1. Chlamydia pneumoniae-specific CSF-restricted oligoclonal IgG bands are associated to a subset of patients with progressive forms of Multiple Sclerosis
Fainardi E1,3, Castellazzi M2, Seraceni S3, Cultrera R3, Granieri E2, Contini C3.
1Neuroradiology Unit, Department of Neurosciences, Azienda Ospedaliera-Universitaria, Arcispedale S. Anna, Ferrara,
2Multiple Sclerosis Center, Section of Neurology, University of Ferrara
3Section of Infectious Diseases, Department of Clinical & Experimental Medicine, University of Ferrara, Italy

2. Background
Multiple Sclerosis (MS) is a presumed autoimmune chronic inflammatory demyelinating disease of the Central Nervous System (CNS) of unknown etiology.
Modified from Hemmer B et al. Nat Rev Neurosci 2002; 3:
Epidemiological studies suggest that MS pathogenesis could be related to an infection superimposed on a predisposing genetic background.

3. Background
IEF
OCB
Regarding laboratory findings, MS is characterized by the presence of oligoclonal IgG bands (OCB) only in cerebrospinal fluid (CSF) and not in the corresponding serum, reflecting an intrathecal synthesis of IgG sustained by few clones of antibody-secreting B cells sequestrated into the CNS
(Correale J, Bassani Molinas DLM J Neurol 2002; 249: )
Serum
CSF

4. Background
Currently, a growing body of data suggests that C. pneumoniae could cause a chronic persistent brain infection acting as a cofactor in the development of the disease.
Modified from Brunham RC, Rey-Ladino J. Nat Rev Immunol 2005; 2:

5. Aim of the study
As an intrathecal oligoclonal antibody response, directed against the causative agent, is usually present in CNS infections, the aim of this study was to verify the distribution of C. pneumoniae-specific CSF-restricted oligoclonal IgG bands (OCB) in MS patients and controls.

6. Patients
Cerebrospinal fluid (CSF) and serum samples were studied from:
41 MS patients: 27 relapsing-remitting (RR), 9 secondary progressive (SP) and 5 primary progressive (PP), grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity.
Sex: 26 females, 15 males
Age (years): 37.8 ± 11.5
Duration of the disease(months): 35.4 ± 51.3 (range = 0-246)
EDSS: 2.3 ± 1.3 (range = 0-6.5)

7. Controls
21 patients with other inflammatory neurological disorders (OIND)
12 females; 9 males; age (years) 50.3 ± 16.1
21 patients with non-inflammatory neurological disorders (NIND)
11 females; 10 males; age (years) 52.8 ± 14
To avoid confounding factors, patients with HIV encephalopathy (OIND), Alzheimer’s disease and cerebrovascular diseases (NIND) were excluded from the study

8. Methods
CSF and serum levels of anti-C. pneumoniae IgG were measured by ELISA technique (Eurospital, Trieste-Italy, cat. Num. 9267).
Reference curve was generated in each assay using the standard serial dilutions by plotting the standard well concentrations, expressed as arbitrary units (AU), versus the OD values.
CSF and serum samples were assayed 1:2 and 1:400 diluted, respectively

9. Methods
Quantitative intrathecal synthesis of anti-C. pneumoniae IgG was determined by Antibody Specific Index (ASI).
The Antibody Specific Index is the ratio between CSF/serum quotient of the specific antibody (Qspec) and the total CSF/serum immunoglobulin quotient, (QIgG), i.e. AI = Qspec / QIgG .
In case of a large intrathecal immunoglobulin synthesis (QIgG > QLim ), QSpec refers to the upper limit of the reference range (QLim ) with
AI = QSpec /QLim to avoid false-negative results.
Abnormal ASI values were considered as ASI > 1.5.

10. Methods
In all patients with C. pneumoniae-specific intrathecally produced antibodies, the presence of C. pneumoniae-specific CSF OCB was assessed by affinity-mediated immunoblot (AMI).
Serum
CSF
Overnight
incubation NC + CP
antigen NC +
Agarose
gel
Nitrocellulose
sheet
Sonicated C.P. EB
IEF run: 75 min.
Affinity mediated immunoblot
Anti-CP specific IgG OCB visualization
IgG immuno-staining

11. Statistic analysis
Statistical analysis was performed by Mann-Whitney U and Chi-square tests (χ2). Bonferroni correction was used for multiple comparisons

12. Results
p =
p = 0.06
Detectable levels of CSF and serum anti-C. pneumoniae IgG were more frequent in total MS and in OIND than in NIND patients.
However, no significant differences were found for these variables among the various groups.

13. Results Serum anti-CP IgG (AU) CSF anti-CP IgG (AU)MS
OIND
NIND MS
OIND
NIND
Accordingly, CSF and serum anti-C. pneumoniae IgG mean levels were higher in total MS and in OIND than in NIND patients.
Also in this case, no significant differences were found for these values among the various groups.

14. Results: Antibody Specific Index (ASI)
ASI values suggestive of C. pneumoniae-specific intrathecal IgG synthesis were present in a small proportion of MS (12/41; 29.3%), OIND (7/21; 33.3%) and NIND (1/21; 4.8%) patients and were significantly more represented (p < 0.05) in total MS and in OIND than in NIND and in SP (p < 0.01) and PP MS (p < 0.05) than in RR MS.

15. Results
No additional statistical differences were observed for CSF and serum anti-C. pneumoniae IgG mean concentrations as well as for ASI values among RR, SP and PP MS and between MS patients with and without clinical and MRI evidence of disease activity.

16. Results: affinity-mediated immunoblot
SP1
SP2
SP3 PP RR
Serum
CSF
Serum
CSF
Serum
CSF
Serum
CSF
Serum
CSF
Among the patients with intrathecally produced anti-C. pneumoniae IgG, C. pneumoniae-specific CSF-restricted OCB were detected only in 3 patients with SP, 1 with PP and 1 with RR MS

17. Conclusions
These findings confirm that the presence of an intrathecal humoral immune response to C. pneumoniae is not selectively restricted to MS, but is shared by several inflammatory neurological conditions.
In addition, our results suggest that an intrathecal production of C. pneumoniae-specific oligoclonal IgG can occur in a subset of patients with MS progressive forms in which a C. pneumoniae chronic persistent brain infection may play an important pathogenetic role.

18. Thank you for your attention