In mice, similar cultures have allowed scientists to make genetically
engineered animals with unprecedented precision. The key is that the cells are
“pluripotent”—they can divide and differentiate to form any tissue type.
Working with a culture of these “embryonic stem cells” held in an
undifferentiated state, geneticists can target genetic manipulations with much
greater precision than would be possible by the haphazard injection of genes
into newly fertilised mouse eggs. They can then inject the genetically
engineered stem cells into an early mouse embryo. This develops into a
“chimeric” mouse in which a proportion of cells in most or all of its tissues
carry the altered genes (see
Figure).

Because those tissues can include the cells that give rise to sperm and eggs,
repeating these experiments in humans would break the biggest taboo in modern
genetics: manipulating the human germline to induce genetic changes that can be
passed down the generations.

The researcher who created the human cell cultures, John Gearhart of Johns
Hopkins University in Baltimore, Maryland, stresses that altering the germline
is not his goal. He hopes that the cells will find a use in conventional gene
therapy, in which the altered genes cannot be inherited, or in creating tissues
that could be used for grafts without the need for drugs to combat rejection. “I
believe what comes out will be extremely beneficial,” he says.

Last week, at the 13th International Congress of Developmental Biology in
Snowbird, Utah, Gearhart revealed that he now has seven separate cell lines
growing in culture.

To create the cultures, Gearhart took cells from fetuses roughly eight weeks
into gestation, which had been aborted at a clinic in Baltimore. The cells came
from a structure called the gonadal ridge, which would have developed into
reproductive organs. As such, they are different from most of the embryonic stem
cells used by mouse geneticists, which usually come from earlier embryos.
However, Gearhart believes his cells have many of the same properties.

One possibility, however, is that Gearhart’s cells grow in culture in an
undifferentiated state not because they are pluripotent, but because they are
cancerous. But Gearhart notes that “marker” proteins carried on the surface of
his cells suggest that they are pluripotent. They can also form clumps of cells
that, in mouse cell cultures, are precursors to further development.

The ultimate test of pluripotency would be to inject the cells into an early
human embryo and show that a healthy human chimera develops—an experiment
that few scientists would regard as ethical. In the absence of these data,
however, other scientists say they are inclined to believe Gearhart’s claim.
“The science is solid and the chromosomes look fine,” says Peter Gruss, a
developmental biologist at the Max Planck Institute for Biophysical Chemistry in
Göttingen, Germany.

Gearhart’s cells could find applications in conventional gene therapy. Gruss
says that experiments with mouse embryonic stem cells show that they can
differentiate into the tissues that give rise to blood cells. If the same is
true for Gearhart’s human cells, it may be possible to use them to treat people
with genetic diseases of the blood.

Gearhart’s main goal is to create cells that could be grown to form tissues
suitable for grafts. If the cells were manipulated to remove genes of the major
histocompatibility complex, which play a central role in the recognition of
foreign tissue by the immune system, this would help solve the problem of
rejection. “These cells would be the best donors,” he claims. But at the back of
people’s minds is the possibility that human embryonic cells could be used to
manipulate the germline. This would raise serious ethical concerns, says David
Shapiro of the Nuffield Council on Bioethics in London, as genetic changes would
be passed down to future generations without their consent.

However, Anne McLaren of the Wellcome Cancer Research Campaign Institute in
Cambridge, who attended the Utah meeting, questions whether anyone would attempt
human germline manipulation. “No one in their right mind would want to do it,”
she says. McLaren notes that couples carrying genetic diseases can instead have
their embryos screened through pre-implantation diagnosis and select a healthy
embryo to take to term.

Mario Capecchi of the University of Utah in Salt Lake City, who pioneered the
use of embryonic stem cells to create genetically engineered mice, says that
using genetic manipulation to develop cells for transplantation would be
acceptable. “But as soon as you do it to create a human being, you’ve crossed
the line.”

For years scientists have tried to understand how three-dimensional branched
structures form in developing organisms. During the 1980s, some tried to develop
mathematical models for the patterns they saw in nature, and a few even came up
with sets of equations to describe them. But these descriptions were based on
simplistic assumptions.

Now Mark Krasnow and his colleagues in the biochemistry department at
Stanford University in California have identified a gene in the fruit fly,
drosophila, that tells the developing tracheal cells exactly where to branch.
The gene—called branchless because mutant fruit flies lacking a
healthy copy have unbranched air sacs—makes its protein only at specific
places outside the tracheal system and, instead of directing the tracheal cells
from within, sends them a “come-hither” signal that tells them where and in
which directions to branch.

The discovery of branchless provides developmental biologists with the
missing piece to a jigsaw puzzle. They knew that, during the early development
of a fruit fly, a grid is laid down in two dimensions by a number of
well-studied genes. But how 3-D structures such as the tracheal system grew
within this grid remained a mystery.

Five years ago, researchers led by Ben-Zion Shilo at the Weizmann Institute
of Science in Israel discovered another gene, called breathless, that
they realised must play an important role in branching. Shilo and his colleagues
found that the protein encoded by breathless is a receptor on cell
surfaces for a molecule known as fibroblast growth factor (FGF), which is known
to serve as a molecular signal for the development and patterning of vertebrate
organs. It still wasn’t clear, though, how the branching system was
orchestrated.

The work by Krasnow and his team ends the confusion. It turns out that the
protein encoded by branchless is FGF itself—the key to fit the
lock. Both proteins are needed, but without branchless, which is
expressed only at specific points, no branching takes place (Cell vol
87, p 1091).

Krasnow’s group screened fruit flies for mutations that prevented tracheal
branching. These mutants had long, unbranched air sacs instead of a complex
filigree of branches, and they died before they had matured beyond the larval
stage. In healthy flies, the researchers found pockets of FGF close to their
tracheal cells.

Krasnow believes that branching mechanisms may have been conserved across a
wide range of species through evolution, so he thinks it plausible that humans
and other vertebrates use similar mechanisms.

The discovery that a single cue can guide the formation of so many branches
is “really exciting”, says Shilo. “Evolution works by tinkering with the same
components in different systems. What is surprising is that not only the
building blocks of the pathways are conserved, but the use of the pathways is
similar in very diverse organisms.” So it would not be surprising, he says, if
the branching of vertebrate lungs and circulation were controlled in a similar
way.

The syndrome is named after the New York dermatologist David Bloom, who first described its symptoms in 1954. Babies with Bloom’s syndrome are born perfectly proportioned but tiny – weighing as little as 0.9 kilograms, even though they are not premature. They rarely grow taller than 1.5 metres and typically die in their teens or twenties from cancers including leukaemia, breast, colon and liver cancer. This susceptibility to cancer is due to frequent chromosome breaks and rearrangements. Bloom’s syndrome affects fewer than 180 people worldwide.

By studying how the syndrome is inherited, researchers tracked down the gene responsible to a region of chromosome 15. That was three years ago; now, a different team including James German and Nathan Ellis of the New York Blood Center and their colleagues at the University of Cincinnati Medical Center, has found a gene within this region that is mutated in people with Bloom’s syndrome (Cell, vol 83, p 655).

The gene, called BLM, contains a sequence similar to one found in a family of bacterial and yeast genes that code for enzymes called RecQ helicases. These enzymes help to unzip double-stranded DNA so that it can be read, copied or repaired. Having examined BLM’s sequence, the researchers believe the enzyme it encodes is used whenever DNA is copied. But its precise role is still unclear, admits Joanna Groden, a member of the team from Cincinnati.

If the researchers can unravel the enzyme’s normal function, and learn why the mutated version allows chromosomes to break and become rearranged, it may be possible to gain insights into the origin of many forms of cancer. “The implications of this discovery will go far beyond those few people with Bloom’s syndrome,” says Richard Kolodner of the Dana Farber Cancer Institute in Boston.

As soon as probes that recognise the mutated form of BLM become available, researchers will be able to determine whether common cancers associated with chromosomal rearrangements – including some forms of leukaemia – are due to sporadically occurring defects in the BLM gene. Eventually, Groden speculates, such tests could also be used to identify tissue that is at risk of becoming cancerous.

All vertebrates show left-right asymmetry, with the heart, spleen, liver and other internal organs forming preferentially on one side of the body. How this asymmetry arises is one of the biggest mysteries in developmental biology. But with two important genes now identified, it may be possible to reveal the molecular pathways involved. “These are the first genes ever described with left-right asymmetry,” says Lewis Wolpert of University College London. “It’s a terrific discovery.”

Developmental biologists have long expected left-right asymmetry to have a genetic basis. Just over 1 in 10 000 people are born with their heart and other organs on the “wrong” side, a genetic condition known as situs inversus. Two years ago, researchers at Baylor College of Medicine in Houston, Texas, stumbled on a clue when they found that mice carrying an additional gene on chromosome 4 developed situs inversus. The added DNA seemed to disrupt a gene controlling left-right asymmetry. But so far the Baylor team has been unable to find the gene involved.

Tabin, Levin and colleagues also made their discovery by accident. They noticed that a protein called Sonic hedgehog, named after the computer game character, was produced asymmetrically in chick embryos about one day after fertilisation, before any anatomical differences between left and right became visible. Biologists call this protein Sonic hedgehog, because it is related to a protein which, if missing, turns fruit fly embryos spiny. Meanwhile, Claudio Stern of Columbia University in New York found a second asymmetrically produced protein in similar-aged chick embryos called activin receptor IIa. This suggested that the body’s left-right asymmetry is in part caused by the asymmetrical activation of the genes encoding these two proteins, called Shh and cAct-RIIa respectively.

To test this idea, the researchers masked this protein asymmetry by adding larger quantities of either Sonic hedgehog or activin receptor IIa to the opposite side of early chick embryos. With either protein, this caused the heart to develop on the left side in half the chicks and on the right in the others (Cell, vol 82, p 803).

Both Sonic hedgehog and activin receptor IIa are signalling molecules, which are produced by certain cells in the developing embryo and cause other cells in their vicinity to grow, migrate or differentiate. Sonic hedgehog controls the formation of patterns in structures as diverse as insect wings, vertebrate limbs and the spinal cord. Activin receptor IIa was first identified in frog embryos as the key molecule instructing cells to form an important layer of tissue called the mesoderm.

The researchers have not yet shown that the same signals influence the development of other asymmetric organs such as the liver and intestines. But Tabin expects that these genes or their close relatives will be involved. He and Levin have already shown that a related gene, cNR-1, is activated asymmetrically not only in pre-heart tissue but also in tissue that forms other organs.

One puzzle is that researchers studying the Shh gene in other vertebrate embryos, such as those of mice and zebrafish, have found no evidence that it is more active on one side of the body than the other. Tabin speculates that this may be because the asymmetric protein production happens for only a short time, and is therefore easy to miss. Alternatively, there might actually be a different mechanism controlling asymmetry in these organisms. Wolpert believes the former explanation. “Evolution is lazy,” he says. “Once it’s got a good technique, it sticks with it.”

The Harvard researchers now hope to identify even earlier developmental signals that bring about the asymmetric activity of the Shh and cAct-RIIa genes. “The real question is, what starts the process?” says Tabin. Many developmental biologists believe that it will be possible to trace left-right asymmetry back to a “handed” molecule that becomes tethered in a fixed orientation relative to the developing embryo’s top-bottom or front-back axis.

American researchers led by Ron Chesser of the University of Georgia’s Savannah River Ecology Laboratory examined the DNA in mitochondria, the energy-producing bodies in the cell cytoplasm. They found 46 mutations in just one gene, the cytochrome b gene, in nine voles taken from the 30-kilometre restricted zone around Chernobyl. When they examined 10 animals from outside the “hot zone”, they found four mutations.

Chesser says he was surprised by the sheer number of mutations. There are “many more mistakes than what was thought to be feasible in a thriving population”, he says. The study raises questions for geneticists about how many mutations a population can tolerate without dying off, he says.

The results also have implications for humans. The Atomic Bomb Casualty Commission spent years looking for any kind of genetic damage in the survivors of the Hiroshima and Nagasaki bombs, says Christopher Wills, an evolutionary geneticist at the University of California in San Diego. They did not report much beyond stillbirths and some cases of cancer, he says. James Crow, a member of the commission’s advisory committee says he has no doubt that genetic changes were induced by the radiation from the bomb “but they were below the threshold of detection”. The new data may help researchers to home in on changes in people exposed to radiation at Chernobyl.

Researchers cannot agree on the natural background rate at which mutations appear in mitochondrial DNA. But even choosing a conservative rate – of, say, 1 in 100 000 per gene per generation – the voles from the contaminated zone have a mutation rate about 40 times that of the background rate, says Chesser. The rate was found to be high enough to cause mutations from one generation to the next.

The team is looking at other genes in mitochondria and in the nucleus to see if the effects are widespread. It would be strange, says Chesser, “for us to have stumbled upon the only gene showing this kind of alteration”.

As long as scientists remain hidden in the reassuring tall grass of the laboratory, they are protected by their jargon and their shared world view. Upon entering the exposed world of popular science writing, however, scientists are faced with a dilemma: how to create an entertaining and educational road map of their discipline.

There are several possible solutions to this problem, but unfortunately Isenberg and Morrow, a rheumatologist at University College London and an immunologist at the Medical College of St Bartholomew’s Hospital, University of London, have chosen to lead us halfway into the thickets of their terminology and leave us there. The result is a barely translated medical text at least two removes from the living, breathing reader.

The topic is not to blame. Autoimmune diseases, which include rheumatoid arthritis, lupus, multiple sclerosis, and some forms of diabetes, affect as many as 1 in 20 people. Sufferers often spend many years struggling with symptoms ranging from painful, swollen joints to gradual, grinding paralysis. Plenty of drama there. And modern biology has come up with a number of promising if barely tested treatments, such as swallowing small amounts of proteins, that could dramatically alter the lives of patients. But these biotechnology-based approaches are relegated to a few pages.

The book’s flaws begin with its structure. Chapter 1 (of six) asks, “What exactly is the immune system?” and Chapter 2 asks, “How does it actually work?” The authors answer with seemingly endless lists of the complex immune system’s many components. The third chapter addresses the factors involved in autoimmunity and the fourth explores how the diseases develop. A more sensible approach would have dealt with each topic disease by disease, since the average reader is likely to come looking for information on a particular illness.

Friendly Fire’s few high spots appear in the last two chapters. We learn, for instance, that the term “diabetes”, first used in the second century AD, means “to run through a siphon”, a reference to the copious amounts of urine produced by diabetics. And rheumatoid arthritis, the authors report, is relatively recent in origin, at least in Europe. Some even believe that early explorers brought it back from the New World, and others claim that Botticelli’s Venus bears the swollen finger joints characteristic of the disease.

In those last two chapters, the authors finally address particular diseases, their symptoms, treatments and complications. Among the most useful features is an informative, if noncomprehensive, list of specific drugs and their side effects.

But the preceding chapters are slow going, as in this typical passage: “Complete or even partial absence of the second component (C2) of complement is a major risk factor for developing autoimmune disease, in particular systemic lupus erythematosus and myositis.” Missing is any explanation of how the reader can use this information, nor is there a clever analogy to explain why this fact interests the physician or basic researcher.

Even worse, the book’s early chapters read like a bad Russian novel: endless characters with long names and no clear reason to be included. Macrophages, eosinophils, T cells and cytokines all make an appearance, and we are apparently expected to memorise their significance until they reappear several chapters later. The guiding, explaining and hand-holding that goes on in a typical żěè¶ĚĘÓƵ feature is sorely lacking.

Other immunologists, however, warn that the mystery of exactly how HIV causes immunodeficiency may be as impenetrable as ever. It is extremely difficult, they point out, to differentiate between these “naive” cells and the “memory” cells that respond to pathogens the body has encountered before. This means that the findings may be much less clear-cut than the Stanford researchers believe.

Roederer and his colleagues have been studying the immune system’s T cells. Naive T cells patrol the blood in search of the particular foreign molecule, or antigen, they are primed to recognise. The immune system generates an almost endless variety of such cells that recognise different foreign molecules – although most of the T cells never encounter their antigen. Those that do, however, trigger an immune response and turn into memory cells – so named because they remember the invader that carried the antigen. This memory can last for decades, as the cells divide through successive generations, enabling the immune system to react rapidly to the pathogen, should it invade again.

The Stanford researchers counted the naive and memory T cells in the blood of 285 people who had been infected with HIV for varying periods of time. Until now, most AIDS researchers have concentrated on the steady decline in numbers of one of the main sub-types of T cells, called CD4 cells, that occurs in HIV-positive people. But in the May issue of the Journal of Clinical Investigation (vol 95, p 2054 and 2061), Roederer and his colleagues claim that behind this general change lurks a more subtle shift in the relative proportions of naive and memory cells.

Healthy adults have equal numbers of naive and memory cells in both the CD4 sub-type and another class called CD8 cells, which kill other cells that are infected with an invading pathogen. Roederer found that naive T cells seem to decline steadily in both cell classes from the early stages of HIV infection. In patients with AIDS, the proportion of naive cells dropped to less than 30 per cent for CD4 cells, and to around 12 per cent for CD8 cells.

The loss of naive CD8 cells was especially surprising, because the total number of CD8 cells initially increases after infection with HIV, as the body battles against the virus. The new finding changes the whole picture of T cell loss in HIV infection, claims Leonore Herzenberg, who with her husband Leonard heads the lab in which Roederer works. “Measurements lumping together all types of CD8 cells failed to resolve the gross imbalance within that population,” she says.

Since the body relies primarily on naive T cells to ward off new infections, the Stanford researchers argue that the disappearance of these cells may be what eventually leaves HIV-infected people defenceless against new pathogens. “Immunodeficiency may result because the naive cells are no longer there,” says Roederer.

Other researchers are sceptical. “This study will help explain the mechanism by which immuno deficiency develops, but whether it has truly found the heart of the problem is not clear,” says Jonathan Kagan, an immunologist at the US National Institute of Allergy and Infectious Diseases in Bethesda, Maryland.

The new results are difficult to interpret, say immunologists, because naive and memory cells are difficult to tell apart. This is done by examining “marker” molecules carried on the surface of T cells which change as naive cells turn into memory cells. The Stanford researchers studied three markers, none of which individually is 100 per cent reliable: naive cells can be made to look like memory cells just by adding proteins that stimulate the cells to divide rapidly – a phenomenon called immune cell activation. Using cell surface markers to separate naive and memory cells, says Kagan, “is like defining me as a liberal because I have long hair”.

Nevertheless, if the Stanford researchers are correct, trials of new treatments for HIV and AIDS are ignoring important information. Researchers testing drugs or therapeutic vaccines in people with HIV try to predict the treatments’ effectiveness by counting their patients’ CD4 cells; if these cells increase, researchers take this as a sign that a treatment may be working.

Roederer says people running clinical trials should routinely monitor their patients’ naive cells. This could be vital for interpreting the results of trials of therapeutic vaccines. To be effective the vaccines might need a certain number of naive cells. If too few naive cells are present, the immune response provoked by a therapeutic vaccine may be too inflexible to cope with HIV’s tendency to mutate. This means that even if the overall results of a trial suggest that a therapeutic vaccine is ineffective, this could be masking the possibility that the vaccine works well in a fraction of patients with higher numbers of naive cells.

MS is just one of many debilitating diseases caused by auto-immune reactions, where the immune system attacks the body’s own tissues. It afflicts about a million people worldwide, two-thirds of them women. People usually first notice the disease’s symptoms in their twenties when immune cells begin to attack the protective sheath wrapped around nerve fibres in the brain and spinal cord. People with MS may eventually lose control over their muscles after repeated cycles of remission and relapse.

Researchers have long speculated that viruses and other infectious agents could trigger MS. Many pathogens are thought to evade the immune system using a cloaking strategy called molecular mimicry. The immune system does not normally attack “self” proteins, so by producing protein fragments, or peptides, that imitate the structure of those in the body’s own tissues, viruses and bacteria may avoid destruction. But about ten years ago, biologists pointed out that this strategy could go awry with disastrous consequences: if the immune system is no longer fooled by a pathogen’s disguise, it could begin to attack both the mimicking pathogen and whichever of the body’s tissues it is imitating.

Proving this hypothesis has been very difficult, as singling out the cloaking peptides among the many thousands of peptides produced by a typical virus or bacterium is a time-consuming and thankless task. The Harvard researchers began by analysing the three-dimensional structure of the small region of the nerve cell sheath protein that is targeted by immune cells in MS patients. Because proteins need not have exactly the same sequence of constituent amino acids to fold up into roughly the same shape, the researchers then had to work out the range of sequences that would mimic the structure of this human peptide.

Once they had done this, Wucherpfennig and Strominger searched through two protein sequence databases and found 600 viral and bacterial sequences that would imitate the key nerve cell sheath peptide. After discarding those from microorganisms that do not infect people, or which only occur in the tropics, where MS is uncommon, the researchers were left with a panel of 129 microbial peptides. They then added these, one by one, to cultures of self-reactive immune system T cells taken from MS patients. These are the cells which attack nerve cell sheaths.

Most of the suspect peptides had no effect on the cultured cells, but seven viral peptides and one peptide from a bacterium made the T cells start dividing. This jump-started cell division is called immune cell activation and is an important component of both normal immune responses and autoimmune reactions.

Wucherpfennig and Strominger suggest that MS begins when a viral or bacterial peptide activates some of the potentially self-reactive T cells that are constantly circulating in the body, but which are usually kept in a suppressed, inactive state. Once activated, the researchers argue, these cells can breach the blood-brain barrier that normally keeps immune cells away from the nervous system. They will then attract the nerve sheath peptide resembling the microbial peptide that originally activated them.

Although researchers have long suspected that viruses or bacteria may cause MS, the identity of the infectious agents implicated by the new study is surprising. MS is a relatively rare disease, but the peptides identified by Wucherpfennig and Strominger come from viruses and a bacterium that are very common. The viruses include the influenza virus, the cold sore virus herpes simplex, and two types of common cold virus called adenoviruses and reoviruses. The Harvard researchers have also implicated two cancer-causing viruses: human papilloma virus, which can cause cervical cancer, and Epstein- Barr virus, which infects about 95 per cent of people and has been linked to several types of tumour. The bacterial peptide found to activate MS patients’ T cells came from Pseudomonas aeruginosa, a common inhabitant of human skin which can infect wounds.

Clearly, simply coming down with flu or a herpes infection does not automatically make a person develop MS, so other factors must also be involved. One such factor may be a genetic predisposition to the disease. żěè¶ĚĘÓƵs already know that the possession of certain immune system genes makes people more likely to develop MS, and it could be that these genes make people prone to autoimmune reactions when faced with microbial molecular mimicry.

Strominger stresses that more work needs to be done to prove that viruses and bacteria trigger MS, and to understand how these infectious agents interact with genetic factors. Nevertheless, he believes that the new research may eventually lead to strategies for preventing MS. “In cases where multiple sclerosis runs in families, one could identify children who have inherited high-risk genes, and give them preventive vaccines,” he says.