By David N. Leff
Editor's note: Science Scan is a roundup of recently published biotechnology-relevant research.
Bacteria don't kill people. It's their endotoxin that perpetrates the lethal septic shock.
When a dying Gram-negative bacterium releases that endotoxic lipopolysaccharide (LPS), the human immune system overkills by secreting two tissue-ravaging cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1). This process has been a perennial - and futile - target for biotechnologists seeking a therapeutic fix for septic shock. (See BioWorld Today, Dec. 21, 1998, p. 1.)
One likely reason clinical trials of antibodies raised against the two doomsday cytokines have failed is that the deadly molecules got in their licks first, before therapy had a chance. Now an article in the current issue of Science, dated July 9, 1999, reports discovery of a third septic shock executioner, which takes its time getting to the killing field. The paper's title: "HMG-1 as a late mediator of endotoxin lethality in mice."
Unlike TNF and IL-1, which responded to an LPS attack in minutes - too soon for effective treatment - mice showed increased levels of the third substance eight to 32 hours after limited exposure to the endotoxin. Delayed antibody treatment diminished LPS' lethality, the Science paper noted.
Suspecting the existence of a third, late, mediator, the co-authors stimulated murine cells with LPS over prolonged time periods. Only after 18 hours did they detect emergence of a 30-kiloDalton protein belonging to the high-mobility-group (HMG) chromosomal protein family. It turned out that macrophages release HMG-1 only after they have been exposed to endotoxin for a relatively long time. Beginning six to eight hours after LPS dosage, their in vitro mouse cells released large amounts of this presumed third cytokine. The team expressed recombinant HMG-1 cDNA in Escherichia coli cells, and used it to generate polyclonal antibodies.
When they administered this antiserum to their mice in a triple-dose regimen - 30 minutes before LPS challenge followed by 12- and 36-hour booster shots - "70 percent of the [challenged] mice survived, as compared with zero percent survival in controls treated with three matched doses of preimmune serum." Moreover, "No late death occurred over two weeks, indicating that anti-HMG-1 did not merely delay the onset of LPS lethality, but provided lasting protection."
As a first step in extrapolating their murine endotoxemia data to human subjects, the co-authors studied eight healthy individuals and 25 sepsis patients critically ill with bacteremia and organ dysfunction. The normal cohort had no HMG-1 in their blood, but it was high in the septic patients, the paper observed, "higher in patients who succumbed as compared to patients with nonlethal infections." It concluded by "suggesting that this protein warrants investigation as a therapeutic target."
Gene Mutation Blocks Enzyme's Protection Against Poisoning By Benzene's Carcinogenic Toxins
Among the 50,000 or so chemicals on the market, the highly toxic - and industrially versatile - hydrocarbon, benzene, ranks 16th. It finds uses in plastics, detergents, dyes, tear gas, and as a multipurpose solvent.
Originally discovered early last century in whale oil, benzene comes today from petroleum and coal tar. It also comes from jet fuel exhaust, charcoal-broiled meat and cigarette smoke. Benzene derivatives are strong carcinogens, linked in particular to acute myeloid leukemia and other blood dyscrasias, as well as bladder cancer. Exposure can lead to buildup of its chemical toxins in the bone marrow, which produces the body's blood cells.
The liver breaks down benzene to phenol (i.e., carbolic acid), hydroquinone and catechol. These metabolites make their way to the bone marrow. There they can be converted to highly poisonous substances, unless blocked by an enzyme called NQO1. It detoxifies these benzene metabolites, notably quinones, and also activates a number of anticancer agents. Extracts of broccoli, cabbage and other cruciferous vegetables are reportedly rich in the protective enzyme. That's the good news. The bad news is that this beneficent enzyme is mutated in the genomes of certain ethnic groups around the world, which therefore are extremely prone to benzene's cancer-causing effects. In Asians, this susceptibility runs as high as 20 percent; in the U.K., as low as 4 percent.
Now a research paper in the current Proceedings of the National Academy of Sciences (PNAS), dated July 6, 1999, reports that benzene derivatives push the NQO1 gene in normal bone marrow cells to express the protective enzyme. No such luck for cells carrying the mutant gene. The paper is titled: "A potential mechanism underlying the increased susceptibility of individuals with a polymorphism in NAD(P)H:quinone oxidoreductase 1 (NQO1) to benzene toxicity."
The article's senior author, pharmaceutical scientist David Ross at the University of Colorado Health Sciences Center in Denver, had in 1992 pinpointed a cytosine-to-thymine point mutation in the NQO1 gene sequence. He and his co-authors determined that its homozygous presence in the chromosomes of both parents conferred the susceptibility to benzene poisoning.
But in 1996 these investigators had disclosed the contrary finding that freshly isolated human bone marrow cells lacked expression of NQO1. In the present PNAS article, Ross and his co-authors offer a mechanism explaining how enzyme protection could occur despite this anomaly.
Mutant Gene Found In Drug-Resistant Sleeping Sickness Bug Paves Way To Diagnosis, Therapy
In those clubby, genteel British whodunits favored by authors Agatha Christie and Dorothy Sayers, the butler usually didn't do it, but the real murderer frequently resorted to arsenic (as in rat poison) for doing in his or her victim. Arsenic also knocks off Trypanosoma brucei, executioner of African sleeping sickness. This endemic, fatal parasitic infection menaces 50 million people across central Africa, and kills 20,000 of them a year. (See BioWorld Today, Feb. 27, 1997, p. 1.)
Arsenic, in various pharmaceutical formulations called arsenicals, is virtually the only drug of choice available to tropical disease doctors treating the sleeping sickness - which often strikes visitors to the Dark Continent, as well as natives. Like the storied rat poison, arsenical drugs are almost as deadly to humans as they are to T. brucei, but they usually work quickly and effectively against the parasite.
When they don't work, it's usually because the tidal wave of drug resistance spreading worldwide has not escaped the sleeping sickness pathogen's notice.
Now scientists at the Swiss Tropical Institute in Basel have identified a gene in T. brucei that looks useful for diagnosing and treating drug-resistant sleeping sickness. When mutated in resistant parasites it makes them vulnerable to arsenicals. The paper reporting this Swiss discovery appears in the current issue of Science, dated July 9, 1999, titled: "A nucleoside transporter from Trypanosoma brucei involved in drug resistance."