Association between glycaemic control and common infections in people with Type 2 diabetes: a cohort study.

AIM: To investigate the impact of glycaemic control on infection incidence in people with Type 2 diabetes. METHODS: We compared infection rates during 2014 in people with Type 2 diabetes and people without diabetes in a large primary care cohort in the UK (the Royal College of General Practitioners Research and Surveillance Centre database). We performed multilevel logistic regression to investigate the impact of Type 2 diabetes on presentation with infection, and the effect of glycaemic control on presentation with upper respiratory tract infections, bronchitis, influenza-like illness, pneumonia, intestinal infectious diseases, herpes simplex, skin and soft tissue infections, urinary tract infections, and genital and perineal infections. People with Type 2 diabetes were stratified by good [HbA1c < 53 mmol/mol (< 7%)], moderate [HbA1c 53-69 mmol/mol (7-8.5%)] and poor [HbA1c > 69 mmol/mol (> 8.5%)] glycaemic control using their most recent HbA1c concentration. Infection incidence was adjusted for important sociodemographic factors and patient comorbidities. RESULTS: We identified 34 278 people with Type 2 diabetes and 613 052 people without diabetes for comparison. The incidence of infections was higher in people with Type 2 diabetes for all infections except herpes simplex. Worsening glycaemic control was associated with increased incidence of bronchitis, pneumonia, skin and soft tissue infections, urinary tract infections, and genital and perineal infections, but not with upper respiratory tract infections, influenza-like illness, intestinal infectious diseases or herpes simplex. CONCLUSIONS: Almost all infections analysed were more common in people with Type 2 diabetes. Infections that are most commonly of bacterial, fungal or yeast origin were more frequent in people with worse glycaemic control.

Longitudinal muscle shows abnormal relaxation responses to nitric oxide and contains altered levels of NOS1 and elastin in uncomplicated diverticular disease.

OBJECTIVE: Recent evidence challenges the 'low-fibre/high-colonic intraluminal pressure' hypothesis of diverticular disease (DD) and raises the possibility that other mechanisms are involved. Although bowel wall smooth muscle is known to be hypercontractile in DD, the nature of its relaxation is unknown. The present study investigated colonic smooth muscle responses to nitric oxide, as well as the smooth muscle contents of neural nitric oxide and elastin associated with the disease. METHOD: Immunohistochemical/image analysis of antibodies to nitric oxide synthase (NOS1), co-localized with protein gene product (PGP) and to elastin, was performed on three histological sections of sigmoid colons from 20 patients (10 DD, 10 controls) following resections for rectal tumours. Organ bath experiments examined smooth muscle responsiveness to nitroprusside, a nitric oxide donor. RESULTS: Uncomplicated diverticular longitudinal muscle showed lower nitric oxide immunoreactivity compared with controls: median percentage surface area of NOS1 over PGP was 26.0% (range 0.5-58.3), controls 45.0% (35.0-70.1; P = 0.018). Median percentage surface area of elastin was elevated, 21.3% (10.6-45.6), controls 8.2% (1.7-13.5; P = 0.0002), together with a low sensitivity to nitroprusside [mean - log EC(50) 5.3 (SD 0.5), controls 6.6 (SD 0.5), difference 1.3, 95% CI 0.8-1.7; P < 0.0001] and there were lower maximum relaxation responses to nitroprusside compared with controls: median percentage (relaxation induced by nitroprussside/contraction induced by bethanecol) was 52.0%, range (20.0-92.0), controls 100.0% (71.0-125.0), P < 0.0001. No statistically significant differences were found in circular muscle, at the sample size studied. CONCLUSION: This study established, for the first time, specific abnormalities in longitudinal muscle relaxation and contents of neural nitric oxide and elastin in uncomplicated DD. These findings may have important implications for both colon structure and function in the disease.

Microarray analysis of BeWo and JEG3 trophoblast cell lines: identification of differentially expressed transcripts.

Trophoblast cell lines are important research tools used as a surrogate for primary trophoblast cells in the study of placental function. Because the cellular origins of transformed trophoblasts are likely to be diverse, it would be of value to understand the unique and shared phenotypes of the cells on a global scale. We have compared two widely used cell lines, BeWo and JEG3, by microarray analysis in order to identify differentially expressed genes. Results indicated that approximately 2700 genes were differentially expressed between the cell lines, with principal differences observed in the biological processes of response to stress, cell adhesion, signal transduction, and protein and nucleobase metabolisms. These data suggest that BeWo and JEG3 cell lines, and perhaps other trophoblast cell lines, are sufficiently dissimilar from each other such that they will be differentially suited for specific experimental paradigms.

Suppression of extravillous trophoblast invasion of uterine spiral arteries by estrogen during early baboon pregnancy.

The present study determined whether estrogen plays a role in regulating invasion and remodeling of the uterine spiral arteries by extravillous trophoblasts during early baboon pregnancy. The level of trophoblast invasion of spiral arteries was assessed on day 60 of gestation (term is 184 days) in baboons untreated or treated on days 25-59 with estradiol or aromatizable androstenedione. The administration of estradiol or androstenedione increased (P<0.01) maternal serum estradiol levels approximately 3-fold above normal. The mean+/-SE percentage of spiral arteries/arterioles invaded by extravillous cytotrophoblasts in estradiol-treated baboons for vessels with diameters of 26-50 microm (0.0+/-0.0), 51-100 microm (1.2+/-0.7) and >100 microm (13.2+/-5.5) was 100%, 90%, and 75% lower (P<0.001), respectively, than in untreated baboons (2.4+/-1.2%; 11.0+/-5.5%, and 54.5+/-8.5%, respectively). Similar results were obtained with androstenedione treatment. However, the distribution of uterine spiral arteries grouped by diameter or number of arteries per basal plate area, i.e. microvessel density, were similar in untreated and estrogen-treated baboons. We suggest, therefore, that the low levels of estrogen exhibited during early primate pregnancy are required to permit normal progression of trophoblast vascular invasion and that the surge in estrogen which occurs during the second-third of normal pregnancy has a physiological role in suppressing further arterial trophoblast invasion. Consequently, we propose that the estrogen-dependent restraint of spiral artery invasion/remodeling ensures optimal blood flow dynamics across the uteroplacental vascular bed to promote normal fetal growth and development.

Influence of maternal diabetes on placental fibroblast growth factor-2 expression, proliferation, and apoptosis.

OBJECTIVE: Type I diabetes mellitus during pregnancy is associated with dysregulation of the oxygen and glucose metabolic pathways, both of which affect placental villous growth and function. Alteration of placental development in women with diabetes may contribute to the increased risk of preeclampsia, macrosomia, or fetal growth restriction. METHODS: To evaluate placental growth in the setting of maternal diabetes, immunohistochemical techniques were used to examine fibroblast growth factor-2 (FGF-2) expression, cell proliferation (Ki67), and apoptosis (Apo-Tag) in placentas from diabetic and nondiabetic patients. RESULTS: Immunostaining for FGF-2 in placentas from diabetic women demonstrated an increase in intensity within the villous stroma and syncytiotrophoblast (P<.05). Associated with these changes in FGF-2 expression, placentas from diabetic women showed no change in villous mitotic activity but did show decreased stromal compartment apoptosis. When expressed as a ratio of Ki67-positive:Apo-Tag-positive nuclei as an index of relative cell turnover, the stromal compartment showed a significant trend towards decreased nuclei turnover (P<.05), suggesting relative tissue growth in diabetic patients. CONCLUSION: Increased FGF-2 expression and decreased stromal cell compartment turnover in the diabetic placenta might be a compensatory mechanism in response to the altered physiologic milieu of maternal diabetes on placental function.

Intracellular potentiation between two second messenger systems may contribute to cholera toxin induced intestinal secretion in humans.

BACKGROUND: Cholera toxin (CT) acts on intestinal epithelial cells both directly and indirectly via activation of a secretory neural reflex. The reflex may release acetylcholine as one of its final neurotransmitters. This opens up the possibility of a third mechanism of action for CT, namely a synergistic interaction between two secretagogues acting on different second messenger systems within the epithelial cell. AIMS: To establish evidence for cholinergic innervation to human ileal epithelial cells and to investigate whether CT potentiates the action of acetylcholine on human intestinal epithelial cells. METHODS: Transverse sections of human ileum were examined for mucosal cholinergic nerves and M3 muscarinic receptors using antibodies raised to choline acetyltransferase and M3 receptors. Short circuit current (Isc) responses and ion flux movements were elicited from T84 epithelial cell monolayers set up in Ussing chambers. RESULTS: Immunohistochemistry of native human ileal mucosa revealed the presence of both cholinergic nerves and muscarinic M3 receptors located to the basolateral domain of epithelial cells. Secretory responses of T84 cell monolayers to acetylcholine were greatly potentiated in the presence of CT. This effect, substituting forskolin for CT, was mirrored by increases in basolateral 86Rb and apical 125I efflux. Charybdotoxin plus apamin reduced both Isc and 86Rb efflux evoked by acetylcholine, in the presence of forskolin. CONCLUSIONS: Human ileal mucosa receives a direct cholinergic innervation to its epithelial cells. Secretory effects of acetylcholine on epithelial cells are augmented in the presence of CT. Such a synergistic response is dependent on optimum opening of basolateral potassium channels by acetylcholine and apical chloride channels by CT. The interaction may contribute to the mechanism of action of cholera toxin induced secretory diarrhoea.

Effect of angiotensin II on ion transport across human Fallopian tube epithelial cells in vitro.

Angiotensin II type 1 receptors have been identified in Fallopian tube epithelia. Polarized confluent human Fallopian tube epithelial cell cultures were used under short-circuit conditions to study the actions of angiotensin II on electrogenic ion transport. The results demonstrate that angiotensin II increases baseline short-circuit current, implying a net transport of negatively charged ions from a basal to apical direction. This effect was inhibited by the selective angiotensin II type 1 receptor antagonist losartan. The effects of angiotensin II on short-circuit current were rapid in onset, brief in duration, and although less than those achieved with ATP, similar in amplitude to those described for other epithelia with angiotensin II. These findings reflect a significant retention of function for these cells in monolayer culture. Immunohistochemistry using the antibody 6313/G2, which is directed against a specific sequence in the extracellular domain of the angiotensin II type 1 receptor, confirmed that the receptor was retained in cultured cells. The results indicate that angiotensin II plays a role in regulating the composition of Fallopian tube secretions.

The effect of cAMP on ion transport in Fallopian tube epithelial cells in vitro.

The coupled movement of ions and water across epithelia determines the composition and volume of fluid present in the lumen of organs. The second messenger cAMP is important in effecting electrolyte and water transport in many transporting epithelia; however, its role in Fallopian tube transport is uncertain. We have conducted electrophysiological studies on Fallopian tube epithelial cell monolayers in Ussing chambers and have demonstrated that exogenously added cAMP and agents that generate its intracellular production results in an increase in short-circuit current consistent with the transport of net electrical charge from a basal to mucosal direction. In contrast to the known effects of ATP in this tissue, the increase in short-circuit current was not explicable in terms of electrogenic chloride secretion as it was not affected by the chloride channel inhibitors, 4-acetamido-4'-isothiocyanostilbene-2,2-disulphonic acid 1 mmol/l (SITS) and frusemide. Instead the current was reduced by the sodium channel inhibitor, amiloride, and was therefore, in part, explicable in terms of electrogenic Na+ absorption. These findings will enhance our understanding of the physiological mechanisms responsible for human Fallopian tubal fluid formation and composition.

Developmental regulation of morphological differentiation of placental villous trophoblast in the baboon.

The present study determined whether morphological differentiation of placental villous cytotrophoblasts into syncytiotrophoblast during primate pregnancy was developmentally regulated and whether oestrogen has a role in this process. Placental volumetric composition of the cytotrophoblast and syncytiotrophoblast was determined by the test-point counting method on days 45-54, 60, 100, and 170 of gestation (term=184 days) in untreated baboons, on day 60 after placental oestrogen production was prematurely elevated by administration of aromatizable androstenedione or oestradiol, and on day 170 after oestrogen production was suppressed by administration of aromatase inhibitor CGS 20267. Cytotrophoblast and syncytiotrophoblast volumes and oestrogen levels increased (P< 0.01) with advancing gestation. Due to the rise in syncytiotrophoblast volume (12-fold) exceeded that of the cytotrophoblast (threefold), the mean (sem) ratio of syncytiotrophoblast and cytotrophoblast volumes increased (P< 0.001) from 3.4 (0.5) ml on day 60 to 12.1 (2.8) ml on day 170. Androstenedione administration elevated serum oestradiol levels threefold (P< 0.01) and increased the ratio of syncytiotrophoblast: cytotrophoblast volumes on day 60 by 50 per cent (P< 0.03) to that normally observed on day 100. However, the ratio of trophoblast volumes was unaltered in oestradiol-treated and CGS 20267-treated baboons. It is concluded that there is a developmental increase in morphological differentiation of the placental villous trophoblast during primate pregnancy and that androstenedione potentially via its conversion to oestrogen has a role in this process.

Imidazolines inhibit secretory responses of rat colonic mucosa to calcium-dependent but not cyclic AMP-dependent secretagogues.

The purpose of this study was to investigate whether imidazolines have an anti-secretory action on intestinal epithelial cells. Muscle-stripped preparations of rat colon and monolayers of T84 human colonic epithelial cells were set up in Ussing chambers for measurement of short-circuit current. In rat colon acetylcholine, histamine, vasoactive intestinal polypeptide and forskolin elicited secretory responses which were recorded as increases in short-circuit current. Secretory responses to acetylcholine were inhibited in a concentration-dependent manner by the imidazolines phentolamine, idazoxan and clonidine. The effect of clonidine was not reversed by pre-incubation of mucosal preparations with yohimbine. Secretory responses to vasoactive intestinal polypeptide were unaffected by the three imidazolines. Phentolamine reduced responses of colonic mucosa to histamine but had no effect on responses to forskolin. Responses to vasoactive intestinal polypeptide and forskolin were significantly reduced in the presence of barium. In T84 cell monolayers phentolamine significantly reduced responses to acetylcholine. Three imidazolines, two with alpha-adrenoceptor-antagonist properties and one with alpha-agonist properties, have anti-secretory effects in rat colonic mucosal preparations. The anti-secretory action appears to discriminate between calcium-dependent and cyclic AMP-dependent secretagogues, inhibiting the former but not the latter.

T(84) epithelial cells respond to 5-hydroxytryptamine when grown in serum-free media.

The aim of this study was to establish the cause of insensitivity of T(84) human colonic epithelial cells to 5-hydroxytryptamine (5-HT). Monolayers of T(84) cells were placed in modified Ussing chambers for measurement of short-circuit current, an index of secretion. When grown in serum-supplemented media, T(84) cells gave secretory responses to acetylcholine and forskolin but not to 5-HT. When grown in AIM V serum-free media, T(84) cells responded to 5-HT. Chromatographic analysis with fluorimetric detection showed high levels of 5-HT (1.8 microM) in the serum. This contamination is probably responsible for subsequent desensitization of T(84) cells to 5-HT.

Screening for antiproliferative actions of mifepristone. Differential endometrial responses of primates versus rats.

This laboratory has previously shown the capability of the antiprogestin, mifepristone, to noncompetitively inhibit estrogen-induced endometrial proliferation in nonhuman primates. In the following study, use of the rat uterine weight bioassay was compared against a primate (Macaca fascicularis) uterine bioassay to identify the noncompetitive/antiproliferative effects of mifepristone. These uterine bioassays were contrasted for reasons of identifying a comparative laboratory rodent model that could substitute for the need to use primate models in the screening of potential antiprogestins, thereby saving time, cost, and primate resources. Results of the primate experiment showed that mifepristone decreased endometrial proliferation in a dose-dependent manner; importantly, this decrease occurred in the presence of sustained physiologic serum 17 beta-estradiol (E2) levels. However, in the rat model, results showed that mifepristone altered uterine wet weight and blotted weight values only in those animals receiving pharmacological doses of E2 (p < 0.05). Based on the results summarized herein, use of this rat uterine weight bioassay as a substitute for primate models is not recommended for screening and identification of "interesting" antiprogestins. Apparently, the endometrial noncompetitive antiestrogenic/antiproliferative effects of mifepristone, observed repeatedly in these laboratory primates, do not operate in the rat uterine tissue.

Inhibition of potassium (KATP) channels reduces the short-circuit current response of rat colonic mucosa to acetylcholine.

Intestinal secretion depends upon electrogenic chloride transport into the gut lumen, which requires maintenance of an electrically negative cell-membrane voltage. We have investigated whether secretory responses of rat colonic mucosa to acetylcholine were sensitive to inhibition of potassium channels and whether selective inhibition could indicate the nature of the channel involved. Rat colonic mucosa was set up in Ussing chambers, short-circuit current responses obtained to acetylcholine, and the sensitivity of such responses to inhibition of potassium channels was investigated. Non-selective potassium-channel blockade by barium induced concentration-dependent inhibition of responses to acetylcholine. Similar inhibitory effects were obtained using 4-aminopyridine and glibenclamide. 5-Hydroxydecanoate and phentolamine also inhibited the increase in short-circuit current. However, a combination of charybdotoxin plus apamin was without effect. We conclude that a basolateral outward movement of potassium ions is required for the secretory action of acetylcholine on rat colonic mucosa. The potassium channel involved seems to be ATP-dependent and calcium-insensitive.

Evidence for a nonneural electrogenic effect of cholera toxin on human isolated ileal mucosa.

Cholera toxin-induced intestinal secretion in intact rats requires a functioning myenteric plexus. The aim of this investigation was to determine whether neural elements were essential for cholera toxin to produce a secretory effect in human isolated ileum. Mucosal preparations were mounted in Ussing chambers. Cholera toxin was applied apically and short-circuit current monitored for 3 hr, at which point forskolin was given. Cholera toxin (10 microg/ml) induced a tetrodotoxin-insensitive increase in short-circuit current in muscle-stripped preparations of human ileum. The increase was not additive with the action of forskolin (25 microM). Cholera toxin exerts a marked nonneural secretory effect in human ileal mucosa in vitro, probably by the same mechanism as forskolin, namely elevation of cyclic AMP.

5-HT1D and 5-HT2B receptors mediate contraction of smooth muscle in human small intestine.

In conclusion, 5-HT has been shown to contract both circular and longitudinal muscle layers of human terminal ileum. By the use of selective antagonists, the receptors mediating these actions have been identified. In the circular muscle of human small intestine, 5-HT-induced contraction is mediated via a receptor of the 5-HT1D sub-type, whereas a receptor of the 5-HT2B sub-type mediates the contractile response to 5-HT of longitudinal muscle layers.

Heterogeneity of 5-HT receptors mediating secretion in the human intestine.

In conclusion, application of 5-HT has been shown to induce fluid secretion in both the small and large intestine of man. By the use of selective antagonists, the receptors mediating these effects have been identified and characterized. 5-HT induces secretion across human ileal mucosa via a receptor of the 5-HT4 sub-type, whereas a receptor of the 5-HT2A sub-type appears to mediate the effect in human sigmoid colon. The response in human ascending colonic mucosa cannot be definitively classified at this time, but may reflect the presence of a heterogenous receptor population.

Human colonic mucosa possesses a mixed population of 5-HT receptors.

The aim of this study was to characterise the 5-HT receptor(s) mediating secretory responses of isolated human colonic mucosa to 5-HT. Sheets of muscle-stripped mucosa from proximal (ascending) and distal (sigmoid) human colon were set up in Ussing chambers for measurement of short-circuit current (Isc). Serosal application of 5-HT led to non-neural, concentration-dependent increases in Isc. Desensitisation to 5-HT was observed with ascending and sigmoid colonic mucosa. Using selective 5-HT antagonists we have shown that 5-HT induces secretion in sigmoid colon via a 5-HT2A receptor. In ascending colon a combination of 5-HT2A and 5-HT4 receptors appears to be involved.

Functional evidence for a 5-HT2B receptor mediating contraction of longitudinal muscle in human small intestine.

Application of 5-hydroxytryptamine induces contraction of longitudinal muscle strips from human terminal ileum. The response was resistant to antagonism by ketanserin, ondansetron or DAU6285, but was non-surmountably antagonized by methysergide. The selective 5-HT2B/2C receptor antagonist, SB 200646A evoked a concentration-dependent, parallel and dextral displacement of the concentration-response curve to 5-HT, yielding a pA2 estimate of 7.17. Application of yohimbine, a 5-HT1 and 5-HT2B receptor antagonist, also induced a rightward shift of the response curve to 5-HT, yielding a pA2 estimate of 8.10. In conclusion, it appears that a 5-HT2B receptor mediates the contractile response of the longitudinal muscle of human small intestine to 5-HT.

Changes in concentrations of hormones, metabolites, and amino acids in plasma of adult horses relative to overnight feed deprivation followed by a pellet-hay meal fed at noon.

Experiment 1 was conducted to characterize the concentrations of prolactin, growth hormone (GH), cortisol, insulin, glucagon, glucose, nonesterified fatty acids (NEFA), urea N, and 10 indispensable amino acids in the plasma of mares (n = 8) and stallions (n = 8) during the last 4 h of a 19-h period of feed deprivation and for 8 h after a noon meal. Experiment 2 was similar to Exp. 1 except that only stallions (n = 8) were used, and they were either fed (n = 4) or not fed (n = 4) at noon in a 2 x 2 Latin square design conducted over two sampling days 7 d apart. In Exp. 1, increases (P < .01) after feeding were observed for plasma concentrations of prolactin, cortisol, insulin, glucagon, glucose, urea N, and all amino acids except methionine; NEFA concentrations decreased (P < .01) after feeding. Episodic increases in GH concentrations were observed for most horses but were not associated with either feeding or gender (P > .1). Plasma urea N concentrations were higher (P < .025) overall in stallions than in mares, and the rise in prolactin concentrations after feeding was greater (P < .01) in stallions than in mares. In Exp. 2, meal-associated increases (P < .01) were observed for plasma concentrations of prolactin, insulin, glucagon, and glucose; NEFA concentrations decreased (P < .01). Except for cortisol, no hormone or metabolite varied with time across days when the stallions were not fed (P > .1), indicating that there was no inherent diurnal or feeding schedule-associated fluctuations in their concentrations. Cortisol concentrations varied (P < .02) over time but did not differ (P > .1) between fed and nonfed stallions. Again, GH concentrations were episodic but did not differ (P > .1) between fed and nonfed stallions. The lack of feeding effects on GH secretion in horses is similar to the response in pigs but differs from that in ruminants, in which GH concentrations generally decline after feeding.

The effect of atrial natriuretic peptides on ion transport by muscle-stripped and full-thickness preparations of rat colon.

The effects of three atrial natriuretic peptides (ANPs) have been tested for their ability to increase ion transport in muscle-stripped and intact preparations of rat colon. Anaritide (fragment 4-28 of human ANP), human ANP and rat atriopeptin III had no effect on short-circuit current in muscle-stripped preparations. Such insensitivity could not be explained by desensitisation, enzymic destruction or non-specific absorption. Short-circuit current was increased by 8-Br-cGMP and NaNP. The pharmacological activity of anaritide was demonstrated by abolition of phenylephrine-induced tone in rat aortic smooth muscle. In contrast to muscle-stripped colon, intact preparations responded with a tetrodotoxin-sensitive increase in short-circuit current to anaritide even though such preparations were less sensitive to acetylcholine and substance P. These results imply that selective neural damage or the lack of certain nerve pathways, caused by complete muscle stripping, will prevent the pro-secretory effects of anaritide in rat colon.