Companies – at least public ones – customarily produce annual reports for shareholders and potential investors. Here is BioWorld’s annual report of newsworthy milestone advances in biotechnology research and development during the calendar year 2000. The selected items, set forth by disease or other category, are keyed primarily to advances reported in scientific journals that were covered in BioWorld Today. Dates of the issue – all from 2000, and all on page 1 unless otherwise noted – follow each item in parentheses.

AUTOIMMUNE DISEASE

Systemic Lupus Erythematosus (SLE)

One day a young woman in her childbearing years notices a weird rash across her face. The red blemish resembles the outspread wings of a butterfly. It’s a hallmark of lupus, the Latin word highlighting the wolfish facial mask of SLE.

Of the 1 million-plus lupus patients in the U.S. today, some 200,000 have advanced multiple-organ failures, wrought by inflammatory autoimmune antibodies. Between 10 percent and 15 percent die within a decade of being diagnosed with the disease. There’s no across-the-board treatment for SLE, only high-dose immunosuppressive corticosteroids that aim at dampening the immune system’s friendly fire.

But hematopoietic stem cell auto-transplantation came to the rescue this year of seven women with aggressive, life-threatening lupus. The seven remain in long-term remission of their wide-ranging symptoms (April 27, Aug. 30).

Mouse models of SLE also came off well. When injected with a soluble receptor for a tumor necrosis factor homologue implicated in B-cell autoimmune disease, they experienced arrest of their lupus symptoms, and lived longer than control mice.

Systemic lupus erythematosus and rheumatoid arthritis (RA) – both autoimmune afflictions – often occur in the same patient. Now they apparently share the same common denominator. It’s a molecule of the immune system called B-lymphocyte Stimulator (BlyS). This naturally occurring growth-factor protein works by binding to immune system B cells. These promote secretion of antibodies – the body’s frontline defense against infection and cancer – not to mention counterattacking constituents of the SLE and RA autoimmune antigenic targets in the patients’ bodies.

When 335 patients with SLE and RA at two academic centers were tested for BlyS in their blood, the level was far higher than in healthy individuals. This finding led the executive vice president for research and development at Human Genome Sciences Inc., in Rockville, Md., to declare, “These new studies provide a clear rationale that we should test the hypothesis that an anti-BlyS antibody may be an effective therapy for SLE and RA” (Dec. 11).

Multiple Sclerosis (MS)

This incurable disease afflicts upwards of 1.1 million victims throughout the world – a fourth to a third of them in the U.S. Why twice as many women as men contract MS, which strikes between 20 and 40 years of age, is another unknown.

For decades, viruses have been indicted – on circumstantial evidence – as perpetrators of multiple sclerosis. One circumstance: MS is six times as prevalent in Winnipeg (50 degrees north latitude) as it is in New Orleans (30 degrees N). By the same token, MS is rare between the sun-drenched equatorial tropics of Cancer and Capricorn.

Now research immunologist Andrew Caton, at the Wistar Institute in Philadelphia, has demonstrated in specially engineered mice that “virus infections, and other kinds of infections like them, can trigger autoimmunity.” But the etiology of MS remains uncertain. “Healthy people,” Caton observed, “get rid of all those T cells and B cells that can recognize their own proteins” (Dec. 19).

The myelin basic protein, of which MS nerve axons are denuded by misguided autoimmune cells, is manufactured mainly in the brain, by glial cells called oligodendrocytes. The amino acid glutamate is the dominant excitatory neurotransmitter in the mammalian central nervous system. A recent finding reports that glutamate’s action is guilty of oligodendrocyte death in MS (Jan. 6).

Two widely reported clinical trials of an experimental drug called APL, altered peptide ligand, showed promising hints of efficacy – “an improvement in neuronal inflammation” – against MS. One enrolled 144 patients at 14 research centers in North America and Europe. The other treated 24 patients at NINDS, the National Institute of Neurological Disorders and Stroke. But NINDS canceled both studies in midcourse for fear of possibly life-threatening allergic side effects in APL’s action (Oct. 4).

Rheumatoid Arthritis (RA)

This chronic, crippling disease savages the proteins in the synovial fluid of joints, much like oil or grease lubricate moving parts in machinery. RA patients in the U.S. number 2.5 million. The disease can lead to severe disablement and – unlike MS – in extreme cases, death.

RA frequently masquerades as Lyme disease, and vice versa.

Until recently, the anonymous perpetrator blamed for RA was called “the rheumatoid factor.” It’s since been fingered as antibodies in the blood of RA patients, which pounce on antigens causing the joint disease. Immunologist Henrik Ditzel at the Scripps Research Institute in La Jolla, Calif., observed: “In a lot of autoimmune diseases, like RA for example, the general belief has been that T cells are the most important immune factor. There is increasing evidence that antibodies, too, are important.

“Neutrophils,” he explained, “are a type of white cell very important for fighting bacterial infections. In RA you have this extra complication where patients, besides the basic arthritic symptoms, also have neutrophil destruction, caused by a separate autoimmune attack.”

Instead of looking in a patient’s blood for antibodies that react to neutrophils, Ditzel’s team used a reactive antibody as bait for fishing out its target autoantigen. Two-thirds of the RA patients, but none of the healthy donors, had that antibody in their bloodstream.

Next they isolated the RNA from a patient’s bone marrow sample, amplified the immunoglobulin chains, and expressed the antibodies on phage displays.

“Then,” Ditzel recounted, “we selected that library on tissue where we believed autoantigens were targeted by these autoantibodies. This method,” he suggested, “can be applied to a lot of other autoimmune diseases, such as diabetes” (Aug. 1).

CANCER

Angiogenesis

Why does a solid tumor resemble an unhealed wound? “Both display angiogenesis, the formation of new blood vessels, as required for replacement tissue to grow properly,” explained molecular geneticist Brad St. Croix, of the Johns Hopkins Oncology Center in Baltimore. “Our studies show that the molecules turned on in these new tumor-induced arterioles, venules and capillaries are also activated in vessels that take part in healing wound injuries.

“This research,” St. Croix pointed out, “presents for the first time fundamental differences between the endothelial cells lining healthy and malignant blood vessels.” He said “it has promise, at least in the long term, for making antitumor therapeutics and diagnostics, as a first step in order to capitalize clinically on the whole concept of tumor antiangiogenesis.”

He made the point that “the angiogenic blood vessels that infiltrate and grow into the tumors are not themselves malignant. They’re actually normal host cells that the tumor pirates, in order to expand. The network of new blood vessels provides a route for cancerous cells to escape and metastasize to different organs.”

St. Croix and his colleagues characterized all the genes in these two cell types, normal and malignant, by a technique called SAGE – serial analysis of gene expression. They now envisage three modes of applying their gene data clinically: diagnosis, chemotherapy and imaging.

“If something is turned on and expressed by a gene on the surface of these tumor endothelial cells, intravenous injection of a drug that’s linked up to an antibody that recognizes this molecule would kill the cells that express it. As for diagnostic potential,” he went on, “If the new blood vessels are secreting factors into the bloodstream, if angiogenesis is detected when it shouldn’t be, that would lead to suspicion of a tumor at that place.” As for imaging, “A tracer linked to an antibody would hook up to something on the surface of tumor endothelial cells, to scan and visualize those tumors under the microscope (Aug. 18).

Brain Tumors

A mouse’s tail is farther from its head than any other part of its body. Yet a gene therapy construct injected into the tail veins of mice previously infected with brain cancer made its way from caudal appendage to cerebral space, and delivered the anticancer transgenes to the malignant tumors.

Glioblastoma multiforme (GBM) is the most common solid tumor in children. Yearly in the U.S., 19,000 new brain cancers are diagnosed. GBM kills 5,000 to 6,000 Americans annually – young and old. It is virtually untreatable and inevitably fatal. Life expectancy after diagnosis averages 12 months. Defying surgery, radiation and chemotherapy, GBM metastasizes widely throughout the brain. The only type of brain cell that can hope to keep up with this loose-cannon migration is the neural stem cell (NSC) – ancestor of all the other cells in the central nervous system.

Harvard neurobiologist Evan Snyder deputized NSCs to track, corner, round up and shoot down the murderous, outlaw GBM cells. He and his colleagues injected adult neural stem cells not only into tail veins but also into remote brain regions and the tumor itself. The antitumor transgenes expressed an enzyme that converts to a powerful oncolytic chemotherapeutic agent, 5-fluorouracil. This gene therapy experiment induced death of surrounding tumor cells, even when these outnumbered the chemo compound 4-to-1 (Nov. 13).

Because glioblastomas are so aggressive and fast growing, they need a copious and expanding blood supply. This means angiogenesis – propagating a de novo network of blood vessels to nourish the budding tumor with oxygen and nutrients.

Urokinase (uPA), a plasminogen activator, is an enzyme intimately involved in tumor angiogenesis. Molecular geneticists have identified an antigenic peptide from within uPA’s sequence that controls the motility and contractility of the endothelial cells that line the tube-like walls of blood vessels. They derived a peptide named A6.

In a preclinical in vivo experiment, the researchers inoculated the brains of nude mice with growing human GBM cells, then treated them with A6, followed by cisplatin – a potent cytotoxic agent that disrupts DNA synthesis. That combo cut down growth of the GBM cells by 90 percent, with no toxic side affects – but did not lead to outright tumor regression (Aug. 21).

Colorectal Cancer

Fat-finger syndrome is a common complaint in people who never mastered touch-typing. Hitting two keys at once on the keyboard will often produce a typographical error, which the word-processor’s spell-checker instantaneously catches, and underlines in red for correction.

This high-tech performance dwindles to triviality compared with the human body’s DNA mismatch-repair system. Whenever DNA slips up and commits a typo, half a dozen DNA repair genes go into action to mend the mistake. But like other genes, these six are themselves susceptible to glitches, which in turn may be susceptible to cancer – in particular colon cancer.

At NIH’s National Human Genome Research Institute (NHGRI), molecular biologists have discovered a seventh colon cancer-susceptibility repair gene, which they named MLH3. Then a group at Roswell Park Cancer Institute in Buffalo, N.Y., found that this MLH3 region of the human genome’s chromosome 14 was lost in approximately 30 percent of all colorectal cancers. Whereupon the NHGRI team set about creating a knockout mouse deprived of its MLH3 gene to relate this mutation to the malignancy (Jan. 13).

Is the generation of colorectal tumors a crapshoot, or Russian roulette with most of the revolver chambers loaded? The experimental pathologists at Roswell Park counted some 11,000 aberrant genomic events per colon carcinoma cell, “considerably more abundant than expected,” they report.

Even less expected, and more disquieting, they also found that precancerous adenomatous polyps “showed similar numbers of events.” To perform this mutational census, they subjected 58 human colon cancer samples and 14 polyps to PCR (polymerase chain reaction) analysis, and concluded: “Together, our results support the model of genomic instability being a cause rather than an effect of malignancy, facilitating vastly accelerated somatic cell evolution, with the observed orderly steps of the colon cancer progression pathway reflecting the consequences of natural selection” (Jan. 10).

Melanoma

People diagnosed with advanced malignant melanoma spend the rest of their lives on a sort of death row, awaiting almost certain execution within five years after therapy. In the U.S., where melanoma is the 5th most common cancer, over 40,000 new cases are diagnosed each year, and more than 7,000 patients die. Surgery is virtually the only treatment of choice, except for high-dose interferon-alpha, introduced about five years ago.

But attendees at the 36th annual meeting of the American Society of Clinical Oncology heard a new note of optimism in prolonging the lease on life of melanoma victims. It took the form of a poster titled, “Post-surgical treatment of clinical stage III melanoma with autologous, hapten-modified vaccine: Expanded sample size and long-term follow-up.”

The presenter, and inventor, of this novel vaccine was David Berd of Thomas Jefferson University in Philadelphia. He and the university have licensed it in exclusivity to AVAX Technologies Inc., of Kansas City, Mo. The firm is embarked on a 20-center, 400-patient Phase III randomized trial, comparing Berd’s vaccine to FDA-approved high-dose interferon (May 24).

INFECTIOUS DISEASES

AIDS

The idea of using HIV – the human immunodeficiency virus – as a DNA delivery vehicle for gene therapy may seem bizarre at first glance. But at second glance, dragooning this AIDS pathogen into a potential therapeutic gig has logic.

HIV belongs to the family of lentiviruses, which means, as the name implies, that they act relatively slowly over time, compared, for example, to the influenza virus, which can inflict symptoms on its victims within hours. What excites gene therapists at the prospect of making HIV a vector for delivering therapeutic genes to defective cells is the fact that lentiviruses, unlike most viral vector candidates, can infect nondividing cells, notably those of nerves, liver and blood-forming stem cells. That gives them a possible leg up in treating, say, brain cancers, and exploiting the ability of liver to churn out useful proteins.

What sparked this far-out concept was the fact that HIV, which mainly attacks rapidly dividing T cells of the immune system, can also infect that system’s macrophages, which are nondividing (Jan. 18).

Later in the year, researchers at NINDS, the National Institute of Neurological Disorders and Stroke, contrived a double gene therapy vector consisting of two lentiviruses – HIV and herpes simplex virus. This construct conveyed a transgene that expressed the visible marker, green fluorescent protein. When they implanted this package into the ventricles of mouse brains, they found the expressed protein not only in the tissues immediately surrounding the ventricle, but in the cerebral cortex, a typical brain tumor target area (Oct. 11).

After an AIDS retrovirus breaks and enters the T cell it has hijacked, the invading HIV hooks up its own DNA to the genome of its target cell, and forces it to replicate the virus big time. Then this swarm of viral progeny bud out of the cell, and spread out to infect more and more neighboring cells.

The now-celebrated multidrug cocktail of highly active antiretroviral therapy (HAART) has tamed HIV from a slow but lethal threat to a costly, pill-popping ordeal. But the AIDS virus is fighting back. Sooner or later, the virologists know, drug resistance will foreclose HAART’s title.

Looking for a new Achilles’ heel to again bring down HIV, drug designers have fixed on a viral enzyme called integrase. This is the molecule that actually hitches the viral strands of DNA onto the target cell’s genome. So integrase looks like a logical site to cut the invading pathogen off at the pass.

Virologists at the Merck Research Laboratories in West Point, Pa., think they may have solved the admitted obstacles to recruiting integrase. Their paper in Science dated Jan. 28, 2000, bears the hopeful title: “Inhibitors of strand transfer that prevent integration, and inhibit HIV-1 replication in cells” (Jan. 28).

On the street, a shiv is a gang member’s blade or razor, used as a weapon. In the lab of clinical retrovirologist Ruth Ruprecht, SHIV denotes a lethal mixture of human and simian immunodeficiency viruses.

To see if her triple-antibody vaccine could prevent mother-to-child transmission of SIV – the simian equivalent of HIV – Ruprecht, at the Dana-Cancer Cancer Institute in Boston, injected the antibodies into the veins of four healthy, pregnant macaque monkeys, four days before delivery and three after. An hour later the dams received intravenous challenge doses of SHIV. Over the next six months, tests showed all four of them to be free of infection.

Shortly after being born, their neonatal offspring swallowed a trio of potent monoclonal antibodies, used as a vaccine against the viruses, followed a few hours later by shots of SHIV. They got this challenge dose by mouth, a route that simulates transmission of the AIDS virus from an infected mother to her infant. A separate cohort of control newborn monkeys got SHIV orally, but no antibody vaccine. Over the next six months the controls suffered persistent SHIV infection while their vaccinated contemporaries had none.

“This is the first demonstration,” Ruprecht pointed out, “that contrary to widely held opinion, neutralizing antibodies actually do work. When used in a synergistic combination, they can be powerful enough to protect against even mucosal viral exposure” (Feb. 8).

Influenza

Like a heavily armed squadron of paratroopers that drops down in advance of defensive ground forces to fight off an invading enemy, the immune system deploys interferon, well ahead of defensive antibodies, to confront and destroy viruses before they can infect a human cell or body.

A case in point, observed virologist Peter Palese, at New York’s Mt. Sinai School of Medicine, “is the influenza virus – a formidable pathogen that kills thousands of people a year in the U.S. alone.” Palese reported finding that the flu virus, long thought devoid of defenses against interferon, does indeed possess interferon antagonist activity, in the form of a protein he named “NS1.”

“In a normal influenza infection,” Palese pointed out, “the virus with a functional NS1 protein in its genome will overcome the host’s interferon response.” To demonstrate this effect, he recounted, “We put flu viruses without NS1 up the noses of mice. The animals got infected, but because the virus is attenuated – weakened – not very pathogenic or virulent, they didn’t get sick. Four weeks later we challenged them with a virulent virus, armed with NS1. The control animals, which had never seen the immunizing pathogen, died, whereas immunized mice were protected.

“The university,” Palese observed, has filed patent applications, claiming the making of vaccines, in the case of influenza, but against other viral infections as well” (March 24).

A young Eskimo woman who died suddenly in November 1918 went to her grave harboring the secret of the greatest serial killer in human history. Her death was one of 72 in the remote Alaskan village of Brevig Mission. She and her stricken neighbors were buried 6-feet deep in the Arctic permafrost.

Fast-forward 79 years to August 1997. That was when a retired pathologist named Johann Hultin exhumed the woman’s remains from its frozen sepulcher. He found her lungs well preserved, and shipped their tissues to Jeffrey Taubenberger, chief of molecular pathology at the U.S. Armed Forces Institute of Pathology in Washington, D.C.

Hultin’s gift was a personal contribution to Taubenberger’s long-term project of sequencing the genome of the hypervirulent influenza virus that perpetrated what he calls “the most extensive infectious disease in history.” Those 72 Alaskan victims were part of a larger statistic: The 1918-19 flu pandemic took the lives of about 675,000 people in the U.S., plus 20 million to 40 million dead the world over.

Taubenberger’s latest progress report, in the Proceedings of the National Academy of Sciences, released May 23, 2000, is titled: “Characterization of the 1918 ‘Spanish’ Influenza virus neuraminidase gene” (May 25).

Malaria

Throughout the world’s tropics, malaria infects half a billion people a year, and kills 2 million to 3 million of them – mostly children. The endemic disease wages war against the human race via airborne stealth bombers – Anopheles mosquitoes – which drop microscopic smart bomblets – Plasmodium falciparum parasites – into the bites the insect inflicts on its victims.

Malariologists got to know this enemy’s heavy pathogenic weaponry well enough to counterattack with chemical warfare. For centuries, the preferred antimalarial drug was quinine. Then in the 1930s, medicinal chemists synthesized the more powerful chloroquine. In fact, it was too powerful for its own good. Not only did chloroquine treat malarial attacks, it prevented them. So people gobbled the drug to stave off the infection. That’s when P. falciparum deployed its secret weapon: drug resistance. Now chloroquine is on the endangered list of antimalarial drugs that the parasite’s multidrug-resistance genes simply shrug off.

One after another, the latest frontline antimalarial compounds have been added to the list of multidrug resistance. It includes mefloquine, halofantrine and a worrisome new drug – artemisinin.

“In malaria,” explained molecular malariologist Alan Cowman, “the drugs that we’ve shown to be threatened are all somewhat related. They have a structure that’s similar but not identical to that of chloroquine. Whereas,” he went on, “artemisinin is totally different. It’s a new compound, developed by the Chinese from the Artemisia plant.”

Cowman is head of infection and immunity at the Walter & Eliza Hall Institute of Medical Research in Melbourne, Australia. He made the point, “It’s really being recommended that artemisinin be used only to treat severe malaria, and not let it get out there too much, so that resistance doesn’t develop too quickly. There’s a company in Europe now,” he observed, “that’s producing large quantities of artemisinin, but not for general availability – to protect it from drug resistance.”

Tuberculosis (TB)

TB and malaria seem to be reading from the same bad news/good news hymnbook. Both are seeing their prime chemotherapeutic compounds wither away under pathogenic drug resistance. And each has found a new and promising agent that may escape this fate.

Mycobacterium tuberculosis has been a fellow traveler with Homo sapiens ever since man took up farming 10,000 years ago, and acquired the deadly bug from his cattle. The infection waxed and waned throughout human history, but seemed terminally vanquished half a century ago with the discovery of a very effective antibiotic called isoniazid. This TB-specific drug emptied tuberculosis sanitoriums, and people began regarding T. tuberculosis as a has-been germ.

But ironically – or inevitably – isoniazid, the first and foremost anti-TB agent, became the first victim of the bacterium’s drug-resistance backlash. And besides the bug’s diabolical anti-isoniazid onslaught, the resurgence of TB in the 1980s has other powerful perps – its own human society. Blame is shared among the HIV/AIDS epidemic, immigration from TB-ridden countries, increased poverty, decline in health care infrastructure and poor patient compliance with onerous but effective treatment regimens.

So once again, tuberculosis is not to be sneezed at – literally. A sneeze, a cough, a kiss, even heavy breathing up close and personal, can waft the airborne pathogen into a mouth or nostril, and thence infect the lung. For AIDS victims, TB has become a leading cause of death (Feb. 9, June 2).

Now for the artemisinin-like good news:

Nitroimidazopyran is not a word that comes trippingly off the tongue, or readily to mind. Yet it bids fair to become the first drug in 30 years to treat successfully all forms of tuberculosis. A precursor to this novel compound was discovered eight years ago in India – one of the Third World countries most sorely afflicted with TB. Microbiologists at Ciba-Geigy’s Pharmaceutical Research Center in Bombay, testing anti-cancer chemicals, found that their nitroimidazopyran showed antitubercular activity instead.

They reported that mice treated with it experienced “significant increase in survival time,” on a par with isoniazid and rifampin, the world’s frontline TB antibiotics.

After Ciba-Geigy shut down its Bombay facility completely, Seattle-based PathoGenesis Corp. (now part of Chiron Corp.) took up the nitroimidazopyran cudgels, and spent the rest of the 20th century developing promising analogues. By June 22, 2000, they were able to report, in Nature of that date, that in vivo testing of their lead compound in mice and guinea pigs showed “reductions of mycobacterial burden in both spleen and lung tissue, comparable to that of isoniazid” (June 22).

DERS

Alzheimer s Disease (AD)

In the little Italian city of Nicastro, some 40 years ago, a large extended family made neurological history. Its members were afflicted with what became known as familial, early onset Alzheimer’s disease (AD). Usually, this neurodegenerative senile dementia strikes its victims in their 70s. The Nicastro family victims came down with AD’s signal symptoms – memory loss, confusion, disorientation – while in their 30s or 40s.

Fast forward to 1995, when Canadian neurobiologists cloned the genes for two key proteins – presenilin-1 and presenilin-2 – which have a lot to answer for in the most severe, Nicastro-like, forms of familial, early onset AD. That’s why they named their new 700-amino-acid protein “nicastrin,” which they have just isolated. It binds to both presenilins, and influences the neuronal pathway that leads to senile neuritic amyloid plaques – the toxic hallmarks wrapped around AD neurons. They are considered principal culprits in nerve cell death, and eventually dementia (Sept. 7).

The maxim “Seeing is believing” has a special meaning in the diagnosis of Alzheimer’s disease. A skilled clinical neurologist, by dint of extensive behavioral testing, can render a verdict for a particular patient that’s better than 90 percent accurate. The remaining 10 percent are confused with the spectrum of other senile dementias that resemble each other – and AD.

Their main difference is that, unlike AD, this mixed bag of other cognitive defects can be medicated with more or less efficacious drugs. So far, there is no approved medicament for efficiently predicting, arresting, slowing or ameliorating the devastating laundry list of Alzheimer’s disease symptoms. The key hallmark of AD is invisible to the eye – even of the most experienced clinician. It’s a peptide called amyloid-beta, which form those plaques around dying nerve cells in the AD brain. The only way those plaques can be seen, and thus a definitive diagnosis of AD confirmed, is by post-mortem examination of the deceased patient’s brain (Aug. 23).

The idea of a vaccine against Alzheimer’s disease seems somehow counter-intuitive. The amyloid plaques and tangles that mark the aging AD brain obviously don’t arise from infectious pathogens (although prions have been suspected). Rather, they derive from that rogue molecule, amyloid-beta, which breaks off from its mother molecule, the amyloid precursor protein, to poison the failing AD neurons that wreak AD’s loss of mental faculties.

Yet as recently as Dec. 21, 2000, three papers in Nature of that date describe preclinical vaccination experiments, using amyloid-beta as the antigenic target. Their authors, from Canada, Scotland and the U.S., all reported that their vaccinated mice were protected from developing memory deficits.

Neuroscientist Dennis Morgan, at the University of South Florida in Tampa, explained: “When we inject the amyloid peripherally, subcutaneously, in the presence of an adjuvant, the animal thinks there’s an infection going on that actually provokes the immune system to make antibodies against the amyloid-beta (Dec. 21).

On this score, a nonsteroidal anti-inflammatory drug named Celebrex, much prescribed against arthritis, is in clinical trials at the UCLA (University of California at Los Angeles) Memory Clinic to see if it prevents brain-function decline, as in AD (May 23).

Drug Addiction

When asked why he kept banging his head against the wall, the weirdo replied, “Because it feels so good when I stop.” The exact opposite is true of hard-drug addicts. Much as they’d like to kick their addiction, they can’t – because it feels so God-awful when they stop.

“Cessation of drug use in chronic opiate abusers produces a severe withdrawal syndrome that is highly aversive,” said research psychiatrist Gary Aston-Jones, at the University of Pennsylvania in Philadelphia. “Clinically,” he explained, “it’s the memory of the physical and aversive responses that drives an addict to make sure he has enough drug around so he doesn’t risk having that horrendous symptom happen again.”

This bi-level horror story is generated in a small, almond-shaped area of the human and rat forebrain, thought to be involved in memory. It’s called the “bed nucleus of the stria terminalis,” (“bed nucleus” for short). This bundle of brain cells is linked to the aversiveness of opiate withdrawal, and hence to perpetuation of addictive, drug-taking behavior.

“We found,“ Aston-Jones recounted, “that noradrenaline-activated beta receptors in the bed nucleus to elicit the aversive response of withdrawal. We also found that if we antagonize its actions at the specific beta receptor in the bed nucleus, we can eliminate those dysphoric effects.” Aston-Jones allowed that the therapeutic applications of his findings for treating presently untreatable opiate addiction are “pretty straightforward.”

He observed, “If we could develop a specific drug that would attack specific beta receptors in that bed nucleus, but didn’t have side effects, those drugs would be very useful. Pharmaceutical companies do that all the time,” he concluded. “That’s what they do for a living. They’re very good at it” (Jan. 27).

Huntington s Disease (HD)

Like his father and grandfather before him, George Sumner Huntington (1850-1916) was a rural general practitioner in the East Hamptons of Long Island, New York. Fresh from medical school, he began in 1872 to describe the inherited neurological disease that now bears his name. Its three clinical hallmarks include jerky, spasmodic, dance-like movements of the limbs, plus facial grimaces; onset at 30 to 40 years of age; and slowly advancing dementia for the next 10 to 20 years until death.

The HD gene, IT-15, was mapped to human chromosome 4 in the 1980s, and the genetic test for the mutated protein it encodes – huntingtin – was approved in 1993. ”When that test became available,” recalled neuropsychiatrist Christopher Ross, at Johns Hopkins University in Baltimore, “we expected a large expression of interest from the 150,000 presymptomatic individuals at risk of HD in the U.S. In fact,” he continued, “only 3,000 asked to be tested, presumably for two reasons: The disease is totally untreatable, so the diagnosis would be useless, and those who tested predictively positive feared they would lose their insurance.

“One of the wonderful things about HD genetic testing,” Ross observed, “is that we can begin examining people years before the onset of disease. It allows us to see in this early stage how the disease manifests itself in destroying the nervous system.”

So he and his colleagues designed a servo-electric contrivance that measured the brain’s motor control in individuals at genetic risk but still free of symptoms. “We hypothesized,” Ross recalled, “that if this automatic limb response were influenced by the brain’s basal ganglia, there might be some detectable defect in its functioning long before any clinical symptoms appeared.” To check out their hunch, they recruited 42 test subjects.

The positive predictive findings of their experiments led Ross to conclude: “The brain is fundamentally a plastic system; it can be trained. Our hope is that now that we think we can anticipate a particular deficit in motor control, we could somehow focus rehabilitation early – teach the brain what’s happening, essentially – years before there is any clinical manifestation of HD” (Feb. 3).

A few years ago, neuroscientists were asking a reasonable question: If fetal brain-tissue transplantation helped Parkinson’s disease patients, why not Huntington’s?

Beginning in 1997, neurosurgeon Thomas Freeman, at the University of South Florida in Tampa, implanted human fetal brain cells into the striatal cerebral regions of seven adult patients with moderate to advanced HD. “It was our hope,” Freeman said, “that the transplants would be able to restore lost neuronal connections in their diseased brains, and thereby slow the disease.”

Just 18 months into that human trial, one of Freeman’s seven patients, a 54-year-old man, died suddenly of a massive heart attack unrelated to his HD surgery progression. “Brain autopsy revealed,” Freeman noted, “that we had graft survival that reproduced regions of typical striatal tissue, which we had transplanted. What’s more, the graft survived for a year and a half without immunosuppression of the patient.

“In five of the six remaining subjects,” he went on, “who have come to one-year follow-up, we got a 20 percent improvement in the HD global rating score. This compares with deterioration before surgery at a rate of 15 [percent] to 20 percent, which is what you’d expect.” The sixth sustained head injury from a fall six weeks after her transplant, so could not be evaluated (Dec.5).

Parkinson s Disease (PD)

Back in the early 1990s, a bunch of drug addicts went on a heroin trip – and ended up in the ditch. The synthetic good stuff they’d bought and binged on turned them into permanent physical zombies, trembling with the shakes, their steps sluggish, their sense of balance erratic, their limbs stiff – the hallmark symptoms of Parkinson’s disease.

It turned out that the synthetic narcotic was contaminated by a potent neurotoxin called 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine – now much better known as MPTP. The brain cells that grim drug disrupted were the very same neurons that make dopamine, the neurotransmitter that keeps bodily movements on an even keel.

Presumably, those hapless heroin junkies had to switch from their favorite narcotic fix to the only drug that treats the symptoms of PD – namely, Levo-dopa. This is the precursor of dopamine, which dwindles with advancing age in the dopaminergic neurons that make it. But L-dopa’s clinical effectiveness also wanes with time.

Now gene therapy may be coming to the rescue of PD with a neuron-nourishing gene called GDNF – glial cell-line-derived neurotrophic factor. Neuroscientist Jeffrey Kordower, at Rush-Presbyterian-St. Luke’s Medical Center in Chicago, inserted the gene for GDNF into primate models of PD.

“Our rationale,” he explained, “was we knew from animal studies that GDNF is a very potent agent for keeping alive those dopaminergic neurons that die in PD. The exciting thing about this approach,” Kordower pointed out, “is that we’re trying to alter the underlying course of the disease, attacking the pathological process rather than replacing something that’s lost – i.e., L-dopa.”

In one typical in vivo experiment, Kordower recounted, “Six doses of the GDNF gene therapy, given to eight aged monkeys, brought their dopamine levels back to those of a young monkey” (Oct. 27).

Monkeys and mice are not the only animal models of Parkinson’s disease. Would you believe the fruit fly, Drosophila melanogaster, as a surrogate simulator of the human disorder?

Molecular neuroscientist Mel Feany, at Harvard-affiliated Brigham and Women’s Hospital in Boston, explained: “We chose Drosophila because we’re particularly interested in identifying the proteins that mediate neurodegeneration in PD. That’s quite difficult; you have to do genetics. And it’s really hard in people, whose life span is 60, 70, 80 or more years. It’s hard in mice, which live about 24 months, but it’s really easy in fruit flies, which die after 60 days. Also flies are good for drug screening.

“To construct our transgenic insects,” Feany recounted, “we spliced the human alpha-synuclein gene into a circular piece of DNA – a plasmid vector – and microinjected that construct into early fruit fly embryos. The overall result,” she summed up, “is that we have produced an excellent model of PD, which replicates the three cardinal manifestations of the human disease: (1) locomotor dysfunction; (2) age-dependent loss of dopaminergic neurons; and (3) abnormal alpha-synuclein protein fibrillar aggregates in those neurons – the so-called Lewy bodies” (March 23).

OTHER DISORDERS

Hemophilia

New therapies for hemophilia chalked up a three-base hit in the first two months of the new millennium. To wit:

The first three patients to receive clotting factor IX by gene therapy all achieved clinical improvement, as an efficacy data bonus in a Phase I trial set to test only safety and dosage. Nature Genetics for March 2000 reported this news in an article titled, “Evidence for gene transfer and expression of factor IX in hemophilia B patients treated with an AAV vector.”

A Keystone, Colo., symposium Jan. 8 on the theme, “Gene therapy: The next millennium,” heard a communiqué from The Immune Response Corp., of Carlsbad, Calif., on “Optimizing the cDNA sequence of a human B-domain-deleted Factor VIII gene,” designed to improve gene therapy of patients with hemophilia A.

And cell biologists at the Uniformed Services University of Medicine in Bethesda, Md., described in the March issue of Nature Biotechnology an indwelling pump, or chamber, that gets around clotting factor shortages. Its title: “In vivo bypass of hemophilia A coagulation defect by factor XIIa implant” (March 2).

Infertility

Every man, woman and child on earth today came into this world as an enemy alien. From the vantage point of a pregnant mother’s immune defenses, we all started life as illegal immigrants.

It’s always been a sweet mystery of life why an embryo within its mother’s womb survives, since half of its proteins come from the father. The saving grace is fetomaternal tolerance – the ability of the mother’s immune defenses to recognize, or at least tolerate, the developing offspring for the sake of the future baby’s survival. Research immunologist Hector Molina, at Washington University in St. Louis, has described one set of proteins that cancels a terminator arm of the immune system’s complement cascade.

He reports this finding in Science dated Jan. 21, 2000, under the title: “A critical role for murine complement regulator Crry in fetomaternal tolerance.” Crry stands for “complement receptor-related gene Y.” The Y chromosome, of course, is the male-determining factor in mammalian reproduction.

The co-authors found that in pregnant mice lacking the Crry protein, the complement cascade attacks and destroys the embryo as if it were something strange and hostile to its mother. They determined that, in order for the embryo to survive, Crry must be expressed on the surface of placental cells. Sperm cells, too, they point out, carry on their surface the same complement-curbing proteins that they found on the placenta.

Molina noted, “There are some conditions in infertility that involve antibodies against sperm, causing multiple miscarriages. And antibodies are among the most potent activators of complement.” His team aims to provide the human equivalents of Crry, in order to prevent these still births (Jan. 26).

Sperm gotta swim (post-ejaculation), strip their tips for action (capacitation), and penetrate the ovum’s wall (acrosome reaction). All these functions depend on a soluble enzyme, adenylyl cyclase (sAC). Its job is to convert adenosine monophosphate (AMP) to cyclic adenosine monophosphate (cAMP) – the second messenger of neural or hormonal activation.

Molecular geneticist Lonny Levin, at Cornell University’s Weill Medical College in New York, is senior author of a paper on this subject in Science dated July 28, 2000. Its title: ”Soluble adenyl cyclase (sAC) is an evolutionarily conserved bicarbonate sensor. “One significant aspect of this finding,” Levin observed, “is that we have a candidate molecule for a male anti-fertility drug, or even a topically applied one.” By inhibiting sAC, he explained, “sperm couldn’t swim, couldn’t capacitate and penetrate post-ejaculation – and would be shooting blanks” (Aug. 8).

Organ Transplantation

For openers, consider some grim statistics concerning the human body’s largest organ – the liver. Besides its imposing size, the liver is uniquely complex. It converts food into the molecules needed for life and growth, produces the blood’s vital clotting factors, and detoxifies poisonous products. Also – like very few tissues in the body – a liver can regenerate itself, after most of its bulk has been lost to trauma or disease.

In the U.S. alone, an estimated 20 million to 25 million people are, or have been, afflicted with liver disease, especially cirrhosis, and more than 40,000 of these patients die each year. The light at the end of their dark tunnel is a liver transplant – by which many are saved but few are chosen. In 1998, 4,318 donor livers were transplanted. But because of organ shortage, some 1,327 end-stage sufferers died that year while on the transplant waiting list.

Molecular biologist Phillippe Leboulch, at Harvard Medical School and Massachusetts Institute of Technology, is also chief scientific officer of Genetix Pharmaceuticals Inc. in Cambridge, Mass. He has devised a gene-transfer method for transplanting cultured liver cells, to give these last-resort hopefuls “a gift of time.” His bridge strategy consists of introducing an oncogene into hepatocytes in vitro, to render their division and expansion immortal. As reported in Science dated Feb. 18, 2000, it works – so far preclinically. The paper’s title: “Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes” (Feb. 22).

In organ transplantation, a patient with Type I diabetes confronts a double whammy from his or her immune system. The injury to which it adds insult is the autoimmune cause of the disease, in which rogue antibodies and T cells destroy their insulin-producing beta cells. The added insult is that attempts to transplant donor islets of Langerhans – the pancreatic beta-cell source of insulin – comes up against the prospect of a lifetime on immunosuppressive drugs to prevent annihilation of the alien islets by that same, too-vigilant immune system.

“In the past 10 years,” observed biomedical engineer Ray Rajotte, at the University of Alberta in Edmonton, “from 1989 to 1998, there were 267 recorded islet transplants. The number of those diabetics who achieved insulin independence for one year was only 8 percent. Their donor islets came in combination with a kidney transplant, and always the problem was the immunosuppressive steroids used in those days.”

A paper in the New England Journal of Medicine dated July 27, 2000, of which Rajotte is senior author, reports on what is now known as “the Edmonton protocol.” Its title introduces that story: “Islet transplantation in seven patients with Type I diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.” Since then, the number of recipients has climbed from seven to 10, and all have become insulin-independent, and remained that way – the longest for 14 months.

The secret? Instead of steroids, the Edmonton protocol administers, at time of transplant, three non-steroidal drugs – tacrolimus, which inhibits T-cell activation, rapamycin, and Roche’s Zenapax anti-interleukin antibody (June 14).

OTHER ADVANCES

Stem Cells (SCs)

“As a general concept, it was generally thought that stem cells in adult tissues generate only the type of cells that are expressed in that particular tissue. For example, blood SCs generate only blood, skin SCs only skin, and brain stem cells only brain cells. But in the last few years we have seen indications that this may not be entirely true.”

So spoke Swedish neuroscientist Jonas Frisen, at the Karolinska Institute in Stockholm, Sweden. “What we have done now,” he related, “is to ask: What if we placed brain stem cells in a different cellular environment? We supposed that the most rich system for a lot of such cellular neighborhoods must be the very early embryo. Because the starting cell there gives rise to all different cell types, there must be inductive signals to make that happen.”

For starters, he and his colleagues chose two embryonic neighborhoods – one murine (mice), the other avian (chicks). First, they injected adult mouse brain SCs into very early mouse embryos. “We could see liver, heart muscle and kidney cells developing from these brain stem cells,” Frisen recounted.

When they injected adult murine brain cells into chick embryos, “we saw that neural stem-cell-derived liver cells produced albumin, and their hearts – fully formed mosaic mouse/chick organs – were beating.”

In 1998, Frisen co-founded Stockholm-based NeuroNova AB to commercialize these stem-cell experiments. The company’s CEO, Anders Haegerstrand, observed, “We are sitting on a pot of gold, in that the human brain stem cell is really useful for understanding stem cell differentiation and function.” To which Frisen added, “The main project we are working on right now with NeuroNova is to specifically generate the type of dopamine-secreting neurons that are lost in Parkinson’s disease” (June 7).

In the 1990s, skin biologist Robert Lavker, at the University of Pennsylvania in Philadelphia, reported that the stem cells that grow hair – as the grandmother cells of any self-renewing tissue – reside in a portion of the hair follicle called the bulge. He postulated that if these were truly SCs, their progeny, the daughter cells, would actually replenish the skin’s neighboring epidermis as well as hair shafts. Lavker and his co-workers supported this point in the August 18, 2000, issue of Cell, under the title: ”Involvement of follicular stem cells in forming not only the follicle but also the epidermis” (Aug. 22).

Long bones, which serve as skeletal scaffolding for the mammalian body, are semi-hollow shafts, stuffed with constantly regenerating blood cells – the process of hematopoiesis. Its main products are the erythrocytes – red blood cells freighted with oxygen and nutrients – plus the array of immune system’s army of T and B lymphocytes, and other defense cells.

This multiservice legion of blood cells is commanded, indeed recruited, by a single general – the bone-marrow stem cell, whose commission was traditionally limited to hematopoiesis. Until a few years ago, scientists did not think mammals produced any new neurons at all after childhood – much less that foreign bone marrow cells might be drafted into such a feat. Prior research had succeeded in morphing bone marrow SC progeny into neuron-resembling cells, but only in vitro. Now, two back-to-back papers in Science dated Dec. 1, 2000, show that bone marrow SCs could indeed express neuron-specific genes.

Their titles: “From marrow to brain: Expression of neuronal phenotypes in adult mice,” reported by Stanford University scientists, and “Turning blood into brain: Cells bearing neuronal antigens generated in vivo from bone marrow,” by researchers at NINDS, the National Institute of Neurological Disorders and Stroke.

“The fact that this process exists,” observed Stanford cell biologist Timothy Brazleton, “means that it may be tweakable. It could have a huge impact potentially,” he added, “on the treatment of all kinds of brain injuries, as well as genetic disorders such as Huntington’s disease or Parkinson’s” (Dec. 13).

Genome Sequencing

In 2000 (see table), an insect and a plant joined the growing list of single-cell organisms that have all but monopolized the field of total genome sequencing. The exception is Caenorhabditis elegans, the lowly roundworm, which, in 1998, became the first fully sequenced multicellular organism. The full genome of Homo sapiens is expected in the next year or two or three.