Effects of alcohol on the human placental GnRH receptor system.

Isolation of human term placental membranes in the presence or absence of protease inhibitors indicated that protease inhibitors significantly reduced the amounts of [(125)I]-labelled gonadotrophin-releasing hormone (GnRH) binding to membrane GnRH-receptors in vitro by approximately 20%. This decrease was largely due to the ethanol used to dissolve the serine protease inhibitor, phenylmethylsulphonylfluoride (PMSF). Ethanol alone decreased the specific binding of [(125)I]-labelled GnRH isoform (IC(50), 7.9 +/- 0.8 mg/ml; n = 6) or agonist tracers (IC(50), 10.0 +/- 1.4 mg/ml; n = 6) to human placental membranes in a dose-dependent manner. Other alcohols also interfered with [(125)I]-GnRH isoform or agonist binding: inhibition increased with increasing carbon chain length and was dependent on the isomeric position of the hydroxyl group. Fractionation of term placental cytosol by gel chromatography demonstrated the presence of a high molecular weight fraction ( approximately 60-70 kDa) which inhibited [(125)I]-GnRH binding to human placental membranes. However, placental cytosol fractions did not cross-react significantly with a specific anti-GnRH antibody. Surprisingly, re-assay of cytosol fractions in the presence of a cocktail of protease inhibitors generated a factor (molecular weight approximately 40-50 kDa) which did cross-react strongly with the GnRH antibody. The generation of this factor was due to the ethanol solvent rather than to the protease inhibitors per se, as treatment of pooled 'latent' cytosol fractions with ethanol alone generated GnRH-like immunoactivity (irGnRH) which competed in parallel with GnRH standard. The amount of irGnRH generated depended on the concentration of ethanol added to the 'latent' cytosol fractions. However, ethanol had no effect on the assay in the absence of cytosol fraction, or with inactive cytosol fractions. Thus, ethanol can perturb the human placental GnRH/GnRH-receptor system in vitro in two distinct ways: by inhibition of GnRH binding to receptor, and by dissociation of complexed endogenous GnRH-like factor(s) from a GnRH-binding protein. It is postulated that high alcohol consumption in vivo may interfere with placental GnRH secretion/action and affect placental secretion of factors important to the establishment and maintenance of pregnancy.     

Clinical and economic impact of enterovirus illness in private pediatric practice

OBJECTIVE: To characterize the acute clinical course and economic burden of nonpolio enteroviral (NPEV) illness in the summer/fall season as seen in private pediatric practice. METHODS: We prospectively studied 380 children aged 4 to 18 years with systemic NPEV syndromes presenting to private suburban pediatric practices. Seventy-three asymptomatic controls were concurrently enrolled. Clinical diagnosis of NPEV illness was based on the presence of fever plus at least one of the following: headache and stiff neck (n = 2); myalgia and malaise (n = 105); nonpuritic maculopapular rash (n = 10); papulovesicular stomatitis (n = 214); papular rash of the hands, feet, and mouth (H/F/M) (n = 30); or pleurodynia (n = 11). Study participants were enrolled during a 4-month time span (July-October, 1994) and followed daily for 14 days. A parent symptom diary card and twice weekly phone contacts by study nurses characterized the illness to include the frequency of health care contacts, the necessity for laboratory tests, medication use, and school/work absenteeism. RESULTS: Three hundred seventy-two (98%) children completed the study; 122 (33%) of the patients were confirmed to be infected with NPEV. Confirmed NPEV infection was more frequently observed in Rochester, NY (85/147 = 58%) than in Scottsdale, AZ (32/224 = 14%). The age group 4 to 12 years comprised 79% to 90% of the enrollees, depending on the syndrome. Median duration of illness and median number of missed days of school/summer camp/work for the enrolled patients was: meningitis (7 days ill, 2 days missed), myalgia/malaise (9 days ill, 3 days missed), rash (6 days ill, 4 days missed), stomatitis (7 days ill, 2 days missed), H/F/M (7 days ill, 1 day missed), and pleurodynia (8 days ill, 3 days missed). Direct medical costs varied from $69 per case to $771 per case and indirect costs, attributable primarily to parent missed work and/or sick-child care, varied from $63 per case to $422 per case for H/F/M and meningitis, respectively. In households, H/F/M spread to 50% of siblings and 25% of parents. CONCLUSIONS: In our study population, NPEV infection: 1) caused sufficient illness to prompt physician visits in summer and fall; 2) occurred more frequently in 4 to 12 year olds than in adolescents; 3) produced various clinical syndromes concurrently during the same months in the same season of a given year; 4) varied in occurrence geographically; 5) was characterized by numerous symptoms of longer duration than previously recognized; and 6) produced a significant economic impact by generating both direct and indirect costs.     

A proof-of-concept study to assess the putative dose response to topical corticosteroid in persistent allergic rhinitis using adenosine monophosphate challenge

INTRODUCTION: The aim of this proof-of-concept study was to assess whether nasal adenosine monophosphate (AMP) challenge may be used to quantify dose response to topical fluticasone propionate (FP) in persistent allergic rhinitis (PER). METHODS: Eligible subjects with PER entered a randomized double-blind crossover study of 2 weeks of intranasal FP at 100 microg or 400 microg daily, with a 2-week placebo washout period before each randomized treatment. Measurements after each washout or treatment comprised: peak nasal inspiratory flow (PNIF) response to nasal AMP (the primary outcome), domiciliary PNIF, the mini rhinoconjunctivitis quality of life questionnaire (miniRQLQ), symptom scores, nasal nitric oxide levels and overnight urinary cortisol:creatinine ratios. RESULTS: Thirteen patients completed per protocol. Maximal PNIF response to AMP was attenuated 0.9% (95% confidence interval -7.1 to 9.0, P=NS) by FP 100 microg, and 12.9% (4.8-20.9, P=0.009) by FP 400 microg. The 400-100 microg difference was 12.0% U (2.6-21.3, P=0.049). None of the other outcomes were responsive enough to detect any significant treatment effects. The standardized response means to FP 400 microg were 81% for AMP challenge, 54% for domiciliary PNIF, 53% for miniRQLQ, 24% for symptom scores and 18% for nasal nitric oxide. No adrenal suppression was detected at either dose. CONCLUSION: FP exhibited dose-related suppression of nasal airway hyperresponsiveness to AMP challenge, but without associated detectable adrenal suppression at the higher dose. Moreover, the AMP response demonstrated the highest signal to noise ratio compared with other outcome measures in PER.     

Apparent alpha-inhibin subunit immunoactivity in porcine and ovine luteal extracts is due to interference by cytosolic proteases in the assay.

Immunoreactive alpha-inhibin (ir-inhibin) was measured in luteal homogenates and subcellular fractions of ovine and porcine corpora lutea (CL) and in pig granulosa cells (GCs), using a sensitive radioimmunoassay specific for the 1-26 amino acid sequence of the N-terminus of the alpha chain of porcine inhibin (p1-26 alpha-inhibin). Inclusion of N-ethylmaleimide (N-EM) and/or EDTA in the immunoassay had no effect on the measurement of p1-26 alpha-inhibin peptide standards, on ir-inhibin levels in ovine follicular fluid and serum, or on ir-inhibin in subcellular fractions of pig GC. Fractionation of porcine GC homogenates on sucrose gradients demonstrated a major particular peak of ir-inhibin (buoyant density, 1.15-1.21 g/cm3) with variable activity in the cytosol. The particulate ir-inhibin peak was released into the cytosol by pretreatment of GC homogenates with the saponin, digitonin, prior to fractionation. Porcine GC extracts contained a protein (M(r) 45,000) which immunoblotted against p1-26 alpha-inhibin antibody. In the absence of inhibitors of proteolysis, apparent ir-inhibin activity was very high in extracts of sheep and pig CL. However, inclusion of N-EM or EDTA in the radioimmunoassay significantly reduced ir-inhibin levels in porcine and ovine CL extracts in a dose-dependent manner. Measurements of peptide tracer integrity indicated that porcine luteal cytosol degraded 125I-labelled p1-26 alpha-inhibin peptide. Subcellular fractionation studies demonstrated high levels of apparent ir-inhibin in luteal cytosol fractions, with only minor activity peaks associated with particulate fractions; however, this material was not releasable by digitonin. Immunoblotting of detergent extracts of porcine luteal particulate fractions failed to demonstrate alpha-inhibin material, and immunocytochemical localization studies of alpha-inhibin in porcine and ovine luteal sections were negative. Our results are consistent with the intracellular packaging/storage of a form of alpha-inhibin (M(r) similar to that of alpha-inhibin subunit precursor) in the porcine granulosa cell. However, luteinization of the porcine follicle was associated with a dramatic fall in ir-inhibin content, and the loss of immunostaining for alpha-inhibin peptides. We conclude that porcine and ovine CL contain little, if any, authentic inhibin. These studies emphasize the importance of excluding proteolytic artefacts when measuring biological peptides in luteal tissue extracts by radioimmunoassay.     

Isoforms of ferritin have a specific cellular distribution in the brain

Ferritin is the major iron storage protein and accounts for the majority of the iron in the brain. Thus, ferritin is a key component in protecting the brain from iron induced oxidative damage. The high lipid content, high rate of oxidative metabolism, and high iron content combine to make the brain the organ most susceptible to oxidative stress. The role of oxidative damage and disruption of brain iron homeostasis is considered clinically important to normal aging and a potential pathogenic component of a number of neurologic disorders including Alzheimer's disease and Parkinson's disease. Little is known, however, of the mechanism by which the brain maintains iron homeostasis at either the whole organ or cellular level. In this study we report the cellular distribution of the two isoforms of ferritin in the brain of adult subhuman primates. A subset of neurons immunolabel specifically for the H-chain ferritin protein, whereas cells resembling microglia are immunolabeled only after exposure to the L-chain ferritin antibody. Only one cell type immunostains for both H-and L-chain ferritin; these cells are morphologically similar and have the same distribution pattern as oligodendrocytes. Neither ferritin isoform is usually detected in astrocytes. These data indicate considerable differences in iron sequestration and use between neurons and glia and among neuronal and glial subtypes. This information will be essential in determining the role of each of these cells in maintaining general brain iron homeostasis and the relative abilities of these cells to withstand oxidative stress. © 1994 Wiley-Liss, Inc.     

Optic disc haemorrhages and vascular abnormalities in a glaucoma population

Purpose: To retrospectively examine the optic disc photographs of a glaucoma population for optic disc haemorrhages, vascular occlusions and vascular abnormalities. Methods: The optic disc photographs of 906 eyes of glaucoma and suspect glaucoma patients were examined. Optic disc photographs were taken annually, where possible, with the follow-up period varying between 1 and 14 years duration (mean, 2.89). Glaucoma patients are regularly reviewed every 4–6 months and glaucoma suspects every 1–2 years, depending on the ophthalmologist. Low-tension glaucoma patients were reviewed more frequently (mean, every 2.6 months). The results of the findings were compared to a control group of 39 subjects with a mean follow-up period of 7 years, using Fisher's exact test. Results: It was found that during the period under review, 7.4% (n= 67) of eyes had optic disc haemorrhages. The highest frequency of optic disc haemorrhages (37.5%) was found in the low tension glaucoma group (P= 0.0001) followed by 11.4% of primary open-angle glaucoma eyes (P= 0.03). In the normal group there were three eyes with optic disc haemorrhages and one with a disc collateral, which constitutes 5.1% vascular changes in this sub-group. Of the study eyes 2.8% had central retinal vein occlusions, 1.3% branch vein occlusion, 1.2% disc vessel abnormalities (loops) and 1.1% disc collaterals. Discrete nerve fibre layer haemorrhages and microaneurysms were found in 0.8% and 1.8% of eyes, respectively. Conclusions: A total of 16.8% of the eyes observed in this study had either disc haemorrhages or vascular changes. The underlying trend of vascular and haemorrhagic changes in glaucoma are demonstrated in this sample, which is in general agreement with previous studies. The high percentage of optic disc haemorrhages in low tension glaucoma is highlighted. The presence of microaneurysms and nerve fibre layer haemorrhages is interesting but of unknown significance.     

A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains.

Immunohistochemical and histochemical staining were performed on Alzheimer's diseased brain tissue obtained at autopsy. The iron-regulatory proteins transferrin and ferritin as well as iron are, in general, found predominantly in oligodendrocytes similar to that previously reported for normal brain tissue. However, in the vicinity of senile plaques, the staining pattern is altered for both proteins and iron. Transferrin is homogenously distributed around the senile plaques and is apparently extracellular. In addition, transferrin is found in astrocytes in the cerebral cortical white matter of the Alzheimer's tissue rather than its normal distribution in oligodendrocytes. A robust ferritin immunoreaction accompanies senile plaques and many blood vessels in the Alzheimer's brain tissue. Although many ferritin-positive oligodendrocytes are present in the Alzheimer's tissue, most of the ferritin-containing cells associated with senile plaques and blood vessels are microglia. Iron can also be demonstrated in the senile plaques. The iron reaction product is observed both diffusely in proximity of the plaques and in cells associated with the plaques. These data strongly suggest a disruption in brain iron homeostasis in Alzheimer's disease as demonstrated by alterations in the normal cellular distribution of iron and the proteins responsible for iron regulation. These data will contribute to understanding both the potential for oxidative damage and the potential for metal neurotoxicity in Alzheimer's disease.     

Humoral response to Mycobacterium tuberculosis antigens in patients with tuberculosis in the Gambia.

OBJECTIVE: To determine and compare the sensitivity and specificity of four common mycobacterial antigens with three RD-1 region antigens in the serological diagnosis of active pulmonary tuberculosis (PTB) in the Gambia. DESIGN: Serum from 300 Gambians (100 with active PTB, 100 of their household contacts, and 100 community controls) was tested using an ELISA method to detect antibodies to seven mycobacterial antigens (three encoded in the RD-1 region [ESAT-6, CFP-10 and Rv3871] and four common [38 kDa, GLU-S, 19 kDa and 14 kDa]). Individuals with active TB were recruited from one of the National Leprosy and TB Control Program clinics in the western region of the Gambia, and neighborhood controls were an age-matched individual living within five houses of the case. RESULTS: The sensitivity of the RD-1 antigens ranged from 34% to 67%, while specificity ranged from 51% to 71%. The sensitivity of the common antigens ranged from 24% to 75% and specificity from 26% to 75%. CONCLUSION: In countries with high rates of TB, such as the Gambia, the clinical utility of serological testing to diagnose active TB remains limited, even with newer antigens encoded in the RD-1 region of Mycobacterium tuberculosis.