Response Killing Research Articles

Diminished neutrophil extracellular trap (NET) formation is a novel innate immune deficiency induced by acute ethanol exposure in polymicrobial sepsis, which can be rescued by CXCL1.

Polymicrobial sepsis is the result of an exaggerated host immune response to bacterial pathogens. Animal models and human studies demonstrate that alcohol intoxication is a key risk factor for sepsis-induced mortality. Multiple chemokines, such as CXCL1, CXCL2 and CXCL5 are critical for neutrophil recruitment and proper function of neutrophils. However, it is not quite clear the mechanisms by which acute alcohol suppresses immune responses and whether alcohol-induced immunosuppression can be rescued by chemokines. Thus, we assessed whether acute ethanol challenge via gavage diminishes antibacterial host defense in a sepsis model using cecal ligation and puncture (CLP) and whether this immunosuppression can be rescued by exogenous CXCL1. We found acute alcohol intoxication augments mortality and enhances bacterial growth in mice following CLP. Ethanol exposure impairs critical antibacterial functions of mouse and human neutrophils including reactive oxygen species production, neutrophil extracellular trap (NET) formation, and NET-mediated killing in response to both Gram-negative (E. coli) and Gram-positive (Staphylococcus aureus) pathogens. As compared with WT (C57Bl/6) mice, CXCL1 knockout mice display early mortality following acute alcohol exposure followed by CLP. Recombinant CXCL1 (rCXCL1) in acute alcohol challenged CLP mice increases survival, enhances bacterial clearance, improves neutrophil recruitment, and enhances NET formation (NETosis). Recombinant CXCL1 (rCXCL1) administration also augments bacterial killing by alcohol-treated and E. coli- and S. aureus-infected neutrophils. Taken together, our data unveils novel mechanisms underlying acute alcohol-induced dysregulation of the immune responses in polymicrobial sepsis, and CXCL1 is a critical mediator to rescue alcohol-induced immune dysregulation in polymicrobial sepsis.

Abstract 5459: Autophagy and senescence as possible mechanisms leading to proliferative recovery and escape from treatment-induced tumor dormancy

Abstract Despite the ability of chemotherapy to eliminate the majority of tumor cells, some are able to escape cell death and proliferate. In this study we evaluated mechanisms of autophagy and senescence, that in theory allow cells to escape apoptosis and might be responsible for cancer recurrence. Autophagy and senescence, either alone or in concert, may result in temporary growth arrest followed by proliferative recovery. With regard to autophagy, its function may be either cytoprotective, where autophagy inhibition results in sensitization to therapy or nonprotective, where autophagy inhibition does not alter sensitivity to chemotherapy and/or ionizing radiation. To determine the mode of autophagy in murine metastatic carcinoma 4T1 cell lines, autophagy was inhibited pharmacologically using chloroquine, and by genetic silencing of the Atg5 autophagy associated protein. Cells were treated with 1 μM Adriamycin, stained with Acridine Orange to assess autophagy by fluorescent microscopy (autophagosomes appear orange-red) and autophagy quantified by Flow Cytometry. Induction and blockade of autophagy by chloroquine was confirmed by Western blotting for the appearance of LC3B and degradation of p62. Senescence was monitored by β-galactosidase staining and quantified by Flow Cytometry based on C12FDG staining; senescence was confirmed based on the induction of p21 and p16. Adriamycin (doxorubicin) exposure resulted in breast tumor cell death as well as prolonged growth arrest; some of the arrested cells eventually recovered and formed colonies. After exposure to doxorubicin, 4T-1 cells were sorted based on intensity of β-galactosidase staining and increased size (senescent cells experience changes in size and morphology). β-galactosidase positive cells were plated and monitored over time during which some of the cells recovered the capacity to proliferate. In separate studies with mouse mammary carcinoma cells, radiation also promoted cell death and prolonged growth arrest from which some cells were able to recover proliferative capacity. Our data suggests that although chemotherapy and radiation induce prolonged growth arrest and senescence, these features are not permanent and cells are able to escape and re-emerge from the senescent state to generate proliferating daughter cells. As pharmacologic inhibition of autophagy did not result in increased sensitivity to doxorubicin in 4T1 cells (or to radiation in the mouse mammary tumor cells) or interfere with proliferative recovery, we postulate that inhibition of senescence associated pathways may block proliferative recovery and/or promote tumor cell killing in response to chemotherapy and/or radiation. Studies in progress are focused on modulation of c-myc and miR34 levels and the IL1/6/8 signaling axis as potential strategies for interference with senescence and suppression of residual (dormant) tumor growth and cancer recurrence. Citation Format: Liliya Tyutyunyk, Theresa Thekkudan, Tareq Saleh, David A. Gewirtz. Autophagy and senescence as possible mechanisms leading to proliferative recovery and escape from treatment-induced tumor dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2017-5459

NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

NADPH oxidase-generated reactive oxygen species (ROS) from immune cells are well known to be important for pathogen killing in response to TLR ligands. Here, we investigated a new aspect of NADPH oxidase in the TLR2/6-induced release of the immunologically relevant GM-CSF by endothelial cells. Stimulation of human endothelial cells with TLR2/6 agonist, MALP-2 (macrophage-activating lipopeptide of 2 kDa), induced NADPH oxidase activation and ROS formation. Inhibition by ROS scavengers and NADPH oxidase inhibitors blocked MALP-2-induced GM-CSF release. NADPH oxidase activators or ROS donors alone did not result in GM-CSF secretion; however, additional superoxide supply augmented MALP-2-induced GM-CSF secretion and restored GM-CSF levels after NADPH oxidase inhibition. MALP-2-dependent NF-ĸB activation was suppressed by NADPH oxidase inhibition, and inhibition of NF-κB completely blunted MALP-2-induced GM-CSF release. Vascular explants from mice that were deficient for the NADPH oxidase subunit p47 phox showed diminished intimal superoxide production and GM-CSF release after ex vivo stimulation with MALP-2. Moreover, an increase in circulating progenitor cells after MALP-2 injection was completely abolished in p47phox-knockout mice. Finally, MALP-2 stimulation increased mRNA expression of the major subunit NADPH oxidase, (Nox)2, in endothelial cells, and Nox2 inhibition prevented MALP-2-induced GM-CSF release. Our findings identify a Nox2-containing NADPH oxidase as a crucial regulator of the immunologic important growth factor GM-CSF after TLR2/6 stimulation in endothelial cells.-Schuett, J., Schuett, H., Oberoi, R., Koch, A.-K., Pretzer, S., Luchtefeld, M., Schieffer, B., Grote, K. NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies

Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8(+) T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1. While chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1.

Troxerutin, a natural flavonoid binds to DNA minor groove and enhances cancer cell killing in response to radiation

Troxerutin, a flavonoid best known for its radioprotective and antioxidant properties is of considerable interest of study due to its broad pharmacological activities. The present study on troxerutin highlights its abilities to bind DNA and enhance cancer cell killing in response to radiation. Troxerutin showed strong binding with calf thymus DNA in vitro. Troxerutin-DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing troxerutin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. DAPI fluorescence was drastically reduced with linear increase in troxerutin concentration suggesting possible binding of troxerutin to DNA minor groove. Further, computational studies of docking of troxerutin molecule on mammalian DNA also indicated possible troxerutin-DNA interaction at minor groove of DNA. Troxerutin was found to mainly localize in the nucleus of prostate cancer cells. It induced cytotoxicity in radioresistant (DU145) and sensitive (PC3) prostate cancer cells. When troxerutin pre-treated DU145 and PC3 cells were exposed to γ-radiation, cytotoxicity as estimated by MTT assay, was found to be further enhanced. In addition, the % subG1 population detected by propidium iodide staining also showed similar response when combined with radiation. A similar trend was observed in terms of ROS generation and DNA damage in DU145 cells when troxerutin and radiation were combined. DNA binding at minor groove by troxerutin may have contributed to strand breaks leading to increased radiation induced cell death.

Defective monocyte oxidative burst predicts infection in alcoholic hepatitis and is associated with reduced expression of NADPH oxidase

ObjectiveIn order to explain the increased susceptibility to serious infection in alcoholic hepatitis, we evaluated monocyte phagocytosis, aberrations of associated signalling pathways and their reversibility, and whether phagocytic defects could...

Abstract 2323: Mutant B-Raf-mediated activation of deubiquitinases in melanoma defines Usp5 as a potential therapeutic target.

Abstract Melanoma is a lethal form of cutaneous cancer which accounts for 80% of skin cancer deaths owing to its highly metastatic behavior and few clinically effective therapies. More than 60% of melanoma patients present with mutations in B-Raf, a serine/threonine kinase that increases proliferation and survival through the constitutive activation of ERK signaling. Inhibition of mutated B-Raf with specific inhibitors like vemurafenib reduces tumor growth and extends patient survival. However, clinical responsiveness to vemurafenib is limited in most melanoma patients, prompting analysis of additional pathways that could be targeted to extend vemurafenib responsiveness or overcome resistance. Towards this goal, we examined the impact of vemurafenib on substrates or cascades activated in B-Raf mutant and wild-type melanoma cells. We were particularly interested in assessing the impact of mutant B-Raf inhibition on deubiquitinases (DUBs), which are overexpressed or activated in many tumor types. DUBs control the stability, activity and localization of multiple proteins and are emerging as therapeutic targets. We found that vemurafenib treatment or lentiviral-mediated B-Raf knockdown reduced the activity of several DUBs in B-Raf mutant melanoma cells, including Usp9x, Usp14 and Usp5. These DUBs were not affected by vemurafenib in w/t B-Raf-expressing cells. Interestingly lentiviral-mediated knockdown of Usp5, but not Usp9x, reduced melanoma growth and increased vemurafenib sensitivity whereas Usp5 overexpression suppressed vemurafenib activity. Inhibition of Usp5 activity by vemurafenib or by Usp5 shRNA KD alone had little effect on melanoma cell apoptosis. However B-Raf inhibition in Usp5 KD cells resulted in >5-fold increased melanoma cell apoptosis. Further, Usp5 KD increased accumulation of p53 and p53-related proteins as well as their transcriptional targets. Our data clearly shows that mutant B-Raf activates Usp5 to suppress apoptotic responsiveness to vemurafenib and kinase inhibition alone was not sufficient to completely suppress Usp5 activity. Silencing studies demonstrate that Usp5 inhibition will enhance cell killing in response to B-Raf inhibition in melanoma. To address that potential, we compared anti-tumor activity in mice treated with vemurafenib and a small molecule DUB inhibitor, WP1130, with activity against Usp5. Treatment with either agent alone reduced tumor growth while mice receiving both vemurafenib and WP1130 had enhanced anti-tumor responses. Together, these results suggest that mutant B-Raf activates Usp5 activity to suppress p53 induction and apoptotic responsiveness to vemurafenib. These observations also suggest that further testing and refinement of Usp5 inhibitors as an additional approach to targeted therapy for melanoma are warranted. Citation Format: Harish Potu, Luke F. Peterson, Monique Verhaegen, Moshe Talpaz, Nicholas J. Donato. Mutant B-Raf-mediated activation of deubiquitinases in melanoma defines Usp5 as a potential therapeutic target. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2323. doi:10.1158/1538-7445.AM2013-2323

A novel murine model to deplete hepatic stellate cells uncovers their role in amplifying liver damage in mice

We have developed a novel model for depleting mouse hepatic stellate cells (HSCs) that has allowed us to clarify their contributions to hepatic injury and fibrosis. Transgenic (Tg) mice expressing the herpes simplex virus thymidine kinase gene (HSV-Tk) driven by the mouse GFAP promoter were used to render proliferating HSCs susceptible to killing in response to ganciclovir (GCV). Effects of GCV were explored in primary HSCs and in vivo. Panlobular damage was provoked to maximize HSC depletion by combining CCl(4) (centrilobular injury) with allyl alcohol (AA) (periportal injury), as well as in a bile duct ligation (BDL) model. Cell depletion in situ was quantified using dual immunofluorescence (IF) for desmin and GFAP. In primary HSCs isolated from both untreated wild-type (WT) and Tg mice, GCV induced cell death in ≈ 50% of HSCs from Tg, but not WT, mice. In TG mice treated with CCl(4) +AA+GCV, there was a significant decrease in GFAP and desmin-positive cells, compared to WT mice (≈ 65% reduction; P < 0.01), which was accompanied by a decrease in the expression of HSC-activation markers (alpha smooth muscle actin, beta platelet-derived growth factor receptor, and collagen I). Similar results were observed after BDL. Associated with HSC depletion in both fibrosis models, there was marked attenuation of fibrosis and liver injury, as indicated by Sirius Red/Fast Green, hematoxylin and eosin quantification, and serum alanine/aspartate aminotransferase. Hepatic expression of interleukin-10 and interferon-gamma was increased after HSC depletion. No toxicity of GCV in either WT or Tg mice accounted for the differences in injury. Activated HSCs significantly amplify the response to liver injury, further expanding this cell type's repertoire in orchestrating hepatic injury and repair.

Functional Characterization of T- and NK-Cells in Chronic Myeloid Leukemia Patients Treated with Interferon-α Monotherapy.

AbstractAbstract 2782 Background:The inhibition of oncogenic BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has significantly improved the prognosis of CML, but the majority of patients need life-long therapy. Before the TKI era, CML patients were treated with interferon-α (IFN-α), and a minor proportion of patients (10–20%) achieved prolonged complete cytogenetic remissions (CCyR). Interestingly, many of the patients in prolonged CCyR were able to discontinue the treatment without imminent disease relapse. As the ultimate goal of current CML therapy is cure, the deeper understanding of the cellular mechanisms behind successful IFN-α discontinuation are of outmost importance. In this study, we analyzed the function of immune effector cells (NK- and T-cells) derived from CML patients in prolonged molecular remission after IFN-α monotherapy in order to understand the putative anti-leukemic effects. METHODS:The study cohort included 13 CML patients treated with IFN-α who were in complete molecular remission (CMR) at the time of blood sampling. Five patients were still on IFN-α therapy (IFN-ON, median duration of the treatment 13.7 years) and 8 had stopped treatment successfully (IFN-OFF) and were off from any treatment (median time without treatment 6.3 years). None of the patients had used any TKIs. Samples from 10 healthy volunteers were used as controls. The cytotoxicity of NK-cells in blood was studied by measuring the direct killing of K562 cells and by degranulation assay (CD107). The cytokine secretion (TNF-α and IFN-g) of NK-cells was measured with flow cytometry after stimulation with K562. The function of T-cells was studied by antibody (OKT3) stimulation and measuring TNF-α and IFN-g production by flow cytometry. In addition, NK-cells were phenotyped by multi-color flow cytometry. RESULTS:IFN-OFF patients had a larger proportion of NK-cells from lymphocytes than IFN-ON patients or healthy controls (IFN-OFF median 23.5 %, IFN-ON 7.0 %, healthy 13.2 %; p=0.0136). Based on the subset analysis, expanded NK-cells in IFN-OFF patients had mature phenotype CD56dimCD62LlowCD27lowCD57high. On the contrary, IFN-ON patients had a larger proportion of immunoregulatory CD56bright NK-cells (20.2% vs. 6.5% in healthy and 3.0% in IFN-OFF, p=0.0035).When the direct killing of K562 cells was studied, NK-cells from healthy controls killed better than NK-cells from either of the patients groups (in healthy 55 % of K562 cells were alive compared to 79% in IFN-ON and 91% in IFN-OFF, p=0.0042). Similar trend was also observed in degranulation assay (median degranulation in healthy controls 8.3 % compared to 4.3 % in IFN-OFF and 3.6 % in IFN-ON group, p= 0.39). The direct killing capability tended to correlate negatively r=-0.60, p=0.07) with the NK-cell proportion (ie. patients with high NK-cell counts had worse killing).Instead of cytotoxicity, NK-cells from IFN-OFF patients seemed to secrete more efficiently cytokines (IFN-γ/TNF-α) than NK-cells from healthy controls, but due to low number of patients no firm conclusions can be made (IFN-OFF median 6.9 %, healthy 0.7 %; p= 0.25).The proportion of potentially cytotoxic CD4+GrB+ T-cells did not significantly differ between the groups, but wide variation was observed between the individual patients (IFN-ON median 9.0 %, IFN-OFF 2.6 %, and healthy controls 3.5 %; p=0.4142). However, the secretion of TNF-α and IFN-γ by CD4+ cells seemed to be increased in IFN-OFF patients when compared to healthy controls (IFN-OFF mean 5.9 %, IFN-ON 3.7 %, healthy 2.5 %; p=0.1055). CONCLUSIONS:CML patients who have been able to discontinue IFN-α therapy (IFN-OFF group) had an expansion of mature CD56dimCD62LlowCD27lowCD57high NK-cells in peripheral blood. Although CD56dim NK-cells have typically been suggested to act as cytotoxic cells, the killing in response to K562 cells was lower in IFN-OFF patients than in controls. It is possible that the expanded NK-cells in IFN-OFF patients represent exhausted terminal stage cells (CD57+) or memory cells that do not react properly against third party target cells (K562). Moreover, they may have an immunoregulatory function instead of cytotoxicity. Further follow-up studies with other patient cohorts (TKI monotherapy and TKI+IFN-α combination therapy treated patients) are ongoing to clarify the issue further. Disclosures:Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

POPULATION DISTRIBUTION, THREATS AND CONSERVATION STATUS OFSemnopithecus priam thersites AT MIHINTALE SANCTUARY, SRI LANKA

The Dry zone grey langur (Semnopithecus priam thersites) is a subspecies endemic to Sri Lanka. In this research investigated the population distribution, threats and conservation status of the S. priam thersites in the contiguous forest of Mihintale Sanctuary in Sri Lanka. Population study done by direct counting and vocal sounds along the permanent line transects. Threats were identified by using a questionnaire survey. A total of 03 troops and least 60 individuals were recorded during the study period from December 2009 to May 2010. These three troops were sighted in Kaludiyapukuna forest (19 individuals), Rajagiri lena forest (21 individuals) and the boundery near kumarasirigama village (18 individuals). The remaining populations living almost completely in disturbed habitats due to human influences. The average group sizes were 16.8±2.65 and 19.14 ±3.76 in forested areas and forests neighboring human habitation respectively and the all three groups were recorded as multimale groups. The troop‟s distribution varied with the food availability and restricted to human habitation and disturbed forest patches. The species is vulnerable to hunting and retaliatory killing in response to crop damage.

PKA knockdown enhances cell killing in response to radiation and androgen deprivation

The therapeutic efficacy of Gem®231, a second generation antisense molecule targeted to the RIα subunit of PKA(RIα) (AS-PKA), administered in combination with androgen deprivation (AD) and radiation therapy (RT), was examined in androgen sensitive (LNCaP) and insensitive (PC3) cell lines. Apoptosis was assayed by Caspase 3 + 7 activity and Annexin V binding. AS-PKA significantly increased apoptosis in vitro from RT (both lines), with further increases in LNCaP cells grown in AD medium. In LNCaP cells, AD increased phosphorylated mitogen activated protein-kinase (pMAPK), which was reduced by AS-PKA relative to the mismatch (MM) controls. AS-PKA also reduced pMAPK levels in PC3 cells. Cell death was measured by clonogenic survival assays. In vivo, LNCaP cells were grown orthotopically in nude mice. Tumor kinetics were measured by magnetic resonance imaging and serum prostate-specific antigen. PC3 cells were grown subcutaneously and tumor volume assessed by caliper measurements. In PC3 xenografts, AS-PKA caused a significant increase in tumor doubling time relative to MM controls as a monotherapy or in combination with RT. In orthotopic LNCaP tumors, AS-PKA was ineffective as a monotherapy; however, it caused a statistically significant increase in tumor doubling time relative to MM controls when used in combination with AD, with or without RT. PKA(RIα) levels in tumors were quantified via immunohistochemical (IHC) staining and image analysis. IHC measurements in LNCaP cells exhibited that AS-PKA reduced PKA(RIα) levels in vivo. We demonstrate for the first time that AS-PKA enhances cell killing androgen sensitive prostate cancer cells to AD ± RT and androgen insensitive cells to RT.

Shining a Light on an Opportunistic Pathogen

Shining a Light on an Opportunistic Pathogen

Targeted and Pharmacologic Inhibition of DNMT3b Sensitizes MDA‐MB‐453 Breast Cancer Cells to Doxorubicin and Paclitaxel

Basal‐like breast cancers frequently express a hypermethylation defect characterized by DNMT hyperactivity and DNMT3b overexpression, which may contribute to chemotherapy resistance and provides a target for development of new treatment strategies. To determine the effect of DNMT3b status on the sensitivity of hypermethylator MDA‐MB‐453 breast cancer cells to doxorubicin hydrochloride (DOX) and paclitaxel (PAX), we employed RNAi‐mediated DNMT3b knockdown (KD) and/or demethylating agent treatment with 5‐aza‐2′‐deoxycytidine (5‐aza). DNMT3b KD reduces the IC50 for DOX from 0.079 μM to 0.048 μM (39% reduction) and for PAX from 0.497 nM to 0.376 nM (24%). Treatment with 250 nM 5‐aza for 7d did not increase efficacy of DOX or PAX, but 7d treatment with 500 nM 5‐aza sensitized cells, reducing the IC50 for DOX to 0.0349 μM (56%) and the PAX to 0.311 nM (37%). 5‐aza treatment of KD cells reduced the IC50 for DOX to 0.0355 μM (55%) and for PAX to 0.313 nM (37%). Inhibition of DNMT3b produced improved cell killing in response to 0.079 μM DOX and 0.497 nM PAX: DNMT3b KD (19% and 10% respectively), 5‐aza (38% and 18%), and combined KD and 5‐aza treatment (39% and 18%). These results show that effectiveness of DOX and PAX can be increased by targeted and pharmacological inhibition of DNMT3b. These results strongly suggest that DNMT3b can be targeted to improve the efficacy of breast cancer chemotherapy. UNC University Cancer Research Fund

Astroglia overexpressing heme oxygenase‐1 predispose co‐cultured PC12 cells to oxidative injury

The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage.

Distribution and Conservation Status of the Arunachal Macaque, Macaca munzala, in Western Arunachal Pradesh, Northeastern India

The recently described Arunachal macaque, Macaca munzala, has to date been reported only from western Arunachal Pradesh, Eastern Himalaya. Our surveys have recorded a total of 35 troops and 569 individuals, probably a conservative estimate, for the macaque population in the Tawang and West Kameng districts of the state. The species appears to be tolerant to anthropogenic habitat change, but is vulnerable to hunting and retaliatory killing in response to crop damage. Data from one part of the area surveyed, however, indicate that the species can attain remarkably high population densities in the absence of hunting. Macaca munzala will need to be protected in human-modified landscapes, and the issues of crop damage and retaliatory persecution must be addressed urgently.

Improved Suicide Gene Therapy: Lentiviral Gene Transfer of Equine Herpes Virus Type 4 Thymidine Kinase into Target Cells.

Herpes virus type 1 thymidine kinase (HSV1-TK) with ganciclovir (GCV) prodrug treatment is the most widely used approach for suicide gene therapy. This ‘suicide' strategy allows direct reduction of tumors and clearance of donor cells should graft-versus-host disease (GvHD) arise after bone marrow transplantation. Given recent clinical outcomes, this suicide approach may also provide a key safety component for therapeutic gene transfer vectors that integrate. Although suicide gene therapy using HSV1-TK-encoding oncoretroviral vectors has been evaluated in the clinic, the success of this approach has been relatively modest. Reasons for this include: low gene transfer efficacy, reduced expression of the suicide gene, and insufficient conversion of substrate. Our goal is to overcome these limitations by using a novel lentiviral vector (LV) encoding an alternative kinase/prodrug combination. The rational for our innovative suicide gene therapy strategy is two-fold: 1) Lentiviral vectors can efficiently transduce not only dividing cells but also non-dividing cells. 2) Applying a faster viral enzyme like equine herpes virus type 4 thymidine kinase (EHV4-TK) could be advantageous as it has been shown to be kinetically superior to HSV1-TK at GCV phosphorylation. The aim of this study is to evaluate whether LV-mediated gene modification of target cells with EHV4-TK can lead to efficient killing following GCV treatment. We first constructed a LV expression system carrying the wild-type EHV4-TK cDNA with an IRES element followed by a truncated form of human CD19 (hCD19Δ). Human CD19 was chosen as a cell surface marker to allow functional titering of virus and for immuno-enrichment of transduced cells prior to infusion since it is not expressed in the T cell lineage. The truncated form lacks the intracellular domain and therefore does not signal. Use of an IRES element can abrogate some variegated expression seen with vectors having dual promoters. The LV was pseudotyped with VSV-g and concentrated by ultracentrifugation. After one infection, Jurkat cells (human T cell leukemia) showed a more than 80% functional and stable transduction efficiency (MOI = 10). Using hCD19Δ as a selective marker, transduced Jurkat cells were enriched to over 95% positive by immuno-affinity sorting. EHV4-TK-transduced Jurkat cells exhibited increased cell killing in response to GCV treatment (the apoptotic cell indexes with or without GCV were 69.4 ± 1.5 % and 18.8 ± 1.7 %, respectively; n=3). Highly efficient transduction (more than 60%) of primary human T cells was accomplished by a three time exposure to virus over 36 hours at MOI of 20. Next, we found that GCV efficiently killed transduced primary human T cells in a dose dependent manner. We are now comparing the efficiency of GCV conversion by HSV1-TK and EHV4-TK using LV-transduced cells that express the similar protein levels. We are also evaluating intracellular levels of GCV metabolites by HPLC. These results demonstrate that our novel suicide gene therapy strategy has significant potential for many clinical applications.

IL-15 alters expression and function of the chemokine receptor CX3CR1 in human NK cells

The chemokine receptor CX3CR1 is thought to regulate inflammation in part by modulating NK cell adhesion, migration, and killing in response to its ligand CX3CL1 (fractalkine). Recent reports indicate that IL-15, which is essential for development and survival of NK cells, may negatively regulate CX3CR1 expression, however, the effects of the cytokine on human NK cell CX3CR1 expression and function have not been fully delineated. Here, we demonstrate that short term culture in IL-15 decreases surface expression of CX3CR1 on cultured CD56 + cells from human blood resulting in diminished chemotaxis and calcium flux in response to CX3CL1. Cells cultured long term in IL-15 (more than five days) completely lost surface expression as well as mRNA and protein for CX3CR1. The effect was specific since mRNA for CCR5 was increased and mRNA for CXCR4 was unchanged in these cells by IL-15. Thus, exogenous IL-15 is a negative regulator of CX3CR1 expression and function in human CD56 + NK cells. The data imply that the use of IL-15 alone to expand NK cells ex vivo for immunotherapy may produce cells impaired in their ability to traffic to sites of inflammation.

The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress.

AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status found in all eukaryotic cells. AMPK is activated by stimuli that increase the cellular AMP/ATP ratio. Essential to activation of AMPK is its phosphorylation at Thr-172 by an upstream kinase, AMPKK, whose identity in mammalian cells has remained elusive. Here we present biochemical and genetic evidence indicating that the LKB1 serine/threonine kinase, the gene inactivated in the Peutz-Jeghers familial cancer syndrome, is the dominant regulator of AMPK activation in several mammalian cell types. We show that LKB1 directly phosphorylates Thr-172 of AMPKalpha in vitro and activates its kinase activity. LKB1-deficient murine embryonic fibroblasts show nearly complete loss of Thr-172 phosphorylation and downstream AMPK signaling in response to a variety of stimuli that activate AMPK. Reintroduction of WT, but not kinase-dead, LKB1 into these cells restores AMPK activity. Furthermore, we show that LKB1 plays a biologically significant role in this pathway, because LKB1-deficient cells are hypersensitive to apoptosis induced by energy stress. On the basis of these results, we propose a model to explain the apparent paradox that LKB1 is a tumor suppressor, yet cells lacking LKB1 are resistant to cell transformation by conventional oncogenes and are sensitive to killing in response to agents that elevate AMP. The role of LKB1/AMPK in the survival of a subset of genetically defined tumor cells may provide opportunities for cancer therapeutics.

Killing of Candida albicans by human salivary histatin 5 is modulated, but not determined, by the potassium channel TOK1.

Salivary histatin 5 (Hst 5), a potent toxin for the human fungal pathogen Candida albicans, induces noncytolytic efflux of cellular ATP, potassium, and magnesium in the absence of cytolysis, implicating these ion movements in the toxin's fungicidal activity. Hst 5 action on Candida resembles, in many respects, the action of the K1 killer toxin on Saccharomyces cerevisiae, and in that system the yeast plasma membrane potassium channel, Tok1p, has recently been reported to be a primary target of toxin action. The question of whether the Candida homologue of Saccharomyces Tok1p might be a primary target of Hst 5 action has now been investigated by disruption of the C. albicans TOK1 gene. The resultant strains (TOK1/tok1) and (tok1/tok1) were compared with wild-type Candida (TOK1/TOK1) for relative ATP leakage and killing in response to Hst 5. Patch-clamp measurements on Candida protoplasts were used to verify the functional deletion of Tok1p and to provide its first description in Candida. Tok1p is an outwardly rectifying, noisily gated, 40-pS channel, very similar to that described in Saccharomyces. Knockout of CaTOK1 (tok1/tok1) completely abolishes the currents and gating events characteristic of Tok1p. Also, knockout (tok1/tok1) increases residual viability of Candida after Hst 5 treatment to 27%, from 7% in the wild type, while the single allele deletion (TOK1/tok1) increases viability to 18%. Comparable results were obtained for Hst-induced ATP efflux, but quantitative features of ATP loss suggest that wild-type TOK1 genes function cooperatively. Overall, very substantial killing and ATP efflux are produced by Hst 5 treatment after complete knockout of wild-type TOK1, making clear that Tok1p channels are not the primary site of Hst 5 action, even though they do play a modulating role.

E2F-1 overexpression sensitizes colorectal cancer cells to camptothecin.

Topoisomerase I inhibitors have been shown to have clinical activity against human colorectal cancer. Previous studies showed that the cytotoxicity of camptothecin, a topoisomerase I inhibitor, occurs mainly in the S -phase of the cell cycle and is protectable by aphidicolin, an inhibitor of replicative DNA polymerase in some camptothecin-sensitive colorectal cells. Transcription factor E2F-1 regulates the G1/S transition, and recent studies have shown that E2F-1 potentiated the cytotoxicity of some cell-cycle-related drugs. Therefore, the present study was designed to investigate the effect of adenovirus-mediated E2F-1 gene transfer on chemosensitivity of colorectal cancer to camptothecin, in vitro and in vivo. Two human colorectal cancer cells, SW620 (mutant p53) and RKO (wild-type p53), were treated with camptothecin, alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ), or E2F-1 (Ad-E2F-1). E2F-1 overexpression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at low doses (less than the LD(20) dose) markedly increased the sensitivity of human colorectal cancer cells to camptothecin in vitro, which is because of induction of apoptosis. Aphidicolin did not have any protective effect on the Ad-E2F-1/camptothecin-mediated cytotoxicity. The level of topoisomerase I expression was not affected by combination treatment as well, suggesting that DNA replication and topoisomerase I activity may not account for the molecular mechanism of cell killing in response to Ad-E2F-1/camptothecin treatment. Fas and Fas ligand expression were not altered by treatment with camptothecin and/or Ad-E2F-1. Moreover, combination of camptothecin and Ad-E2F-1 has an additive antitumor effect in an in vivo nude mouse xenograft model. When combined with camptothecin, E2F-1 adenovirus therapy resulted in a 95.7% decrease in tumor size compared to control groups (P<.05). These results suggest a chemosensitization strategy that may have clinical utility in human colorectal cancer.