Luminescent PLGA Nanoparticles for Delivery of Darunavir to the Brain and Inhibition of Matrix Metalloproteinase-9, a Relevant Therapeutic Target of HIV-Associated Neurological Disorders.

Human immunodeficiency virus (HIV) can independently replicate in the central nervous system (CNS) causing neurocognitive impairment even in subjects with suppressed plasma viral load. The antiretroviral drug darunavir (DRV) has been approved for therapy of HIV-infected patients, but its efficacy in the treatment of HIV-associated neurological disorders (HAND) is limited due to the low penetration through the blood-brain barrier (BBB). Therefore, innovations in DRV formulations, based on its encapsulation in optically traceable nanoparticles (NPs), may improve its transport through the BBB, providing, at the same time, optical monitoring of drug delivery within the CNS. The aim of this study was to synthesize biodegradable polymeric NPs loaded with DRV and luminescent, nontoxic carbon dots (C-Dots) and investigate their ability to permeate through an artificial BBB and to inhibit in vitro matrix metalloproteinase-9 (MMP-9) that represents a factor responsible for the development of HIV-related neurological disorders. Biodegradable poly(lactic-co-glycolic) acid (PLGA)-based nanoformulations resulted characterized by an average hydrodynamic size less than 150 nm, relevant colloidal stability in aqueous medium, satisfactory drug encapsulation efficiency, and retained emitting optical properties in the visible region of the electromagnetic spectrum. The assay on the BBB artificial model showed that a larger amount of DRV was able to cross BBB when incorporated in the PLGA NPs and to exert an enhanced inhibition of matrix metalloproteinase-9 (MMP-9) expression levels with respect to free DRV. The overall results reveal the great potential of this class of nanovectors of DRV for an efficacious treatment of HANDs.

Marine pharmacology: therapeutic targeting of matrix metalloproteinases in neuroinflammation.

Alterations in matrix metalloproteinase (MMP) expression and activity are recognized as key pathogenetic events in several neurological disorders. This evidence makes MMPs possible therapeutic targets. The search for substances that can inhibit MMPs is moving progressively toward the screening of natural products. In particular, marine bioprospecting could be promising for the discovery of marine natural products with anti-MMP activities. Despite recent advances in this field, the possibility of using marine MMP inhibitors (MMPIs) for the treatment of neuroinflammation is still under-investigated. Here, we review the latest findings in this promising research field and the potential that marine MMPIs can have in the management and treatment of various neurological diseases.

In vitro downregulation of matrix metalloproteinase-9 in rat glial cells by CCR5 antagonist maraviroc: therapeutic implication for HIV brain infection.

BACKGROUND: Matrix metalloproteinases (MMPs) released by glial cells are important mediators of neuroinflammation and neurologic damage in HIV infection. The use of antiretroviral drugs able to combat the detrimental effect of chronic inflammation and target the exaggerated MMP activity might represent an attractive therapeutic challenge. Recent studies suggest that CCR5 antagonist maraviroc (MVC) exerts immunomodulant and anti-inflammatory activity beyond its anti-HIV properties. We investigated the in vitro effect of MVC on the activity of MMPs in astrocyte and microglia cultures. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultures of rat astrocytes and microglia were activated by exposure to phorbol myristate acetate (PMA) or lypopolysaccharide (LPS) and treated in vitro with MVC. Culture supernatants were subjected to gelatin zymography and quantitative determination of MMP-9 and MMP-2 was done by computerized scanning densitometry. MMP-9 levels were significantly elevated in culture supernatants from both LPS- and PMA-activated astrocytes and microglia in comparison to controls. The treatment with MVC significantly inhibited in a dose-dependent manner the levels and expression of MMP-9 in PMA-activated astrocytes (p<0,05) and, to a lesser extent, in PMA-activated microglia. By contrast, levels of MMP-2 did not significantly change, although a tendency to decrease was seen in PMA-activated astrocytes after treatment with MVC. The inhibition of levels and expression of MMP-9 in PMA-activated glial cells did not depend on cytotoxic effects of MVC. No inhibition of MMP-9 and MMP-2 were found in both LPS-activated astrocytes and microglia. CONCLUSIONS: The present in vitro study suggests that CCR5 antagonist compounds, through their ability to inhibit MMP-9 expression and levels, might have a great potential for the treatment of HIV-associated neurologic damage.

Apolipoprotein(a) inhibits lipopolysaccharide-induced IL-6 secretion in human astrocytoma cell line by interfering with lipopolysaccharide signaling.

OBJECTIVE: To examine the role of lipoprotein(a) [Lp(a)] on the inflammatory response of cells in the nervous system by investigating its effect on lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) secretion. MATERIALS AND METHODS: Human astrocytoma U373 cells were treated with recombinant apolipoprotein(a) [r-apo(a)] A10K (175-11 nM), alone or in combination with LPS (100 and 10 ng/ml). IL-6 levels were evaluated by immunoblotting. Statistical analysis was performed by one-way ANOVA. RESULTS: r-apo(a) caused dose-dependent inhibition of LPS-induced IL-6 secretion (100 ng/ml LPS, p = 0.0205; 10 ng/ml LPS, p = 0.0005). Pre-treatment of cells with 88 nM r-apo(a), rinsing, and activation with 10 ng/ml LPS did not reverse the inhibition (p = 0.0048), which could be reversed by supplementation with excess serum (5-20%) (p = 0.0454) or recombinant CD14 (2.0-0.05 µg/ml) (p = 0.0230). CONCLUSIONS: Our data indicate that apo(a) plays a natural anti-endotoxin role which relies on its interference with cell-associated and serum components of LPS signaling.

Matrix metalloproteinase dysregulation in HIV infection: implications for therapeutic strategies.

The emerging role of immune activation and inflammation in the pathogenesis of human immunodeficiency virus (HIV) disease has stimulated the search for new approaches for managing HIV infection. Recent evidence suggests that an imbalance between matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of MMPs (TIMPs) might contribute to HIV-associated pathology by inducing remodelling of the extracellular matrix. Here, we discuss the evidence and the potential mechanisms for altered MMP or TIMP function in HIV infection and disease. Furthermore, we outline the possible medical implications for the use of compounds that target MMP activity, and we propose that antiretroviral drugs, particularly HIV protease inhibitors (PIs), and compounds with anti-inflammatory properties, such as statins, natural omega-3 fatty acids and tetracyclines, which inhibit MMP function, might represent useful therapeutic approaches to mitigate potential MMP-related damage during HIV infection.

Antiretroviral therapy inhibits matrix metalloproteinase-9 from blood mononuclear cells of HIV-infected patients.

OBJECTIVE: To investigate whether antiretroviral therapy (ART) influences the release of matrix metalloproteinase (MMP)-9 from peripheral blood mononuclear cells (PBMC) of HIV-infected individuals. DESIGN: Culture supernatants were collected from PBMC isolated from 46 HIV-infected subjects and 19 healthy donors (HD). Among the HIV-infected subjects, 30 were receiving ART therapy, while 16 were naive for any ART treatment. METHODS: Zymography of culture supernatants was followed by determination of MMP-9 using computered scanning densitometry. MMP-9 net enzymatic activity was determined by the sensitive fluorescent-activated substrate conversion assay (FASC) to complement the zymography data. RESULTS: MMP-9 was significantly elevated in culture supernatants from PBMC of ART-naive subjects in comparison with HD. In the supernatants from 30 HIV-infected subjects receiving ART therapy, MMP-9 was significantly lower than that in those from ART-naive subjects. Analysis by the reverse transcriptase polymerase chain reaction indicated that MMP-9 expression was increased in ART-naive subjects in comparison with HD but ART induced a decrease of MMP-9 expression to levels comparable with those of HD. FASC used as a functional assay showed conversion of fluorescent gelatine in ART-naive subjects, indicating the presence of active MMP-9. By contrast, in both HD and ART-treated subjects, there was no MMP-9 activity, indicating that MMP-9 was completely blocked by binding to its natural tissue inhibitor TIMP-1. CONCLUSIONS: The present findings show for the first time that ART can reduce the capacity of PBMC from HIV-infected patients to secrete increased amounts of MMP-9.

Autoimmune myelofibrosis: report of three cases and review of the literature.

Autoimmune myelofibrosis is a distinct clinicopathological entity, recognizing immunopathogenetic mechanisms and occurring isolately or in association with systemic and/or organ-specific autoimmune diseases. It results in chronic cytopenias, and is defined by a pattern including bone marrow, peripheral blood, serological and clinical features. It has to be distinguished from other disorders having myelofibrosis. Among these, the most relevant differential diagnosis is with chronic idiopathic myelofibrosis, particularly when disclosing autoimmune clinical and/or laboratory features as epiphenomenon related to a secondary immune-dysregulation. Here we report on 3 patients admitted because of chronic cytopenias. In all of them, the clinicopathological evaluation essentially demonstrated myelofibrosis, not clustered megakaryocytes, reactive lymphoid infiltration in marrow biopsies, absence of significant tear-drop poikilocytosis and leukoerythroblastosis on peripheral blood smears, normal-sized spleen, positive autoimmune serology. The resulting patterns met the diagnosis of autoimmune myelofibrosis occurring isolately in a patient and associated with Sjögren's syndrome or concomitant Sjögren's syndrome and Hashimoto's thyroiditis in the other two, respectively. Transient improvements in cytopenias and unmodified myelofibrosis were observed following corticosteroid treatment. It is noteworthy the lack of a specific therapy, being the underlying pathophisiology of myelofibrosis still unclear. In conclusion, increased awareness of the clinicopathological pattern identifying autoimmune myelofibrosis is recommended in order to improve basic and clinical knowledge of this emerging entity.

Matrix metalloproteinase-9 and tissue inhibitors of matrix metalloproteinase-1 in plasma of patients co-infected with HCV and HIV.

BACKGROUND: Accelerated progression of hepatic fibrosis has been shown in patients co-infected with hepatitis C virus (HCV) and HIV. Liver fibrosis is a dynamic process in which the altered balance between matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) may play a major role. METHOD: The involvement of MMP-9 and TIMP-1 in HCV liver disease progression in patients co-infected with HIV was evaluated. Plasma concentrations of human MMP-9 and TIMP-1 were assessed in 76 HIV-infected patients; 27 were co-infected with HCV and 49 were HCV negative. 18 healthy donors were included as controls. RESULTS: Patients with HIV infection exhibited a striking increase in TIMP-1 levels; this is more evident in patients with advanced CD4 depletion. There was no elevation in the plasma concentrations of the MMP-9. The highest levels of TIMP-1 were found in the HIV/HCV co-infected patients. The values of TIMP-1 in HIV-infected patients with chronic HCV hepatitis were significantly higher than in HIV-positive individuals without HCV infection, even including those with low CD4 count. No significant differences were seen in the MMP-9 levels. CONCLUSION: These findings suggest that the altered balance between circulating MMP-9 and TIMP-1 during HIV infection may play an important role in exacerbating liver fibrosis progression in patients co-infected with HCV.

T-cell response to myelin basic protein and lipid-bound myelin basic protein in patients with multiple sclerosis and healthy donors.

An autoimmune T-cell response to myelin proteins is thought to be involved in the pathogenesis of multiple sclerosis (MS) and myelin basic protein (MBP) is the most widely studied potential target antigen. We investigated the T-cell response to MBP in MS patients and controls using two different molecular forms of the protein: the classical hydrophilic MBP (lipid-free MBP, LF-MBP) and a lipid-bound, native-like preparation of MBP isolated in a molecular form retaining the binding to all myelin lipids (lipid-bound-MBP, LB-MBP). Short term T-cell lines (TCL) were generated using either LF- or LB-MBP and tested for their reactivity to the in vitro stimulating antigen. No differences were detected between MS patients and healthy donors in the percentage of T-cell cultures responsive to the LF-MBP. In contrast, the number of LB-MBP reactive cultures was higher in MS patients than in controls. This difference was almost entirely due to the presence of high numbers of LB-MBP-specific TCL in MS patients which did not cross-react with LF-MBP and were not present in healthy subjects. LB-MBP may represent a novel antigen worth to be investigated in MS.