March 07, 2010 U-M scientists identify reservoirs where HIV-infected cells can lie in wait Targeting these reservoirs of latent cells may open door to new treatments.
ANN ARBOR, Mich. - University of Michigan scientists have identified a new reservoir for hidden HIV-infected cells that can serve as a factory for new infections. The findings, which appear online today in Nature Medicine, indicate a new target for curing the disease so those infected with the virus may someday no longer rely on AIDS drugs for a lifetime.
``Antiviral drugs have been effective at keeping the virus at bay. However once the drug therapy is stopped, the virus comes back,'' says senior author of the study Kathleen L. Collins, M.D., Ph.D., associate professor of both internal medicine and microbiology and immunology at the U-M Medical School.
In people infected with HIV (human immunodeficiency virus), the virus that causes AIDS, there's an unsolved problem with current anti-viral drugs. Though life-saving, they cannot root the virus out of the body. Infected cells are able to live on, undetected by the immune system, and provide the machinery for the virus to reproduce and spread.
This has led researchers to believe HIV-infected cells may lay-in-wait somewhere in the body. Important new research by U-M has discovered that bone marrow, previously thought to be resistant to the virus, can contain latent forms of the infection.
``This finding is important because it helps explain why it's hard to cure the disease,'' Collins says. ``Ultimately to cure this disease, we're going to have to develop specific strategies aimed at targeting these latently infected cells.''
``Currently people have to take anti-viral drugs for their entire life to control the infection,'' she says. ``It would be easier to treat this disease in countries that don't have the same resources as we do with a course of therapy for a few months, or even years. But based on what we know now people have to stay on drugs for their entire life.''
Using tissue samples, U-M researchers detected HIV genomes in bone marrow isolated from people effectively treated with antiviral drugs for more than six months.
While further studies are needed to demonstrate that stem cells can harbor the HIV virus, the study results confirm that HIV targets some long-lived progenitor cells, young cells that have not fully developed but mature into cells with special immune functions. When active infection occurs the toxic effects of the virus kill the cell even as the newly made viral particles spread the infection to new target cells.
``Our finding that HIV infects these cells has clear ramifications for HIV disease because some of these cells may be long-lived and could carry latent HIV for extended periods of time,'' she says. ``These HIV cell reservoirs can be induced to generate new infections.''
The new research gives a broader view of how HIV overwhelms the body's immune system and devastates its ability to regenerate itself.
Globally more than 30 million people are infected with HIV, including millions of children. Improvements have been made since the 1990s in the way the disease is treated that has led to an 85 percent to 90 percent reduction in mortality.
``Drugs now available are effective at treating the virus, making HIV more of a chronic disease than a death sentence,'' Collins says. ``This has made a huge impact in quality of life, however only 40 percent of people worldwide are receiving anti-viral drugs and unfortunately that means that not everybody is benefiting.''
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Additional authors: Christoph C. Carter, Adewunmi Onafuwa-Nuga, Lucy A. McNamara, James Riddell IV, and Dale Bixby, all of U-M; and Michael R. Savona, University of Texas Health Science Center, San Antonio, Texas, formerly of U-M Heath System.
Funding: National Institutes of Health. The work of first author Carter was supported by the Wellcome Foundation, U-M Molecular Mechanisms in Microbial Pathogenesis Training Grant and a Rackham Predoctoral Fellowship, and McNamara's work was supported by a National Science Foundation Predoctoral Fellowship and a Bernard Maas Fellowship.
Key words: polymerase chain reaction, outer surface protein A gene, B. burgdorferi DNA
Abstract The diagnosis of persistent Lyme disease has depended on the detection of serum antibody to Borrelia burgdorferi. Patients may lose their immune response over time or it may be abrogated by antimicrobial therapy. These case reports describe patients with chronic Lyme disease and a reactive bone marrow polymerase chain reaction (PCR). After appropriate and aggressive treatment, specific DNA may persist in sequestered sites such as bone marrow.
Introduction Diagnosis of Lyme disease by laboratory methods continues to be difficult. Most tests rely on the production of IgM and/or IgG antibody to Borrelia burgdorferi. Antibody production may be abrogated by antimicrobial agents1 or may not occur at all. IgM and IgG antibody responses may fluctuate over time. Complicating these immune responses is the lack of reliability of many of the commercially available serological tests.2,3 Only recently4 has standardization of confirmatory Western blot testing been discussed in an organized forum and recommendations published for the use of proficiency panels.5 There is a need for methods to directly detect B. burgdorferi in body fluids. Gene amplification, notably polymerase chain reaction (PCR), satisfies the requirements for both sensitivity and specificity. Several reports document the advantages of PCR for detection of DNA in synovial fluid6 cerebrospinal fluid7,8 as well as other body fluids such as blood and urine.9 Relatively few, if any reports, have cited bone marrow as a diagnostic specimen.
The following case reports describe patients who had a reactive PCR for B. burgdorferi DNA in the bone marrow and positive B. burgdorferi antibody tests.
Case #1
This 51-year-old female was seen for the first time in April 1989 with a Lupus-like illness. There were insufficient clinical and laboratory data to fulfill the American Rheumatology Association criteria for Systemic Lupus Erythematosus (SLE). She was treated with nonsteroidal anti-inflammatory agents. From October 1989 to January 1991, she reported frequent respiratory infections, sinusitis, increasing joint pains, headaches, and fatigue. She was started on Plaquenil (hydroxychloroquine sulfate) for autoimmune arthritis in March 1991, but was symptomatic through June of 1991 with frequent upper respiratory infections on intermittent antimicrobial agents. By September 1991, she complained of severe headaches, fatigue, nausea, joint swelling, and urinary symptoms. She was again placed on various regimens of oral antimicrobials in addition to Plaquenil and tapering doses of steroids.
In June 1992 she was admitted to the hospital with chest pain (left hilar) and aorticopulmonary adenopathy. All of her laboratory tests were within normal limits, except antinuclear antibodies (ANA), which were present at a titer of 1:40. The gallium scan was positive in the left hilum and mediastinum. An abdominal CT scan was unremarkable. A biopsy of a mediastinal lymph node revealed noncaseating granulomas, which were atypical for sarcoidosis. Progressive improvement was seen through January of 1993 when she developed an acute influenza-like illness associated with severe arthritis and neck stiffness. The patient was started on oral Biaxin (clarithromycin) and Suprax (cefixime) for presumptive seronegative Lyme disease. Initiation of treatment was followed by a flare of an acute necrologic syndrome including vertigo, headaches, and profound fatigue. She became progressively worse until March 1993 when she began intravenous antimicrobial (IV) therapy with Claforan (cefotaxime). By that time her ANA was repeatedly negative but she experienced multiple Herxheimer reactions on Claforan requiring adjustment of dosage.
After 2 months of IV antimicrobial agents, her condition improved. She had no additional antimicrobials until March 1994 when she developed gradually recurring symptoms of headaches, joint pains, fevers, paresthesia, profound fatigue, and difficulty with word-finding. The ANA's remained either negative or weakly positive. An ELISA antibody test for Lyme disease was performed with a commercial kit and was reactive on June 2, 1994 but was not confirmed by Westernblotting. She was maintained on Zithromax (azithromycin) and Plaquenil. She continued to have severe fatigue, requiring total bed rest. Immunological studies were normal except for IgG and lambda monoclonal gammopathy. In September 1994, a bone marrow biopsy was performed. The PCR for B. burgdorferi DNA was reactive. She was again started on IM 1.2 mu BiCillin (penicillin) on October 3, 1994 and oral Zithromax 250 mg BID. The patient was maintained on the above regimen until clinical remission with progressive improvement of all her symptoms. Repeat immunological studies continue to be within normal limits.
Case #2
This 59-year-old female was seen for the first time on April 8, 1994 after being treated by another physician for Lyme disease. She had multiple influenza-like illnesses in 1992 through 1993 and was treated with short courses of oral antibiotics. She went to Spain in 1993. This trip was followed by the onset of severe pain and weakness. The patient had been a professional singer and could no longer sing. When tested for Lyme disease, the IgM Western blot was reactive. She was started on treatment for Lyme disease in August 1993 consisting of IV Claforan (Cefotaxime) for 8 weeks. She initially felt better but then relapsed. Intravenous ampicillin was administered for 1 month followed by Claforan (9 g/d) for 2 weeks which was reduced to 6 g/d. By May of 1994, there was lack of response and she was started on IV vancomycin. The patient responded well but relapsed when vancomycin was discontinued. Vancomycin was restarted in June of 1994 and the patient responded well.
Therapy was continued through July followed by pulse vancomycin and BiCillin LA (penicillin). By August 1994, she was very symptomatic with fevers and arthritis. The IV vancomycin was stopped and Plaquenil started in addition to intramuscular (IM) BiCillin. The patient was noted to have hypogammaglobulinemia and anemia and treated with IV gammaglobulin and transfusions. The patient did well until October 1994 when both symptoms and anemia recurred. The consulting hematologist performed a bone marrow biopsy that was unremarkable except for a positive PCR (B. burgdorferi). By November 1994 she had recurrent fevers and arthritis. Her anemia is stable on Epogen (epoetin alpha) and her strength is improving on Plaquenil. She has had two gammaglobulin transfusions for recurrent hypogammaglobulinemia. Throughout this time, both her IgG and IgM Westem blots have remained consistently positive. The patient now reports that she is feeling better.
Discussion B. burgdorferi DNA was detected from bone marrow samples of these patients. The DNA was amplified with specific primers for the highly conserved OspA (outer surface protein A) gene sequence of B. burgdorferi. The amplification and detection system used detected 10 copies of the OspA gene in the sample. The unique OspA primers are highly specific for B. burgdorferi and do not cross-react with closely related organisms such as B. hermsii. Known positive serum and urine samples, as well as a standard curve of B. burgdorferi DNA are included in each assay to confirm assay sensitivity and reproducibility throughout the sample processing, amplification and detection steps. Since PCR can be adversely affected by inhibitors of Taq polymerase commonly found in clinical specimens, all samples are required to pass a strict inhibition test that consists of the amplification, and detection of a small amount of a known DNA sequence added to an aliquot of the test sample. Samples that fail the inhibition test are repurified and retested. Negative controls included a series of buffer controls, extracts from normal human cells, and negative patient material. All negative controls must test negative before the PCR process can be validated. Amplification products were detected using a proprietary microtiter-based capture hybridization assay for the OspA gene. This system generates a strong signal with greater sensitivity than conventional hybridization formats. Southern blot hybridization with nonradioactive alkaline phosphatase probes specific for the OspA sequence, was used as a confirmatory method for amplicon detection. To assure the validity of the sensitive PCR system, stringent quality control practices were observed. Sample processing, preparation, and reagent formulation functions were carried out in separate areas within the laboratory dedicated exclusively for PCR. Reagents were pretested and prealiquoted for one time usage. Further precautions included the use of uracil-N-glycosylase to minimize amplicon cross-contamination. In cases such as these, the presence of B. burgdorferi DNA can be detected in patients with active infection. Body fluids have been shown to contain small levels of Borrelia, which may be difficult to detect by culture, but may be detectable with the use of the PCR assay that is capable of detecting as little as the equivalent of less than 10 organisms per mL of fluid.9 This laboratory performs more than 2500 PCR tests per month for a variety of infectious agents and has never had a contamination incident. Manak et al10 suggested that PCR reactivity could be cyclical. That is, B. burgdorferi DNA may be released into the circulation in small amounts at unpredictable time intervals but apparently associated with antibiotic therapy. They also reported that reactive PCR tests may be observed in patients with no detectable antibody to B. burgdorferi and who fulfilled the Centers for Disease Control's criteria for Lyme disease. Of 16 patients with chronic Lyme disease and a reactive PCR, only 4 were seropositive.
The significance of B. burgdorferi DNA in the bone marrow is unclear. Some investigators believe that B. burgdorferi is an intracellular bacterium. It is possible that the bone marrow serves as a reservoir for the Lyme disease agent and that sporadic appearance of nucleic acid in the circulation occurs after a DNA shower from the bone marrow, possibly stimulated by antimicrobial induced cell lysis. Thus, the bone marrow biopsy, albeit invasive, may prove to be a clinically significant procedure in a patient whose signs and symptoms are not sufficiently focused to rule out other diseases.
Unlike many suspected Lyme disease patients, these 2 individuals had a serologic diagnosis of Lyme disease and received extensive and seemingly adequate antibiotic treatment over a several-year period. They developed hematologic abnormalities, which resulted in a bone marrow biopsy. Symptoms of Lyme disease persisted in spite of aggressive treatment.
This report demonstrates several phenomena:
Despite aggressive IV and oral antimicrobial treatment, B. burgdorferi may persist in sequestered areas of the body such as bone marrow.
The yield on PCR testing for Lyme disease may be enhanced by testing bone marrow specimens or, by extrapolation, testing leukocytes, ie, buffy coat, where the organism may reside intracellularly protected from immune defenses. The bone marrow provides a specimen resource rich in nucleated cells, which may harbor the spirochete in dormant or active form. We clearly do not advocate this invasive testing for all patients but rather those who are refractory to standard therapy and have concomitant immunologic or hematologic abnormalities.
There does not appear to be a clear correlation between reactive Lyme serology and PCR positivity, although one patient had a persistently positive IgG and IgM Western blot and the other a positive ELISA test. In conclusion, these case reports suggest that bone marrow may be an important site for detection of B. burgdorferi DNA.
Address correspondence to Lesley Fein, 1099 Bloomfield Avenue, West Caldwell, NJ 07006.
References 1. Dattwyler RJ, Volkman DJ, Luff BJ, et al. Dissociation of specific T and B lymphocyte responses to Borrelia burgdorferi. N Engl J Med. 1988;319:1441-1446.
2. Bakken LL, Case KL, Callister SM, et al. Performance of 45 laboratories participating in a proficiency testing program for Lyme disease serology. JAMA. 1992;268:891-895. 3. Fister RD, Weymouth LA, McLaughlin JC, et al. Comparative evaluation of three products for the detection of Borrelia burgdorferi antibody in human serum. J Clin Microbial. 1989;27:2834-2837. 4. Second National Conference on Serologic Diagnosis of Lyme disease. Association of State and Territorial Public Health Laboratory Directors; Centers for Disease Control and Prevention; Michigan Dept. of Health, Dearborn, Ml. 1994. 5. Tilton RC, Sand MN, Manak M. Determination of sensitivity, specificity, and interpretive criteria with use of commercially available performance panels. Clin Infect Dis. 1997;25(Suppl 1):S31-S35. 6. Nocton JJ, Dressler R, Rutledge B, et al. Detection of Borrelia burgdorferi by polymerase chain reaction in synovial fluid from patients with Lyme arthritis. N Engl J Med. 1994;330:229-234. 7. Hupertz HI, Schmidt H, Karch H. Detection of Borrelia burgdorferi by nested polymerase chain reaction in cerebrospinal fluid and urine of children with neuroborreliosis. Eur J Pediatr. 1993;152:414-417. 8. Lebeck AM, Hansen J. Detection of Borrelia burgdorferi DNA in urine samples and cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by polymerase chain reaction. J Clin Microbiol. 1992;30:1646-1653. 9. Schmidt BL. PCR in laboratory diagnosis of human Borrelia burgdorferi infections. Clin Microbiol Rev. 1997;10:185-201. 10. Leibling MR, Nishio NJ, Rodriguez A. The polymerase chain reaction for the detection of Borrelia burgdorferi in human body fluids. Arthr Rheum. 1993;36:665-675. 11.Manak MM, Gonzalez GV, Stanton S, et al. Use of PCR assays to monitor the clearance of Borrelia burgdorferi DNA from blood following antibiotic therapy.
J Spirochetal Tick-borne Dis. 1997;4:11-20
Written by Shantell M. Kirkendoll
-------------------- Neil Posts: 697 | From Tucson, AZ USA | Registered: Apr 2002
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very interesting studies, thanks.
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Vermont_Lymie
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Good find! I wonder why the major media picked up the HIV story, which I had seen, but not the lyme story. Oh, just noticed the 1997 date. Thanks for posting it.
Not a surprise to find spirochetes in bone marrow, but sorry to hear it.
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Pinelady
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An injection called VGV-L has been developed to treat Lyme disease, an illness that saw a 100% increase in CDC-confirmed cases from 1991 to 2006. Animal studies have been completed with positive results. We are now in the process of introducing our research to the FDA. The proposed therapy, like several others in our R&D p...ipeline, is based on TPT and uses synthetic peptides to "trick" cells, making them vulnerable to the body's natural immune response mechanism. http://www.news-medical.net/news/20100212/Viral-Genetics-Company-update.aspx For those that don't know Luc Montaigne(Aids) works for this company.
-------------------- Suspected Lyme 07 Test neg One band migrating in IgG region unable to identify.Igenex Jan.09IFA titer 1:40 IND IgM neg pos 31 +++ 34 IND 39 IND 41 IND 83-93 + DX:Neuroborreliosis Posts: 5850 | From Kentucky | Registered: Dec 2008
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sparkle7
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I'm no scientist but HIV is a retro virus while Bb is bacterial or maybe parasite...
I don't think there's a big similarity between the 2 in regards to the type of pathogen that they are. I'm not sure what else may be sequestered in the bone marrow or if this is significant.
Herpes can live in the nerves & there's some thought that Bb may cause dementia in the brain cells... There are also alot of toxins that are in the environment that can be contained by fat cells in the body or effect the hormones. I've seen animations on YouTube of how mercury effects nerve cells. I'm sure there are other examples.
Like I said - I'm no scientist. It's just that when you make a comparison of the 2 illnesses as being in the bone marrow - people can get panicked. I'm not sure if it means anything or if this is something we have to worry about.
Thanks for the info in any case. Thanks Pinelady - too - for the info on peptides.
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Pinelady
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I think the key word was trick the cells. They know HIV hides as does Lyme.
No I'm not going to rush out and get it when it becomes available, but they are working on things
like this, which is good to know. It may be that old adage-If you can't beat um -join um.
Play the borrelia at their own game.
-------------------- Suspected Lyme 07 Test neg One band migrating in IgG region unable to identify.Igenex Jan.09IFA titer 1:40 IND IgM neg pos 31 +++ 34 IND 39 IND 41 IND 83-93 + DX:Neuroborreliosis Posts: 5850 | From Kentucky | Registered: Dec 2008
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disturbedme
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Unlike many suspected Lyme disease patients, these 2 individuals had a serologic diagnosis of Lyme disease and received extensive and seemingly adequate antibiotic treatment over a several-year period. They developed hematologic abnormalities, which resulted in a bone marrow biopsy. Symptoms of Lyme disease persisted in spite of aggressive treatment.
Augh, kind of sounds like me.... I have hematological abnormalities... well, I have for a long time... over 14 years.... since I was bitten by the tick... but the problem is, we are not sure if it's due to lyme or not and now I am going through a TON of testing by Johns Hopkins to figure out if I have fatal blood genetic diseases, etc., etc., etc.
It's crazy and scary and just a pain in the butt!
I've had one bone marrow biopsy but now I have to have another.
This is a very interesting post.... I would like to send my bone marrow off to IGeneX for testing for Bb, but not sure how to go about it... that, and not sure if Johns Hopkins would be all that willing to send some of my bone marrow to IGeneX for me!!!! LOL!
-------------------- One can never consent to creep when one feels an impulse to soar. ~ Helen Keller
My Lyme Story Posts: 2965 | From Land of Confusion (bitten in KS, moved to PA, now living in MD) | Registered: Jun 2007
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Pinelady
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If they would not give you enough for a Independent lab/second opinion I don't think I would get it done.
-------------------- Suspected Lyme 07 Test neg One band migrating in IgG region unable to identify.Igenex Jan.09IFA titer 1:40 IND IgM neg pos 31 +++ 34 IND 39 IND 41 IND 83-93 + DX:Neuroborreliosis Posts: 5850 | From Kentucky | Registered: Dec 2008
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disturbedme
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Pinelady, I wouldn't have it done either, except for the fact that they are testing me again for a genetic fatal disease. I was negative the first time but now they are telling me that sometimes you can have a negative and still be positive... so they are testing again... I can't really say no to that, after they tell you something like THAT and have you freaked out/scared all over again.
-------------------- One can never consent to creep when one feels an impulse to soar. ~ Helen Keller
My Lyme Story Posts: 2965 | From Land of Confusion (bitten in KS, moved to PA, now living in MD) | Registered: Jun 2007
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