Anti-human RP105 sera induces lymphocyte proliferation.

Cellular environment dictates whether antigen binding to the B lymphocyte receptor together with co-stimulatory molecules will result in proliferation, anergy, or apoptosis. Murine RP105 is a member of the leucine-rich repeat family of proteins, which is specifically expressed on mature B cells. Monoclonal antibodies to the murine RP105 induce proliferation and protect B cells from apoptosis, suggesting an important regulatory role in murine B lymphocyte function. We identified a human RP105 homolog and mapped the gene to chromosome 5q12.3-13.1. Tissue distribution analysis shows that the transcript is found predominately in lymphoid tissues including spleen, tonsils, appendix, and peripheral blood leukocytes. Polymerase chain reaction analysis of isolated primary human cell populations confirms that mRNA exists in spleen B lymphocytes and monocytes but not T lymphocytes. Western blot analysis demonstrates specific expression of human RP105 in human B lymphocytes. Murine anti-human RP105 sera was generated using DNA immunization. The antisera contained antibodies that recognized and bound to human B lymphocytes from both spleen and peripheral blood as assessed by flow cytometry. Assessment of biological function showed that human peripheral blood leukocytes incubated with anti-RP105 sera were induced to proliferate as measured by tritiated thymidine incorporation. Moreover, anti-CD40 and interleukin-4-treated cells but not those exposed to anti-RP105 sera produced soluble CD23, suggesting distinct functional roles. This is the first demonstration of both the existence of RP105 protein on human B lymphocytes and its role in the regulation of B lymphocyte activation.

Effects of thyrotropin and cholera toxin on the thyroidal adenylate cyclase-adenosine 3',5'-monophosphate system.

The present experiments examined the relationship between cholera toxin and TSH stimulation of the adenylate cyclase system in bovine thyroid tissue. Preincubation of thyroid slices for 20 min at 4 C with a maximal concentration of cholera toxin (100 microgram/ml) did not impair the subsequent stimulation of cAMP by submaximal amounts of TSH (1 mU/ml) during a 5-min incubation at 37 C. Incubation of cholera toxin or TSH with mixed gangliosides, followed by the addition of thyroid slices resulted in inhibition of the cholera toxin but not the TSH stimulation of cAMP formation. Previous exposure of thyroid slices to TSH induced refractoriness to subsequent stimulation of cAMP formation by TSH, but the response to cholera toxin was unchanged. NAD is necessary for cholera toxin, but not TSH, stimulation of adenylate cyclase. In the absence of NAD, cholera toxin inhibited the effect of maximal concentrations of TSH and prostaglandin E1 on adenylate cyclase activity but had no effect on NaF stimulation. In the presence of NAD, the stimulation of adenylate cyclase activity of bovine thyroid plasma membranes by a maximal amount of TSH was not influeced by maximal amounts of cholera toxin. Cholera toxin had a biphasic action on the binding of [125I]iodo-TSH, with low concentrations enhancing and high concentrations inhibiting binding. TSH augmented the binding of [125I]iodo-cholera toxin over the range of 1-100 mU/tube. Cholera toxin at 10 microgram/ml maximally inhibited binding. In addition to the requirement for ribosylation of adenylate cyclase, the present results indicate that the mechanisms of action of TSH and cholera toxin on cAMP formation are different.

Effect of increased circulating thyroid-stimulating hormone on in vitro thyroid-stimulating hormone stimulation of thyroid and adipose tissues.

Hormones have been shown to regulate the number and/or binding properties of their own receptors. The present studies examined the effect of chronic increased endogenous TSH levels, induced by tapazole or thyroidectomy, on in vitro TSH responsiveness and binding in thyroid and adipose tissues. The results showed that TSH and prostaglandin E1 significantly increased cAMP levels in the thyroids of weight- and age-matched controls, whereas thyroids from tapazole-treated rats responded only to prostaglandin E1. Iodide organification was also measured, and the thyroids from tapazole-treated rats showed a significantly reduced effect of TSH compared to weight- and age-matched controls, although stimulation by dibutyryl cAMP was equivalent in all three groups. TSH or epinephrine stimulation of cAMP and glucose oxidation was equivalent in adipose tissue from control and hypothyroid rats. There was a significant 50% reduction in the number TSH-binding sites in thyroids from tapazole-treated rats: the affinity remained unchanged. [125I]TSH binding to adipose tissue plasma membranes was similar in control and hypothyroid groups. These studies demonstrate that elevated levels of TSH appear to regulate the number of TSH receptors in thyroid, but not adipose, tissue.

Affinity labelling of the human uterine progesterone receptor with 21-, 16 alpha- and 11 alpha-bromoacetoxyprogesterones.

This report describes the use of 21-, 16 alpha- and 11 alpha -[2'-3H]bromoacetoxyprogesterone as affinity labels to characterize the human uterine progesterone receptor (HPR). These three derivatives can bind to and displace progesterone bound to the HPR. This affinity labelling was inhibited by an excess of radioinert progesterone and could not be demonstrated if bovine serum albumin was used in place of the HPR. Bromoacetic acid alone did not affinity label the HPR. Polyacrylamide gel electrophoresis under denaturing conditions showed that all three derivatives bound to a 45,000 molecular weight protein.

Transferrin binds specifically to pachytene spermatocytes.

We have examined the secretion and binding of transferrin to rat testicular cells. The only testicular cells found to secrete transferrin were the Sertoli cells (control 549 +/- 6; FSH 1020 +/- 17 ng/day X 10(6) cells, mean +/- SEM). The Sertoli cells also contained specific binding sites for transferrin with a Kd of 2.0 X 10(-9)M. Of the other testicular cells examined only fractions rich in pachytene spermatocytes possessed specific transferrin-binding sites. Late pachytene spermatocytes (97% pure) bound [125I]iodotransferrin with a similar affinity as Sertoli cells (Kd 1.7 X 10(-9)M). Fractions of early and mid pachytene spermatocytes contained transferrin-binding sites with a higher affinity (Kd 0.3 X 10(-9)M). This is the first report of a protein that has specific binding sites on germ cells.

Regulation of transferrin secretion by human Sertoli cells cultured in the presence or absence of human peritubular cells.

Transferrin represents 1-4% of the total proteins secreted by human Sertoli cells, and the amount secreted by the cells was maximal after a 4- to 5-day period in culture, the time chosen for each medium change. During this first 4- to 5-day period, the addition of FSH, insulin, (Bu)2cAMP, and isobutylmethylxanthine had no effect on transferrin secretion; however, from days 4-5 to 8-10, each of the above compounds significantly stimulated transferrin secretion compared to control values. Testosterone (in the absence or presence of insulin) had no effect. Transferrin secretion increased for the first 5 days in culture, with a similar magnitude in the presence or absence of the above stimulators, and thereafter declined, more so in untreated cultures. These results suggest that these agents do not stimulate, but, rather, limit the decline in transferrin secretion. When human peritubular cells were cocultured with Sertoli cells, transferrin secretion was significantly elevated compared to that by Sertoli cells alone. Interestingly, in the cocultures (Bu)2cAMP stimulated transferrin secretion when added for the first 4- to 5-day culture period. Human fibroblasts or spent medium from the peritubular cell cultures did not mimic the effect found when peritubular and Sertoli cells were cocultured. These results provide evidence that peritubular cells play a critical role in regulating human Sertoli cell function.

Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase: a potential new risk factor for coronary artery disease.

A specific and robust immunoassay for the lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), platelet-activating factor acetylhydrolase, is described for the first time. The immunoassay was used to evaluate possible links between plasma Lp-PLA(2) levels and atherosclerosis risk amongst susceptible individuals. Such an investigation was important because Lp-PLA(2) participates in the oxidative modification of low density lipoprotein by cleaving oxidised phosphatidylcholines, generating lysophosphatidylcholine and oxidised free fatty acids. The majority of Lp-PLA(2) was found associated with LDL (approximately 80%) and, as expected, enzyme levels were significantly positively correlated to LDL cholesterol. Plasma Lp-PLA(2) levels were significantly elevated in patients with angiographically proven coronary artery disease (CAD) when compared with age-matched controls, even though LDL cholesterol levels did not differ significantly. Indeed, when included in a general linear model with LDL cholesterol and other risk factors, Lp-PLA(2) appeared to be an independent predictor of disease status. We propose, therefore, that plasma Lp-PLA(2) mass should be viewed as a potential novel risk factor for CAD that provides information related to but additional to traditional lipoprotein measurements.

Reactivity and assay restriction profiles of monoclonal and polyclonal antibodies to acid phosphatases: a preliminary study.

The development of secure diagnostic immunoassays requires, among others, rigorous characterisation of potential antibody reagents. The reactivity profiles of seven antibodies (six monoclonal [MAb] and one polyclonal [PAb]) with putative specificity for tartrate-resistant acid phosphatase (TRAP) and/or osteoclasts were evaluated in enzyme-linked immunosorbent assay (ELISA) and/or immunocytochemistry. MAbs 2H1, 4E6 and 5Cl demonstrated assay restriction: exhibiting reactivity only in ELISA. The remaining three MAbs (G211D, G312G and V35B) and the PAb 8023 recognised recombinant TRAP (rTRAP) in ELISA and native acid phosphatases in selected tissues and cell lines. The latter were cytochemically assessed for both tartrate-sensitive acid phosphatase (TSAP) and TRAP. V35B showed reactivity against the monocytic leukaemia cell line U937 and guinea pig kidney tissue (both TSAP+ and TRAP+) and ECV304 (TSAP+) cells. Interestingly, the reactivity of MAb G211D co-localised with TRAP activity in the membrane of osteoclasts but also detected cytoplasmic components in U937 cells and human embryonic lung fibroblasts (TRAP+ and TRAP+). G211D exhibited immunoreactivity against placental trophoblasts (positive for total AP). Intriguingly, MAbs 2H1, 4E6, 5Cl and PAb 8023 cross-reacted with potato acid phosphatase in ELISA, suggesting reactivity to conformationally similar epitopes. Thus, some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5Cl and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.

Evidence that both long-acting thyroid stimulator and long-acting thyroid stimulator-protector stimulate the human thyroid gland.

Thyroid-stimulating immunoglobulins were prepared from two potent sera, one contained long-acting thyroid stimulator (LATS) and the other contained both LATS and LATS-protector (LATS-P). The potencies of the immunoglobulin G (IgG) preparations were estimated in the McKenzie assay. The accumulation of cyclic AMP in mouse thyroid lobes was stimulated only by LATS--IgG; LATS-P--IgG was inactive. In contrast, both LATS-IgG and LATS-P--IgG were equally effective in slices of human thyroid.

Effects of human thyroid-stimulating hormone and immunoglobulins on adenylate cyclase activity and the accumulation of cyclic AMP in human thyroid membranes and slices.

The activation of adenylate cyclase and the accumulation of cyclic AMP resulting from the action of human thyroid-stimulating hormone (TSH), long-acting thyroid stimulator (LATS) or LATS-protector (LATS-P) have been investigated in preparations of human thyroid membranes and slices. Human TSH significantly increased adenylate cyclase activity in membranes from non-toxic goitres whereas LATS and LATS-P had no consistent effect. However, pre-incubation of goitrous membranes with LATS--immunoglobulin G inhibited the effect of TSH on adenylate cyclase. When thyroid membranes were prepared from the glands of patients with Graves's disease neither TSH nor thyroid-stimulating immunoglobulins (TSIg) stimulated adenylate cyclase significantly. Whether from non-toxic goitres or thyrotoxic tissue, the concentration of TSH needed to induce half of the maximum response was lower in thyroid slices than in membranes. Both LATS and LATS-P significantly stimulated the accumulation of cyclic AMP in slices of goitrous tissue but thyrotoxic tissue slices did not respond. In goitrous slices, submaximum concentrations of TSH and TSIg caused additive responses in the accumulation of cyclic AMP but TSIg did not increase the maximum response to TSH.

Effects of interferon on the cAMP-adenylate cyclase system of human, mouse, and bovine thyroid tissue.

This report studied the action of interferon on the thyroidal adenylate cyclase-cAMP system. It was found that human interferon did not increase cAMP levels in human or bovine thyroid slices during a 60-min incubation. Mouse interferon also had no effect on cAMP levels in mouse thyroidal lobes over the 60-min incubation, nor did it increase adenylate cyclase activity in mouse homogenates.

Transferrin and gonadal dysfunction in man.

Transferrin concentrations were quantitated in the seminal fluid of normal, oligozoospermic, and azoospermic patients and related to other known parameters of testicular function. Transferrin concentration in the semen of patients 2 months after vasectomy (13.2 +/- 1.8 micrograms/ml) was significantly less than that obtained from pregnancy-proven donors (65.6 +/- 10.1 micrograms/ml). This indicates that approximately 80% of the seminal fluid transferrin is derived from the testes. The concentration of transferrin in semen from patients with azoospermia (14.4 +/- 1.8 micrograms/ml), severe oligozoospermia (17.5 +/- 1.7 micrograms/ml), and moderate oligozoospermia (21.8 +/- 4.3 micrograms/ml) was significantly lower than normospermic groups. Serum follicle-stimulating hormone (FSH) was measured in a group of infertile patients; those having an elevated FSH had a significantly lower concentration of semen transferrin, 14.1 +/- 1.6 micrograms/ml, compared with patients who had FSH levels within the normal range (33.7 +/- 5.3 micrograms/ml). It is possible that the underlying cause in decreased spermatogenesis associated with both an elevated FSH and a decreased transferrin concentration is impaired Sertoli cell function.

Peritoneal fluid plasminogen activator activity in endometriosis and pelvic adhesive disease.

Plasminogen activator activity in PF was assayed by the fibrinolysis method described by Strickland and Beers. In 45 patients studied, there were no discernible differences according to whether patients had endometriosis and/or pelvic adhesive disease. No differences were detected according to when in the menstrual cycle the sample of PF was obtained. These data are in concordance with a previous report and taken together suggest that there is no difference in fibrinolytic mechanisms in PF in patients with or without endometriosis and/or pelvic adhesive disease, when compared with control subjects. If such differences exist, they may be present in the tissues, per se, but are not discernible in PF.

Results of the Cox-Maze III/IV procedure in patients over 75 years old who present for cardiac surgery with a history of atrial fibrillation.

AIM: Elderly patients with atrial fibrillation (AF) present a special challenge. Despite the documented advantage in ablating AF, the addition of the procedure may add complexity and potentially impact patient outcome. This study explored the impact of the Cox-Maze III/IV procedure on elderly patients experiencing AF who present for cardiac surgery. METHODS: Forty-four patients aged ≥ 75 with concomitant surgery underwent the Cox-Maze III/IV procedure for AF. These patients were followed using our extensive longitudinally designed registry to include health related quality of life (HRQL). Late death was captured by the Social Security Index and the National Death Index. RESULTS: The mean age for this sample was 79.5 ± 3 years and mean additive euroSCORE was 9 ± 2.1 (high risk). The majority of patients with the Cox-Maze procedure underwent concomitant valve surgery (N. = 41, 93%). There was a low incidence of STS measured perioperative outcomes in this group. NSR rates at six months were 90% (26/29) and 85% (23/27) at 12 months for the ablation group. There were no embolic strokes and major bleeding events occurred in only two patients. By Kaplan-Meier analysis, two-year cumulative survival was 89.6% and there was only one operative mortality in this group (2.3%). CONCLUSION: Addition of the Cox-Maze III/IV procedure in patients ≥ 75 years may add to the complexity of the surgical procedure, but does not increase the operative risk. Age should not be the only discriminating factor when considering the Cox-Maze III/IV procedure for patients aged ≥ 75 years who present for cardiac surgery while experiencing atrial fibrillation.

The definition of chronic lung disease in patients undergoing cardiac surgery: a comparison between the Society of Thoracic Surgeons and the American Thoracic Society/European Respiratory Society Classifications.

AIM: Early and late outcomes following cardiac surgery may be adversely affected in patients with chronic lung disease (CLD) and the presence of CLD is definition dependent. The purpose of this study was to compare the Society of Thoracic Surgeons (STS) definitions for CLD to the modified American Thoracic Society (ATS)/European Respiratory Society (ERS) definitions in diagnosing and classifying CLD among a cohort of cardiac surgery patients. METHODS: A prospectively-designed study whereby high risk patients for CLD presenting for non-emergent cardiac surgery and had a history of asthma, a 10 or more pack year history of smoking or a persistent cough were included. All patients underwent spirometry testing within two weeks of surgery. The presence and severity of CLD was coded two times: 1) STS definitions with spirometry; 2) ATS/ERS guidelines. The rate of misclassification was determined using concordance and discordance rates. Sensitivity analysis of the STS spirometry definitions was calculated against the ATS/ERS definitions and respective classifications. RESULTS: The discordant rate for the STS spirometry driven definitions versus the ATS/ERS definitions was 21%. Forty patients (21%) classified as no CLD by the STS spirometry definition were found to have CLD by the ATS/ERS definition. The STS classification had 68% sensitivity (84/124) when identifying any CLD and only 26% sensitivity (14/54) when identifying moderate CLD. CONCLUSION: The current STS spirometry driven definitions for CLD did not perform as well as the ATS/ERS definitions in diagnosing and classifying the degree of CLD. Consideration should be given to using the ATS/ERS definitions.

Identification of histidine and methionine residues in the active site of the human uterine progesterone receptor with the affinity labels 11 alpha- and 16 alpha-(bromoacetoxy)progesterone.

The affinity labels 11 alpha- and 16 alpha-(bromo[2'-3H]acetoxy)progesterone (BAP) react covalently with amino acids present in the progesterone binding site of the human uterine progesterone receptor. Hydrolysis of the affinity labeled receptor followed by separation and analysis of the amino acid products demonstrated the sites of affinity labeling. The 11 alpha-BAP alkylates the 1-position of a histidine residue. The 16 alpha-BAP alkylates the 3-position of histidine, and a methionine residue. Affinity labeling did not occur in the presence of excess progesterone, and under the optimum conditions for affinity labeling of the receptor, heat-denatured receptor, bovine serum albumin, and 20 beta-hydroxysteroid dehydrogenase were not affinity labeled. This is the first report of the identification of specific amino acid residues in the binding site of a steroid hormone receptor.

Fasciola hepatica: the effects of neuropharmacological agents upon in vitro motility.

The effects of a wide range of neuropharmacological agents on the motility in vitro of Fasciola hepatica have been determined using an isometric transducer system. The neuromuscular blocking agents tubocurarine and decamethonium cause a long-term stimulation of the basal activity of the fluke. Acetylcholine causes an inhibition of activity. This effect is mimicked by the cholinergic agonists carbachol and nicotine, antagonised by the cholinergic blocking agents atropine and mecamylamine, and potentiated by eserine, a cholinesterase inhibitor. With nicotine and atropine the effects are accompanied by an increase in muscle tone at a concentration of 1 X 10(-2) M. Noradrenaline and adrenaline also cause some inhibition of activity, an effect antagonised by guanethidine, which blocks the release of noradrenaline. In contrast, dopamine stimulates fluke motility, whilst its antagonist dihydroergotamine causes an inhibition of activity. The monoamine oxidase inhibitors iproniazid and p-chloromercuribenzoic acid induce a stimulation of activity; with the latter there is an increase in muscle tone at a concentration of 1 X 10(-3) M. The amine depleting agents chloroamphetamine and reserpine, and the monoamine uptake inhibitors desipramine and nortriptyline produce an inhibition of fluke activity, as does the serotonin uptake inhibitor fluoxetine. High concentrations of chloroamphetamine (1 X 10(-2) M) and the uptake inhibitors (1 X 10(-3) M and above) also induce an increase in muscle tone. Serotonin causes a marked stimulation of motility. The pharmacological evidence is consistent with a neurotransmitter role of acetylcholine (inhibitory), dopamine (excitatory), and noradrenaline (inhibitory). The status of serotonin is discussed.

Hymenolepis diminuta: the mechanism of egg hatching.

The fine structure and histochemistry of the envelopes surrounding the mature oncosphere of Hymenolepis diminuta have been investigated by transmission electron microscopy and light microscope observations of JB-4 embedded material. The fate of these envelopes during the hatching sequence in vitro has been followed by phase-contrast and scanning electron microscopy. Hatching is considered to comprise 4 stages. Stage 1 involves the mechanical breakage and removal of the shell and the outer cytoplasmic layer of the inner envelope; Stage 2 the activation of the oncosphere and the swelling of the gelatinous layer of the inner envelope; and Stage 3 the digestion and rupture of the embryophore. This is accomplished both by secretions from the penetration gland and by the action of external digestive enzymes, together with hook activity. Trypsin is more effective than amylase in digesting the embryophore. Stage 4 involves the enzymatic weakening of the gelatinous layer which helps the oncosphere to tear itself free with its hooks. Amylase is more effective than trypsin in attacking the gelatinous layer. On emergence from the gelatinous layer, the oncosphere is still enveloped by the 'oncospheral membrane', although this covering is soon lost. Once activated, the oncosphere is capable of completing the hatching sequence by itself, without the addition of enzymes. This process is a lengthy one, however, taking up to 2 h, and the expenditure of its glandular and energy reserves makes successful gut penetration by the oncosphere unlikely.