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Bevilacqua G, Sobel ME, Liotta LA, Steeg PS. Association of low NM23 RNA levels in human primary infiltrating ductal breast carcinomas with lymph node involvement and other histopathological indicators of high metastatic potential. Cancer Res 1989; 49: 5185-90. 6. Hennessy C, Henry JA, May FE, Westley BR, Angus B, Lennard TW.

5.

Expression of the antimetastatic gene nm23 in human breast cancer: an association with good prognosis. J Natl Cancer Inst 1991; 83: 281-85. 7. Leone A, Flatow U, King CR, et al. Reduced tumor incidence, metastatic potential, and cytokine responsiveness of NM23 transfected melanoma cells. Cell 1991; 65: 25-35. 8. Leone A, McBride W, Weston A, et al. Somatic allelic deletion of nm23 in human cancer. Cancer Res 1991; 51: 2490-93. 9. Haut M, Steeg PS, Wilson JK, Markowitz SD. Induction of nm23 gene expression in human colonic neoplasms and equal expression in colon tumors of high and low metastatic potential. J Natl Cancer Inst 1991; 83: 712-16. 10. Mehta CR, Patel NR, Senchauduri P. Importance sampling for estimating exact probabilities in permutational inference. J Am Stat Assoc 1988; 83: 999-1005.

ADDRESSES Departments of Surgery (K H. Cohn, MD, F Wang, MD, L. G Patterson, BS, M R Arnold, BS, J. Weimar, BS), Medicine (W. B. Solomon, MD), and Preventive Medicine and Community Health (J. G. Feldman, Dr PH), State University of New York Health Science Center at Brooklyn; Departments of Surgery (K. H. Cohn, F Wang, L G Patterson, M. R. Arnold, J Weimar) and Pathology (F DeSoto-LaPaix, MD), Brooklyn VA Medical Center, Brooklyn, New York; and Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA (A T Levy, MS, A. Leone, PhD, P.S Steeg, PhD). Correspondence to Dr Kenneth H. Cohn, Department of Surgery (112), Brooklyn VA Medical Center, 800 Poly Place, Brooklyn, New York 11209, USA

Reaction of human

non-collagenous polypeptides with coeliac disease autoantibodies

We

six human noncollagenous protein molecules that specifically bind to serum IgA from patients with coeliac disease, and which as a combination can act as true antigen to reticulin antibodies. In affinity chromatography, the purified human protein molecules removed antibodies against reticulin and endomysium from serum samples of coeliac disease patients. We postulate that an autoimmune mechanism operates in generating the jejunal damage in gluten-sensitive enteropathy and that the human protein molecules described here act as self-antigens in the disease.

identified

and

purified

The molecular mechanisms leading to morphological damage in the small intestine in coeliac disease are unknown. Because serum from untreated coeliac disease patients reacts with many human tissues,1 including the jejunum,1-3 we postulated that antibodies are generated against the patient’s own tissue material. These tissue antibodies recognise one or more antigens associated with collagen fibres, but not the collagen itself4 Here, we describe human non-collagenous protein molecules which react with autoantibodies in active coeliac disease.

We chose fetal lung as starting material, because strong fluorescence of tissue fibres was detected when binding of serum IgA from coeliac disease patients was measured by an indirect immunofluorescence-microscopic method.1 This tissue had never been in contact with gliadin. The tissues were obtained from routine necropsy material and the study protocol was approved by the ethical committee of the University Hospital of Tampere. The lung tissue was digested with collagenase (600 units per g tissue) in potassium phosphate buffer (pH 62) containing 01 mmol/1 dithiothreitol, 0-05% sodium dodecylsulphate, and 04 mol/1 guanidinium-HCI. After centrifugation the supernatant was applied to a 45 x 1.8cm gel filtration column and fractions of 1ml were collected. The protein material in each fraction was then radioiodinated. We tested binding to IgA purified from serum of two patients with active coeliac disease. The fractions which contained reactive material were applied to a preparative highperformance liquid chromatography (HPLC) gel filtration column. The fractions that showed binding to the patient IgA were then applied to a preparative HPLC ion-exchange column. The final purification was carried out by preparative isoelectric focusing. 20 ug of the polypeptide mixture was applied, either untreated or after 1 h incubation with 50 mmol/1 dithiothreitol, to 1 mm thick polyacrylamide isoelectric focusing gel with pH gradient 3-5-95. After 1 ’5 h at 20 W the gel was stained. To evaluate binding of the polypeptides to reticulin (ARA) and endomysium (EMA) antibodies, 100 ul samples of serum from ten patients with active coeliac disease and known concentrations of antigliadin antibodies (AGA), ARA, and EMA were applied to affinity chromatography columns consisting of polymer beads bound to crude gliadin or to the mixture of the purified coeliac disease autoantigen proteins (CDAP). Unbound material was eluted with potassium phosphate buffer and collected for measurements of AGA (by an enzyme-linked immunosorbent assay) and ARA and EMA (by indirect immunofluorescence microscopy with rat kidney and liver [ARA] and monkey

oesophagus [EMA]

as

substrates’,’).

Six non-collagenous proteins reacting with coeliac disease patient serum IgA were obtained, with isoelectric points of 4-97, 5-15,6-95,7-49,7-64, and 7-75 and molecular weights of 18-5-37-0 kDa (measured by gel filtration). The polypeptides with isoelectric points 7-49 and 7 75 were no longer visible after dithiothreitol treatment which indicates disulphide bonds between polypeptide chains. The other polypeptides seem to be monomeric. The purity of each was ensured by analytical isoelectric focusing. The purified human proteins (CDAP) attached to the affinity gel beads apparently removed all ARA and EMA from serum of each of the ten coeliac disease patients (see table), whereas titres of AGA were not affected by the CDAP column. By contrast, the gliadin-attached gel did not bind ARA and EMA, but removed most AGA from the BINDING OF

IgA AGA, ARA, AND EMA TO GLIADIN AND CDAP IN AFFINITY CHROMATOGRAPHY

*For AGA m ELISA unlts/ml; for ARA and EMA, reciprocal of highest dilution that gave positive fluorescence

725

patient serum samples. Thus, in active coeliac disease there are serum antibodies against these six protein molecules. The antigens do not cross-react with AGA. We have now identified human protein molecules which, in genetically determined individuals, become autoantigens when gluten ingestion starts. The antigen (CDAP) is composed of several non-collagenous proteins; patient serum IgA specifically bound all these molecules during the purification process. These human molecules seem to have homologues in both rat and monkey tissues. The high sensitivity and disease specificity of ARA and EMA in coeliac disease and dermatitis herpetiformis’ 3,5,6 suggest that the triggering antigens have a pathogenetic role. Serum reticulin antibodies can precede jejunal damage in coeliac disease, and are markers also for the latent form of the disease. Their importance is emphasised by the fact that positivity for these antibodies is genetically determined. They not only predict undiagnosed coeliac disease in healthy first-degree relatives of coeliac disease patients, but also detect a small number of relatives with normal jejunal mucosa but with coeliac-type HLA haplotypes (Al, B8, DR3) (unpublished observations). The purification of coeliac-disease-specific autoantigens will allow study of their role in the pathogenesis of coeliac disease and dermatitis herpetiformis. Our fmdings suggest an autoimmune process. Gamma/delta T-cell-receptorbearing lymphocytes are thought to have an important role in autoimmune disorders?,8 Jejunal specimens from coeliac disease patients contain many more of these cells than do normal jejunal specimens. Gamma/delta T cells are present in substantial numbers in the jejunal mucosa at the latent stage of the disease.9 Specific HLA class II molecules on antigen-presenting cells, especially DQ, could present self-peptides, thus activating the autoreactive T-cell population. CDAP could be such a self-antigen. These autoantigens are presented to alpha/beta cells, leading to the production of the autoantibodies ARA and EMA. In this hypothetical autoimmune model gliadin would be the aetiological agent and CDAP the putative autoantigen. The Coeliac Disease Study Project, Tampere, is supported by the Academy of Finland (M. M. and grant number 1061129).

M, Holm K, Collin P, Savilahti E. Increase of gamma/delta T cell receptor bearing lymphocytes in normal small bowel mucosa of a patient with latent coeliac disease. Gut (in press).

9. Maki

ADDRESS: Department of Clinical Sciences, University of Tampere, Finland (M. Maki, MD, O Hällstrom, MD, A. Marttinen, PhD) Correspondence to Dr Markku Maki, Department of Clinical Sciences, University of Tampere, Teiskontie 35, PO Box 607, SF-33101 Tampere, Finland.

Prevention of chronic Pseudomonas aeruginosa colonisation in cystic fibrosis

by

early treatment

To assess whether chronic pulmonary colonisation with Pseudomonas aeruginosa in cystic fibrosis is preventable, 26 patients who had never received

anti-pseudomonas chemotherapy were randomly allocated to groups receiving either no antipseudomonas chemotherapy or oral ciprofloxacin and aerosol inhalations of colistin twice daily for 3 weeks, whenever Ps aeruginosa was isolated from routine sputum cultures. During the 27 months of the trial, infection with Ps aeruginosa became chronic in significantly fewer treated than untreated subjects (2 [14%] vs 7 [58%]; p

Reaction of human non-collagenous polypeptides with coeliac disease autoantibodies.

We identified and purified six human noncollagenous protein molecules that specifically bind to serum IgA from patients with coeliac disease, and whic...
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