SYNOVIAL FLUID T CELLS AND 65 kD HEAT-SHOCK PROTEIN

856 cultures showed no or

growth of salmonella, shigella, campylobacter,

yersinia.

We conclude that this patient had a typhoid-fever-like syndrome following Ty21a vaccine. Department of Internal Medicine, Free University Hospital, 1007 MB Amsterdam, Netherlands

A. BEISHUIZEN T. W. VAN HAEFTEN

R, Furer E. Isolation and characterization of gal E mutants Ty21a of Salmonella typhi: a candidate strain for a live oral typhoid vaccine. J Infect Dis 1975; 141: 553-58. 2. Levine MM, Ferrecio L, Black RE, Germanier R, Chilean Typhoid Committee. Large scale Ty21a live oral typhoid vaccine in enteric-coated capsule formulation Lancet 1987; i: 1049-52 3. Gilman RH, Homick RB, Woodward WE, et al. Immunity in typhoid feverevaluation of Ty21a: an epimerase less mutant of S. typhi as a live oral vaccine. J Infect Dis 1977; 136: 717-23. 4. Wahdan MH, Sene C, Censier Y, Sallam S, Germanier R. A controlled field trial of live Salmonella typhi strain Ty21a oral vaccine against typhoid; three year results. J Infect Dis 1982; 145: 292-96. 5. Levine MM, Black RE, Ferrecio C, et al. The efficacy of attenuated Salmonella typhi oral vaccine strain Ty21a evaluated in controlled field trials In: Holmgren J, Lindberg H, Mollby R, eds. Development of vaccines and drugs against diarrhoea. Lund: Student Literature, 1986: 90-101. 6. Black RE, Levine MM, Young C, et al. Immunogenicity of Ty21a attenuated Salmonella typhi given wiuth sodium bicarbonate or in enteric-coated capsules. Devl Biol Stand 1983; 53: 9-14. 7. Levine MM, Ferreccio C. Typhoid vaccines. Lancet 1988; i: 173. 1. Germanier

SYNOVIAL FLUID T CELLS AND 65 kD HEAT-SHOCK PROTEIN

SIR,-Mr Res and his colleagues (Aug 27, p 478) describe the recognition by synovial fluid T lymphocytes of a 65 kD antigen from Mycobacterium bovis. As noted in their discussion, it is not clear whether T-cell recognition of this antigen is related to cell-mediated immunity for mycobacteria because the 65 kD antigen is found in a wide variety of bacteria and is conserved.1,2 We have confirmed that the 65 kD antigen is stimulatory for synovial fluid T lymphocytes and we have examined the ability of bacterial antigens from non-mycobacterial sources to elicit similar responses. Although the 65 kD mycobacterial antigen has been strongly implicated in experimental adjuvant arthritis of rats,3,4 in man the strongest link between immune responses to bacterial antigens and arthritis occurs in reactive arthritis. In this disease, we and others have demonstrated synovial fluid T cell recognition of the organisms that "trigger" reactive arthritis. However, synovial T cells from some patients with other arthropathies can also respond to these bacteria.’ We have examined synovial and peripheral blood T cells from 31 patients with inflammatory arthropathy for their proliferative responses not only to mycobacterial antigens but also bacteria associated with reactive arthritis. We used recombinant ill protein of M leprae (95% homologous to the M bovis protein),8 purified protein derivative (PPD) from M tuberculosis, and heat-killed or irradiated suspensions of salmonella, campylobacter, and yersinia. The patients had reactive arthritis (5), rheumatoid arthritis (16), Still’s disease (2), systemic lupus (1), or to

03

spondyloarthropathy (8). Our findings (to be presented in detail elsewhere) accord with Res and colleagues’ in that a high proportion of the patients (17/31) PROLIFERATIVE RESPONSES OF SYNOVIAL FLUID AND PERIPHERAL

responded to 65 kD (response being defmed as a stimulation index at day 6 of more than 3 and 3H-thymidine incorporation above 2000 disintegrations per min); a similar proportion (18/31) also responded to salmonella. Responses to 65 kD and salmonella were confined to synovial fluid lymphocytes; responses to PPD were recorded by both synovial fluid and peripheral blood T cells, though the synovial cell responses were invariably greater. When the synovial T cell responses to 65 kD, PPD, and salmonella were compared, a strong correlation was found between 65 kD and salmonella (r=0’63, p= 0-002) but not between 65 kD and PPD responses (r=0’ll, p=0’6). Although the largest responses to salmonella and 65 kD were recorded for reactive arthritis, some others reacted just as strongly and the correlation between salmonella and 65 kD responses remains significant even when the reactive cases are excluded. The recognition of both 65 kD and salmonella within synovial fluid, and the dissociation of the 65 kD and PPD responses are illustrated by an additional patient (table) with an acute spondyloarthropathy probably psoriatic, and no clinical evidence for a preceding infection by enteric organisms. These observations add weight to Res and colleagues’ suggestion that 65 kD is an important stimulatory antigen for T cells within the joint, and they show that recognition of 65 kD can accompany recognition of enteric bacteria rather than mycobacteria. This relation was most easily seen in reactive arthritis where the immune response to 65 kD may be elicited by the antigenic challenge of the enteric organism. In the non-reactive arthritides the role of bacterial infection is less clear, though there has been speculation on the relation between arthritis and bowel flora in both rheumatoid arthritis and spondyloarthropathy.9 65 kD is a heat- shock protein and is likely to have conserved epitopes that could be shared with the heat-shock proteins of other mammals. Since these proteins may be produced in the synovium during an acute inflammatory responses 65 kD-specific T cell responses initiated by different bacteria might be perpetuated by antigenically cross-reactive "self’ heat-shock proteins.

J. S. H. GASTON Department of Rheumatology, University of Birmingham, Birmingham B15 2TJ

P. F. LIFE L. BAILEY P. A. BACON

Young DB, Ivanyi J, Cox JH, Lamb JR. The 65 kD antigen of mycobacteria a common bacterial protein? Immunol Today 1987; 8: 215-19. 2. Thole JER, Hindersson P, de Bruyn J, et al. Antigenic relatedness of a strongly immunogeneic 65 kDa mycobactenal protein antigen with a similarly sized ubiquitous bacterial common antigen. Microb Pathogen 1988; 4: 71-83. 3. Cohen IR, Holoshitz J, van Eden W, Frenkel A. T lymphocyte clones illuminate pathogenesis and effect therapy of experimental arthritis. Arthritis Rheum 1985,28: 1.

841-45. Eden W, Thole JER, van der Zee R, et al. Cloning of the mycobacterial epitope recognized by T lymphocytes m adjuvant arthritis. Nature 1988; 331: 171-73 5. Gaston JSH, Life P, Bailey L, Consalvey S, Bacon PA. Recognition of bacterial antigens by synovial fluid T cells of patients with reactive arthritis Arthritis Rheum 1988; 31 (suppl): C91. 6. Ford DK, da Roza DM, Schulzer M. Lymphocytes from the site of disease but not blood lymphocytes indicate the cause of arthritis. Ann Rheum Dis 1984, 44: 701-10 7. Gaston JSH, Life P, Bailey L, Bacon PA. Synovial fluid lymphocyte responses to bacterial antigens. Br J Rheumatol 1988; 27: 44s. 8. Lamb FI, Kingston AE, Estrada-G I, Colston MJ. Heterologous expression of the 65 kilodalton antigen of mycobacterium leprae and munne T-cell responses to the gene product. Infect Immun 1988; 56: 1237-41. 9. Ebringer A, Shipley M, eds. Pathogenesis of ankylosing spondylitis and rheumatoid arthritis Br J Rheumatol 1988; 27 (suppl II). 1-178. 10. Polla BS. A role for heat shock proteins in inflammation? Immunol Today 1988, 9: 134-36. 4.

van

BLOOD LYMPHOCYTES FROM PATIENT WITH ACUTE SPONDYLOARTHROPATHY

SiR,—Interest in the mycobacterial 65 kD heat-shock protein (HSP65) has intensified following the discovery of its cross-reaction with cartilage and its association with adjuvant arthritis.’ Mr Res and colleagues measured the in vitro proliferative response to various mycobacterial antigens of synovial fluid and peripheral blood mononuclear cells from patients with arthritis. TheB conclude that responsiveness to 65 kD was not specific 10 rheumatoid arthritis but was found in all types of early chrome arthritis. Here, we present our findings. Synovial specimens from patients with arthritis were obtained J: joint replacement surgery, snap frozen, and stored at --70CC Control specimens were obtained at necropsy. Sections,,7I-lID of

857 tissue from 5 patients with rheumatoid arthritis, 2 with osteoarthritis, and from 4 controls were labelled with monoclonal antibodies to stress proteins by the indirect immunoperoxidase technique. We used an antibody directed against the 65 kD mycobacterial protein (TB78)Z and two antibodies directed against human stress proteins, one specific for the highly inducible 72 kD antigen (C92)3 and the other (N27) recognising (by immunoprecipitation or western blotting) both the highly inducible and the constitutive (73 kD) molecules (Dr W. J. Welch, Cold Spring Harbor Laboratory, personal communication). The protein distribution was very different. Anti-HSP65 stained the fibrinous intimal layer in the rheumatoid arthritis specimens. The human specific antibodies (C92 and N27) labelled intracellularly, the most intense reaction being with synovial intimal cells and capillary endothelial cells. Enhanced binding was noted in all specimens from arthritic patients. Thus, human synovial tissue binds HSP65 antibodies; these results are in agreement with the in-vitro work of Res et al. However, the nature of this binding is consistent with the labelling of immune complexes trapped in the fibrinous tissue of the joint. In contrast, the human-specific antibodies showed cytoplasmic labelling which was enhanced in arthritic patients, suggesting up-regulation of the synthesis of the 72 kD protein in response to inflammation. I. L. McLEAN V. R. WINROW ARC Bone and Joint Research Unit, London Hospital Medical College, P. I. MAPP London El 2AD; A. H. CHERRIE and MRC Tuberculosis J. R. ARCHER and Related Infections Unit, Hammersmith Hospital, London W12 D. R. BLAKE Eden W, Holoshitz J, Nevo Z, Frenkel A, Klajman A, Cohen IR Arthritis induced by a T-lymphocyte clone that responds to Mycobacterium tuberculosis and to cartilage proteoglycans. Proc Natl Acad Sci USA 1985; 82: 5117-20. 2 Coates ARM, Hewitt J, Allen BW, Ivanyi J, Mitchison DA. Antigenic diversity of Mycobacterium tuberculosis and Mycobacterium bovis detected by means of monoclonal antibodies. Lancet 1981; ii: 167-69. 3. Welch WJ, Suhan JP. Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J Cell Biol 1986; 103: 2035-52. 1

distribution in West Africa, inheritance, haematology, similarities or differences from ovalocytosis in other populations, or any possible protection from malaria it may offer have not been studied. Most patients have normal or slightly reduced haemoglobin (Hb) concentrations despite extremely abnormal red-cell appearances; but I have seen one Nigerian infant aged two years with ovalocytosis, Hb 15 g/dl, and intense Plasmodium falciparum

parasitaemia. A systematic search

in Zambia of 100 blood films and observations over three years of many films from patients with miscellaneous disorders did not reveal a single subject with

ovalocytosis. Darwinian selection for the gene or genes governing ovalocytosis populations of Nigeria, where P falciparum malaria is holoendemic or hyperendemic. There is also a selective survival of HbAS childrenbut not in Zambia, where malaria is generally hypoendemic or mesoendemic and the frequency of the HbS-gene low.

may have started in

Flat E33, Du Cane Court, London SW17 7JJ 1. Osamo

NO, Photiades DP, Haddock DRW. Hereditary elliptocytosis in Nigerians. Nigerian J Med Sci 1979; 2: 141-46. 2. Fleming AF, Harrison KA, Briggs ND, et al. Anemia in young primigravidae in the guinea savanna of Nigeria: sickle-cell trait gives partial protection against malaria Ann Trop Med Parasitol 1984; 78: 395-404. 3. Fleming AF, Storey J, Molineaux L, et al. Abnormal hemoglobins in the sudan savanna of Nigeria I prevalence of hemoglobins and relationships between sickle-cell trait, malaria and survival. Ann Trop Med Parasitol 1979; 73: 162-72

PERCEPTION OF DRUG HAZARDS

van

NIFEDIPINE AND NOCTURIA

SIR,-A recent survey by a regional centre for adverse drug reaction monitoring has collected 12 cases of increased nocturnal micturitions (7 men and 5 women) among 157 patients treated with nifedipine (30-60 mg per day) for periods up to several months. Except for 1 case the men did not have prostatic enlargement, which confirms Williams and Donaldson’s findings.’ A 73-year-old man has been operated on without success for a bladder neck dysfunction. In all the 12 cases, this increased nocturnal micturition (4 per night on average) disappeared within a few days after

stopping nifedipine. In 2 cases readrninistration of the drug was followed by a return of nocturia. In 5 patients another calciumblocking agent (nicardipine) could be substituted without reactivation of the side-effect. Individual cases of acute urine retention have also been reportedThe mechanism of these urinary disturbances could be the result of the addition of diuretic effect to a detrusor hypocontractility, both consequences of calcium channel

blocking. Centre Régional de Pharmacovigilance,

Hôpital Pasteur, 06002 Nice, France

G. MIGNOT G. BERNARD R. M. CHICHMANIAN

ALAN F. FLEMING

SiR,—At the autumn meeting of the British Association for Psychopharmacology (Galway, Ireland, Sep 15-17, 1988) practitioners were concerned about the unjustified withdrawal of drugs from the market and were skeptical about the real importance of the haematotoxicity of mianserin. Dr Inman’s paper on this point (July 9, p 90) comforts those who, after fair study of available data, have concluded that the haematotoxicity of mianserin has been overestimated.l It also illustrates the fact that, even among scientific researchers, the perception of risks may be biased.z I would like to give another illustration of the inaccurate perception of drug hazards. During the first four years after mianserin was marketed, there was only one published case of agranulocytosis From 1960 to 1961, at the beginning of imipramine use, there were nine published case reports of agranylocytosis. Such an observation acquires particular significance when one recalls that postmarketing surveillance systems were not developed in 1960 (the year of the thalidomide tragedy) and that doctors were then more concerned with efficacy than with safety. Groupe de Recherche en Pharmacovigilance, 19 Rue de la Glacière, 75013 Paris, France

MARC GIRARD

1 Girard 2

M Le rapport risque/bénefice des médicaments. l’exemple des antidépresseurs L’Encéphale 1988, 14: 97-99. Inman WHW Risks in medical intervention balancing therapeutic risks and benefits

PEM News 1984, 2: 15-37. DA, Hale AS. Mianserin and

3 Curson

agranulacytosis. Br Med J 1979, 1: 378-79.

WHO’S FOR THE THORACIC DUCT? 1. Williams G, Donaldson RM. Nifedipine and noctuna. Lancet 1986, i: 738. 2 Eicher JC, Chalopin JM, Tanter Y. Nicardipine et appareil urinaire L’association

Française des Centres de Pharmacovigilance, Dijon, Oct 1-2, 1987

OVALOCYTOSIS AND MALARIA

SIR,-Your Sept 10 editorial on ovalocytosis and malaria is inaccurate in stating that hereditary ovalocytosis has been found almost nowhere other than in South-East Asia, and certainly not in Africa.

Haematologists have been aware for many years that a benign form of ovalocytosis or elliptocytosis is seen in 2-3% of the population

in both southern and northern

Nigeria. 1-2It is

sufficiently common for laboratory scientists and medical students to

be

taught

to

recognise

the appearance on blood films, but its

SiR,-Being a gentleman, I must contradict your peripatetic correspondent (Sept 3, p 562). The thoracic duct is alive and well. As a schoolboy, I thought that nerve impulses were like radio waves-transmitted through the tissues of the body to receivers placed in the various organs-and the discovery of nerves themselves came as a great disappointment, though not, in this context, a shock. (I am still not entirely convinced that these nerves do what physiologists tell us they do.) Your contributor seems to have a similar view about the ephemeral or imaginary nature of the thoracic duct. Is it that most medical students never bother much about the posterior mediastinum? Perhaps this region always comes at the end of term, just before examinations. In any case here, in