午夜福利1000集合

THEY are minuscule egg-shaped structures mere billionths of a metre across, dwarfed by the tiniest living cell and smaller than many viruses. They have a hard bony shell, replicate like a living organism and are wiped out by antibiotics and radiation, yet seem to lack DNA. Some say they are infectious microbes, possibly even an unknown form of life, able to cause diseases ranging from Alzheimer鈥檚 to atherosclerosis. Others say they are simply harmless crystals.

Welcome to the topsy-turvy world of nanobacteria. Once described as the 鈥渃old fusion of microbiology鈥�, the very existence of these microbes has been denied or ridiculed by mainstream opinion for nearly a decade and their proponents branded mavericks. Just like cold fusion, though, nanobacteria have refused to go away and now 鈥� under the new guise of 鈥渃alcifying nanoparticles鈥� 鈥� they are making a renewed bid for scientific respectability. The stakes are high. If diseases long thought incurable are actually caused by nanobacteria, they could be prevented with vaccines, or treated with antibiotics.

The story began in the early 1990s when Olavi Kajander, a biochemist at the University of Kuopio in Finland, was carrying out some important, if unexciting, work on cell-culture contaminants. In 1993 he reported finding a hitherto unknown contaminant in cow serum: tiny, self-replicating spheres which he tentatively called 鈥溾��. He and a colleague, Neva 脟ift莽ioglu, went on to find the same particles in the blood of cows and humans, as well as in supposedly sterile blood products. In 1997, the duo claimed to have sequenced a small piece of DNA extracted from the particles which proved they were a new type of bacterium.

In 1998 the rumpus kicked off. The trigger was a paper in which Kajander and 脟ift莽ioglu claimed not only that nanobacteria were alive, but also that they were the cause of kidney stones (, vol 95, p 8274). The pair had noticed that their nanobacteria often cocooned themselves in thick coats of calcium phosphate 鈥� the material in some types of kidney stone. So they examined 30 human kidney stones and found that all of them contained signs of nanobacteria. Putting two and two together, they proposed that these microbes were the cause of the stones.

To say this went against the grain is an understatement. Kidney stones are an example of 鈥減athological calcification鈥�, in which tissue becomes clogged with deposits of calcium phosphate. The root cause of this has long been a mystery, but it is widely considered to be a purely chemical process. Now here was a team arguing that it was caused by infection with a class of organisms that many doubted was even real.

That wasn鈥檛 all. Calcification is also linked with other chronic and often fatal diseases, including atherosclerosis (hardening of the arteries), cancer, dementia and arthritis. If nanobacteria could cause kidney stones, why not these diseases too? In a commentary in the same issue (p 7846), Dennis Carson of the University of California at San Diego proposed exactly that.

These ideas sparked an immediate uproar. Critics quickly pointed out that these supposed bacteria weren鈥檛 big enough to be alive. At between 50 and 100 nanometres across, they are widely considered too small to house the machinery a living organism needs.

Researchers began trying to replicate Kajander and 脟ift莽ioglu鈥檚 research, with scant success. The most convincing rebuttal came from a group led by microbiologist John Cisar at the National Institutes of 午夜福利1000集合 in Bethesda, Maryland. He successfully isolated nanobacteria-like particles from human saliva and propagated them in the lab. However, he was unable to detect any proteins or genetic material associated with them. What is more, the DNA sequence Kajander used to classify nanobacteria wasn鈥檛 new but belonged to a common contaminant of cell cultures. Cisar concluded that the calcification attributed to so-called nanobacteria was nothing more than crystallisation triggered by non-living particles (Proceedings of the National Academy of Sciences, vol 97, p 11511).

As far as most researchers were concerned, that was that. Kajander and 脟ift莽ioglu were branded mavericks and their funding slashed. But interest in nanobacteria did not diminish 鈥� it grew.

In the past few years, the tide has started turning in Kajander and 脟ift莽ioglu鈥檚 favour. Other researchers, most notably a team from the Mayo Clinic in Rochester, Minnesota, have replicated and extended many of the pair鈥檚 findings. Nanobacteria-like particles have been isolated from numerous diseased tissues, and several groups now share the view that they are the cause of calcification. There is even preliminary evidence that drugs designed to knock out nanobacteria can cure a disease associated with calcification.

The turnaround began in 2004, when the Mayo team, led by nephrologist John Lieske and physiologist Virginia Miller, published a report supporting many of Kajander and 脟ift莽ioglu鈥檚 early findings. They collected diseased arteries and heart valves from surgical waste and found that many contained spheres matching the description of nanobacteria. They went on to show that these spheres replicated in culture and stained positive for DNA (American Journal of Physiology 鈥� Heart and Circulatory Physiology, vol 287, p H1115).

The Mayo group also tried another method to interrogate the nanoparticles for signs of life. Called spectromicroscopy, this technique relies on generating an optical signature for microbes according to how they absorb and reflect light. The Mayo team compared the signature of the nanoparticles with those from two types of bacteria known to build calcium phosphate shells. They found clear similarities but, crucially, the nanoparticles lacked a DNA signature, though Miller says this is probably an artefact of the set-up. 鈥淭his was our first experiment,鈥� she says.

Perhaps unsurprisingly, this research failed to convince the hard-core sceptics. But it helped the atmosphere thaw a little. By 2006, nanobacteria were considered respectable enough for the organisers of the annual Experimental Biology meeting to invite Kajander, 脟ift莽ioglu and the Mayo team to San Francisco to discuss their research.

Alive without DNA?

At that meeting the Mayo team reported yet more evidence. They had cultivated nanobacteria from kidney stones and extracted DNA, RNA and four proteins that closely resemble other bacterial proteins. 鈥淚 think that [the proteins] would be evidence supporting that they are a microorganism,鈥� says Lieske cautiously, 鈥渂ut one can still argue that it is a contaminant, so it is not definitive proof.鈥� And the DNA? 鈥淪till inconclusive,鈥� says Lieske. The quantity they collected was minuscule, providing little material for further analysis. Lieske and his colleagues now aim to collect more DNA and sequence it. By culturing nanobacteria in different nutrient solutions, they think they can grow them without their bony shells, making it easier to extract any DNA that might be there.

Another unexpected boost came from the discovery of nanoscale microbes in Richmond mine, California. They were archaea rather than bacteria, but were smaller than the theoretical minimum size for living cells (, vol 314, p 1933).

Despite these advances, many observers remain unconvinced. One sceptic is David Relman, a microbiologist who heads the infectious diseases section at Stanford University. He says that none of Lieske鈥檚 assays are definitive tests for life, and that their positive results can all be explained by non-living processes.

So what would be definitive proof? 鈥淒NA or RNA,鈥� says Fredric Coe, a nephrologist at the University of Chicago. 鈥淲ith modern genomics, if you have really got important DNA, you are going to track it down. That鈥檚 what I think happens when you are onto something real.鈥� Coe also points out that the Richmond mine nanoarchaea were quickly sequenced, proving that size doesn鈥檛 matter when it comes to plucking out a genome. 鈥淭hose are really small and they yielded immediately to modern science,鈥� he says.

In the absence of a genome, the argument over whether nanobacteria are living or non-living will rumble on. Curiously, however, Kajander and 脟ift莽ioglu have washed their hands of the debate 鈥� at least for now. They have stopped using the word 鈥渘anobacteria鈥� in favour of the more ambiguous term 鈥渃alcifying nanoparticles鈥� or CNPs.

鈥淲e are aware that the name 鈥榥anobacteria鈥� is wrong,鈥� concedes 脟ift莽ioglu, now the science director of in Tampa, Florida, which she and Kajander set up in 2003. 鈥淲e don鈥檛 know if this is final but it seems like they don鈥檛 have genomic DNA.鈥�

Kajander stresses that the new name doesn鈥檛 mean they have abandoned the idea that the spheres are alive. 鈥淚 think that we are talking about a new form of primordial life,鈥� he says. 鈥淚t can鈥檛 be classified with the existing groups of life.鈥� Even so, he now considers the debate to be something of a sideshow. The real issue, he says, is whether the nanoparticles have a role in disease.

鈥淚 think that we are talking about a new form of primordial life鈥�

To that end, the past few years have seen a series of suggestive findings. Scientists in China, India, Turkey, Germany and the US, some of whom have ties to Nanobac, have found CNPs in gallstones, kidney stones, diseased coronary arteries and certain tumours. There is also some early, though controversial, evidence that pathological calcification can be cured with antibiotics.

In 2003, Kajander was carrying out an early clinical trial involving 13 patients with a range of afflictions including coronary artery disease, rheumatoid arthritis and chronic fatigue. All had tested positive for CNP markers, and the trial aimed to find out whether a drug cocktail based on the antibiotic tetracycline could reduce these markers.

A few of the patients had a condition called chronic pelvic pain syndrome, which is caused by inflammation of the prostate. CPPS is hard to treat but, surprisingly, the drug cocktail seemed to help. This led Kajander to suggest that CPPS was related to calcification of the prostate, caused by nanobacteria. The data has not been published in a peer-review journal, but is included in a 2006 .

He took the idea to urologist Daniel Shoskes, a CPPS specialist at the Cleveland Clinic Florida in Weston. Prostate calcification is often seen in patients with CPPS but is considered irrelevant to the disease, so Shoskes was doubtful. 鈥淎 lot of older men have calcification,鈥� he says. 鈥淚t鈥檚 always considered a normal consequence of ageing and doesn鈥檛 have any role in the symptoms.鈥� Even so, Shoskes agreed to test Nanobac鈥檚 drug. He gave it to 15 CPPS patients who had failed all other therapies and found that after three months, 12 of them had improved significantly (Journal of Urology, vol 173, p 474). Shoskes describes the result as 鈥渁n interesting observation鈥� but says a full-scale clinical trial is needed. Nanobac is moving in this direction: in January the company met the FDA to set the wheels in motion.

Shoskes, now at the Glickman Urological Institute in Cleveland, Ohio, has since joined Nanobac鈥檚 medical advisory board and is still using the drug. 鈥淚t doesn鈥檛 help everyone but continues to help a lot of people,鈥� he says. He is also studying prostate calcification to see whether it is associated with CPPS.

So far so good, but this still doesn鈥檛 answer a key question: are CNPs involved?

To find out, the Mayo team has embarked on another round of experiments, this time funded by Nanobac. In April, they reported preliminary results at the Experimental Biology 2007 meeting in Washington DC.

Team member Maria Kraemer harvested nanoparticles from human aneurysms and injected them into adult male rabbits whose carotid arteries had been stripped of their cellular lining to simulate the damage seen in early atherosclerosis. After 35 days the rabbits鈥� carotid arteries were badly calcified and had significantly narrowed compared with controls. This is at least preliminary evidence that CNPs contribute to atherosclerosis, says Kraemer. Lieske is also looking for signs of kidney stone formation in those same rabbits.

Research into CNPs has come a long way since 1998. Even arch-critics are now prepared to accept that the spheres exist and can replicate. Even so, supporters have more to do.

One unanswered criticism is that CNPs are so common in human tissue that you could blame them for almost everything. 鈥淚t would be like saying that oxygen is the cause of disease,鈥� says nephrologist David Goldfarb at the New York University Medical Center. In 2004, Goldfarb reviewed the evidence for the role of nanobacteria in kidney stone formation (Nephron Physiology, vol 98, p 48). 鈥淭he ubiquity also allows a sweeping, amazingly broad theory that this is the cause of calcification in every organ,鈥� he says.

Cisar, too, is sticking to his guns. Not only is he convinced that CNPs are not alive, he doesn鈥檛 see any new evidence that they cause disease. He believes that calcification is not the cause of diseases such as atherosclerosis, but an effect of the underlying disease process.

Coe agrees. He studies kidney calcification with transmission electron microscopes and says he has never seen any sign of CNP involvement. 鈥淵ou see gorgeous structures 鈥� the more you magnify, the stranger and more beautiful they are 鈥� but we know exactly what they are: they are crystallisation,鈥� he says.

Cisar is also openly suspicious of Nanobac. 鈥淥ne has to acknowledge that there are commercial interests here,鈥� he says. 鈥淭hese people are selling a product.鈥� 脟ift莽ioglu retorts that going into the private sector was the only way to continue the research. 鈥淲hen the only funding comes from investors who believe in your science, you take this opportunity,鈥� she says. 鈥淲ho wouldn鈥檛?鈥�

Perhaps the greatest barrier to acceptance is a set of rules laid down in the 19th century by German physician Robert Koch. Known as Koch鈥檚 postulates, they remain the gold standard for proving a link between an infectious agent and a disease. To satisfy Koch鈥檚 postulates, a putative disease-causing agent must pass four tests. First, the organism must be found in animals suffering from the disease. Then it must be possible to isolate and grow it in the lab. The lab-grown microbe must produce the same disease when inoculated into healthy animals, and finally, the organism must be isolated once more from the sick animal.

When it comes to CNPs, Koch鈥檚 postulates have not been fully satisfied, though Lieske and Miller are arguably close. 鈥淲e have identified CNPs in diseased tissues, we can cultivate them from diseased tissue and the recent studies have shown that CNPs localise to diseased arteries and promote disease,鈥� says Miller. All that is missing is to recover nanoparticles from the newly infected rabbits.

Also, as Kajander and 脟ift莽ioglu are quick to point out, prions fail to satisfy Koch鈥檚 postulates despite now being widely accepted as the cause of scrapie, BSE, CJD and several other brain diseases. It took decades for the prion hypothesis to be accepted by mainstream scientific opinion. Perhaps CNPs will be the same.

The view is gradually gaining ground. 鈥淭his is not a case of the emperor鈥檚 new clothes,鈥� says Goldfarb. 鈥淭here is something there, but exactly what it is isn鈥檛 clear to me.鈥�