Novel hydrazone compounds with broad-spectrum antiplasmodial activity and synergistic interactions with antimalarial drugs.

The development of novel antiplasmodial compounds with broad-spectrum activity against different stages of Plasmodium parasites is crucial to prevent malaria disease and parasite transmission. This study evaluated the antiplasmodial activity of seven novel hydrazone compounds (referred to as CB compounds: CB-27, CB-41, CB-50, CB-53, CB-58, CB-59, and CB-61) against multiple stages of Plasmodium parasites. All CB compounds inhibited blood stage proliferation of drug-resistant or sensitive strains of Plasmodium falciparum in the low micromolar to nanomolar range. Interestingly, CB-41 exhibited prophylactic activity against hypnozoites and liver schizonts in Plasmodium cynomolgi, a primate model for Plasmodium vivax. Four CB compounds (CB-27, CB-41, CB-53, and CB-61) inhibited P. falciparum oocyst formation in mosquitoes, and five CB compounds (CB-27, CB-41, CB-53, CB-58, and CB-61) hindered the in vitro development of Plasmodium berghei ookinetes. The CB compounds did not inhibit the activation of P. berghei female and male gametocytes in vitro. Isobologram assays demonstrated synergistic interactions between CB-61 and the FDA-approved antimalarial drugs, clindamycin and halofantrine. Testing of six CB compounds showed no inhibition of Plasmodium glutathione S-transferase as a putative target and no cytotoxicity in HepG2 liver cells. CB compounds are promising candidates for further development as antimalarial drugs against multidrug-resistant parasites, which could also prevent malaria transmission.

Soluble Insulin Receptor Levels in Plasma, Exosomes, and Urine and Its Association With HIV-Associated Neurocognitive Disorders.

Background: HIV-associated neurocognitive disorders (HAND) are one of the HIV-associated comorbidities affecting 20-50% of the people with HIV (PWH) infection. We found that the soluble insulin receptor (sIR) levels in plasma and cerebrospinal fluid (CSF) were significantly higher in HIV-infected women. The mechanism of sIR release into the plasma remains unknown, but the detection of the sIR in exosomes may uncover novel mechanisms of sIR secretion from HIV-infected cells and its contribution to HIV disease progression and HAND development. Quantification of sIR in urine may represent a less invasive and more accessible diagnostic tool. Our objective was to quantify sIR levels in plasma, plasma-derived exosomes, and urine, and evaluate their association with HAND and renal function. Methods: We measured full-length sIR in the plasma and urine of 38 controls and 76 HIV-infected women by ELISA, and sIR, HIV-1 Tat, and reactive oxygen species (ROS) in exosomes by flow cytometry. Results: Plasma and exosomes with sIR were significantly higher in HIV-infected women when compared with controls and HAND. Exosomal sIR positively correlated with exosomal ROS and exosomal HIV-1 Tat in HIV-infected women. Exosomal ROS was significantly higher in HIV-infected women with more symptomatic cognitive impairment. Plasma-derived exosomes exhibited significantly higher levels of astrocyte (GFAP) and neuronal (L1CAM) markers in HIV-infected women, confirming the presence of circulating CNS-derived exosomes in the blood of HIV-infected women. Urine sIR positively correlated with eGFR in controls, but not in HIV-infected women, regardless there was no significant difference in renal function as determined by the estimated glomerular filtration rate (eGFR, p = 0.762). In HIV-infected women, higher plasma sIR correlated with lower urine sIR that could suggest sIR retention in blood or decreased renal filtration. Discussion: Higher plasma sIR levels and their correlation with ROS in plasma-derived exosomes with HAND suggest a combined role of metabolic disturbances, oxidative stress, exosome release, and cognitive decline. Communication between CNS and periphery is compromised in PWH, thus plasma-derived exosomes may shed light on disrupted cellular mechanisms in the brain of PWH. High plasma and low urine sIR levels could suggest sIR retention in blood or decreased renal filtration.

Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages.

HIV-associated neurocognitive disorders (HAND) are prevalent despite combined antiretroviral therapy (cART), affecting 52% of people living with HIV. Our laboratory has demonstrated increased expression of cathepsin B (CATB) in postmortem brain tissue with HAND. Increased secretion of CATB from in vitro HIV-infected monocyte-derived macrophages (MDM) induces neurotoxicity. Activation of cannabinoid receptor type 2 (CB2R) inhibits HIV-1 replication in macrophages and the neurotoxicity induced by viral proteins. However, it is unknown if CB2R agonists affect CATB secretion and neurotoxicity in HIV-infected MDM. We hypothesized that HIV-infected MDM exposed to CB2R agonists decrease CATB secretion and neurotoxicity. Primary MDM were inoculated with HIV-1ADA and treated with selective CB2R agonists JWH-133 and HU-308. HIV-1 p24 and CATB levels were determined from supernatants using ELISA. MDM were pre-treated with a selective CB2R antagonist SR144528 before JWH-133 treatment to determine if CB2R activation is responsible for the effects. Neuronal apoptosis was assessed using a TUNEL assay. Results show that both agonists reduce HIV-1 replication and CATB secretion from MDM in a time and dose-dependent manner and that CB2R activation is responsible for these effects. Finally, JWH-133 decreased HIV/MDM-CATB induced neuronal apoptosis. Our results suggest that agonists of CB2R represent a potential therapeutic strategy against HIV/MDM-induced neurotoxicity.

Plasticity of GluN1 at Ventral Hippocampal Synapses in the Infralimbic Cortex.

Although the infralimbic cortex (IL) is not thought to play a role in fear acquisition, recent experiments found evidence that synaptic plasticity is occurring at ventral hippocampal (vHPC) synapses in IL during auditory fear acquisition as measured by changes in the N-methyl-D-aspartate (NMDA) receptor-mediated currents in male rats. These electrophysiological data suggest that fear conditioning changes the expression of NMDA receptors on vHPC-to-IL synapses. To further evaluate the plasticity of NMDA receptors at this specific synapse, we injected AAV particles expressing channelrhodopsin-EYFP into the vHPC of male and female rats to label vHPC projections with EYFP. To test for NMDA receptor changes in vHPC-to-IL synapses after fear learning, we used fluorescence-activated cell sorting (FACS) to quantify synaptosomes isolated from IL tissue punches that were positive for EYFP and the obligatory GluN1 subunit. More EYFP+/GluN1+ synaptosomes with greater average expression of GluN1 were isolated from male rats exposed to auditory fear conditioning (AFC) than those exposed to context and tones only or to contextual fear conditioning (CFC), suggesting that AFC increased NMDA receptor expression in males. In a second experiment, we found that pairing the tones and shocks was required to induce the molecular changes and that fear extinction did not reverse the changes. In contrast, females showed similar levels of EYFP+/GluN1+ synaptosomes in all behavioral groups. These findings suggest that AFC induces synaptic plasticity of NMDA receptors in the vHPC-to-IL projection in males, while female rats rely on different synaptic mechanisms.

Angiotensin II Induces Differentiation of Human Neuroblastoma Cells by Increasing MAP2 and ROS Levels.

INTRODUCTION: The roles of angiotensin II (Ang II) in the brain are still under investigation. In this study, we investigated if Ang II influences differentiation of human neuroblastoma cells with simultaneous activation of NADPH oxidase and reactive oxygen species (ROS). Moreover, we investigated the Ang II receptor type involved during differentiation. METHODS: Human neuroblastoma cells (SH-SY5Y; 5 × 105 cells) were exposed to Ang II (600 nM) for 24 h. Differentiation was monitored by measuring MAP2 and NF-H levels. Cell size and ROS were analyzed by flow cytometry, and NADPH oxidase activation was assayed using apocynin (500 µM). Ang II receptors (ATR) activation was assayed using ATR blockers or Ang II metabolism inhibitors (10-7 M). RESULTS: (1) Cell size decreased significantly in Ang II-treated cells; (2) MAP2 and ROS increased significantly in Ang II-treated cells with no changes in viability; (3) MAP2 and ROS decreased significantly in cells incubated with Ang II plus apocynin. (4) A significant decrease in MAP2 was observed in cells exposed to Ang II plus PD123.319 (AT2R blocker). CONCLUSION: Our findings suggest that Ang II influences differentiation of SH-SY5Y by increasing MAP2 through the AT2R. The increase in MAP2 and ROS were also mediated through NADPH oxidase with no cell death.

Release of Soluble Insulin Receptor From Neurons by Cerebrospinal Fluid From Patients With Neurocognitive Dysfunction and HIV Infection.

Previously, we found that high levels of soluble insulin receptor (sIR) in the cerebrospinal fluid (CSF) of an HIV-infected women cohort were associated with the presence and severity of HIV-associated neurocognitive disorders (HAND). In this study we investigated if CSF from this population, HIV-1 Tat, and selected cytokines induces sIR secretion from human neuronal cells. Twenty-three (23) HIV-seropositive women stratified by cognitive status and five HIV- seronegative women were evaluated. Soluble IR levels were measured in the extracellular medium of neuronal cells (SH-SY5Y) that were exposed (for 24 h) to the CSF of patients. The levels of sIR, HIV-1 Tat, and cytokine levels (IL-2, IL4, IL-6, IFNγ, TNFα, and IL-10) were quantified in the CSF of participants by ELISA and flow cytometry. Neuronal secretion of sIR was measured after exposure (24 h) to HIV-1 Tat (0.5-250 nM), or specific cytokines. The effects of TNFα and HIV-1 Tat on sIR secretion were also evaluated in the presence of R7050 (TNFα antagonist; 10 nM). Neurons exposed to the CSF of HIV-infected women had higher sIR levels according to the severity of neurocognitive impairment of the participant. Increased CSF sIR levels were associated with the presence and severity of HAND and were positively correlated with CSF HIV-1 Tat levels in HIV-infected women with cognitive impairment. CSF levels of IL-2, IFNγ, and TNFα were significantly increased with HAND. However, only TNFα (5 pg/mL) and HIV-1 Tat (100 nM) induced a significant increase in neuronal sIR secretion after 24 h exposure, an effect that was antagonized when each were combined with R7050. Our data suggests that TNFα and HIV-1 Tat from the CSF of HIV-infected women may regulate the secretion of sIR from neuronal cells and that the effect of HIV-1 Tat on sIR secretion may depend on TNFα receptor activation.

Losartan counteracts the effects of cardiomyocyte swelling on glucose uptake and insulin receptor substrate-1 levels.

A growing body of evidence demonstrates an association between Angiotensin II (Ang II) receptor blockers (ARBs) and enhanced glucose metabolism during ischemic heart disease. Despite these encouraging results, the mechanisms responsible for these effects during ischemia remain poorly understood. In this study we investigated the influence of losartan, an AT1 receptor blocker, and secreted Ang II (sAng II) on glucose uptake and insulin receptor substrate (IRS-1) levels during cardiomyocyte swelling. H9c2 cells were differentiated to cardiac muscle and the levels of myogenin, Myosin Light Chain (MLC), and membrane AT1 receptors were measured using flow cytometry. Intracellular Ang II (iAng II) was overexpressed in differentiated cardiomyocytes and swelling was induced after incubation with hypotonic solution for 40min. Glucose uptake and IRS-1 levels were monitored by flow cytometry using 2-NBDG fluorescent glucose (10µM) or an anti-IRS-1 monoclonal antibody in the presence or absence of losartan (10-7M). Secreted Angiotensin II was quantified from the medium using a specific Ang II-EIA kit. To evaluate the relationship between sAng II and losartan effects on glucose uptake, transfected cells were pretreated with the drug for 24h and then exposed to hypotonic solution in the presence or absence of the secreted peptide. The results indicate that: (1) swelling of transfected cardiomyocytes decreased glucose uptake and induced the secretion of Ang II to the extracellular medium; (2) losartan antagonized the effects of swelling on glucose uptake and IRS-1 levels in transfected cardiomyocytes; (3) the effects of losartan on glucose uptake were observed during swelling only in the presence of sAng II in the culture medium. Our study demonstrates that both losartan and sAng II have essential roles in glucose metabolism during cardiomyocyte swelling.

Angiotensins and Huntington's Disease: A Study on Immortalized Progenitor Striatal Cell Lines.

Neurons from mouse models of Huntington's disease (HD) exhibit altered electrophysiological properties, potentially contributing to neuronal dysfunction and neurodegeneration. The renin-angiotensin system (RAS) is a potential contributor to the pathophysiology of neurodegenerative diseases. However, the role of angiotensin II (Ang II) and angiotensin (1-7) has not been characterized in HD. We investigated the influence of Ang II and angiotensin (1-7) on total potassium current using immortalized progenitor mutant huntingtin-expressing (Q111) and wild-type (Q7) cell lines. Measurements of potassium current were performed using the whole cell configuration of pCLAMP. The results showed that (1) the effect of Ang II administered to the bath caused a negligible effect on potassium current in mutant Q111 cells compared with wild-type Q7 cells and that intracellular administration of Ang II reduced the potassium current in wild type but not in mutant cells; (2) the small effect of Ang II was abolished by losartan; (3) intracellular administration of Ang II performed in mutant huntingtin-expressing Q111 cells revealed a negligible effect of the peptide on potassium current; (4) flow cytometer analysis indicated a low expression of Ang II AT1 receptors in mutant Q111 cells; (5) mutant huntingtin-expressing striatal cells are highly sensitive to Ang (1-7) and that the effect of Ang (1-7) is related to the activation of Mas receptors. In conclusion, mutant huntingtin-expressing cells showed a negligible effect of Ang II on potassium current, a result probably due to the reduced expression of AT1 receptors at the surface cell membrane. In contrast, administration of Ang (1-7) to the bath showed a significant decline of the potassium current in mutant cells, an effect dependent on the activation of Mas receptors. Ang II had an intracrine effect in wild-type cells and Ang (1-7) exerted a significant effect in mutant huntingtin-expressing striatal cells.

Characterization of a Dual Rac/Cdc42 Inhibitor MBQ-167 in Metastatic Cancer.

The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell-cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC50, 1,100 nmol/L) inhibits cancer cell migration and viability and reduces tumor growth, metastasis, and angiogenesis in vivo Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC50 values of 103 and 78 nmol/L, respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of STAT3, without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial-to-mesenchymal transition by a loss of cell polarity and inhibition of cell surface actin-based extensions to ultimately result in detachment from the substratum. Prolonged incubation (120 hours) in MBQ-167 decreases metastatic cancer cell viability with a GI50 of approximately 130 nmol/L, without affecting noncancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167-mediated G2-M cell-cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by approximately 90%. In conclusion, MBQ-167 is 10× more potent than other currently available Rac/Cdc42 inhibitors and has the potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42. Mol Cancer Ther; 16(5); 805-18. ©2017 AACR.

Measurement of Cardiac Angiotensin II by Immunoassays, HPLC-Chip/Mass Spectrometry, and Functional Assays.

The molecular mechanisms related to the effect of angiotensin II, its level on cardiac tissues, as well as its overexpression represent an important aspect of cardiovascular pharmacology and pathology. Severe alterations of cardiac functions are induced by hypertension including activation of circulating and local cardiac renin angiotensin systems. In this chapter, we are providing the methods and materials necessary for further investigation of this important topic.

Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade.

The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15µM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic.

Soluble insulin receptor as a source of insulin resistance and cognitive impairment in HIV-seropositive women.

Insulin resistance occurs in HIV-infected individuals and is associated with HIV-associated neurocognitive disorders (HAND). However, the mechanisms involved are not well understood. Previously, we showed a correlation between soluble insulin receptor (sIR) and HAND. Here, we investigated if binding of free insulin to sIR and soluble insulin-like growth factor-1 receptor (sIGF1-R) levels are associated with sIR in HAND. Thirty-four (34) HIV-seropositive women stratified by cognitive status and five HIV-seronegative women were evaluated. In a subgroup of 20 HIV positive and 5 donors, binding of plasma insulin to sIR was determined by ELISA assay of residual insulin levels in plasma immuno-depleted with anti-IR-monoclonal antibody-Sepharose beads. sIR and sIGF1-R levels were determined by ELISA. Nonparametric statistics were used. Higher percentages of insulin bound to sIR significantly correlated with sIR levels and were associated with HAND status. Higher levels of plasma sIGF1-R had a positive correlation with sIR levels (p = 0.011) and were associated with HAND (p = 0.006). No correlations were observed with age, viral-immune profile, antiretroviral therapy, or TNF. This study suggests that changes in sIGF1-R levels and insulin binding to sIR may contribute to HAND.

The α7-nicotinic receptor is upregulated in immune cells from HIV-seropositive women: consequences to the cholinergic anti-inflammatory response.

Antiretroviral therapy partially restores the immune system and markedly increases life expectancy of HIV-infected patients. However, antiretroviral therapy does not restore full health. These patients suffer from poorly understood chronic inflammation that causes a number of AIDS and non-AIDS complications. Here we show that chronic inflammation in HIV+ patients may be due to the disruption of the cholinergic anti-inflammatory pathway by HIV envelope protein gp120IIIB. Our results demonstrate that HIV gp120IIIB induces α7 nicotinic acetylcholine receptor (α7) upregulation and a paradoxical proinflammatory phenotype in macrophages, as activation of the upregulated α7 is no longer capable of inhibiting the release of proinflammatory cytokines. Our results demonstrate that disruption of the cholinergic-mediated anti-inflammatory response can result from an HIV protein. Collectively, these findings suggest that HIV tampering with a natural strategy to control inflammation could contribute to a crucial, unresolved problem of HIV infection: chronic inflammation.

Cathepsin B and cystatin B in HIV-seropositive women are associated with infection and HIV-1-associated neurocognitive disorders.

OBJECTIVE: HIV-1-associated neurocognitive disorders (HAND) is triggered by immune activation of brain cells and remain prevalent during progressive viral infection despite antiretroviral therapy. Cathepsins and cystatins are lysosomal proteins secreted by macrophages and microglia, and may play important roles in neuroregulatory responses. Our laboratory has shown increased secretion and neurotoxicity of cathepsin B from in-vitro HIV-infected monocyte-derived macrophages, and increased expression in postmortem brain tissue with HIV encephalitis and HAND. We hypothesized that cystatin B and cathepsin B could represent potential biomarkers for HAND. METHODS: Monocytes, plasma, and cerebrospinal fluid (CSF) from retrospective samples from 63 HIV-seropositive Hispanic women were selected for this study. These were stratified as 27 normal, 14 asymptomatic, and 22 HIV dementia, and as 14 progressors and 17 nonprogressors. Samples were evaluated for cystatins B and C and cathepsin B expression and activity. RESULTS: Increased cathepsin B and cystatins B and C were found in plasma of HIV-seropositive women. Higher intracellular expression of cathepsin B and cystatin B were found in monocytes from women with HIV-associated dementia (P < 0.05). Significant increase in cystatin B concentration in CSF was found in women with dementia compared with HIV-seropositive asymptomatic women. CONCLUSION: These results demonstrate that dysregulation of cystatin B-cathepsin B system is operative in HIV-associated neurocognitive impairment and suggests that intracellular expression of cystatin B and cathepsin B in monocytes could be potential candidate biomarkers for HIV dementia, whereas increased cathepsin B and cystatins B and C in plasma are potential candidate markers of chronic HIV-1 activation.

Aliskiren, at low doses, reduces the electrical remodeling in the heart of the TGR(mRen2)27 rat independently of blood pressure.

METHODS: The influence of chronic administration of low doses of aliskiren (5 mg/kg/day, i.p.) for a period of eight weeks on cardiac electrophysiological and structural remodeling was investigated in transgenic (TGR)(mRen-2)27 rats. Cardiac and plasma angiotensin II (Ang II) levels were determined by ELISA before and after administration of the drug. Moreover, histological, electrophysiological and echocardiographic studies were performed in controls and at the end of eight weeks of aliskiren administration. RESULTS: 1) The cardiac Ang II levels were significantly reduced while the plasma Ang II levels were not significantly decreased in rats treated with low doses of aliskiren; 2) echocardographic studies showed a decrease of left ventricle diameter (LVD), left ventricle posterior wall thickness (LVPW), left ventricle end diastolic volume (LVEDV) and increased ejection fraction (EF); 3) aliskiren improved the impulse propagation, increased the cardiac refractoriness and reduced the incidence of triggered activity; 4) perivascular and interstitial fibrosis were greatly reduced, which explains the increase in conduction velocity. All these effects of aliskiren were found independently of blood pressure, suggesting that the beneficial effect of aliskiren was related to an inhibition of the local cardiac renin angiotensin system; and 5) the effect of mechanical stretch on action potential duration, conduction velocity and spontaneous rhythmicity was changed by aliskiren, supporting the hypothesis presented here that the beneficial effect of the drug on cardiac remodeling is related to a decreased sensitivity of cardiac muscle to mechanical stress.

Soluble and cell-associated insulin receptor dysfunction correlates with severity of HAND in HIV-infected women.

BACKGROUND: Blood sugar metabolism abnormalities have been identified in HIV-infected individuals and associated with HIV-associated neurocognitive disorders (HAND). These abnormalities may occur as a result of chronic HIV infection, long-term use of combined antiretroviral treatment (CART), aging, genetic predisposition, or a combination of these factors, and may increase morbidity and mortality in this population. OBJECTIVE: To determine if changes in soluble and cell-associated insulin receptor (IR) levels, IR substrate-1 (IRS-1) levels, and IRS-1 tyrosine phosphorylation are associated with the presence and severity of HAND in a cohort of HIV-seropositive women. METHODS AND RESULTS: This is a retrospective cross-sectional study using patient database information and stored samples from 34 HIV-seropositive women and 10 controls without history of diabetes from the Hispanic-Latino Longitudinal Cohort of Women. Soluble IR subunits [sIR, ectodomain (α) and full-length or intact (αß)] were assayed in plasma and CSF samples by ELISA. Membrane IR levels, IRS-1 levels, and IRS-1 tyrosine phosphorylation were analyzed in CSF white cell pellets (WCP) using flow cytometry. HIV-seropositive women had significantly increased levels of intact or full-length sIR in plasma (p<0.001) and CSF (p<0.005) relative to controls. Stratified by HAND, increased levels of full-length sIR in plasma were associated with the presence (p<0.001) and severity (p<0.005) of HAND. A significant decrease in IRS-1 tyrosine-phosphorylation in the WCP was also associated with the presence (p<0.02) and severity (p<0.02) of HAND. CONCLUSIONS: This study provides evidence that IR secretion is increased in HIV-seropositive women, and increased IR secretion is associated with cognitive impairment in these women. Thus, IR dysfunction may have a role in the progression of HAND and could represent a biomarker for the presence and severity of HAND.

Proteomic analyses of monocytes obtained from Hispanic women with HIV-associated dementia show depressed antioxidants.

PURPOSE: Monocyte ingress into the brain during progressive human immunodeficiency virus (HIV-1) infection parallels the severity of cognitive impairments. Although activated monocyte phenotypes emerge in disease, the functional correlates of these cells remain unresolved. EXPERIMENTAL DESIGN: To this end, we studied the proteome of blood-derived monocytes obtained from Hispanic women with the most severe form of HIV-associated neurocognitive disorders, HIV-associated dementia (HAD). Monocytes isolated from peripheral blood mononuclear cells by CD14+ immunoaffinity column chromatography were >95% pure. Cells were recovered from four patients without evidence of cognitive impairment and five with HAD and analyzed by 2-D DIGE and tandem MS. RESULTS: Importantly, ADP ribosylhydrolase, myeloperoxidase, thioredoxin, peroxiredoxin 3, NADPH, and GTPase-activating protein were all downregulated in HAD. In regards to myeloperoxidase, thioredoxin, and peroxiredoxin 3, these changes were validated in an additional cohort of 30 patients by flow cytometry. CONCLUSIONS AND CLINICAL RELEVANCE: We conclude that deficits in monocyte antioxidants lead to neuronal damage through the loss of hydrogen peroxide scavenging capabilities; thus exposing neurons to apoptosis-inducing factors. Altered monocyte functions therefore may contribute to the development and progression of cognitive impairment.

Prolonged exposure of cardiac cells to renin plus angiotensinogen reduces intracellular renin in the failing heart. On the role of angiotensin II-AT1 complex internalization.

UNLABELLED: To investigate the influence of prolonged exposure of cardiac cells to renin plus angiotensinogen (Ao) on intracellular renin levels, myocytes were isolated from the ventricle of cardiomyopathic hamsters(TO-2) and incubated in Krebs solution containing renin(128 pmol Ang ml/min) plus Ao (110 pmol Ang I generated by renin to exhaustion) for a period of 24 h. Membrane-bound and intracellular AT1 receptors levels as well as intracellular renin were studied using immunological methods and quantified by flow cytometry. The results indicated: a) intracellular renin levels were higher in the failing heart at an advanced stage of the disease (8 months) than in age-matched controls; b) the intracellular renin levels were significantly reduced in cells exposed to renin (128 pmol Ang I.ml/min) plus angiotensinogen (Ao)(110 pmol Ang I generated by renin to exhaustion) for a period of 24 h; c) incubation of the cardiomyocytes with renin (128 pmol Ang I.ml/min) alone did not reduced the intracellular renin levels; d) the fall of the intracellular renin level was related to the formation of angiotensin II (Ang II) at the surface cell membrane and internalization of the Ang II-AT1 complex because losartan (10(-7) M) added to the incubation medium containing renin plus Ao, blocked the internalization of AT1 and suppressed the decline of the intracellular renin levels; e) no internalization of renin or renin secretion was found in these experiments. IN CONCLUSION: prolonged exposure of cardiac cells to renin plus Ao (24 h) reduced intracellular renin levels through the internalization of Ang II-AT1 complex and inhibition of renin expression.

Eplerenone inhibits the intracrine and extracellular actions of angiotensin II on the inward calcium current in the failing heart. On the presence of an intracrine renin angiotensin aldosterone system.

UNLABELLED: The influence of chronic administration of eplerenone on the intracrine as well as on the extracellular action of angiotensin II (Ang II) on L-type inward calcium current was investigated in the failing heart of cardiomyopathic hamsters (TO-2).For this, eplerenone (200 mg/kg/day) was administered orally to 2 month-old cardiomyopathic hamsters for a period of 3 months. Measurements of the peak inward calcium current (I(Ca)) was performed in single cells under voltage clamp using the whole cell configuration. The results indicated that eplerenone suppressed the intracrine action of Ang II (10(-)(8) M) on peak I(Ca) density. Moreover, the intracellular dialysis of the peptide did not change the time course of I(Ca) inactivation in animals treated chronically with eplerenone. The extracellular administration of Ang II (10(-)(8) M) incremented the peak I(Ca) density by only 20+/-8% (n=30) compared with 38+/-4% (n=35) (P<0.05) obtained in age-matched cardiomyopathic hamsters not exposed to eplerenone. Interestingly, the inhibitory of eplerenone (10(-7) M) on the intracrine action of Ang II was also found, in vitro, but required an incubation period of, at least, 24 h. The inhibitory action of eplerenone on the intracellular action of Ang II was partially reversed by exposing the eplerenone-treated cells to aldosterone (10 nM) for a period of 24 h what supports the view that: a) the mineralocorticoid receptor(MR) was involved in the modulation of the intracrine action of the peptide; b) the effect of eplerenone on the intracrine as well as on the extracellular action of Ang II was related ,in part, to a decreased expression of membrane-bound and intracellular AT1 receptors. IN CONCLUSION: a) eplerenone inhibits the intracrine action of Ang II on inward calcium current and reduces drastically the effect of extracellular Ang II on I(Ca); b) aldosterone is able to revert the effect of eplerenone; c) the mineralocorticoid receptor is an essential component of the intracrine renin angiotensin aldosterone system.