An antibody against Siglec-15 promotes bone formation and fracture healing by increasing TRAP+ mononuclear cells and PDGF-BB secretion.

Osteoporosis (OP) is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures. Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed. We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation. We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment. The secretion of platelet-derived growth factor-BB (PDGF-BB), the number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells, and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice. The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP+ cells. These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP+ mononuclear cells. To test the therapeutic potential of the Siglec-15 neutralizing antibody, we injected the antibody in an ovariectomy-induced osteoporotic mouse model, which mimics postmenopausal osteoporosis in women, and in two fracture healing models because fracture is the most serious health consequence of osteoporosis. The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis. Of note, the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models. Thus, the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture.

Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy.

Overexpression of the B7-H1 (PD-L1) molecule in the tumor microenvironment (TME) is a major immune evasion mechanism in some patients with cancer, and antibody blockade of the B7-H1/PD-1 interaction can normalize compromised immunity without excessive side-effects. Using a genome-scale T cell activity array, we identified Siglec-15 as a critical immune suppressor. While only expressed on some myeloid cells normally, Siglec-15 is broadly upregulated on human cancer cells and tumor-infiltrating myeloid cells, and its expression is mutually exclusive to B7-H1, partially due to its induction by macrophage colony-stimulating factor and downregulation by IFN-γ. We demonstrate that Siglec-15 suppresses antigen-specific T cell responses in vitro and in vivo. Genetic ablation or antibody blockade of Siglec-15 amplifies anti-tumor immunity in the TME and inhibits tumor growth in some mouse models. Taken together, our results support Siglec-15 as a potential target for normalization cancer immunotherapy.

Evaluation of the Ability of Immune Humanized Mice to Demonstrate CD20-Specific Cytotoxicity Induced by Ofatumumab.

CD20 monoclonal antibodies are well-established therapeutics for the treatment of B-cell malignancies. Several mechanisms of target cell killing occur from anti-CD20 therapy, including complement-dependent cytotoxicity (CDC) cell lysis and antibody-dependent cell-mediated cytotoxicity. Human Fc receptors (FcRs) are required to mediate these functions and are either not present or not cross-reactive in mice and most animal species. In contrast, some nonhuman primates have cross-reactive FcR; however, their cellular expression and function may differ from humans. Therefore, we tested bone marrow-liver-thymus (BLT) humanized mice to determine if they could recapitulate the pharmacokinetics (PKs), pharmacodynamics, and potential toxicities of ofatumumab, for which CDC is the predominant mechanism of action. Ofatumumab-treated BLT mice depleted B cells in a dose-dependent manner in all tissues sampled and recapitulated the PKs observed in humans, suggesting that BLT mice can mediate the CDC effector mechanism associated with biological drug products.

Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-ß-α NDPSase fold differentiates NDPSases from ADPRases.

Stoichiometric parameters of HIV-1 entry.

During HIV type 1 (HIV-1) entry, trimers of gp120 bind to CD4 and either the CCR5 or CXCR4 coreceptor on the target cell. The stoichiometric parameters associated with HIV-1 entry remain unclear. Important unanswered questions include: how many trimers must attach to CD4 molecules, how many must bind coreceptors, and how many functional gp120 subunits per trimer are required for entry? We performed single round infectivity assays with chimeric viruses and compared the experimental relative infectivity curves with curves generated by mathematical models. Our results indicate that HIV-1 entry requires only a small number of functional spikes (one or two), that Env trimers may function with fewer than three active subunits, and that there is no major difference in the stoichiometric requirements for CCR5 vs. CXCR4 mediated HIV-1 entry into host cells.

Increasing extracellular protein concentration reduces intracellular antiretroviral drug concentration and antiviral effect.

The objective of this study was to develop an in vitro pharmacodynamic (PD) system to test the impact of protein binding on antiretroviral (ARV) drug effect and intracellular ARV distribution. CD4(+) T cells were isolated from peripheral blood mononuclear cells (PBMCs) and exposed to varying and physiologically relevant concentrations of human serum albumin (HSA) and the ARV drugs efavirenz (EFV), raltegravir (RAL), etravirine (ETR), and enfuvirtide (ENF). The effect of varying extracellular protein concentration on the intracellular distribution of EFV, RAL, and ETR was assessed using ultraperformance liquid chromatography tandem mass spectrometry. HIV infectivity was assessed using an HIV-1 reporter virus expressing an Env-green fluorescent protein (GFP) and quantified using flow cytometry. Increasing extracellular HSA concentration was associated with increased relative infectivity for all drugs tested as well as decreased intracellular concentrations for EFV, RAL, and ETR. Median-effect plots indicate linearity between log10 antiviral effect (fraction of virus affected divided by fraction unaffected) and log10 intracellular drug concentration. The median [interquartile range (IQR)] slope (m) of the median-effect plots was 2.97 (2.26-5.85) for EFV, 3.52 (3.11-3.74) for ETR, and 2.39 (2.15-3.74) for RAL. The intracellular ARV concentrations associated with half-maximal antiviral effect (IC50) of EFV, ETR, and RAL were 1.2 (0.51-5.39), 39.06 (30.10-51.76), and 4.67 (3.91-5.02) ng/ml, respectively. This study demonstrates a significant reduction in cell penetration and antiviral effect of highly bound ARVs due to increasing extracellular concentration of HSA. This study is therefore the first to demonstrate experimentally how protein binding impacts intracellular distribution and the efficacy of ARVs.

A quantitative basis for antiretroviral therapy for HIV-1 infection.

Highly active antiretroviral therapy (HAART) has dramatically decreased mortality from HIV-1 infection and is a major achievement of modern medicine. However, there is no fundamental theory of HAART. Elegant models describe the dynamics of viral replication, but a metric for the antiviral activity of drug combinations relative to a target value needed for control of replication is lacking. Treatment guidelines are based on empirical results of clinical trials in which other factors such as regimen tolerability also affect outcome. Why only certain drug combinations control viral replication remains unclear. Here we quantify the intrinsic antiviral activity of antiretroviral drug combinations. We show that most single antiretroviral drugs show previously unappreciated complex nonlinear pharmacodynamics that determine their inhibitory potential at clinical concentrations. We demonstrate that neither of the major theories for drug combinations accurately predicts the combined effects of multiple antiretrovirals. However, the combined effects can be understood with a new approach that considers the degree of independence of drug effects. This analysis allows a direct comparison of the inhibitory potential of different drug combinations under clinical concentrations, reconciles the results of clinical trials, defines a target level of inhibition associated with treatment success and provides a rational basis for treatment simplification and optimization.

Ler interdomain linker is essential for anti-silencing activity in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) expresses a type III secretion system (T3SS) required for pathogenesis. Regulation of the genes encoding the T3SS is complex; two major regulators control transcription, the silencer H-NS, and the related H-NS-like protein Ler. Our laboratory is interested in understanding the molecular differences that distinguish the anti-silencer Ler from H-NS, and how Ler differentially regulates EPEC virulence genes. Here, we demonstrate that mutated Ler proteins either containing H-NS alpha-helices 1 and 2, missing from Ler, or truncated for the 11 aa C-terminal extension compared with the related H-NS protein, did not appreciably alter Ler function. In contrast, mutating the proline at position 92 of Ler, in the conserved C-terminal DNA binding motif, eliminated Ler activity. Inserting 11 H-NS-specific amino acids, 11 alanines or 6 alanines into the Ler linker severely impaired the ability of Ler to increase LEE5 transcription. To extend our analysis, we constructed six chimeric proteins containing the N terminus, linker region or C terminus of Ler in different combinations with the complementary domains of H-NS, and monitored their in vivo activities. Replacing the Ler linker domain with that of H-NS, or replacing the Ler C-terminal, DNA binding domain with that of H-NS eliminated the ability of Ler to increase transcription at the LEE5 promoter. Thus, the linker and C-terminal domains of Ler and H-NS are not functionally equivalent. Conversely, replacing the H-NS linker region with that of Ler caused increased transcription at LEE5 in a strain deleted for hns. In summary, the interdomain linker specific to Ler is necessary for anti-silencing activity in EPEC.