PARP-3 and APLF function together to accelerate nonhomologous end joining

PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf−/− B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions

Fighting viral infections and virus-driven tumors with cytotoxic CD4+ T cells

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors

Suppression of steady-state, but not stimulus-induced NF-kappaB activity inhibits alphavirus-induced apoptosis.

Recent studies have established cell type- specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-kappaB. In each of these studies, inhibitors of NF-kappaB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-kappaB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-kappaB activation and apoptosis in cultured cell lines. To address whether Sindbis virus- induced NF-kappaB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-kappaB activity. Complete suppression of virus-induced NF-kappaB activity neither prevents nor potentiates Sindbis virus-induced apoptosis. In contrast, inhibition of NF-kappaB activity before infection inhibits Sindbis virus-induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-kappaB activity, regulates expression of gene products required for Sindbis virus-induced death. Furthermore, we show that in the same cell line, NF-kappaB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-kappaB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-kappaB activity relative to the death stimulus

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

A latent ability to persist: differentiation in Toxoplasma gondii

A critical factor in the transmission and pathogenesis of Toxoplasma gondii is the ability to convert from an acute disease-causing, proliferative stage (tachyzoite), to a chronic, dormant stage (bradyzoite). The conversion of the tachyzoite-containing parasitophorous vacuole membrane into the less permeable bradyzoite cyst wall allows the parasite to persist for years within the host to maximize transmissibility to both primary (felids) and secondary (virtually all other warm-blooded vertebrates) hosts. This review presents our current understanding of the latent stage, including the factors that are important in bradyzoite induction and maintenance. Also discussed are the recent studies that have begun to unravel the mechanisms behind stage switching

Interferon regulatory factor 2 protects mice from lethal viral neuroinvasion.

The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study, we demonstrate that IFN regulatory factor 2 (IRF2), an ISG and a negative regulator of IFN signaling, influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2-/- mice after peripheral inoculation. Irf2-/- mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2-/- mouse brains despite the presence of peripheral neutralizing antibodies, suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell-deficient μMT mice are significantly more susceptible to viral infection, yet WT B cells and serum are unable to rescue the Irf2-/- mice. Collectively, our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections

Studies on early cellular responses during Epstein-Barr virus infection

The human gamma-herpesvirus Epstein-Barr virus (EBV) has been implicated in the pathogenesis of a broad spectrum of lymphoid and epithelial cell malignancies. A characteristic property of the virus is the capacity to establish a non-productive growthpromoting infection in B-lymphocytes. Although the induction of cell proliferation is a key feature in oncogenesis, it is not sufficient for full malignancy. In the work presented in this thesis my colleagues and I have asked whether the virus might contribute to oncogenesis by triggering additional events that are required for tumor progression. Replicative immortality is dependent on the activation of mechanisms that maintain the integrity of telomeres. Malignant cells achieve this by activating telomerase or a recombination-dependent pathway known as alternative lengthening of telomeres (ALT). We observed multiple signs of telomere dysfunction consistent with the activation of ALT in newly EBV infected Blymphocytes. These include accumulation of telomere-associated promyelocytic leukemia nuclear bodies (APBs), telomeric-sister chromatid exchange (T-SCE), and low expression of telomere associated proteins such as TRF1, TRF2, POT1, and ATRX, pointing to telomere de-protection as possible cause of telomere damage. The early phase of EBV induced B-cell immortalization is characterized by the accumulation of DNA damage and activation of a DNA damage response (DDR) that limits the efficiency of growth transformation. By comparing the response of B-lymphocytes infected with EBV or stimulated with a potent Bcell mitogen, we found that significant higher levels of damage occur in EBV infected blasts due to stronger and sustained accumulation of reactive oxygen species (ROS). Quenching of ROS did not affect the kinetics and magnitude of viral gene expression but dramatically decreased the efficiency of B-cell transformation, which correlated with selective downregulation of the viral LMP1 and the phosphorylated form of the cellular transcription factor STAT3. Analysis of the mechanism by which high levels of ROS support LMP1 expression revealed selective inhibition of viral microRNAs that target the LMP1 transcript. Viral products that are delivered to the infected cells by the incoming virions are likely to play important roles in regulating the cellular response to infection. One of such products, the large tegument protein BPLF1, is a cysteine protease with potent ubiquitin and NEDD8-specific deconjugase activities. We found that targeting of the deneddylase activity of BPLF1 to nucleus of productively infected cells requires processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious viruses. Collectively, the findings summarized in this thesis provide new insights on the capacity of EBV to contribute to tumor initiation and progression by triggering events, such as oxidative stress and ALT, that favor the acquisition of both genomic instability and replicative immortality. Regulation of viral functions by the cellular response to danger signals delivered by incoming virions may further contribute to the remodeling of the host cell environment allowing successful infection