Impact of chromium dinicocysteinate supplementation on inflammation, oxidative stress, and insulin resistance in type 2 diabetic subjects: an exploratory analysis of a randomized, double-blind, placebo-controlled study.

BACKGROUND: Chromium dinicocysteinate (CDNC) is a unique chromium complex consisting of chromium, niacin, and L-cysteine. Previous preclinical and clinical studies support the safety and efficacy of CDNC in modulating oxidative stress, vascular inflammation, and glycemia in type 2 diabetes. OBJECTIVE: Herein, we report the results of several exploratory analyses conducted on type 2 diabetic subjects who previously participated in a 3-month randomized, double-blind, placebo-controlled trial and were treated with only metformin as standard diabetic care in addition to receiving the test supplementations. DESIGN: Results from 43 metformin users, who were randomly assigned to receive either placebo (P, n=13), chromium picolinate (CP, 400 µg elemental Cr(3+)/day, n=12), or CDNC (400 µg elemental Cr(3+)/day, n=18), were analyzed for blood markers of vascular inflammation, insulin resistance, and oxidative stress at baseline and at 3 months of supplementation. RESULTS: A statistically significant decrease in insulin resistance in the CDNC-supplemented cohort compared to placebo (p=0.01) was observed at 3 months. The CDNC group also demonstrated a significant reduction in insulin levels (p=0.03), protein carbonyl (p=0.02), and in TNF-α (p=0.03) compared to the placebo group. The CP group only showed a significant reduction in protein carbonyl levels (p=0.03) versus placebo. CONCLUSIONS: When controlling for diabetes medication, CDNC supplementation showed beneficial effects on blood markers of vascular inflammation, insulin resistance, and oxidative stress compared to placebo. The findings suggest that CDNC supplementation has potential as an adjunct therapy for individuals with type 2 diabetes.

Efficacy and tolerability of an undenatured type II collagen supplement in modulating knee osteoarthritis symptoms: a multicenter randomized, double-blind, placebo-controlled study.

BACKGROUND: Undenatured type II collagen (UC-II) is a nutritional supplement derived from chicken sternum cartilage. The purpose of this study was to evaluate the efficacy and tolerability of UC-II for knee osteoarthritis (OA) pain and associated symptoms compared to placebo and to glucosamine hydrochloride plus chondroitin sulfate (GC). METHODS: One hundred ninety one volunteers were randomized into three groups receiving a daily dose of UC-II (40 mg), GC (1500 mg G & 1200 mg C), or placebo for a 180-day period. The primary endpoint was the change in total Western Ontario McMaster Universities Osteoarthritis Index (WOMAC) from baseline through day 180 for the UC-II group versus placebo and GC. Secondary endpoints included the Lequesne Functional Index (LFI), the Visual Analog Scale (VAS) for pain and the WOMAC subscales. Modified intent-to-treat analysis were performed for all endpoints using analysis of covariance and mixed model repeated measures, while incremental area under the curve was calculated by the intent-to-treat method. RESULTS: At day 180, the UC-II group demonstrated a significant reduction in overall WOMAC score compared to placebo (p = 0.002) and GC (p = 0.04). Supplementation with UC-II also resulted in significant changes for all three WOMAC subscales: pain (p = 0.0003 vs. placebo; p = 0.016 vs. GC); stiffness (p = 0.004 vs. placebo; p = 0.044 vs. GC); physical function (p = 0.007 vs. placebo). Safety outcomes did not differ among the groups. CONCLUSION: UC-II improved knee joint symptoms in knee OA subjects and was well-tolerated. Additional studies that elucidate the mechanism for this supplement's actions are warranted. TRIAL REGISTRATION: CTRI/2013/05/003663 ; CTRI/2013/02/003348 .

Safety and toxicological evaluation of Meratrim®: an herbal formulation for weight management.

Meratrim is a unique dietary ingredient consisting of extracts from Sphaeranthus indicus flower heads and Garcinia mangostana fruit rind. Clinical studies have demonstrated that Meratrim is effective and well-tolerated in weight management. Herein we assessed the broad spectrum safety of Meratrim in a battery of in vitro and animal toxicological studies including a sub-chronic repeated-dose 13-week oral toxicity study to determine the no-observable-adverse-effect-level (NOAEL). The LD50 levels of Meratrim in Sprague-Dawley (SD) rats, as determined by the acute oral and dermal toxicity studies, were >5000 and >2000 mg/kg body weight, respectively. The primary skin and eye irritation tests classified Meratrim as non-irritating to the skin and mildly irritating to the eye. Genotoxicity studies showed that Meratrim is non-mutagenic. In the repeated-dose 13-week oral toxicity study, SD rats were orally gavaged with Meratrim at 0, 250, 500 or 1000 mg/kg/day. No morbidity, mortality, or significant adverse events were observed either during the course of the study or on the 13th week. The NOAEL of Meratrim was concluded to be 1000 mg/kg of body weight/day in male and female SD rats. These results, combined with the tolerability of Meratrim in the human clinical trials, demonstrate the broad spectrum safety of Meratrim.

Undenatured type II collagen (UC-II®) for joint support: a randomized, double-blind, placebo-controlled study in healthy volunteers.

BACKGROUND: UC-II contains a patented form of undenatured type II collagen derived from chicken sternum. Previous preclinical and clinical studies support the safety and efficacy of UC-II in modulating joint discomfort in osteoarthritis and rheumatoid arthritis. The purpose of this study was to assess the efficacy and tolerability of UC-II in moderating joint function and joint pain due to strenuous exercise in healthy subjects. METHODS: This randomized, double-blind, placebo-controlled study was conducted in healthy subjects who had no prior history of arthritic disease or joint pain at rest but experienced joint discomfort with physical activity. Fifty-five subjects who reported knee pain after participating in a standardized stepmill performance test were randomized to receive placebo (n = 28) or the UC-II (40 mg daily, n = 27) product for 120 days. Joint function was assessed by changes in degree of knee flexion and knee extension as well as measuring the time to experiencing and recovering from joint pain following strenuous stepmill exertion. RESULTS: After 120 days of supplementation, subjects in the UC-II group exhibited a statistically significant improvement in average knee extension compared to placebo (81.0 ± 1.3º vs 74.0 ± 2.2º; p = 0.011) and to baseline (81.0 ± 1.3º vs 73.2 ± 1.9º; p = 0.002). The UC-II cohort also demonstrated a statistically significant change in average knee extension at day 90 (78.8 ± 1.9º vs 73.2 ± 1.9º; p = 0.045) versus baseline. No significant change in knee extension was observed in the placebo group at any time. It was also noted that the UC-II group exercised longer before experiencing any initial joint discomfort at day 120 (2.8 ± 0.5 min, p = 0.019), compared to baseline (1.4 ± 0.2 min). By contrast, no significant changes were seen in the placebo group. No product related adverse events were observed during the study. At study conclusion, five individuals in the UC-II cohort reported no pain during or after the stepmill protocol (p = 0.031, within visit) as compared to one subject in the placebo group. CONCLUSIONS: Daily supplementation with 40 mg of UC-II was well tolerated and led to improved knee joint extension in healthy subjects. UC-II also demonstrated the potential to lengthen the period of pain free strenuous exertion and alleviate the joint pain that occasionally arises from such activities.

MicroRNA: implications in HIV, a brief overview.

MicroRNAs (miRNAs) are 20-22 nucleotide length noncoding RNA molecules that represent key regulators of many normal cellular functions. miRNAs undergo two processing steps which transform a long primary transcript into the mature miRNA. Available literatures demonstrate the association between alterations in the expression of miRNAs and the progression of numerous human disorders. Even though significant advances have been made, many fundamental questions about their expression and function still remain unanswered. Identifying factors that block the negative action of drugs of abuse on the miRNAs could help in identifying new therapeutic strategies. In this review, we briefly discuss the importance of miRNAs on HIV, strategies used by virus to avoid the cells' antiviral miRNA defenses, and how HIV might control and regulate host cell genes by encoding viral miRNAs.

Human immunodeficiency virus type 1 clade B and C gp120 differentially induce neurotoxin arachidonic acid in human astrocytes: implications for neuroAIDS.

HIV-1 clades (subtypes) differentially contribute to the neuropathogenesis of HIV-associated dementia (HAD) in neuroAIDS. HIV-1 envelop protein, gp120, plays a major role in neuronal function. It is not well understood how these HIV-1 clades exert these neuropathogenic differences. The N-methyl-D: -aspartate (NMDA) receptor-reduced glutamine synthesis could lead to secretion of neurotoxins such as arachidonic acid (AA) which plays a significant role in the neuropathogenic mechanisms in neuroAIDS. We hypothesize that clade B and C gp120 proteins exert differential effects on human primary astrocytes by production of the neurotoxin arachidonic acid. Our results indicate that clade B gp120 significantly downregulated NMDA receptor gene and protein expression, and level of glutamine while increasing expression of prostaglandin E2 (PGE(2)) and thromboxane A2 receptor (TBXA(2) R) compared to HIV-1 clade C gp120 protein. Thus, our studies for the first time demonstrate that HIV-1 clade B-gp120 protein appears to induce higher levels of expression of the neuropathogenic molecule cyclooxygenase-2 (COX-2)-mediated arachidonic acid by-products, PGE(2), and TBXA(2) R compared to HIV-1 clade C gp120 protein. These studies suggest that HIV-1 clade B and C gp120 proteins may play a differential role in the neuropathogenesis of HAD in neuroAIDS.

HIV-1 Tat upregulates expression of histone deacetylase-2 (HDAC2) in human neurons: implication for HIV-associated neurocognitive disorder (HAND).

Histone deacetylases (HDACs) play a pivotal role in epigenetic regulation of transcription and homeostasis of protein acetylation in histones and other proteins involved in chromatin remodeling. Histone hypoacetylation and transcriptional dysfunction have been shown to be associated with a variety of neurodegenerative diseases. More recently, neuron specific overexpression of HDAC2 has been shown to modulate synaptic plasticity and learning behavior in mice. However, the role of HDAC2 in development of HIV-associated neurocognitive disorders (HAND) is not reported. Herein we report that HIV-1 Tat protein upregulate HDAC2 expression in neuronal cells leading to transcriptional repression of genes involved in synaptic plasticity and neuronal function thereby contributing to the progression of HAND. Our results indicate upregulation of HDAC2 by Tat treatment in dose and time dependant manner by human neuroblastoma SK-N-MC cells and primary human neurons. Further, HDAC2 overexpression was associated with concomitant downregulation in CREB and CaMKIIa genes that are known to regulate neuronal activity. These observed effects were completely blocked by HDAC2 inhibition. These results for the first time suggest the possible role of HDAC2 in development of HAND. Therefore, use of HDAC2 specific inhibitor in combination with HAART may be of therapeutic value in treatment of neurocognitive disorders observed in HIV-1 infected individuals.

Effect of methamphetamine on expression of HIV coreceptors and CC-chemokines by dendritic cells.

UNLABELLED: The United States is currently experiencing an entangled epidemic of HIV infection and use of different drugs of abuse, especially of methamphetamine (Meth). Blood monocyte-derived dendritic cells (DC) are the first line of defense against HIV-1 infection, and are the initial target of HIV-1 infection in injection drug users. DC-SIGN present on dendritic cells is the first molecule that facilitates HIV-1 infection independent of CD4 or HIV coreceptors. AIMS: The aim of this study was to evaluate whether Meth acts as a cofactor in the pathogenesis of HIV-1 infection. MAIN METHODS: Monocyte derived DCs, obtained from normal subjects were cultured with and without Meth±HIV-1B, followed by analyzing the gene and protein expression by real-time quantitative polymerase chain reaction (RT-PCR) and fluorescence-activated cell-sorting analyses, respectively. KEY FINDINGS: Our results show that Meth significantly enhances HIV infection, and downregulates the gene expression of chemokines and costimulatory molecules with reciprocal upregulation of HIV coreceptors and DC-SIGN by dendritic cells. SIGNIFICANCE: Better understanding of the role of Meth in HIV-1 disease susceptibility and the mechanism through which Meth mediates its effects on HIV-1 infection may help to devise novel therapeutic strategies against HIV-1 infection in Meth using HIV-1 infected population.

Implications of ER stress, the unfolded protein response, and pro- and anti-apoptotic protein fingerprints in human monocyte-derived dendritic cells treated with alcohol.

BACKGROUND: Dendritic cells (DCs) are responsible for the activation of T cells and B cells. There is accumulating evidence that psychoactive substances such as alcohol can affect immune responses. We hypothesize that this occurs by modulating changes in proteins triggering a process known as unfolded protein response (UPR). This process protects cells from the toxic effects of misfolded proteins responsible for causing endoplasmic reticulum (ER) stress. Although much is known about ER stress, little is understood about the consequences of ethanol use on DC's protein expression. METHODS: In this study, we investigated alterations in the proteins of human monocyte-derived dendritic cells (MDDC) treated with 0.1% of alcohol by two-dimensional (2D) gel electrophoresis followed by liquid chromatography-tandem mass spectrometry, protein identification, and confirmation at the gene expression level by qRT-PCR. RESULTS: Proteomes of related samples demonstrated 32 differentially expressed proteins that had a 2-fold or greater change in expression (18 spots were up-regulated and 14 were down-regulated), compared to the control cultures (untreated cells). Alcohol significantly changed the expression of several components of the UPR stress-induced pathways that include chaperones, ER stress, antioxidant enzymes, proteases, alcohol dehydrogenase, cytoskeletal and apoptosis-regulating proteins. qRT-PCR analyses highlighted the enhanced expression of UPR and antioxidant genes that increased (18 hours) with alcohol treatment. CONCLUSION: Results of these analyses provide insights into alcohol mechanisms of regulating DC and suggest that alcohol induced specifically the UPR in DC. We speculate that activation of a UPR by alcohol may protect the DC from oxidant injury but may lead to the development of alcohol-related diseases.

Interactive role of human immunodeficiency virus type 1 (HIV-1) clade-specific Tat protein and cocaine in blood-brain barrier dysfunction: implications for HIV-1-associated neurocognitive disorder.

In recent years, increasing interest has emerged to assess the human immunodeficiency virus type 1 (HIV-1) clade C viral pathogenesis due to its anticipated spread in the United States and other western countries. Previous studies suggest that clade C is less neuropathogenic than clade B; however, the underlying mechanism is poorly understood. Additionally, the interactive role of drugs of abuse such as cocaine on clade C-associated neuropathogenesis has not been reported. In the current study, we hypothesize that HIV-1 clade-specific Tat proteins exert differential effects on blood-brain barrier (BBB) integrity and cocaine further differentially aggravates the BBB dysfunction. We evaluated the effect of Tat B and Tat C and/or cocaine on the BBB integrity using an in vitro model constructed with primary human brain microvascular endothelial cells (HBMECs) and astrocytes. The BBB membrane integrity was measured by transendothelial electrical resistance (TEER) and paracellular permeability was measured by fluorescein isothiocyanate (FITC)-dextran transport assay and monocytes transmigration across the BBB. Results indicate that Tat B disrupts BBB integrity to a greater extent compared to Tat C and cocaine further differentially exacerbates the BBB dysfunction. This BBB dysfunction was associated with altered expression of tight junction proteins zona occuldens (ZO-1) and junctional adhesion molecule (JAM)-2. Thus, these results for the first time delineate the differential role of Tat B and Tat C and/or cocaine in BBB dysfunction, which may be correlated with the clade-specific differences observed in HIV-1-associated neurological disorders.

Magnetic nanoformulation of azidothymidine 5'-triphosphate for targeted delivery across the blood-brain barrier.

Despite significant advances in highly active antiretroviral therapy (HAART), the prevalence of neuroAIDS remains high. This is mainly attributed to inability of antiretroviral therapy (ART) to cross the blood-brain barrier (BBB), thus resulting in insufficient drug concentration within the brain. Therefore, development of an active drug targeting system is an attractive strategy to increase the efficacy and delivery of ART to the brain. We report herein development of magnetic azidothymidine 5'-triphosphate (AZTTP) liposomal nanoformulation and its ability to transmigrate across an in vitro BBB model by application of an external magnetic field. We hypothesize that this magnetically guided nanoformulation can transverse the BBB by direct transport or via monocyte-mediated transport. Magnetic AZTTP liposomes were prepared using a mixture of phosphatidyl choline and cholesterol. The average size of prepared liposomes was about 150 nm with maximum drug and magnetite loading efficiency of 54.5% and 45.3%, respectively. Further, magnetic AZTTP liposomes were checked for transmigration across an in vitro BBB model using direct or monocyte-mediated transport by application of an external magnetic field. The results show that apparent permeability of magnetic AZTTP liposomes was 3-fold higher than free AZTTP. Also, the magnetic AZTTP liposomes were efficiently taken up by monocytes and these magnetic monocytes showed enhanced transendothelial migration compared to normal/non-magnetic monocytes in presence of an external magnetic field. Thus, we anticipate that the developed magnetic nanoformulation can be used for targeting active nucleotide analog reverse transcriptase inhibitors to the brain by application of an external magnetic force and thereby eliminate the brain HIV reservoir and help to treat neuroAIDS.

AZT 5'-triphosphate nanoformulation suppresses human immunodeficiency virus type 1 replication in peripheral blood mononuclear cells.

Inefficient cellular phosphorylation of nucleoside and nucleotide analog reverse transcriptase inhibitors (NRTIs) to their active nucleoside 5'-triphosphate (NTPs) form is one of the limitations for human immunodeficiency virus (HIV) therapy. We report herein direct binding of 3'-azido-3'-deoxythymidine-5'-triphosphate (AZTTP) onto magnetic nanoparticles (Fe(3)O(4); magnetite) due to ionic interaction. This magnetic nanoparticle bound AZTTP (MP-AZTTP) completely retained its biological activity as assessed by suppression of HIV-1 replication in peripheral blood mononuclear cells. The developed MP-AZTTP nanoformulation can be used for targeting active NRTIs to the brain by application of an external magnetic force and thereby eliminate the brain HIV reservoir and help to treat NeuroAIDS.

Upregulation of serotonin transporter by alcohol in human dendritic cells: possible implication in neuroimmune deregulation.

BACKGROUND: Alcohol is the most widely abused substance and its chronic consumption causes neurobehavioral disorders. It has been shown that alcohol affects the function of immune cells. Dendritic cells (DC) serve as the first line of defense against infections and are known to accumulate neurotransmitters such as 5-hydroxytryptamine (5-HT). The enzyme monoamine oxidase-A (MAO-A) degrades 5-HT that is associated with clinical depression and other neurological disorders. 5-HT is selectively transported into neurons through the serotonin transporter (SERT), which is a member of the sodium- and chloride-dependent neurotransmitter transporter (SLC6) family. SERT also serves as a receptor for psychostimulant recreational drugs. It has been demonstrated that several drugs of abuse such as amphetamine and cocaine inhibit the SERT expression; however, the role of alcohol is yet to be elucidated. We hypothesize that alcohol can modulate SERT and MAO-A expression in DC, leading to reciprocal downregulation of 5-HT in extracellular medium. METHODS: Dendritic cells were treated with different concentrations (0.05% to 0.2%v/v) of alcohol for 24-72 hours and processed for SERT and MAO-A expression using Q-PCR and Western blots analysis. In addition, SERT function in DC treated with alcohol both in the presence and absence of imipramine, a SERT inhibitor was measured using 4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide uptake assay. 5-HT levels in culture supernatant and intracellular 5-hydroxy indole acetic acid (5-HIAA) and cyclic AMP were also quantitated using ELISA. RESULTS: Dendritic cells treated with 0.1% alcohol for 24 hours showed significant upregulation of SERT and MAO-A expression compared with untreated DC. We also observed that 0.1% alcohol enhanced the function of SERT and decreased extracellular 5-HT levels compared with untreated DC cultures, and this was associated with the elevation of intracellular 5-HIAA and cyclic AMP levels. CONCLUSIONS: Our study suggests that alcohol upregulates SERT and MAO-A by elevating cyclic AMP, which may lead to decreased concentration of 5-HT in the extracellular medium. As 5-HT is a major neurotransmitter and an inflammatory mediator, its alcohol-mediated depletion may cause both neurological and immunological deregulation.

Differential regulation of indoleamine-2,3-dioxygenase (IDO) by HIV type 1 clade B and C Tat protein.

Previous studies have demonstrated that infection with HIV-1 clades might differentially contribute to the neuropathogenesis of HIV-1-associated dementia (HAD). HIV-1 transactivator regulatory protein (Tat) plays a major role in the process of disruption of neuronal function. It is not well understood how these HIV-1 subtypes exert different neuropathogenic effects. Activation of indoleamine-2,3-dioxygenase (IDO), the rate-limiting enzyme of the kynurenine pathway, leads to increased tryptophan catabolism and the generation of neurotoxins such as kynurenine (KYN). It is known that KYN plays a crucial role in the neuropathogenesis of HAD. We hypothesize that HIV-1 clade B and C Tat proteins might exert differential effects on human primary astrocytes by the upregulation of the IDO gene and protein expression as well as its activity and production of the neurotoxin KYN. RNA extracted from human primary astrocytes treated with either HIV-1 clade B and C Tat proteins was reverse transcribed and analyzed by quantitative real-time PCR to determine IDO gene expression. In addition, the enzymatic activity of IDO and the concentration of KYN were measured in cell lysates and culture supernatants. Our results indicate that HIV-1 clade B Tat protein significantly upregulated the IDO gene and protein expression, IDO enzyme activity, as well as KYN concentration compared to HIV-1 clade C Tat protein. Thus, our studies for the first time demonstrate that HIV-1 clade B Tat protein in human primary astrocytes appears to increase the level of neuropathogenic agents, such as IDO and KYN, as compared to HIV-1 clade C Tat protein. These results provide further evidence that the prevalence of HAD may be correlated with the difference in clades of HIV-1.

Methamphetamine enhances HIV-1 infectivity in monocyte derived dendritic cells.

The US is currently experiencing an epidemic of methamphetamine (Meth) use as a recreational drug. Recent studies also show a high prevalence of HIV-1 infection among Meth users. We report that Meth enhances HIV-1 infectivity of dendritic cells as measured by multinuclear activation of a galactosidase indicator (MAGI) cell assay, p24 assay, and LTR-RU5 amplification. Meth induces increased HIV-1 infection in association with an increase in the HIV-1 coreceptors, CXCR4 and CCR5, and infection is mediated by downregulation of extracellular-regulated kinase (ERK2) and the upregulation of p38 mitogen-activated protein kinase (MAPK). A p38 inhibitor (SB203580) specifically reversed the Meth-induced upregulation of the CCR5 HIV-1 coreceptor. The dopamine D2 receptor antagonist RS +/- sulpiride significantly reversed the Meth-induced upregulation of CCR5, demonstrating that the Meth-induced effect is mediated via the D2 receptor. These studies report for the first time that Meth fosters HIV-1 infection, potentially via upregulating coreceptor gene expression. Further, Meth mediates its regulatory effects via dopamine receptors and via downregulating ERK2 with a reciprocal upregulation of p38 MAPK. Elucidation of the role of Meth in HIV-1 disease susceptibility and the mechanism through which Meth mediates its effects on HIV-1 infection may help to devise novel therapeutic strategies against HIV-1 infection in high-risk Meth-using HIV-1-infected subjects.