Marc W. Halterman

The paradox of the neutrophil's role in tissue injury

The neutrophil is an essential component of the innate immune system, and its function is vital to human life. Its production increases in response to virtually all forms of inflammation, and subsequently, it can accumulate in blood and tissue to varying degrees. Although its participation in the inflammatory response is often salutary by nature of its normal interaction with vascular endothelium and its capability to enter tissues and respond to chemotactic gradients and to phagocytize and kill microrganisms, it can contribute to processes that impair vascular integrity and blood flow. The mechanisms that the neutrophil uses to kill microorganisms also have the potential to injure normal tissue under special circumstances. Its paradoxical role in the pathophysiology of disease is particularly, but not exclusively, notable in seven circumstances: 1) diabetic retinopathy, 2) sickle cell disease, 3) TRALI, 4) ARDS, 5) renal microvasculopathy, 6) stroke, and 7) acute coronary artery syndrome. The activated neutrophilˈs capability to become adhesive to endothelium, to generate highly ROS, and to secrete proteases gives it the potential to induce local vascular and tissue injury. In this review, we summarize the evidence for its role as a mediator of tissue injury in these seven conditions, making it or its products potential therapeutic targets.

Oncolytic viral therapy for human colorectal cancer and liver metastases using a multi-mutated herpes simplex virus type-1 (G207)

G207 is a multi‐mutated, replication‐competent type‐1 herpes simplex virus designed to target, infect, and lyse neurological tumors. This study examines the feasibility of using G207 in the treatment of human colorectal cancer and defines the biological determinants of its antitumor efficacy. This virus was tested on five human colorectal cancer cell lines in vitro to determine efficacy of infection and tumor cell kill. These results were correlated to measures of tumor cell proliferation. In vivo testing was performed through direct injections of G207 into xenografts of human colorectal cancer tumors grown in flanks of athymic rats. To evaluate an alternate method of administration, hepatic portal vein infusion of G207 was performed in a syngeneic model of liver metastases in Buffalo rats. Among the five cell lines tested, infection rates ranged between 10% and 90%, which correlated directly with S‐phase fraction (8.6%–36.6%) and was proportional to response to G207 therapy in vitro (1%–93%). Direct injection of G207 into nude rat flank tumors suppressed tumor growth significantly vs. control (0.58 ± 0.60 cm3 vs. 9.16 ± 3.70 cm3, P<0. 0001). In vivo tumor suppression correlated with in vitro effect. In the syngeneic liver tumor model, portal infusion resulted in significant reduction in number of liver nodules (13 ± 10 nodules in G207‐treated livers vs. 80 ± 30 nodules in control livers, P<0.05). G207 infects and kills human colorectal cancer cells efficiently. In vitro cytotoxicity assay and tumor S‐phase fraction can be used to predict response to treatment in vivo. This antineoplastic agent can be delivered effectively by both direct tumor injection and regional vascular infusion. G207 should be investigated further as therapy for colorectal cancer and liver metastases.—Kooby, D. A., Carew, J. F., Halterman, M. W., Mack, J. E., Bertino, J. R., Blumgart, L. H., Federoff, H. J., Fong, Y. Oncolytic viral therapy for human colorectal cancer and liver metastases using a multi‐mutated herpes simplex virus type‐1 (G207). FASEB J. 13, 1325–1334 (1999)

The paradox of the neutrophil’s role in tissue injury: a review

The neutrophil is an essential component of the innate immune system, and its function is vital to human life. Its production increases in response to virtually all forms of inflammation, and subsequently, it can accumulate in blood and tissue to varying degrees. Although its participation in the inflammatory response is often salutary by nature of its normal interaction with vascular endothelium and its capability to enter tissues and respond to chemotactic gradients and to phagocytize and kill microrganisms, it can contribute to processes that impair vascular integrity and blood flow. The mechanisms that the neutrophil uses to kill microorganisms also have the potential to injure normal tissue under special circumstances. Its paradoxical role in the pathophysiology of disease is particularly, but not exclusively, notable in seven circumstances: 1) diabetic retinopathy, 2) sickle cell disease, 3) TRALI, 4) ARDS, 5) renal microvasculopathy, 6) stroke, and 7) acute coronary artery syndrome. The activated neutrophilˈs capability to become adhesive to endothelium, to generate highly ROS, and to secrete proteases gives it the potential to induce local vascular and tissue injury. In this review, we summarize the evidence for its role as a mediator of tissue injury in these seven conditions, making it or its products potential therapeutic targets.

HIF-1α and p53 Promote Hypoxia-Induced Delayed Neuronal Death in Models of CNS Ischemia

Brain ischemia is a cause of substantial morbidity and mortality during the later decades of life. In light of this, many studies have used in vitro and in vivo models of acute necrosis to test candidate therapeutic agents. More recently, the existence of a genetically programmed component of ischemic death has become widely accepted. We have used molecular genetic approaches to investigate the potential link between hypoxia-induced gene transcription and the delayed death of ischemic neurons. Hypoxia-induced gene expression is an evolutionarily conserved response comprising both transcriptional activation and posttranscriptional and posttranslational stabilization events. Members of the PER-ARNT-SIM (PAS) family of basic helix-loop-helix transcription factors have been shown to regulate hypoxic transcripts in nonneuronal cultured lines. However, evidence for ischemic activation of PAS proteins within the neuronal compartment or possible involvement in neuronal death is lacking. The tumor-suppressor protein p53 is a known transcriptional activator within the central nervous system that is clearly involved in the pathologic response to ischemia. This article will provide data that implicate the coordinate activities of p53 and the PAS protein HIF-1alpha in driving ischemia-induced delayed neuronal death. Background regarding mechanisms of ischemic neuronal death will also be provided with special attention paid to the role of de novo gene expression in promoting this pathologic sequence. The identification of the HIF-1alpha/p53-mediated signaling pathway in neurons highlights a novel target toward which anti-ischemic neuroprotective drug discovery can be applied.

Selective Infection and Cytolysis of Human Head and Neck Squamous Cell Carcinoma with Sparing of Normal Mucosa by a Cytotoxic Herpes Simplex Virus Type 1 (G207)

This study evaluates inhibition of human squamous cell carcinomas (SCCs) by a replication-competent multimutated herpes simplex virus type 1 (G207). Infectivity and cytotoxicity of the G207 virus were evaluated in vitro in seven human SCC cell lines. In vivo effects of the G207 virus on human tumor xenografts in an athymic rat model were then investigated by injecting established tumors with 1 x 10(7) virus particles and monitoring tumor growth. In addition, oral cavity tumors in immunocompetent hamster were infected with the G207 virus by selective intraarterial perfusion and the tumor response was monitored. In vitro studies demonstrated infection rates, measured 24 hr after exposure, exceeding 40% at an MOI of 2 in five of seven human SCC cell lines. Cytotoxic effects, as measured by percent cell death on day 5, exceeded 90% in five of seven SCC cell lines. In vivo inhibition of tumor growth in an athymic rat model was seen (p < 0.005) and in two of the cell lines a complete clinical response was seen in 12 of 14 tumors. In the hamster model, selective intraarterial perfusion with G207 virus showed selective infection of the tumor cells, with sparing of the adjacent normal mucosa, which leading to significant suppression of tumor growth (p < 0.005). The G207 virus displayed efficient and selective cytotoxicity and tumor growth inhibition against human SCC and may prove useful as a therapeutic agent for head and neck SCC.

Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers

Recently developed helper virus-free methods of herpes simplex virus (HSV) amplicon vector packaging provide stocks that are virtually devoid of the cytotoxic component normally associated with traditional helper virus-based packaging methods. These approaches involve cotransfection of amplicon plasmid DNA with either a five-cosmid set or a bacterial artificial chromosome (BAC) that contains the HSV genome without its cognate pac signals. Helper virus-free amplicon packaging produces low-titer stocks (<105 expressing particles/ml) that exhibit a high frequency of pseudotransduction. In an effort to enhance amplicon titers, we introduced in trans a genomic copy of the virion host shutoff (vhs) protein-encoding gene UL41 into both cosmid- and BAC-based packaging strategies. Cotransfection of this plasmid with the amplicon and packaging reagents results in a 10-fold higher amplicon titer, and stocks that do not exhibit the pseudotransduction phenomenon. To further enhance packaging efficiency, the HSV transcriptional activator VP16 was introduced into packaging cells 1 day before the packaging components. Pre-loading of packaging cells with VP16 led to an additional enhancement of amplicon titers, an effect that did not occur in the absence of vhs. Increased helper virus-free amplicon titers resulting from these modifications will make in vivo transduction experiments more feasible.

Antitumor efficacy of regional oncolytic viral therapy for peritoneally disseminated cancer

Oncolytic viral therapy is a promising new method of cancer treatment. Peritoneal dissemination of cancer is a common and fatal clinical condition seen in many malignancies, with few effective therapies available. G207, a multimutated replication-competent herpes simplex virus type-1, effectively treats disseminated peritoneal cancer. This study evaluates viral proliferation and subsequent tumoricidal effects in vitro and in vivo after regional viral delivery. In vitro studies demonstrate that G207 efficiently kills five human gastric cancer cell lines, and that permissiveness to viral replication is correlated with cytotoxicity. In a murine xenograft model of human gastric carcinomatosis, peritoneal delivery of G207 effectively kills tumor and prolongs survival. Data from quantitative PCR characterizes peritoneal clearance of virus after intraperitoneal injection, and identifies G207 replication within tumor cells in vivo, similar to in vitro proliferation. Further analysis of various organs confirms that G207 does not replicate within normal tissue after peritoneal delivery. Wild-type KOS viral replication was also demonstrated in vivo, with significant toxicity secondary to dissemination and encephalitis. In vivo viral proliferation of G207 is restricted to tumor cells, is correlated with in vitro assays, and is an important mechanism of anticancer efficacy.

The Endoplasmic Reticulum Stress Response Factor CHOP-10 Protects against Hypoxia-induced Neuronal Death

Hypoxia-induced gene expression is a critical determinant of neuron survival after stroke. Understanding the cell autonomous genetic program controlling adaptive and pathological transcription could have important therapeutic implications. To identify the factors that modulate delayed neuronal apoptosis after hypoxic injury, we developed an in vitro culture model that recapitulates these divergent responses and characterized the sequence of gene expression changes using microarrays. Hypoxia induced a disproportionate number of bZIP transcription factors and related targets involved in the endoplasmic reticulum stress response. Although the temporal and spatial aspects of ATF4 expression correlated with neuron loss, our results did not support the anticipated pathological role for delayed CHOP expression. Rather, CHOP deletion enhanced neuronal susceptibility to both hypoxic and thapsigargin-mediated injury and attenuated brain-derived neurotrophic factor-induced neuroprotection. Also, enforced expression of CHOP prior to the onset of hypoxia protected wild-type cultures against subsequent injury. Collectively, these findings indicate CHOP serves a more complex role in the neuronal response to hypoxic stress with involvement in both ischemic preconditioning and delayed neuroprotection.

Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector.

To develop a reproducible gene transfer method for the murine CNS we evaluated delivery of various gene vehicles using mechanical or manual stereotaxic intracranial inoculation. A microprocessor controlled microsyringe pump (The World Precision Instruments/UltraMicroPump) programmable for volume, rate and syringe size and designed to dispense nanoliter and picoliter volumes was compared to a standard manual deliver method. Gene transfer efficiency of two viral vectors, two synthetic cationic lipid molecules, and naked DNA were evaluated in mice injected unilaterally in two brain regions. Animals received 1 microl over 10 min. of either HSVlac (1 x 10(5) b.f.u), AdLac (1 x 10(5) p.f.u), Tfx-10 or Tfx-20 (2.6 microg DNA in 2.0 microl Tfx; 1:1 charge ratio of DNA to liposome), or naked DNA (HSVlac plasmid, 10 microg/microl). After 4 days, animals from each group were perfused and tissue prepared for X-gal histochemical detection of beta-galactosidase expression. Blue cells were observed in the HSV, Adenovirus, and Tfx-20 groups only at the injection site in animals injected using the UMP. Animals injected manually exhibited fewer blue cells and positive cells were not restricted to the injection site. To quantify expression, tissue punches harvested from the injection sites as well as other brain regions were analyzed using a chemiluminescent reporter assay to detect beta-galactosidase (Galacto-Light). These data indicated increased activity in all animals injected with a lacZ containing vector via the UMP as compared to manual delivery: A 41% increase in the expression levels of beta-gal in HSVlac infected animals (p = 0.0029); a 29% increase in Adlac infected animals (p = 0.01); a 56% increase in Tfx-10 transduced animals (p = 0.04); a 24% increase in Tfx-20 transduced animals (p = 0.01); and a 69% increase in naked DNA gene transfer (p = 0.05). Total beta-galactosidase activity was greatest in HSVlac infected mice followed by Adlac > Tfx-20 > Tfx-10 = naked DNA.

The role of the THY1 gene in human ovarian cancer suppression based on transfection studies

In our recent studies, the expression of the THY1 gene encoding a 25-28 kDa glycoprotein located at 11q23-q24, was found to be associated with complete tumor suppression of the ovarian cancer cell line SKOV-3 after the transfer of chromosome 11. These studies raised the possibility that THY1 maybe a candidate tumor suppressor gene for ovarian cancer. To investigate this, the complete cDNA sequence for THY1 was cloned and transfected into SKOV-3 ovarian cancer cells. The expression of THY1 in the transfectants was confirmed by Northern blot analysis, immunocytochemistry, and flow cytometry. Both SKOV-3-THY1 and SKOV-3-null cells were inoculated subcutaneously into severe combined immunodeficiency (SCID) mice to determine in vivo tumorigenicity. THY1 transfectants formed tumors, but overall tumor growth rate and tumor size was significantly reduced compared with their null counterparts. To further correlate THY1 expression with tumorigenicity, the THY1 antisense was transfected into the nontumorigenic clone, 11(C)9-8, which resulted in restoration of tumorigenicity. These data indicate that THY1 expression alone cannot suppress tumorigenicity; however, abrogation of THY1 expression from nontumorigenic cells can restore tumorigenesis. Taken together, the data suggest that THY1 is necessary but not sufficient to suppress ovarian tumorigenicity. Therefore, THY1 can be designated as a putative tumor suppressor gene for human ovarian cancer.