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The development of effective combined anti-retroviral therapy (cART) led to a significant reduction in the death rate associated with human immunodeficiency virus type 1 (HIV-1) infection. However, recent studies indicate that considerably more than 50% of all HIV-1 infected patients develop HIV-1-associated neurocognitive disorder (HAND). Microglia are the foremost cells infected by HIV-1 in the central nervous system (CNS), and so, are also likely to contribute to the neurotoxicity observed in HAND. The activation of microglia induces the release of pro-inflammatory markers and altered secretion of cytokines, chemokines, secondary messengers, and reactive oxygen species (ROS) which activate signalling pathways that initiate neuroinflammation. In turn, ROS and inflammation also play critical roles in HAND. However, more efforts are required to understand the physiology of microglia and the processes involved in their activation in order to better understand the how HIV-1-infected microglia are involved in the development of HAND. In this review, we summarize the current state of knowledge about the involvement of oxidative stress mechanisms and role of HIV-induced ROS in the development of HAND. We also examine the academic literature regarding crucial HIV-1 pathogenicity factors implicated in neurotoxicity and inflammation in order to identify molecular pathways that could serve as potential therapeutic targets for treatment of this disease.KEY MESSAGESNeuroinflammation and excitotoxicity mechanisms are crucial in the pathogenesis of HAND.CNS infiltration by HIV-1 and immune cells through the blood brain barrier is a key process involved in the pathogenicity of HAND.Factors including calcium dysregulation and autophagy are the main challenges involved in HAND., Graphical Abstract

Ongoing with current combinations of antiretroviral drugs for the treatment of Human Immunodeficiency Virus (HIV) infection can successfully maintain long-term suppression of HIV-1 replication in plasma. Still, none of these therapies is capable of extinguishing the virus from the long-lived cellular reservoir, including monocyte-derived macrophages (MDM), that means the principal obstacle to HIV cure. MDM are widely distributed in all tissues and organs, including central system nervous (CNS) where they represent the most frequent HIV-infected cells that means the principal obstacle to HIV cure. Current FDA-approved antiretroviral drugs target viral reverse transcriptase, protease, integrase, and entry processes (coreceptor or fusion blockade). It is desirable to continue to develop new antiretrovirals directed against alternative targets in the virus lifecycle in order to further optimize therapeutic options, overcome resistance to existing medications, and potentially contribute to the elimination of viral reservoirs. This review provides a comprehensive overview of the activity of antiretroviral drugs (classical and upcoming) in monocytes-derived macrophages (MDM). Defining the antiviral activity of these drugs in this important cellular HIV-1 reservoir provides crucial hints about their efficacy in HIV-1 infected patients.

Candida albicans is a commensal fungus that causes systemic infections in immunosuppressed patients. In order to deal with the changing environment during commensalism or infection, C. albicans must reprogram its proteome. Characterizing the stress-induced changes in the proteome that C. albicans uses to survive should be very useful in the development of new antifungal drugs. We studied the C. albicans global proteome after exposure to hydrogen peroxide (H2O2) and acetic acid (AA), using a data-independent acquisition mass spectrometry (DIA-MS) strategy. More than 2,000 C. albicans proteins were quantified using an ion library previously constructed using data-dependent acquisition mass spectrometry (DDA-MS). C. albicans responded to treatment with H2O2 with an increase in the abundance of many proteins involved in the oxidative stress response, protein folding, and proteasome-dependent catabolism, which led to increased proteasome activity. The data revealed a previously unknown key role for Prn1, a protein similar to pirins, in the oxidative stress response. Treatment with AA resulted in a general decrease in the abundance of proteins involved in amino acid biosynthesis, protein folding, and rRNA processing. Almost all proteasome proteins declined, as did proteasome activity. Apoptosis was observed after treatment with H2O2 but not AA. A targeted proteomic study of 32 proteins related to apoptosis in yeast supported the results obtained by DIA-MS and allowed the creation of an efficient method to quantify relevant proteins after treatment with stressors (H2O2, AA, and amphotericin B). This approach also uncovered a main role for Oye32, an oxidoreductase, suggesting this protein as a possible apoptotic marker common to many stressors. IMPORTANCE Fungal infections are a worldwide health problem, especially in immunocompromised patients and patients with chronic disorders. Invasive candidiasis, caused mainly by C. albicans, is among the most common fungal diseases. Despite the existence of treatments to combat candidiasis, the spectrum of drugs available is limited. For the discovery of new drug targets, it is essential to know the pathogen response to different stress conditions. Our study provides a global vision of proteomic remodeling in C. albicans after exposure to different agents, such as hydrogen peroxide, acetic acid, and amphotericin B, that can cause apoptotic cell death. These results revealed the significance of many proteins related to oxidative stress response and proteasome activity, among others. Of note, the discovery of Prn1 as a key protein in the defense against oxidative stress as well the increase in the abundance of Oye32 protein when apoptotic process occurred point them out as possible drug targets.

The current use of combined antiretroviral therapy (cART) is leading to a significant decrease in deaths and comorbidities associated with human immunodeficiency virus type 1 (HIV-1) infection. Nonetheless, none of these therapies can extinguish the virus from the long-lived cellular reservoir, including microglia, thereby representing an important obstacle to curing HIV. Microglia are the foremost cells infected by HIV-1 in the central nervous system (CNS) and are believed to be involved in the development of HIV-1-associated neurocognitive disorder (HAND). At present, the pathological mechanisms contributing to HAND remain unclear, but evidence suggests that removing these infected cells from the brain, as well as obtaining a better understanding of the specific molecular mechanisms of HIV-1 latency in these cells, should help in the design of new strategies to prevent HAND and achieve a cure for these diseases. The goal of this review was to study the current state of knowledge of the neuropathology and research models of HAND containing virus susceptible target cells (microglial cells) and potential pharmacological treatment approaches under investigation.

Dopamine and its 5 receptors, which are grouped into two families (D1-like and D2-like), modulate functions at a systemic level in both the central nervous system and periphery. The central nervous system and the immune system are the main adaptive systems, which participate in a continuous and functional crosstalk to guarantee homeostasis. On binding to its 5 dopamine receptors, dopamine acts as a co-regulator of the immune system, contributing to the interaction of the central nervous system and inflammatory events and as a source of communication between the different immune cells. Dopaminergic perturbations in the central nervous system are observed in several neurological and psychiatric disorders. Schizophrenia is one of the most common mental disorders with a poorly understood pathoaetiology that includes genetic and environmental components that promote alterations in the dopaminergic system. Interestingly, abnormalities in dopamine receptors expression in lymphocytes of schizophrenia patients have been reported, often significantly correlating with the severity of the psychotic illness. Here, we review the current literature regarding the dopaminergic system in human lymphocytes and its alterations in schizophrenia., Highlights 1. The existence of DA in the bloodstream suggests the presence of dopaminergic components that modulate functions at a systemic level; therefore, its effects are not limited to the CNS and the signalling in the neuronal dopaminergic system should be independent from that of the peripheral systems. 2. The effects by DA-mediated activation of different DRs on immune cells show different sensitivities to DA, but binding profiles of DA on T cells are similar to those in neuronal membranes, suggesting receptors act similarly to those found in neurons. 3. All DRs are expressed on the LYM membrane. However, more detailed information is required on the expression patterns of DR in immune cells in healthy conditions and in pathologies. 4. DA has been observed to influence LYM functions acting in a variety of important processes, like cytokine secretion, cell adhesion, chemotaxis, and cytotoxicity. 5. In human LYM, DA on D1-like receptors decreases oxidative metabolism and apoptosis, activates the selective secretion of IL-10 and TNFα, and facilitates NK cells. In contrast, most of the immunostimulatory DA effects on LYM depend on stimulation of D2-like receptors including activation, proliferation, differentiation, and suppression of NK cells. 6. To date, an altered expression or signalling of neurotransmitter receptors is observed in immune cells during psychiatric disorders and, consequently, these cells also markedly respond to antipsychotics. 7. Numerous technologies have been used in search of biomarkers for SCZ. However, after a century of studying SCZ their application in psychiatry remains rare and there are currently no validated biomarkers for the diagnosis and prognosis of patients with SCZ or the prediction of treatment efficacy.

Background and objectives: To enter the target cell, HIV-1 binds not only CD4 but also a co-receptor &beta, chemokine receptor 5 (CCR5) or &alpha, chemokine receptor 4 (CXCR4). Limited information is available on the impact of co-receptor usage on HIV-1 replication in monocyte-derived macrophages (MDM) and on the homeostasis of this important cellular reservoir. Materials and Methods: Replication (measured by p24 production) of the CCR5-tropic 81A strain increased up to 10 days post-infection and then reached a plateau. Conversely, the replication of the CXCR4-tropic NL4.3 strain (after an initial increase up to day 7) underwent a drastic decrease becoming almost undetectable after 10 days post-infection. The ability of CCR5-tropic and CXCR4-tropic strains to induce cell death in MDM was then evaluated. While for CCR5-tropic 81A the rate of apoptosis in MDM was comparable to uninfected MDM, the infection of CXCR4-tropic NL4.3 in MDM was associated with a rate of 14.3% of apoptotic cells at day 6 reaching a peak of 43.5% at day 10 post-infection. Results: This suggests that the decrease in CXCR4-tropic strain replication in MDM can be due to their ability to induce cell death in MDM. The increase in apoptosis was paralleled with a 2-fold increase in the phosphorylated form of p38 compared to WT. Furthermore, microarray analysis showed modulation of proapoptotic and cancer-related genes induced by CXCR4-tropic strains starting from 24 h after infection, whereas CCR5 viruses modulated the expression of genes not correlated with apoptotic-pathways. Conclusions: In conclusion, CXCR4-tropic strains can induce a remarkable depletion of MDM. Conversely, MDM can represent an important cellular reservoir for CCR5-tropic strains supporting the role of CCR5-usage in HIV-1 pathogenesis and as a pharmacological target to contribute to an HIV-1 cure.

The local renin-angiotensin system (RAS) and insulin-like growth factor 1 (IGF-1) have been involved in longevity, neurodegeneration and aging-related dopaminergic degeneration. However, it is not known whether IGF-1 and angiotensin-II (AII) activate each other. In the present study, AII, via type 1 (AT1) receptors, exacerbated neuroinflammation and dopaminergic cell death. AII, via AT1 receptors, also increased the levels of IGF-1 and IGF-1 receptors in microglial cells. IGF-1 inhibited RAS activity in dopaminergic neurons and glial cells, and also inhibited the AII-induced increase in markers of the M1 microglial phenotype. Consistent with this, IGF-1 decreased dopaminergic neuron death induced by the neurotoxin MPP+ both in the presence and in the absence of glia. Intraventricular administration of AII to young rats induced a significant increase in IGF-1 expression in the nigral region. However, aged rats showed decreased levels of IGF-1 relative to young controls, even though RAS activity is known to be enhanced in aged animals. The study findings show that IGF-1 and the local RAS interact to inhibit or activate neuroinflammation (i.e. transition from the M1 to the M2 phenotype), oxidative stress and dopaminergic degeneration. The findings also show that this mechanism is impaired in aged animals.

The neuroprotective effects of menopausal hormonal therapy in Parkinson's disease have not yet been clarified, and it is not known whether there is a critical period. Estrogen induced significant protection against 6-hydroxydopamine-induced dopaminergic degeneration when administered immediately or 6 weeks, but not 20 weeks after ovariectomy. In the substantia nigra, ovariectomy induced a decrease in levels of estrogen receptor-α and increased angiotensin activity, NADPH-oxidase activity, and expression of neuroinflammatory markers, which were regulated by estrogen administered immediately or 6 weeks but not 20 weeks after ovariectomy. Interestingly, treatment with angiotensin receptor antagonists after the critical period induced a significant level of neuroprotection. In cultures, treatment with 1-methyl-4-phenylpyridinium induced an increase in astrocyte-derived angiotensinogen and dopaminergic neuron death, which were inhibited by estrogen receptor α agonists. In microglial cells, estrogen receptor β agonists inhibited the angiotensin-induced increase in inflammatory markers. The results suggest that there is a critical period for the neuroprotective effect of estrogen against dopaminergic cell death, and local estrogen receptor α and renin-angiotensin system play a major role.

A paucity of information is available on the activity of protease inhibitors (PI) in chronically-infected monocyte-derived macrophages (MDM) and on the kinetics of viral-rebound after PI removal in vitro. To fill this gap, the activity of different concentrations of amprenavir (AMP) was evaluated in chronically-infected MDM by measuring p24-production every day up to 12 days after drug administration and up to seven days after drug removal. Clinically-relevant concentrations of AMP (4 and 20 μM) drastically decreased p24 amount released from chronically-infected MDM from Day 2 up to Day 12 after drug administration. The kinetics of viral-rebound after AMP-removal (4 and 20 μM) showed that, despite an initial increase, p24-production over time never reached the level observed for untreated-MDM, suggesting a persistent intracellular drug activity. In line with this, after AMP-removal, human immunodeficiency virus 1 (HIV-1) infectivity and intracellular the p24/p55 ratio (reflecting virion-maturation) were remarkably lower than observed for untreated MDM. Overall, AMP shows high efficacy in blocking HIV-1 replication in chronically-infected MDM, persisting even after drug-removal. This highlights the role of protease inhibitors in preventing the establishment of this important HIV-1 reservoir, thus reducing viral-dissemination in different anatomical compartments.

Previous studies have shown that the brain renin-angiotensin system may play a major role, via angiotensin type 1 (AT1) receptors, in the regulation of neuroinflammation, oxidative stress and progression of dopaminergic degeneration. Angiotensin-induced activation of the microglial nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex and microglial Rho-kinase are particularly important in this respect. However, it is not known whether crosstalk between Rho-kinase and NADPH-oxidase leads to microglial activation. In the present study, we found that, in the substantia nigra of rats, NADPH-oxidase activation was involved in angiotensin-induced Rho-kinase activation, which, in turn, was involved in angiotensin-induced NADPH-oxidase activation. In N9 microglial cell line and primary microglial cultures, a crosstalk signaling between NADPH-oxidase and Rho-kinase occurred in a positive feedback fashion during angiotensin-induced microglial activation. Angiotensin-induced NADPH-oxidase activation and superoxide generation led to NF-кB translocation and Rho-kinase activation. Rho-kinase activation was involved in regulation of NADPH-oxidase activation via p38 mitogen-activated protein kinase. Moreover, Rho-kinase activation, via NF-кB, upregulated AT1 receptor expression in microglial cells through a feed-forward mechanism. NADPH-oxidase and Rho-kinase pathways are known to be responsible for major components of the microglial response, such as changes involving microglial motility and phagocytosis, generation of superoxide, and release of inflammatory cytokines. The present results show that both pathways are linked by a common mechanism that may constitute a basic means of coordinating the microglial response.

The small GTP-binding protein Rho plays an important role in several cellular functions. RhoA, which is a member of the Rho family, initiates cellular processes that act on its direct downstream effector Rho-associated kinase (ROCK). ROCK inhibition protects against dopaminergic cell death induced by dopaminergic neurotoxins. It has been suggested that ROCK inhibition activates neuroprotective survival cascades in dopaminergic neurons. Axon-stabilizing effects in damaged neurons may represent another mechanism of neuroprotection of dopaminergic neurons by ROCK inhibition. However, it has been shown that microglial cells play a crucial role in neuroprotection by ROCK inhibition and that activation of microglial ROCK mediates major components of the microglial inflammatory response. Additional mechanisms such as interaction with autophagy may also contribute to the neuroprotective effects of ROCK inhibition. Interestingly, ROCK interacts with several brain factors that play a major role in dopaminergic neuron vulnerability such as NADPH-oxidase, angiotensin, and estrogen. ROCK inhibition may provide a new neuroprotective strategy for Parkinson’s disease. This is of particular interest because ROCK inhibitors are currently used against vascular diseases in clinical practice. However, it is necessary to develop more potent and selective ROCK inhibitors to reduce side effects and enhance the efficacy.

Although the renin-angiotensin system (RAS) was classically considered as a circulating system that regulates blood pressure, many tissues are now known to have a local RAS. Angiotensin, via type 1 receptors, is a major activator of the NADPH-oxidase complex, which mediates several key events in oxidative stress and inflammatory processes involved in the pathogenesis of major aging-related diseases. Several studies have observed the presence of RAS components in the basal ganglia, and particularly in the nigrostriatal system. In the nigrostriatal system, RAS hyperactivation, via NADPH-oxidase complex activation, exacerbates oxidative stress and the microglial inflammatory response and contributes to progression of dopaminergic degeneration, which is inhibited by angiotensin receptor blockers and angiotensin converting enzyme inhibitors. Several factors may induce an increase in RAS activity in the dopaminergic system. A decrease in dopaminergic activity induces a compensatory upregulation of local RAS function in both dopaminergic neurons and glia. In addition to its role as an essential neurotransmitter, dopamine may also modulate microglial inflammatory responses and neuronal oxidative stress via RAS. Important counterregulatory interactions between angiotensin and dopamine have also been observed in several peripheral tissues. Neurotoxins and proinflammatory factors may also act on astrocytes to induce an increase in RAS activity independent or before the loss of dopamine. Consistent with a major role of RAS in dopaminergic vulnerability, increased RAS activity has been observed in the nigra of animal models of aging, menopause and chronic cerebral hypoperfusion, which also showed higher dopaminergic vulnerability. Manipulation of the brain RAS may constitute an effective neuroprotective strategy against dopaminergic vulnerability and progression of Parkinson’s disease.

Several recent studies have shown that activation of the RhoA/Rho-associated kinase (ROCK) pathway is involved in the MPTP-induced dopaminergic cell degeneration and possibly in Parkinson's disease. ROCK inhibitors have been suggested as candidate neuroprotective drugs for Parkinson's disease. However, the mechanism responsible for the increased survival of dopaminergic neurons after treatment with ROCK inhibitors is not clear. We exposed primary (neuron-glia) mesencephalic cultures, cultures of the MES 23.5 dopaminergic neuron cell line and primary mesencephalic cultures lacking microglial cells to the dopaminergic neurotoxin MPP+ and the ROCK inhibitor Y-27632 in order to study the effects of ROCK inhibition on dopaminergic cell loss and the length of neurites of surviving dopaminergic neurons. In primary (neuron-glia) cultures, simultaneous treatment with MPP+ and the ROCK inhibitor significantly reduced the loss of dopaminergic neurons. In the absence of microglia, treatment with the ROCK inhibitor did not induce a significant reduction in the dopaminergic cell loss. Treatment with the ROCK inhibitor induced a significant decrease in axonal retraction in primary cultures with and without microglia and in cultures of the MES 23.5 neuron cell line. In conclusion, inhibition of microglial ROCK is essential for the neuroprotective effects of ROCK inhibitors against cell death induced by the dopaminergic neurotoxin MPP+. In addition, ROCK inhibition induced a direct effect against axonal retraction in surviving neurons. However, the latter effect was not sufficient to cause a significant increase in the survival of dopaminergic neurons after treatment with MPP+.

In vitro and in vivo models of Parkinson's disease were used to investigate whether TNF-α plays a major role in the enhancement of the microglial response and dopaminergic degeneration induced by brain angiotensin hyperactivity. Treatment of primary mesencephalic cultures with low doses of the neurotoxin MPP(+) induced a significant loss of dopaminergic neurons, which was enhanced by cotreatment with angiotensin II and inhibited by TNF-α inhibitors. Treatment of primary cultures with angiotensin induced a marked increase in levels of TNF-α, which was inhibited by treatment with angiotensin type-1-receptor antagonists, NADPH-oxidase inhibitors and NFK-β inhibitors. However, TNF-α levels were not significantly affected by treatment with angiotensin in the absence of microglia. The microglial origin of the angiotensin-induced increase in TNF-α levels was confirmed using dopaminergic (MES 23.5) and microglial (N9) cell lines. Inhibition of the microglial Rho-kinase activity also blocked the AII-induced increase in TNF-α levels. Treatment of the dopaminergic cell line with TNF-α revealed that NFK-β activation mediates the deleterious effect of microglial TNF-α on dopaminergic neurons. Treatment of mice with MPTP also induced significant increases in striatal and nigral TNF-α levels, which were inhibited by angiotensin type-1-receptor antagonists or NFK-β inhibitors. The present results show that microglial TNF-α plays a major role in angiotensin-induced dopaminergic cell death and that the microglial release of TNF-α is mediated by activation of angiotensin type-1 receptors, NADPH-oxidase, Rho-kinase and NFK-β.

Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption. Proteomic studies in legumes have increased significantly in the last years but few studies have been performed to date in P. vulgaris. We report here a proteomic analysis of bean seeds by two-dimensional electrophoresis (2-DE). Three different protein extraction methods (TCA–acetone, phenol and the commercial clean-up kit) were used taking into account that the extractome can have a determinant impact on the level of quality of downstream protein separation and identification. To demonstrate the quality of the 2-DE analysis, a selection of 50 gel spots was used in protein identification by mass spectrometry (MALDI-TOF MS and MALDI-TOF/TOF). The results showed that a considerable proportion of spots (70%) were identified in spite of incomplete genome/protein databases for bean and other legume species. Most identified proteins corresponded to storage protein, carbohydrate metabolism, defense and stress response, including proteins highly abundant in the seed of P. vulgaris such as the phaseolin, the phytohemagglutinin and the lectin-related α-amylase inhibitor.