Hyperhomocysteinemia is detrimental to pregnancy in mice and is associated with preterm birth.

Elevated levels of homocysteine produce detrimental effects in humans but its role in preterm birth is not known. Here we used a mouse model of hyperhomocysteinemia to examine the relevance of homocysteine to preterm birth. The mouse carries a heterozygous deletion of cystathionine ß-synthase (Cbs(+/-)). Gestational period was monitored in wild type and Cbs(+/-) female mice. Mouse uterine and placental tissues, human primary trophoblast cells, and human myometrial and placental cell lines were used to determine the influence of homocysteine on expression of specific genes in vitro. The activity of BKCa channel in the myometrial cell line was monitored using the patch-clamp technique. We found that hyperhomocysteinemia had detrimental effects on pregnancy and induced preterm birth in mice. Homocysteine increased the expression of oxytocin receptor and Cox-2 as well as PGE2 production in uterus and placenta, and initiated premature uterine contraction. A Cox-2 inhibitor reversed these effects. Gpr109a, a receptor for niacin, induced Cox-2 in uterus. Homocysteine upregulated GPR109A and suppressed BKCa channel activity in human myometrial cells. Deletion of Gpr109a in Cbs(+/-) mice reversed premature birth. We conclude that hyperhomocysteinemia causes preterm birth in mice through upregulation of the Gpr109a/Cox-2/PGE2 axis and that pharmacological blockade of Gpr109a may have potential in prevention of preterm birth.

AZFa protein DDX3Y is differentially expressed in human male germ cells during development and in testicular tumours: new evidence for phenotypic plasticity of germ cells.

BACKGROUND: DDX3Y (DBY), located within AZoospermia Factor a (AZFa) region of the human Y chromosome (Yq11), encodes a conserved DEAD-box RNA helicase expressed only in germ cells and with a putative function at G1-S phase of the cell cycle. Deletion of AZFa results most often in germ cell aplasia, i.e. Sertoli-cell-only syndrome. To investigate the function of DDX3Y during human spermatogenesis, we examined its expression during development and maturation of the testis and in several types of testicular germ cell tumours (TGCTs), including the pre-invasive carcinoma in situ (CIS) precursor cells which are believed to originate from fetal gonocytes. METHODS: DDX3Y protein expression was analysed during development in different tissues by western blotting. The localization of DDX3Y in normal fetal and prepubertal testis tissue of different ages as well as in a series of distinct TGCT tissue samples (CIS, classical seminoma, spermatocytic seminoma, teratoma and embryonal carcinoma) was performed by immunohistochemistry. RESULTS: Germ cell-specific expression of DDX3Y protein was revealed in fetal prospermatogonia but not in gonocytes and not before the 17th gestational week. After birth, DDX3Y was expressed at first only in the nuclei of Ap spermatogonia, then also in the cytoplasm similarly to that seen after puberty. In CIS cells, DDX3Y was highly expressed and located predominantly in the nuclei. In invasive TGCT, significant DDX3Y expression was found in seminomas of the classical and spermatocytic type, but not in somatically differentiated non-seminomas, consistent with its germ-cell specific function. CONCLUSIONS: The fetal germ cell DDX3Y expression suggests a role in early spermatogonial proliferation and implies that, in men with AZFa deletion, germ cell depletion may begin prenatally. The strong expression of DDX3Y in CIS cells, but not in gonocytes, indicates phenotypic plasticity of CIS cells and suggests partial maturation to spermatogonia, likely due to their postpubertal microenvironment.

Analysis of gene expression in normal and neoplastic human testis: new roles of RNA.

Large-scale methods for analysing gene expression, such as microarrays, have yielded a wealth of information about gene expression at the mRNA level. However, expression of alternative transcripts, together with the presence of a wide range of largely undescribed RNA transcripts combined with regulation from the RNA interference pathway, may cause misinterpretations when trying to base conclusions from expression data derived from studies at the mRNA level. With HLXB9, PRM1, DICER and E2F1 as examples, we here show a range of situations that can occur when investigating gene expression, and give recommendations for the complementary methods that can verify gene expression data from large-scale studies, as well as give new information regarding the regulation of specific genes. Especially, we show that the absence of a protein despite high expression of the corresponding mRNA can be caused by expression of miRNAs targeting the mRNA. Additionally, we show through cloning the presence of both known and new miRNAs in the testis emphasizing the necessity for following up mRNA expression data by investigating expression at the protein level.

Antenatal dexamethasone treatment leads to changes in gene expression in a murine late placenta.

Antenatal steroids like dexamethasone (DEX) are used to augment fetal lung maturity and there is a major concern that they impair fetal growth. If delivery is delayed after using antenatal DEX, placental function and hence fetal growth may be compromised even further. To investigate the effects of DEX on placental function, we treated 9 pregnant C57/BL6 mice with DEX and 9 pregnant mice were injected with saline to serve as controls. Placental gene expression was studied using microarrays in 3 pairs and other 6 pairs were used to confirm microarray results by semi-quantitative RT-PCR, real-time PCR, in situ hybridization, western blot analysis and Oligo ApopTaq assay. DEX-treated placentas were hydropic, friable, pale, and weighed less (80.0+/-15.1mg compared to 85.6.8+/-7.6mg, p=0.05) (n=62 placentas). Fetal weight was significantly reduced after DEX use (940+/-32mg compared to 1162+/-79mg, p=0.001) (n=62 fetuses). There was >99% similarity within and between the three gene chip data sets. DEX led to down-regulation of 1212 genes and up-regulation of 1382 genes. RT-PCR studies showed that DEX caused a decrease in expression of genes involved in cell division such as cyclins A2, B1, D2, cdk 2, cdk 4 and M-phase protein kinase along with growth-promoting genes such as EGF-R, BMP4 and IGFBP3. Oligo ApopTaq assay and western blot studies showed that DEX-treatment increased apoptosis of trophoblast cells. DEX-treatment led to up-regulation of aquaporin 5 and tryptophan hydroxylase genes as confirmed by real-time PCR, and in situ hybridization studies. Thus antenatal DEX treatment led to a reduction in placental and fetal weight, and this effect was associated with a decreased expression of several growth-promoting genes and increased apoptosis of trophoblast cells.

Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours.

AIMS: NANOG is a key regulator of embryonic stem cell (ESC) self-renewal and pluripotency. Our recent genome-wide gene expression profiling study of the precursor of testicular germ cell tumours, carcinoma in situ testis (CIS), showed close similarity between ESC and CIS, including high NANOG expression. In the present study we analysed the protein expression of NANOG during normal development of human testis and in a large series of neoplastic/dysgenetic specimens. METHODS AND RESULTS: We detected abundant expression of NANOG in CIS and in CIS-derived testicular tumours with marked differences; seminoma and embryonal carcinoma were strongly positive, differentiated somatic elements of teratoma were negative. We provide evidence for the fetal origin of testicular cancer as we detected strong expression of NANOG in fetal gonocytes up to gestational week 20, with subsequent down-regulation occurring earlier than for OCT-4. We detected no expression at the protein level in normal testis. CONCLUSIONS: NANOG is a new marker for testicular CIS and germ cell tumours and the high level of NANOG along with OCT-4 are determinants of the stem cell-like pluripotency of the preinvasive CIS cell. Timing of NANOG down-regulation in fetal gonocytes suggests that NANOG may act as a regulatory factor up-stream to OCT-4.

Do environmental factors play a role in the aetiology of carcinoma in situ testis and the testicular dysgenesis syndrome?

The hypothesis of the Testicular Dysgenesis Syndrome (TDS), first suggested in 2001, propose that several disorders of the male reproductive system such as infertility, hypospadias, cryptorchidism and testicular cancer are all symptoms of TDS, which is most likely initiated during early foetal development, and may be provoked by external factors such as endocrine disruptors in addition to genetic predisposition. Testicular germ cell tumours (TGCTs), considered the most severe symptom of TDS, have increased in incidence during the last 60 years, to become the most common malignancy in young Caucasian men aged 17-45 years. TGCTs of young men originate from carcinoma in situ (CIS) cells. In the last few years, progress has been made identifying candidate genes involved in the neoplastic development of CIS, which may elucidate the timing of the initiation of CIS, currently thought to originate in foetal life from primordial germ cells or early gonocytes. Histological dysgenetic features are frequently seen in testes affected with the TDS components testis cancer or cryptorchidism. A TDS-like phenotype can be induced in male rats by in utero exposure to high concentrations of dibutyl phthalate (DBP) suggesting that ubiquitously present environmental endocrine disruptors may play a role in the aetiology of human TDS. So far, no animal model has been able to mimick all the symptoms of TDS including TGCTs although CIS-like cells have been found in a spontaneous testicular neoplasm in a rabbit.

Paroxetine for social anxiety and alcohol use in dual-diagnosed patients.

The objective of this study was to compare the efficacy and tolerability of paroxetine to matched placebo in adults with co-occurring social anxiety disorder and alcohol use disorder. Outcome measures included standardized indices of social anxiety and alcohol use. Fifteen individuals meeting DSM-IV criteria for both social anxiety disorder and alcohol use disorder were randomized to treatment. Paroxetine (n = 6) or placebo (n = 9) was given in a double-blind format for 8 weeks using a flexible dosing schedule. Dosing began at 20 mg/d and increased to a target dose of 60 mg/d. There was a significant effect of treatment group on social anxiety symptoms, where patients treated with paroxetine improved more than those treated with placebo on both the Clinical Global Index (CGI) and the Liebowitz Social Anxiety Scale (Ps < or = 0.05). On alcohol use, there was not a significant effect of treatment on quantity/frequency measures of drinking, but there was for the CGI ratings (50% paroxetine patients versus 11% placebo patients were improvers on drinking, P < or = 0.05). This pilot study suggests that paroxetine is an effective treatment for social anxiety disorder in individuals with comorbid alcohol problems, and positive treatment effects can be seen in as little as 8 weeks. Further study is warranted to investigate its utility in helping affected individuals reduce alcohol use.

Alcohol and substance abuse.

Substance abuse and dependence is a public health problem with far-reaching societal implications. The acute toxicity of substances of abuse and medical consequences of chronic use are substantial. On a more optimistic note, a great deal of progress has been made in understanding and treating substance use disorders. Expanding knowledge concerning the neurobiology of substances of abuse and substance use disorders has led to a growth in pharmacotherapeutic treatment options. A growth in understanding of behavioral processes, motivational issues, and processes of behavioral change has been important in designing new and increasingly more effective psychosocial treatments. A growing body of evidence indicates that the treatment of substance use disorders can be effective, making early diagnosis and treatment or referral increasingly important.

Oral nutritional supplementation for the alcoholic patient: a brief overview.

Approximately 1.5 million Americans seek treatment for alcoholism annually. Malnutrition is frequently observed in the alcoholic population due to several factors, including poor dietary intake, alcohol-associated gastrointestinal problems, decreased nutrient absorption and enhanced excretion of trace elements. The administration of multivitamins with minerals and folic acid is commonplace. The present paper reviews basic nutrient deficits commonly encountered in alcoholic individuals and describes the rationale and basic guidelines for oral replacement therapy.

Cocaine dependence with and without post-traumatic stress disorder: a comparison of substance use, trauma history and psychiatric comorbidity.

This study examined the relationship between substance use, trauma history, post-traumatic stress disorder (PTSD), and psychiatric comorbidity in a treatment seeking sample of cocaine dependent individuals (N = 91). Structured clinical interviews revealed that 42.9% of the sample met DSM-III-R criteria for lifetime PTSD. Comparisons between individuals with and without lifetime PTSD revealed that individuals with PTSD had significantly higher rates of exposure to traumatic events, earlier age of first assault, more severe symptomatology, and higher rates of Axis I and Axis II diagnoses. The results illustrate a high incidence of PTSD among cocaine dependent individuals. Routine assessment of trauma history and PTSD may assist in the identification of a subgroup of cocaine users in need of special prevention and treatment efforts.

Substance abuse and bipolar comorbidity.

Bipolar disorder and substance abuse commonly co-occur. In fact, as many as 50% of individuals with bipolar disorder have been found to have a lifetime history of substance abuse or dependence. This article discusses the very important comorbidity of bipolar disorder as it is complicated by substance abuse, focusing on the prevalence, course, diagnostic considerations and treatment.

The role of stress in alcohol use, alcoholism treatment, and relapse.

Addiction to alcohol or other drugs (AODs) is a complex problem determined by multiple factors, including psychological and physiological components. Stress is considered a major contributor to the initiation and continuation of AOD use as well as to relapse. Many studies that have demonstrated an association between AOD use and stress have been unable to establish a causal relationship between the two. However, stress and the body's response to it most likely play a role in the vulnerability to initial AOD use, initiation of AOD abuse treatment, and relapse in recovering AOD users. This relationship probably is mediated, at least in part, by common neurochemical systems, such as the serotonin, dopamine, and opiate peptide systems, as well as the hypothalamic-pituitary-adrenal (HPA) axis. Further exploration of these connections should lead to important pharmacological developments in the prevention and treatment of AOD abuse. Studies indicate that treatment techniques which foster coping skills, problem-solving skills, and social support play a pivotal role in successful treatment. In the future, individualized treatment approaches that emphasize stress management strategies in those patients in whom a clear connection between stress and relapse exists will become particularly important.

Posttraumatic stress disorder and cocaine dependence. Order of onset.

To investigate differences between patients whose posttraumatic stress disorder (PTSD) preceded their cocaine dependence and vice versa, 33 patients with comorbid PTSD and cocaine dependence were divided into two groups: one in which the traum and PTSD occurred before onset of cocaine dependence (primary PTSD) and one in which the PTSD occurred after cocaine dependence was established (primary cocaine). In the primary-PTSD group, the trauma was generally childhood abuse. In the primary-cocaine group, the trauma was generally associated with the procurement and use of cocaine. In the primary PTSD group, there were significantly more women, more other Axis I diagnoses, more Cluster B and C Axis II diagnoses, and more benzodiazepine and opiate use. In the primary-cocaine group, there was a trend toward more cocaine use in the previous month. Significant clinical differences between these two groups may warrant different types of treatment or differing treatment emphasis.

Diagnosis of personality disorders in cocaine-dependent individuals.

The authors explored the relationship between personality disorder (PD) and cocaine use. Diagnostic interviews for PD were performed at baseline and 12 weeks (study termination) in 47 cocaine-dependent individuals entering a pharmacologic treatment trial; 68% met criteria for a PD at baseline, 51% at study termination. In comparing baseline and termination PD, 22 individuals had no change in the number of PDs, 6 had more, and 17 had fewer. The group with an increase in the number of PD diagnoses had more positive urine drug screens than either of the other groups and more cocaine use by self-report. Personality disorders appear to be common in cocaine-dependent individuals but may be affected by cocaine use and withdrawal.

Cocaine-like effects of intravenous procaine in cocaine addicts.

Pharmacological treatments that alter dopaminergic functioning have not lessened cocaine use in addicted patients. Non-dopaminergic mechanisms may therefore be important in the chronic use of cocaine. Procaine, like cocaine, is a local anesthetic, but has only 1% of cocaine's affinity for the dopamine reuptake receptor. In order to assess the subjective effects of procaine and its similarity to cocaine, we administered procaine to nine cocaine-dependent subjects. Patients 2-3 weeks abstinent were administered placebo, low dose procaine (0.46 mg/kg), and high dose procaine (1.84 mg/kg procaine) over a single 2-hour session. Patients were assessed for craving and similarity to cocaine experience and were administered the Symptom Checklist 90 Revised (SCL90R). High dose procaine was identified as similar to cocaine and induced significant cocaine craving. High dose procaine also induced significant elevations in somatization, obsessive-compulsive symptoms, phobic anxiety, interpersonal sensitivity, anxiety, positive symptoms and global severity (from the SCL90R). Our findings suggest that procaine shares subjective effects similar to cocaine, despite a much lower affinity for the dopamine reuptake receptor. Procaine may be a useful tool to explore non-dopaminergic mechanisms of cocaine's reinforcing and addictive properties.