to discriminate VT positive O157 strains. The O157:H8 strains did not produce VT. All gave localised attachment to HEp-2 cells, associated with a positive fluorescence-actin staining test, and all hybridised with the E coli attaching and effacing (eae) probe. In addition to the difference in VT production, O157:H8 strains could be distinguished from VT positive O157 strains by their beta-glucuronidase activity, their failure to produce enterohaemolysin, and their lack of hybridisation with the CVD419 probe derived from a plasmid in an O157:H7 strain. The 0157:H8 strains had in vitro properties characteristic of the class of E coli that causes attaching and effacing lesions in epithelial intestinal cells. They may therefore be considered a putative cause of diarrhoea but their prevalence remains to be established. Several O157:H8 strains failed to ferment sorbitol in agar plates and therefore could be misidentified as VT positive O157 strains. Confirmatory tests for VT production are needed when O157 strains are isolated from faeces.


Phenotypic methods for speciating clinical Aeromonas isolates. To establish the suitability of currently available phenotypic methods for speciation of clinical Aeromonas isolates in diagnostic microbiology laboratories. Using 62 Aeromonas spp, three schemes based on biochemical reactions were compared: a series of conventional tests; a system based on the suicide phenomenon, comprising two tubes in total; and a commercially available test, API 20 NE, augmented with a plate assay for beta haemolysin production. The whole cell and outer membrane protein (OMP) profiles of strains were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE), according to the results of the above schemes, to determine the intra-species homogeneity. Ninety per cent of strains were identified satisfactorily according to conventional criteria. For these strains, agreement was obtained using the suicide phenomenon and API schemes in 93% and 88% of cases, respectively. The three schemes concurred for 82% of strains. Whole cell protein profiles were unsuitable for comparing strains within a species. However, OMP patterns were similar for 89% of A caviae and 63% of A hydrophila. Phenospeciation of clinical Aeromonas isolates by the scheme based on the suicide phenomenon is simple to perform and accurate, and suitable for use in the diagnostic laboratory. OMP profiles are potentially useful for confirming the identity of A caviae and most A hydrophila, but not A sobria.


Immunohistological detection of human cytotoxic/suppressor T cells using antibodies to a CD8 peptide sequence. To evaluate whether cytotoxic/suppressor T cells can be detected in paraffin wax embedded human tissue samples using antibodies to a synthetic CD8 peptide sequence. Polyclonal and monoclonal antibodies were raised against a 13 amino acid peptide sequence from the cytoplasmic portion of the alpha chain of the human CD8 molecule. These antibodies specifically detected the native form of the CD8 polypeptide when tested by immunoprecipitation with radiolabelled T cells, and gave the expected staining pattern for cytotoxic/suppressor T cells in cryostat sections. Being raised in rabbits, the polyclonal antibodies were also useful for double labelling for CD8 in conjunction with monoclonal antibodies. CD8 positive cells could also be detected in paraffin wax embedded tissues. This was achieved without prior treatment of the sections if the tissue had been fixed in Bouin's fixative. When tissues had been exposed to conventional formalin fixation, preliminary microwave treatment was required. These findings provide further evidence that antibodies against leucocyte associated antigens, capable of reacting on paraffin wax embedded tissue, can be produced by immunisation with synthetic peptide sequences.


Comparison between histochemical and immunohistochemical methods for diagnosis of sporotrichosis. To compare the efficacy of histochemical and immunohistochemical methods in detecting forms of Sporothrix schenckii in tissue. Thirty five cutaneous biopsy specimens from 27 patients with sporotrichosis were stained by histochemical haematoxylin and eosin, periodic acid Schiff, and Gomori's methenamine silver methods and an immunohistochemical (avidin-biotin complex immunoperoxidase) (ABC) technique associated with a newly produced rabbit polyclonal antibody anti-Sporothrix schenckii. A total of 29 (83%) cases were positive by the ABC method used in association with anti-Sporothrix schenckii rabbit polyclonal antibodies. Histochemical methods, using silver staining, periodic acid Schiff, and conventional haematoxylin and eosin detected 37%, 23%, and 23% of forms of S schenckii, respectively. The ABC technique was significantly more reliable than periodic acid Schiff and silver staining techniques. It is concluded that immunostaining is an easy and rapid method which can efficiently increase the accuracy of the diagnosis of sporotrichosis in human tissue.


Audit of bone marrow trephines.

Audit of bone marrow trephines. To establish criteria of adequacy for bone marrow trephine biopsy specimens and to audit the quality of trephines performed at the Christie Hospital, Manchester. Trephines (n = 767) performed over 12 months were reviewed. Their lengths, and the lengths of their constituent parts (soft tissue, cortex, crushed marrow and interpretable marrow) were measured. The mean performance of each operator was calculated. Criteria of adequacy were established by a review of the published findings and an analysis of the relation between trephine length and the rate of infiltration by tumour. Before processing, the average trephine was 1.59 cm long. Trephines shrunk by 25% during processing. In histological sections the average length was 1.15 cm, consisting of 0.09 cm of soft tissue, 0.04 cm of cortex, 0.26 cm of disrupted marrow and 0.74 cm of interpretable marrow. A large number of operators were taking trephine biopsy specimens and their performance varied considerably. Review of the published findings suggested that the minimum adequate length is in the range 1.5 cm to 2.0 cm. The analysis of the relation between length of trephine and the rate of positivity for neoplasia yielded a minimum adequate length of 1.2 cm in section (1.6 cm before processing). Fifty eight per cent of the trephines were inadequate by this criterion. There was a tendency for the Jamshidi needle to produce a longer trephine than the Islam needle. According to objective criteria, at the Christie Hospital, many operators are producing a high proportion of inadequate bone marrow trephines.


Lipopeliosis: fat induced sinusoidal dilatation in transplanted liver mimicking peliosis hepatis. A distinct peliosis-like lesion arose in the liver allograft of a 51 year old man. This lesion was caused by necrotic, fat-laden hepatocytes that released fat globules into the sinusoids. These then became strikingly distended with cysts, thus mimicking peliosis hepatitis. It is suggested that this lesion be called lipopeliosis.


Splenic lymphoma with villous lymphocytes in two sisters. Splenic lymphoma with villous lymphocytes is a new entity characterised by the presence of atypical lymphocytes in the peripheral blood and bone marrow, and splenic infiltration in the white and red pulp. Cell membrane markers are those of a B mature cell, and no particular chromosomal abnormalities have been associated with this disease. A case of this rare lymphoma occurred in two sisters. Histological examination of splenic tissue was identical in both cases, with the same immunological surface markers, although the clinical and laboratory features were different. Karyotype analysis showed an abnormal pattern in one case; no environmental causative factor could be detected. Familial cases of other lymphoproliferative disorders have been reported, but no consistent common link has been found. It is suggested that further reports of this lymphoma, including cytogenetic and molecular studies, may provide a better understanding of the aetiology.


Measurement of placental alkaline phosphatase activity in benign and malignant pleural effusions. The usefulness of placental alkaline phosphatase (PLAP) as a diagnostic marker of malignancy was assessed in pleural fluid from 60 patients with effusions. Pleural fluid PLAP activities were measured by an enzyme linked immunoassay (ELISA) using the two monoclonal antibodies H17E2 and H317. Similar values were found in groups of patients with primary bronchial tumours (n = 12), secondary malignancies (n = 23), and "benign" conditions (n = 25). The highest values were found in a small subgroup of patients with metastatic ovarian carcinoma. However, the production of this enzyme by normal lung makes the measurement of PLAP in pleural fluid unhelpful as a diagnostic aid to distinguish "benign" from malignant effusions.


Backprocessing paraffin wax blocks for subgross examination. A method called "backprocessing", for subgross examination of sections, ranging between 50 microns and 1 mm in thickness using a dissecting microscope was developed. It requires either archival or current diagnostic breast tissue. In this study tissue is assumed to have been already embedded in paraffin wax for immediate diagnostic purposes. The advantages of backprocessing include the ability to select valuable or rare diagnostic tissue from archival histopathological material, and the preservation of histological structure should further examination at 5 microns thickness be required at a later date.


Microwaves improve chromosome G-banding in fresh blood and bone marrow. A simple technique for obtaining good chromosomal G-banding on fresh blood and bone marrow is presented. The usual technique is modified by using a microwave oven before staining the preparations with Wright's stain.


Eosinophilic bodies in pyloric and Brunner's gland cells. A previously unreported cell phenotype occurred in the pyloric and Brunner glands in two gastrectomy specimens. The cells were characterised by homogeneous, eosinophilic material in the cytoplasm. The eosinophilic material had an abnormally strong reactivity for Cystatin C, a protein found recently in the normal secretion of pyloric and Brunner's gland cells. The reason for the apparent cytoplasmic accumulation of cystatin C in the two patients described remains unclear.


Reassessment of the rate of fixative diffusion. The diffusion of fixatives is slow. Early work using plasma gels and animal tissues showed the distance penetrated by a fixative to be a simple function of the fixation time but this relation has not been established in human tissues. The rates of diffusion into whole human spleens were measured for three primary fixatives over periods ranging from one to 25 days. A positive correlation was demonstrated between penetration distance (mm) and fixation time (hours). The diffusion rates were slower than those in previous studies. These results have possible implications for the handling of surgical specimens.


Cytoblock preparations for examination of cervical and other cells. There are a number of antibodies which may be of value in the investigation of cervical smears, effusion, and cells grown in monolayer culture. The Shandon Cytoblock method was used to prepare discs of such cells suitable both for diagnosis and for a variety of other techniques.


Does lithium dose prediction improve treatment efficiency? Prospective evaluation of a mathematical method. An attempt was made to determine whether an algorithm for lithium dose prediction allows earlier or more economical attainment of a therapeutic steady state than does the usual "clinical titration." Twelve patients were treated using an algorithm developed by Zetin and associates and compared with 21 patients treated by clinical titration. Ten of the 12 algorithm-treated patients (83%) had reached the desired lithium blood level by the seventh day, whereas only 8 of the 21 clinical titration patients (38%) had done so. This difference is significant. In addition, the Zetin algorithm performed safely: the 2 dose prediction errors led to underdosing. Some of the relevant literature is reviewed.


Characteristics of long-term alprazolam users in the community. The widespread use of benzodiazepines remains a source of concern to the medical profession and the general public, especially as newer compounds come on the market. Our goal was to characterize long-term alprazolam users in the community and to determine whether such use represented abuse or behavioural dependence. We conducted three community surveys to learn about the natural history of long-term alprazolam use. Current long-term alprazolam users (those using the drug for 3 months or longer) were recruited on three separate occasions 1 year apart by identical newspaper advertisements in the metropolitan Toronto area. All respondents were mailed a questionnaire with a stamped, addressed return envelope. Our data from 312 respondents show that: (1) the majority of patients have a substantial history of prior medication use for symptom control (65%), (2) dose escalation is not a characteristic of long-term use, (3) patients change their initial pattern of regular use to one of symptom control only when required, (4) most physicians do not discuss discontinuation of the drug with their patients, (5) patients frequently try to stop their drug use (with a median of 2 attempts) and often report symptoms upon discontinuation, and (6) patients perceive a need for medication use and indicate that alprazolam is effective (75%). We conclude that some patients persistently use alprazolam but that this use does not represent abuse or behavioral dependence.


Clonidine treatment of hyperactive and impulsive children with autistic disorder. Many autistic children have associated problems of inattention, impulsivity, and hyperactivity that limit the effectiveness of educational and behavioral interventions. Few controlled psychopharmacologic trials have been conducted in autistic children to determine which agents may be effective for these associated features. Eight male children (8.1 +/- 2.8 years) with autistic disorder, diagnosed by DSM-III-R criteria, completed a placebo-controlled, double-blind crossover trial of clonidine. Subjects were included in the study if they had inattention, impulsivity, and hyperactivity that was excessive for their developmental level. Subjects had not tolerated or responded to other psychopharmacologic treatments (neuroleptics, methylphenidate, or desipramine). Teacher ratings on the Aberrant Behavior Checklist irritability, stereotypy, hyperactivity, and inappropriate speech factors were lower during treatment with clonidine than during treatment with placebo. Attention deficit disorder with hyperactivity: Comprehensive Teacher's Rating Scale ratings were not significantly improved during the study, except for oppositional behavior. Parent Conners Abbreviated Parent-Teacher Questionnaire ratings significantly improved during clonidine treatment. Clonidine led to increased ratings of the side effects of drowsiness and decreased activity. Clinician ratings (Children's Psychiatric Rating Scale Autism, Hyperactivity, Anger and Speech Deviance factors; Children's Global Assessment Scale; Clinical Global Impressions efficacy) of videotaped sessions were not significantly different between clonidine and placebo. Clonidine was modestly effective in the short-term treatment of irritability and hyperactivity in some children with autistic disorder.


Fluoxetine: activating and sedating effects at multiple fixed doses. Serotonin uptake inhibitors are generally considered activating antidepressants. To assess rates and temporal patterns of activation and sedation as well as dose-effect relationships, adverse event data were evaluated from a fixed-dose study comparing placebo and fluoxetine 5, 20, and 40 mg/day in the treatment of major depressive disorder (N = 363) and two fixed-dose studies pooled together comparing placebo and fluoxetine 20, 40, and 60 mg/day in the treatment of major depressive disorder (N = 746). The adverse events nervousness, anxiety, agitation, and insomnia were considered indicative of activation; somnolence and asthenia were considered indicative of sedation. Activation and sedation were both statistically significant (p less than or equal to 0.05) treatment-emergent phenomena, but dose-effect relationships differed. Activation rates were relatively stable between 5 and 40 mg/day, and then increased at 60 mg/day. Sedation rates increased linearly to 40 mg/day and then were comparable at 40 and 60 mg/day. Discontinuations for either phenomenon were uncommon. The temporal patterns of first occurrences and persistence of activation and sedation differed. First occurrences of activation peaked early and declined over time with all doses. First occurrences of sedation also peaked early with all doses, but there may have been greater variability in first occurrences of sedation over time with lower doses. Persistent occurrences of sedation may decline less over time than persistent occurrences of activation.


Clomipramine plasma levels among depressed outpatients in Benin, west Africa: drug compliance and comparison with Caucasian patients. A 75 mg/day clomipramine treatment was prescribed for 4 weeks to 92 outpatients with major depression at the Neuropsychiatric Clinic of the National University Hospital of Cotonou in Benin, West Africa. Among them, only 42 followed the treatment during the 4 weeks and had a clomipramine and desmethylclomipramine plasma level measure (gas chromatography technique). The rate of noncompliance appeared high: 10 patients had no trace and another 2 had only traces of antidepressant in their plasma. For the remaining 29, a comparison with 29 Caucasian patients treated by clomipramine (the two samples are matched for sex and age, and the variables of weight, dosage and drug association are controlled) shows no significantly higher plasma levels in the Beninese sample.


Acute and chronic administration of trazodone in the treatment of disruptive behavior disorders in children. We report the results of an open trial of trazodone in the treatment of severe behavioral disturbances in a sample of 22 hospitalized children previously found to be unresponsive to other treatments. Response to treatment was assessed by overall clinical criteria and improvements in individual symptom dimensions during the inpatient hospitalization. Thirteen children (67%) were found to benefit from the introduction of trazodone. Aggressive, impulsive behaviors were symptoms most frequently improved by this agent. Three of those found to be nonresponders actually worsened in symptomatology. A follow-up interview of the parents was conducted 3-14 months after discharge from the inpatient unit, for those children who initially responded to trazodone administration. The results of this interview suggest that the effect of trazodone was persistent for a prolonged period of time after the initial inpatient trial. Trazodone appears to be of value in the management of severe behavioral disturbances in children. The possible mechanism of action of trazodone is discussed.


Discontinuation of alprazolam after long-term treatment of panic-related disorders. Discontinuation of alprazolam after long-term treatment of 142 patients with panic-related disorders was examined in five study sites using a telephone interview. The majority (67%) of patients interviewed discontinued alprazolam for a period of at least 3 days after a gradual dosage reduction schedule over a 4-week period at the end of the long-term treatment study. A marked difference among the study sites in percentages of patients discontinuing therapy with alprazolam suggests that physician intervention played an important role in determining the ability of patients successfully to discontinue use of alprazolam: 90% and 95% of patients ceased therapy at two sites whereas only 21%, 38%, and 66% of patients discontinued therapy at the other three sites. The mean daily dosage for patients who continued using alprazolam decreased from 5.1 mg/day at the end of the long-term segment to 2.7 mg/day at the time of the poststudy interview. This decline indicates a lack of tolerance to the therapeutic effectiveness of alprazolam over an extended period of time.


Adverse cutaneous reactions associated with fluoxetine strategy for reintroduction of this drug in selected patients. Four patients treated with fluoxetine alone developed generalized urticaria. One also suffered polyarthritis. These reactions subsided when fluoxetine was stopped. After 6-12 months, because of progressive psychiatric problems, fluoxetine was reintroduced using a desensitization protocol. In each case this was well tolerated, and fluoxetine has been continued without adverse affects for 3-10 months.


An electron microscopical study of calcitonin gene-related peptide-like immunoreactive innervation of the anterior pituitary in the dog. Our previous studies have demonstrated the presence of a considerable number of substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive (LI) nerve fibers in the anterior pituitary in several mammalian species. The present study investigated the ultrastructure of the CGRP-LI innervation of this gland in the dog. The CGRP-LI nerve fibers were unmyelinated, with a wealth of varicosities containing both small clear synaptic vesicles and large dense-cored vesicles. They were found to be in direct contact with every cell type of the anterior pituitary. However, only on corticotropes and somatotropes were CGRP-LI synaptic contacts identified. Most of them were asymmetrical in type. Occasional symmetrical synaptic contacts were also found. It is considered likely that direct neural factors may play a role in the regulation of the anterior pituitary.


Enkephalin-immunoreactive neurons in the parvicellular subdivisions of the paraventricular nucleus project to the external zone of the median eminence. The enkephalin-immunoreactive neurons that project to the external zone of the median eminence were identified on thin paraffin and thick vibratome sections using a combination of retrograde labeling with peripherally administered Fluoro-Gold and immunocytochemistry. The vast majority of the enkephalin-immunoreactive neurons that project to the external zone of the median eminence (ME) reside in the paraventricular nucleus (PVN) of the hypothalamus. Within the PVN, the majority of these hypophysiotropic neurons are located in the medial parvicellular subdivision, while a smaller number can be detected in the anterior and the periventricular subdivisions. Although many enkephalin-immunoreactive neurons are present in other hypophysiotropic areas of the hypothalamus, such as the medial preoptic area, the anterior periventricular area, and the arcuate nucleus, only a few of these can be retrogradely labeled from the ME. These results provide morphological evidence for the key role of paraventricular enkephalin-immunoreactive neurons in the regulation of neuroendocrine functions.


Development of callosal connections in the sensorimotor cortex of the hamster. To investigate the development of corpus callosal connectivity in the hamster sensorimotor cortex, we have used the sensitive axonal tracer 1,1 dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI), which was injected either in vivo or in fixed brains of animals 3-6 days postnatal. First, to study changes in the overall distribution of developing callosal afferents we made large injections of DiI into the corpus callosal tract. We found that the anterogradely labeled callosal axons formed a patchy distribution in the contralateral sensorimotor cortex, which was similar to the pattern of adult connectivity described in earlier studies of the rodent corpus callosum. This result stands in contrast to previous retrograde studies of developing callosal connectivity which showed that the distribution of callosal neurons early in development is homogeneous and that the mature, patchy distribution arises later, primarily as a result of the retraction of exuberant axons. The initial patchy distribution of callosal axon growth into the sensorimotor cortex described in the present study suggests that exuberant axons destined to be eliminated do not enter the cortex. In addition, small injections of DiI into developing cortex resulted in homotopic patterns of callosal topography in which reciprocal regions of sensorimotor cortex are connected, as has been shown in the adult. Second, to study the radial growth of callosal afferents we followed the extension of individual callosal axons into the developing cortex. We found that callosal axons began to invade the contralateral cortex on about postnatal day 3, with little or no waiting period in the callosal tract. Callosal afferents then advanced steadily through the cortex, never actually invading the cortical plate but extending into layers on the first day that they could be distinguished from the cortical plate. The majority of callosal axons grew radially through the cortex and did not exhibit substantial branching until postnatal day 8, the age when the cortical plate disappears and callosal afferents reach the outer layer of cortex. This mode of radial growth through cortex prior to axon branching could serve to align callosal afferents with their radial or columnar targets before arborizing laterally.


Postnatal development of the ipsilateral corticospinal component in rat spinal cord: a light and electron microscopic anterograde HRP study. Most developmental and regeneration studies using the corticospinal tract (CST) as a model are focussed on its major contralateral component in the ventralmost part of the dorsal funiculus. However, for a correct interpretation of the data in these and future studies, a complete anatomical description of the rat CST, including its ipsilateral uncrossed component, is a prerequisite. In this study we used anterograde horseradish-peroxidase (HRP) tract-tracing at the light and electron microscopic level to describe the development of the uncrossed ipsilateral CST component. In the far caudal portions of the medulla oblongata, most of the CST fibers decussate dorsally and medially to assume positions in the ventralmost part of the dorsal funiculus. However, a small number of labeled fibers continue uncrossed in a position just lateral to the ventral median fissure. Labeled ipsilateral CST fibers caudally extend into the ventral funiculus of the third thoracic segment at postnatal day 1 (P1), into the seventh thoracic segment at P3, and into the first lumbar segment at P5 and P7. After the first postnatal week labeled ipsilateral CST fibers gradually disappear from upper lumbar, lower-, and mid-thoracic spinal cord levels, respectively. From P14 through adulthood the ipsilateral CST projection can not be visualized below upper thoracic spinal cord levels. Quantitative analysis of the amount of label along the length of the outgrowing ipsilateral CST revealed a characteristic staggered pattern of outgrowth: a small number of fibers take the lead and additional fibers are successively added. The outgrowth of labeled ipsilateral CST fibers is mainly restricted to spinal cord white matter. Occasionally a few outgrowing fibers into the adjacent cervical spinal gray can be observed, however, only between P3 and P7. Electron microscopic observations at the cervical enlargement (fifth cervical segment) demonstrate that the first labeled ipsilateral CST fibers, at P1, are characterized by the presence of growth cone-like enlargements at their distal endings. The process of myelination of labeled ipsilateral CST axons starts at about day P14. Although myelination seems to be largely completed at P28, labeled unmyelinated axons are still present in the adult ipsilateral CST component.


Timing and duration of dihydrotestosterone treatment affect the development of motoneuron number and morphology in a sexually dimorphic rat spinal nucleus. The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic motor nucleus in the rat lumbar spinal cord. SNB motoneurons and their perineal target muscles are present in adult males, but reduced or absent in adult females. This dimorphism is due to the presence of androgens during development. Perinatal treatment of females with testosterone (T), or a combination of dihydrotestosterone (DHT) and estrogen (E+D females) from embryonic (E) day 16 through postnatal (P) day 5, results in a masculine number of SNB motoneurons and the retention of the target muscles. Perinatal treatment with estrogen alone does not masculinize the SNB; prenatal treatment with DHT alone from E17-E22 results in a feminine number of SNB motoneurons and a significantly altered motoneuron morphology and connectivity. To determine if masculinization of the SNB involves the interaction of estrogen and DHT or results from a longer exposure to DHT alone, the number, morphology, and connectivity of SNB motoneurons in females treated with DHT both pre- and post-natally (from E16-P5) were examined. At E22, DHTP (E16-P5) females have SNB motoneuron numbers identical to E+D and normal females, but far fewer than normal males, thus indicating that T is essential for prenatal masculinization. After E22, SNB motoneuron number declines precipitously in normal females but remains stable in DHTP (E16-P5) females and E+D females, which do not differ from normal males at P10. These results demonstrate that DHT can completely masculinize SNB motoneuron number without any synergistic actions with estrogen, and suggest that the development of SNB motoneuron number is strictly an androgen-mediated event. In adulthood, horseradish peroxidase histochemistry reveals that the connectivity, dendritic length, and soma size of SNB motoneurons in DHTP (E16-P5) females are identical to those of normal males but differ significantly from those of DHTP (E17-E22) females. These data suggest that the altered connectivity in DHTP (E17-E22) females is not simply a hormone-specific effect, but the result of a truncated hormone exposure. Thus, DHT can fully masculinize SNB morphology and connectivity if given during the appropriate period of development. It is suggested that while T may be required to masculinize the SNB prenatally, DHT may be involved in masculinizing postnatal aspects of SNB development.


Cholecystokinin concentrations and peptide immunoreactivity in the intact and deafferented medullary dorsal horn of the rat. To further address the hypothesis that cholecystokinin (CCK) in the medullary dorsal horn (MDH) arises from intrinsic or higher-order neurons, CCK-8-specific radioimmunoassay (RIA) and immunohistochemical (IHC) experiments were carried out in adult rats after trigeminal tractotomy. RIA of punches from deafferented superficial layers of the MDH revealed no significant change in CCK levels vs. the control right side. In this same area, IHC revealed modest reductions in CCK, gastrin, and substance P staining. Calcitonin gene-related peptide (CGRP) staining was reduced substantially. Gastrin immunoreactive cell bodies, present normally in inner lamina II, were reduced in number. RIA and IHC methods were also used to assess MDH CCK concentrations in adult rats subjected to left infraorbital nerve section at birth. The left medulla contained significantly higher levels of CCK than the control right medulla (1.27 +/- 0.19 vs. 0.97 +/- 0.11 ng/mg protein). IHC revealed a dense band of CCK-like staining in laminae I and II ipsi- and contralateral to the lesion. Thus, neonatal deafferentation elevates medullary CCK. To determine if the neonatal lesion-induced increase in medullary CCK is due to primary afferent or higher-order reorganization, RIA and IHC experiments were run after infraorbital nerve section at birth and trigeminal tractotomy in adulthood. RIA revealed no significant change in CCK levels caudal to the tractotomy, although they were higher than control levels in 9 of 12 cases. IHC revealed modest reductions in CCK, substance P, and gastrin staining that resembled the reductions observed in tractotomy-alone cases. These data suggest that 1) most MDH CCK is of non-primary afferent origin, 2) gastrin immunoreactivity in layer II probably originates in CCK-containing cells intrinsic to layer II, the expression of which is dependent upon trigeminal primary afferent input, 3) neonatal V deafferentation induces increased CCK in the superficial MDH, reflecting reorganized intrinsic or higher-order inputs, and 4) higher-order substance P in the MDH is robust.


Neuronal and synaptic composition of the mediodorsal thalamic nucleus in the rat: a light and electron microscopic Golgi study. The distribution and dendritic domain of neurons in each segment of the mediodorsal thalamic nucleus (MD) have been studied in the rat with the Golgi technique. In addition, a combined Golgi method-electron microscopic (Golgi-EM) study was undertaken to determine the distribution of morphologically distinct synapse types along the dendrites of individual identified neurons in MD. All the subdivisions or "segments" of MD (medial, central, lateral) contained both stellate and fusiform cells. The dendritic domain of both types of cells was predominantly restricted to the same segment of MD that contained the cell body of the neuron. Typical stellate neurons were found near the center of each segment, with radiating dendrites that extended to but not across the boundaries of the segment. Fusiform cells were usually located close to the segmental or nuclear boundaries and tended to have dendrites oriented parallel to those borders; again, the dendrites tended not to extend across borders between segments or at the outer edge of MD. In the medial segment of MD many fusiform cells had especially bipolar dendritic configurations, generally with a dorsoventral orientation. Because no small neurons were identified that might correspond to thalamic interneurons, all the impregnated cells in MD are presumed to be thalamocortical projection neurons. These results indicate that cells and their major dendrites are confined to a single segment of MD, with little dendritic overlap across segmental or nuclear borders. The segments of MD may therefore be considered to be relatively independent subnuclei. The distribution of the four types of synapses previously identified in MD (Kuroda and Price, J. Comp. Neurol., 303:513-533, 1991) was determined along several identified dendrites studied with the Golgi-EM method. Primary dendrites were contacted mostly by large axon terminals, including both large, round vesicle (LR) terminals and large, pleomorphic vesicle (LP) terminals, as well as a few small to medium sized terminals with pleomorphic vesicles (SMP). No small terminals with round vesicles (SR terminals) were observed to make synapses with primary dendrites. Secondary and tertiary dendrites received synapses from all types of axon terminals. Higher order dendrites were contracted predominantly by SR boutons, but they also carried some LR and SMP terminals. In addition, SMP boutons were often found to form symmetric contacts with cell somata.


Nerve ring of the hypostome in hydra. I. Its structure, development, and maintenance. The anatomy and developmental dynamics of the nerve ring in the hypostome of Hydra oligactis were examined immunocytochemically with an antiserum against a neuropeptide and with neuron-specific monoclonal antibodies. The nerve ring is unique in the mesh-like nerve net of hydra. It is a distinct neuronal complex consisting of a thick nerve bundle running circumferentially at the border between the hypostome and tentacle zone. Immunostaining showed that the nerve ring was heterogeneous and contained at least four different subsets of neurons. During head regeneration and budding, the nerve ring appeared only after the nerve net of ganglion and sensory cells had formed. Every epithelial cell is continuously displaced with neurons toward either head or foot in an adult hydra. However, the ectoderm in the immediate vicinity of, and including, the nerve ring constitutes a stationary zone that is not displaced. Tissue immediately above this zone is displaced toward the tip of the hypostome, while tissue below is displaced along the tentacles. Correspondingly, the production of new neurons in the ring as measured by their differentiation kinetics is much slower than in surrounding areas. Thus, the nerve ring is static and stable in contrast to the dynamic features of the nerve net of hydra.


Immunocytochemical localization of kynurenine aminotransferase in the rat striatum: a light and electron microscopic study. Kynurenine aminotransferase is the biosynthetic enzyme for kynurenic acid, an antagonist of excitatory amino acid receptors. Because of the possible role of kynurenic acid in basal ganglia diseases, the distribution of kynurenine aminotransferase immunoreactivity was examined in the adult rat striatum at the light and electron microscopic levels. Kynurenine aminotransferase immunoreactivity was detected in glial cells and in neurons. The preadsorption control vastly reduced or eliminated specific staining at both the light and electron microscopic levels. Kynurenine aminotransferase positive glial cells were abundant and contained a robust and homogeneous distribution of reaction product in both the nucleus and cytoplasm. The majority of neurons, both medium and large, were immunostained and exhibited granular kynurenine aminotransferase immunoreactivity in the cytoplasm of somata and proximal dendrites. At the ultrastructural level, kynurenine aminotransferase immunoreactive astrocytic processes were apparent throughout the neuropil where they often encircled capillaries and surrounded axospinous synapses. Reaction product was associated with the cytoplasmic matrix, filaments, rough endoplasmic reticulum, and the nucleus. In neurons, the majority of label occurred in round membrane-bound cytoplasmic organelles located adjacent to the Golgi apparatus, rough endoplasmic reticulum, and the cell or nuclear membranes. Cisternae and vesicles were identifiable in some of the labeled profiles. Polyribosomes and rough endoplasmic reticulum were also labeled. These data provide an anatomical basis for biochemical studies that have suggested the presence of striatal kynurenine aminotransferase in both astrocytes and neurons.


Antibody to NGF inhibits collateral sprouting of septohippocampal fibers following entorhinal cortex lesion in adult rats. We have used an antiserum raised against mouse 2.5S NGF to examine the involvement of endogenous neurotrophins in the collateral sprouting of septohippocampal fibers in the adult rat brain. The antiserum was administered intraventricularly. Immunocytochemical techniques indicated that the injected antibodies penetrated into brain tissue that included the basal forebrain, cortex, striatum, corpus callosum, and hippocampus. Unilateral lesioning of the entorhinal cortex was done to evoke the sprouting of the cholinergic septohippocampal fibers. At 8 days postlesion, the sprouting was much advanced, as evidenced by an increase in density of the acetylcholinesterase (AChE) staining in the outer molecular layer (OML) of the dentate gyrus and by the associated increase in the absolute number of AChE-positive fibers in the OML. As well, there was a widening of the inner molecular layer (IML), interpreted as being due to sprouting of noncholinergic axons in that region. In rats injected daily with anti-NGF or anti-NGF Fab fragments, no increase in AChE density, or in the population of AChE-positive fibers, was observed in the OML. In contrast, the widening of the IML seemed to be unaffected by the anti-NGF treatment. No changes were observed in the AChE related parameters in the dentate gyrus of nonlesioned animals treated similarly for 8 days with anti-NGF; there was, however, a decrease of choline acetyltransferase (ChAT) immunostaining in the ChAT-positive cells of the basal forebrain. Our findings and the confirmation that our polyclonal anti-NGF also recognizes other members of the NGF neurotrophin family, specifically brain-derived neurotrophic factor and neurotrophin-3, indicate that at least one of these neurotrophins plays a key role in the collateral sprouting of the cholinergic septohippocampal fibers (but not that presumed to occur within the IML) following an entorhinal cortex lesion.


Course of the fiber pathways to pons from parasensory association areas in the rhesus monkey. The course of the fiber pathways to pons from parasensory association areas in the rhesus monkey was investigated by injection of tritiated amino acids and the technique of autoradiography. Results confirm the projection to pons from parasensory association areas in the temporal, parietal, and occipital lobes and extend these observations to include the posterior parahippocampal gyrus. The findings reveal that the white matter of the posterior limb of the internal capsule above the midpoint of the lateral geniculate nucleus, and at the medial aspect of the lateral geniculate nucleus, comprise common regions through which these corticopontine fibers lead to the basis pontis. The fibers demonstrate a certain degree of topographic organization in the posterior limb of the capsule above the lateral geniculate nucleus and also in the cerebral peduncle. Taken together with previous observations concerning the termination patterns of these associative corticopontine projections, it would appear that the corticopontine system consists of segregated and partially overlapping pathways, which are to some extent distinguishable anatomically at each stage of their trajectory from origin to destination. Furthermore, the existence of a common area through which all parasensory associative input to pons is transmitted suggests that a precisely located lesion in this part of the corticopontocerebellar circuit may disrupt the cerebellar access to higher order information derived from the parasensory associative regions.


Vasoactive intestinal peptide (VIP) in magnocellular neurons of the hypothalamo-neurohypophysial system of the mink (Mustela vision) is co-localized with vasopressin or oxytocin. The distribution of vasoactive intestinal peptide (VIP) was analysed in perikarya of the mink hypothalamus with immunohistochemistry and, surprisingly, a large population of magnocellular VIP-immunoreactive neurons was present in the paraventricular and supraoptic nuclei as well as in accessory hypothalamic nuclei. From perikarya in the paraventricular as well as supraoptic nuclei, a large number of VIP immunoreactive nerve fibers was observed to enter the hypothalamo-neurohypophysial tract. Within the median eminence, a high density of VIP-immunoreactive nerve fibers was present in the external and internal zones. Fibers in the external zone of the median eminence were endowed with varicosities and perivascular terminals, while fibers in the internal zone were smooth and without terminal specializations. From the internal zone of the median eminence, fibers coursed via the infundibular stalk to terminate in perivascularly situated terminals in the neurohypophysis. In addition, a substantial number of small VIP-immunoreactive perikarya was observed within the suprachiasmatic nucleus. These perikarya were immunoreactive to neither vasopressin nor neurophysin. To elucidate the co-existence of VIP-immunoreactivity with vasopressin, oxytocin or neurophysin, a sequential double immunoperoxidase procedure to localize antigens with diaminobenzidine and benzidine dihydrochloride as chromagens was performed. From these experiments it was evident that VIP in nearly all magnocellular hypothalamo-neurohypophysial neurons co-existed with neurophysin. Based on a semi-quantitative estimate, half the VIP-immunoreactive magnocellular perikarya co-stored vasopressin, while another half co-stored oxytoxin. The present study describes the presence of a large population of VIP-containing neurons in the hypothalamo-neurohypophysial system of the mink. These findings raise evidence that within the mink, VIP may be involved in neurohypophysial physiology.