By David N. Leff

Companies at least public ones customarily produce annual reports for shareholders and potential investors. Here is BioWorld Today¿s annual report of newsworthy milestone advances in biotechnology research and development during the calendar year 1999. The selected items, set forth by disease or other category, are keyed primarily to coverage in BioWorld Today. Dates of issue all from 1999, and all on page 1 unless otherwise noted follow each item in parentheses.

Autoimmune Disease

¿We have met the enemy, and they are ours,¿ signaled Admiral Oliver Perry, after his historic naval victory in the War of 1812. Developers of successful vaccines and antibiotics might well adopt Perry¿s boast in their never-ending battle against infective pathogens.

¿We have met the enemy, and he is us,¿ declared Pogo, the comic-strip creation of artist Walt Kelly, commenting on the human condition. His epigram neatly fits the roster of autoimmune diseases, in which the immune system¿s antibodies and killer T cells attack organs, tissues and cells in their own bodies.

Multiple Sclerosis

Multiple sclerosis (MS) is one such disorder. It strikes twice as many women as men. So do rheumatoid arthritis and systemic lupus erythematosus. The reasons for this sex bias may include such factors as pregnancy, oral contraception, seasonal viral infection and menstruation, noted a report by the National Multiple Sclerosis Society (March 2).

Central to MS is a layered lipoprotein called myelin basic protein (MBP), which sheaths the axons leading outward from the brain¿s cerebral cortex neurons. That myelin coating enhances the message-carrying ability of axons. It¿s often compared to the insulation on telephone and cable TV wires. In MS, those axons are patchily denuded of myelin.

An ubiquitous protein in the body, calpain by name, is suspected of targeting myelin for destruction at the behest of rogue autoimmunity cells. Scientists at the Medical University of South Carolina in Charleston buttressed this hypothetical calpain-MS connection by comparing tissue samples from patients who had died of the disease with similar cerebral specimens from Alzheimer¿s and Parkinson¿s disease samples as controls. Calpain ranked 90.1 percent higher in the MS specimens than in either of the controls. Many companies on both sides of the Atlantic are actively searching for drugs to block calpain¿s inimical activity (Oct. 12).

MS is a ¿global¿ that is, a brain-wide affliction. An animal model called the mutant shiverer mouse goes through life with a chronic case of the shakes, for lack of the glial brain cells (called oligodendrocytes) that manufacture myelin basic protein. Pediatric neurologists at Harvard-affiliated Boston Children¿s Hospital injected cloned neural stem cells into the brain ventricles of newborn shiverer mice. When these MS animal models grew up, their brains contained widely disseminated oligodendrocytes busily making myelin. And 60 percent of the mutant tremblers evinced near-normal behavior. If all goes well with ongoing monkey studies, the team proposes to treat myelin-deficient human newborns diagnosed with those neural stem cells (June 8).

Rheumatoid Arthritis

A visitor from outer space who looked at TV commercials and pharmacy shelves might conclude that every human earthling suffers from arthritis. Suffering and swelling are the hallmarks of arthritic joints. Some five dozen prescription drugs treat the ailment; all are essentially pain-killers. The only out-and-out cure is prosthetic joint replacement.

Now rheumatologists at DuPont Pharmaceuticals in Wilmington, Del., describe an enzyme aggrecanase which is partly culpable of cartilage degradation. The discovery, they predict, ¿gives us a protocol therapeutic target to deter, or actually stop, the disease as a whole¿ (June 7).

Of 18 mice turned into faithful mimics of human rheumatoid arthritis (RA), only eight developed mild, late-onset disease. The other 10 escaped even this kind fate. All 18 had been lapping up an extract of green tea, in which scientists at Case Western Reserve University in Cleveland found that a protein called catechin exerted anti-inflammatory effects in cancer. But 17 of 18 RA animal models, given only water to drink, came down with the swollen, painful eroded joints that mark the authentic human autoimmune disease. The polyphenolic catechin proteins in green tea, the researchers reported, are found only in that version of the beverage (April 16).

CANCER

Angiogenesis

This strategy of fighting tumors by starving them of their blood supply remained an academic and industrial buzzword throughout 1999. Following the 1998 media frenzy brought on by discovery of endostatin and angiostatin by surgeon Judah Folkman at Boston Children¿s Hospital, new anti-angiogenic products and processes continue to proliferate. As of August 99, the National Cancer Institute noted that 20 angiogenesis inhibitors were in clinical trials for cancer patients in the U.S. seven of which had reached Phase III.

Folkman himself cautioned that ¿it will take at least three separate anti-angiogenesis strategies prevention, intervention and regression to stop tumor growth.¿ In the prevention stage of animal trials, he reported, endostatin and angiostatin reduced angiogenic switching by some 60 percent. All told, the two inhibitors, plus two newer agents, ¿added at least two weeks to the animals¿ survival, but differed in tumor-diminishing efficacy¿ (May 3).

A chemical engineer at Massachusetts Institute of Technology reasoned that blood-free cartilage must be warding off angiogenesis by its own anti-blood-cell molecules, to preserve the joint¿s glassy-smooth surface. He identified one such protein as troponin, a potent anti-angiogenic factor (March 18).

Brain

It takes a newborn human baby¿s cerebral neurons something like two months to migrate from their point of origin in the evolving brain to their ultimate mission control center. The protein encoded by a gene called slit guides the brain-cell bodies and their axons along this developmental route. Neuroscientists at Washington University in St. Louis pointed out that such a molecule ¿might be useful for controlling the unwanted migration of brain tumor cells, without having to stick a needle into the target area¿ (July 22).

Breast

Living with cancer, rather than dying of it, is the take-home concept of a molecule just eight amino acids long, developed at McGill University in Montreal. It¿s derived from urokinase plasminogen activator (UPA), a clot-busting drug used to de-block thrombosed arteries. The neuroscientists tested their stubby peptide in rats and mice harboring implanted human breast tumors. After six weeks of twice-daily UPA injections into the animals¿ abdominal cavity, the tumors had shrunk more than 90 percent, and metastases to lymph nodes were down 80 percent. The authors saw no toxicity or visible toxicity. ¿We will treat cancer as a manageable disease,¿ they concluded. ¿We will teach patients to live with their metastases for many a year¿ (April 14).

Pregnancy, nursing, weaning and virginity all show up in the waxing and waning human mammary glands. Molecular biologists at the National Institutes of Health (NIH) have engineered a strain of mouse that mimics the cycle of human breast responses. Their aim is to figure out how the mutated BRCA1 gene causes mammary carcinoma in humans. The NIH team sees its unique animal model as ¿an important tool in future work on tumors and their progression.¿

In their first workout, the breast-cancer surrogate mice revealed a hitherto unknown pathway leading from a healthy caretaker BRCA1 tumor suppressor to a tumorigenic array of genes presided over by the sinister mutant p53 oncogene. The researchers are now using their animals to hunt for mammary-gland-specific drugs, and report ¿getting some quite exciting data¿ (May 6).

Human hair shafts also reflect bodily changes. Breast cancer seems to correlate with X-ray-scattering patterns of hair. An Australian biophysicist analyzed by high-energy X-ray beam the molecular structure of pubic hairs from women with mammary carcinoma. Of 23 samples, 100 percent showed the characteristic intensity rings, while 86 percent of hair from 48 women not suspected of the malignancy had normal patterns (March 4).

Colorectal

Most colon tumors sprout from benign polyps that blossom along the inner walls of the large intestine. To detect and remove these growths before they turn malignant, gastroenterologists have three therapeutic options all of them turn-offs for too many patients. Least onerous is occult fecal blood testing, from a smear on a piece of paper. Traces of blood signify one or more polyps that have bled, as they often do, when bruised by passing food particles.

Such a positive sign calls for sigmoidoscopy internal visual inspection, and on-the-spot polyp excision by a flexible, illuminated optical tube that scopes the lower one-third of a suspect colon. Finally, if warranted, comes colonoscopy a full court press that scrutinizes the entire 5- or 6-foot length of the colon.

No wonder all too many people shun or put off these procedures as embarrassing or distressing. That avoidance of timely monitoring contributes to the grim statistic that colorectal cancer mortality ranks second only to lung cancer deaths in the U.S.

Enter a start-up biotech company in Maynard, Mass., named Exact Laboratories Inc. Its so-called ¿no-touch¿ method relies on the solid stool that traverses the colon from end to end conceptually, the body¿s own colonoscope. In a pilot trial of 40 colon cancer patients at the Mayo Clinic, the Exact test correctly called 90 percent, compared to the conventional fecal-blood returns of 25 percent to 50 percent. The Mayo is now recruiting a 5,000- patient, 10-center trial to start in 2000 (May 21).

Another strategy against colon cancer relies on an enzyme called MAP kinase. This master regulator of mammalian cell growth is always turned on in tumor cells. Biologists at the Parke-Davis Division of Pharmacological Research in Ann Arbor, Mich., developed an oral inhibitor of the enzyme; it causes tumor cells to revert to non-malignancy. In one key experiment, the scientists implanted fragments of human colon carcinomas under the skin of nude mice, then fed them their anticancer compound for 14 days. This treatment curbed tumor growth by 53 percent to 79 percent. When it was stopped, the cancers staged a temporary comeback, until the next round of therapy put them down again. The company aims to start Phase I human trials sometime in 2000 (July 21).

Melanoma

Malignant melanoma represents only 5 percent of all skin cancers in the U.S., but it accounts for 75 percent of all deaths 6,500 annually in recent years and its incidence is climbing fast. Treatment usually involves surgical removal of suspected melanotic skin blemishes, before they have time to metastasize lethally in visceral organs. And that¿s a dicey race against time.

Research dermatologists at the University of Vienna, Austria, noted that the Ras protein, expressed by the ras oncogene, is abnormal, hence tumor-promoting, in 25 percent of human cancers. They pitted a recently discovered antagonist to Ras, code-named FTS, against human melanoma cells implanted in SCID (immunity-defenseless) mice. The Viennese scientists reported reduction of Ras in the malignant cells, after treatment with the novel compound. ¿Our findings,¿ they concluded, ¿stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma¿ (Dec. 6).

At NIH¿s National Cancer Institute (NCI), several hundred patients with metastatic melanoma sign up each year for the latest therapies that have worked well in tumor-ridden mice. Some of these clinical volunteers appear with bleached, pigmentless white patches on their skin and hair a condition called vitiligo. The NCI oncologists noticed that participants whose melanoma went away with treatment almost always had vitiligo a benign, if unsightly, side effect. So the team screened a melanoma cell library for antigens related to pigment cells, and made a vaccine out of five selected genes they discovered.

Vaccinated mice carrying aggressively spreading melanoma cells resisted tumor growth. ¿We¿re not on the threshold,¿ observed the lead NCI investigator, ¿but we¿re going to push it hard, because if human beings are anything like mice and we really are this may also work better in people than existing therapies¿ (March 24).

Prostate

Troublesome urination is the first sign of BPH benign prostatic hypertrophy. Correctable by surgery, it may or may not progress from non-malignant to cancerous. Prostate cancer (PC) itself then starts when the prostate gland grows larger, but remains within its capsule. Then it jumps this reservation to become invasive and later metastatic. At this point, PC tumor cells infiltrate bone or lymph nodes, driven by androgen i.e., the maleness hormone, testosterone. Surgical castration can cut off the testicular source of testosterone, which temporarily slows androgen-dependent PC. Later, metastasis kicks in again, but this time its tumors are androgen-independent, and death intervenes.

This multistage process usually takes many years, which makes monitoring problematical. In recent years, the introduction of prostate-specific antigen (PSA) has proven of limited use, in the early stages. A fast-growing animal model, ideally a PC-mimicking mouse was urgently needed, and provided a few years ago by researchers at the University of California, Los Angeles. They licensed the patented rodent to UroGenesis Inc., of Santa Monica, Calif.

By seeding surrogate PC animals with human tumor cells, the firm¿s scientists discovered a prostate-specific, cell-surface antigen that tracked the disease at high levels from its inception to androgen-independence, to death.

They named it STEAP for ¿Six-Transmembrane Epithelial Antigen of the Prostate¿ and found that it faithfully replicates every stage of prostate disease. Hence, UroGenesis expects that STEAP, or similar antigens, can be targeted to a patient as either a drug or its antibody, and probably eliminate all prostate cells at all disease stages, including the benign.

INFECTIOUS DISEASES

AIDS

Despite its phenomenal success at abating HIV symptoms and forestalling AIDS death, the triple-drug anti-HIV cocktail called HAART highly active antiretroviral therapy has two strikes against it: One, the virus will eventually develop resistance to those drugs; and two, the HIV particles break and enter their intended immune-system cells before HAART can do them in. So virologists and drug designers are seeking a strategy to cut the invading virions off at the pass before they can penetrate their target cells.

Just before the viral envelope¿s gp160 approaches its intended port of entry, it cleaves into two sub-particles, gp120 and gp41. The smaller segment goes through kinky contortions of shape, preliminary to fusing the virion and target-cell membranes. This briefly exposes a deep structural pocket that has drug designers scrambling to create inhibitors of that gp41 protein fragment.

Structural biologists at the Whitehead Institute of Biomedical Research in Cambridge, Mass., have now identified compounds that bind to this cavity. These may serve for starters at developing small-molecule oral drugs that can bar HIV¿s entry into cells (Oct. 4).

Another HIV molecule that goes along for the ride into the virion¿s target cell victims is named TAT transactivating protein. In the hands of molecular oncologists at Washington University in St. Louis, TAT became a do-gooder protein. The scientists borrowed that sequence from HIV¿s genome in order to smuggle outsize proteins into cells for therapeutic purposes. They were seeking to break the logjam that frustrates drug designers looking for ways to squeeze bulky candidate compounds through cell walls.

Most drug discovery companies ignore anything larger than 500 Daltons (units of atomic mass) because they couldn¿t enter cells with sufficient efficiency to treat the disease. That¿s why pharmaceutical companies throw away 70 percent of the substances they test because they¿re too big.

The St. Louis team chose the 1,000-amino-acid beta-galactosidase enzyme, weighing in at 120,000 Daltons, and coupled it to a stripped-down 11-amino acid segment of TAT. Injected into the abdominal cavities of mice, this protein package penetrated every mouse cell type tested. It should solve the main problem of gene therapy, the authors suggest, namely, getting a vector to deliver its DNA payload to cells of any size ( Sept. 3).

Influenza

Every year in mid-February, vaccinologists from the U.S., UK, Australia and Japan gather around a table at the World Health Organization (WHO) headquarters in Geneva, Switzerland. There they go over the influenza virus sequence data collected by laboratories all over the world. From this and the antigenic responses seen in each lab, they select the viral strain that will be used for this year¿s vaccine. Crystal-balling the amino-acid sequence changes that the flu virus is likely to perpetrate bases this vaccine designing by educated guesses on the pathogen¿s past behavior pattern. Vaccinologists would love to find ways of predicting the viral transmutations, rather than playing catch-up year after year.

Such a way is on the way. Virologists at the U.S. Centers for Disease Control (CDC) in Atlanta have teamed with evolutionary biologists at the University of California, Irvine. The CDC scientists noticed that 18 amino acids in the viral genome appear to change their sequences more often than others. For 11 annual flu seasons from 1986-87 to 1996-97 the group tracked the footprints of viruses from all over the world, and construed the evolution of their family trees. The CDC representative plans to present this phylogenetic approach to this year¿s WHO flu-vaccine commission meeting (Dec. 8).

Every year at the onset of ¿flu season,¿ around Nov. 1, children and oldsters the most vulnerable to influenza are urged to get their annual flu shots. Current vaccines consist of inactivated influenza virus A and B strains in the same injection. Strain A is the more virulent, more apt to mutate, and gives rise to more lethal outbreaks.

A virologist at the University of Wisconsin in Madison has constructed a flu virus from plasmid DNA, which, unlike the attenuated virus, is not potentially pathogenic, and which allows introducing mutations at will into the viral genome. The university has applied to patent his invention, and is now in the market for one or more investment-minded biotech partners (Aug. 6).

Tuberculosis

Humankind enters the third millennium of the common era with a global population that¿s just turned 6 billion. Fully one-third of this number is chronically infected with Mycobacterium tuberculosis, which kills three million of them a year. Their only protection against catching tuberculosis (TB) consists of a vaccine, BCG by name, which consists of a hodgepodge of the mycobacterium¿s protein antigens. One weakness of BCG vaccine is that antibodies it generates against those antigens can¿t seem to get rid of them.

A new anti-TB DNA vaccine, created by mycobacteriologists at Britain¿s Medical Research Council (MRC) in London, picks up in immune potency where BCG leaves off. For one thing, it contains only one antigenic dart board, instead of BCG¿s wimpish melange of antibody targets. Another surprising difference: Instead of merely preventing the infection, the DNA immunization dramatically reduced M. tuberculosis body count, thus reversing the infection so far in mice and guinea pigs.

In a key in vivo experiment, the MRC team infected mice with high doses of highly infective bacteria, followed by chemotherapy plus DNA vaccination. The animals seemed completely cured of their TB. A while later they received injections of corticosteroids (which suppress immune responses). There were far fewer mycobacteria in the animals¿ lungs and spleens compared with controls, including BCG vaccine as a placebo.

If further preclinical experiments in guinea pigs and rabbits hold up, the MRC scientists expect to try their DNA vaccine on people within a year or two. They foresee two clinical trial cohorts: HIV-positive individuals, who are particularly susceptible to TB, and patients infected with multidrug resistance pathogens that negate the effectiveness of antibiotics (July 15).

NEUROLOGICAL DISORDERS

Alzheimer¿s Disease

Neuroscientists examining the brains of people who died with Alzheimer¿s disease (AD) can barely make out in electron microscope images tiny clumps of matter that wrap around the dying neurons of the disease. These lifeless autopsy pictures of amyloid plaques are the hallmarks that confirm a diagnosis of Alzheimer¿s. If only they could be studied in living brains, those telltale plaques might provide early diagnosis, and the basis for designing drugs to deal with the disorder.

MRI magnetic resonance imaging can and does visualize human brains in vivo in hospitals, but its resolution akin to magnification is barely 1 cubic millimeter, and only 2-dimensional. Plaques are 1,000 to 10,000 times smaller. Enter MRM magnetic resonance microscopy. This is a souped-up prototype version of MRI, under development at Duke University in Durham, N.C. When researchers there imaged the donated brains of five patients who had died with AD, they discerned ¿what appeared to be small, black, 3-dimensional spheres, about 63 microns in diameter. There were so many of them that they could almost establish a row of spheres in the intra-cellular space surrounding the neurons.¿

Having proved their MRM technique in vitro, the Duke team is moving into AD prognosis in living patients. They await delivery of a special magnetic coil now under development, and expect it to be ready for preclinical use on mice by the end of 2000. If the plaque load correlates with behavioral changes, they will try to get drugs that decrease plaque in human AD brains (Nov. 23).

Parkinson¿s Disease

One of the deadliest poisonous mushrooms is Amanita muscaria, better known as the Destroying Angel or Death Cap. Its lethal ingredient is an alkaloid, muscarine, which can activate certain specific receptors of the powerful brain neurotransmitter, acetylcholine.

In Parkinson¿s disease (PD), neurons secreting another neurotransmitter, dopamine, die off, and patients must receive L-dopa, which stimulates dopamine receptors. But this therapy wanes with time, leading neurologists to replace it with drugs that antagonize certain muscarine receptors, and rev up that dwindling dopamine secretion.

A molecular biologist at the NIH pointed out, ¿In Parkinson¿s disease, you have dopamine neurons that die in the brain¿s substantia nigra. Muscarinic antagonists are frequently used to treat PD, after the primary dopa therapy diminishes. But these cause adverse side effects. If we could pinpoint that specific brain receptor on which these muscarinic antagonists exert their beneficial effects in PD, we might be able to develop such selective drugs, and get rid of those side effects.¿

Jointly with the [Eli] Lilly Research Laboratories, the NIH team tested this concept in PD-mimicking knockout mice, Now they are seeking a balance between dopamine and acetylcholine in that brain region, where an imbalance ¿is the underlying evil in Parkinson¿s disease¿ (Aug. 31).

Rhesus monkeys given certain toxic brain chemicals can closely duplicate the irreversible neuronal and motor-control symptoms of PD. Neuroscientists at Harvard University injected dopamine-secreting fetal pig neurons into these primates¿ brains

As a rule, the mammalian immune defenses dispatch killer molecules of the complement cascade system to terminate such intruding foreign cells. The Harvard investigators sidetracked this immune rejection threat by genetically engineering their xenotransplanted porcine brain cells to express a protein (provided by Alexion Pharmaceuticals Inc.) that inhibits the recipient¿s complement system. They dosed the PD-mimicking primates systemically with Alexion antibodies, which clobbered those cell-killing complement molecules.

¿This study,¿ the team leader observed, ¿represents the first successful engraftment of pig neurons into primates¿ (Oct. 26).

Huntington¿s Disease

Far less common than AD or PD though by no means rare Huntington¿s disease (HD) is far more insidious. Its devastating mix of dementia and uncontrollable movements kick in only after its victim has passed his young adulthood. Not knowing earlier in life if he is afflicted with this inherited disease, he may well have married, and sired offspring who in turn won¿t know if HD looms in their future until its too late.

Although the huntingtin gene and the Huntingtin protein it expresses are both known, nothing is known of how that protein functions. At Harvard-affiliated Brigham and Women¿s Hospital in Boston, one neurosurgeon played a hunch that the brain neurons wasting away in HD die off by programmed cell death apoptosis. A prime perpetrator of apoptosis is a well-known enzyme called caspase-1. Having found that caspase also plays this role in other brain diseases stroke, amyotrophic lateral sclerosis (Lou Gehrig¿s disease), and head injury, he set about testing caspase-1 activity in a mouse model of HD.

These inborn time-bomb mice come into the world healthy, but begin to lose brain and body weight by 5 weeks to 7 weeks of age. At 9 weeks, HD mice start to stagger, shudder and have epileptic seizures, leading to death. By knocking out the cell-slaying caspase-1 gene with an inhibiting compound, the scientists delayed the animals¿ symptoms and demise, ¿suggesting that this family of drugs would be useful for the treatment of human HD a disease for which at present there¿s no effective therapy¿ (May 20).

Drug Addiction

Unlike the above neurological diseases, addiction to drugs of abuse from alcohol and nicotine to amphetamines, heroin and cocaine is in a sense a self-inflicted disorder. Smokers hooked on nicotine struggle to get off the hook by buying into an expensive cafeteria of treatments, from nicotine patches and chewing gum to group therapy. A majority of these wannabe quitters stay addicted.

Fruit flies aren¿t addiction-proof, either. Biologists at the University of Virginia in Charlottesville found that genes involved in the insects¿ circadian body clocks have non-circadian responses to cocaine. Flies as well as animals given repeated doses of the psychostimulant developed an enhanced reaction to the drug; they got sensitized to it. ¿The importance of sensitization,¿ the scientists point out, ¿is that it¿s been linked to the addictive process in humans, as well as potentially to diseases such as schizophrenia.¿

To study mutated strains of the fruit flies¿ body-clock genes, they exposed the insects to repeated doses of volatalized freebase that is, crack cocaine, then measured their behavior by videotape. That data suggested that those genes may incite susceptibility to cocaine addiction. That is, the mutated ones did not become sensitized to the drug; the functionally intact ones did. The Virginia scientists¿ overriding conclusion is that ¿addiction is likely to be a disease state rather than a criminal behavior.¿

¿This exciting new research,¿ commented the director of the National Institute on Drug Abuse, ¿has given us a clue to the specific genetic mechanisms that influence vulnerability to addiction. They could be the basis for predicting who is most at risk for addiction, and thus become a major aid in preventing this national health problem¿ (Aug. 13).

Molecular psychiatrists at Yale University in New Haven, Conn., think they may be coming close to an addiction remedy. It¿s based on a protein called deltaFos-beta, which is induced in the brain by chronic exposure to a drug of abuse, but not by acute exposure. To probe what role the protein plays relative to addiction, they made genetic mutant mice in which they could turn on the gene for deltaFos-beta in the same neurons where cocaine induces it. These in vivo experiments confirmed that deltaFos-beta does bring on relatively long-lived sensitization that is, addiction.

They point out that the protein ¿can be targeted for the development of novel anti-addiction drugs¿ (Sept. 21).

Not all drugs of abuse are illicit illegal. Alcohol (ethanol) and nicotine are licit, but can and do become addictive. Thus, nearly one in 13 adult Americans are ethanol-dependant, that is, alcoholics, at a social cost of more than $167 billion dollars annually. And male alcoholics outnumber females 4 to 1. This statistic points a finger at the male-only Y chromosome in the human genome. In fact, population geneticists at NIAAA, the National Institute of Alcohol Abuse and Alcoholism, point out that ¿some forms of alcoholism are nearly totally sex-limited. And the Y chromosome is the only genetic element that fathers, not mothers, bequeath to their sons, not daughters.¿ Armed with this chromosomal smoking gun, the NIAAA scientists set out to see if a tendency to alcohol abuse is also passed down with the Y.

To map these variant markers on the Y chromosomes of men possessing putative genetic traits for alcoholism called for a highly uniform human population. The team found it in Finland. All impulsive criminals in that country tended to have alcohol problems. DNA analyzed from 359 adult male Finnish criminals and their families found that the Y chromosomes did affect their addictive vulnerability. Next the binational task force will try to localize the specific Y genes involved (April 9).

Spinal Cord Injury

A severed spinal cord, if not fatal, generally sentences its victim to a wheelchair for life. Trying to repair these neural life-lines is a major focus of neurosurgery. At the Harvard-affiliated Children¿s Hospital in Boston, current hopes are pinned on a small molecule named inosine, which occurs in most cells of the body. It¿s a purine nucleoside, indirectly implicated in DNA and RNA synthesis.

Neurosurgeons at the hospital report repairing an experimental spinal-cord wound that interrupted commands between nerve cells in the brain¿s cerebral cortex down axons the length of the spine to control lower-limb movements in rats. In humans, these axons activate the muscles of fingers, hands, legs and feet. ¿Somehow,¿ the lead investigator said, ¿using inosine, we were able to get the axons to revert to a growth state where they think they¿re kids again, and start doing again what they did back in their developmental youth, which was growing axonal branches. After 14 days, all but one of the 14 rats treated with inosine showed signs of collateral sprouting of axons from the uninjured to the injured side of the corticospinal tract. The number of new axons ranged up to 2,500 per treated animal, compared to 28 to 170 seen in controls.¿

Boston Life Sciences Inc., which partially funded this work, plans clinical trials of inosine late in 2000, to treat stroke and spinal cord injury (Nov. 10).

And four African green monkeys with surgically severed spinal cords received successful xenotransplants of pig cells specialized in sheathing axons with myelin. This feat was performed at Yale University in New Haven, Conn., jointly with Alexion Pharmaceuticals Inc. ¿The pronounced survival and myelination observed in the lesioned primate spinal cord,¿ the team pointed out, ¿offers hope for the potential application of this approach in spinal cord injury patients.¿ (Oct. 26).

OTHER DISORDERS

Cardiovascular

Angina pectoris is a sharp twinge of pain down the left arm, across shoulders, abdomen and backbone. It is nature¿s heads-up warning that the patient¿s heart is gasping for breath that is, oxygen. In a healthy person, coronary arteries deliver oxygen-rich blood to the heart. In atherosclerosis, those vessels get clogged with clots of atherosclerotic plaque. Those blockages can lead to ischemia and death from heart attack, unless the arteries are reopened, or replaced by bypass surgery.

Cardiologists have noted that patients who suffered an episode of angina 24 to 48 hours prior to the heart attack did better than those who did not. This preconditioning effect decreased deaths in hospital, and the reprieve lasted five years. Scientists at the University of Louisville in Kentucky sought in 321 mice the basis for this counterintuitive phenomenon.

To mimic the preconditioning phase, they alternately tightened and loosened sutures tied around the animals¿ coronary arteries, causing ischemia. First of all, they found that mice endowed with a gene encoding inducible nitric oxide synthase (iNOS) which generates NO were protected from heart attacks by these brief ischemic episodes. NO is responsible for preconditioning. Animals lacking the iNOS gene were completely defenseless. The Kentucky cardiologists are now working to develop therapeutic drugs based on this finding (Sept. 29).

Diets rich in fats, sugar, and above all cholesterol, are prime risk factors for atherosclerosis, the main cause of heart disease. Now a team of French cardiovascular researchers add two new risk factors to that list namely, bacteria and viruses, which abound in the ambient environment. The scientists, at France¿s National Institute of Health and Medical Research (INSERM) in Paris, fed two cohorts of mice the same high-cholesterol and cocoa-butter chow, guaranteed to bring on atherosclerosis. One group inhabited germ-free cages supplied with filtered air; the other half lived in habitats open to their surroundings, hence, exposed to airborne pathogens.

The team noted that the open-air animals¿ immune systems sent anti-inflammatory cytokines, specifically, interleukin-10, to counteract the infections that hit them. They then constructed a tribe of 14 knockout mice prone to atherosclerosis, but unable to synthesize IL-10. As the animals¿ arterial plaques grew, they injected mice with vectors delivering the IL-10 gene. After 16 weeks on the clot-promoting diet, IL-10-minus mice housed in pathogen-free cages had lesions on their aortas three times larger than did similarly caged IL-10-plus animals. But the IL-10-minus ones housed in open-air cages had a 30-fold increase in lesion size.

The researchers concluded that ¿IL-10 inhibits many cellular processes that could play an important role in atherosclerotic plaque progression. Our data may open the way for identifying novel therapeutic strategies to combat this common and fatal disease¿ (Oct. 25).

Hemophilia

When a boy born without the blood-clotting Factor VIII incurs severe bleeding episodes, he receives transfusions of the missing protein. But his immune system has never seen Factor VIII, so it duly sends antibodies to attack what looks to it like a foreign invading molecule. Recent data show that 25 percent of hemophiliacs suffer this grim practical joke of nature. It¿s the bane of physicians who treat the disease, and a tricky challenge to research hematologists and immunologists.

Belgian scientists at the University of Louvain created mice lacking the murine Factor VIII gene. Then they injected 13 of the baby hemophilic animals with a gene-therapy vector that delivered the human Factor VIII gene to their livers, which manufactures the coagulation protein. Six of the mice expressed ¿physiologic or higher¿ levels of the immunologically alien human factor. Five of the rescued murine hemophiliacs survived a usually lethal bleeding test in which 1-centimeter tips were clipped from their tails. Two of these success-story animals have retained their human clotting factor expression for more than 14 months. But six of the seven mice that failed to express the gene developed anti-Factor VIII antibodies (Sept. 7).

In June 1999, a 50-year-old retiree named Don Miller became the world¿s first hemophilia patient to receive gene therapy for his disease. At the University of Pittsburgh, an extra-short needle injected a vector-driven Factor VIII gene sequence into a vein on his hand. The Phase I/II trial was followed a day later by a similar gene therapy treatment for Factor IX clotting factor deficiency on a patient at Children¿s Hospital in Philadelphia. This study plans to enroll nine to 12 participants, with an interval of several weeks between each, to monitor any problems.

Because even a stubby hypodermic needle can provoke uncontrolled bleeding in these hemophilic patients, each received a prior injection of Factor VIII and IX, respectively, which raised their blood levels transiently to 100 percent of that of healthy individuals (June 15).

Infertility

The latest upgrade of in vitro fertilization (IVF) is ICSI intracytoplasmic sperm injection. This maneuver gets around male infertility (literally) caused by sperm deficiency owing to deletions on the male-only Y chromosome. A loophole in those subnormal sperm counts enables urologists to fish out individual sperm cells from upstream in the testis. ICSI specialists then microinject them into individual ova in vitro, and insert this single-cell embryo into the mother¿s fallopian tube to launch pregnancy.

The technique carries one built-in uncertainty: If the desired offspring is a boy, will he inherit his father¿s infertility? The jury is still out, but the answer seems to be ¿yes.¿ In which case, a molecular geneticist at the Massachusetts Institute of Technology proposes that between puberty and early adulthood, men with the Y-chromosome deletion may produce normal amounts of sperm. ¿In that case, the physician might consider harvesting the boy¿s sperm when he¿s younger, and save it until he¿s ready to start a family¿ (July 6).

In female infertility, one little-noticed culprit turns out to be the thymus gland. This small organ in the lower neck and upper chest serves as a finishing school for immune-system antibodies. During pregnancy, the thymus shrivels to a fraction of its normal size, presumably to prevent excess antibodies newly formed in the bone marrow from attacking the growing fetus as a foreign invader. Receptors for the progesterone hormone a master molecule of female reproduction have the chore of whittling the thymus to a nubbin as pregnancy gets going.

At Baylor College of Medicine in Houston, molecular endocrinologists grew mice lacking the progesterone receptor (PR). They noticed that in these PR-minus knockouts, the thymus didn¿t regress under the influence of pregnancy. They concluded that PR is a must for fertilization and delivery of the fetus. These scientists suggest that ¿our findings are definitely a major part of the fetal immune tolerance, and therefore it¿s quite likely that this is a pathway that would be defective in the 10 percent or so of women who have fertility problems. That¿s a diagnostic potential for fertility clinics (Oct. 19).¿

OTHER ADVANCES

Stem Cells

Well over 200 victims of Parkinson¿s disease (PD) have received brain transplants of human fetal nerve cells. The scarcity of such tissues besides ethical and legal scruples drives the search for alternative sources of such replacement neurons. A team of Swedish-American neuroscientists, partnered with two U.S. biotech companies CytoTherapeutics Inc., of Lincoln, R.I., and Geron Corp., of Menlo Park, Calif. report progress in this quest.

As their starting material, they obtained forebrain tissue from two human embryos. From these samples, they cultured neural stem cells to yield at least 10 million copies. Then the team injected 100,000-cell aliquots into the brains of 20 adult rats. Their cerebral targets centered on areas of the hippocampus and striatum, which are critical to motor and cognitive functions lost in PD and other neurodegenerative diseases.

After settling in for several weeks, the implants showed ¿a growth rate similar to other human progenitor cell cultures.¿ The authors concluded that ¿this culture system may provide an almost unlimited source of human neural progenitors, as an alternative to primary embryonic brain tissues for intracerebral transplantation¿ (Aug. 2).

What neural stem cells are to the brain, hematopoietic stem cells are to the blood. At Thomas Jefferson University in Philadelphia, a pioneer hematologist and biochemist explained: ¿The pluripotent stem cell is the lifetime source of all blood cells. They differentiate into progenitor cells, which in turn form the various terminal cells carried by the blood vessels.¿ He and his colleagues nailed down the elusive hematopoietic stem cell in vivo first in mice, then in fetal sheep. ¿Stem cells,¿ he pointed out, ¿produce an enormous amount of blood cells during our lifetime. What we need to do is make them replicate in the laboratory, then differentiate down to the terminal cells, which would be utilized for clinical blood transfusion¿ (Sept. 8).

GENOME SEQUENCING

How many genes in a human genome? Estimates keep rising; the outside guess has now reached 140,000. An international DNA-mapping consortium hopes to complete sequencing that genome in the next few years. A U.S. company, Celera Genomics, announced its intention to finish the job by the end of this year.

By the end of last year 1999 the consortium had taken a first bite out of that enormous human genome: It sequenced human chromosome 22 all 33.4 megabases of it (Dec. 13).