Chlamydia pneumoniae and multiple sclerosis: no significant association

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Research Update

TRENDS in Microbiology Vol.9 No.4 April 2001

Chlamydia pneumoniae and multiple sclerosis: no significant association Jean C. Tsai and Donald H. Gilden The cause of multiple sclerosis (MS) is unknown. Despite indications from epidemiological and identical-twin studies that MS is infectious, no virus or other infectious agent has been tightly linked to disease. The isolation of Chlamydia pneumoniae from the cerebrospinal fluid (CSF) of MS patients and the detection of both Chlamydia-specific DNA and antibody in MS CSF have been reported. Other analyses of brain and CSF have shown no significant difference in C. pneumoniae-specific DNA or antibody between MS and control subjects. Recent work has revealed intrathecal production of C. pneumoniae-specific IgG in only 24% of MS patients compared with 5% of control patients. More importantly, the major CSF oligoclonal bands from MS patients did not react to C. pneumoniae.

Multiple sclerosis (MS) is a common, exclusively human, chronic demyelinating disease of the brain and spinal cord that occurs in nearly all parts of the world. In the USA alone, approximately 300 000 individuals are afflicted. Common neurological features of MS include weakness, tingling and sometimes pain in the extremities or trunk; disturbances of vision, speech and gait; bladder and bowel incontinence; impotence; and impairment of memory and other higher cognitive functions. In patients under age 40, MS attacks are most commonly relapsing–remitting, but after age 40, neurological disease is usually progressive. Although the cause of MS is unknown, two leading theories have emerged, one implicating infectious agents, probably viral, and the other implying that the disease is produced by a host immune response to an infectious agent or autoantigen. The mechanisms proposed include: (1) lysis of oligodendrocytes (which produce myelin) by a replicating pathogen; (2) destruction of infected cells by the immune system; (3) non-specific (bystander) damage to myelin by an activated immune system; and (4) autoimmunity related or unrelated to infection. The etiology of MS is a complex question, given that combinations of these putative

mechanisms could contribute to the clinical presentation of the disease. Microorganisms and MS

No less than 17 different microorganisms have been associated with MS, but none has been tightly linked to disease. Many of the viruses implicated cause persistent or latent infections and can reactivate, raising the additional question of how virus at different stages of infection interacts with the immune system. In the past decade, infections with two human herpesviruses have been associated with MS: Epstein–Barr virus (EBV) infection, which causes infectious mononucleosis, can be followed by demyelinating disease1, and DNA and antibody directed against human herpesvirus 6 (HHV-6), the cause of roseola, have been detected in MS brain and cerebrospinal fluid (CSF)2. The detection of fingerprints of these two ubiquitous viruses known to be latent in blood B- (EBV) or T- (HHV-6) cells is intriguing as the primary encounter with either virus usually occurs before or during puberty, the same time that epidemiological evidence indicates MS patients acquire the disease-causing agent3. However, HHV-6 is present not only in brain and CSF of MS patients, but also in neoplastic and normal brain tissue4, suggesting that the detection of the virus reflects its reactivation from latency in blood T cells trafficking through the brain. Furthermore, the results of an in situ hybridization study for EBVspecific RNA in ten MS brains were entirely normal5. Finally, the mechanism by which reactivated virus can cause disease is unknown. Overall, conclusive evidence for a causal relationship between any virus and MS is lacking. Chlamydia pneumoniae and MS

The latest organism to be implicated in MS is Chlamydia pneumoniae, a Gramnegative, obligate intracellular bacterium. The genus Chlamydia consists of two human pathogens: C. pneumoniae and Chlamydia trachomatis. A third species, Chlamydia psittaci, primarily infects birds. C. pneumoniae infection occurs both

endemically and in epidemics, causing mild respiratory symptoms and occasionally atypical pneumoniae. Serum antibody can often be found at five years of age, and titers climb rapidly until age 15 (Refs 6,7). In the general population, ~50% of adults are seropositive for C. pneumoniae by middle age8. Much about the life cycle of Chlamydia is interesting and relevant to its possible role in MS. It attaches to a putative hostcell ligand as an infectious elementary body, is then taken up by endocytosis, and converts to a non-infectious, reproductive reticulate body. Inclusions of bacteria are released during host cell lysis. Chlamydia is also thought to persist in macrophages, and probably contributes to the chronic course of most chlamydial infections9. Cell-culture models have revealed that surviving C. psittaci-infected cells are resistant to superinfection and express changes in membrane surface proteins10,11. Persistent Chlamydia can exhibit low metabolic activity, especially in an immune-active environment, and growth is restricted in infected cells treated with interferon (IFN)-γ (Refs 12,13). Isolation and identification of C. pneumoniae

The standard methods to identify C. pneumoniae are cell culture, PCR amplification of bacterial DNA, and detection of bacteria-specific antigen or antibody. Yet these tests have not been consistently successful in diagnosing acute infection. Intracellular C. pneumoniae replicates poorly, partly as a result of conversion to a non-infectious reticulate body that does not grow in cell culture. Furthermore, Chlamydia is readily inactivated in tissue during freeze–thaw cycles. Serum antibodies are detected by microimmunofluorescence (MIF), complement fixation (CF) and ELISA. Although MIF is generally considered to be specific and sensitive, detection of antibody in CSF by MIF has been problematic14 and requires an experienced microscopist. In 1999, Sriram and colleagues reported the isolation of C. pneumoniae from the CSF of 65% of MS patients

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Research Update

compared with 11% of controls afflicted with other neurological disease; they also detected antibody to Chlamydia in the CSF of MS patients, and amplified the Chlamydia major outer membrane protein (MOMP)-encoding gene by nested PCR from the CSF of 97% of MS patients compared with 18% of controls15. However, despite extensive recent efforts worldwide to confirm the association of Chlamydia infection with MS, the accumulated data do not support the findings of Sriram (Table 1). Layh-Schmitt et al.16 isolated Chlamydia from two of eight MS CSF samples, but found no significant increase in serum IgG by MIF. Those investigators also successfully amplified the ompA gene from the CSF of only 23% of MS patients, a four-times lower incidence than that reported by Sriram et al.15 Two additional analyses revealed no correlation between C. pneumoniae infection and MS (Refs 17,18). Neither study detected amplifiable C. pneumoniae DNA in MS or control CSF, or peripheral blood mononuclear cells. Serum IgG against C. pneumoniae was detected in a similar proportion of MS and control samples, and the paired serum:CSF antibody ratio was greater than 100:1 in both groups. No complement-fixing antibody to C. pneumoniae was found, and Boman’s laboratory could not isolate C. pneumoniae from MS CSF. Other studies analyzed paraffinembedded or cryogenically preserved brain and CSF (Refs 19,20). Using PCR techniques similar to those of Sriram et al.15, another developed by Meijer et al.21, or the procedures of Tong and Sillis22, none of the investigators was able to amplify either the C. pneumoniae 16s-rDNA or the MOMP-encoding gene from MS or control brains and CSF. Controls were rigorous, and included nonMS neurological disease brains and CSF, as well as brains from patients without neurological disease, or trauma patients without any documented disease. Proper controls appeared to ensure the reproducibility and sensitivity of the PCR techniques and to validate the negative results. For example, Hammerschlag and colleagues19 designed weakly positive controls using purified bacteria and tissue samples spiked with supernatants from infected cells, thus ruling out potential PCR inhibitors. Furthermore, the Chlamydia Research Laboratory at SUNY http://tim.trends.com

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Table 1. Current research on the relationship between Chlamydia pneumoniae and MSa Samples

Controls

PCR

Antibodies

Culture

Ref.

37 CSFs

27 ONDs

MOMP gene MS 97% ONDs 18%

ELISA MS 86%

MS 65% ONDs 11%

15

30 CSFs + 17 possible MS

56 ONDs

ompA gene 7/30 (p< 0.001)

MIF Not significant

MS 2/8 No ONDs

16

48 CSFs

51 ONDs

All negative

MIF MS 68% ONDs 82%

All negativeb

17

10 brains (paraffin embedded) 27 CSFs

10 OND brains 36 OND CSFs

16s-rDNA All negative

ND

ND

20

29 CSFs 7 ONDs + blood mononuclear cells

MOMP gene All negative

C. psittaci antigen All negative by CF in CSF

ND

18

25 brains (55 frozen samples)

5 ONDs 11 nonneurological disease controls

All negative for MOMP gene (confirmed by Ref. 17)

ND

All negativeb

19

Serum/CSF 46 definite MS 12 possible MS

35 OINDs 27 ONDs

ND

C. pneumoniae specific-IgGc 24% MS, 5% controls

ND

24

aAbbreviations:

CF, complement fixing; CSF, cerebrospinal fluid; MOMP, major alternative membrane protein; MS, multiple sclerosis; ND, not determined; OINDs, other (non MS) inflammatory neurological diseases; ONDs, other neurological diseases. bAnalysis performed in the Chlamydia Research Laboratory at SUNY, Brooklyn. cThe major CSF oligoclonal IgG bands from MS patients with intrathecal IgG-production to C. pneumoniae did not react to C. pneumoniae by IEF-western as seen by isolectric focusing and subsequent affinity-mediated immunoblot (IEF-western) towards purified elementary bodies and reticulate bodies of C. pneumoniae.

Brooklyn failed to isolate C. pneumoniae from any of the brain samples sent by Hammerschlag’s laboratory19. Concluding remarks

One technique that has successfully linked infectious agents to neurological diseases is the ability to demonstrate that the oligoclonal bands (OGBs) in CSF are specific for the infectious agent causing the disease (reviewed in Ref. 23). Most recently, the possible role of C. pneumoniae in MS was studied not only for the presence of intrathecal IgG production against C. pneumoniae but also to determine whether the oligoclonal IgG in MS CSF was directed against C. pneumoniae. Analysis of serum/CSF samples from 120 subjects revealed intrathecal production of C. pneumoniaespecific IgG in 24% of MS patients, compared with 5% of control patients with other inflammatory or non-inflammatory disease. More importantly, the major CSF oligoclonal bands from MS patients did not react to C. pneumoniae24. The same studies also showed that the IgG in the CSF of control patients with neuroborreliosis

reacted strongly with Borrelia burgdorferi. It can be concluded that, although a trial is under way to treat MS patients with the antibiotic roxithromycin25, a rapidly accumulating body of research in the past 1–2 years does not support the original findings of Sriram et al.15 Acknowledgements

We thank Cathy Allen for manuscript preparation. References 1 Bray, P.T. et al. (1992) Demyelinating disease after neurologically complicated Epstein–Barr virus infection. Neurology 42, 278–282 2 Challoner, P.B. et al. (1995) Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc. Natl. Acad. Sci. U. S. A. 92, 7440–7444 3 Kurtzke, J.F. (1965) On the time of onset in multiple sclerosis. Acta Neurol. Scand. 41, 140–158 4 Cuomo, L. et al. (2001) Human herpesvirus 6 infection in neoplastic and normal brain tissue. J. Med. Virol. 63, 45–51 5 Hilton, D.A. et al. (1994) Absence of Epstein–Barr virus RNA in multiple sclerosis as assessed by in situ hybridisation. J. Neurol. Neurosurg. Psychiatry 57, 975–976 6 Aldous, M.B. et al. (1992) Seroepidemiology of Chlamydia pneumoniae TWAR infection in

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Research Update Seattle families, 1966–1979. J. Infect. Dis. 166, 646–649 Camm, A.J. and Gupta, S. (1999) Chronic Infection, Chlamydia and Coronary Heart Disease, Kluwer Academic Publishers Kuo, C. et al. (1995) Chlamydia pneumoniae (TWAR). Clin. Microbiol. Rev. 8, 451–461 Barron, A.L. (1988) Microbiology of Chlamydia, CRC Press Moulder, J.W. et al. (1981) Attachment defect in mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci. Infect. Immun. 34, 285–291 Moulder, J.W. et al. (1982) Association between resistance to superinfection and patterns of surface protein labeling in mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci. Infect. Immun. 35, 834–839 Byrne, G.I. and Faubion, C.L. (1982) Lymphokine-mediated microbistatic mechanisms restrict Chlamydia psittaci growth in macrophages. J. Immunol. 128, 469–474 de la Maza, L.M. et al. (1985) The anti-chlamydial and anti-proliferative activities of recombinant murine interferon-α are not dependent on tryptophan concentrations. J. Immunol. 135, 4198–4200 Koskiniemi, M. et al. (1996) Chlamydia pneumoniae associated with central

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nervous system infections. Eur. Neurol. 36, 160–163 Sriram, S. et al. (1999) Chlamydia pneumoniae infection of the central nervous system in multiple sclerosis. Ann. Neurol. 46, 6–14 Layh-Schmitt, G. et al. (2000) Evidence for infection with Chlamydia pneumoniae in a subgroup of patients with multiple sclerosis. Ann. Neurol. 47, 652–655 Boman, J. et al. (2000) Failure to detect Chlamydia pneumoniae in the central nervous system of patients with MS. Neurology 54, 265 Pucci, E. et al. (2000) Lack of Chlamydia infection of the central nervous system in multiple sclerosis. Ann. Neurol, 48, 399–400 Hammerschlag, M.R. et al. (2000) Is Chlamydia pneumoniae present in brain lesions of patients with multiple sclerosis? J. Clin. Microbiol. 38, 4274–4276 Morré, S.A. et al. (2000) Is Chlamydia pneumoniae present in the central nervous system of multiple sclerosis patients? Ann. Neurol. 48, 399 Meijer, A. et al. (1998) Detection of microorganisms in vessel wall specimens of the abdominal aorta: development of a PCR assay in the absence of a gold standard. Res. Microbiol. 149, 577–583 Tong, C.Y. and Sillis, M. (1993) Detection of Chlamydia pneumoniae and Chlamydia psittaci

in sputum samples by PCR. J. Clin. Pathol. 46, 313–317 23 Gilden, D.H. et al. (1996) The search for virus in multiple sclerosis brain. Multiple Sclerosis 2, 179–183 24 Derfuss, T. et al. Intrathecal antibody production against Chlamydia pneumoniae in multiple sclerosis is part of a polyspecific immune response. Brain (in press) 25 Treib, J. et al. (2000) Multiple sclerosis and Chlamydia pneumoniae. Ann. Neurol. 47, 408

Jean C. Tsai Dept of Microbiology and the Neuroscience Graduate Group, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104, USA. Donald H. Gilden* Depts of Neurology and Microbiology, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Mail Stop B-182, Denver, CO 80262, USA. *e-mail: [email protected]

Techniques & Applications

It’s easy to build your own microarrayer! Arthur Thompson, Sacha Lucchini and Jay C.D. Hinton DNA microarrays are becoming the tool of choice for microbial gene-expression profiling and genotypic analysis. The construction of a gridding robot for the ‘in-house’ production of microarrays is a choice worth considering, and offers distinct advantages over other options in terms of cost effectiveness and scale. Having built our own robot, we want to dispel some of the myths that might be associated with such a project, as well as provide practical advice for potential builders in the UK and Europe.

Microarrays are becoming the pre-eminent technology for the investigation of functional genomics. Many laboratories and research centres are deciding whether to invest in microarraying technology by setting up ‘in-house’ facilities or purchasing commercial pre-printed arrays, which can cost ~£500 each. One factor that can influence this decision is that a significant number of microarrays is required to obtain one set of publishable data. It is not possible to re-use microarrays for fluorescent DNA applications, and the results from several

microarray experiments are required to produce robust data1. Microarrays are produced by specialized gridding robots2, which are readily available from several US and European companies for upwards of £50 000. The machines differ in terms of the accessories, which include the type and number of print pins (4–48), the number of slides that can be printed in a single run, and the inclusion of cooled plate stackers, and humidity or temperature controls. An attractive alternative is the construction of your own microarraying robot. This is surprisingly easy, and offers a cheap and effective way to produce microarrays at a density of up to 40 000 spots per microscope slide. The total cost to build this robot is in the region of £18 000 plus the cost of at least 20 printing pins (£100 each). Plans for this robust and accurate basic machine, which was designed by Joe DeRisi and Pat Brown at Stanford University, are freely available at: http://cmgm.stanford.edu/pbrown/mguide/ index.html. The site includes clear building instructions, and the details of suppliers of specific parts, as well as the software (free to academic institutions and available at a

small cost to industrial concerns) for programming the microarraying robot and the analysis of microarray data. Inspiration

We are currently one of the two institutions within the UK to have built our own DNA microarrayer according to the Stanford design, the other location being at the MRC Toxicology Unit, University of Leicester (http://www.le.ac.uk/cmht/twg1/ array-fp.html). The construction of our microarrayer was inspired by a Cold Spring Harbor course attended by A.T. from 20 October to 2 November 1999 entitled ‘Making and Using DNA Microarrays’, where a group of 16 participants constructed four microarrayers within two days3. This article is intended to address the reservations held by most molecular biologists about pursuing a DIY project of this type. We hope to dispel the impression that self-build is an intimidating undertaking. A ‘state of the art’ engineering workshop is certainly not required; all that is needed is a little patience, and some familiarity with the use of a screwdriver and wire stripper. As Joe DeRisi says ‘a

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