Use of post-mortem human synaptosomes for studies of metabolism and transmitter amino acid release

Synaptosomes have been prepared from human brain obtained at autopsies carried out up to 24 h postmortem (p.m.). They showed generally good retention of morphology, as well as accumulation of tissue potassium and linear rates of oxygen uptake. In response to veratrine depolarization they showed increased respiration rate, decreased tissue potassium content and the specific release of transmitter amino acids. Regression analysis indicated that metabolically and functionally active preparations may be obtained up to ca. 25 h p.m. Preparations obtained from patients dying with brain injury were inactive.     

Diagnosis of Neisseria gonorrhoeae infections in women by using the ligase chain reaction on patient-obtained vaginal swabs.

The increased sensitivities of nucleic acid amplification tests such as ligase chain reaction (LCR) have the potential to simplify specimen collection for gonorrhea diagnosis. In this study patients took their own vaginal swab specimens for gonorrhea culture and LCR testing. Immediately following specimen collection by patients, a trained clinician obtained endocervical swab specimens for the same tests. By using LCR to diagnose gonorrhea, 54 (17.5%) of 309 patients had positive tests. Forty-five patients with positive cervical LCR tests also had positive vaginal LCR tests; for one patient, only a cervical LCR specimen was positive, and for eight patients, only vaginal specimens were positive. For specimens from patients whose gonorrhea cultures were positive, all vaginal swab specimens were positive by LCR and 42 (91%) of 46 cervical swab specimens were positive by LCR. LCR-positive specimens from eight patients with negative cultures (four with positive vaginal specimens only, one with a positive cervical specimen only, and three with positive vaginal and cervical specimens) were further evaluated with unrelated probe sets for gonococcal pilin B. Following resolution of the discrepancies between culture-negative and LCR-positive specimens, a diagnosis of gonorrhea could be confirmed for 52 of 54 patients with positive LCR tests. LCR testing with vaginal swabs was 100% sensitive and 99.6% specific and had a positive predictive value of 98.1% and a negative predictive value of 100%. In this study LCR testing of vaginal swab specimens obtained by patients themselves was significantly more sensitive for gonorrhea diagnosis of women than cervical LCR or culture (100% versus 84.6% for cervical LCR or culture; Mantel-Haenszel chi-square test result, 8.58; P = 0.003).     

A new microcellular cytotoxicity test based on calcein AM release

We present a microtest for cell-mediated immunity, based on the use of the Tarasaki tray and calcein AM vital dye. The number of target cells needed has been reduced to 500 per test with a corresponding tenfold reduction in the number of effector cells needed. Results were read at the rate of 1 second per test using a fluorimeter attached to a microscope. Each reaction was also confirmed visually with the use of ethidium bromide as a counterstain for dead cells. The calcein AM dye used to stain the living cells was shown to have a low spontaneous leakage rate—less than 15% in 4 hours at 37°C. Dilutions of targets stained by calcein AM had a linear relationship with measured fluorescence values. NK cells, LAKs, and CTLs were readily detectable by this microtest. Quantitation of killing and kinetic analysis was readily performed with this test system. A significant positive correlation to ⁵¹Cr-release results was found. We conclude that the microtest should find wide application in studies of cell-mediated immunity.     

Endothelin receptor expression in human decidua

The endothelins are signalling peptides that act via two receptors, ET(A) and ET(B). In the human endometrium, endothelin receptors have been demonstrated in glands and stroma and have been shown to vary during the course of the menstrual cycle. The present study was undertaken to determine whether or not expression of endothelin receptors changes during pregnancy or after administration of exogenous progestagens. The expression of the receptors was correlated with the appearance of basement membrane components during decidualization of the endometrial stroma. Decidual specimens (n = 15) were obtained during the first trimester of pregnancy and 10 at term. Sixteen pairs of endometrial biopsies were obtained from women with menorrhagia before and after exposure to exogenous progestagens. A total of 15 hysterectomy specimens were used as controls for the expression of stromal basement membrane proteins in the absence of decidualization. Autoradiography was carried out with selective ligands for ET(A) ([125I]-PD 151242) and ET(B) ([125I]-BQ3020). The distribution of ligand binding was then compared with the distribution of laminin alpha2 light chain and collagen IV. ET(A), ET(B), laminin alpha2 light chain, and collagen IV were expressed in stromal decidual cells in the first trimester of pregnancy. ET(B) was also found on endometrial glandular epithelium. Quantitative macro-autoradiography and multiple regression analysis demonstrated a highly significant positive correlation (P < 0.001) between expression of ET(B) and laminin alpha2 light chain. In the third trimester qualitative examination suggested a reduction of ET(A) in the stroma. Progestagen-induced decidua exhibited a similar pattern to that found in first trimester decidua. This study has demonstrated up-regulation of ET(B) during the progesterone- dependent process of decidualization and suggests a paracrine or autocrine role for endothelins in the decidua.      

Permanent impairment of embryo development by hydrosalpinges

Recent reports suggest a deleterious effect of hydrosalpinges on pregnancy outcome for in-vitro fertilization (IVF) and improvement following surgical treatment. We compared the effect of hydrosalpinx on pregnancy outcome in 286 patients having 348 IVF cycles and followed the development of untransferred embryos for 7 days to determine if hydrosalpinges affect oocyte quality or embryo development. The delivery rate per retrieval was significantly lower for patients with hydrosalpinx, but was restored by surgical treatment to that of patients without hydrosalpinx. However, the implantation rate per embryo transferred and normal blastulation of untransferred embryos, which were significantly decreased in patients with hydrosalpinx, and growth arrest and degeneration of untransferred embryos, which were significantly increased compared to patients without hydrosalpinx, were not restored by surgical treatment of hydrosalpinges. We conclude that surgical treatment of hydrosalpinges decreases early pregnancy loss and improves pregnancy outcome, possibly by diminishing reversible deleterious effects exerted on the endometrium. As we have seen in our laboratory, hydrosalpinges may have a permanent negative influence on ovarian function, follicular development and oocyte quality since implantation of transferred embryos and normal blastulation of untransferred embryos remain low, and in-vitro growth arrest and degeneration remain high despite surgical treatment of hydrosalpinges.      

Variability of clarithromycin and erythromycin susceptibility tests with Haemophilus influenzae in four different broth media and correlation with the standard disk diffusion test.

Four separate laboratories performed antimicrobial susceptibility tests with 40 Haemophilus influenzae isolates, each tested in triplicate. Erythromycin and a new macrolide, clarithromycin (A-56268; TE-031), were tested by the disk diffusion method, by the agar dilution procedure in two different media, and by broth microdilution tests in four different media. Erythromycin MICs for 90% of the strains were 16 micrograms/ml in Mueller-Hinton broth with 3% lysed horse blood and NAD, 4.0 micrograms/ml in hemophilus test medium, and 2.0 micrograms/ml in supplemented Schaedler broth or in the fastidious broth medium from Beckman Instruments, Inc. Clarithromycin MICs were generally 1 doubling dilution greater than erythromycin MICs in each of the media. Erythromycin disk tests corresponded best with MICs determined in the fastidious broth medium. In that same medium, clarithromycin MICs were about 1 doubling dilution greater than what would be expected from the results of disk tests. Because there were fewer growth failures, hemophilus test medium is recommended for microdilution tests with H. influenzae. Incubation of all tests for a full 24 h without an increased CO2 atmosphere was needed to achieve maximal precision of the tests. Interlaboratory and intralaboratory reproducibility of all tests was satisfactory.     

Differential effects of the antioxidant alpha-lipoic acid on the proliferation of mitogen-stimulated peripheral blood lymphocytes and leukaemic T cells

The effects of the antioxidant alpha-lipoic acid (LA) on the proliferation of mitogen-stimulated human peripheral blood lymphocytes (HPBL) were investigated in comparison to its effects on the proliferation of two leukaemic T cell lines, Jurkat and CCRF-CEM. At low mM concentrations, LA inhibited in a dose-dependent manner DNA synthesis of HPBL stimulated with either phorbol myristate acetate (PMA) in combination with ionomycin (IoM), or phytohaemagglutinin (PHA). At similar concentrations, LA inhibited the proliferation of Jurkat and CCRF-CEM cells. However, LA was preferentially cytotoxic to the leukaemic cell lines. The selective toxicity of LA to Jurkat cells was shown by electron microscopy (EM) to be due to the induction of apoptosis. Furthermore, LA had different effects on the secretion of interleukin-2 (IL-2) and steady-state levels of IL-2 mRNA in mitogen-stimulated HPBL depending on the mitogens used. LA dramatically increased the induction of IL-2 mRNA and IL-2 protein secretion in PMA/IoM-stimulated HPBL, whereas it inhibited these in HPBL stimulated with PHA. The differential effects of LA on normal and leukaemic T lymphocytes may indicate a new route towards development of therapeutic agents.     

A new presenilin Alzheimer's disease case confirms the helical alignment of pathogenic mutations in transmembrane domain 5

In a case of familial early onset Alzheimer's disease, a mutation was detected in exon 7 of the presenilin 1 gene at codon 226 with a resultant amino acid change from leucine (CTC) to arginine (CGC) (L226R). This is a novel finding, yet is consistent with the previously reported mutations at codons 222, 229, 233 and 237 in transmembrane domain 5 which show a helical alignment of mutations in this domain. We conclude that the cause of Alzheimer's disease in this patient is an authentic PS1 gene abnormality responsible for the patient's early onset Alzheimer's disease.