By David N. Leff
Editor's note: Science Scan is a roundup of recently published biotechnology-related research:
Some cynics say that the once — and endless — target of biotechnology research is endotoxic shock. It's proven to be a moving target, in which the Gram-negative bacteria that cause this often-fatal blood infection have time and again thwarted therapeutic stratagems. (See BioWorld Today, Nov. 11, 1998, p. 1.)
Bringing endotoxic shock to heel is a much desired goal, with at least 20,000 deaths a year in the U.S., and perhaps a million worldwide.
Now, scientists at the University of Texas Southwestern Medical Center, in Dallas, report a new clue: a gene that normally prevents endotoxic shock. Their paper, in Science, dated Dec. 11, 1998, bears the shorthand title, "Defective LPS signaling in C3H/Hej and C57BL/10ScCr mice: Mutations in Tlr4 Gene." Its senior author is immunologist Bruce Beutler.
LPS is the bull's-eye of that moving bacterial target. It stands for lipopolysaccharide, the powerful toxin that those pathogens release from their cell walls.
As invading bacteria, such as Salmonella, pump out the LPS toxin, the body's immune system promptly counterattacks by deploying selected white blood cells. These send in anti-shock troops, cytokines such as tumor necrosis factor (TNF), which itself can be highly toxic in too large a quantity. Nevertheless, says Beutler's paper, "timely recognition of LPS by cells of the innate [i.e., early-warning, non-specific] immune system permits effective clearance of a Gram-negative infection before it becomes widely disseminated."
"The whole idea," he told BioWorld Today, "is that early in infection it is very important for the immune system to sense LPS, because then it realizes that the host is being invaded. If you are completely blind to endotoxin, the bacteria grow without restraint to a point that you develop overwhelming infection, and then you die of shock caused by other things."
Those two mouse strains with the long license-plates carry mutated Lps genes, which express LPS. Exploring those mutations, the co-authors discovered a gene called "toll-like receptor" (Tlr4), and concluded that "Lps is identical to Tlr4."
"The mutation," Beutler explained, "makes the mice impervious to any dose of endotoxin you want to give them, but it makes them very likely to develop sepsis. If you inject them with only one or two Salmonella typhimurium bugs, they'll die, whereas normal mice — which can sense LPS — will resist challenge by even tens of thousands of organisms. Discovery of the gene may enable creation of a test to screen for people with Tlr4 genetic defects. If so, we could protect susceptible individuals with antibiotics, eliminating some, and perhaps most, cases of endotoxic shock before they begin."
Mouse Model Of Sickle Cell Anemia Offers A New Way To Test Potential Therapies
Sickle cell anemia (SCA) is one of the commonest genetic diseases worldwide.
As in other disorders, a faithful animal model is essential to developing diagnostic and therapeutic strategies.
The most recent of numerous attempts to implant human genes that express human hemoglobin in mouse models is reported in the current Proceedings of the National Academy of Sciences (PNAS), dated Dec. 8, 1998. Its title is, "Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb beta8 globin yeast artificial chromosome: A mouse model of sickle cell anemia." Its senior author is Karin Gaensler at the University of California, San Francisco.
"Exclusive" is the operative word here. The co-authors' successful strategy depended on totally eliminating the functional murine alpha- and beta-globin genes, and replacing them with the human sequences. Their mice have not only irreversibly sickled cells in their peripheral blood, but hemolytic anemia, reticulocytosis and other phenotypic features of SCA. They "provide a novel model for assessing future therapies for SCA," the paper concludes.
Applying NMR Spectroscopy To Two HIV Genomic Structures Turns Up Possible Therapeutic Targets
In its grim task of killing off immune-system T cells, the human immunodeficiency virus (HIV) employs a protein in its genome called Vpr. This arrests T lymphocytes at a checkpoint in their cell cycle, and induces wholesale apoptosis. Vpr interacts specifically with a human DNA protein domain. Overexpression of this domain alleviates that cell-cycle arrest, and may be a good target for therapeutics.
As reported in Nature Structural Biology for December 1998, in a paper titled "Structure of a target of the HIV-1 Vpr protein," molecular biologists at the University of California, Los Angeles, with co-authors at Amgen Inc., of Thousand Oaks, Calif., used nuclear magnetic resonance (NMR) to determine the structure of this domain. They suggest that it "provides a rational basis for designing mutants to define the precise Vpr binding site."
In a separate article, titled "Molecular Mimicry in HIV reverse transcription," Nature Structural Biology reports on the viral makeover from DNA to RNA before integration into the host's DNA can occur. This reverse transcription has led to useful AIDS drugs, but more information on the steps involved "could yield additional useful targets," the co-authors, at Stanford University, Calif., point out.
One such step produces pairing of RNA sequences, "and the resulting RNA-RNA duplex fits in the active site of reverse transcriptase." Again using NMR, the Stanford team investigated a promising loop in the duplex, for more efficient initiation of the DNA-RNA conversion.
Dynavax Immunostimulatory DNA Sequence Knocks Out Asthma-Provoking Cytokines In Mice
Lastly, more data on the asthma story in BioWorld Today, Dec. 18, 1998, p. 1:
A paper in the Journal of Immunology, dated Dec. 15, 1998, is bears the title "Immunostimulatory DNA sequences [ISS-DNA] inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice." Its senior author is immunologist Eyal Raz, chief scientific officer of Dynavax Technologies Corp., in Berkeley, Calif.
The company's president and CEO, Dino Dina, told BioWorld Today that Dynavax's proprietary ISS-DNA "exerts a direct suppressive action on the cytokines that make IL-4 and IL-13, which leads to complete ablation of asthmatic symptoms." Moreover, Dina said, "these sequences generate an opposite immune response, which makes the mice immune to the next challenge."