The Impact of Nutritional Supplementation on Donor Kidneys During Oxygenated Ex Vivo Subnormothermic Preservation.

Evidence suggests that nutritional supplementation during normothermic ex vivo perfusion improves organ preservation. However, it is unclear whether the same benefit is observed during room temperature (subnormothermic) oxygenated perfusion. In this study, we tested the impact of providing complete nutrition during subnormothermic perfusion on kidney outcomes. Methods: Porcine kidneys were recovered after 30 min of cross clamping the renal artery in situ to simulate warm ischemic injury. After flushing with preservation solution, paired kidneys were cannulated and randomly assigned to perfusion with either (1) hemoglobin-carrier hemoglobin-based oxygen carrier or (2) hemoglobin-based oxygen carrier + total parenteral nutrition (TPN) for 12 h at 22 °C. To mimic reperfusion injury, all kidneys were reperfused with whole blood for an additional 4 h at 37 °C. Kidney function and damage were assessed. Results: Kidneys preserved with or without TPN performed equally well, showing similar renal function postreperfusion. Histological findings indicated similar levels of damage from apoptosis staining and acute tubular necrosis scores in both groups. Additionally, markers of renal damage (KIM-1) and inflammation (IL-6; high-mobility group box 1) were similar between the groups. Conclusions: Unlike other studies using normothermic oxygenated perfusion platforms, nutritional supplementation does not appear to provide any additional benefit during ex vivo kidney preservation over 12 h evaluated by whole blood-based reperfusion method at subnormothermic temperature. Further study should include a kidney autotransplant model to assess the role of TPN in vivo.

Carbon monoxide mechanism of protection against renal ischemia and reperfusion injury.

Carbon monoxide is quickly moving past its historic label as a molecule once feared, to a therapeutic drug that modulates inflammation. The development of carbon monoxide releasing molecules and utilization of heme oxygenase-1 inducers have shown carbon monoxide to be a promising therapy in reducing renal ischemia and reperfusion injury and other inflammatory diseases. In this review, we will discuss the developments and application of carbon monoxide releasing molecules in renal ischemia and reperfusion injury, and transplantation. We will review the anti-inflammatory mechanisms of carbon monoxide in respect to mitigating apoptosis, suppressing dendritic cell maturation and signalling, inhibiting toll-like receptor activation, promoting anti-inflammatory responses, and the effects on renal vasculature.

Comparison of Centrifugal and Pulsatile Perfusion to Preserve Donor Kidneys Using Ex Vivo Subnormothermic Perfusion.

Objective We have previously demonstrated benefits of kidney preservation utilizing an oxygenated subnormothermic ex vivo perfusion platform. Herein, we aim to compare pulsatile versus centrifugal (steady and uniform flow) perfusion with the goal of optimizing renal preservation with these devices. Materials and methods: Pig kidneys were procured following 30 min of warm ischemia by cross-clamping both renal arteries. Paired kidneys were cannulated and underwent either: oxygenated pulsatile or centrifugal perfusion using a hemoglobin oxygen carrier at room temperature with our ex vivo machine perfusion platform for 4 hr. Kidneys were reperfused with whole blood for 4 hr at 37° C. Renal function, pathology and evidence of inflammation were assessed post-perfusion. Results: Both pump systems performed equally well with organs exhibiting similar renal blood flow, and function post-reperfusion. Histologic evidence of renal damage using apoptosis staining and acute tubular necrosis scores was similar between groups. This was corroborated with urinary assessment of renal damage (NGAL 1) and inflammation (IL-6), as levels were similar between groups. Conclusion: In our porcine model with added warm ischemia simulating the effects of reperfusion after transplantation, pulsatile perfusion yielded similar renal protection compared with centrifugal perfusion kidney preservation. Both methods of perfusion can be used in ex vivo kidney perfusion systems.

Renal Protection Against Ischemia Reperfusion Injury: Hemoglobin-based Oxygen Carrier-201 Versus Blood as an Oxygen Carrier in Ex Vivo Subnormothermic Machine Perfusion.

BACKGROUND: The optimal method of oxygen delivery to donor kidneys during ex vivo machine perfusion has not been established. We have recently reported the beneficial effects of subnormothermic (22°C) blood perfusion in the preservation of porcine donation after circulatory death kidneys. Since using blood as a clinical perfusate has limitations, including matching availability and potential presence of pathogen, we sought to assess hemoglobin-based oxygen carrier (HBOC-201) in oxygen delivery to the kidney for renal protection. METHODS: Pig kidneys (n = 5) were procured after 30 minutes of warm in situ ischemia by cross-clamping the renal arteries. Organs were flushed with histidine tryptophan ketoglutarate solution and subjected to static cold storage or pulsatile perfusion with an RM3 pump at 22°C for 4 hours with HBOC-201 and blood. Thereafter, kidneys were reperfused with normothermic (37°C) oxygenated blood for 4 hours. Blood and urine were subjected to biochemical analysis. Total urine output, urinary protein, albumin/creatinine ratio, flow rate, resistance were measured. Acute tubular necrosis, apoptosis, urinary kidney damage markers, neutrophil gelatinase-associated lipocalin 1, and interleukin 6 were also assessed. RESULTS: HBOC-201 achieved tissues oxygen saturation equivalent to blood. Furthermore, upon reperfusion, HBOC-201 treated kidneys had similar renal blood flow and function compared with blood-treated kidneys. Histologically, HBOC-201 and blood-perfused kidneys had vastly reduced acute tubular necrosis scores and degrees of terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining versus kidneys treated with cold storage. Urinary damage markers and IL6 levels were similarly reduced by both blood and HBOC-201. CONCLUSIONS: HBOC-201 is an excellent alternative to blood as an oxygen-carrying molecule in an ex vivo subnormothermic machine perfusion platform in kidneys.

Subnormothermic Oxygenated Perfusion Optimally Preserves Donor Kidneys Ex Vivo.

INTRODUCTION: The current methods of preserving donor kidneys in nonoxygenated cold conditions minimally protect the kidney against ischemia-reperfusion injury (IRI), a major source of complications in clinical transplantation. However, preserving kidneys with oxygenated perfusion is not currently feasible due to the lack of an ideal perfusion mechanism that facilitates perfusion with blood at warm temperature. Here, we have designed an innovative renal pump circuit system that can perfuse blood or acellular oxygen carrier under flexible temperatures, pressures, and oxygenation. We have tested this apparatus to study optimal conditions of storage of our porcine model of donation after cardiac death (DCD) kidneys. METHODS: Porcine kidneys were retrieved after 30 minutes of cross-clamping renal pedicles in situ. Cessation of blood mimics postcardiac death in humans and simulates DCD warm ischemic injury. Procured kidneys were flushed and subjected to static cold storage (SCS) for 4 hours. For warm perfusion, kidneys were cannulated for pulsatile oxygenated perfusion with blood:PlasmaLyte for 4 hours at 15 °C, 22 °C, and 37 °C. To mimic posttransplant scenario, all kidneys were reperfused with blood for an additional 4 hours at 37 °C. RESULTS: Compared with all other groups, 22 °C perfusion resulted in significant reduction of acute tubular necrosis (ATN), apoptosis, kidney damage markers, Toll-like receptor signaling, and cytokine production. It was associated with maximal renal blood flow and urine output. Kidneys stored at 15 °C thrombosed within 2 hours under this condition. Martius Scarlet Blue staining confirmed that 22 °C was the optimal temperature to minimize hemorrhage and blood clots. CONCLUSION: Our novel study shows that oxygenated perfusion at near-room-temperature provides optimal donor kidney storage conditions.

Machine preservation of donor kidneys in transplantation.

With increasing demands for 'less than ideal' kidneys for transplantation, machine perfusion of kidneys has been utilized to improve the preservation of kidneys during storage. Hypothermic machine perfusion (HMP) of renal allografts has been shown to reduce delayed graft function rates in both expanded criteria and donation after cardiac death renal allografts. However, the beneficial impact upon long-term graft function is unclear. There has been emerging evidence that both subnormothermic (room temperature) and normothermic machine perfusion (NMP) of allografts have beneficial effects with regards to early graft function, survival and injury in pre-clinical and early clinical studies. Additionally, machine perfusion allows functional assessment of the organ prior to transplantation. Ultimately, the greatest benefit of machine perfusion may be the ability to treat the organ with agents to protect the graft against ischemia reperfusion injury, while awaiting transplantation.

CORM-401 Reduces Ischemia Reperfusion Injury in an Ex Vivo Renal Porcine Model of the Donation After Circulatory Death.

BACKGROUND: Carbon monoxide (CO) inhalation protects organ by reducing inflammation and cell death during transplantation processes in animal model. However, using CO in clinical transplantation is difficult due to its delivery in a controlled manner. A manganese-containing CO releasing molecules (CORM)-401 has recently been synthesized which can efficiently deliver 3 molar equivalents of CO. We report the ability of this anti-inflammatory CORM-401 to reduce ischemia reperfusion injury associated with prolonged cold storage of renal allografts obtained from donation after circulatory death in a porcine model of transplantation. METHODS: To stimulate donation after circulatory death condition, kidneys from large male Landrace pig were retrieved after 1 hour warm ischemia in situ by cross-clamping the renal pedicle. Procured kidneys, after a brief flushing with histidine-tryptophan-ketoglutarate solution were subjected to pulsatile perfusion at 4°C with University of Wisconsin solution for 4 hours and both kidneys were treated with either 200 µM CORM-401 or inactive CORM-401, respectively. Kidneys were then reperfused with normothermic isogeneic porcine blood through oxygenated pulsatile perfusion for 10 hours. Urine was collected, vascular flow was assessed during reperfusion and histopathology was assessed after 10 hours of reperfusion. RESULTS: We have found that CORM-401 administration reduced urinary protein excretion, attenuated kidney damage markers (kidney damage marker-1 and neutrophil gelatinase-associated lipocalin), and reduced ATN and dUTP nick end labeling staining in histopathologic sections. CORM-401 also prevented intrarenal hemorrhage and vascular clotting during reperfusion. Mechanistically, CORM-401 appeared to exert anti-inflammatory actions by suppressing Toll-like receptors 2, 4, and 6. CONCLUSIONS: Carbon monoxide releasing molecules-401 provides renal protection after cold storage of kidneys and provides a novel clinically relevant ex vivo organ preservation strategy.

IL-37 inhibits IL-18-induced tubular epithelial cell expression of pro-inflammatory cytokines and renal ischemia-reperfusion injury.

Cytokines and chemokines produced by tubular epithelial and infiltrating cells are critical to inflammation in renal ischemia-reperfusion injury. IL-37, a newly described IL-1 family member, inhibits IL-18-dependent pro-inflammatory cytokine production by its binding to IL-18 receptors and IL-18 binding protein. The potential role of IL-37 in renal ischemia-reperfusion injury is unknown. Here we found that exposure of tubular epithelial cells to exogenous IL-37 downregulated hypoxia and the IL-18-induced expression of TNFα, IL-6, and IL-1ß. Importantly, human PT-2 tubular epithelial cells have inducible expression of IL-37. Moreover, pro-inflammatory cytokine expression was augmented in IL-37 mRNA-silenced tubular epithelial cells and inhibited by transfection with pCMV6-XL5-IL-37. In a mouse ischemic injury model, transgenic expression of human IL-37 inhibited kidney expression of TNFα, IL-6, and IL-1ß and improved mononuclear cell infiltration, kidney injury, and function. Thus, human tubular epithelial cells express the IL-18 contra-regulatory protein IL-37 as an endogenous control mechanism to reduce inflammation. Augmenting kidney IL-37 may represent a novel strategy to suppress renal injury responses and promote kidney function after renal ischemic injury and transplantation.

Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells.

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562(MLN), R-U937(MLN)) were selected. R-K562(MLN) and R-U937(MLN) cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme's affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562(MLN) cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.

Co-expression of α9ß1 integrin and VEGF-D confers lymphatic metastatic ability to a human breast cancer cell line MDA-MB-468LN.

INTRODUCTION AND OBJECTIVES: Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9ß1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9ß1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. RESULTS: A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9ß1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9ß1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9ß1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells. CONCLUSION: Differential capacity for VEGF-D production and α9ß1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.

Telomere-mediated chromosomal instability triggers TLR4 induced inflammation and death in mice.

BACKGROUND: Telomeres are essential to maintain chromosomal stability. Cells derived from mice lacking telomerase RNA component (mTERC-/- mice) display elevated telomere-mediated chromosome instability. Age-dependent telomere shortening and associated chromosome instability reduce the capacity to respond to cellular stress occurring during inflammation and cancer. Inflammation is one of the important risk factors in cancer progression. Controlled innate immune responses mediated by Toll-like receptors (TLR) are required for host defense against infection. Our aim was to understand the role of chromosome/genome instability in the initiation and maintenance of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: We examined the function of TLR4 in telomerase deficient mTERC-/- mice harbouring chromosome instability which did not develop any overt immunological disorder in pathogen-free condition or any form of cancers at this stage. Chromosome instability was measured in metaphase spreads prepared from wildtype (mTERC+/+), mTERC+/- and mTERC-/- mouse splenocytes. Peritoneal and/or bone marrow-derived macrophages were used to examine the responses of TLR4 by their ability to produce inflammatory mediators TNFalpha and IL6. Our results demonstrate that TLR4 is highly up-regulated in the immune cells derived from telomerase-null (mTERC-/-) mice and lipopolysaccharide, a natural ligand for TLR4 stabilises NF-kappaB binding to its promoter by down-regulating ATF-3 in mTERC-/- macrophages. CONCLUSIONS/SIGNIFICANCE: Our findings implied that background chromosome instability in the cellular level stabilises the action of TLR4-induced NF-kappaB action and sensitises cells to produce excess pro-inflammatory mediators. Chromosome/genomic instability data raises optimism for controlling inflammation by non-toxic TLR antagonists among high-risk groups.

Inhibition of poly (ADP-Ribose) polymerase-1 in telomerase deficient mouse embryonic fibroblasts increases arsenite-induced genome instability.

BACKGROUND: The telomerase enzyme is a viable target for anti-cancer therapy given the innate differences in telomerase activity between tumour cells and normal somatic cells. However, the time lag between telomerase inhibition and telomeres becoming critically short to trigger cell death, allows cancer cells to acquire drug resistance. Inhibition of DNA repair pathways along with telomerase could be an alternative strategy to enhance anti-tumour effects and circumvent the possibility of drug resistance. Poly (ADP-Ribose) Polymerase-1 (PARP-1), an important DNA damage sensor and a DNA repair factor, has important roles in maintaining telomeres and chromosomal stability. In this study, the effects of combined inhibition of PARP-1 and telomerase in mouse embryonic fibroblasts (MEFs) following sodium arsenite exposure (a carcinogen and potent DNA damaging agent), were evaluated. RESULTS: Inhibition of PARP in telomerase deficient MEFs induced an increase in arsenite-induced DNA damage as compared to control cells. Combined inhibition also resulted in enhanced genomic instability, demonstrated by elevated micronuclei induction and chromosomal aberrations with decreased cell survival. In addition, telomerase inhibition in PARP-1 deficient MEFs led to greater telomere shortening and increased genomic instability. CONCLUSIONS: Our study demonstrated that the co-inhibition of PARP-1 and telomerase in MEFs rendered cells more susceptible to DNA damaging agents. Hence, these results offer support for the use of combined inhibition of PARP-1 and telomerase as a strategy to minimise the problems associated with long-term telomerase inhibition in cancer therapeutics.

Relationship between cyclooxygenase-2 and human epidermal growth factor receptor 2 in vascular endothelial growth factor C up-regulation and lymphangiogenesis in human breast cancer.

Both cyclooxygenase (COX)-2 and human epidermal growth factor receptor (HER)-2 promote breast cancer progression; however, the relationship between the two molecules remains unclear. We utilized human breast cancer tissues and cell lines to examine whether COX-2 and HER-2 played independent or interdependent roles in vascular endothelial growth factor (VEGF)-C up-regulation and lymphangiogenesis. A paired correlation of immunodetectable levels of COX-2, VEGF-C, and HER-2 proteins and lymphovascular density (LVD; D2-40-immunolabeled) in 55 breast cancer specimens revealed a positive correlation between COX-2 and HER-2 irrespective of clinicopathological status. However COX-2 alone positively correlated with LVD. In 10 independent specimens, mRNA levels showed a positive correlation between HER-2 and COX-2 or VEGF-C but not LYVE-1 (lymphovascular endothelial marker). These findings implicate COX-2, but not HER-2, in breast cancer-associated lymphangiogenesis. Manipulation of the COX-2 or HER-2 genes in breast cancer cell lines varying widely in COX-2 and HER-2 expression revealed a direct role of COX-2 and an indirect COX-2 dependent role of HER-2 in VEGF-C up-regulation: (i) high VEGF-C expression in high COX-2/low HER-2 expressing MDA-MB-231 cells was reduced by siRNA-mediated down-regulation of COX-2, but not HER-2; (ii) integration of HER-2 in these cells simultaneously up-regulated COX-2 protein as well as VEGF-C secretion; and (iii) low VEGF-C secretion by high HER-2/low COX-2 expressing SK-BR-3 cells was stimulated by COX-2 overexpression. These findings of the primary role of COX-2 and the COX-2-dependent role of HER-2, if any, in VEGF-C up-regulation and lymphangiogenesis suggest that COX-2 inhibitors may abrogate lymphatic metastasis in breast cancer irrespective of HER-2 status.

Environmental toxicogenomics: a post-genomic approach to analysing biological responses to environmental toxins.

Environmental genomics has revolutionised how researchers can study the molecular basis of adverse effects of environmental toxicants. It is expected that the new discipline will afford efficient and high-throughput means to delineate mechanisms of action, risk assessment, identify and understand basic pathogenic mechanisms that are critical to disease progression, predict toxicity of unknown agents and to more precisely phenotype disease subtypes. Previously, we have demonstrated the potential of environmental genomics in a toxicant exposure model and, perhaps, this might become a crucial tool in biological response marker or biomarker discovery. To illustrate how toxicogenomics can be useful, we provide here an overview of some of the past and potential future aspects of environmental genomics. The present article also reviews the principles and technological concerns, and the standards and databases of toxicogenomics. In addition, applications of toxicogenomics in drug target identifications and validation strategies are also discussed.

Translocation of VP1686 upregulates RhoB and accelerates phagocytic activity of macrophage through actin remodeling.

Here, we report that Vibrio parahaemolyticus induces a rapid remodeling of macrophage actin and activates RhoB GTPase. Mutational analysis revealed that the effects depend on type III secretion system 1 regulated translocation of a V. parahaemolyticus effector protein, VP1686, into the macrophages. Remodeling of actin is shown to be necessary for increased bacterial uptake followed by initiation of apoptosis in macrophages. This provides evidence for functional association of the VP1686 in triggering an eat-me-and-die signal to the host.

Short dysfunctional telomeres impair the repair of arsenite-induced oxidative damage in mouse cells.

Telomeres and telomerase appear to participate in the repair of broken DNA ends produced by oxidative damage. Arsenite is an environmental contaminant and a potent human carcinogen, which induces oxidative stress on cells via the generation of reactive oxygen species affecting cell viability and chromosome stability. It promotes telomere attrition and reduces cell survival by apoptosis. In this study, we used mouse embryonic fibroblasts (MEFs) from mice lacking telomerase RNA component (mTERC(-/-) mice) with long (early passage or EP) and short (late passage or LP) telomeres to investigate the extent of oxidative damage by comparing the differences in DNA damage, chromosome instability, and cell survival at 24 and 48 h of exposure to sodium arsenite (As3+; NaAsO2). There was significantly high level of DNA damage in mTERC(-/-) cells with short telomeres as determined by alkaline comet assay. Consistent with elevated DNA damage, increased micronuclei (MN) induction reflecting gross genomic instability was also observed. Fluorescence in situ hybridization (FISH) analysis revealed that increasing doses of arsenite augmented the chromosome aberrations, which contributes to genomic instability leading to possibly apoptotic cell death and cell cycle arrest. Microarray analysis has revealed that As3+ treatment altered the expression of 456 genes of which 20% of them have known functions in cell cycle and DNA damage signaling and response, cell growth, and/or maintenance. Results from our studies imply that short dysfunctional telomeres impair the repair of oxidative damage caused by arsenite. The results will have implications in risk estimation as well as cancer chemotherapy.

VP1686, a Vibrio type III secretion protein, induces toll-like receptor-independent apoptosis in macrophage through NF-kappaB inhibition.

Vibrio parahaemolyticus, causative agent of human gastrointestinal diseases, possesses several virulent machineries including thermostable direct hemolysin and type III secretion systems (TTSS1 and -2). In this report, we establish that TTSS1-dependent secretion and translocation of a V. parahaemolyticus effector protein VP1686 into the cytosol induces DNA fragmentation in macrophages. We performed yeast two-hybrid screening to identify the molecules involved in VP1686-mediated cell death pathways and showed that nuclear factor RelA p65/NF-kappaB physically interacts with VP1686. To understand the impact of this interaction on the NF-kappaB DNA binding activities in infected macrophages, we analyzed a series of deletion mutants for the TTSS and its secreted proteins. Induction of DNA binding activity of NF-kappaB was significantly suppressed, and increased macrophage apoptosis has been associated with V. parahaemolyticus strain, which contains both VP1686 and TTSS1. Macrophages lacking Toll-like receptor adaptor molecules MyD88 (myeloid differentiation primary response protein 88) or TRIF (TIR domain-containing adapter-inducing interferon beta) showed similar sensitivity to VP1686. As a consequence of NF-kappaB suppression, microarray analysis has revealed that VP1686 translocation alerted the expression of many genes that have known functions in cellular responses to apoptosis, cell growth, and transcriptional regulation. Our results suggest an important role for Vibrio effector protein VP1686 that activate a conserved apoptotic pathway in macrophages through suppression of NF-kappaB activation independent of Toll-like receptor signaling.

Modifying toll-like receptor 9 signaling for therapeutic use.

Toll-like receptor (TLR) 9 recognizes synthetic oligodeoxynucleotides (ODN) containing unmethylated deoxycytidyl-deoxyguanosine (CpG) motifs and mimics the immunostimulatory activity of bacterial DNA. Both innate and adaptive immune systems are activated through TLR9 signaling and thus its synthetic agonists or inhibitors have potential significance as a target for therapeutic use in immunological disorders. Interestingly, TLR9 found in the dendritic cells and B cells produce differential outcome in response to structurally distinct CpG-ODNs. While one class of CpG-ODN activates B cells and produce immunoglobulin, other can either redirect plasmacytoid dendritic (pDC) cells to secrete high level of IFNalpha or myeloid dendritic cells (mDC) to produce Th1-like cytokines and chemokines necessary for asthma control. This review focuses on potential use of various synthetic CpG to modify TLR9 signaling for therapeutic treatment of multiple diseases including cancer, asthma, allergy and systemic lupus erythematosus (SLE).

Transcriptional silencing of the mouse mammary tumor virus promoter through chromatin remodeling is concomitant with histone H1 phosphorylation and histone H3 hyperphosphorylation at M phase.

We examined histone phosphorylation and their effects on glucocorticoid receptor (GR)-mediated activation of the mouse mammary tumor virus promoter (MMTV) in synchronized cells. In vivo protein expression studies suggest that both histones H1 and H3 are highly phosphorylated in mitotic-arrested cells in which GR is unable to remodel chromatin and recruit transcription factor NF1 to the promoter. Postmitotic cells show an open chromatin structure and efficient binding of NF1 to the promoter accompanied by reversing histone H1 and H3 phosphorylation level. In contrast, the acetylation status of histone H3 and H4 did not change in either condition. These results suggest that hyperphosphorylation of histone H1 and H3 leads to inhibition of GR-mediated chromatin remodeling and inactivation of MMTV by preventing the association of transcription factors to the promoter in vivo.

Lack of poly(ADP-ribose) polymerase-1 gene product enhances cellular sensitivity to arsenite.

Arsenite (As3+) has long been known to induce cancer and other degenerative diseases. Arsenite exerts its toxicity in part by generating reactive oxygen species. Identification of genetic factors that contribute to arsenic mutagenicity and carcinogenicity is critical for the treatment and prevention of arsenic exposure in human population. As poly(ADP-ribose) polymerase (PARP) is critical for genomic DNA stability, role of PARP-1 was evaluated in arsenic-induced cytotoxic and genotoxic effects. Our study revealed that telomere attrition, probably owing to arsenite-induced oxidative stress, was much more pronounced in PARP-1-/- mouse embryonic fibroblasts (MEF; 40%) compared with PARP-1+/+ MEFs (10-20%). Correlation observed between telomere reduction and apoptotic death in PARP-1 null cells strongly indicates that the telomere attrition might be a trigger for enhanced apoptotic death after arsenite treatment. Elevated DNA damage detected by alkaline comet assay points to an impaired repair ability of arsenite-induced DNA lesions in PARP-1-/- MEFs. Consistent with elevated DNA damage, increased micronuclei induction reflecting gross genomic instability was also observed in arsenite-treated PARP-1-/- MEFs. Microarray analysis has revealed that arsenite treatment altered the expression of about 311 genes majority of which have known functions in cellular responses to stress/external stimulus and cell growth and/or maintenance. Our results suggest an important role for PARP-1 gene product in the maintenance of chromosome-genome stability in response to arsenite-induced DNA damage.