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Extrahepatic Manifestations
of Hepatitis C Virus Infection
Service de Médecine Interne, et CNRS UMR 7087 Université Pierre et Marie Curie
Centre National de Référence Maladies Autoimmunes
Hôpital La Pitié-Salpêtrière, Paris, FRANCE
Pr. Patrice CACOUB
Manifestation Prevalence
certainly associated with HCV %-------------------------------------------------------• Vasculitis (PAN, cryoglobulinemia) 5-40 • Fatigue 35-54• Arthralgia-myalgia 25-35• Sicca syndrome 10-25• Autoantibodies 10-40• Thrombocytopenia 20-40• Lymphoma ?
3
• Hepatitis • Cirrhosis• Hepatocarcinoma
• Cryoglobulinemia• Auto-Ab• B-NHL
HepatocyteChoo. Science 1989
Lymphocyte
Zignego. J Hepatol 1992
Ferri. Blood 1993
Hepatitis C Virus Chronic Infection: Two Main Target Cells
Cryoglobulinémies mixtes
Saadoun, Arch Intern Med, 2006
Infection VHC +++
5
Endothelial cells
Cryoprecipitation
6
Pathogenesis of
cryoglobulinae
mic vasculitis
Roccatello, D. et al. Nephrol. Dial. Transplant. 2004
7
Skin Purpura
Membrano-proliferative Glomerulonephritis CNS Vasculitis
Neuropathy
Cryoglobulinemia-Systemic Vasculitis
10
HCV Mixed Cryoglobulinemia & Digestive Tract
Mesenteric artery stenosis
Intestinal wall thickening
Terrier B et al, GUT 2011
Distal Polyneuropathy 80%
Cacoub P et al, AIDS 2005
Mixed Cryoglobulin and Neuropathy
• Chronic progressive course,
• Distal, symetric, axonal PN, mainly
sensory and painful
• Few extra neurological signs :
purpura
• Severe liver involvement
• Moderate inflammatory syndrome
- important peri-vascular infiltrate of lymphocyte- around small vessels i.e. venules, capillaries- no PMN, no destruction of the vascular wall
Mixed Cryoglobulin and Distal Polyneuropathy
Peripheral Nerve Biopsy
Cryoglobulinemic Membrano-Proliferative Glomerulonephritis
Central Nervous System Involvement in HCV-
Cryoglobulinemia Vasculitis
HCV-vasculitis HCVControls
(n=40) (n=11) (n=36)--------------------------------------------------------------------------------------Gender (F/M) 23/17 6/5 20/16Age (yrs) 59 ± 13 56 ± 10
58 ± 12WMHS 7.0 ± 9.9 0.9 ± 1.8 *2.0 ± 3.1
PVHS 2.5 ± 3.1 0.4 ± 0.5 * 0.8 ±
1.4
NCFD 2.2 ± 1.8 0.9 ± 0.8 * -
--------------------------------------------------------------------------------------* P<0.01WMHS: White Matter Hypersignals PVHS: Periventricular HypersignalsNCFD: Number of Cognitive Function Deficiency
Casato M et al, J Hepatol 2004
17
Age at disease onset 54 ± 13 (29-72) Female/Male ratio 3 Purpura 98% Weakness 98% Arthralgias 91% Arthritis (non-erosive) 8% Raynaud's phenomenon 32% Sicca syndrome 51% Peripheral neuropathy 81% Renal involvement 31% B-cell non-Hodgkin's lymphoma 11% Hepatocellular carcinoma 3%
Ferri C, Mascia MT, Saadoun D, Cacoub P. 2009
Demographic & Clinical Features of 250 Mixed Cryoglobulinemic Patients
18
HCV Core Protein in Skin Vascular Structures
Who’s the culprit ?
Cellular Infiltrate in HCV-Vasculitis
19
Detection of Genomic Viral RNA in Nerve and Muscle of Patients with HCV
Neuropathy
Inflammatory vascular lesions in 26/30 (87%) patients
Positive-strand genomic HCV RNA detected in 10/30 patients (muscle 9, nerve 3)
Negative-strand replicative HCV RNA never detected
--> HCV neuropathy probably results from virus-triggered immune-mediated mechanisms rather than direct nerve infection and in situ replication
Authier JF et al, Neurology, 2003
20
A Major Role for T Cell Immunity in HCV-Vasculitis
Abnormal T lymphocytes distribution
Predominant T lymphocytes infiltration in vasculitis lesions
MHC-II polymorphism (DR11)
Th1 cytokines profile in vasculitis lesions
Deficit in Treg lymphocytes
21
Quantitative Deficit in Treg Lymphocytes (CD4+CD25+) in HCV-Systemic Vasculitis
Boyer O, Saadoun D et al, Blood 2004
22
23
Before treatmentOn treatmentEarly F/u Late F/U3
4
5
6
CD
25
hig
h (
% o
f C
D4
+)
4 4
5
6
Before
treat.
On Treat.
Early F/U
Late F/U.
**†
**†
-CR
-NR/PR
0
10
20
30
40
CD25h
igh
(ce
lls/μl)
†*
BeforeTreat.
CR NR/PRAfter Treat.
C
After Treat.
A
Complete clinical response of HCV-vasculitis to anti-viral treatment is
associated with an increase in CD4+CD25high levels
24
0 20 40 60 80 1000.0
0.2
0.4
CD25high (cells /μl)C
4 (
g/l
)
R²-0.16, p<0.005
0 20 40 60 80 1000
1
2
3
CD25high (cells /μl)
Cry
og
lob
uli
ns
(g
/l)
R²-0.1, p<0.005
Correlation between Immune Response and Treg Lymphocytes in HCV MC Vasculitis
25
Chronic HCV infection
Poly- oligoclonal
B-cell expansion
AutoantibodiesRF - IC
Mixed cryoglobulins
Cryoglobulinemic vasculitis
Monoclonal B-cellproliferation
Overt lymphoma
HCV eradication
Immunosuppressors
Chemotherapy
Plasma exchange
Steroids
26
HCV Treatment Efficacy in HCV-Vasculitis%
im
pro
vem
en
t
Zuckerman, J Rheumatol 2000. Naarendorp, J Rheumatol 2001. Cacoub, Arthritis Rheum 2002, Zaja F, Blood 2003. Sansonno D, Blood 2003 , Cacoub, Arthritis Rheum 2005, Saadoun, Arthritis Rheum 2007
27
Predictive Factors of Clinical Response to HCV Therapy in Mixed Cryoglobulinemia
VasculitisMultivariate Analysis
Odds ratio [95%CI]
p
• Renal involvement 0.27 [0.08-0.87]
0.02
• Renal insufficiency (GFR<70) 0.18 [0.05-0.67]
0.01
• Daily proteinuria > 1g 0.32 [0.09-1.11]
0.05
• Early virological response 3.53 [1.18-10.59]
0.02
28
Rationale for Rituximab
treatment in cryoglobulinemic
vasculitis
Rocatello D, Nephrol Dial Transplant, 2004Roccatello, D. et al. Nephrol. Dial. Transplant. 2004
29
Treatment of Mixed Cryoglobulinemia Resistant
to Interferon with Rituximab*
Sansonno D et al, Zaja F et al, Blood 2003
31
10
20
30
40
50
60
70
80
90
MONTHS
100
6 12
15 (93.7)
13 (81.2)
12 (75)
1 2 3 4 5 7 8 9 1011 24 36 48
10 (62.5)
6 (37.5)
Cryoglobulinemia Vasculitis: Response Maintenance after Discontinuation of
Rituximab
Sansonno D et al, 2007
32
HCV Vasculitis: a Two-Faces Disease
…Needs a Two Faces Treatment Strategy
Rituximab
PegIFN plus Ribavirin
34
RITUXIMAB (375 mg/m²)
Time (months)0 1
RIBAVIRIN (600-1200 mg/d)
PEGYLATED INTERFERON 2b (1.5 μg/Kg/wk)
12
Rituximab plus Peg-IFNα2b-Ribavirin in Refractory HCV-Related Systemic
Vasculitis
2
Saadoun D et al, Ann Rheum Dis 2008
37
38
Outcome of HCV-MC pts according to treatment
Parameters All PegIFN-ribavirin RTX-PegIFN-
ribavirinn=93 n=55 n=38 P
Time clinical response, months
6.8 ± 4.7
8.4 ± 4.75.4 ± 4.0
0.004
Clinical response
CR68
(73.1) 40 (72.7) 28 (73.7) 0.98PR 22 (23.6) 13 (23.6) 9 (23.7)NR 3 (3.2) 2 (3.6) 1 (2.6)Relapse 17 (18.3) 10 (18.1) 7 (18.4)
Immunological response
CR49
(52.7) 24 (43.6) 26 (68.4) 0.001PR 35 (37.6) 25 (45.4) 10 (26.3)NR 8 (8.6) 6 (10.9) 2 (5.2)Relapse 17 (18.3) 10 (18.1) 7 (18.4)
Virological response
SVR55
(59.1) 33 (60) 22 (57.9) 0.94Death 5 (5.4) 2 (3.6) 3 (7.9) 0.70
39
Course of kidney parameters in HCV-MC patients according to the type of treatment
PegIFN-ribavirin RTX-PegIFN-
ribavirinn=10 p n=21 p
CR kidney involvement 4 (40) 17 (80.9) 0.04Creatininemia (µmol/l)Baseline 150 ± 30 217 ± 47EOF 169 ± 44 0.28 136 ± 27 0.03GFR (ml/min)Baseline 58 ± 7 42 ± 5EOF 59 ± 9 0.41 57 ± 4 0.01Daily Proteinuria (gr/d)Baseline 3.1 ± 0.9 3 ± 1EOF 1.2 ± 0.5 0.046 0.4 ± 0.1 <0.001Hematuria (n,%)Baseline 10 (100) 19 (90.5)EOF 2 (20) 2 (10.5) <0.001
40
Antiviral therapy alone decreases the
memory B cells
n=38 n=55
Saadoun D et al, Blood 2010
41
Antiviral therapy alone decreases the memory B
cells
Antiviral therapy plus Rituximab
decrease naive B-cells
Saadoun D et al, Blood 2010
44
Time Course of HCV Viral Load
Terrier B et al. Arthritis Rheum 2009
45
46
• If failure or CI of PegINF/riba: RTX alone• Place to be defined for PegIFN/Riba/Previr
48
Years
Overa
ll su
rviv
all
Overall Survival of 151 HCV-Vasculitis Patients
Terrier B et al. Arthritis Rheum 2010
49
Years
Overa
ll su
rviv
all
Overall Survival of 151 HCV-Vasculitis Patients
Terrier B et al. Arthritis Rheum 2010
32 deaths after a median follow-up of 54
months (IQR 26-89)
Causes of death:- Infection (n=10)
- Cirrhosis (n=10; 4 HCC)- Non-HCC neoplasia (n=4)
- Cardiovascular (n=4)- Renal failure (n=2)
- Vasculitis (n=2)- Unknown (n=2)
50
Baseline Prognostic Factors of HCV-Vasculitis Patients
51
• Metavir fibrosis score:HR = 10.8 (3.63-32.14),
P<0.0001
• Five Factor Score:HR = 2.49 (1.29-4.8),
P=0.007
Liver Fibrosis and Five Factor Scores are Associated with a Poor Prognosis in HCV
vasculitis Patients Multivariate Analysis
52
Multivariate analysis
- Metavir fibrosis score:HR 10.8 (3.63-32.14), P<0.0001-FFS:HR 2.49 (1.29-4.8), P=0.007
Metavir Fibrosis
FFS F0-F2 F3-F4
0 1.0
1 2.49
> 1 6.2
FFS is a good predictorof outcome
Interaction Between Liver Fibrosis and Five Factor Score in HCV-Vasculitis
Patients
53
Multivariate analysis
- Metavir fibrosis score:HR 10.8 (3.63-32.14), P<0.0001-FFS:HR 2.49 (1.29-4.8), P=0.007
Metavir Fibrosis
FFS F0-F2 F3-F4
0 1.0 10,8
1 2.49 10,25
> 1 6.2 9,74
FFS is a good predictorof outcome
Interaction Between Liver Fibrosis and Five Factor Score in HCV-Vasculitis
Patients
No more prognostic
value of FFS
54
Use of Peg-IFN/riba had a positive prognostic impact
HR = 0.34 (0.16-0.67)
Prognostic Factors
During follow-up
After adjustment on vasculitis severity
• Negative impact of immunosuppressantsHR = 4.05 (1.75-9.36), P=0.001
•… but not of corticosteroidsHR = 1.79 (0.77-4.16), P=0.17
55
Reversible Quantitative Deficit in Treg Lymphocytes (CD4+CD25+) in HCV-Systemic
Vasculitis
Before treatmentOn treatmentEarly F/u Late F/U3
4
5
6
CD
25
hig
h (
% o
f C
D4
+)
4 4
5
6
Before
treat.
On Treat.
Early F/U
Late F/U.
**†
**†
-CR
-NR/PR
After Treat.
A
0 20 40 60 80 1000
1
2
3
CD25high (cells /μl)
Cry
og
lob
uli
ns
(g
/l)
R²-0.1, p<0.005
0 20 40 60 80 1000.0
0.2
0.4
CD25high (cells/μl)
C4
(g/l )
R²-0.16, p<0.005
56
57
Effects of Low-Dose Interleukin-2 on Levels of CD4-Treg (c) and CD8-Treg (sq) in Patients with HCV-Vasculitis, According to
Treatment Course.
58
Effects of Low-Dose Interleukin-2 on Levels on the Ratio of Treg Cells to the sum of Effector T Cells CD4 + CD8 in Patients with
HCV-Vasculitis, According to Treatment Course.
59
60
Effects of Low-Dose Interleukin-2 on Levels on CD19+ total B Cells (c) and Marginal-Zone B Cells (sq) in Patients with HCV-Vasculitis,
According to Treatment Course.
Baseline C1 C2 C3 C4 Post IL-2 Baseline C1 C2 C3 C4 Post IL-2 Baseline C1 C2 C3 C4 Post IL-2 Baseline C1 C2 C3 C4 Post IL-2
Baseline C1 C2 C3 C4 Post IL-20
10
20
30
Baseline C1 C2 C3 C4 Post IL-20
10
20
30
Baseline C1 C2 C3 C4 Post IL-20
10
20
30
Baseline C1 C2 C3 C4 Post IL-20
10
20
30
PurpuraNeuropathy
ArthralgiaFatigueKidney Involvement
CD
4+Tr
eg
(%
)C
LIN
ICA
LR
ES
PO
NS
ETemporal Effects of Low-Dose Interleukin-2 on Clinical
Features, Levels of Regulatory T Cells, and Cryoglobulin for Each Study Patient
62
BEFORE IL-2 AFTER IL-2CCL3CCL3L1CCL3L3
IL1ACCL20
IL6CLECL1CD79A
BLKCCL4L2
EBF1CCL4L1CXCR5
IER3
CXCR7OLR1PDE48PTGS2IL1B
BAFFR
4-1BBL
PLAURNLRP3RIPK2ATF3
NAMPT-PBEF1
TNFRSF21-DR6ETS2
MAPK3K8-COT
GOS2
CD83
Up Down Khi2 test
Inflammation 0 251 1,30E-40
Immune Response 16 684 3,40E-94
Lymphocyte 77 555 7,00E-49
Cell Cycle 1701 208 1,50E-138
Control 226 343 2,50E-01
Autoimmune & transplantation pathologies
0 46 7,60E-09
Inflammatory infectious diseases 6 242 7,60E-36
Other diseases 190 211 4,15E-02
Saadoun D et al. NEJM 2011
Anti-inflammatory Effects of Low-Dose Interleukin-2 Revealed through Unsupervised Transcriptome Analyses of PBMCs.
63
Regulatory T Cell Recovery in HCV-Vasculitis through Low-Dose IL-2
Treatment
We provide the first evidence of Treg recovery through low-dose IL-2 therapy in a human autoimmune disease.
Low-dose IL-2 dramatically increases CD4+CD25highCD127– Foxp3+ Treg cells that are functional
Treg expansion persists after IL-2 therapy.
IL-2 therapy was well tolerated with no flare of vasculitis.
Saadoun D et al. NEJM 2011
64
The Yin and Yang of IL-2-Mediated Immunotherapy
Balance of Pathogenic Effector T Cells and Regulatory T Cells
Bluestone JA, NEJM 2011
Manifestation Prevalence
certainly associated with HCV %-------------------------------------------------------• Vasculitis (PAN, cryoglobulinemia) 5-40 • Fatigue 35-54• Arthralgia-myalgia 25-35• Sicca syndrome 10-25• Autoantibodies 10-40• Thrombocytopenia 20-40• Lymphoma ?
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007
% of patients
n = 1614
% of controls
n = 412
Fatigue without depression
Fatigue with depression
Depression without fatigue
No fatigue and no depression
Total
48
5
2
45
100
0.7
0
0
99.3
100
Fatigue without EM
Fatigue with EM
EM without fatigue
No fatigue and no EM
Total
19
35
21
25
100
0.5
0.2
3.4
96
100
Association between fatigue, depression and clinical extrahepatic manifestations (EM)
Poynard T et al. J Viral Hep, 2002
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007Multivariate analysisMultivariate analysis
Fatigue (moderate or severe) in comparison to absence of fatigue was associated with:
• female gender,
• age > 50 years,
• cirrhosis or many septa,
• purpura. Independently of these associations, fatigue
(moderate-severe) was associated with : arthralgia, myalgia, paresthesia, sicca sd & pruritus.
Poynard T et al. J Viral Hep, 2002Poynard T et al. J Viral Hep, 2002
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007Prevalence of fatigue at baseline and at 18 months follow-up in treated
and untreated patients
Baseline 18 months 18 months vsbaseline
Non treated (n=72) No fatigue Moderate Severe
39 %35 %26 %
42 %39 %19 %
P = 0.74
Sustained responders(n=82) No fatigue Moderate Severe
41 %37 %22 %
69 %24 %7 %
P < 0.001
Relapsers (n= 47) No fatigue Moderate Severe
45 %43 %13 %
40 %45 %15 %
P = 0.68
Non responders (n= 224) No fatigue Moderate Severe
40 %42 %18 %
46 %40 %14 %
P = 0.18
Poynard T et al. J Viral Hep, 2002
Manifestation Prevalence
certainly associated with HCV %-------------------------------------------------------• Vasculitis (PAN, cryoglobulinemia) 5-40 • Fatigue 35-54• Arthralgia-myalgia 25-
35• Sicca syndrome 10-25• Autoantibodies 10-40• Thrombocytopenia 20-40• Lymphoma ?
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007
0%5%
10%
15%20%25%30%
35%40%
Sustained responders (n = 83)
Impact of Treatment on Extra hepatic Manifestations in HCVpatients.
At Baseline and 18 months Follow-up in Responders.
Cacoub P et al. J Hepatol 2002
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007
0%5%
10%15%20%25%30%35%40%
Sustained responders (n = 83) Non responders - RNA + (n = 348)
Cacoub P et al. J Hepatol 2002
Impact of Treatment on Extra hepatic Manifestations in HCVpatients.
At Baseline and 18 months Follow-up in Responders.
Manifestation Prevalence
certainly associated with HCV %-------------------------------------------------------• Vasculitis (PAN, cryoglobulinemia) 5-40 • Fatigue 35-54• Arthralgia-myalgia 25-35• Sicca syndrome 10-25• Autoantibodies 10-40• Thrombocytopenia 20-40• Lymphoma ?
Auto-antibody production in chronic HCV infection.
0
10
20
30
40
50
60
70
%
A-nuclearA-phospholipidA-thyroglobulinA-smooth muscle≥ one auto-Ab≥ three auto-Ab
Pawlotsky JM, Hepatology 1994. Pawlotsky JM, Ann Intern Med 1994.Prieto J, Hepatology 1996. Cacoub P, J Rheumatol 1997. Cacoub P, Medicine 2000.
Extrahepatic manifestations associated with HCV infection.(Prospective study in 321 HCV patients)
Autoantibody Number %
----------------------------------------------------- Antinuclear 124 41
• A-nucleosome 6 2
• A-DNA 8 3
• A-histone 9 3
• A-ENA 10 3
Cacoub P et al. Medicine 2000; 79: 47-56
Manifestation Prevalence
certainly associated with HCV %-------------------------------------------------------• Vasculitis (PAN, cryoglobulinemia) 5-40 • Fatigue 35-54• Arthralgia-myalgia 25-35• Sicca syndrome 10-25• Autoantibodies 10-40• Thrombocytopenia 20-40• Lymphoma ?
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007
B-cell-Non Hodgin’s LymphomaB-cell-Non Hodgin’s Lymphoma
Hepatitis C virusHepatitis C virus
2462 tested2462 tested
13.5 % positive • vs 0-5 % in controlsvs 0-5 % in controls
• vs 5 % in other malignant vs 5 % in other malignant hemopathyhemopathy
469 tested469 tested
0 - 39 %
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007Effects of alpha-interferon on HCV+/SLVL course
After 6 months of IFN alpha treatment in SLVL/HCV+: Complete clinical hematologic response (spleen size < 12
cm, lymphocytosis <4500/mm3, No cytopenia ):
---> 7/9 HCV RNA negative Partial clinical hematologic response
(spleen size or lymphocytosis decrease >50%) :
---> 2/9 HCV RNA +
Hermine O. et al, N Engl J Med 2002; 347: 89-94
HCV antibodies : B-NHL (< 3%) vs SLVL (15%)HCV antibodies : B-NHL (< 3%) vs SLVL (15%)
----> Splenic lymphoma with villous lymphocytes may be associated with HCV infection
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007
Median Follow-up of 3 years (2-5)
6 Complete Responses ---> HCV RNA still negative6 Complete Responses ---> HCV RNA still negative
1 relapse off therapy at 1 year,1 relapse off therapy at 1 year,
• associated with positivity of HCV RNA. associated with positivity of HCV RNA.
• second CR following IFN & negativity HCV RNAsecond CR following IFN & negativity HCV RNA
2 Partial Responses 2 Partial Responses
• CR after Combination of Interferon and Ribavirin CR after Combination of Interferon and Ribavirin
• PR after Interferon and Ribavirin PR after Interferon and Ribavirin
Hermine O. et al, N Engl J Med 2002; 347: 89-94
Effects of alpha-interferon on HCV+/SLVL course
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007HCV negative / SLVL Patients Treated with Alpha-Interferon
Median age 65 (54-72)Median age 65 (54-72)
Prior therapy (2/6), chemotherapy (1), splenectomy(1)Prior therapy (2/6), chemotherapy (1), splenectomy(1)
Splenomegaly (4/6)Splenomegaly (4/6)
Hyperlymphocytosis Median 25,000 (500-100.000)Hyperlymphocytosis Median 25,000 (500-100.000)
Cytopenia (2/6)Cytopenia (2/6)
Cryoglobulinemia or rheumatoid factor (0/6)Cryoglobulinemia or rheumatoid factor (0/6)
Alpha-Interferon 3 M IU x 3/W during 6 monthsNo response
Hermine O. et al, N Engl J Med 2002; 347: 89-94
Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007
Conclusion
Extrahepatic manifestations of HCV
infection are frequent, and may be cured
by HCV treatment :
• Systemic vasculitis (cryoglobulinemia,
PAN)
• Fatigue
• Arthralgia - myalgia - arthritis (±)
• Auto-antibodies (?)
• Splenic lymphoma with villous
lymphocytes
• Thrombocytopenia
82
S. Caillat-Zucman, Paris
P. Ghillani, Paris D. Klatzmann, Paris L. Musset, Paris M. Rosenzwajg, Paris
D. Saadoun, Paris D. Sene, Paris B. Terrier, Paris G. Géri, Paris P. Hausfater, Paris O. Lidove, Paris A. Gatel, St Brieuc J-M. Léger, Paris N. Limal, Paris T. Maisonobe, Paris JC Piette, Paris
Thanks
L. Alric, Toulouse M. Bourlière, Marseille P. Halfon, Marseille S. Pol, Paris T. Poynard, Paris V. Thibault, Paris Les membres du
GERMIVIC
L. Calabrese, Cleveland
M. Casato, Roma C. Ferri, Modena G. Kerr, Washington E. Sasso, Seattle JA. Schifferli, Basel V. Soriano, Madrid