HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.
The cellular prion protein (PrPC) is essential for the pathogenesis and transmission of prion diseases. PrPC is bound to the plasma membrane via a glycosylphosphatidylinositol anchor, although a secreted, soluble form has also been identified. Previously we reported that PrPC is subject to ectodomain shedding from the membrane by zinc metalloproteinases with a similar inhibition profile to those involved in shedding the amyloid precursor protein. Here we have used gain-of-function (overexpression) and loss-of-function (small interfering RNA knockdown) experiments in cells to identify the ADAMs (a disintegrin and metalloproteinases) involved in the ectodomain shedding of PrPC. These experiments revealed that ADAM9 and ADAM10, but not ADAM17, are involved in the shedding of PrPC and that ADAM9 exerts its effect on PrPC shedding via ADAM10. Using dominant negative, catalytically inactive mutants, we show that the catalytic activity of ADAM9 is required for its effect on ADAM10. Mass spectrometric analysis revealed that ADAM10, but not ADAM9, cleaved PrP between Gly228 and Arg229, three residues away from the site of glycosylphosphatidylinositol anchor attachment. The shedding of another membrane protein, the amyloid precursor protein β-secretase BACE1, by ADAM9 is also mediated via ADAM10. Furthermore, we show that pharmacological inhibition of PrPC shedding or activation of both PrPC and PrPSc shedding by ADAM10 overexpression in scrapie-infected neuroblastoma N2a cells does not alter the formation of proteinase K-resistant PrPSc. Collectively, these data indicate that although PrPC can be shed through the action of ADAM family members, modulation of PrPC or PrPSc ectodomain shedding does not regulate prion conversion.
Mitochondrial dysfunction has been recognized as an early event in Alzheimer's disease (AD) pathology, preceding and inducing neurodegeneration and memory loss. The presence of cytochrome c (CytC) released from the mitochondria into the cytoplasm is often detected after acute or chronic neurodegenerative insults, including AD. The carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) was identified among a library of drugs as an inhibitor of CytC release and proved to be neuroprotective in Huntington's disease and stroke models. Here, using neuronal and glial cell cultures, in addition to an acute model of amyloid beta (Aβ) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aβ, we analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer's amyloid. MTZ prevented DNA fragmentation, CytC release and activation of caspase 9 and caspase 3 induced by Aβ in neuronal and glial cells in culture through the inhibition of mitochondrial hydrogen peroxide production. Moreover, intraperitoneal administration of MTZ prevented neurodegeneration induced by intra-hippocampal Aβ injection in the mouse brain and was effective at reducing caspase 3 activation in neurons and microglia in the area surrounding the injection site. Our results, delineating the molecular mechanism of action of MTZ against Aβ-mediated mitochondrial dysfunction and caspase activation, and demonstrating its efficiency in a model of acute amyloid-mediated toxicity, provide the first combined in vitro and in vivo evidence supporting the potential of a new therapy employing FDA-approved CAIs in AD.
Indian rhesus macaques (Macaca mulatta, MM) are routinely used in preclinical studies to evaluate therapeutic antibodies and candidate vaccines. The efficacy of these interventions in many cases is known to rely heavily on the ability of antibodies to interact with a set of antibody Fc gamma receptors (FcγR) expressed on innate immune cells. Yet, despite their presumed functional importance, MM antibody receptors are largely uncharacterized, posing a fundamental limit to ensuring accurate interpretation and translation of results from studies in this model. Here we describe the binding characteristics of the most prevalent allotypic variants of MM FcγR for binding to both human and MM IgG of varying subclasses. The resulting determination of the affinity, specificity, and glycan sensitivity of these receptors promises to be useful in designing and evaluating studies of candidate vaccines and therapeutic antibodies in this key animal model, and exposes significant evolutionary divergence between humans and macaques.
Background: Tissue non-specific alkaline phosphatase (TNAP) has been shown to promote the neurotoxicity of extracellular tau which contributes to the spread of pathology in Alzheimer’s disease (AD). Objective: To investigate changes in TNAP activity in the hippocampus in both sporadic and familial AD, and to examine whether changes in neuronal TNAP are reflected systemically by looking at changes in plasma TNAP activity in AD. Methods: We measured the activity of TNAP in the hippocampus in sporadic AD, familial AD and appropriate age-matched controls, and in an ageing series (age: 25–88 years) of brains. In addition, we measured TNAP activity in plasma from 110 AD and 110 non-demented control participants. Results: TNAP activity was significantly increased in the hippocampus in sporadic (by 56%; p = 0.038) and familial AD (by 121%; p = 0.042) compared with the age-matched controls. However, there was no correlation of TNAP activity with age. Furthermore, plasma TNAP activity was increased in AD (by 13%; p = 0.018) and inversely correlated with cognitive function (rs = –0.211; p = 0.027). Conclusion: Together, these data indicate that TNAP is increased in both sporadic and familial AD but not in the aged brain, indicating that the increase is likely a consequence of AD-associated changes in the brain. The neuronal change in TNAP is reflected in an increase in plasma TNAP in AD and is inversely correlated with cognitive function.
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