SEMA4D compromises blood–brain barrier, activates microglia, and inhibits remyelination in neurodegenerative disease

AbstractMultiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by immune cell infiltration of CNS, blood–brain barrier (BBB) breakdown, localized myelin destruction, and progressive neuronal degeneration. There exists a significant need to identify novel therapeutic targets and strategies that effectively and safely disrupt and even reverse disease pathophysiology. Signaling cascades initiated by semaphorin 4D (SEMA4D) induce glial activation, neuronal process collapse, inhibit migration and differentiation of oligodendrocyte precursor cells (OPCs), and disrupt endothelial tight junctions forming the BBB. To target SEMA4D, we generated a monoclonal antibody that recognizes mouse, rat, monkey and human SEMA4D with high affinity and blocks interaction between SEMA4D and its cognate receptors. In vitro, anti-SEMA4D reverses the inhibitory effects of recombinant SEMA4D on OPC survival and differentiation. In vivo, anti-SEMA4D significantly attenuates experimental autoimmune encephalomyelitis in multiple rodent models by preserving BBB integrity and axonal myelination and can be shown to promote migration of OPC to the site of lesions and improve myelin status following chemically-induced demyelination. Our study underscores SEMA4D as a key factor in CNS disease and supports the further development of antibody-based inhibition of SEMA4D as a novel therapeutic strategy for MS and other neurologic diseases with evidence of demyelination and/or compromise to the neurovascular unit

Autonomous Targeting of Infectious Superspreaders Using Engineered Transmissible Therapies

Infectious disease treatments, both pharmaceutical and vaccine, face three universal challenges: the difficulty of targeting treatments to high-risk ‘superspreader’ populations who drive the great majority of disease spread, behavioral barriers in the host population (such as poor compliance and risk disinhibition), and the evolution of pathogen resistance. Here, we describe a proposed intervention that would overcome these challenges by capitalizing upon Therapeutic Interfering Particles (TIPs) that are engineered to replicate conditionally in the presence of the pathogen and spread between individuals — analogous to ‘transmissible immunization’ that occurs with live-attenuated vaccines (but without the potential for reversion to virulence). Building on analyses of HIV field data from sub-Saharan Africa, we construct a multi-scale model, beginning at the single-cell level, to predict the effect of TIPs on individual patient viral loads and ultimately population-level disease prevalence. Our results show that a TIP, engineered with properties based on a recent HIV gene-therapy trial, could stably lower HIV/AIDS prevalence by ∼30-fold within 50 years and could complement current therapies. In contrast, optimistic antiretroviral therapy or vaccination campaigns alone could only lower HIV/AIDS prevalence by <2-fold over 50 years. The TIP's efficacy arises from its exploitation of the same risk factors as the pathogen, allowing it to autonomously penetrate superspreader populations, maintain efficacy despite behavioral disinhibition, and limit viral resistance. While demonstrated here for HIV, the TIP concept could apply broadly to many viral infectious diseases and would represent a new paradigm for disease control, away from pathogen eradication but toward robust disease suppression

Ion currents and proliferation in MCF-7 human breast cancer cells.

Previous work in our laboratory has demonstrated that in order to proliferate MCF-7 human breast cancer cells have to undergo a change of the membrane potential from a very depolarized to a more hyperpolarized. That change occurs at a hypothetical regulatory point located in early G1 phase, D control point. Potassium (K) channel antagonists quinidine, linogliride, glibenclamide, 4-aminopyridine (4-AP) and tetraethylammonium (TEA) inhibited MCF-7 cell proliferation. Among those blockers three (quinidine, linogliride and glibenclamide) also produced a reversible G0/G1 arrest. On the basis that these three drugs are antagonists of ATP-sensitive K (KA{dollar}\\sb{lcub}\\rm ATP{rcub}{dollar}) channels, we concluded that in MCF-7 cells it is the activation of K{dollar}\\sb{lcub}\\rm ATP{rcub}{dollar} channels at the D control point that promotes progression through G1 phase. The goal of our work was to identify in MCF-7 cells ion current(s) whose activation is required for passage through early G1 phase. To achieve this goal we used whole-cell and single-channel configurations of patch-clamp technique. We expected that the ion current-regulator of the G1 progression would: (1) have a reversal (i.e., zero current) potential near the equilibrium potential for K{dollar}\\sp+{dollar} ({dollar}-{dollar}84 mV); (2) be blocked by physiological concentrations of intracellular ATP; (3) be blocked by linogliride, glibenclamide and quinidine, but not by TEA. Our study demonstrated that MCF-7 cells contain ATP-sensitive K{dollar}\\sp+{dollar} current with the pharmacological profile of the regulator of G1 progression. The conductance density of the current was very low in cells arrested in early G1 phase (prior to D control point) with quinidine that further confirmed the involvement into G1 progression. We also identified in MCF-7 cells a small-conductance (8.5 pS) channel whose reversal potential was close to the equilibrium potential for K{dollar}\\sp+.{dollar} Furthermore, the channel was inhibited by quinidine. We concluded that this channel underlies the current-regulator of G1 progression

Mutation of the Methylated tRNA [Formula: see text] Residue A58 Disrupts Reverse Transcription and Inhibits Replication of Human Immunodeficiency Virus Type 1

Cellular tRNA [Formula: see text] serves as the primer for reverse transcription of human immunodeficiency virus type 1 (HIV-1). tRNA [Formula: see text] interacts directly with HIV-1 reverse transcriptase (RT), is packaged into viral particles, and anneals to the primer-binding site (PBS) of the HIV-1 genome in order to initiate reverse transcription. Residue A58 of tRNA [Formula: see text] , which lies outside the PBS-complementary region, is posttranscriptionally methylated to form 1-methyladenosine 58 (M(1)A58). This methylation is thought to serve as a pause signal for plus-strand strong-stop DNA synthesis during reverse transcription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we investigated the role of tRNA [Formula: see text] residue A58 in the replication cycle of HIV-1 in living cells. We have developed a mutant tRNA [Formula: see text] derivative, tRNA [Formula: see text] A58U, in which A58 was replaced by U. This mutant tRNA was expressed in CEM cells. We demonstrate that the presence of M(1)A58 is necessary for the appropriate termination of plus-strand strong-stop DNA synthesis and that the absence of M(1)A58 allows RT to read the tRNA sequences beyond residue 58. In addition, we show that replacement of M(1)A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRNA primer residue A58 in the reverse transcription process. Inhibition of reverse transcription with mutant tRNA primers constitutes a novel approach for therapeutic intervention against HIV-1