CD4 mimetics sensitize HIV-1-infected cells to ADCC

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.Worldwide, it is estimated that more than 35 million people are living with HIV. In 2013 alone, around 2.1 million people became newly infected with HIV, and 1.5 million people died from AIDS (1). Measures to prevent HIV-1 transmission are desperately needed. Prevention of HIV-1 transmission and progression likely requires approaches that can specifically target and eliminate HIV-1-infected cells. Interestingly, there is increasing evidence supporting a role of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) in controlling HIV-1 transmission and disease progression (2–8). Analysis of the correlates of protection in the RV144 vaccine trial suggested that increased ADCC activity was linked with decreased HIV-1 acquisition (9), and Abs with potent ADCC activity were isolated from some RV144 vaccinees (10). Recent studies reported that the viral accessory proteins Nef and Vpu protect HIV-1-infected cells from anti-HIV-1 envelope (Env)-mediated ADCC responses (11–14). Importantly, we and others reported that Env in the CD4-bound conformation was preferentially targeted by ADCC-mediating Abs and sera from HIV-1-infected individuals (11, 12, 15, 16), which represent a significant proportion of anti-Env Abs elicited during natural HIV infection (11, 17). However, the vast majority of circulating HIV-1 strains worldwide express functional Nef and Vpu proteins, which limit the exposure of CD4-induced (CD4i) Env epitopes at the surface of infected cells, likely preventing ADCC responses.Theoretically, agents promoting the CD4-bound Env conformation should expose CD4i epitopes that are readily recognized by ADCC-mediating Abs and sera from infected individuals (11, 12, 15, 16, 18), resulting in the sensitization of HIV-1-infected cells to ADCC. Importantly, modulating Env conformation at the surface of HIV-1-infected cells has become feasible as a result of the availability of small CD4-mimetic compounds (CD4mc). The prototypes of such compounds, NBD-556 and NBD-557, were discovered in a screen for inhibitors of gp120-CD4 interaction (19). These small-molecule ∼337-Da compounds and recent derivatives (DMJ-I-228, JP-III-48) bind in the Phe-43 cavity (20–22), a highly conserved ∼150-ų pocket in the gp120 glycoprotein located at the interface of the inner domain, outer domain, bridging sheet, and CD4 receptor (23). CD4mc block gp120-CD4 interaction and induce thermodynamic changes in gp120 similar to those observed during CD4 or soluble CD4 (sCD4) binding (24). Accordingly, these small molecules, as well as sCD4, can promote the transition of Env to the CD4-bound conformation, thus sensitizing HIV-1 particles to neutralization by otherwise nonneutralizing CD4i Abs (17, 25). Additional strategies using scaffolded miniproteins targeting critical gp120 elements required for CD4 interaction allowed the identification of CD4 mimetics with nanomolar affinity for gp120 (26). One of these variants, M48U1, displayed remarkably potent neutralization of three HIV-1 isolates (27). Its crystal structure in complex with HIV-1 gp120 was recently solved, showing that M48U1 engages the Phe-43 cavity in a manner similar to that of CD4 (28); thus, M48U1 might induce gp120 to adopt the CD4-bound conformation and expose CD4i epitopes. Previous studies exploring the antiviral properties of CD4mc were performed on viral particles (17, 25, 27). However, whether these compounds are able to engage the large amounts of Env present at the surface of infected cells and modulate Env conformation in a way that allows exposure of ADCC-mediating epitopes is currently not known. In this study, we show that CD4mc strongly sensitize HIV-1-infected primary CD4 T cells to ADCC mediated by sera, cervicovaginal fluids, and breast milk from HIV-1-infected individuals, as well as help eliminate infected, ex vivo-expanded primary CD4 T cells from HIV-1-infected individuals. Therefore, CD4mc possess three valuable complementary antiviral properties: direct inactivation of viral particles, sensitization of viral particles to neutralization by otherwise nonneutralizing Abs, and sensitization of HIV-1-infected cells to ADCC-mediated killing.     

NMR structure and dynamics of the agonist dynorphin peptide bound to the human kappa opioid receptor

The structure of the dynorphin (1–13) peptide (dynorphin) bound to the human kappa opioid receptor (KOR) has been determined by liquid-state NMR spectroscopy. ¹H and ¹⁵N chemical shift variations indicated that free and bound peptide is in fast exchange in solutions containing 1 mM dynorphin and 0.01 mM KOR. Radioligand binding indicated an intermediate-affinity interaction, with a K_d of ∼200 nM. Transferred nuclear Overhauser enhancement spectroscopy was used to determine the structure of bound dynorphin. The N-terminal opioid signature, YGGF, was observed to be flexibly disordered, the central part of the peptide from L5 to R9 to form a helical turn, and the C-terminal segment from P10 to K13 to be flexibly disordered in this intermediate-affinity bound state. Combining molecular modeling with NMR provided an initial framework for understanding multistep activation of a G protein-coupled receptor by its cognate peptide ligand.G protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins in the human genome and play a critical role in human physiology by initiating signal transduction in response to extracellular stimuli (1, 2). Since 2007, 89 GPCR crystal structures have been reported, including receptors in inactive and active states, as well as the beta-2 adrenergic receptor (β₂-AR) bound to heterotrimeric G proteins (3). NMR spectroscopy has revealed that the intrinsic conformational heterogeneity of GPCRs is influenced by ligand pharmacology, membrane composition, and effector interactions (4–6). These structural biology studies have provided atomic-resolution insights of systems defined by dynamic structural rearrangements that are correlated with diverse cellular and physiological outcomes.The classic opioid receptors (δ/κ/μ) are GPCRs activated in response to binding enkephalin-like peptide agonists and are the primary targets of widely prescribed pain medications (7). The kappa opioid receptor (KOR) and its cognate peptide dynorphin are implicated in neuronal pathways associated with addiction, pain, reward, mood, cognition, and perception (8, 9). Nonselective KOR antagonists such as naltrexone have been prescribed for alcohol dependence with limited efficacy in humans, and next-generation KOR antagonists continue to be developed to treat drug addiction and other disorders. Although much is known regarding the antagonist-bound, inactive state of GPCRs, including the crystal structure of JDTic-bound KOR, the interaction of these receptors with neuropeptide agonists remains largely unknown (10). Peptide agonist-bound structures have thus far been limited to a conformationally stabilized neurotensin receptor, likely corresponding to a low-energy peptide-receptor state (11–13).Dynorphin was discovered by Goldstein and Chavkin as the endogenous activating neuropeptide for KOR, with a “low-resolution” structural model of interaction proposed to PNAS in 1981 (14, 15). Dynorphins are derived from the precursor prodynorphin, with dynorphin A(1–17), dynorphin B(1–13), and alpha neoendorphin sharing a highly conserved N-terminal sequence and charge distribution (16). Dynorphin A(1–13) was shown to act as an agonist on opioid kappa receptors in vivo (17). Physiological activation of KOR is mainly associated with unwanted effects such as dysphoria, anhedonia, and hallucinations, and a current hypothesis in the field is that KOR functionally selective ligands may produce analgesia without dysphoria (18, 19). Functional selectivity has emerged as the leading model to understand the ability of a ligand to activate a subset of signaling cascades, providing a framework for developing next-generation drugs with rationally designed pharmacological profiles (20).The seminal work of Schwyzer in the 1970s and 1980s led to a model of KOR activation by dynorphin that proceeds via a multistep binding mechanism (14, 21, 22). Thereby, low- to intermediate-affinity binding states of dynorphin correspond to binding to cell-surface membranes or to extracellular loops of the GPCR. A “message–address” paradigm has been formulated based on structure–activity relations observed with dynorphin analogs (21–26). Accordingly, the N-terminal YGGF “message” sequence, which is common to all opioid peptides, was found to be responsible for receptor activation. A C-terminal “address” sequence was further found to contribute via electrostatically driven interactions to KOR subtype specificity. In the context of this paradigm, the present study yields intriguing data on the N-terminal segment of the KOR-bound opioid peptide dynorphin. The methods used, NMR in solution and molecular dynamics simulations, enabled us to define structural ensembles of KOR-bound dynorphin and to characterize internal peptide motions in the presently prepared low-affinity receptor-bound state.     

Reference Intervals,Intraindividual and Interindividual Variability,and Reference Change Values for Hematologic Variables in Laboratory Beagles

In research and development studies for human and veterinary medicine, relevant comparators for interpreting clinical pathology results are matched with concurrent control animals. However, reference intervals (RI) provide a comparator database and important aids for interpreting clinical pathology data, especially in laboratory beagle dogs. Furthermore, RI incorporate biologic variation, which includes analytical, intraindividual, and interindividual variation. No studies to date have established RI and studied the effect of biologic variation on hematologic variables in a large group of laboratory dogs. The purpose of this retrospective study was to establish hematologic RI for laboratory beagles according to international recommendations and estimate the effect of biologic variation in routinely measured hematologic analytes by using the databank at a pharmaceutical center. Blood specimens from 340 healthy beagles (age, 9 to 36 mo) were evaluated by using a flow-cytometry-based hematology analyzer. RI and their 90% confidence intervals were established by using a nonparametric method. Effects of sex, age, and weight were investigated. Weight had no effect on any analyte. RBC, Hgb, Hct, MCV, MCH, RBC distribution width, and platelet count increased with age, whereas WBC count decreased. The only clinically relevant effect of sex was observed for platelets, which were lower in male beagles than in female and warranted 2 different RI. The calculated index of individuality showed that population-based RI were appropriate for almost all hematologic analytes, as might be expected for a homogeneous group of laboratory beagles.Abbreviation: CLSI, Clinical and Laboratory Standards Institute; RCV, reference change value; RI, reference intervalsRoutine hematology is a basic examination that is performed in laboratory beagles to monitor health and evaluate the toxicologic potential of various drugs, pollutants, or treatments. A recent statistical survey showed that 3000 laboratory dogs were used for scientific purposes in 2010 in France,²⁷ half of them for toxicological studies and the other half for safety evaluations. The interpretation of clinical pathology results primarily is based on the statistical comparison of results from controls with those of treated animals. This constitutes only a crude use of the data, because it does not take into account basic knowledge of reference intervals (RI) and inter- and intraindividual factors of variation of the analytes.Recently, population-based RI for hematologic variables have been determined according to international recommendations in various breeds of companion dogs by using modern cell counters, which are partially or completely based on flow cytometry.^4,6,28,34 These efforts demonstrated an occurrence of breed effects, which had not been previously established. Paradoxically, similar studies involving modern hematology analyzers and techniques for determining RI in laboratory beagles have not been published. Today, the most frequently cited hematologic canine RI³⁰ are taken from textbooks published in the 1960s and 1970s,^31-33 and the reference values were obtained from both mongrels and beagles, both kept under laboratory animal conditions. Similarly, previous studies done on large populations of beagles used for biomedical research were focused on blood reference values obtained from animals of different age classes.^{1-3,13,26,35,43} To our knowledge, the latest report of comprehensive hematology and chemistry analyte RI in laboratory beagles was published in 1986⁴³ and involved a now-outdated analyzer. In consequence, none of these studies meets the currently available international recommendations of the International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute,⁸ because the preanalytical and analytical conditions, animal population characteristics, and statistical procedures are not fully documented. Because laboratory beagles typically are young animals, with low genetic variability and bred under fully controlled conditions, we surmised that the RI of other breeds would not be transferrable and that interindividual variability would be lower than that in companion dogs.Moreover, information regarding the biologic variation of an analyte is important to consider before reference values are determined. If the intraindividual variability is considerably less than the interindividual variability, a population-based RI is not sufficiently sensitive to detect changes in a subject over time.²⁴ Although biologic variation has been acknowledged and accommodated in human medicine over the last 15 y,^10,17,29 corresponding data in veterinary medicine remain scant.⁴⁰ A Danish team interested in animal biologic variation, particularly in dogs,^21,23,42 published a study 16 y ago on the variability of some hematologic analytes, especially in beagles.²² The conclusions were that population-based RI were relevant for RBC count, Hct, Hgb, and WBC count and that subject-based RI consequently were unnecessary.²²We therefore performed the current study to investigate the biologic variation of hematologic analytes from laboratory beagles by using the database of control dogs of a pharmaceutical company. The objectives were: to establish an a posteriori procedure for the determination of population-based RI according to the American Society of Veterinary Clinical Pathology¹² and the International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute;⁸ to estimate indexes of individuality; to consider the use of population-based compared with subject-based reference values; and to estimate the reference change value (RCV) to identify the significance of any change between consecutive results.     

Inhibition of Kupffer cell activity induces hepatic triglyceride synthesis in fasted rats, independent of lipopolysaccharide challenge

BACKGROUND: Lipopolysaccharides (LPS), cleared from the blood by Kupffer cells, induce hypertriglyceridemia. AIMS: To test the hypothesis that GdCl(3), through inhibition of large Kupffer cell activity, modulates LPS-induced hyperlipidemia in rats. METHODS: Male Wistar rats received a single intravenous injection of GdCl(3)(10 mg/kg) or saline, 24 h before intraperitoneal LPS (1.5 mg/kg) administration. Serum and hepatic lipids as well as activity of key enzymes controlling fatty acid synthesis and esterification in liver tissue were measured. The incorporation of labeled precursors into lipids was assessed in cultured precision-cut liver slices. RESULTS: GdCl(3) does not prevent hypertriglyceridemia occurring in LPS-treated rats. Surprisingly, GdCl(3) per se is able to promote triglycerides accumulation in the liver tissue, an effect related to an increase in hepatic fatty acid esterification. Such an effect also occurs in rats receiving a dietary supplementation with glycine (5%) known to inhibit Kupffer cell secretory capacity. CONCLUSIONS: Large Kupffer cell inhibition does not prevent LPS-induced hypertriglyceridemia and even leads to a metabolic shift of fatty acids towards their esterification and accumulation in the liver tissue, suggesting that Kupffer cells play a role in the regulation of lipid metabolism of the adjacent hepatocytes, independent of any inflammatory stimulus.     

BRCA1 and BRCA2 Mutations in Ethnic Lebanese Arab Women With High Hereditary Risk Breast Cancer

Purpose.

Breast cancer is the most common malignancy among women in Lebanon and in Arab countries, with 50% of cases presenting before the age of 50 years.

Methods.

Between 2009 and 2012, 250 Lebanese women with breast cancer who were considered to be at high risk of carrying BRCA1 or BRCA2 mutations because of presentation at young age and/or positive family history (FH) of breast or ovarian cancer were recruited. Clinical data were analyzed statistically. Coding exons and intron-exon boundaries of BRCA1 and BRCA2 were sequenced from peripheral blood DNA. All patients were tested for BRCA1 rearrangements using multiplex ligation-dependent probe amplification (MLPA). BRCA2 MLPA was done in selected cases.

Results.

Overall, 14 of 250 patients (5.6%) carried a deleterious BRCA mutation (7 BRCA1, 7 BRCA2) and 31 (12.4%) carried a variant of uncertain significance. Eight of 74 patients (10.8%) aged ≤40 years with positive FH and only 1 of 74 patients (1.4%) aged ≤40 years without FH had a mutated BRCA. Four of 75 patients (5.3%) aged 41–50 years with FH had a deleterious mutation. Only 1 of 27 patients aged >50 years at diagnosis had a BRCA mutation. All seven patients with BRCA1 mutations had grade 3 infiltrating ductal carcinoma and triple-negative breast cancer. Nine BRCA1 and 17 BRCA2 common haplotypes were observed.

Conclusion.

Prevalence of deleterious BRCA mutations is lower than expected and does not support the hypothesis that BRCA mutations alone cause the observed high percentage of breast cancer in young women of Lebanese and Arab descent. Studies to search for other genetic mutations are recommended.     

Clinical and genomic analysis of a randomised phase II study evaluating anastrozole and fulvestrant in postmenopausal patients treated for large operable or locally advanced hormone-receptor-positive breast cancer

Background:

The aim of this study was to assess the efficacy of neoadjuvant anastrozole and fulvestrant treatment of large operable or locally advanced hormone-receptor-positive breast cancer not eligible for initial breast-conserving surgery, and to identify genomic changes occurring after treatment.

Methods:

One hundred and twenty post-menopausal patients were randomised to receive 1 mg anastrozole (61 patients) or 500 mg fulvestrant (59 patients) for 6 months. Genomic DNA copy number profiles were generated for a subgroup of 20 patients before and after treatment.

Results:

A total of 108 patients were evaluable for efficacy and 118 for toxicity. The objective response rate determined by clinical palpation was 58.9% (95% CI=45.0–71.9) in the anastrozole arm and 53.8% (95% CI=39.5–67.8) in the fulvestrant arm. The breast-conserving surgery rate was 58.9% (95% CI=45.0–71.9) in the anastrozole arm and 50.0% (95% CI=35.8–64.2) in the fulvestrant arm. Pathological responses >50% occurred in 24 patients (42.9%) in the anastrozole arm and 13 (25.0%) in the fulvestrant arm. The Ki-67 score fell after treatment but there was no significant difference between the reduction in the two arms (anastrozole 16.7% (95% CI=13.3–21.0) before, 3.2% (95% CI=1.9–5.5) after, n=43; fulvestrant 17.1% (95%CI=13.1–22.5) before, 3.2% (95% CI=1.8–5.7) after, n=38) or between the reduction in Ki-67 in clinical responders and non-responders. Genomic analysis appeared to show a reduction of clonal diversity following treatment with selection of some clones with simpler copy number profiles.

Conclusions:

Both anastrozole and fulvestrant were effective and well-tolerated, enabling breast-conserving surgery in over 50% of patients. Clonal changes consistent with clonal selection by the treatment were seen in a subgroup of patients.     

AMP-activated protein kinase is dispensable for maintaining ATP levels and for survival following inhibition of glycolysis,but promotes tumour engraftment of Ras-transformed fibroblasts

Lactic acid generated by highly glycolytic tumours is exported by the MonoCarboxylate Transporters, MCT1 and MCT4, to maintain pHi and energy homeostasis. We report that MCT1 inhibition combined with Mct4 gene disruption severely reduced glycolysis and tumour growth without affecting ATP levels. Because of the key role of the 5′-AMP-activated protein kinase (AMPK) in energy homeostasis, we hypothesized that targeting glycolysis (MCT-blockade) in AMPK-null (Ampk^−/−) cells should kill tumour cells from ‘ATP crisis’. We show that Ampk^−/−-Ras-transformed mouse embryonic fibroblasts (MEFs) maintained ATP levels and viability when glycolysis was inhibited. In MCT-inhibited MEFs treated with OXPHOS inhibitors the ATP level and viability collapsed in both Ampk^+/+ and Ampk^−/− cells. We therefore propose that the intracellular acidification resulting from lactic acid sequestration mimicks AMPK by blocking mTORC1, a major component of an ATP consuming pathway, thereby preventing ‘ATP crisis’. Finally we showed that genetic disruption of Mct4 and/or Ampk dramatically reduced tumourigenicity in a xenograft mouse model suggesting a crucialrolefor these two actors in establishment of tumours in a nutrient-deprived environment. These findings demonstrated that blockade of lactate transport is an efficient anti-cancer strategy that highlights the potential in targeting Mct4 in a context of impaired AMPK activity.