Recent studies have shown that the gut microbiome changes brain function, behavior, and psychiatric and neurological disorders. The Gut–Brain Axis (GBA) provides a neuronal pathway to explain this. But exactly how do commensal bacteria signal through the epithelial layer of the large intestine to activate GBA nerve afferents? An in vitro model is described. We differentiated two human cell lines: Caco2Bbe1 into mature epithelium on 0.4-micron filters and then SH-SY5Y into mature neurons in 24-well plates. These were co-cultured by placing the epithelium-laden filters 1 mm above the neurons. Twenty-four hours later they were tri-cultured by apical addition of 107Lactobacillus rhamnosus or Lactobacillus fermentum which settled on the epithelium. Alone, the Caco2bbe1 cells stimulated neurite outgrowth in underlying SH-SY5Y. Beyond this, the lactobacilli were well tolerated and stimulated further neurite outgrowth by 24 h post-treatment, though not passing through the filters. The results provide face validity for a first-of-kind model of transepithelial intestinal lumen-to nerve signaling. The model displays the tight junctional barrier characteristics found in the large intestine while at the same time translating stimulatory signals from the bacteria through epithelial cells to attracted neurons. The model is easy to set-up with components widely available.

Juicing vegetables is thought to be an anticancer treatment. Support exists for a rank order of anticancer greens (kale > dandelion > lettuce > spinach) based on degrees of bioavailability of different phytochemicals, also offset by some noxious molecules (i.e., calcium-oxalate). We developed a new in vitro transepithelial anti-neuroblastoma model system. The juices were diluted as predicted once in the small intestine. They were applied to apical Caco-2Bbe1 cells atop dividing SH-SY5Y neuroblastoma cells, and changes in transepithelial electrical resistance (TEER) and cell growth were considered with juice spectroscopies. Studied first in monoculture, kale and dandelion were the most cytostatic juices on SH-SY5Ys, lettuce showed no effect, and high (4.2%) spinach was cytotoxic. In co-culture, high (4.2%) kale was quickest (three days) to inhibit neuroblastoma growth. By five days, dandelion and kale were equally robust. Lettuce showed small anti-proliferative effects at five days and spinach remained cytotoxic. Spinach’s cytotoxicity corresponded with major infrared bands indicative of oxalate. Kale juice uniquely induced reactive oxygen species and S-phase cell cycle arrest in SH-SY5Y. The superiority of kale and dandelion was also apparent on the epithelium, because raising TEER levels is considered healthy. Kale’s unique features corresponded with a major fluorescent peak that co-eluted with kaempferol during high performance liquid chromatography. Because the anticancer rank order was upheld, the model appears validated for screening anticancer juices.

Obesity is one of the largest current public health problems. Recent studies suggest that persistent changes in the intestinal microbiota (dysbiosis) can eventually lead to obesity. A stable core of intestinal microbiota exists, primarily composed of the phyla Firmicutes and Bacteroidetes, but their proportions can be altered by antibiotics. Such changes appear to not only alter host energy consumption but also modify host satiety mechanisms. Our study evaluated possible changes in the gut microbiota caused by oral administration of tetracycline, both alone or in combination with Lactobacillus gasseri in rats. Zoometric analyses were conducted and qPCR of fecal samples were analyzed to allow comparison before and during treatment regarding Firmicutes Bacteroidetes proportions. The results showed increased weight and body mass index (BMI) in animals treated with tetracycline alone (P < 0.05) when compared to the group that received tetracycline with probiotic, except for BMI in phase two when there was no statistical significance. Molecular analysis showed that after animals were treated with tetracycline, Firmicutes predominated over Bacteroidetes bacteria, which was coincident with increased weight and BMI. Probiotic addition may have minimized tetracycline dysbiosis, preventing excessive weight gain. Changes in microbiota caused by antibiotics have been shown to be an important factor related to childhood obesity. Microbiological manipulation of microbiota can play an important role in weight control, especially with antibiotic acting microbiota. More studies are needed to elucidate this mechanism.

L-Arginine pathway metabolites appear to play differential roles in the pathogenesis of major depressive disorder (MDD). Studies have revealed an antidepressant and anxiolytic effect of agmatine and putrescine. Possible mechanisms of these effects include inhibition of nitric oxide synthase and N-methyl-D-aspartate receptors. The present study sought to determine whether MDD is associated with altered levels of arginine metabolites and whether these metabolites are associated with depression, anxiety and stress severity. Seventy seven MDD patients 21-65 years of age with a minimum score of 18 on the Hamilton Depression Scale, and 27 age and sex matched healthy controls (HC) were included. Patients with uncontrolled physical diseases, abnormal routine lab tests, other psychiatric diagnoses, or under psychotropic medication were excluded. HC subjects were recruited from the community. Rating instruments included Hamilton Depression and Anxiety Scales, Beck Depression and Anxiety Inventory and Perceived Stress Scale. Fasting blood was drawn between 8:30 and 11:00 a.m. and High Performance Liquid Chromatography (HPLC) was used to measure plasma arginine metabolites. ADMA (Asymmetrical dimethylarginine) and putrescine were significantly lower while SDMA (Symmetric dimethylarginine), agmatine and ornithine were significantly higher in MDD patients (p(<)0.05). Depression, anxiety and stress severity were negatively correlated with ADMA and putrescine (p(<)0.05). Stress was positively correlated with citrulline, NOHA (N-omega-hydroxy-nor-1-arginine), SDMA, agmatine and ornithine (p(<)0.05). Lower putrescine levels predicted depression diagnosis (p = 0.039) and depression severity (p = 0.003). Low ADMA level predicted depression severity as well. Arginine pathway metabolites are associated with the pathophysiology of depression. Putrescine may be a biomarker to predict MDD.

Safety concerns over cell-derived pharmaceutical products being manufactured in supplements of fetal bovine serum (FBS) have ignited pleas to replace FBS. Herein, four newly marketed alternatives to FBS were compared: a xeno-free product called Cell-Ess®, a human platelet lysate marketed as GroPro®, and two mixtures of adult bovine serum varying in their proportions of neonatal growth factors, called Liporo® and FetalGro®. An endothelial cell line (C2BBe1) and a neuronal cell line (SHSY5Y) near confluency in media with 10% FBS were selectively scraped and taken through a 25-day step-wise algorithm to replace FBS, and another human endothelial cell line (HRA-19) was studied to replicate C2BBe1. Cells were stained, counted, and compared for viability, migration, and spheroids. The C2BBe1 and HRA-19 cell lines failed to proliferate in 10% Cell-Ess® but grew in 10% GroPro® or 10% FetalGro® reasonably well compared to reference 10% FBS. With SH-SY5Y, only FetalGro® approached FBS's efficacy. These were all inferior to 11 different branded lots of FBS (positive controls), but five days into switching just amongst the FBS brands, 4 of 11 supported less proliferation than reference FBS in endothelial HRA-19 (p < 0.004). Moreover, neurospheres were enriched in two branded lots of FBS and FetalGro® (each p < 0.004), neurospheres being an unwanted phenotype for any neuronal cell application. Because platelet-derived GroPro® stood out amongst the non-FBS growth supplements to allow proliferation without inducing spheroids, it seems the best (mindful that the cells still grew slower in it compared to FBS). While no perfect replacement was found amongst the alternatives to FBS, the algorithm for switching should be useful in future testing of new alternatives to FBS as the need arises to switch from FBS and expand pharmaceutical products with safety for human use.

Background: Noncardiac chest pain (NCCP) is defined as persistent angina-like chest pain with no evidence of cardiac disease. There is some controversy about the long-term morbidity and mortality outcomes of NCCP patients. Many studies have found no significant differences in death rates in chest pain patients without coronary artery disease compared to the general population. However, studies that include longer follow-up periods and a better characterization of the NCCP population reveal a twofold elevation in the relative risk of adverse cardiac events over 5-26 years. This review sought to identify studies in relation to cardiovascular and psychological prognosis of NCCP patients. Methods: PubMed database and reference lists from relevant publications were reviewed. Inclusion criteria were systematic reviews, prospective studies, and retrospective surveys from 1970 to 2011. Search terms were as follows: chest pain, noncardiac chest pain, nonspecific chest pain, unexplained chest pain, prognosis of noncardiac chest pain, prognosis of angina with normal angiography, and angina with normal coronary arteries. Results: Studies supporting worse outcome (cardiac morbidity and mortality; n=16) included 173,875 patients with mean age 57 and mean length of follow-up 7.5 years. Studies supporting good outcome (n=25) included 244,998 patients with age 50 and length of follow-up 5 years. Articles supporting poor psychological outcome (n=9) included 3,987 patients and length of follow-up 2 years. Conclusion: There are mixed data on long-term morbidity, cardiovascular adverse events, and mortality of NCCP patients. Some studies provide supporting evidence for poor outcome, while others provide evidence for positive outcome. However, many patients with NCCP have prolonged psychosocial comorbidity. The heterogeneity of NCCP and study populations limited definitive conclusions. However, many patients with NCCP have psychiatric morbidity and poor quality of life. Several questions remain about NCPP with respect to the psychopathology and pathophysiology of this condition. Whether NCCP patients have good or bad outcome requires careful risk stratification.

1.1 Objectives: Test the prevailing hypothesis that depressive illness is associated with platelet hyperactivity and that treatment with an atypical antipsychotic with established antidepressant efficacy – quetiapine - will normalize platelet activity. 1.2 Methods: Forty-seven outpatients with major depressive disorder (MDD) and 27 healthy controls (HC) without evidence of cardiovascular disease were enrolled. Behavioral rating scales and medical tests preceded baseline assessments of (1) platelet-rich plasma (PRP) aggregometry and (2) whole blood flow cytometry (P-selectin surface labeling). The measures were repeated in those MDD subjects who completed 8 weeks (n=27) or 12 weeks (n=19) of treatment with quetiapine. 1.3 Results: Untreated MDD compared to HC subjects displayed more platelet aggregation when PRP was stirred agonist-free (p=0.021). Other platelet measurements at baseline such as in vitro agonist-stimulated PRP aggregometry or P-selectin expression by flow cytometry did not distinguish MDD from HC subjects. After 8 weeks on quetiapine, a reduced (now normal) agonist-free aggregatory response to stirring (p=0.035) was observed. By 12 weeks the aggregometry response to arachidonic acid (AA) was also lowered (p=0.016 vs. pretreatment; p=0.001 vs. HC). Other agonist additions (ADP, epinephrine, or collagen) failed to distinguish MDD from HC. There were no significant associations between mood rating scores and any form of platelet activity at any time point on quetiapine. 1.4 Limitations: High percentage of dropouts attributable to dose-related side effects limited the post-treatment assessments. 1.5 Conclusions: The hypothesis that untreated depression is associated with more active platelets, was confirmed, but this finding was confined to the “resting”, or agonist-free state. Quetiapine treatment normalized this resting activity and led to a lower-than-normal response to AA-induced aggregation after 12 weeks of treatment. These findings confirm that 8 weeks treatment with quetiapine can normalize at least one form of platelet hyperactivity, but the lower-than-healthy response to AA after 12 weeks on quetiapine warrants further study.

Agmatine is an endogenous brain metabolite, decarboxylated arginine, which has neuroprotective properties when injected intraperitoneally (i.p.) into rat pups following hypoxic-ischemia. A previous screen for compounds based on rat brain lysates containing agmatinase with assistance from computational chemistry, led to piperazine-1-carboxamidine as a putative agmatinase inhibitor. Herein, the neuroprotective properties of piperazine-1-carboxamidine are described both in vitro and in vivo. Organotypic entorhinal-hippocampal slices were firstly prepared from 7-day-old rat pups and exposed in vitro to atmospheric oxygen depletion for 3 h. Upon reoxygenation, the slices were treated with piperazine-1-carboxamidine or agmatine (50 μg/ml agents), or saline, and 15 h later propidium iodine was used to stain. Piperazine-1-carboxamidine or agmatine produced substantial in vitro protection compared to post-reoxygenated saline-treated controls. An in vivo model involved surgical right carotid ligation followed by exposure to hypoxic-ischemia (8 % oxygen) for 2.5 h. Piperazine-1-carboxamidine at 50 mg/kg i.p. was given 15 min post-reoxygenation and continued twice daily for 3 days. Cortical agmatine levels were elevated (+28.5 %) following piperazine-1-carboxamidine treatment with no change in arginine or its other major metabolites. Histologic staining with anti-Neun monoclonal antibody also revealed neuroprotection of CA1-3 layers of the hippocampus. Until endpoint at 22 days of age, no adverse events were observed in treated pups’ body weights, rectal temperatures, or prompted ambulation. Piperazine-1-carboxamidine therefore appears to be a neuroprotective agent of a new category, agmatinase inhibitor.

Agmatine (decarboxylated arginine) has been known as a natural product for over 100 years, but its biosynthesis in humans was left unexplored owing to long-standing controversy. Only recently has the demonstration of agmatine biosynthesis in mammals revived research, indicating its exceptional modulatory action at multiple molecular targets, including neurotransmitter systems, nitric oxide (NO) synthesis and polyamine metabolism, thus providing bases for broad therapeutic applications. This timely review, a concerted effort by 16 independent research groups, draws attention to the substantial preclinical and initial clinical evidence, and highlights challenges and opportunities, for the use of agmatine in treating a spectrum of complex diseases with unmet therapeutic needs, including diabetes mellitus, neurotrauma and neurodegenerative diseases, opioid addiction, mood disorders, cognitive disorders and cancer.

This study describes the construction and preliminary analysis of a database of summary level genetic findings for bipolar disorder from the literature. The database is available for noncommercial use at http://bioprogramming.bsd.uchicago.edu/BDStudies/. This may be the first complete collection of published gene-specific linkage and association findings on bipolar disorder, including genome-wide association studies. Both the positive and negative findings have been incorporated so that the statistical and contextual significance of each finding may be compared semi-quantitatively and qualitatively across studies of mixed technologies. The database is appropriate for searching a literature populated by mainly underpowered studies, and if ‘hits’ are viewed as tentative knowledge for future hypothesis generation. It can serve as the basis for a mega-analysis of candidate genes. Herein, we discuss the most robust and best replicated gene findings to date in a contextual manner.

Selective serotonin reuptake inhibitors (SSRI) are a first‐line treatment option for mood disorders such as depression (D). They inhibit neuronal reuptake of serotonin and result in depleted serotonin stores in the dense bodies of platelets. It is hypothesized that depressive illnesses cause platelet activation and endothelial dysfunction, which could be modulated by the use of SSRIs. To validate this hypothesis an institutional‐based co‐morbidity of D and cardiovascular (CV) study was undertaken. 25 healthy control subjects and 25 patients meeting the DSM‐IV criteria for major depressive disorder were recruited in this study. Blood samples were collected at baseline and then 4 and 8 weeks following treatment with Escitalopram. Agonist‐induced platelet aggregation showed varying degrees of inhibition of collagen, ADP, arachidonic acid and epinephrine‐induced platelet aggregation. Agonist Induced Percent Platelet Aggregation BL 4 weeks 8 weeks Collagen 71.5±22.4 63.4±35.1 74.7±21.3 AA 64.1±35.1 67.1±25.1 55.7±36.1 ADP 48.2±30.9 33.0±26.7 52.4±31.0 EPI 55.6±29.2 37.4±25.8 50.4±32.3 Saline 6.9±4.5 9.4±7.8 5.0±3.9 These results indicate that treatment with SSRIs may inhibit platelet aggregation by some agonists, which may have an impact in the prevention of atherothrombotic and CV disease. SSRIs might alter hemostasis in some patients by inhibiting platelet function.

Background Disturbances in affective state are known to cause platelet activation, which may relate to evidence linking major depression and coronary artery disease. The platelet activation biomarker, P-selectin, exists in various blood fractions that demarcate steps of platelet activation. If circulating platelets are more activated in depressed patients, the literature suggests that amongst antidepressant classes, the SerotoninSelective Reuptake Inhibitors (SSRIs) are best able to normalize this abnormality. Serotonin/ Norepinephrine Reuptake Inhibitors (SNRIs), like venlafaxine, have not been studied in this regard. Aim of the Study To determine the status of activated P-selectin (platelet surface-bound and/or released forms) in depressed patients before and after 8 weeks of treatment with the SNRI, venlafaxine. Methods Baseline (untreated) subjects were major depressives (n= 23) and age/sex-matched healthy controls (n= 17) with no evidence of coronary artery disease. A subgroup of the depressed cohort (n= 15) was restudied after 4 and 8 weeks on venlafaxine. P-selectin levels were compared to two biomarkers not known to change during platelet activation: the membranous fibronectin-signaling protein, IRAS, and the antigen of the GPIIb/IIIa receptor, CD61. Results At baseline, both activated forms of P-Selectin were high in the depressives, but only the soluble form showed statistical significance (p= 0.03) versus healthy controls. Venlafaxine treatment led to mood normalization based on reduced Hamilton Depression scores (p< 0.0001), while the level of soluble P-selectin was non-significantly lowered (p= 0.13). Platelet membranous IRAS and CD61 levels were normal at baseline but down-regulated after 4 and 8 weeks of treatment (p = 0.01 each). Conclusion High levels of soluble P-selectin were identified in depression, indicative of platelet activation. Venlafaxine treatment had minimal effect on soluble P-selectin but had clear effects on platelet IRAS and CD61. Therefore, platelet activation does not readily normalize with mood correction after 8 weeks on venlafaxine, but other platelet effects seem to occur.

Stress-related psychiatric disorders (eg, major depression, bipolar disorder, anxiety disorders, and posttraumatic stress disorder) have been variably associated with pervasive loss of homeostasis in the autonomic nervous system. This loss, often referred to as allostatic load, leads to dysregulation of the autonomic nervous system with sympathetic overdrive and vagal tone diminution. An efferent vagal pathway has been identifi ed, the cholinergic anti-infl ammatory pathway, which is believed to exert an anti-infl ammatory action. When vagal tone is diminished, infl am- mation becomes disinhibited and one would expect specifi c infl ammation biomarkers to become elevated in the brain and the periphery. This provides a plausible mechanism for the elevated blood levels of proinfl ammatory cytokines and chemo- kines previously reported in depressed patients compared with healthy controls by other groups and ours (Piletz et al, 2009). When proinfl ammatory cytokines become chronically elevated, endothelial dysfunction eventually ensues, ultimately leading to atherosclerosis and atherothrombosis. Of interest is whether this proinfl ammatory status of depression can be reversed by antide- pressant treatment and/or whether the proinfl ammatory changes normalize when the depressed mood is alleviated. Our previous study using venlafaxine-XR (a serotonin-norepinephrine reuptake inhibitor) to treat depression suggested that the elevated plasma proinfl ammatory cytokines in depression were not normalized after 8 weeks despite mood normalization (Piletz et al, 2009). Other studies have suggested that selective serotonin

High levels of pro-inflammatory biomarkers have been reported in depression. In the present study, five pro-inflammatory biomarkers were measured in the blood of patients with major depressive disorder (MDD). Biomarker levels were compared to age- and sex-matched healthy subjects. Patients with MDD had significantly higher baseline levels of tumour necrosis factor-alpha (TNFalpha, P=0.04), interleukin-1beta (IL1beta, P=0.03), and monocyte chemotactic protein-1 (MCP-1; P=0.02) compared to controls. There were no differences between groups in levels of cell determinant-40 ligand (CD40L) and C-reactive protein (CRP). A subset of the MDD patients consented to undergo treatment with venlafaxine (an SNRI: at lower doses a selective serotonin reuptake inhibitor; at higher doses also a norepinephrine reuptake inhibitor) for 8 weeks. By week 8, all treatment completers had responded therapeutically. However, levels of TNFalpha, IL1beta, and MCP-1 remained elevated. A concave quadratic equation described the associations between plasma venlafaxine concentrations and IL1beta (P=0.03), TNFalpha (P=0.09), and MCP-1 (P=0.02), suggesting that these biomarkers may have become selectively lowered in the serotonergic dose range of venlafaxine. This is the first report of venlafaxine's possible effect on pro-inflammatory biomarkers.

13 Nitric Depressed mood is known to be an independent risk factor for cardiovascular disease. Nitric oxide (NO)-mediated oxidative stress has been linked to endothelial dysfunction preceding car- diovascular disease (CVD). In this study, we sought to determine if the metabolic con- version of l-arginine to NO might be altered in the plasma of depressed patients as a possible early-warning sign of future CVD, and whether treating depression might affect this pathway favor-ably. To do this, the following fi ve plasma biomarkers were mea- sured: (1) asymmetric-dimethylarginine (ADMA; an endogenous inhibitor of the NO synthases), (2) total nitrite (a biomarker of endogenous NO production), (3) agmatine (an alternative metabolite of arginine and putative stress-related transmitter), (4) myeloperoxidase (an enzyme leading to peroxynitrite produc- tion), or (5) nitrotyrosine (an index of peroxynitrite levels). No baseline differences were observed for any of these biomarkers between depressed patients (n = 23) and matched healthy con-trols (n = 17). An ancillary fi nding was that nitrotyrosine cova- ried with body mass index ( P = .03). Fourteen of the depressed patients were then treated for 8 weeks with venlafaxine, a serotonin-norepinephrine reuptake inhibitor (SNRI). All 14 patients responded by achieving improved Hamilton Depression rating scores < 10. After 8 weeks on venlafaxine, lowered plasma levels of agmatine ( P = .02) and myeloperoxidase ( P = .02) were observed

Objectives Major Depressive Disorder is an established independent risk factor for cardiovascular disease, but the precise pathophysiological mechanism remains obscure. The nitric oxide branch of arginine metabolism has been linked to vascular homeostasis. Methods Five plasma biomarkers of the nitric oxide branch of arginine metabolism were quantified in depressed patients (n = 22) and healthy controls (n = 17): total nitrite, nitrotyrosine, asymmetric dimethylarginine, agmatine, and myeloperoxidase. Thirteen of the depressed patients were restudied after 4-8 weeks of mood normalization with venlafaxine, a mixed serotonin/norepinephrine reuptake blocker. Results None of the biomarkers were altered in depressed patients compared to controls. However, treatment reduced agmatine and myeloperoxidase levels (p=0.02 each). A clear but nonsignificant rise in total nitrite and nitrotyrosine was also observed at week-4. Conclusions Despite no changes at pre-treatment, the reductions in agmatine and myeloperoxidase may result from serotonin and/or noradrenaline changes occurring with venlafaxine antidepressant therapy.