Surface antigens of human trophoblasts

Single cells and cell aggregates of human trophoblasts were studied by immunofluorescence and mixed haemadsorption for the presence of different surface antigens. Results indicate that fresh trophoblast cells do not express HL-A in detectable amounts but carry human chorionic gonadotropin (HCG) and Con-A receptors. Fresh cells also reacted with antisera to B₂-microglobulin but after outgrowth to a monolayer, this molecule was detected at a low frequency from different individuals; the reactions were weak. In contrast, the outgrowth cells seemed to carry HCG. It was not possible to demonstrate any significant adsorption of maternal immunoglobulin to fresh trophoblast cells. Surface antigens of trophoblast cells in suspension were slightly redistributed by antibodies whereas the cells in aggregate showed no signs of redistribution.     

Treatment of distal colitis with local anaesthetic agents

The results of clinical and experimental studies on topical treatment of distal colitis with local anaesthetic agents are summarized. The original observation was an adrenergic hyperinnervation of the inflamed mucosa (hyperinnervation hypothesis). In order to silence local nervous reflexes, the mucosa was treated topically with 2% lidocaine gel. The clinical results are promising and no side effects have been observed. The relapse rate is relatively high and related to the duration of treatment. In studies of experimental colitis a potential antagonism between harmful adrenergic nerves (vasoconstrictor substances and proinflammatory cytokines) and mucosa-protective visceral afferents (antiinflammatory cytokines) in the pathogenesis of colitis is intriguing. Other studies have emphasized the importance of neutrophils for causing damage to the colon epithelium (neutrophil hypothesis) and local anaesthetics have potent effects on several steps of the inflammatory response in addition to the nervous blockade.     

Short term complications of the tension free vaginal tape operation for stress urinary incontinence in women

Objective To assess the prevalence of intra- and post-operative complications with the tension free vaginal tape operation for female urinary incontinence.
Design Non-control case series.
Setting University Hospital.
Population One hundred and seventy-seven operations on women who were diagnosed urodynamically to have genuine stress incontinence. In 26 cases (15%) there was symptomatic urge incontinence.
Main outcome measures Intra- and post-operative complications in relation to individual surgeons and mode of anaesthesia (local or spinal), and continence at short term follow up.
Results Bladder or urethral perforation occurred in 26 cases (15%) and three operations were abandoned for these reasons. A failure to void after the first 24 hours was registered in 35 (20%); 21 (12%) had to undergo urethral dilatation while five patients (2.8%) had persistent urinary retention which required excision of the sling. These problems were significantly associated to the experience of surgeon. In seven cases (4%) haemorrhage required intravaginal tamponade. In three (1.7%) sling rejection occurred post-operatively. When followed up six to eight weeks post-operatively, 154 patients (88%) reported subjective cure, 21 (11%) significant improvement and two (1%) no improvement. The use of spinal anaesthesia increased the frequency of peroperative bladder perforation but affected neither the incidence of post-operative bladder obstruction nor the outcome at follow up.
Conclusions This study identifies short term complications which relate partly to the experience of the individual surgeon. Nevertheless the 88% subjective cure rate was independent of these factors.     

Sound and vibration: effects on infants' heart rate and heart rate variability during neonatal transport

Aim: To measure the effect of sound and whole‐body vibration on infants’ heart rate and heart rate variability during ground and air ambulance transport. Methods: Sixteen infants were transported by air ambulance with ground ambulance transport to and from the airports. Whole‐body vibration and sound levels were recorded and heart parameters were obtained by ECG signal. Results: Sound and whole‐body vibration levels exceeded the recommended limits. Mean whole‐body vibration and sound levels were 0.19 m/s² and 73 dBA, respectively. Higher whole‐body vibration was associated with a lower heart rate (p < 0.05), and higher sound level was linked to a higher heart rate (p = 0.05). The heart rate variability was significantly higher at the end of the transport than at the beginning (p < 0.01). Poorer physiological status was associated with lower heart rate variability (p < 0.001) and a lower heart rate (p < 0.01). Infants wearing earmuffs had a lower heart rate (p < 0.05). Conclusions: Sound and whole‐body vibration during neonatal transport exceed recommended levels for adults, and sound seem to have a more stressful effect on the infant than vibrations. Infants should wear earmuffs during neonatal transport because of the stress‐reducing effect.     

Glucose- and hormone-induced cAMP oscillations in α- and β-cells within intact pancreatic islets

OBJECTIVE

cAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown.

RESEARCH DESIGN AND METHODS

The subplasma-membrane cAMP concentration ([cAMP]_pm) was recorded in α- and β-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and β-cells.

RESULTS

In islets exposed to 3 mmol/L glucose, [cAMP]_pm was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP]_pm, often with oscillations synchronized among β-cells. Whereas glucagon also induced [cAMP]_pm oscillations in most α-cells, <20% of the α-cells responded to GLP-1. Elevation of the glucose concentration to 11–30 mmol/L in the absence of hormones induced slow [cAMP]_pm oscillations in both α- and β-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in the β-cells but not caused by the changes in [Ca²⁺]_i. The transmembrane adenylyl cyclase (AC) inhibitor 2′5′-dideoxyadenosine suppressed the glucose- and hormone-induced [cAMP]_pm elevations, whereas the preferential inhibitors of soluble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-β-triol perturbed cell metabolism and lacked effect, respectively.

CONCLUSIONS

Oscillatory [cAMP]_pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]_pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.Cyclic AMP and Ca²⁺ are key messengers in the regulation of insulin and glucagon secretion from pancreatic β- and α-cells, respectively, by nutrients, hormones, and neural factors. Glucose stimulation of insulin secretion involves uptake and metabolism of the sugar in the β-cells, closure of ATP-sensitive K⁺ channels, and depolarization-induced Ca²⁺ entry generating oscillations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that trigger periodic exocytosis of secretory granules (1,2). This process is amplified by mechanism(s) acting distal to the elevation of Ca²⁺ (3). cAMP promotes exocytosis by facilitating the generation of Ca²⁺ signals (4,5), by sensitizing the secretory machinery to Ca²⁺ (4,6), and by stimulating mobilization and priming of granules via protein kinase A– and Epac-dependent pathways (7,8).Measurements of the cAMP concentration beneath the plasma membrane ([cAMP]_pm) in individual INS-1 β-cells showed that glucagon-like peptide-1(7-36)-amide (GLP-1) induces [cAMP]_pm elevation, often manifested as oscillations (9). Glucose has been regarded to only modestly raise islet cAMP, supposedly by amplifying the effect of glucagon (10), but single-cell cAMP recordings have recently shown that glucose alone induces marked elevation of cAMP in MIN6 β-cells (11,12) and primary mouse β-cells (12,13). Although one study reported that the glucose-induced cAMP response depends on elevation of [Ca²⁺]_i (11), other studies show only partial or no Ca²⁺-dependence of the cAMP signal (12,13). Like hormone stimulation, glucose induces oscillations of [cAMP]_pm, and coordination of the [cAMP]_pm and [Ca²⁺]_i elevations generates pulsatile insulin release (12,14).There are 10 isoforms of cAMP-generating adenylyl cyclases (ACs) with different regulatory properties, nine of which are transmembrane (tm) proteins stimulated by Gα_s and the plant diterpene forskolin. Such tmACs mediate the cAMP-elevating action of glucagon and incretin hormones (15). β-cells express multiple tmACs (16), and the Ca²⁺-stimulated AC8 has been proposed to be particularly important for integrating hormone- and depolarization-evoked signals (17). Soluble AC (sAC) is the only isoform that lacks transmembrane domains. It is insensitive to forskolin and G-proteins but stimulated by bicarbonate (18) and Ca²⁺ (19). Although sAC was first found in the testis, it also seems to be expressed in other tissues and was recently proposed to be involved in glucose-induced cAMP production in insulin-secreting cells (20).Like insulin secretion, exocytosis of glucagon from the α-cells is triggered by an increase of [Ca²⁺]_i (21). Glucagon release is stimulated by absence of glucose and is maximally inhibited when the sugar concentration approaches the threshold for stimulation of insulin secretion (22). Under hypoglycemic conditions, glucagon secretion is also stimulated by adrenaline, which raises [Ca²⁺]_i and [cAMP]_pm via α₁- and β-adrenergic mechanisms (23,24). There are fundamentally different ideas about the mechanisms underlying glucose inhibition of glucagon secretion, including paracrine influences from β- and δ-cells (25–29) and direct actions of glucose on the α-cells, resulting in depolarization- (30) or hyperpolarization-mediated (22) inhibition of exocytosis. Apart from the inhibitory effect of glucose, we observed that very high glucose concentrations unexpectedly stimulate glucagon secretion (31). The stimulatory component may be important under physiological conditions because the hormone is released in pulses from rat (32) and human (33) islets. Glucose thus causes alternating periods of stimulation and inhibition resulting in time-average reduction of glucagon secretion. Ca²⁺ is probably not the only messenger in glucose-regulated glucagon release (29). Like for insulin secretion, cAMP is believed to promote glucagon release by enhancing intracellular Ca²⁺ mobilization, Ca²⁺ influx through the plasma membrane, and mobilization of secretory granules (23,24,34,35). However, it has also been suggested that cAMP-mediated reduction of N-type Ca²⁺ currents can explain the inhibitory effect of GLP-1 on glucagon secretion (36).Until now, nothing was known about cAMP kinetics in α-cells and all information on primary β-cells was based on studies of dispersed islet cells. However, as a result of gap junctional coupling and paracrine influences, the electrophysiological characteristics and [Ca²⁺]_i signaling in intact islets differ considerably from those in dispersed β-cells (2). Therefore, the aim of the current study was to clarify how glucose, glucagon, and GLP-1 affect cAMP signaling in α- and β-cells within intact islets of Langerhans.     

Prolactin regulation of the expression of TNF-alpha, IFN-gamma and IL-10 by splenocytes in murine multiple low dose streptozotocin diabetes

Previously, the hormone prolactin (PRL) has been found to protect against development of type 1 diabetes induced by multiple injections of streptozotocin (STZ) in mice. To further investigate this effect of PRL, C57BL/Ks mice were injected intraperitoneally with STZ (40 mg/kg body weight) or NaCl for 5 days and PRL (4 mg/kg body weight) or NaCl for 14 days. On day 15, splenocytes were isolated from the in vivo treated mice. Spleen cell preparations depleted in erythrocytes and macrophages were stained for cytoplasmic TNF-alpha, IFN-gamma and IL-10 and analyzed with flow cytometry. Isolated spleen cells were also cultured (RPMI 1640+10% fetal bovine serum) for 24 h. Thereafter, cytokine mRNA expression by the spleen cells was measured by real-time PCR and cytokine secretion determined by enzyme linked immunosorbent assay (ELISA). Freshly isolated spleen cell preparations from PRL and STZ+PRL treated animals seemed to have an increased frequency of IL-10 positive cells compared to controls. In cultured spleen cells isolated from STZ treated mice, IFN-gamma and IL-10 mRNA expression was up-regulated. PRL treatment down-regulated the mRNA expression of these cytokines and also TNF-alpha in the splenocytes obtained from animals treated with STZ. The accumulation of these cytokines in the cultures of the explanted splenocytes showed only minor differences between the experimental groups. Overall, the data seems to favor the view that PRL enhanced a Th2 response, which may reflect the preventive effect of PRL against development of multiple low dose STZ diabetes in mice.     

Assessing testicular germ cell DNA damage in the comet assay; introduction of a proof-of-concept

The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell-specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource-efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.