Plague Time, continued, Chronic Fatigue

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If simian virus 40 was spread by polio vaccines, what about HIV?
A destructive Lyme vaccine?
Spirochete persistence
e-coli spreads


``Study Finds Heart-Muscle Viruses Cause Chronic-Fatigue Syndrome

TORONTO -- Viruses that insidiously damage heart muscle may be the cause of chronic-fatigue syndrome, a mysterious malady that many physicians have written off as a psychological condition, according to a provocative new study by an infectious disease expert.

At a major scientific conference here, Martin Lerner, a doctor at William Beaumont Hospital in Royal Oak, Mich., presented data on a series of patients, including himself, who developed the debilitating condition and were later treated, with apparent success, with potent antiviral drug regimens.

"It's an infectious disease," that primarily attacks the heart, Dr. Lerner declared at a meeting of the American Society of Microbiology.

Dr. Lerner said daylong cardiac monitoring found that 95% of chronic-fatigue patients he and his research team tested in two separate small studies had abnormal electrocardiograms indicative of heart damage.

Dr. Lerner said he suspects the heart damage is caused by Epstein-Barr virus and cytomegalovirus, both long implicated in the condition.

The damage to the heart occurred, he believes, when the viruses were held in partial check by the patients' immune systems. Though the immune systems appear to have kept the viruses from reproducing, Dr. Lerner said partial bits of the viruses that were being produced appear to be causing heart damage ...

A test for the hard-to-diagnose syndrome would represent a significant clinical advance.

Chronic-fatigue syndrome now is diagnosed by a rough checklist of symptoms and a process of elimination. The key clinical finding is that patients have persistent or relapsing fatigue for six months or more.

In fact, doctors have argued whether the syndrome is a disease at all, and even if it is, exactly how prevalent it might be. By some estimates, the syndrome affects about six in every 100,000 people.

After implicating viruses as the cause of the syndrome, Dr. Lerner tested possible treatments.

Many of the patients, including Dr. Lerner, who were infected by Epstein-Barr virus regained cardiac function and returned to normal life after taking high doses of valacyclovir (brand name Valtrex), an antiviral drug made by Glaxo Wellcome PLC, for several months.

Patients with cytomegalovirus received ganciclovir (Cytovene), another antiviral drug made by Roche Holding AG.

Dr. Lerner became interested in chronic-fatigue syndrome when he fell ill in 1988 at age 58. He thought at first that he had heart disease, and an examination by doctors confirmed that his heart was weak.

Later, he suspected there was more to the picture. In 1996, he began antiviral drug therapy and his heart function returned to normal.

The smoking gun in Dr. Lerner's investigation came from patient samples of heart tissue. The viruses had weakened their hearts by scrambling the normally well-ordered muscle fibers ... ''

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``Growing Medical Fear Over Possible Carcinogenic Virus

SAN FRANCISCO -- A growing number of medical researchers fear that a monkey virus that contaminated polio vaccine given to tens of millions of Americans in the 1950s and '60s may be causing rare human cancers.

For four decades, government officials have insisted that there is no evidence the simian virus called SV40 is harmful to humans. But in recent years, dozens of scientific studies have found the virus in a steadily increasing number of rare brain, bone and lung-related tumors -- the same malignant cancer SV40 causes in lab animals.

Even more troubling, the virus has been detected in tumors removed from people never inoculated with the contaminated vaccine, leading some to worry that those infected by the vaccine might be spreading SV40.

The discovery of SV40 in human tumors has generated intense debate, pitting government health officials, who are convinced that the virus is harmless, against researchers from Boston to China who now suspect SV40 may be a human carcinogen. At stake are millions of research dollars and potential medical treatments for those afflicted with the cancers SV40 may be causing.

In April, [2001] more than 60 scientists met in Chicago to discuss the controversial virus and how it works to defeat certain cells' natural defenses against cancer.

"I believe that SV40 is carcinogenic (in humans)," said Dr. Michele Carbone of Loyola University Medical Center in Maywood, Ill. "We need to be creating therapies for people who have these cancers, and now we may be able to because we have a target - SV40."

But scientists at the National Cancer Institute [NCI] say their studies show almost no SV40 in human tumors and no cancer increase in people who received the contaminated vaccine.

"No one would dispute there's been a widespread, very scary exposure to the population of potentially cancer-causing virus," said Dr. Howard Strickler, NCI's chief investigator. "But none of our studies and other major analyses have shown an inkling of an effect on the population."

Critics charge, however, that the few studies done by the government are scientifically flawed and that health officials have downplayed the potential risks posed by SV40 ever since they learned in 1961 that the virus contaminated the polio vaccine and caused tumors in rodents.

"How long can the government ignore this?" asked Dr. Adi Gazdar, a University of Texas Southwestern Medical Center cancer researcher. "The government has not sponsored any real research. Here's something possibly affecting millions of Americans, and they're indifferent."

"Maybe they don't want to find out." The recent SV40 discoveries come at a time of growing concern over the dangers posed by a range of animal viruses that have crossed the species barrier to humans, including HIV, which scientists now believe came from chimpanzees and ultimately caused the AIDS epidemic ...

During the first half of the 20th century, polio struck down hundreds of thousands of people, leaving many paralyzed - some in iron lung machines - and killing others. The worst year was 1952, when more than 57,000 polio cases were reported in the United States. Three thousand died.

Then on April 12, 1955, Dr. Jonas Salk, a slightly built, soft-spoken researcher from Pittsburgh, mounted the podium at the University of Michigan and announced that he had developed a vaccine. That afternoon, the government licensed the vaccine for distribution.

Salk's vaccine was made by growing live polio virus on kidney tissue from Asian rhesus monkeys. The virus was then killed with formaldehyde. When the vaccine was injected in humans, the dead virus generated antibodies capable of fending off live polio ...

Four years later, Bernice Eddy, a researcher at the National Institutes of Health, noticed something strange while looking through her microscope. Monkey kidney cells - the same kind used to make the vaccine - were dying without apparent cause.

So she tried an experiment. She prepared kidney extracts from eight to 10 rhesus monkeys and injected tiny amounts under the skin of 23 newborn hamsters. Within nine months, "large, malignant, subcutaneous tumors" appeared on 20 of the animals.

On July 6, 1960, concerned that a monkey virus might be contaminating the polio vaccine, Eddy took her findings to Dr. Joseph Smadel, chief of the NIH's biologics division. Smadel dismissed the tumors as harmless "lumps."

The following year, however, at a Merck laboratory in Pennsylvania, Dr. Maurice Hilleman and Dr. Ben Sweet isolated the virus. They called it simian virus 40, or SV40, because it was the 40th virus found in rhesus kidney tissue ...

At the same time, an oral polio vaccine developed by virologist Albert Sabin was in final trials in Russia and Eastern Europe, where tens of millions had been inoculated, and it was about to be licensed in the United States. Unlike the Salk vaccine, the oral version contained a live but weakened form of polio virus and promised lifelong immunity.

But U.S. Public Health Service officials were worried. Tests had found SV40 in both the Sabin and Salk vaccines - it was later estimated that as much as a third of the Salk vaccine was tainted - and that SV40 was causing cancer in lab animals.

In the spring of 1961, they quietly met with the agency's top vaccine advisers. The agency found no evidence that the virus had been harmful to humans, but in May, the officials ordered manufacturers to eliminate SV40 from all future vaccine.

New procedures were adopted to neutralize the tainted polio virus seed stock and SV40-free African green monkeys were used to produce the bulk vaccine instead of rhesus monkeys.

But officials did not recall contaminated Salk vaccine - more than a year's supply - still in the hands of the nation's doctors.

And they did not notify the public of the contamination and SV40's carcinogenic effect on newborn hamsters ...

The first public disclosure that the Salk vaccine was contaminated came in the New York Times on July 26, 1961. A story on Page 33 reported that Merck and other manufacturers had halted production until they could get a monkey virus out of the vaccine.

When asked to comment, the U.S. Public Health Service "stressed" there was no evidence the virus was dangerous ...

In Boston, two researchers stumbled on something disturbing. Dr. Robert Garcea and his assistant, Dr. John Bergsagel, were using a powerful new tool called polymerase chain reaction, or PCR, to look for a pair of common human viruses in children's brain tumors.

But a different DNA footprint kept popping up in more than half the tumors. They finally realized they were seeing SV40.

For more than a decade, scientists had reported sporadic findings of SV40-like proteins in human tumors. But the earlier tests were primitive and the results suspect. PCR, however, is capable of amplifying infinitesimal fragments of DNA, which makes detections far more credible.

The findings were troubling. The researchers noted in their published report that the children were too young to have received the contaminated vaccine. But somehow the virus had infected them and embedded itself in their tumors.

That same year, Michele Carbone was surprised to find a milky, rindlike tumor in a laboratory hamster at the National Institutes of Health in Bethesda, Md.

The animal was one of a group given an SV40 injection directly into their hearts. Sixty percent of those hamsters developed the fatal cancer called mesothelioma.

Carbone, a postdoctoral fellow at the institute, knew that SV40 caused tumors in hamsters but only in specific locations where large doses of virus were injected. Here the mesothelial membrane lining the lungs apparently became cancerous from minuscule amounts of SV40 shed by the tip of the needle on the way to the hamsters' hearts.

So he tried another experiment, this time injecting SV40 directly into the thin mesothelial walls of another group of hamsters. Within six months, every animal developed mesothelioma.

Carbone was puzzled. Mesothelioma is a rare cancer. Few human cases were reported before the 1950s, but its incidence had been increasing steadily, reaching several thousand cases a year in the United States by 1988.

Studies had linked mesothelioma to asbestos exposure - with tumors usually appearing many decades later. Yet 20 percent of victims had no asbestos exposure.

Carbone decided to use PCR to test 48 human mesotheliomas stored at the NIH. He was stunned: 28 of them contained SV40.

PCR unleashed a wave of SV40 discoveries. By the end of 1996, dozens of scientists reported finding SV40 in a variety of bone cancers and a wide range of brain cancers, which had risen 30 percent over the previous 20 years.

Then, Italian researchers reported finding SV40 in 45 percent of the seminal fluid and 23 percent of the blood samples they had taken from healthy donors.

That meant SV40 could have been spreading through sexual activity, from mother to child, or by other means, which could explain how those never inoculated with the contaminated vaccine, such as the Boston children, were being infected ...

Dr. Howard Strickler ... led a study using PCR on 50 mesotheliomas from Armed Forces hospitals across the country. And he found no SV40.

Although the findings bolstered the government's long- standing position that SV40 did not appear to be a health risk, federal officials decided to convene a conference on the virus.

In January 1997, 30 scientists gathered at the National Institutes of Health in Maryland. Garcea, Carbone and others presented their evidence showing SV40 in tumors and pleaded for research funding.

Strickler presented his mesothelioma study, as well as new research he had just completed ...

This new study compared 20 years of cancer rates of people born between 1947 and 1963, and therefore likely to have been exposed to the contaminated polio vaccine, with people born after 1963, whom they believed weren't exposed.

The study found no significant difference between the two groups.

But when Susan Fisher read Strickler's ... study in the Journal of the American Medical Association, she fired off a letter of protest to the publication.

An epidemiologist at Loyola University Medical Center in Maywood, Ill., Fisher challenged the study's methodology, calling it "an error in judgment" and misleading.

Using the same 20-year national cancer database for the two groups, Fisher compared people of the same age - "because these cancers are highly correlated with age" - and she came up with very different results.

Studying 18- to 26-year-olds who probably had been exposed to the contaminated vaccine, Fisher found a 19.6 percent greater incidence of the two major brain cancers linked to SV40 when compared with the incidence in people the same age who were not exposed. She also found 16.6 percent more bone cancers and 178 percent more mesotheliomas among those exposed to the vaccine.

But Fisher cautioned against comparing the two groups. She argued that if SV40 is being transmitted and circulating in the population, then many people in the "unexposed" group would also be carrying the virus and that would undermine the comparison.

For years, researchers had believed that all SV40-contaminated Salk vaccine made between 1955 and 1963 had been used or discarded.

Then in 1999, Carbone was contacted by a former public health director in Oak Park, Ill., who said he had seven sealed vials of vaccine dated October 1955 in a refrigerator in his basement.

Carbone, who had left the NIH and joined the faculty at Loyola University Medical Center, ran tests on the vaccine and made a startling discovery: Not only was the vaccine contaminated, it contained a second form of the virus - an "archetypal" SV40 strain.

Although manufacturers switched from rhesus monkeys to SV40-free green African monkeys to grow the bulk vaccine in 1961, they have continued to use potentially contaminated polio seed stock grown on the rhesus monkeys tissue to start the bulk vaccine process.

Manufacturers checked the purity of their vaccine with a series of 14- day tests to detect whether any SV40 slipped through.

But when Carbone replicated the tests, he found that the second, slower-growing "archetypal" strain took 19 days to emerge.

It was possible, Carbone noted in a published report, that this second strain of SV40 had been evading manufacturers' screening procedures for years - and infecting vaccine recipients after 1962.

Meanwhile, a new study led by Strickler had bogged down in bitter internal conflict.

After the NIH's 1997 conference, nine laboratories were recruited to participate in a government-sponsored study to determine if tests were really finding SV40 in tumors or whether earlier detections were the result of laboratory contamination.

Carbone and other researchers considered the study unnecessary. A similar multilab study led by Dr. Joseph Testa of Philadelphia had just been completed, and it virtually eliminated the contamination theory. The prestigious journal Cancer Research published Testa's findings in 1998.

But Strickler pressed on. An independent laboratory in Maryland prepared mesothelioma samples for nine participants.

When tests revealed almost no SV40 in the tumor samples, some participants questioned the preparation methods used by the Maryland lab. They also challenged Strickler's written conclusion implying that contamination had caused the earlier findings of SV40 in tumors.

If Strickler was right, the earlier SV40 detections were probably the result of stray SV40 in the labs. But critics argued that the study was scientifically flawed and should be scrapped.

The dispute became so contentious that FDA officials were forced to intervene and a neutral arbitrator assigned to mediate.

Finally, in early 2000, more than two years after the study was initiated, a carefully rewritten report emerged for publication.

It concluded that contamination was an unlikely explanation for earlier SV40 findings. Then it struggled to explain the discrepancy between earlier detections of SV40 in about half of all mesotheliomas tested and the fact that the nine labs found the virus in only slightly more than 1 percent of the study's tumor specimens.

The report noted that discrepancy might be because of the inefficiency of the method used by the Maryland lab to recover viral DNA - like the genetic sequences of SV40 - from the mesothelial tissue to create the test samples.

The Maryland lab also had inadvertently contaminated some of the laboratory controls and "theoretically" could have contaminated others.

The report concluded by calling for further research. Despite the study's ambivalent conclusions and technical problems, the NCI submitted it to Cancer Research, the journal that had published Testa's study.

It was rejected. In laboratories around the world, researchers continued to find SV40 in a widening range of tumors that now included pulmonary, pituitary and thyroid cancers and some lymphomas.

Meanwhile, an NCI investigator named Dr. David Schrump was able to gut a common respiratory virus and use it to deliver genetic material called "antisense" into SV40-infected mesothelial cells and stop the cells' malignant growth.

His discovery, which was patented by the government, strongly suggested that SV40 contributed to mesothelioma and that a treatment might be possible.

Then in August, Carbone and several colleagues published a major study providing a "mechanistic" explanation of how SV40 contributes to the uncontrolled growth of mesothelial cells. The key, they found, was the large number of "tumor suppressor" proteins found in the mesothelial cells that makes them unusually susceptible to SV40.

In most human cells, they said, the virus reproduces itself and kills the infected cell in the process. But in mesothelial cells, SV40 is especially attracted to the "tumor suppressor" proteins and binds to them, knocking them out of action. The virus then lives on in the cell.

The result, they said, is a rate of malignant cell transformation in tissue cultures 1,000 times higher than has ever been observed.

In a paper published in the Proceedings of the National Academy of Science, Carbone further explained that asbestos fibers appear to act as a co-carcinogen in mesothelioma by somehow suppressing the immune system's response, which is designed to kill the infected cells.

Carbone and others believed that the time had come for another conference on the virus he calls "a perfect little war machine."

In April, [2001] more than 60 scientists gathered on a warm weekend at the University of Chicago's downtown conference center. Despite numerous faxes and certified letters inviting him, Strickler declined to attend.

Carbone opened the conference by confronting the question of whether SV40 is present in humans.

"Sixty-two papers from 30 laboratories from around the world have reported SV40 in human tissues and tumors," he said. "It is very difficult to believe that all of these papers, all of the techniques used and all of the people around the world are wrong."

For two days, scientists from as far away as China and New Zealand presented the results of their studies, with almost every speaker concluding that SV40 was present in the tissues they examined.

One of the newest discoveries came from Dr. Jeffrey Kopp, an NIH scientist who reported finding SV40 in a high percentage of patients with kidney disease. The virus was also present, he said, in 60 percent of a new "collapsing" type of renal disease that was unknown before 1980 but has since increased rapidly in incidence.

There were also reports on efforts to develop a vaccine, recently funded by the NCI, that would allow the immune system to target and eliminate SV40.

At times, the meeting took on almost revivalist overtones as scientist after scientist said he or she was initially very skeptical of SV40's presence in human tumors but was now a believer.

"I was a hard sell," said Testa, the Philadelphia geneticist who conducted the first multilaboratory tests, noting that the study had convinced him.

Gazdar, the cancer researcher from Texas, showed a slide describing his own transformation: "Nonbeliever (arrow) Believer (arrow) Zealot."

The conference concluded with a consensus among the leading scientists that SV40's presence in human tumors was no longer in question. They were more circumspect about the virus's possible role in causing cancer.

If SV40 is a human carcinogen, they said, the virus probably requires interaction with other cancer-causing substances like asbestos.

Dr. Janet Butel from Baylor Medical College in Houston said that it simply might be too soon to make a determination, citing the many years it has taken to establish that other viruses cause cancer.

But even renowned tumor biologist George Klein from Sweden said he was impressed by Carbone and Schrump's work.

"This strongly suggests that the virus plays a role (in causing tumors)," said Klein, a former chairman of the Nobel Assembly.

In May, shortly after the conference, Strickler's multilab study was published in a small journal called Cancer Epidemiology, Biomarkers & Prevention.

Carbone and other SV40 experts dismissed the study. "A garbage paper in a garbage journal," said Garcea, now on the faculty at the University of Colorado School of Medicine.

But Strickler strongly defends the study. He said it was the first to use strict controls not used in other studies. He acknowledged, however, that the study "doesn't prove that SV40 is not out there."

Strickler, who now teaches at Albert Einstein School of Medicine in New York, said he remains skeptical about whether SV40 has infected humans, a suspicion he says that is shared by the broader scientific community.

But a recent NCI statement acknowledges that there is evidence to suggest that SV40 "may be associated with human cancer." The statement, released last month, also said that SV40's interaction with "tumor suppressor proteins" indicates "possible mechanisms that could contribute to the development of cancer."

Top NCI officials declined to be interviewed on the record for this report ... ''

See article

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Infect Immun 2000 Feb;68(2):658-663

``Occurrence of Severe Destructive Lyme Arthritis in Hamsters Vaccinated with Outer Surface Protein A and Challenged with Borrelia burgdorferi.

Croke CL, Munson EL, Lovrich SD, Christopherson JA, Remington MC, England DM, Callister SM, Schell RF

Wisconsin State Laboratory of Hygiene, University of Wisconsin, Madison, Wisconsin 53706.

Arthritis is a frequent and major complication of infection with Borrelia burgdorferi sensu stricto.*

The antigens responsible for the induction of arthritis are unknown.

Here we provide direct evidence that a major surface protein, outer surface protein A (OspA), can induce arthritis.

Hamsters were vaccinated with 30, 60, or 120 mug of recombinant OspA (rOspA) in aluminum hydroxide and challenged with B. burgdorferi sensu stricto isolate 297 or C-1-11.

Swelling of the hind paws was detected in 100, 100, and 50% of hamsters vaccinated with 30, 60, or 120 mug of rOspA, respectively.

In addition, arthritis developed in 57% of hamsters vaccinated with a canine rOspA vaccine after infection with B. burgdorferi sensu stricto.

When the canine rOspA vaccine was combined with aluminum hydroxide, all vaccinated hamsters developed arthritis after challenge with B. burgdorferi sensu stricto.

Histopathologic examination confirmed the development of severe destructive arthritis in rOspA-vaccinated hamsters challenged with B. burgdorferi sensu stricto.

These findings suggest that rOspA vaccines should be modified to eliminate epitopes of OspA responsible for the induction of arthritis.

Our results are important because an rOspA vaccine in aluminum hydroxide was approved by the Food and Drug Administration for use in humans. ''

PMID: 10639430

* sensu stricto

Definition: "In the narrow sense," a phrase used to modify taxonomic designations when there is some debate about which individuals or populations truly belong in a given species or other taxon.

"Sensu stricto" means that the taxonomic label is being used in the narrow sense, including only those individuals that everyone agrees belong within it.

Also Known As: s.s.

Example: Homo erectus "sensu stricto" refers to those remains from Java that Eugene Dubois originally called Pithecanthropus erectus, as well as those from elsewhere in Asia that most or all researchers would include in the same species.

Homo erectus "sensu lato" includes those European and African fossils that some palaeoanthropologists prefer to assign to H. ergaster, H. antecessor, and H. heidelbergensis.

See reference

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14th International Scientific Conference on Lyme Disease & Other Tick-Borne Disorders

Day 1 - April 23, 2001

Fighting Back: How B burgdorferi Persists

Julie Rawlings, MPH, and Harry Goldhagen


That spirochetes tend to persist in the human body has been demonstrated in both syphilis, caused by Treponema pallidum, and Lyme disease, caused by Borrelia burgdorferi. What accounts for this ability to evade or suppress an effective immune response? According to Charles Pavia, PhD,[1] of the New York College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, New York, there are at least 6 potential explanations:

1) antigenic variation (this is seen with the Borrelia species that cause tick-borne relapsing fever) or differential expression of antigens (especially the outer surface proteins; with B burgdorferi, only OspC is expressed during mammalian infection)

2) production of an outer protective coat (eg, capsule, as seen with T pallidum)

3) atypical forms (eg, cyst-like variants)

4) incomplete immune response (eg, insufficient antibody , T-cell , or phagocytic response)

5) deranged host immune response (eg, host-, tick-, or spirochete-derived immunosuppressive factors)

6) other evasive factors (eg, motility)


Is there evidence that any of these mechanisms allow B burgdorferi to persist in the human body? As of now, not much. However, there have been a few suggestive studies in animals that support immune suppression as a possible explanation.

For instance, a study by Chiao and colleagues[2] showed that B burgdorferi is capable of suppressing the immune response. When sonicated Borrelia were added to lymphocytes, the ability of the lymphocytes to proliferate -- a measure of the immune system's ability to respond to an infectious challenge -- was inhibited.

A similar study by Giambartolomei and coworkers[3] showed that Borrelia can stimulate interleukin-10 (IL-10) production, a down regulator of the immune system. In this series of experiments, heat-killed B burgdorferi caused peripheral blood mononuclear cells of humans and rhesus monkeys to produce this cytokine.

Another study, by Keane-Myers and Nickell,[4] found that B burgdorferi could suppress T-cell responses in mice, specifically T-helper cells.

Even the tick itself may play a role in immunosuppression. Urioste and colleagues[5] showed that the saliva of Ixodes dammini ticks contains an uncharacterized substance that can suppress the immune response, specifically suppressing lymphocyte proliferation and other markers of immune system activity.

Looking at the issue of immune suppression from the other side -- that is, by boosting the immune response with the use of cytokines -- Zeidner and colleagues[6] showed that tumor necrosis factor alpha (TNF-alpha), IL-2, and interferon-gamma could suppress B burgdorferi infection in mice.

By contrast, it appears that infection with B burgdorferi can also over stimulate the immune system, and this may explain many of the symptoms of both acute and chronic Lyme disease. For instance, Lim and colleagues [7] showed that CD4+ T cells play a role in the arthritis seen in the hamster model of Lyme disease.


A second mechanism that may be part of the B burgdorferi survival repertoire is evasion of the immune response. Antigenic variation[8] -- the periodic alteration of surface proteins resulting in the presentation of new antigens that the immune system has not seen before -- has been a successful strategy for tick-borne relapsing fever Borrelia, and it is possible that such a mechanism occurs in B burgdorferi as well.

Richard T. Marconi, PhD,[9,10] of the Medical College of Virginia at Virginia Commonwealth University, Richmond, has examined potential candidate proteins for antigenic variation in Lyme disease. The most likely candidate, based on his research, is outer surface protein E (OspE).

The genes for these surface-exposed lipoproteins have 2 hypervariable domains (the variation is due to insertions and deletions in the gene) as well as repeat regions (which can lead to recombination with other genes, and thus new antigens).

OspE variation has been shown to occur in vivo, at least in mice. A number of variants have been shown to be antigenically distinct and to arise at different times during infection.

By contrast, the OspF genes are stable; that is, they do not show the variability seen with OspE genes. However, different members of the OspF family are expressed at different times during infection, suggesting that this surface protein may also play a role in antigenic variation.

Other investigators have suggested that VlsE may be involved in immune evasion as well, since VlsE genes exhibit both variation and mutation during infection.However, Dr. Marconi's work suggests that the resulting VlsE are not antigenically distinct.

In other words, even though the VlsE genes mutate during infection, the changes in the second or third generation VlsE proteins are not sufficient to evade the antibodies produced against the first generation of VlsE surface protein. Therefore, he believes it is unlikely VlsE plays a prominent role in immune evasion.


A different way for an organism to avoid being killed is to destroy the harmful compounds before they do their damage! For example, many bacterial species produce beta-lactamases. These enzymes degrade beta-lactam antibiotics before they block bacterial cell-wall synthesis.

It appears that B burgdorferi may have its own approach to fighting back. John T. Skare, PhD,[11] of Texas A&M University Health Science Center, College Station, examined one such battle tactic Borrelia use against humans: resisting oxidative stress. (Oxidative stress is the use of hydrogen peroxide, superoxide, and other active oxygen radicals to kill invading organisms.)

Dr. Skare's group has examined a gene called BB0647 to see what role it may play in resisting oxidative stress. Originally, this gene was thought to resemble a ferric uptake regulator, or Fur.

Oddly enough, though, Borrelia don't require iron. This puzzling finding prompted Dr. Skare to search databases for genes that more closely resemble BB0647. He found that the most homologous gene is one that produces a peroxide regulatory protein, PerR. This protein controls the expression of catalases and peroxidases, enzymes that play a major role in defending cells against oxidative stress.

To confirm these findings, Dr. Skare showed that B burgdorferi containing an active form of the BB0647 gene was indeed able to resist hydrogen peroxide, around 3000 times better than a strain containing a mutated form of the gene.


How does B burgdorferi know when to express certain proteins? For instance, when B burgdorferi is inside ticks, at approximately 23C, the surface of the spirochete is covered with OspA and OspB. But when the organism gets into mammals, at an average temperature of 35-37C, OspC is the predominant surface antigen. So, does temperature affect gene expression?

Dr. Scott Samuels, PhD,[12] of the University of Montana, Missoula, presented some highly technical research on how the Lyme disease spirochete manages this transformation. The process appears to be mediated by supercoiling of DNA. (For a description and pictures of DNA supercoiling, see the handy Web site from the Mount Sinai School of Medicine.[13])

Supercoiled DNA can be visualized as a coiled phone cord; the more you twist it, the more tightly wrapped it becomes. Untwisting the DNA opens up the loops and coils.

It turns out that the degree of supercoiling is determined by the temperature: at the lower temperature of ticks, B burgdorferi DNA is more supercoiled, and the OspC gene is expressed at only a low level. By contrast, at the higher temperatures seen in mammals, DNA is less supercoiled, and the OspC gene is expressed at higher levels. The next step for Dr. Samuels' team is to determine what is affecting expression of the other 2 surface protein genes.


Borrelia have evolved in numerous ways to avoid the immune system. The better we can understand these molecular defense strategies, the sooner we can develop better approaches to diagnosis and treatment.


Pavia C. Mechanisms of persistency of the Lyme disease spirochete. Program and abstracts of the 14th International Scientific Conference on Lyme Disease and Other Tick-Borne Disorders; April 21-23, 2001; Hartford, Connecticut.

Chiao JW, Pavia C, Riley M, et al. Antigens of Lyme disease of spirochaete Borrelia burgdorferi inhibits antigen or mitogen-induced lymphocyte proliferation. FEMS Immunol Med Microbiol. 1994;8:151-155. Abstract available at:


Giambartolomei GH, Dennis VA, Philipp MT. Borrelia burgdorferi stimulates the production of interleukin-10 in peripheral blood mononuclear cells from uninfected humans and rhesus monkeys. Infect Immun. 1998;66:2691-2697. Available at:


Keane-Myers A, Nickell SP. T cell subset-dependent modulation of immunity to Borrelia burgdorferi in mice. J Immunol. 1995;154:1770-1776. Abstract available at:


Urioste S, Hall LR, Telford SR 3rd, Titus RG. Saliva of the Lyme disease vector, Ixodes dammini, blocks cell activation by a nonprostaglandin E2-dependent mechanism. J Exp Med. 1994;180:1077-1085. Abstract available at:


Zeidner N, Dreitz M, Belasco D, Fish D. Suppression of acute Ixodes scapularis-induced Borrelia burgdorferi infection using tumor necrosis factor-alpha, interleukin-2, and interferon-gamma. J Infect Dis. 1996;173:187-195. Abstract available at:


Lim LC, England DM, Glowacki NJ, et al. Involvement of CD4+ T lymphocytes in induction of severe destructive Lyme arthritis in inbred LSH hamsters. Infect Immun. 1995;63:4818-4825. Available at:


Introduction to Antigenic Variation, from the Laboratory of Molecular Parasitology, The Rockefeller University. Available at:


Marconi RT. Comparative analysis of the immune evasion systems employed by the Lyme disease spirochetes. Program and abstracts of the 14th International Scientific Conference on Lyme Disease and Other Tick-Borne Disorders; April 21-23, 2001; Hartford, Connecticut.

Sung SY, McDowell JV, Carlyon JA, Marconi RT. Mutation and recombination in the upstream homology box-flanked OspE-related genes of the Lyme disease spirochetes result in the development of new antigenic variants during infection. Infect Immun. 2000;68:1319-1327. Available at:


Skare J. How Borrelia burgdorferi responds to oxidative stress. Program and abstracts of the 14th International Scientific Conference on Lyme Disease and Other Tick-Borne Disorders; April 21-23, 2001; Hartford, Connecticut.

Samuels S. Regulation of outer surface protein gene expression. Program and abstracts of the 14th International Scientific Conference on Lyme Disease and Other Tick-Borne Disorders; April 21-23, 2001; Hartford, Connecticut. Supercoiling, from the Department of Biomathematical Sciences, Mount Sinai School of Medicine. Available at:



The Lyme Disease Foundation (LDF), in their brochure entitled "LDF Frequently Asked Questions About Lyme Disease" lists the following nine reasons for false negative Lyme disease test results.

1. Antibodies against Bb are present, but the laboratory is unable to detect them. [Borrelia burgdorferi (Bb) is the Lyme disease bacteria.]

2. Antibodies against Bb may NOT be present in detectable levels in a patient with Lyme disease because the patient is currently on, or has recently taken, antibiotics. The antibacterial effect of antibiotics can reduce the body's production of antibodies.

3. Antibodies against Bb may NOT be present in detectable levels in a patient with Lyme disease because the patient is currently on or has previously taken anti-inflammatory steroidal drugs These can suppress a person's immune system, thus reducing or preventing an antibody response.

4. Antibodies against Bb may NOT be present in detectable levels in a patient with Lyme disease because the patient's antibodies may be bound with the bacteria with not enough free antibodies available for testing.

[For this reason, some of the worst cases of Lyme disease test negative -- too much bacteria for the immune system to handle.]

5. Antibodies against Bb may NOT be present in detectable levels in a patient with Lyme disease because the patient could be immunosuppressed for a number of other reasons, and the immune system is not reacting to the bacteria.

6. Antibodies against Bb may NOT be present in detectable levels in a patient with Lyme disease because the bacteria has changed its makeup (antigenic shift) limiting recognition by the patient's immune system.

7. Antibodies against Bb may NOT be present in detectable levels in a patient with Lyme disease because the patient's immune response has not been stimulated to produce antibodies, i.e., the blood test is taken too soon after the tick-bite (8-6 weeks).

Please do not interpret this statement as implying that you should wait for a positive test to begin treatment.

8. Antibodies against Bb may NOT be present in detectable levels in a patient with Lyme disease because the laboratory has raised its cutoff too high.

9. Antibodies against Bb may NOT be present in detectable levels in a patient with Lyme disease because the patient is reacting to the Lyme bacteria, but is not producing the "right" bands to be considered positive.

Lyme Disease Foundation
1 Financial Plaza
Hartford, CT 06103
fax (860)525-TICK
Lyme Disease National Hotline (800)886-LYME


Andrew R. Pachner, Wei-Fen Zhang, Henry Schaefer, Susan Schaefer, and Tim O'Neill.

Journal of Clinical Microbiology, November 1998;36:3243-3247

Ideally a diagnosis of infection of the central nervous system (CNS) is made by culture of the etiologic pathogen, but Borrelia burgdorferi, the causative agent of Lyme neuroborreliosis (LNB), is rarely cultured from the cerebrospinal fluid (CSF).

[Polymerase Chain Reaction] PCR and measurement of specific antibody in the CSF [cerebrospinal fluid] also have their limitations.

The role of available assays for LNB [Lyme neuroborreliosis] has not been studied carefully in a comparative investigation.

There is a need to assess the reliability of assays and to increase the ability to document active infection in the CNS. The recent development of the nonhuman primate (NHP) model of LNB [Lyme neuroborreliosis] allowed us to address this need in a faithful model of human LNB.

In this study we compared the abilities of PCR and culture to detect the presence of spirochetes in the CSF [cerebrospinal fluid] and brain tissue of infected NHP [nonhuman primates] and related these measures of infection to the development of anti-B. burgdorferi antibody.

We also tested a bioassay, the mouse infectivity test (MIT), in this model.

Fourteen of 16 CSF [cerebrospinal fluid]s from four NHP [nonhuman primates] were positive by at least one of these techniques.

Detection of spirochetes in the CSF [cerebrospinal fluid] by PCR, the MIT [mouse infectivity test], and culture was INVERSELY related to the concomitant presence of anti-B. burgdorferi antibody intrathecally [from the spine]. The performance of any particular test was associated with the strength of the host immune response.

In early CNS infection, when anti-B. burgdorferi antibody had not yet appeared, or in immunocompromised hosts, the MIT [mouse infectivity test] compared favorably to culture and PCR for infected NHP [nonhuman primates]; antibody in the CSF [cerebrospinal fluid] was the most useful assay for immunocompetent NHP [nonhuman primates].

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``Spread of Antibiotic-Resistant E. coli From Animals to People May Be Common

LONDON -- Antibiotic-resistant Escherichia coli is commonly spread from animals to people in the Netherlands, according to a report in the June [2001] issue of the Journal of Antimicrobial Chemotherapy.

Dr. E. E. Stobberingh and colleagues from the University Hospital Maastricht in the Netherlands analyzed the prevalence of resistance in faecal E. coli in 47 turkeys and 50 broilers commonly given antibiotics and in 25 laying hens that were infrequently treated with antibiotics.

To examine the "possible dissemination of resistant E. coli or resistance genes from these poultry populations to humans," the researchers examined faecal samples from individuals who had contact with these animals. This included 47 turkey farmers, 51 broiler farmers, 25 laying-hen farmers, 47 turkey slaughterers and 46 broiler slaughterers ...

Turkeys and broilers had a significantly higher prevalence of resistant E. coli than the laying-hen population. Turkey and broiler farmers and turkey and broiler slaughterers had a higher resistance to nearly all antibiotics than laying-hen farmers ... ''

J Antimicrob Chemother 2001;47:763-771.

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