CD8+ cells in liver and expression of hereditary haemochromatosis (HH)

143

Genetics, genetic diseases, gene therap)

1 P/C10/005 ]

CDS’ CELLS HEREDITARY

1 P/C10/007 1

IN LIVER AND EXPRESSION HAEMOCHROMATOSIS (HH)

OF

E. M. P. Card SO’. M. de S a”. R. Hultcrantz’. ‘Depts of Gastroenterologt and HepiyAogy, Karolinska Hospital, Stockholm, Sweden; “Molecular Pathology and Immunology, Abel Salazar Institute for Biomedical Sciences, Porto, Portugal. Although most patients with HH have the same mutations in the HFB gene, the phenotypic expression varies. Recent work indicates that HH patients with low blood CD8’ T-lymphocytes have higher iron stores. We have investigated if low CD8’ cells in the liver are seen in patients with higher iron stores and fibrosis. . wand 37 HH patients (30/7 male/females, 20- 73yrs), all C282Y +I+; 4 had cirrhosis, 8 fibrosis, 25 normal liver histology. Mean iron stores, was 7g (2.5148). Six normal livers were used as controls. The %CD8’ and %CD4+ Tlymphocytes in blood was determined by FACS. CD8* cells were quantified by immunohistcchemistry and morphometry in sections from paraffin embedded liver tissue (37 before, 6 after treatment). Results: In HH patients the count was 0.5-13.6 CD8’ cells/urn’ in the lobules. The controls had 2.8-11.5 CD8’ cellsAm?. The number of CD8’ cells in the liver was correlated ‘with the percentage of CD8’ T-lymphocytes in blood (r=O.64, p=O.OOl). There was no difference in CD8’ cells in liver biopsies before and after treatment. Them was no correlation between age and CD8’ cells. There was a correlation between liver CD8’ cells and iron stores (r= -0.51, p=O.Ol). Patients with low CD8’ cells also had more fibrosis and patients with cirrhosis had the lowest CD8+ count. QB&&B: Our findings clearly show that iron accumulation and subsequent fibrosis is related to CD8’ cells in the liver in patients with HH.

NORMAL BETA-ZMICROGLOBULIN (B2m) GENE IN PATIENTS WITH HEMOCHROMATOSIS AND NO EVIDENCE OF LINKAGE TO CHROMOSOME 6 M.Sampietro, L.Lupica. N.Corbetta, M.Amato, A.Casatta, A.Pizzuti”. L.Laurenzi*, SFargion, G.FioreIli. Zstituto di

Medicina Interm e Fisiopatologia Medica; ‘Istituto di Neurologia; *Istituto di lgiene e Medicina Preventiva; IRCCS Ospedale Maggiore - Universith di Milatw, Milano, Italy

HFE, a candidate gene for genetic hemochromatosis (GH) has recently been identified and a single mutation (C282Y) is present in the homozygous state in the large majority of patients. HFE has the typical structure of MHC class I genes. The mutation of cystein 282 disrupts the binding site for g2m in the a3 domain of the protein. This structural abnormality, and the existence of an animal model -the p2rn knockout mouse- reproducing the features of human GH, raised the possibility that the p2m gene could be involved in cases of GH with no evidence of linkage to chromosome 6. We selected 8 Italian male patients with a clinical picture of GH and the following features: a) homozygosity for the wild-type allele at position 282 of HFE; b) absence of HLA-A3 (which is part of the ancestral GH haplotype); c) no evidence of linkage to the MHC complex. In each patient the serum levels of B2m were determined and the fi2rn gene was searched for mutations by a combination of polymerase chain rection (PCR), single strand conformation polymorphism (SSCP) analysis, restriction analysis and Southern blotting. Serum p2m levels were normal in all patients. Southern blotting of multiple restrictions of DNA allowed to rule out gross insertions/deletions. The normal size of PCR products and the results of SSCP analysis allowed to exclude the presence of small insertions /deletions or point mutations. In summary, the results of our study do not support the hypothesis of p2m involvement in GH cases lacking evidence of linkage to chromosome 6.

VASOACTIVE COMPOUNDS INCREASE THE EFFICIENCY ADENOVIRAL GENE TRANSFER TO PRIMARY LIVER TUMORS

OF

R. Bilbao. C. @an. M. Drozdzik. M.J. Paiares, J. Prieto. Dpt. Internal MedicincMedical School and Clinica Universitaria. University of Navarra. Pamplona. Spain. An

obstacle

penetration

for the success of gene therapy

vasoactive compmmds (histamine on

the

transduction

adenovirus.

(Hist),

efficiency

of

induced

interleukine

2(IL2)

hepatocellular

(AdCMVlacZ)

of non-tumoral

expressing

IacZ

in rats by diethylnitrosoamine

rats were divided

into seven groups. Group and hepatic artery (H.A.)

I and

Once HCC

before AdCMVlacZ

rats were sacrificed. a lobule

tumoral

Neoplastlc

in size),

major (EM)

vessels

II received

administration

portions

of

lobes).

wtth

absent

fenestratlon

EM of

as: Foci

the

AdCMVlacZ

GrupoIV(hist

with or without

0,4mgtAdCMVlacZ,

H.A.) H.A.)

Grubo VI‘(AII40n~+AdCMVlacZ,‘H.A.j VII

(IL2

lesions and

less HCC’

analyzed

by

structure

endothelium, Penetration of

electron

presence

of

basal

of these abnormal

tumoral

lesions

using

vasoactive treatment was as follows,

Gruoo V (AI1 200netAdCMVlacZ.

G~po

size)

abnormal

Transduction

impatred.

(small in

were

vessel wall.

was

via

V,, VI and VII

showed

membrane and collagen fibers surrounding AdCMVlacZ

a

The rest of

only AdCMVlacZ

lobules

Samples

&t&s:

was

40 ng of AI1 and IO’ units of

lesions were classified

liver

HCC

via hepatic artery. Two days later

Neophsfrc nodules (several

and X gal staining.

by and

was established,

Group 111, IV,

vessels

by

cells

A recombinant

vessel morphology.

respectively.

to

II (AH))

(HCC)

gene was produced.

received 2 mg of Hist., 0.4 mg of Hist., 200 ng of AII, IL2 respectively

(involving

and angiotensin

vessels. Methods:

reporter

administratton.

portal vein (P.V.)

microscopy

tumors

the effect of three

carcinoma

group of animals werv sacrificed to study the tumoral

than

of solid

The first hvo compounds are known to increase vascular permeability

the third to cause vasoconstriction adenovirus

is the resistance

by gene therapy vectors. In this study, we investigated

IO’UtAdCMVlacZ,

H.A.)

155% 1 1 35% 1

,

40%

1 22%

10%

1 10%

Conclusions: I) In HCC the abnormal vessel structure represents a physical barrier for transduction of the tumor with viral vectors. 2)Transduction is more efftcient using hepatic artery route than portal vein. 3) Vasoactive agents can increase the efficiency of tumor transduction.

MHC CLASS-I ASSOCIATIONS IN IRON OVERLOAD NEW LINK BETWEEN THE H63D MUTATION, HLAA29 AND “NON-CLASSICAL” HAEMOCHROMATOSIS G Porte’.‘. H Alves3, P Rodriaues2. JM CabedalX2.C Portal’. A Ruivo’, B Justica2, R Wall? and M De Sousa’ 1. Santo Antonio General Hospital 4050-Porto, Portugal; 2. Abel Salazar Institute for the Biomedical Sciences 4050-Porto, Portugal; 3. North Histocompatibility Center 4200-Porto, Portugal; 4. Mercator Genetics, Inc., 4040 Menlo Park, California 94025, USA The present study consists of an analysis of the frequencies of HFE mutations and HLA-A and B antigens in patients with different forms of iron overload compared to the frequencies found in normal subjects. The study population included: 71 normal individuals; 39 genetically and clinically well characterised patients with genetic haemochromatosis (HH); and 25 patients with “non-classical” forms of iron overload (NCH), excluding secondary haemochromatosis. HLA typing was done by the standard microlymphotoxicity test and HFE genotyping by Oligonucleotide Ligation Assay (OLA). The gene frequencies found for the C282Y and H63D mutations of HFE were respectively: 0.03 and 0.23 in normal individuals, 0.86 and 0.04 in HH patients, and 0.08 and 0.48 in NCH patients. An expected significant association was found between the C282Y mutation, I&A-A3 and HLA-B7 in HH patients. A new association was observed between the H63D mutation, HLAA29 and HL,A-B44 in NCH patients.These findings reinforce the evidence for the involvement of MHC class-1 itself in iron metabolism, supporting the notion of a physiological role for the immunological system in the regulation of iron load.