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DIR Papers of the Month

By Robin Arnette
October 2006

p38 MAPK May Offer Treatment for Viral Hemorrhagic Fevers

Viral-induced hemorrhagic fevers such as Lassa Fever, Dengue, Marburg and Ebola cause high morbidity and mortality in humans and have the potential to be used as biological weapons. Currently, there are no FDA-approved treatments for these illnesses, except for yellow fever, which has a viable vaccine. But researchers from NIEHS and the University of Chicago Pritzker School of Medicine may have found a possible treatment for these illnesses.

The results of the study appeared in the July 2006 issue of the Journal of Clinical Immunology and demonstrated how endothelial cells (ECs) were activated and eventually impaired after infection by a virus. Previous work done by this group had established that viral-induced inflammation was mediated by activation of p38 Mitogen-Activated Protein Kinase (MAPK)-a protein kinase that performs a crucial step in relaying signals from the plasma membrane to the nucleus-and others in the field had reported that ECs were involved in viral hemorrhagic fevers. Nevertheless, the exact mechanism of viral-induced hemorrhagic fever remained unknown, particularly what caused vascular permeability and barrier dysfunction in ECs.

The researchers grew human lung ECs in culture and infected them with either bluetongue virus (BTV), the agent that causes hemorrhagic fever in ruminants, or double-strand RNA (dsRNA), a common viral structure that is present in some viruses or may form during the lifetime of the virus. Infected cells were tested using trans-endothelial electrical resistance (TER), which determines vascular permeability. In both instances, BTV or dsRNA-treated cells exhibited decreased TER, which is an indication of increased vascular permeability. To determine whether p38 MAPK was involved in this vascular permeability, ECs were pretreated with a p38 MAPK inhibitor, exposed to BTV or dsRNA, and continuously measured using TER. The results indicated that inhibiting p38 MAPK blocks the reduction in TER, therefore suggesting that the p38 MAPK pathway is involved with vascular permeability.

The results of this study have far-reaching implications with regard to therapeutic approaches to treating viral-induced hemorrhagic fevers.

Citation: Chiang ET, Persaud-Sawin D, Kulkarni D, Garcia JGN, Imani F. 2006. Bluetongue virus and double-stranded RNA increase human vascular permeability: role of p38 MAPK. J Clin Immunol 26(4):406-416.

Estrogen Receptor-α Suppresses Development of Testis-Specific Cells in Mouse Ovaries

The tendency of ovaries to produce estrogens and testes to produce androgens is determined by the types of specialized cells and enzymes that arise during development of the fetus. In the August issue of Endocrinology, researchers from NIEHS and the Centre Hospitalier Universitaire de Quebec have determined that one of the estrogens, estradiol, may be an important factor in ensuring that Leydig cells do not inappropriately develop in the ovaries of females. Leydig cells are specific to the testes of males and responsible for testosterone production.

The team studied three groups of female mice: a line of mice developed by this research group that lacked the estrogen receptor (αER), a transgenic line with elevated levels of luteinizing hormone and wild-type mice with normal ovaries. Using Northern blotting, radioimmunoassay and in-situ hybridization, the investigators determined that the ovaries of female mice lacking αER produced enormous amounts of the male androgen testosterone. These mice also showed masculine-like characteristics. Further study indicated that ovaries from the aER null females possessed an enzyme, hydroxysteroid (17b)-dehydrogenase 3 (HSD3), that is normally found only in testes and is necessary for the final step in the production of testosterone. Using transmission electron microscopy, the researchers found that the ovaries of these mutant female mice had Leydig cells similar to those that produce testosterone in the testes of males. Therefore, the investigators discovered a form of hormonal and cellular "sex-reversal" in females that occurs when the aER is removed.

These data are the first to demonstrate that αER may play an important role in the proper development of the ovaries in females. By repressing Leydig cells and the presence of the male-specific enzyme, HSD3, αER ensures that females are not exposed to excessive amounts of testosterone that could lead to a loss of feminine characteristics. The researchers suggested that αER is integral to the proper development and maintenance of the female phenotype.

Citation: Couse JF, Yates MM, Rodriguez KF, Johnson JA, Poirier D, Korach KS. 2006. The intraovarian actions of estrogen receptor-α are necessary to repress the formation of morphological and functional Leydig-like cells in the female gonad. Endocrinology 147(8):3666-3678.

Identifying Changes in the Rat Serum Proteome During Acetaminophen-Induced Liver Injury

Exposure to xenobiotic compounds produces a myriad of pathologies in living organisms, and may lead to damage in vital organs and tissues. Of all of the bodily fluids, one offers the best source for scientists to monitor organ or tissue injury: blood. Currently, many researchers are mapping the soluble human blood elements-serum or plasma proteome-in an attempt to better understand various diseases, but little of the technology in serum proteome mapping has been applied to studying damage in the liver. To this end, researchers from NIEHS and the Large Scale Biology Corporation, Germantown, Md., measured changes in the serum proteome during acute liver injury and recovery in rats. Lab rats were exposed to a high dose of the analgesic acetaminophen, which causes a liver pathology in rats similar to that seen in humans after accidental overdose.

The investigators performed proteomic analysis of rat serum at various times over a five-day period after acetaminophen exposure using gel electrophoresis, mass spectrometry and antibody arrays. They typically monitored over 800 serum proteins and identified over 50 serum proteins as significantly altered during liver damage. Changes in the serum proteome reflected the animal's reaction to liver injury by evoking changes in acute phase response, coagulation, protein degradation, intermediary metabolism and scavenger proteins in the general blood circulation. These changes in serum proteins accompanied an inflammatory response that helped recruit reparative immune cells from blood into the liver. In addition, an increase in serum catalase activity acted as an antioxidant that could provide a critical defense to the body's blood vessels during liver repair.

NIEHS researchers believe that tracking serum proteome changes in experimental animals provides a means to gain insight into how the body mounts a collective tissue response for recovery from acute liver injury that represents processes with relevance for other human hepatic diseases.

Citation: Merrick BA, Bruno ME, Madenspacher JH, Wetmore BA, Foley J, Pieper R, Zhao M, Makusky AJ, McGrath AM, Zhou JX, Taylor J, Tomer KB. 2006. Alterations in the rat serum proteome during liver injury from acetaminophen exposure. J Pharmacol Exp Ther 318(2):792-802.

Drug-Induced NAG-1 Inhibits Tumor Growth

In an article published in the August 2006 issue of The Journal of Pharmacology and Experimental Therapeutics, researchers from NIEHS, NCI, EPA, University of Tennessee-Knoxville and the University of Occupational and Environmental Health, Kitakyushu, Japan, studied the interaction of a potential anticancer drug and a gene with anti-tumor properties. The team confirmed that nonsteroidal anti-inflammatory drug-activated gene (NAG-1) suppresses tumor growth in mice, and that the expression of NAG-1 can be controlled with anticancer drugs. These findings are important in the fight against cancer because drugs that target NAG-1 may lead to the development of new cancer treatments.

The investigators used 2-(4-amino-3-methylphenyl)-5-fluorobenzothaizole (5F203) to determine whether it induced NAG-1 expression in human breast cancer cells. The compound is the active moiety of Phortress, an experimental anticancer drug currently undergoing phase I clinical trials in cancer patients in the UK. Western analysis using NAG-1 antibodies indicated that treatment with 5F203 induced NAG-1 expression, and this expression was dependent on time of incubation and drug concentration.

Since most of the information about NAG-1 is based on in vitro experiments, the team wanted to confirm that the up-regulation of NAG-1 is associated with inhibition of tumor development in vivo. They injected MCF-7 (breast cancer cell line) cells into controls and treated nude mice by injecting 15 mg/kg or 10 mg/kg of 5F203. The investigators then measured the size of the mammary tumors produced and used polymerase chain reaction to measure NAG-1 levels in the tumors. The median tumor weight in treated mice decreased by up to 75%, and needle biopsies of the tumors taken 6 and 24 hours after 5F203 injection indicated a dose-dependent increase of NAG-1 expression compared to controls. Taking all of the data into account, treating mice with 5F203 increased NAG-1 expression and inhibited tumor growth in vivo. This outcome indicates that NAG-1 is an important mediator for the activity of 5F203 and provides a possible treatment for cancer.

Citation: Martinez JM, Sali T, Okazaki R, Anna C, Hollingshead M, Hose C, Monks A, Walker NJ, Baek SJ, Eling TE. 2006. Drug-induced expression of nonsteroidal anti-inflammatory drug-activated gene/macrophage inhibitory cytokine-1/prostate-derived factor, a putative tumor suppressor, inhibits tumor growth. J Pharmacol Exp Ther 319(2):899-906.



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