Diazabicyclo analogues of maraviroc: synthesis, modeling, NMR studies and antiviral activity.

Two diazabicyclo analogues of maraviroc, in which the azabicyclooctane moiety is replaced by diazabicyclooctane or diazabicyclononane, were synthesized and tested, through a viral neutralization assay, on a panel of six pseudoviruses. The diazabicyclooctane derivative maintained a significant infectivity reduction power, whereas the diazabicyclononane was less effective. Biological data were rationalized through a computational study that allowed the conformational preferences of the compounds to be determined and a correlation between the inhibitory activity, the bridge length of the bicycle, and the rotational barrier around dihedral angle τ7 to be hypothesized. A high-field NMR analysis supported the modeling results.

Virus like particle based strategy to elicit HIV-protective antibodies to the alpha-helic regions of gp41.

Natural antibodies to gp41 inhibit HIV-1 replication through the recognition of two different regions, corresponding to the leucine zipper motif in the HR1 alpha-helix and to another motif within HR2 region, hosting 2F5 and 4E10 epitope. This study aimed at reproducing such protective responses through VLP vaccination. Six regions covering the alpha-helical regions of gp41 were conjugated to the surface of AP205 phage-based VLPs. Once administered in mice via systemic or mucosal route, these immunogens elicited high titers of gp41-specific IgG. Immunogenicity and HIV infectivity reduction were obtained either with HR2 regions or with peptides where aminoacid strings were added to either the C-terminus or N-terminus of core epitope in HR1 region. Antibody-dependent cell cytotoxicity (ADCC) activity was induced by one of the HR2 epitopes only. These results may have relevant implications for the development of new vaccinal approaches against HIV infection.

Passively transmitted gp41 antibodies in babies born from HIV-1 subtype C-seropositive women: correlation between fine specificity and protection.

HIV-exposed, uninfected (EUN) babies born to HIV-infected mothers are examples of natural resistance to HIV infection. In this study, we evaluated the titer and neutralizing potential of gp41-specific maternal antibodies and their correlation with HIV transmission in HIV-infected mother-child pairs. Specific gp41-binding and -neutralizing antibodies were determined in a cohort of 74 first-time mother-child pairs, of whom 40 mothers were infected with HIV subtype C. Within the infected mother cohort, 16 babies were born infected and 24 were PCR negative and uninfected at birth (i.e., exposed but uninfected). Thirty-four HIV-uninfected and HIV-unexposed mother-child pairs were included as controls. All HIV-positive mothers and their newborns showed high IgG titers to linear epitopes within the HR1 region and to the membrane-proximal (MPER) domain of gp41; most sera also recognized the disulfide loop immunodominant epitope (IDE). Antibody titers to the gp41 epitopes were significantly lower in nontransmitting mothers (P < 0.01) and in the EUN babies (P < 0.005) than in HIV-positive mother-child pairs. Three domains of gp41, HR1, IDE, and MPER, elicited antibodies that were effectively transmitted to EUN babies. Moreover, in EUN babies, epitopes overlapping the 2F5 epitope (ELDKWAS), but not the 4E10 epitope, were neutralization targets in two out of four viruses tested. Our findings highlight important epitopes in gp41 that appear to be associated with exposure without infection and would be important to consider for vaccine design.

Acute lipophilicity-dependent effect of intravascular simvastatin in the early phase of focal cerebral ischemia.

The acute effects of simvastatin lactone (lipophilic) and simvastatin acid (hydrophilic) on transient focal ischemia were assessed using the isolated guinea pig brain maintained in vitro by arterial perfusion. This new model of cerebral ischemia allows the assessment of the very early phase of the ischemic process, with the functional preservation of the vascular and neuronal compartments and the blood-brain barrier (bbb). The middle cerebral artery was transiently tied for 30 min followed by reperfusion for 60 min. Statins (nanomolar doses) were administered by intravascular continuous infusion starting 60 min before ischemia induction. Brain cortical activity and arterial vascular tone were continuously recorded. At the end of the experiment immunoreactivity for microtubule-associated protein 2 (MAP-2), expression of survival kinases (ERK and Akt) and total anti-oxidant capacity were assayed. Brains treated with simvastatin lactone showed i) reduced amplitude and delayed onset of ischemic depressions, ii) preservation of MAP-2 immunoreactivity, iii) activation of ERK signaling in the ischemic hemisphere and iv) increase in whole-brain anti-oxidant capacity. Treatment with the bbb-impermeable simvastatin acid was ineffective on the above-mentioned parameters. Vascular resistance recordings and Akt signaling were unchanged by any statin treatment. Our findings suggest that intravascular-delivered simvastatin exerts an acute lipophilicity-dependent protective effect in the early phase of cerebral ischemia.

Activation of cerebral endothelium is required for mononuclear cell recruitment in a novel in vitro model of brain inflammation.

Brain inflammation is a common event in the pathogenesis of several neurological diseases. It is unknown whether leukocyte/endothelium interactions are sufficient to promote homing of blood-borne cells into the brain compartment. The role of mononuclear cells and endothelium was analyzed in a new experimental model, the isolated guinea-pig brain maintained in vitro by arterial perfusion. This preparation allows one to investigate early steps of brain inflammation that are impracticable in vivo. We demonstrate by confocal microscopy analysis that in vitro co-perfusion of pro-inflammatory agents and pre-activated fluorescent mononuclear cells induced endothelial expression of selectins and intracellular adhesion molecule-1 in correspondence of arrested mononuclear cells, and correlates with a moderate increase in blood-brain barrier permeability. Separate perfusion of pro-inflammatory agents and mononuclear cells induced neither mononuclear cell adhesion nor adhesion molecule expression. We demonstrate that co-activation of mononuclear cells and cerebral endothelium is an essential requirement for cell arrest and adhesion in the early stages of experimental cerebral inflammation.

Induction of murine mucosal CCR5-reactive antibodies as an anti-human immunodeficiency virus strategy.

The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4+ cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4+ lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4+ cells from both humans and untreated mice, (iii) inhibit Mip-1beta chemotaxis of CD4+ CCR5+ lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.

Catecholamine-induced regulation in vitro and ex vivo of intralymphocyte ionized magnesium.

Despite the importance of the adrenergic activity and of the metabolism of magnesium in some important cardiovascular pathologies, very little is known about how intracellular ionized magnesium (Mgi2+) is regulated by catecholamines. We made an in-vitro study of the variations in the concentration of ionized magnesium in human lymphocytes using the fluorescent probe furaptra in response to different catecholamines. We also made an ex-vivo study of the changes in intracellular ionized magnesium in lymphocytes in 20 subjects with essential arterial hypertension, 10 treated with 120 mg/d of propranolol and 10 with placebo. Norepinephrine and isoproterenol significantly decrease Mgi2+ and this effect is blocked by beta-blockers but not by alpha-blockers. The EC50 of the effect of norepinephrine is within the range of concentrations physiologically present in plasma. The substitution of extracellular sodium with choline blocks the decrease in intracellular ionized magnesium induced by norepinephrine, which leads us to suppose that the magnesium-reducing effect of catecholamines is a result of the activation of a Na+-Mg2+ exchanger. We were not able to demonstrate any change in intracellular ionized magnesium after 1 and 17 days of active treatment in essential hypertensives. The impossibility of demonstrating ex vivo the mechanism of catecholamine-mediated regulation that is evident in vitro is perhaps due to our experimental conditions or to substances which in vivo inhibit the action of the catecholamines on magnesium, such as insulin and/or glucose.

A new prospective against HIV infection: induction of murin CCR5-downregulating antibodies.

Natural resistance to HIV is widely growing in humans. An example of an extremely efficacious resistance is represented by exposed seronegative (ESN) subjects, i.e. individuals who, despite repeated sexual and/or parenteral exposure to HIV, remain seronegative and apparently uninfected. A small group within ESN produces anti-CCR5 antibodies which cause antigen down-modulation and a CCR5 minus phenotype. It has been previously demonstrated that a single conformed extracellular domain (corresponding to first cystein loop) of CCR5 is recognized by ESN antibodies. In order to verify the possibility to induce and reproduce infection-protecting anti-CCR5 antibodies in individuals at high risk of HIV infection, we generated immunogens containing the relevant CCR5 peptide. Since the first cysteine loop of human CCR5 is identical in sequence to its mouse homologue, mice were immunized according to an intra-peritoneal procedure with CCR5 peptide loop, #90-103. Anti-CCR5-responses elicited in mice did share the same specificity and functions as human anti-CCR5 immunoglobulins previously identified in ESN cohorts. In particular, murine IgG and IgA: 1. Specifically recognize both mouse and human CCR5. 2. Down-modulate CCR5 expression on CD4+ cells of both untreated mice and human. 3. Downregulate "in vivo" peripheral CCR5 expression on mice CD4+CCR5+ cells. 4. Inhibit CD4+ CCR5+ lymphocytes chemotaxis. These findings show that CCR5-mediated effects on CD4+ cells can be achieved in mice both "in vitro" and "in vivo". Therefore, novel immune strategies aimed at generating partial or complete immune protection through anti-CCR5 downregulation at genital mucosa could be elicited successfully also in monkey and eventually in humans.

The effect of HAART on humoral immune response in primary HIV-1 infected patients.

The effect of Highly Active Antiretroviral Therapy (HAART) on binding and neutralizing antibody responses to human immunodeficiency virus type-1 (HIV-1) during primary and chronic infection was investigated. Seven patients HAART treated during primary infection, six HAART treated during chronic infection and five patients treated only with ZVD (Zidovudine) were analysed. HAART inhibited the development of anti env antibodies during primary infection. Administering HAART during primary infection usually did not substantially affect the development of weak neutralizing antibody responses against autologous virus. However, we demonstrated that very early treatment, during seroconversion, induce in some cases, a strong neutralizing antibodies against autologous virus. These results may be relevant for understanding how HAART may elicit a strong protective responses and may be useful in developing new strategies designed to achieve a long term control of the HIV infection.

Immune activation in africa is environmentally-driven and is associated with upregulation of CCR5. Italian-Ugandan AIDS Project.

BACKGROUND: HIV infection in Africa is associated with immune activation and a cytokine profile that stimulates CCR5 expression. We investigated whether this immune activation is environmentally driven; if a dominant expression of CCR5 could indeed be detected in African individuals; and if R5 HIV strains would be prevalent in this population. METHODS: Freshly drawn peripheral blood mononuclear cells from HIV-uninfected African and Italian individuals living in rural Africa, from HIV-uninfected Africans and Italians living in Italy, and from HIV-infected African and Italian patients were analysed. Determinations of HIV coreceptor-specific mRNAs and immunophenotype analyses were performed in all samples. Virological analyses included virus isolation and characterization of plasma neutralizing activity. FINDINGS: Results showed that: immune activation is detected both in Italian and African HIV-uninfected individuals living in Africa but not in African subjects living in Italy; CCR5-specific mRNA is augmented and the surface expression of CCR5 is increased in African compared with Italian residents (CXCR4-specific mRNA is comparable); R5-HIV strains are isolated prevalently from lymphocytes of African HIV-infected patients; and plasma neutralizing activity in HIV-infected African patients is mostly specific for R5 strains. CONCLUSIONS: Immune activation in African residents is environmentally driven and not genetically predetermined. This immune activation results in a skewing of the CCR5 : CXCR4 ratio which is associated with a prevalent isolation of R5 viruses. These data suggest that the selection of the predominant virus strain within the population could be influenced by an immunologically driven pattern of HIV co receptor expression.

HIV neutralizing IgA in exposed seronegative subjects recognise an epitope within the gp41 coiled-coil pocket.

Human immunodeficiency virus (HIV)-specific IgA can be detected in cervical secretions, saliva, and sera of HIV-infected and HIV-uninfected individuals with a known exposure to the virus. IgA from HIV-uninfected exposed seronegative individuals (ESN) neutralize in vitro primary strains of HIV-1. We analyzed the epitopes of HIV recognized by serum HIV-specific IgA of ESN individuals to identify the antigenic correlates of HIV neutralization in exposed-uninfected subjects, and to verify whether different epitopes would be recognized by HIV-specific IgA of ESN and of HIV-infected patients. Results confirmed that HIV-neutralizing IgA are detected in sera of ESN and showed that neutralization of primary HIV strains is mediated by the recognition of different epitopes in HIV-infected patients and ESN. Thus, whereas IgA of HIV+ individuals recognize epitopes expressed both within gp120 and gp41, IgA of ESN exclusively bind to gp41-expressed epitopes. Epitope mapping revealed that the epitope recognized by serum IgA of ESN on gp41 is restricted to aa 581-584 (LQAR) and corresponds to coiled coil pocket in the alpha helic region. In contrast, the epitope seen by IgA of HIV-infected patients on gp41 is identified by two regions; the first is contained within the cystein loop (aa 589-618), the second correspond to C terminal region in the extra membrane region of gp 41 (aa 642-673). Thus, we have identified and characterized the epitopes that mediate neutralization of HIV in individuals in whom infection does not occur despite multiple exposures to the virus. These results have important implications for the development of a new therapy against HIV infection.

Early production of HIV-1 neutralising antibodies in patients following highly active antiretroviral treatment (HAART) during primary HIV infection.

We investigate the effects of highly active antiretroviral therapy (HAART) on humoral immune responses during a 24-month follow up of 15 HIV patients with acute primary HIV infection. The patients were divided into three groups on the basis of the therapeutic protocol they were following at the time of entry: a) five naive patients (untreated or treated with only ZDV or AZT); b) five patients following a triple combination of ZDV+ lamivudine (3TC)+ saquinovir (SQV); and c) five patients on a four-drug combination of ZDV+3TC+SQV+ ritonavir (RTV). The results show that the early introduction of HAART greatly reduces plasma viremia levels and restores the number of CD4 cells. A significant correlation was found between anti HIV neutralising activity and the four-drug, but not the three-drug combination. The reduction in infectivity was directed against viruses of different clades and associated with immunoglobulin fractions. Moreover, the neutralising antibodies in the HAART-treated patients appeared after two weeks of treatment and remained stable throughout the 24 months of follow up. The early appearance of neutralising antibodies represent an important component of immune responses during primary HIV infection, may contribute towards immune reconstitution in patients on HAART, and give further information that may be useful in developing new strategies designed to eradicate the disease.

CCR5-reactive antibodies in seronegative partners of HIV-seropositive individuals down-modulate surface CCR5 in vivo and neutralize the infectivity of R5 strains of HIV-1 In vitro.

Exposure to HIV does not necessarily result in infection. Because primary HIV infection is associated with CCR5-tropic HIV variants (R5), CCR5-specific Abs in the sera of HIV-seronegative, HIV-exposed individuals (ESN) might be associated with protection against infection. We analyzed sera from ESN, their HIV-infected sexual partners (HIV+), and healthy controls (USN) searching for CCR5-specific Abs, studying whether incubation of PBMC with sera could prevent macrophage inflammatory protein 1 beta (Mip1 beta) (natural ligand of CCR5) binding to CCR5. Results showed that Mip1 beta binding to CCR5 was not modified by sera of either 40 HIV+ or 45 USN but was greatly reduced by sera of 6/48 ESN. Binding inhibition was due to Abs reactive with CCR5. The CCR5-specific Abs neutralized the infectivity of primary HIV isolates obtained from the corresponding HIV+ partners and of R5-primary HIV strains, but not that of CXCR4-tropic or amphitropic HIV strains. Immunoadsorption on CCR5-transfected, but not on CXCR4-transfected, cells removed CCR5-specific and virus-neutralizing Abs. Epitope mapping on purified CCR5-specific Abs showed that these Abs recognize a conformational epitope in the first cysteine loop of CCR5 (aa 89-102). Affinity-purified anti-CCR5-peptide neutralized the infectivity of R5 strains of HIV-1. Anti-CCR5 Abs inhibited Mip1beta-induced chemotaxis of PBMC from healthy donors. PBMC from two ESN (with anti-CCR5 Abs) were CCR5-negative and could not be stimulated by Mip1beta in chemotaxis assays. These results contribute to clarifying the phenomenon of immunologic resistance to HIV and may have implications for the development of a protective vaccine.

Anti-cell antibodies in exposed seronegative individuals with HIV type 1-neutralizing activity.

Despite repeated exposures to HIV-1, some individuals remain seronegative. This study reports that sera from a fraction of exposed seronegative (ESN) subjects showed HIV-neutralizing activity; 5 of 17 ESN sera and none of 17 controls neutralized two different HIV-1 primary isolates (range of neutralizing titers: 1/20 to 1/60). The neutralizing activity was associated with the IgG fraction of 4 of 4 neutralizing ESN sera. Moreover, in 11 of 17 and 9 of 17 ESN sera (but none of the control sera) we found antibodies against HLA class I and CD4, respectively. One of the ESN sera (EU22) neutralized efficiently the primary virus derived from the seropositive partner and showed a good broadly cross-reactive neutralization. Immunoadsorption of two IgG fractions from EU19 and EU22 on peripheral blood mononuclear cells (PBMC) removed virus-neutralizing antibodies. The correlations between the ESN status and neutralizing activity (p<0.05), anti-HLA antibodies (p<0.0002), and anti-CD4 antibodies (p<0.001) were statistically significant. However, there was no statistically significant correlation between neutralizing activity and either anti-HLA or anti-CD4 antibodies. It can therefore be said that exposure to HIV-1 without seroconversion is, in some individuals, associated with HIV-neutralizing antibodies (not directed against viral antigens) and/or with anti-cell autoantibodies, which are possibly specific for cellular antigens involved in the infection/entry process.

Intralymphocyte free magnesium in patients with primary aldosteronism: aldosterone and lymphocyte magnesium homeostasis.

It is known that hyperaldosteronism has been associated with magnesium deficiency, yet there are no data on the intracellular concentration of ionized magnesium ([Mg(2+)(i)]) in subjects with primary aldosteronism (PA). We measured intralymphocyte free magnesium ([Mg(2+)(i)]) and intralymphocyte free calcium ([Ca(2+)(i)]) in 16 patients with PA and 26 normotensive control subjects (NCs). [Mg(2+)(i)] and [Ca(2+)(i)] were also measured in blood lymphocytes incubated in vitro with aldosterone, according to a fluorimetric method. In subjects with PA, [Mg(2+)(i)] was significantly lower than that in NCs (mean+/-SD; PA 203+/-56 micromol/L, NCs 291+/-43 micromol/L, 95% confidence interval 57 to 119, P=0.001). In the patients, [Ca(2+)(i)] did not prove to be statistically different from that of NCs (mean+/-SD; PA 47.2+/-10.6 nmol/L, NCs 53.2+/-11 nmol/L). The lymphocytes exposed to the action of aldosterone showed a significant reduction in [Mg(2+)(i)] (n=15, NCs 271+/-28 micromol/L, aldosterone treatment 188+/-39 micromol/L, P=0.001, 95% confidence interval 57 to 108). The dose-effect curve of aldosterone on [Mg(2+)(i)] showed an EC(50) value of approximately 0.5 to 1 nmol/L aldosterone. The reduction in [Mg(2+)(i)] mediated by aldosterone is antagonized by the receptor inhibitor of aldosterone; it is inhibited by inhibitors of protein synthesis and is not measurable when the lymphocytes are incubated in an Na(+)-free medium. The data are consistent with the hypothesis that aldosterone affects the cellular homeostasis of magnesium, probably through modification of the activity of the Na(+)-Mg(2+) antiporter.

Human immunodeficiency virus (HIV)-specific IgA and HIV neutralizing activity in the serum of exposed seronegative partners of HIV-seropositive persons.

The presence and activity of human immunodeficiency virus (HIV)-specific antibodies were analyzed in the sera of 15 sexually exposed seronegative persons who had systemic HIV-specific cell-mediated immunity and IgA-mediated mucosal immunity and in their HIV-infected partners. The HIV-positive subjects had HIV-specific serum IgG and IgA; the seronegative persons had HIV-specific serum IgA in the absence of IgG. Testing of the seronegative persons 1 year after the interruption of at-risk sex showed that no IgG seroconversion had occurred and that HIV-specific IgA serum concentrations had declined. Serum from the HIV-exposed seronegative persons was analyzed for the ability to neutralize primary HIV-1 isolates. Neutralizing activity was detected in 5 of 15 sera and in 2 cases was retained by serum-purified IgA. Thus, the immunologic picture for resistance to HIV infection should include HIV-specific cell-mediated immunity as well as HIV-specific IgA-mediated mucosal and systemic immunity.

Preliminary communication on intralymphocyte ionized magnesium in hypertensive patients under treatment with beta-blockers.

We measured free intralymphocyte magnesium (Mgi) in 10 hypertensive subjects (HS) before and after treatment with atenolol (A) and in 16 normotensive control subjects (NC). We also carried out in vitro studies on human lymphocytes to test the effect of catecholamines and beta-antagonists on Mgi. Mgi in HS was not statistically different before and after the treatment with A (M +/- SD, basal: 227 +/- 31 + mumol/l; A: 236 +/- 47 mumol/l; NS). Mgi in NC proved not statistically different as compared to HS without treatment (M +/- SD, NC: 232 +/- 35 mumol/l; HS before treatment: 227 +/- 31 mumol/l; NS). The Mgi of the lymphocytes treated in vitro with noradrenaline (NA) significantly decreased in comparison with the control (M +/- SD, basal: 244 +/- 26 mumol/l; NA: 198 +/- 25 mumol/l; p = 0.0001), whereas it did not change after incubation with NA and propranolol (P) (M +/- SD, basal: 238 +/- 32 mumol/l; NA + P: 239 +/- 30 mumol/l; NS). On the other hand Mgi decreased significantly after incubation with NA and A (M +/- SD, basal: 255 +/- 21 mumol/l; NA + A: 202 +/- 34 mumol/l). Our in vivo data show that the Mgi of HS remains unvaried after treatment with A, and that there is not statistical difference between Mgi of NC and of HS before treatment. The in vitro study demonstrated that catecholamines cause a significant reduction in Mgi. This effect is only inhibited by a non-selective beta-blocker such as P. These results seem to explain the ineffectiveness of treatment with atenolol on our patients' Mgi.

Intralymphocyte free magnesium and plasma triglycerides.

To evaluate the relative effect of hypertension and plasma triglycerides on intralymphocyte magnesium we measured ionized intralymphocyte magnesium (Mg(i)) concentration by means of a fluorimetric method based on the dye Furaptra in 4 groups of subjects: 18 normotensive normotriglyceridemic controls (NTNC), 9 hypertriglyceridemic normotensive patients (HTN), 8 hypertriglyceridemic essential hypertensive patients (HTEH), 17 normotriglyceridemic essential hypertensive patients (NTEH). Hypercholesterolemic, diabetic patients and alcoholics were excluded from the study. Mg(i) was found to be statistically reduced (ANOVA test F=10.41, P=0.0001) in both HTN and HTEH (M+/- SD, HTN: 0.235 +/- 0.01, HTEH: 0.236 +/- 0.01 mmol/l) as compared to both NTNC and NTEH (M +/- SD, NTNC: 0.294 +/- 0.008, NTEH: 0.297 +/- 0.009 mmol/l). A statistically significant negative correlation was found in the population as a whole between Mg(i) and plasma triglycerides (n=52, R= -541, P=0.00004). Our data suggest that hypertriglyceridemia per se and possibly the so-called plurimetabolic syndrome is characterized by low intralymphocyte free magnesium.