Pronounced reduction of cutaneous Langerhans cell density in recently diagnosed type 2 diabetes.

Immune-mediated processes have been implicated in the pathogenesis of diabetic polyneuropathy. Langerhans cells (LCs) are the sole dendritic cell type located in the healthy epidermis and exert tolerogenic immune functions. We aimed to determine whether alterations in cutaneous LC density and intraepidermal nerve fiber density (IENFD) are present in patients with recently diagnosed type 2 diabetes. Skin biopsy specimens from the distal leg from 96 type 2 diabetic patients and 75 healthy control subjects were used for quantification of LC density and IENFD. LCs and IENFs were labeled using immunohistochemistry. Nerve conduction studies, quantitative sensory testing, and neurological examination were used to assess peripheral nerve function. LC density was markedly reduced in the diabetic group compared with the control group, but did not correlate with reduced IENFD or peripheral nerve function. Multivariate linear regression analysis revealed a strong association between LC density and whole-body insulin sensitivity in women but not in men with diabetes. Prospective studies should establish whether the pronounced reduction of cutaneous LCs detected in recently diagnosed type 2 diabetes could promote a cutaneous immunogenic imbalance toward inflammation predisposing to polyneuropathy and foot ulcers.

Hormones in the mentally disturbed brain: steroids and peptides in the development and treatment of psychopathology.

One of the more fascinating recent discoveries in neuroscience is the widespread influence of hormones on brain regions and functions underlying pathological behaviors. A story is unfolding that points to critical roles played by hypothalamic - pituitary - gonadal (HPG) and hypothalamic - pituitary - adrenal (HPA) axes on a startling array of mental disorders, from depression to dementia. The influence of peptides and steroids does not end with hormones released from the two axes, however. It is now clear that the brain has adapted, "highjacked" is more descriptive, HPG and HPA hormones for uses unrelated to their original functions in reproduction and responses to stress. Findings of neuromodulatory effects of HPA and HPG hormones on monoamine, GABA, glutamate and opioid pathways and of hormone receptors and enzymes involved in hormone synthesis, particularly of steroids, in the hippocampus, amygdala and other subcortical brain regions provide the brain with multiple evolutionary means to adapt to new functions. The complexity of the metabolic cascade for the steroids also leaves open mechanisms by which endogenous errors and exogenous chemicals could be involved in the etiology of psychopathologies. The planned review will examine the recent literature for evidence of steroidal and peptidergic influences on basic biological functions and on mood disorders, anxiety and PTSD, schizophrenia, substance abuse and dementia. Emphasis will be placed on animal models, although findings with patient populations will be prominently included. Special attention will be paid to novel pathways by which the precursors and metabolites of sex steroids can influence psychopathologies. We also will speculate on promising treatments with hormone modulators that may be useful in mollifying the symptomology of the mental disorders.

Effect of long-term exposure at suprazero temperatures on activity and viability of human ovarian cortex.

The experiment was designed to determine the optimal time and temperature for long-distance transport. Prolonged suprazero temperature exposure of ovarian tissue for 26 hours has no negative influence on follicle quality.

Cryobanking of human ovarian tissue for anti-cancer treatment: comparison of vitrification and conventional freezing.

The first case of cryopreservation of human ovarian tissue with good survival of follicles after warming was described in 1996. Childbirth after cryopreservation of ovarian tissue is now a reality. Cryopreservation of ovarian tissue can be performed using one of two methods: conventional ("slow") freezing and cryopreservation by direct plunging into liquid nitrogen (so called vitrification or "rapid" freezing). Comparative investigations of vitrification and conventional freezing performed on mammalian ovarian tissue are limited, and authors present different conclusions. The higher effectiveness of vitrification in comparison with conventional freezing for human oocytes and embryos was shown, whereas data on human ovarian tissue are limited. The aim of different studies was to compare the safety and effectiveness of conventional freezing and vitrification of human ovarian tissue. Below we shortly summarize the results of some investigations with different conclusions. The discussion on the post-warming quality of follicles as well as on the problems of microbial contamination of cells in liquid nitrogen at vitrification is presented. In our opinion, for cryopreservation of human ovarian tissue, conventional freezing is more promising than vitrification.

Human ovarian tissue preservation: is vitrification acceptable method for assisted reproduction?

The aim of this study was to evaluate the most successful vitrification protocol. The ovarian tissue pieces were randomly distributed into seven groups including fresh control. Each experimental group was divided into three subgroups according to the following cooling modes: a) in 1.8 ml cryo-vials with 1ml vitrification medium, b) in 1.8 ml cryo-vials with 0.1 ml vitrification medium, or c) by direct dropping with 0.05 ml vitrification medium into liquid nitrogen. The best results were observed in the protocol using 2.62 M dimethylsulphoxide + 2.6 M acetamide + 1.31 M propylene glycol + 0.0075M polyethylene glycol in combination with direct dropping of ovarian tissue pieces into liquid nitrogen. The vitrified and rewarmed samples after in vitro culture with this protocol showed 86 percent normally developed follicles, compared with 92 percent in fresh non-treated control. The concentrations of hormones in spent medium from culture of the same samples were 319 pg/ml for 17beta-estradiol and 2.6 ng/ml for progesterone compared with fresh non-treated control (253 pg/ml and 6 ng/ml, respectively). The results obtained by vitrification of ovarian tissue with this protocol were compatible with those of the fresh ovarian tissue.

Effects of testosterone on hormonal content and calcium-dependent basal secretion in female rat pituitary cells.

In vivo and in vitro effects of elevated androgens on agonist-induced gonadotropin secretion have been addressed previously. Here we investigated the effects of testosterone on hormonal content and basal (in the absence of agonists) hormone release in pituitary lactotrophs, somatotrophs and gonadotrophs from female rats. Furthermore we tested the hypothesis that testosterone action is dependent on the pattern of spontaneous and Bay K 8644 (a L-type calcium channel agonist) -induced calcium signalling. Mixed anterior pituitary cells were cultured in steroid containing or depleted media, and testosterone (1pM to 10nM) was added for 48h. Cells were studied for their spontaneous and Bay K 8644-induced calcium signalling pattern and total hormone levels (release and hormonal content). In lactotrophs, somatotrophs and gonadotrophs testosterone did not affect the pattern of spontaneous calcium signalling. Bay K 8644-induced calcium signalling and hormone release were not affected by testosterone. In both steroid-depleted and -containing medium, testosterone inhibited prolactin (PRL), luteinizing hormone (LH) and growth hormone (GH) cellular content and release in a dose-dependent manner, with IC(50)s in a sub-nanomolar concentration range. These results indicate that testosterone inhibits basal hormone release from lactotrophs, somatotrophs and gonadotrophs without affecting intracellular calcium signalling. This action of testosterone is not dependent on the presence of other steroid hormones.

Actions of gonadotropin-releasing hormone analogues in pituitary gonadotrophs and their modulation by ovarian steroids.

Recently, GnRH antagonists (GnRHant) like cetrorelix and ganirelix have been introduced in protocols of controlled ovarian hyperstimulation for assisted reproductive techniques to prevent premature luteinizing hormone (LH) surges. Here we tested, whether the actions of cetrorelix and the GnRH agonist (GnRHag) triptorelin in gonadotrophs are dependent on the steroid milieu. Furthermore, we characterized the actions of cetrorelix and triptorelin on LH secretion and the total LH pool. Female rat pituitary cells were treated either with 0.1 nM triptorelin for 1, 2, 4 and 6 days or for 1, 3, 5 and 6 h or with 1, 10 or 100 nM cetrorelix for 1, 2, 3 and 5 h or for 10 min. Cells were stimulated for 3h with different concentrations of GnRH (10 pM-1 microM). For analysis of the total LH pool, which is composed of stored and released LH, cells were lysed with 0.1% Triton X-100 at -80 degrees C overnight. To test, whether the steroid milieu affects the actions of cetrorelix and triptorelin, cells were incubated for 52 h with 1 nM estradiol (E) alone or with combinations of 100 nM progesterone (P) for 4 or 52 h, respectively. Cells were then treated with 0.1 nM triptorelin for 9 h or 1 nM cetrorelix for 3 h and stimulated for 3 h with different concentrations of GnRH (10 pM-1 microM). The suppressive effect of triptorelin on LH secretion was fully accomplished after 3 h of treatment, for cetrorelix only 10 min were sufficient. The concentration of cetrorelix must be at least equimolar to GnRH to block LH secretion. Cetrorelix shifted the EC50s of the GnRH dose-response curve to the right. Triptorelin suppressed total LH significantly (from 137 to 36 ng/ml) after 1 h in a time-dependent manner. In contrast, only high concentrations of cetrorelix increased total LH. In steroid treated cells the suppressive effects of triptorelin were more distinct. One nanomolar cetrorelix suppressed GnRH-stimulated LH secretion of cells not treated with steroids from 10.1 to 3.5 ng/ml. In cells, additionally treated with estradiol alone or estradiol and short-term progesterone, LH levels were higher (from 3.5 to 5.4 or 4.5 ng/ml, respectively). In cells co-treated with estradiol and progesterone for 52 h LH secretion was only suppressed from 10.1 to 9.5 ng/ml. Steroid treatments diminished the suppressive effect of cetrorelix on LH secretion. In conclusion, the depletion of the total LH pool contributes to the desensitizing effect of triptorelin. The actions of cetrorelix and triptorelin are dependent on the steroid milieu.

Limits of learning enhancements with nicotine in old male rats.

Findings with young adult humans and animal models suggest that nicotine may serve both neuroprotective and cognition enhancing roles in old animals. A pair of experiments was conducted to examine drug-induced modification of the cholinergic nicotinic receptor subtype on rates of learning by young and aged rats. In experiment I males (4-7 months or 20-25 months old) were administered nicotine (0.0, 0.3 or 0.7 mg/kg injected s.c. daily) and tested in both a T-maze non-spatial discrimination paradigm and a hole board spatial task. Nicotine failed to improve acquisition by young animals on either task. Nicotine also failed to improve non-spatial learning by old animals. However, both dosages of nicotine improved performance by the old males in the spatial paradigm. In experiment II, a 5-choice serial discrimination paradigm designed to better evaluate visual attention and spatial working memory in aging was used. Groups of old male rats were administered nicotine or mecamylamine (2 or 8 mg/kg), an antagonist of the nicotinic cholinergic receptor. Results were that the 0.3 mg nicotine group learned the task fastest and achieved the highest learning asymptote. Both learning rates and final levels of performance were worst in the 8 mg mecamylamine group. However, the 2 mg mecamylamine rats were the equals of the control group and both reached a higher asymptote than the 0.7 mg nicotine group. These data suggest that healthy old animals can accrue benefits from nicotinic activation but that the benefits are complex, being limited to certain dosages and to specific cognitive skills.

Treatment strategies in PCOS patients.

Polycystic ovary syndrome (PCOS), with a prevalence of up to 7%, is the most common endocrinopathy in women of reproductive age. It is a complex metabolic-endocrine disorder with severe long-term health consequences, such as a higher risk of type 2 diabetes and cardiovascular diseases. According to prospective studies, women with PCOS have abnormal glucose tolerance and diabetes mellitus in 31.0-35.0% and 7.5-10.0% respectively. This risk is 2-3 times higher than normal. Insulin resistance plays a key role in the pathophysiology of this syndrome, and this makes the use of oral antidiabetic drugs most compelling. The majority of studies have shown amelioration of typical symptoms such as hyperandrogenism and cycle irregularities following the use of oral anti-diabetics, and ovulation and pregnancy rates increased. Furthermore, these drugs might be cardioprotective by improving insulin sensitivity and reducing the risk for type 2 diabetes. The best-investigated drug is metformin. Metformin is not approved for PCOS treatment in Germany and is a class B drug in pregnancy. In sterile PCOS patients, clomiphene citrate is still the first choice. The combination of clomiphene with metformin and lifestyle changes such as weight reduction and exercise might be superior to clomiphene alone. This article covers the use of different oral anti-diabetic drugs in the treatment of PCOS, and their influence on fertility and long-term health.

Chronic fluoxetine suppresses circulating estrogen and the enhanced spatial learning of estrogen-treated ovariectomized rats.

We are interested in developing animal models to evaluate cognitive processes as influenced by the interplay of steroidal hormones and drugs commonly used in psychotherapy. Two experiments with female rats were conducted to evaluate the interaction of estrogen with the serotonin specific reuptake inhibitor (SSRI) fluoxetine on spatial learning and memory and on the endocrine system. In experiment 1, estrogen (50 microg estradiol benzoate/kg body weight) was administered SC to young adult, ovariectomized (OVX) rats either alone or in combination with fluoxetine (2 mg/kg SC). After a month, the groups were compared with appropriate OVX and gonadally intact controls on trials to criterion in a hole board spatial memory task using massed training trials. Experiment 2 was a dose-response study of the influence of fluoxetine (0.5-5 mg/kg) on circulating estrogen in OVX, estrogen treated females. Results were that the OVX females administered estrogen only reached the learning criterion significantly faster than the other groups. All other groups, including the estrogen + fluoxetine animals, performed no better than the controls. Combining fluoxetine with estrogen also lowered circulating estrogen titers, with the least estrogen reductions being in the group receiving the highest dosage of fluoxetine. No differences among groups were found on measures of activity in an open field or for anxiety in a plus maze. Conclusions were that administration of estrogen improved spatial learning and memory in OVX rats, whereas concurrent fluoxetine exposure suppressed the levels of estrogen in circulation and eliminated the gains in spatial performance obtained from chronic estrogen exposure.

Gonadotropin-releasing hormone antagonists: pharmacology and clinical use in women.

Gonadotropin-releasing hormone (GnRH) antagonists are now widely used in protocols of patients with controlled ovarian hyperstimulation to treat infertility. By competitively binding to the pituitary GnRH receptor, they lead to a rapid suppression of gonadotropins and consecutively sex hormones. In the past, GnRH agonists have been exclusively used for these patients, with the disadvantage of an initial rise of gonadotropins--the flare-up effect. Several trials comparing the agonistic and antagonistic analogs of GnRH found no significant differences in oocyte quality, fertilization and pregnancy rates. Slightly lower implantation and pregnancy rates, and estradiol levels, in patients treated with GnRH antagonists has raised concern about eventual extrapituitary adverse effects. However, no convincing evidence has yet been found for any detrimental ovarian effects of GnRH antagonists. The lower rate of ovarian hyperstimulation syndrome, a potentially severe disadvantage of infertility treatment, is a positive feature of GnRH antagonists. The key point is that GnRH antagonists have been proven to be as effective and safe as GnRH agonists. This broadens the spectrum of indications for GnRH antagonists to sex hormone-dependent disorders like endometriosis, uterine fibroids, and gynecological cancers such as breast and ovarian cancer.