Assessing the infiltration of immune cells in the upper trachea mucosa after infectious laryngotracheitis virus (ILTV) vaccination and challenge.

The types of immune cells that populate the trachea after ILTV vaccination and infection have not been assessed. The objective of this study was to quantify CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells that infiltrate the trachea after vaccination with chicken embryo origin (CEO), tissue culture origin (TCO), and recombinant herpesvirus of turkey-laryngotracheitis (rHVT-LT) vaccines, and after challenge of vaccinated and non-vaccinated chickens with a virulent ILTV strain. Eye-drop vaccination with CEO, or TCO, or in ovo vaccination with rHVT-LT did not alter the number of CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells in the trachea. After challenge, the CEO vaccinated group of chickens showed swift clearance of the challenge virus, the mucosa epithelium of the trachea remained intact, and a limited number of CD4+, CD8α+, and CD8ß+ cells were detected in the upper trachea mucosa. The TCO and rHVT-LT vaccinated groups of chickens showed narrow viral clearance with moderate disruption of the trachea epithelial integrity, and a significant increase in CD4+, CD8α+, CD8ß+, and TCRγδ+ cells infiltrated the upper trachea mucosa. Non-vaccinated challenged chickens showed high levels of viral replication, the epithelial organization of the upper trachea mucosa was heavily disrupted, and the predominant infiltrates were CD4+, TCRγδ+, and MRC1LB+ cells. Hence, the very robust protection provided by CEO vaccination was characterized by minimal immune cell infiltration to the trachea mucosa. In contrast, partial protection induced by the TCO and rHVT-LT vaccines requires a prolonged period of T cell expansion to overcome the established infection in the trachea mucosa.

Reactive oxygen species regulate nucleostemin oligomerization and protein degradation.

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.

Cyclopentenyl cytosine induces senescence in breast cancer cells through the nucleolar stress response and activation of p53.

The induction of senescence has emerged as a potentially important contributor to the effects of chemotherapeutic agents against tumors. We have demonstrated that depletion of CTP induced by cyclopentenyl cytosine (CPEC; NSC 375575), a specific inhibitor of the enzyme CTP synthetase, induces irreversible growth arrest and senescence characterized by altered morphology and expression of senescence-associated ß-galactosidase activity in MCF-7 breast cancer cells expressing wild-type p53. In contrast, differentiation in the absence of senescence resulted from CPEC treatment in MDA-MB-231 breast cancer cells that express a mutated p53. Both senescence of MCF-7 cells and differentiation of MDA-MB-231 cells were prevented by repletion of CTP through the cytidine salvage pathway. Senescence in MCF-7 cells was associated with a G(2)- and S-phase arrest, whereas differentiation in MDA-MB-231 cells was associated with arrest in G(1) phase at 5 days. Mechanistic studies revealed that CTP depletion induced a rapid translocation of nucleolar proteins, including nucleostemin and nucleolin into the nucleoplasm. This nucleolar stress response resulted in a sustained elevation of p53 and the p53 target genes, p21 and Mdm2, in cells with wild-type p53. Furthermore, short interfering RNA-induced knockdown of p53 in MCF-7 cells treated with CPEC prevented cellular senescence and increased apoptotic cell death. We conclude that CTP depletion and the resulting nucleolar stress response results in a senescence-like growth arrest through activation of p53, whereas cells with mutated p53 undergo differentiation or apoptotic cell death.

A Cell Line Adapted Infectious Laryngotracheitis Virus Strain (BΔORFC) for in ovo and Hatchery Spray Vaccination Alone or in Combination with a Recombinant HVT-LT Vaccine.

To produce more-stable, live attenuated vaccines for infectious laryngotracheitis virus (ILTV), deletion of genes related to virulence has been extensively pursued. Although its function remains unknown, the open reading frame C (ORF C) is among the genes potentially associated with viral virulence that is nonessential for replication in vitro. Earlier results indicated that the ILT virus with deletion of the ORF C gene (BΔORFC) was suitable and safe for eye drop administration but was not sufficiently attenuated for in ovo administration. The objective of this study was to evaluate the safety and protection efficacy of a cell line-adapted, gene-deleted strain (BΔORFC) of ILTV when administered in ovo and/or spray (SP) by itself, or in combination with the recombinant HVT-LT (rHVT-LT) vaccine. Results indicated that vaccination with the BΔORFC strain, either by itself or in combination with an rHVT-LT vaccine, did not affect hatchability, and only marginal signs of respiratory distress were recorded for groups of chickens that received the BΔORFC strain via SP. The replication and seroconversion induced by the BΔORFC strain after in ovo and SP administration was very limited, whereas the replication of the rHVT-LT vaccine was delayed when combined with the BΔORFC strain in ovo. Compared to rHVT-LT or BΔORFC when administered alone, dual vaccination with rHVT-LT + BΔORFC was more effective in mitigating clinical signs of the disease and reducing challenge virus load in the trachea. To our knowledge, this study provides the first proof of concept that ILTV strains can be sufficiently attenuated for early vaccination in ovo or at hatch; also, this study documented the benefits of using a dual (recombinant and live attenuated) hatchery vaccination strategy for ILTV.

Una cepa del virus de la laringotraqueítis infecciosa adaptada a una línea celular (BΔORFC) para vacunación in ovo y en aerosol en incubadora aplicada por sí sola o en combinación con una vacuna recombinante para laringotraqueítis con el vector HVT. Para producir vacunas vivas atenuadas más estables contra el virus de la laringotraqueítis infecciosa (ILTV), se ha buscado ampliamente la eliminación de genes relacionados con la virulencia. Aunque su función sigue siendo desconocida, el marco de lectura continuo C (ORF C) se encuentra entre los genes potencialmente asociados con la virulencia viral que no es esencial para la replicación in vitro. Resultados anteriores indican que el virus de la laringotraqueítis infecciosa con deleción del gene ORF C (BΔORFC) era adecuado y seguro para la administración ocular, pero no estaba lo suficientemente atenuado para su administración in ovo. El objetivo de este estudio fue evaluar la seguridad y la eficacia de la protección de una cepa del virus de la laringotraqueítis infecciosa con deleción genética y adaptada a una línea celular (BΔORFC) cuando se administra in ovo y/o en aerosol por sí sola, o en combinación con una vacuna recombinante con el vector HVT (vacuna rHVT-LT). Los resultados indicaron que la vacunación con la cepa BΔORFC, ya sea sola o en combinación con la vacuna rHVT-LT, no afectó la incubabilidad, y solo se registraron signos marginales de dificultad respiratoria para los grupos de pollos que recibieron la cepa BΔORFC por aspersión. La replicación y seroconversión inducida por la cepa BΔORFC después de la administración in ovo y por aspersión fue muy limitada, mientras que la replicación de la vacuna rHVT-LT se retrasó cuando se combinó con la cepa BΔORFC in ovo. En comparación con las vacunas rHVT-LT o BΔORFC administradas por sí solas, la vacunación dual con rHVT-LT + BΔORFC fue más eficaz para mitigar los signos clínicos de la enfermedad y reducir la carga del virus de desafío en la tráquea. Hasta donde se conoce, este estudio proporciona la primera prueba del concepto de que las cepas del virus de la laringotraqueítis infecciosa pueden atenuarse lo suficiente para la vacunación temprana in ovo o en la incubadora. Además, este estudio documentó los beneficios de utilizar una estrategia de vacunación de incubadora dual (vacuna recombinante y viva atenuada) para la laringotraqueítis infecciosa.

Activation of Cytotoxic Lymphocytes and Presence of Regulatory T Cells in the Trachea of Non-Vaccinated and Vaccinated Chickens as a Recall to an Infectious Laryngotracheitis Virus (ILTV) Challenge.

While the protective efficacy of the infectious laryngotracheitis virus (ILTV) vaccines is well established, little is known about which components of the immune response are associated with effective resistance and vaccine protection. Early studies have pointed to the importance of the T cell-mediated immune responses. This study aimed to evaluate the activation of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and to quantify the presence of regulatory T cells (Tregs) in the larynx-trachea of chickens vaccinated with chicken embryo origin (CEO), tissue culture origin (TCO) and recombinant Herpesvirus of Turkey-laryngotracheitis (rHVT-LT) vaccines after challenge. Our results indicated that CEO vaccine protection was characterized by early CTLs and activated CTLs enhanced responses. TCO and rHVT-LT protection were associated with a moderate increase in resting and activated CTLs followed by an enhanced NK cell response. Tregs increase was only detected in the non-vaccinated challenged group, probably to support healing of the severe trachea epithelial damage. Taken together, our results revealed main differences in the cellular immune responses elicited by CEO, TCO, and rHVT-LT vaccination in the upper respiratory tract after challenge, and that activated CTLs rather than NK cells play a main role in vaccine protection.

Statin treatment prevents the development of pulmonary arterial hypertension in a nonhuman primate model of HIV-associated PAH.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by pulmonary vascular remodeling, elevated pulmonary arterial pressure, and right heart failure. Human immunodeficiency virus (HIV)-infected individuals have a higher incidence of PAH than the non-HIV infected population and evidence suggests a role for systemic and pulmonary inflammation in the pathogenesis of HIV-associated PAH. Due to their pleiotropic effects, including immune-modulatory and anti-inflammatory effects, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been considered for the treatment of PAH, with conflicting results. The effects of statins on HIV-associated PAH have not been specifically evaluated. We have developed a non-human primate (NHP) model of HIV-associated PAH that closely mimics HIV-PAH using simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta). We determined that treatment of healthy macaques with atorvastatin prior to and throughout SIV infection prevented the development of SIV-associated PAH. Additionally, SIV-infected macaques that initiated atorvastatin treatment during the early chronic disease stage had reduced incidence of PAH compared to untreated animals. Statin treatment reduced inflammatory mediators TGF-ß, MIP-1α, and TNF-α and the numbers of CD14dimCD16+ non-classical monocytes, and CD14+CCR7-CD163-CD206+ alveolar macrophages previously shown to be associated with SIV-PAH. These results support the concept that statins reduce inflammatory processes that contribute to PAH and may provide a safe and effective prophylactic strategy for the prevention of PAH in HIV-infected individuals.

Novel mechanism of arenavirus-induced liver pathology.

Viral hemorrhagic fevers (VHFs) encompass a group of diseases with cardinal symptoms of fever, hemorrhage, and shock. The liver is a critical mediator of VHF disease pathogenesis and high levels of ALT/AST transaminases in plasma correlate with poor prognosis. In fact, Lassa Fever (LF), the most prevalent VHF in Africa, was initially clinically described as hepatitis. Previous studies in non-human primate (NHP) models also correlated LF pathogenesis with a robust proliferative response in the liver. The purpose of the current study was to gain insight into the mechanism of liver injury and to determine the potential role of proliferation in LF pathogenesis. C57Bl/6J mice were infected with either the pathogenic (for NHPs) strain of lymphocytic choriomeningitis virus (LCMV, the prototypic arenavirus), LCMV-WE, or with the non-pathogenic strain, LCMV-ARM. As expected, LCMV-WE, but not ARM, caused a hepatitis-like infection. LCMV-WE also induced a robust increase in the number of actively cycling hepatocytes. Despite this increase in proliferation, there was no significant difference in liver size between LCMV-WE and LCMV-ARM, suggesting that cell cycle was incomplete. Indeed, cells appeared arrested in the G1 phase and LCMV-WE infection increased the number of hepatocytes that were simultaneously stained for proliferation and apoptosis. LCMV-WE infection also induced expression of a non-conventional virus receptor, AXL-1, from the TAM (TYRO3/AXL/MERTK) family of receptor tyrosine kinases and this expression correlated with proliferation. Taken together, these results shed new light on the mechanism of liver involvement in VHF pathogenesis. Specifically, it is hypothesized that the induction of hepatocyte proliferation contributes to expansion of the infection to parenchymal cells. Elevated levels of plasma transaminases are likely explained, at least in part, by abortive cell cycle arrest induced by the infection. These results may lead to the development of new therapies to prevent VHF progression.