1^st Workshop of the International society of Behcet's disease (ISBD) on Pathophysiology and Treatment of Behçet’s Disease, Kühtai, AUSTRIA, April 2-5, 2003

Introduction

C.G. Barnes (UK), President of the International Society for Behçet’s Disease (ISBD)

For this meeting of two Working groups of the ISBD, the background to our present knowledge is reviewed, including: (1) The original description of BD by Hippocrates in the 5th century BC and the 20th century description with the adoption of the eponymous title of "Behçet’s Disease", (2) the lack of a specific diagnostic test but noting the pathergy test and the association with HLA-B51, (3) the description of the multisystem manifestations of the disease and the prevalence of these manifestations including the differences in different parts of the world, and (4) the pathological classification of BD as a vasculitis.

Nevertheless the diagnosis and classification of the condition depends on the acumen of the physician in diagnosis of the individual patient in the routine clinical situation, and the classification of groups of patients with the disease for inclusion into clinical studies and trials. The older “diagnostic schemes” (e.g. Japanese, Mason & Barnes, O’Duffy, Dilsen) should be discarded and the International Classification of BD used as an entry criterion for clinical studies and trials.

The natural history of BD still requires further study with particular reference to being able to predict the type of disease (mucocutaneous, ocular, neuro- etc), the localization of manifestations, the likelihood of recurrences and their duration, and the overall severity of the disease in the individual patient.

Basic research – pathogenesis (? bacterial), immunology (e.g. CD4+ CD28- T cells), and gender association (prevalence, severity and response to treatment) – must continue and may procede more quickly if coordinated on a multi-center, multi-national basis.

Therapeutic trials continue but to date there are surprisingly few controlled studies (e.g. colchicine, azathioprine and interferon a). It is imperative that future trials be properly controlled, and open trials reserved for very preliminary “pilot” studies. Controlled trials (either single or double blind; versus placebo or other comparison drug) are required at present in particular for: IFN a - optimum dosage regimen with regard to both efficacy and tolerance (side effects), anti TNF a, and possibly of antibiotics. Again these may be progressed faster on a coordinated multi-center, multi-national basis but with the prior determination of updated entry criteria and outcome measures.

The ISBD, as a medical research society, and through its Working Groups, should be a vehicle for the organization and co-ordination of studies – single center, multi-center in a single country or multicentre on a multinational basis. The Working Groups for Clinical trials and Treatment and for Basic Research should work towards the co-ordination of clinical studies and trials with agreed: (1) entry criteria – diagnostic classification and disease manifestations & activity, (2) outcome measures, and (3) protocols including statistical methodology. Multinational coordination through the ISBD may lead to the necessary availability of appropriate funding and secretarial assistance for such trials and studies.

1. THE Clinical FOCUS

Input and discussion of the group after the lectures are added in cursive style.

Perspectives of the "Working group on drug trials including collaborative trials" of the ISBD and rationale for new clinical trials

S. Assaad-Khalil, Head of the Working Group, University Hospital, Alexandria, Egypt

The goals of the working group are (1) to contact and recruit members, (2) to contact other working groups and propagate their work particularly in the context of established criteria of diagnosis and disease activity in Behçet’s Disease (BD), (3) to exchange experience via direct contact, home page, journal and/or newsletter, (4) to plan collaborative trials, (5) to contact pharmaceutical companies and research institutes for possible cooperation in clinical trials, (6) to prepare a workshop on Clinical Trials at ISBD congresses, and (7) to support and alert patients concerning treatment modalities.

Additional clinical trials are needed. From both the physicians' and the patients' perspective, current therapy does not satisfy the goals of treatment, e.g. preventing blindness in ocular BD, preventing morbidity and mortality of neurologic and vascular events, improving the quality of life. Serious adverse effects may result from current therapies. Prof. S. Assaad-Khalil reported the results of a retrospective evaluation of therapeutic agents commonly used in BD, which he had recently carried out in 127 patients (110 males, 17 females; 20 – 65 years old) randomly selected from the Alexandria BD registry to assess the efficacy of different therapeutic agents in daily practice for a mean duration of 11.07 years by the same group of observers. A special focus was made on the ocular sequelae of the disease, with detailed ocular documentation carried out in 254 eyes (visual acuity, intraocular pressure, state of the lens, presence of uveitis, retinal vessels and optic nerve). 16.6% of BD patients lost effective vision and another 17.6% of the patients resulted in reduced vision lower than 6/60. Uveitis was found in 37.4%, lens opacity in 44.9%, retinal vessel affection occurred in 23.3% and optic nerve affection in 29.9%. In conclusion, it can be seen that eye sequelae are still very devastating in BD, with a delay in diagnosis having a significant deleterious effect on the outcome of the disease. There is still no ideal therapeutic regimen resulting in full remission of BD and preventing its sequelae. At present, combination therapy seems to be the most appropriate approach when considering efficacy and safety. However, there is a great need for a controlled and masked multicenter trial to re-evaluate the efficacy and safety of the different therapeutic modalities on a long term basis.

Investigators must continue to have strong ethical commitments to patients in designing clinical trials. Trials serving patients, patients' life, health and well-being is our goal. There should be actual representation of the patients in the design of the clinical trials. A prospective study design, predetermined specific primary end points and blinding should ensure the integrity of the study. Improvement should be quantified as objectively and accurately as possible, putting into consideration patient's quality of life, and measuring all adverse effects. Statistics should depend on strict criteria for statistical significance, avoiding post-hoc analysis.

Indeed there are other experiences with similar results of at least 30% non-responsiveness to current therapy in clinical routine practice. There is a need for unified protocols, coordination of protocols via the working group, unified activity quantification and unified criteria of effectiveness.

ABSTRACTS OF PRESENTATIONS

Cytotoxic Drugs in ocular lesions of Behcet's Disease

F. Davatchi, Shahram F, Chams H, Nadji A, Jamshidi AR, Chams C, Akbarian M, Gharibdoost F, Sedigh M, and Sadeghi B., Behcet’s Unit, Rheumatology Research Center, Tehran University for Medical Sciences, Tehran, Iran

Introduction: Cytotoxic drugs are the first line treatment for ophthalmologic manifestations of Behcet’s Disease (BD) despite the advent of the new biological agents. The latter are to be used in intractable inflammatory attacks as they seem to be efficient in few case studies. Cytotoxic drugs are affordable, easy to use, effective and safe, even in long-term use. They have to be combined to steroids (0.5 mg prednisolone/kg/day as attack dose, then tapering gradually to the patient’s need). All the cytotoxic drugs that were used were effective (ACR 1966, APLAR 2000): Cyclophosphamide was used as oral (OCP, 2 mg/kg/daily), classic pulse (PCP, 1 g/m²/monthly), or low dose pulse (LDP, 0.5 g/m²/monthly). Weekly methotrexate was used as 7.5 mg/weekly (MTX) or 15 mg/weekly (HMTX). Chlorambucil was used as 0.2 mg/kg/daily (CHL), Cyclosporin as 5 mg/kg/daily (CyA), and Azathioprine as 2 mg/kg/daily (AZA). Combination therapy with LDP and MTX or with LDP and AZA was also used. A double blind control study of PCP with steroids versus steroids alone showed that the combination of PCP and steroids were significantly more effective than steroids alone.

PCP was used in 335 patients, LDP in 182, OCP in 39, MTX in 349, HMTX in 33, CHL in 87, CyA in 22, AZA in 113, LDP-MTX in 97, and LDP-AZA in 19 patients. The mean improvement of Visual Acuity (VA) was 0.5/10 (Snellen Chart) with PCP, 0.8 with LDP, 1.1 with OCP, 0.7 with MTX, 0.8 with CHL, 1.6 with CyA, 1.2 with AZA, 0.4 with HMTX, 0.5 with LDP-MTX, and 1.1 with LDP-AZA. The percentage of improved eyes were 45% for PCP, 52% for LDP, 50% for OCP, 46% for CHL, 47% for CyA, 59% for AZA, 55% for HMTX, 48% for LDP-MTX, and 58% for LDP-AZA.

As all treatment methods with cytotoxic drugs were efficient and had approximately the same efficiency (percentage of eyes with improved VA), all data were pulled together. The advantage to put the data together was to show the result of treatment in long run in the real life. Some patients who were resistant to the given treatment were switched to another treatment and if again non effective to a third or forth treatment. In pulled data the results before the first treatment were compared to those after the last treatment.

Materials and Methods: Patients who had an active posterior uveitis and/or retinal vasculitis were selected for this study. They were 978 patients. Among them, 277 received more than one treatment. The mean duration of eye lesions was 54 months (SD 42.1), with the maximum duration of 271 months. The mean follw-up time was 52 months (SD 40.8) with the maximum of follow up of 261 months. Comparison was made by the Student paired t test. Confidence interval (CI) at 95% was calculated for percentages. VA was calculated on a Snellen chart on a scale of 10 on 10. The Activity Indexes (AI) for different compartment of eyes were calculated according to Ben Ezra.

Results: The mean VA of all eyes was 3.8. It improved to 4.7 after the treatment (t 9.544, p< 0.000001). Improved eyes were 52% (CI 2.4), 18% were unchanged, and 30% were aggravated. The mean AI of anterior uveitis improved from 2.5 to 0.8 (t 18.595, p< 0.000001). Improved eyes were 77% (CI 2.7), 4% were unchanged, and 19% were aggravated. The mean AI of posterior uveitis improved from 2.1 to 0.9 (t 27.039, p< 0.000001). Improved eyes were 74% (CI 2.3), 10% were unchanged, and 16% were aggravated. The mean AI of retinal vasculitis improved from 2.5 to 1.4 (t 11.661, p< 0.000001). Improved eyes were 62% (CI 2.9), 12% were unchanged, and 216% were aggravated.

To see if the cytotoxic drugs maintained their efficacy over the time, patients were divided in different groups according to their treatment duration. The percentage of improved eyes remained the same, even in the group of patients where the duration of treatment exceeded 9 years.

Conclusion: The least improved parameter was the visual acuity, which reflects not only the inflammatory index of the eye, but also the chronicity index (cataract, vitreous organization, hemorrhage, vessel necrosis of retina, neovascularization, and optic nerve atrophy). Seventy percent of the eyes improved or maintained their VA, which is quite remarkable for this disease.

Prior treatments could affect the results of a survey of drug combinations. Prospective studies could support the findings of this survey performed in patients of clinical routine practice.

The use of interferon-alfa in Behcet`s Disease –Review of the Literature

I. Kötter, I. Günaydin, M. Zierhut, N. Stübiger, University Hospital, Dept. of Internal Medicine II and Ophthalmology II, Tübingen, Germany

Objective: To evaluate the efficacy and safety of interferon-alfa for the treatment of Behçet`s Disease and discuss its possible mechanisms of action.

Methods: Reports published until July 2002 in all languages were identified by the PubMed database and the Behçet`s Disease conference proceedings and abstract booklets. The indexing items used were Behçet and interferon.

Results: Thirty-two original reports and four selected abstracts were included in the analysis. Systemic IFNalfa was administered to 405 patients. Two hundred and sixteen patients with acute ocular disease were treated with IFNalfa. Two hundred and ninety eight patients received IFN alfa2a, 141 IFNalfa2b. 85.6% of the patients with mucocutaneous symptoms, 95.8% with arthritis and 95.6% with uveitis exhibited a partial or complete response. Higher IFN doses were more effective than low dose regimens and led up to 56% long term remissions after dicontinuation of IFNalfa. IFNalfa2a apparently was superior to IFNalfa2b with more complete remissions, but this probably was due to a bias caused by the larger number of patients treated with IFNalfa2a. Side effects were dose dependent and similar to those occurring in patients with hepatitis C.

Conclusions: Although the comparability of the studies is hampered due to different study designs, it can be concluded, that IFNalfa is effective for the treatment of BD. It was effective even in resistant posterior uveitis, where long-term remissions with preservation of visual acuity could be achieved. In contrast, for mucocutaneous symptoms, ,only partial remissions were reported.

There is still concern about the high prevalence of possible side effects and the different dosages when compared to the experience from Berlin. There is agreement about using the BenEzra uveitis score for future studies.

Efficacy of recombinant human interferon-alfa2a on ocular and extra-ocular manifestations of Behçet`s Disease and influence on cells of the immune system – Results of an open four center trial

I. Kötter, M. Treusch, R. Vonthein, M. Zierhut, A. Eckstein, T. Ness, I. Günaydin, B. Grimbacher, S. Blaschke, H.H. Peter, N. Stübiger, University Hospital, Dept. of Internal Medicine II and Ophthalmology II, Tübingen, Germany

Background: Behçet`s Disease (BD) is a multisystem vasculitis of unknown origin. Standard treatment comprises systemic immunosuppressive agents. In a study primarily designed for refractory ocular disease we additionally evaluated the efficacy of recombinant human interferon-alpha2a (rhIFN-alpha2a) for the extra-ocular manifestations.

Methods: Fifty patients were included in the study. RhIFN-alpha2a was applied at a dose of 6 Million units subcutaneously daily. Dose reduction was performed according to a decision tree until discontinuation. Disease activity was evaluated by the Behcet`s Disease Activity Scoring System and the Uveitis Scoring System. In parallel, peripheral blood mononuclear cells (PBMC) from 14 patients and 10 healthy controls were isolated, stained with four different fluorescent dyes and measured with a fluorescence activated cell sorter (FACS). Statistical analysis was performed by ANCOVA and Welsh test.

Results: Response rate of the ocular manifestations was 92%. Visual acuity rose significantly from 0.56 to 0.84 at week 24 (p<0.0001). Posterior uveitis score of the affected eyes fell by 46% in one week (p<0.001). Mean BD activity score fell in a dose-dependent fashion by 1.2 points in the first week (p<0.0001) and from 5.8 to 3.3 at week 24. After a mean observation period of 36.4 months, 17 patients are off treatment and disease free for 29.5 months (mean). In the other patients maintenance dosage is 3 million units 3 times weekly. Whereas extra-ocular manifestations such as genital ulcerations, arthritis and skin lesions remitted under IFN, this was the case only for 36% of oral aphthous ulcers. The lymphocytes subpopulations showed a significant increase of gamma-delta positive T-cells and NK cells in the patients before treatment when compared to healthy controls. Under IFN treatment, they decreases significantly and almost reached the level of the control group. Additionally, monocytes and B cells increased.

Conclusions: RhIFN-alpha2a is effective in ocular BD, resulting in significant improvement of vision and complete remission of ocular vasculitis in the majority of the patients. It is also effective for the extra-ocular manifestations of the disease, although less so for oral aphthous ulcers. A participation of gamma-delta positive T-cells and NK cells in the pathogenesis of BD is implicated, their decrease may explain the mechanism by which IFN-alpha exerts its therapeutic effects, whereas the increase of monocytes and B cells may be responsible for side effects of IFN such as flu-like syndrome and autoimmune phenomena.

There is concern about the side-effects and a consensus on the dosage to use. What about including patients not fulfilling the ISBD-criteria?

Ad hoc presentation of ophthalmologic results of the above mentioned study

M. Zierhut, University Hospital, Tübingen, Germany

The beneficial and the adverse effect of combining IFN with steroids is discussed. Combinations of IFN with other immunosuppressive therapy appears to antagonize the formal effects of the IFN. Overall there are discrepant views in the center of Tübingen and Berlin, both in Germany.

Ad hoc presentation of treatment of ocular BD manifestations with IFN in Berlin

L. Krause, Benjamin Franklin Hospital, Free University of Berlin, Germany

Especially the combination of IFN with high versus low-dose steroids needs further investigation.

TNF & anti-TNF Agents in Behçet’s Disease

K.T. Calamia, MAYO Clinic Jacksonville, FL, USA

The rapid response and effectiveness of anti-TNF agents in the treatment of many immune mediated inflammatory disorders draws comparison to the response of rheumatoid arthritis to cortisone, first witnessed over 50 years ago by Philip Hench and colleagues at the Mayo Clinic. It is now known that many of the anti-inflammatory effects of corticosteroids are due to their inhibition of TNF production. A new dimension of efficacy is possible with TNF agents, however, in that inhibition of disease progression is now possible. Might these agents be considered the corticosteroids of the new mellenium?

Like rheumatoid arthritis and Crohn’s disease, Behçet’s disease is believed to be associated with a Th1-mediated immune response (Frassinito, 1999; Melikoglu, 2002). Increased levels of TNFa are found in Behçet’s disease (Hamzaoui, 1990) and has provided support for the empiric use of anti-TNFa therapies used in a number of published cases and small case series.

Hassard (2001) reported rapid and dramatic improvement in gastrointestinal and extra intestinal symptoms and findings of the disorder after treatment with infliximab. Similar response was seen in two other patients treated by Travis (2001). This experience was followed by the report of Robertson (2001) of a patient free of oral and genital ulcerations for the first time in ten years after 3 infusions of infliximab. Remission of mucocutaneous symptoms for one year followed 2 infusions of infliximab in a patient previously uncontrolled by multiple immunosuppressive agents (Goossens, 2001). Mucocutaneous lesions remitted with infliximab in a patient with Behçet’s disease associated with rheumatoid arthritis (Rozenbaum, 2002). At EULAR 2002, Turkish investigators reported the results of the first double-masked, placebo-controlled study (n=40) of anti-TNF therapy with etanercept in mucocutaneous Behçet’s disease (Melikoglu, 2002). This agent suppressed disease manifestations with resurgence after the drug was discontinued.

Additional recent reports of anti-TNF therapies in Behçet’s disease were presented in 2002 at EULAR, at the 10th International Conference on Behçet’s Disease (ISBD) in Berlin, and at the American College of Rheumatology (ACR) meeting in New Orleans.

The experience with anti-TNFa treatments for the ocular manifestations of Behçet’s disease has been growing and very positive. Sfikakis (2001) reported the benefits of infliximab in 5 patients with panuveitis in Behçet’s disease. This included 2 patients treated with infliximab therapy without an increase in conventional treatment. The follow-up of these 2 patients was reported at the meeting of the ISBD in June 2002. The experience of these Greek investigators was updated in their report at the ACR meeting in October 2002. Their success with infliximab monotherapy for acute ocular inflammation in Behçet’s disease was reported in 14 patients. The authors suggested that the dramatic and rapid response in these patients would favor the use of this agent over conventional therapy. Positive responses to anti-TNF agents in ocular Behçet’s disease have been documented in numerous case reports in the literature or presentations at international meetings.

A recent review has examined the evidence to support a role for anti-TNF therapies in Behçet’s disease (Sfikakis, 2002). Recent additional reports of the use of anti-TNF agents include successful treatment of Behçet’s ileocolitis with infliximab (Kram, 2003) and treatment of recalcitrant cerebral vasculitis in Behçet disease with infliximab (Licata, 2003).

Significant differences exist between currently available anti-TNFa agents, including mechanism of TNFa inhibition, avidity, half life, immunogenicity, ability to bind lymphotoxin (TNFb), ability to bind membrane bound TNFa, as well as the mode and frequency of administration. One or more of these differences may account for the variable efficacy of these agents in certain diseases, such as Crohn’s disease, and the variable side-effect profile of these drugs. We believe that it is appropriate to study all available agents, including adalimumab for their efficacy and safety in Behçet’s disease. The efficacy of these new biologic agents for the more serious manifestations of Behçet’s disease, in particular, should be investigated.

The group agrees that prospective and randomized trials are necessary to evaluate TNF-blockers in BD.

A review on disease activity scores in Behcet`s disease (BD)

M. Baltaci, Department of Dermatology and Venerology, University of Innsbruck, Austria

Currently there are no laboratory markers that correlate well with the clinical findings in BD. Therefore assessment of disease activity has to be based on history of clinical features. There have been attempts to develop disease activity measurements. The scheme in use by H. Yazici (1984; evaluation depends on features found on the day the patient attends clinic), the Iranian Behcet`s disease Dynamic Measure (IBDDAM; F. Davatchi 1991; evaluation depends on accurate history of symptoms up to 12 months prior to the date of assessment) and the European scheme (1991; initially developed in the UK by Chamberlain-Barnes-Silman incorporating features of the scheme used by H. Yazici). In all three forms scoring of disease activity is based on clinical features only.

A study, which compared IBDDAM and European scheme (Bhakta B. et al. In: Godeau P, Wechsler B, Behcet`s disease. Excerpta medica 1993) shows that agreement between clinicians in scoring features was greater when the standard period was 28 days, as in the European form, compared with the Iranian form in which a variable time period is taken. Despite greater variability in scoring with the Iranian form, the opinion of the clinicians was that both forms had good aspects and that an internationally accepted activity form could be derived from them without great difficulty.

In 1994 a workshop was held in Leeds (UK) to arrive at a consensus view about the contents of the activity form with emphasis on the need for clarity and consistency for potential use by clinicians worldwide. The Behçet's Disease Current Activity Form (BDCAF) was developed, which depends on

accurate history of clinical features present during the month prior to the date of assessment. Clinical features in BD vary considerably over time; in order to document this variation, new clinical features present over the preceding 28 days are scored. This represents a compromise between assessing disease activity based on clinical features on day of assessment, which may be unrepresentative of overall disease activity and clinical features present over a longer time period, as in the IBDDAM, which reduces reliability in terms of accurate recall of symptoms by patients. In BDCAF disease activity rating for oral and genital ulceration, and skin lesions relies solely on the duration of symptoms and does not take into account the size or number of lesions present (which might also reflect activity).

A study fom Bhakta BB et al (Rheumatology 1999) shows that BDCAF is an easy-to-complete and reliable method of assessing and documenting clinical activity in BD-patients for use in routine clinical practice, that it has a good interobserver reliability for general disease activity and that there is difficulty in reliable scoring of uncommon manifestations such as large-vessel involvement, GI inflammation and nervous system involvement.

A Turkish study (Hamuryudan et al. Rheumatology 1999) to examine interobserver and intra-observer reliability of the Turkish version of BDCAF shows a good intra- and interobserver agreement for oro-genital ulcers and eye involvement, a poor agreement between and within observers for their overall impression and individual low kappa scores for erythema nodosum, vascular -, CNS - and gastrointestinal involvement. A local disease activity index (Lee ES et al. Book of Abstracts. 10th International Congress on Behçet's disease, Berlin, 2002) was developed by Koreans that attempted to overcome cultural differences. It also excludes any terms which could be biased, such as fatigue or headache.

Although changes in body weight and temperature may indicate systemic activity, they were neither specific nor sensitive markers for disease activity. Also hemoglobin and erythrocyte sedimentation rate do not correlate with activity. Other possible markers for disease activity could be sIL2-R (Alpsoy E et al. J Dermatol 1998), IL-8 (Zouboulis CC et al. Arch Dermatol Res. 2000), plasma lipoprotein a (Gurbuz O et al. Eur J Ophthalmol 2001), serum beta 2-microglobulin (Aygunduz M et al. Rheumatol Int 2002) and others.

For the future we propose a setting of a general clinical score, an organ specific clinical score, laboratory assessments and imaging techniques (like MR for CNS involvement, CT/MR-angiogram, ¹⁸F-FDG-PET for vasculitis, ...), which enables us to distinguish between chronic or degenerative changes and active lesions whenever possible. Disease related overall damage would be the total of the cumulative old damage and the damage by active disease.

There is consensus agreement to use the BenEzra activity form in future trials of ocular manifestations of BD. There is a need of a new and simpler, yet comprehensive activity form. Fatigue may be an adverse effect of therapy (such as IFN) and thus should not be put in the activity form, whereas quality of life and subjective patients' opinions should be integrated. More details are to be included in BD patients with elevated erythrocyte sedimentation rate of unknown origin.

Presentation of study porposals

Proposals for studies in various stages of preparation were presented to the group for discussion. These included a proposal to further evaluate the efficacy and safety of an anti-TNF agent in BD in a controlled, multi-center, multi-national trial, presented by K. Calamia, USA. S. Assaad-Khali, Egypt, enlisted the opinions of the group to assist in his preparation of a protocol to investigate the effects of another anti-TNF agent in the treatment of ocular manifestations of BD. In both cases, successful acquisition of funding for these studies remains a critical component for success. A planned, multi-center study comparing IFN-a to cyclosporine in ophthalmic disease was presented by I. Kötter, Germany.

2. THE Basic Research FOCUS

Behçet’s Disease: From innate to adaptive immunity

H. Direskeneli, Head of the Basic Group, Department of Rheumatology, Marmara University Medical School, Istanbul, Turkey

Both innate and adaptive immune systems are activated in Behcet’s Disease (BD) with a pro-inflammatory and Th1 type cytokine profile. BD might be linked to a specific, primary immune abnormality with a genetic mutation effecting an adhesion molecule, a pro-inflammatory cytokine/chemokine or a transcription or regulatory factor, which predisposes to early or more intense neutrophil and T cell responses. Increased neutrophil responses to urate crystals and fMLP or superantigen-drived interferon-g response of T cells suggest a model with this characteristics which also explain the “Pathergy” or “skin urate” tests. MEFV gene mutations, suggested to be specific for Familial Mediterranean Fever, which decrease the expression of an anti-inflammatory protein “pyrine” from neutrophils, are also described in BD from Turkey.

However, adaptive immune system is also crucial in BD with possibly both external (streptoccocal, HSV) and internal antigens driving the pathogenic tissue T cell infiltrations. Heat shock proteins 60 and 70 can also activate innate immune system directly with toll-like receptors-2 and 4 and provide both an early innate activation and prolonged T cell response. The diverse manifestations of BD responding to different therapeutical agents also suggest the role of organ-specific antigens (Retinal-S antigen in uveitis) or genetic predispositions (Factor V Leiden in thrombosis) in different BD clinical subsets.

Better characterisation of pathogenic immune cell-subsets, systemic and local antigens and abnormal cell-activation mechanisms may help to develop more specific and less toxic immuno-therapeutic approaches to still unsatisfactorily treated BD in the future.

Acknowledgement: The studies by the author and his collaborators are supported with grants from Turkish Scientific and Technical Council (TUBITAK) and Marmara University Research Funds.

The question arises what component of all the interesting immunological theories effects most the clinical outcome of Behcet's disease. There is need to extrapolate scientific findings on patient care. Despite the immuological similarities between BD and Familial Mediterranean Fever a coincidence of both of them is rarely seen in the clinic.

Migration of dendritic cells into the lymphatics - the Langerhans cell example

N. Romani, P. Stoitzner, S. Ebner, H. Stössel, S. Holzmann, G. Ratzinger, P. Fritsch, Department of Dermatology and Venerology, University of Innsbruck, Austria

Dendritic cells, including Langerhans cells of the epidermis and the mucous membranes, are key leukocytes for the initiation of adaptive immune responses as well as for the maintenance of peripheral tolerance. In the former regard they may well play an important, but as yet unrecognized role in the pathogenesis of Behcet's disease. Epidermal Langerhans cells may serve as a paradigm for their counterparts in the mucosae. These cells efficiently take up (microbial) antigens, they process them into immunogenic MHC/peptide complexes, and they transport this form of antigen to the lymph nodes via lymphatic vessels. Depending on the milieu where Langerhans cells have encountered antigen (inflammatory vs. non-inflammatory/steady-state) they make T cells proliferate and acquire effector functions (immunity) or render them unresponsive or even delete them (tolerance), respectively. In addition, plasmacytoid dendritic cells, a recently characterized type of dendritic cells may also directly trigger innate responses, e.g., by secretion of type I interferons in response to virus. Studying the pathways and the regulation of dendritic cells migration might help to unravel a possible involvement of dendritic cells in Behcet's disease.

We present observations on the physical obstacles that dendritic cells migrating in the skin have to overcome until they reach dermal lymphatic vessels. Furthermore, we show that migration is critically dependent on the function of matrix metalloproteinases, in particular MMP-2 and MMP-9. It becomes evident that Langerhans cells indeed carry antigens (including self antigens such as melanosomes or apoptotoic bodies or tumor antigens such as particular cytokeratins) through the lymphatics. Given these observations it may be worth to study Langerhans cells and dermal dendritic cells in Behcet's disease.

There is disparity between the time of migration of the dendritic cells with the clinically observed time for heeling of mucocutaneous ulcers. There is a need for studies on dendritic cells in Behcet's disease.

Viral infection of retinal pigment epithelium – a possible role for initiation of Behcet’s disease

E.U. Irschick, H.P. Huemer, C. Larcher, R. Sgonc, W. Göttinger, Department of Ophthalmology, Institute for Hygiene and Sozialmedizin and the³Institute of Pathophysiology, University of Innsbruck, Austria

The in vitro susceptibility of human retinal pigment epithelial cells (RPE) to representative members of different groups of human pathogenic viruses was investigated in this study. Subacute viral infection is known to change the phenotype of infected cells, thereby causing immune-mediated tissue damage. Downregulation of cell surface antigens provides a means of long-term survival of viruses and persistant infection. Therefore the aim of this study was to investigate the capacity of in vitro infection with viruses and the expression fo different cell surface molecules on human RPE cells following viral infection with special emphasis on those having immune regulatory functions. Primary cultures of RPE cells were infected with various viruses. We found infection with different neurotropic viruses, respiratory viruses and enteroviruses whereas no infection was observed with lymphotropic viruses. Cytomegalovirus (CMV) downregulated MHC-class I antigens on RPE, whereas Coxsackie virus (CVB) and HSV did not alter MHC-class I antigen expression. No induction of class II antigens was observed in RPE cells infected with CVB, HSV or CMV. Adhesion molecule ICAM-1 (CD54) was slightly increased after virus infection and the other cell surface molecules did not alter.

Several common human viruses could infect RPE cells. As even animal viruses, such as pseudorabies virus could infect these cells, it might be possible, that transient infections with animal viruses could act as a trigger for „autoimmune“ retinal diseases - and under certain circumstances like genetic predisposition or immunologic disorders this could lead to Behcet’s disease in the eye.

There is a need for a multicentric cooperation to support these findings. For ocular histological samples, specimens may be available in the UK.

Regulation of inflammatory CD28^- T-helper cells by HLA-class I molecules: a new cellular model for Behcet´s disease?

C. Duftner, C. Goldberger, E. Märker-Hermann, M. Schirmer, Innsbruck/Austria and Wiesbaden/Germany

From immunogenetical studies we learnt, that BD is associated with HLA-B*51, and to a lesser extent with HLA-B*2702. There may even be a role of MICA genes in the pathogenesis of BD. Various of these MHC class I molecules can be recognized by NK receptors on NK and NK-T-cells independently from peptides. These NK cell receptors may be activating (DS) or inhibitory (DL). Interestingly, increased percentages of CD4+CD16+ and CD4+CD56+ T-cell subsets have already been described in BD patients.

In patients with rheumatoid arthritis and ankylosing spondylitis, unusual pro-inflammatory and cytotoxic CD4+ T cells marked by the lack of the costimulatory molecule CD28 express stimulatory NK cell receptors on their surface. In rheumatoid arthritis, MHC-class I recognizing NK receptors are even considered as disease risk genes. In CD4+CD28- T cells from patients with ankylosing spondylitis we could recently show functional NK cell features and an enrichment of these cells in the CD4⁺CD25⁺ T-cell compartment by co-stimulation with HLA-B27 transfected cells.

We hypothesize that CD4⁺CD28^- T-cells as markers of a chronic inflammatory process are also elevated in BD patients, express MHC class I recognizing NK receptors, and thus recognize HLA-B*51 via NK receptors. This mechanism could explain the chronicity of BD as an MHC class I associated disease.

Interesting results since this mechanism would be peptide-independent. Thus bacteria or viruses may be needed at the disease onset, but not during the later course of the disease. This mechanism could be responsible for the perpetuation and the chronicity of this MHC class I associated disease.

Immunosuppressive effects of Gemcitabine in the HSV-induced Behcet’s Disease like mouse model

S. Sohn, M. Lutz, H.J. Kwon, G. Konwalinka, S. Lee, M. Schirmer, Seoul/Korea, Erlangen/Germany, and Innsbruck/Austria

Objective: To study effects and side-effects of gemcitabine (2',2'-difluorodeoxycytidine, dFdC), a pyrimidine synthesis inhibitor, on skin lesions of a herpes simplex virus-induced Behçet’s disease (BD)-like mouse model.

Methods: Dose-escalation studies with dFdC were performed in ICR mice with intraperitoneal application over 5 days. After inoculation of ICR mice with herpes simplex virus and classification as having BD according to a revised Japanese classification, 18 BD-mice were randomly assigned to placebo, 0.06 or 0.12 mg of dFdC /day over 5 days. Serum levels of interleukin (IL)-4, IL-6, IL-10, tumor necrosis factor-a and interferon-g were determined using ELISA assays.

Results: After application of 3 µg dFdC over 5 days, alanine aminotransferase increased (p = 0.032), but all other kidney and liver parameters were unchanged. In BD-mice, 5 days of dFdC treatment with 0.06 or 0.12 mg of dFdC /day resulted in a dose-dependent improvement of cutaneous manifestations by more than 60% (p = 0.017). There was no significant change of cytokine levels and none of the cytokine levels correlated with response to treatment.

Conclusion: DFdC shows promising effects to improve cutaneous lesions in the herpes simplex virus-induced BD-like mouse model. In this animal model, effects of dFdC on the cytokine profile remained inconclusive.

The study should be prolonged. The questions arises whether there is an increased dose necessary for ocular lesions and how reliable this animal model is to extrapolate on human situations? Based on the safety and clinical efficacy in this animal model, clinical trials on patients should be initiated.

We regret that the following lecture had to be cancelled as a consequence of the war in Iraq:

Streptococcal Antigen in the Pathogenesis of Behcet’s Disease

F. Kaneko, H. Yanagihori, M. Tojo, E. Isogai, S.N. Lin, K. Oguma, Department of Dermatology, Fukushima Medical University School of Medicine, Fukushima, Department of Preventive Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu and Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, Medical School, Okayama, Japan

Patients with Behcet’s disease (BD) are highly associated with HLA-B51 immunogenetically (1) and tend to be involved with chronic-infectious foci, such as tonsillitis and dental caries, by non-pathogenic streptococci in the oral cavity. BD patients were suggested to be hypersensitive to streptococci and we immunohistologically demonstrated the deposits of streptococcal-related antigen at infiltrated cells which were adhering to the vascular walls in erythema nodosum (EN)-like lesions (2). The Japanese Research Group for BD also demonstrated that BD patients showed greater hypersensitivity against streptococcal antigens than normal healthy controls and that the BD symptoms were frequently induced by the skin tests using these antigens and the treatment of the dental caries (3). Streptococcus sanguis was dominantly isolated from the infectious foci and the strain strongly adhered to the epithelial cells of the oral membrane which might be correlated with chemotactic activity of neutrophils in the BD lesions (4). Attempt of cloning and sequencing of bes-1 gene of S. sanguis isolated from BD patients was made and it has been found that the amino acid sequence of bes-1 gene has more than 60% of homology with the human intraocular peptide brn-3b which is a POU domain expressed in the retinal ganglion cells (5). On the other hand, heat shock protein-65 kDa (HSP-65) derived from microbial organisms which had homology with human HSP-60 was shown to be cross-reactive to the serotype of S.sanguis found in BD patients (6). Recently we have recognized the antibody cross-reactivity against human HSP-60 peptide (336-351) which might stimulate T-lymphocytes of BD patients (7,8).

In order to explain more precisely about the relationship between S. sanguis and BD symptoms, we attempted to find bes-1 gene in the various lesions and to detect the antibodies against both bes-1 synthetic peptides and recombinant HSP-60/65 of S. sanguis in sera of BD patients.

Methology: We performed polymerase chain reaction (PCR) and PCR in situ hybridization (PCR-ISH) on the samples of BD lesions obtained by punch biopsy and controls using nested primers, which amplify the S. sanguis genomic region coding for bes-1, including the brn-3b homologous site. We also evaluated the antibody responses against bes-1 peptides and recombinant HSP-60/65.

Results: We detected the presence of bes-1 DNA in the samples of EN-like eruptions, and oral and genital aphthous lesions by PCR analysis. PCR-ISH also revealed bes-1 DNA gene located in the nuclei of the cells adhering to the vessel walls and macrophages infiltrated in EN-like lesions, whereas the antibodies against both bes-1 peptides and recombinant HSP-60/65 protein have not been detected in sera of these patients.

Conclusions: The presence of bes-1 DNA in macrophages infiltrated in the various lesions of BD patients suggests that the infectious foci by S. sanguis in the oral cavity are deeply correlated with the various lesions in BD patients. It is speculated that the clinical symptoms appear by the internalization of bes-1 DNA to macrophages infiltrated, as an extrinsic factor, in BD patients who are associated with HLA-B51-related gene as an intrinsic factor. However, it is not clear how S. sanguis infection is correlated with HLA-B51-related gene as the genetic background in BD patients.

References: (1) Ohno S, et al.: J Rheumatol 3,1,1975; (2) Kaneko F, et al.: Br J Dermatol 113,303,1985; (3) Mizushima Y, et al.: J Rheumatol 16,506, 1989; (4) Isogai E, et al.: Bifidobct Microflora 9,27,1990; (5) Yoshikawa K, et al.: Zent bl Bacteriol 287, 449,1998; (6) Lehner T, et al.: Infect Immun 59, 1434, 1991; (7) Kaneko S, et al.: Clin Exp Immunol 108, 204, 1997; (8) Isogai E, et al.: J Applied Research 2, 1, 2002

3. SUMMARY

C.G. Barnes, S. Assaad-Khalil and M. Schirmer

In BD, research work is continuing at a great pace, for example:

- Use of interferon a in ocular manifestations of Behçet’s Disease

- Pathogenesis / immunology of Behçet’s Disease as an immune mediated inflammatory disease

What do we need in the near future?

1. There is a NEED for coordination of activities, which can be offered by the ISBD Working Groups

2. There is a NEED for agreed entry criteria into clinical studies: Participants agreed that the International Classification for Behçet’s Disease should be used until a better / revised / updated scheme is available (ACTION Prof. Davatchi)

3. There is a NEED for agreed clinical outcome measures

- For ocular manifestations participants agreed to the use of the BenEzra scheme (ACTION Dr. Kötter)

- For mucocutaneous lesions participants agreed to the use of a simplified IBBDAM and BDCAF scheme (ACTION Prof. S. Assaad-Khalil & Clinical Trials Working Group)

- Other manifestations should be recorded as they occur

4. There is a NEED for a unified protocol for clinical trials

5. There is a NEED for statistical input in the planning stage before any trial is started. Prof. Davatchi offered very generous assistance from his unit – to be communicated to Prof. Silman and Prof. Martus (from the Epidemiology Working Group of the ISBD).

Which studies are required in the next future? On the basis of agreed entry criteria ...

- BASIC STUDIES (ACTION Prof. Direskeneli) on pathogenesis / immunology and gender associations

- CLINICAL DEFINITIONS FOR Behçet’s DISEASE (see above) on disease subtypes, recurrence, duration and severity of lesions

- THERAPEUTIC STUDIES

as open “pilot” OR comparison single or double blind studies vs. placebo or other alternatives (use of cyclosporine or corticosteroids as comparison regimen to be discussed) on IFN a dosage regimen in ocular Behçet’s disease, possible drug combinations (e.g. IFN a ± steroids; anti-TNF a ± methotrexate) and the use of antibiotics

Protocols presented and amended

- IFN a vs. Cyclosporine in ophthalmic disease – Dr. I. Kötter (Tübingen, Germany)

- anti-TNF a in ophthalmic disease – Prof. Assaad-Khalil (Alexandria, Egypt)

- anti-TNF a in mucocutaneous lesions – Prof. Calamia (Jacksonville, USA)

Which support can be offered by the ISBD and their working groups? The ISBD Working Groups can help to coordinate, offer statistical help (as mentioned above), and will start an international data base for collection of clinical cases (ACTION Prof. Davatchi) and promotion of research activities open for international collaborations (ACTION Working group on drug trials)

In conclusion, we must not just talk, debate, agree and disagree, but return home and be active in progressing these projects, and meet again to review progress during the next International Conference on Behçet’s Disease in Antalya, October 27-31, 2004

In the final discussion round, the following last-minute proposals come up: An International Registry with the aim to re-evaluate the classification criteria and to define disease subsets will be initiated (Prof. F. Davatchi). Prof. Direskeneli will try to find informations and options for support by international agencies. New protocols will arise to study efficacy and safety of antibiotic therapy of BD (Prof. S. Assaad-Khalil) and a pilot trial to study gemcitabine for treatment of skin diseases (Prof. S. Assaad-Khalil and Prof. M. Schirmer).

Last not least, we want to thank everybody for his effort to participate in this workshop, for his input and helpful discussions !

S. Assaad-Khalil, C.G. Barnes, H. Direskeneli, K.T. Calamia, M. Schirmer on behalf of the "Working group on drug trials including collaborative trials" and the "Basic group"