No abstract

Colorectal cancers are significant causes of morbidity and mortality and existing therapies often perform poorly for individuals afflicted with advanced disease. Oncolytic virotherapy is an emerging therapeutic modality with great promise for addressing this medical need. Herein we describe the in vivo testing of recombinant variants of the tanapoxvirus (TPV). Recombinant viruses were made ablated for either the 66R gene (encoding a thymidine kinase), the 2L gene (encoding a TNF-binding protein), or both. Some of the recombinants were armed to express mouse chemotactic protein 1 (mCCL2/mMCP-1), mouse granulocyte-monocyte colony stimulating factor (mGM-CSF), or bacterial flagellin (FliC). Tumors were induced in athymic nude mice by implantation of HCT 116 cells and subsequently treated by a single intratumoral injection of one of the recombinant TPVs. Histological examination showed a common neoplastic cell type and a range of immune cell infiltration, necrosis, and tumor cell organization. Significant regression was seen in tumors treated with virus TPV/Δ2L/Δ66R/fliC, and to a lesser extent the recombinants TPV/Δ2L and TPV/Δ66R. Our results suggest that oncolytic recombinants of the TPV armed with activators of the innate immune response may be effective virotherapeutic agents for colorectal cancers in humans and should be explored further to fully realize their potential.

The opening of intracellular potassium channels is a common mechanism of action for a set of anti-hypertensive drugs that includes the hair-growth-inducing agent minoxidil. Recent work suggests potassium channel openers (PCOs) also influence hair growth. Correlative studies demonstrate that a series of PCOs including minoxidil, pinacidil, P-1075, an active pinacidil analog, RP-49,356, cromakalim, and nicorandil maintain hair growth in cultured vibrissa follicles. Studies using balding stumptail macaques verify that minoxidil, P-1075, and cromakalim but not RP-49,356 stimulate hair growth. The definition of potassium channels and documentation of drug effects on these channels is classically done using electrophysiologic techniques. Such studies require the identification and isolation of target cells. Both these are among the unsolved problems in the area of hair biology. Estimating K+ flux using 86Rb+ as a K+ tracer is an accepted method of assessing potassium channel conductance in other organ systems. Both pinacidil and RP-49,356 induce measurable Rb+ flux in isolated vibrissa follicles and a hair epithelial cell line whereas neither minoxidil nor minoxidil sulfate had measurable effects. Potassium channels have been studied successfully in other organ systems using specific pharmacologic blockers for the various channel subtypes. Blockers including glyburide, tetraethylammonium, and procaine failed to inhibit minoxidil stimulation of cultured follicles. The current explosion of knowledge on potassium channel biology, cloning of channels, and continued progress in hair biology promise to clarify the role of K+ ions in the control of hair follicles.

The evolutionarily successful poxviruses possess effective and diverse strategies to circumvent or overcome host defense mechanisms. Poxviruses encode many immunoregulatory proteins to evade host immunity to establish a productive infection and have unique means of inhibiting DNA sensing-dependent type 1 interferon (IFN-I) responses, a necessity given their dsDNA genome and exclusively cytoplasmic life cycle. We found that the key DNA sensing inhibition by poxvirus infection was dominant during the early stage of poxvirus infection before DNA replication. In an effort to identify the poxvirus gene products which subdue the antiviral proinflammatory responses (e.g., IFN-I response), we investigated the function of one early gene that is the known host range determinant from the highly conserved poxvirus host range C7L superfamily, myxoma virus (MYXV) M062. Host range factors are unique features of poxviruses that determine the species and cell type tropism. Almost all sequenced mammalian poxviruses retain at least one homologue of the poxvirus host range C7L superfamily. In MYXV, a rabbit-specific poxvirus, the dominant and broad-spectrum host range determinant of the C7L superfamily is the M062R gene. The M062R gene product is essential for MYXV infection in almost all cells tested from different mammalian species and specifically inhibits the function of host Sterile α Motif Domain-containing 9 (SAMD9), as M062R-null (ΔM062R) MYXV causes abortive infection in a SAMD9-dependent manner. In this study we investigated the immunostimulatory property of the ΔM062R. We found that the replication-defective ΔM062R activated host DNA sensing pathway during infection in a cGAS-dependent fashion and that knocking down SAMD9 expression attenuated proinflammatory responses. Moreover, transcriptomic analyses showed a unique feature of the host gene expression landscape that is different from the dsDNA alone-stimulated inflammatory state. This study establishes a link between the anti-neoplastic function of SAMD9 and the regulation of innate immune responses.

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.