Defining genetic diversity of rhesus macaque Fcγ receptors with long-read RNA sequencing.

Fcγ receptors (FcγRs) are membrane-bound glycoproteins that bind to the fragment crystallizable (Fc) constant regions of IgG antibodies. Interactions between IgG immune complexes and FcγRs can initiate signal transduction that mediates important components of the immune response including activation of immune cells for clearance of opsonized pathogens or infected host cells. In humans, many studies have identified associations between FcγR gene polymorphisms and risk of infection, or progression of disease, suggesting a gene-level impact on FcγR-dependent immune responses. Rhesus macaques are an important translational model for most human health interventions, yet little is known about the breadth of rhesus macaque FcγR genetic diversity. This lack of knowledge prevents evaluation of the impact of FcγR polymorphisms on outcomes of preclinical studies performed in rhesus macaques. In this study we used long-read RNA sequencing to define the genetic diversity of FcγRs in 206 Indian-origin Rhesus macaques, Macaca mulatta. We describe the frequency of single nucleotide polymorphisms, insertions, deletions, frame-shift mutations, and isoforms. We also index the identified diversity using predicted and known rhesus macaque FcγR and Fc-FcγR structures. Future studies that define the functional significance of this genetic diversity will facilitate a better understanding of the correlation between human and macaque FcγR biology that is needed for effective translation of studies with antibody-mediated outcomes performed in rhesus macaques.

Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses.

Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies.

IgG Binding Characteristics of Rhesus Macaque FcγR.

Indian rhesus macaques (Macaca mulatta) are routinely used in preclinical studies to evaluate therapeutic Abs and candidate vaccines. The efficacy of these interventions in many cases is known to rely heavily on the ability of Abs to interact with a set of Ab FcγR expressed on innate immune cells. Yet, despite their presumed functional importance, M. mulatta Ab receptors are largely uncharacterized, posing a fundamental limit to ensuring accurate interpretation and translation of results from studies in this model. In this article, we describe the binding characteristics of the most prevalent allotypic variants of M. mulatta FcγR for binding to both human and M. mulatta 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 Abs in this key animal model and exposes significant evolutionary divergence between humans and macaques.

The carbonic anhydrase inhibitor methazolamide prevents amyloid beta-induced mitochondrial dysfunction and caspase activation protecting neuronal and glial cells in vitro and in the mouse brain.

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 (Aß) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aß, 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 Aß 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 Aß 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 Aß-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.

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques.

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 role of Fc receptors in HIV infection and vaccine efficacy.

PURPOSE OF REVIEW: In this review, the roles of Fc-gamma receptor polymorphisms are discussed in regards to HIV-1 vaccine efficacy, HIV acquisition, and disease progression. In addition, the significance of the neonatal immunoglobulin G (IgG) Fc receptor and potential effects of the aggregated immunoglobulin A Fc receptor (FcalphaR) are addressed. RECENT FINDINGS: Fc receptors undoubtedly play an important role in antibody-mediated action in HIV infection and vaccines. Several studies have determined an association between polymorphic variants of Fc-gamma-RIIA and Fc-gamma-RIIIA in the acquisition and progression of HIV-1 infection, and in responses to vaccination regimens. A rather complex relationship exists between the relative affinity of these molecules and their impact on HIV disease acquisition and progression and HIV vaccine efficacy. SUMMARY: The discrepancies between different investigations of the role of Fc receptor polymorphisms appear to derive from the complex nature of the Fc receptor functions, including factors such as epistatic interactions and the race, sex, age, and relative risk behavior of the investigated individuals. Furthermore, Fc receptors in nonhuman primates (NHPs), the key model to study an AIDS-like disease in an animal model, appear to be even more diverse than in humans, and the function of these proteins has not been extensively explored. Given the critical role of Fc receptors in antibody-mediated function in humans and NHP, more investigations are needed to fully understand and exploit these functions for vaccine design.

Alkaline phosphatase is increased in both brain and plasma in Alzheimer's disease.

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 (r(s) = -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.

Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein.

The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. PrP(C) is bound to the plasma membrane via a glycosylphosphatidylinositol anchor, although a secreted, soluble form has also been identified. Previously we reported that PrP(C) 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 PrP(C). These experiments revealed that ADAM9 and ADAM10, but not ADAM17, are involved in the shedding of PrP(C) and that ADAM9 exerts its effect on PrP(C) 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 Gly(228) and Arg(229), three residues away from the site of glycosylphosphatidylinositol anchor attachment. The shedding of another membrane protein, the amyloid precursor protein beta-secretase BACE1, by ADAM9 is also mediated via ADAM10. Furthermore, we show that pharmacological inhibition of PrP(C) shedding or activation of both PrP(C) and PrP(Sc) shedding by ADAM10 overexpression in scrapie-infected neuroblastoma N2a cells does not alter the formation of proteinase K-resistant PrP(Sc). Collectively, these data indicate that although PrP(C) can be shed through the action of ADAM family members, modulation of PrP(C) or PrP(Sc) ectodomain shedding does not regulate prion conversion.

Crystal structure of the TetR/CamR family repressor Mycobacterium tuberculosis EthR implicated in ethionamide resistance.

Ethionamide has been used for more than 30 years as a second-line chemotherapeutic to treat tuberculosis patients who have developed resistance to first-line drugs, such as isoniazid (INH) and rifampicin. Activation of the pro-drug ethionamide is regulated by the Baeyer-Villiger monooxygenase EthA and the TetR/CamR family repressor EthR, whose open reading frames are separated by 75 bp on the Mycobacterium tuberculosis genome. EthR has been shown to repress transcription of the activator gene ethA by binding to this intergenic region, thus contributing to ethionamide resistance. We have determined the crystal structure of EthR, to 1.7A resolution, revealing a dimeric two-domain molecule with an overall architecture typical for TetR/CamR repressor proteins. A 20A long hydrophobic tunnel-like cavity in the "drug-binding" domain of EthR is occupied by two 1,4-dioxane molecules, a component of the crystallisation buffer. Comparing the present structure to those of the homologues Staphylococcus aureus QacR and Escherichia coli TetR leads to the hypothesis that the hydrophobic cavity constitutes a binding site for an as yet unknown ligand that might regulate DNA-binding of EthR.