HE LANCE
`
`BOSTON, MASS. AND LONDON • SATURDAY AUGUST 27 1988
`
`VOL II FOR 1988
`
`No 8609
`
`ORIGINAL ARTICLES
`Safety, Immunogenicity, and Efficacy of Recombinant Live Oral Cholera
`Vaccines, CVD 103 and CVD 103-HgR
`M. M. Levine, MD, J. B. Kaper, PH 13, Deirdre Herrington, MD, Julian Ketley, PH D,
`Genevieve Losonsky, MD, C. 0. Tacket, MD, Ben Tall, Ps, Stanley Cryz, PH D
`Use of Recombinant Granulocyte-macrophage Colony Stimulating Factor in
`the Brazil Radiation Accident
`Anna Butturini, MD, P. C. De Souza, R. P. Gale, MD, J. M. Cordiero, MD,
`D. M. Lopes, MD, Carlos Neto, MD, C. B. Cunha, MD, C. E. P. De Souza, W. G. Ho,
`D. G. Tabak, MD, J. M. Sanpai, MD, Amihay Burla, MD
`Does Sc1-70 Modulate Collagen Production in Systemic Sclerosis?
`Angeline Douvas, Nip
`
`Synovial Fluid T Cell Reactivity against 65 kD Heat Shock Protein of
`Mycobacteria in Early Chronic Arthritis
`P. C. M. Res, M sc, C. G. Schaar, F. C. Breedveld, MD, Willem van Eden, MD,
`J. D. A. van Embden, PH D, Prof I. R. Cohen, MD, R. R. P. de Vries, PH D
`Reduced Felodipine Bioavailability in Patients taking Anticonvulsants
`S. Capewell, MRCP, S. Freestone, MRCP, J. A. J. H. Critchley, MRCP, A. Pottage, FRCP,
`Prof L. F. Prescott, FRCP
`
`467 (cid:9)
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`471 (cid:9)
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`475 (cid:9)
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`478 (cid:9)
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`480 (cid:9)
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`510
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`510
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`No Clinical Signs 14 Years after HIV-2
`Transmission via Blood Transfusion (cid:9)
`Dr Gerard Dufoort and others
`Short-term Buspirone Treatment in
`Disinhibition with Dementia (cid:9)
`Dr J. W. G. Tiller and others
`Bacterial Antigenic Cross-reactions
`and Haemolytic Uraemic Syndrome 510
`Dr Henrik Chart and others
`Prazosin Contraindicated in Patients
`with Narcolepsy (cid:9)
`Dr Christian Guilleminault and others
`Sleeping Position and SIDS (cid:9)
`Dr Susan Beal
`Anxiety and Panic during Magnetic
`Resonance Scans (cid:9)
`Dr S. C. Brennan and others
`Anosmia (cid:9)
`Dr Allan Knight
`Psoriasis and Cyclosporin Withdrawal (cid:9)
`Dr A. V. Powles and others
`
`Delayed Seroconversion in
`Legionnaire's Disease (cid:9)
`Dr R. Monfort and others
`Energy Expenditure in Children with
`Cystic Fibrosis (cid:9)
`Dr P. B. Pencharz and others
`Fish Oil and Ischaemic Heart Disease
`in Greenland (cid:9)
`Dr Peter Bjerregaard, Dr Jem Dyerberg
`Salt and Pregnancy-induced
`Hypertension (cid:9)
`Dr J. A. Millar
`Is Alzheimer's Disease Distinct from
`Normal Ageing? (cid:9)
`Dr Carol Brayne, Dr Paul Calloway
`Measurement of Cardiac Output (cid:9)
`Dr John Rawles
`Renal Failure during Dissolution of
`Gallstones by Methybutyl Ether (cid:9)
`Prof H. Savolainen
`Hydrocortisone Myopathy (cid:9)
`Dr M. R. J. Sury and others
`Single Photon Emission Computed
`Tomography in Diagnosis of Herpes
`Simplex Encephalitis (cid:9)
`Dr Roderick Duncan and others;
`Dr T. Nara and others
`Continuous Intrapartum Measurement
`. of Fetal Oxygen Saturation (cid:9)
`Dr N. Johnson, Dr R. J. Lilford
`Pulse Oximetry and
`Methaemoglobinaemia (cid:9)
`Dr P. Govaert and others
`The Khartoum Floods and Diarrhoeal
`Diseases (cid:9)
`Dr Paul Shears
`Fetal Haemoglobin Measurement in
`the Assessment of Red Cell
`Isoimmunisation (cid:9)
`Mr D. G. Altman, B SC
`Human Herpesvirus-6 infection and
`Disease (cid:9)
`Prof G. R. F. Krueger
`Chemotherapy and the Circulating
`Progenitor Cell Compartment (cid:9)
`Dr C. D. L. Reid and others
`Coronary Thrombolysis and
`Myocardial Salvage by Tissue
`Plasminogen Activator (cid:9)
`Dr P. L. Thompson
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`511
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`512
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`NEWSPAPER
`
`NOTES AND NEWS
`Going up in Smoke
`Chemobylia
`Pneurnocystis carinii Finds its Identity
`A Three-horse Race?
`Feedback on Prescribing for GPs
`Wishful Drinking
`Parotid Cysts and HIV Infection
`AIDS Counselling
`Confidentiality of Illustrative Clinical
`Records
`Cervical Smear Technique
`Inside the Black Boxes
`
`522
`522
`522
`522
`523
`523
`523
`523
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`523
`523
`523
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`507
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`508 (cid:9)
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`508 (cid:9)
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`509
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`509 (cid:9)
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`REVIEWS
`Notices of Books (cid:9)
`
`HOSPITAL PRACTICE
`Improved Recovery and Reduced
`Postoperative Stay after
`Therapeutic Suggestions during
`General Anaesthesia (cid:9)
`Carlton Evans, B sc,
`P. H. Richardson, PH D
`
`OCCASIONAL SURVEY
`Gastro-oesophageal Reflux and
`Respiratory Disorders in Adults
`Jon Goldman, MRCP,
`J. R. Bennett, FRCP
`
`INTERNATIONAL PHYSICIANS
`FOR THE PREVENTION OF
`NUCLEAR WAR
`Nuclear Weapons Test Ban 1988
`Michael McCally, MD,
`C. K. Cassel, MD
`
`PERSONAL PAPER
`Air Travel and Thrombotic
`Episodes: the Economy,Class
`Syndrome
`J. M. Cruickshank, FRCP,
`Prof Richard Gorlin, MD,
`Prof Bryan Jennett, FRCS
`ROUND THE WORLD
`Japan
`United States
`
`IN ENGLAND NOW
`
`MEDICINE AND THE LAW
`Wrong Blood Transfusion and
`Rhesus Incompatibility (cid:9)
`Bad Professional Relations and Risks
`to Patients
`
`INTERNATIONAL DIARY
`OBITUARY
`Charles Grant Clark (cid:9)
`William Valentine Mayneord
`CORRECTIONS
`Wolff's Headache and other Head
`Pain; 5th edition
`Stomach Cancer Cluster in Mexico
`
`482
`
`491
`
`Resection of Haematogenous
`Metastases (cid:9)
`Chemotherapy of Leprosy (cid:9)
`Food and H, Blockade (cid:9)
`Economics of the Illicit Drug
`Market in the UK
`Williams Syndrome—
`the Enigma Continues (cid:9)
`
`485
`487
`488
`
`489
`
`490
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`493 (cid:9)
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`LETTERS TO THE EDITOR
`Non-A, Non-B Hepatitis Transmission
`by Intravenous Immunoglobulin
`Dr P. E. Williams and others
`What Causes Diabetic Renal Failure?
`Dr P. G. McNally and others
`Are Neonatal Necropsies Useful in
`Developing Countries?
`495 (cid:9)
`Prof Meharban Singh and others
`/4 Epidemiology of Listeriosis, England
`and Wales
`Dr S. M. Hall and others
`Lack of Effect of Topical Retinoic Acid
`on Sebum Excretion Rate in Acne
`Dr W. J. Cunliffe, Dr S. Macdonald-Hull
`Isotretinoin Dose and Teratogenicity
`Dr E. J. Lammer and others
`Dependence Potential of
`Benzodiazepines
`Dr W. W. Fleischhacker
`Benzodiazepines and Convulsions
`Dr J. R. Robertson and others
`Reversal of Hepatic Coma with
`Flumazenil with Improvement in
`Visual Evoked Potentials
`Dr D. A. Burke and others
`In-utero Platelet Transfusion for
`Alloimmune Thrombocytopenia (cid:9)
`Dr U. Nicolini and others
`Guthrie Cards for Detection of Point
`Mutations in Phenylketonuria
`Dr S. Lyonnet and others
`Bone-marrow Transplantation for
`Severe Genetic Anaemia
`Prof J. R. Hobbs
`Triptorelin to Prevent Hysterectomy in
`Patients with Leiomyomas
`Dr H. A. I. M. Van Leusden
`Value of Necropsy in AIDS
`Dr T. A. Jacobson;
`Dr P. E. Hay and others
`Zidovudine Overdose
`Dr M. Hargreaves and others
`Simplified Confirmatory HIV Testing
`Dr Andrew Day, Dr P. P. Mortimer
`
`497 (cid:9)
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`500 (cid:9)
`500 (cid:9)
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`500 (cid:9)
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`Editorial Office: 46 Bedford Square, London WC1B 3SL, England. Tel:
`01-436 4981. The Lancet, North American Edition published weekly by
`Little, Brown and Company, 34 Beacon St., Boston, Mass. 02108. Annual
`subscription in U.S. $75.00, in Canada $85.00; resident and intem rate
`(U.S. and Canada) $48.50; Single copy $7.00. Second class postage paid
`at Boston, Mass., and at additional mailing offices. © The Lancet Ltd.,
`27 August 1988. Postmaster: Send address changes to The Lancet, 34
`Beacon St, Boston, Mass. 02108 (617) 227-0730, ext 516. ISSN 0099-5355
`THE WHOLE OF THE LITERARY MATTER IN THE LANCET IS COPYRIGHT
`
`LI tit 3253411. 033
`'. 101.83 1238 (cid:9)
`9023071 LVOF
`(MOFP DI VI SION
`I/NUOMS UNIT
`OC
`liASdING rtiN (cid:9)
`
`20540
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`Ex. 1047 - Page 1
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`(cid:9)
`

`

`Olvizgot4
`
`The Lancet • Saturday 27 August 1988
`
`SAFETY, IMMUNOGENICITY, AND EFFICACY
`OF RECOMBINANT LIVE ORAL CHOLERA
`VACCINES, CVD 103 AND CVD 103-HgR
`
`MYRON M. LEVINE'
`DEIRDRE HERRINGTON'
`GENEVIEVE LOSONSKY'
`BEN TALL'
`
`JAMES B. KAPER'
`JULIAN KETLEY'
`CAROL 0. TACKET1
`STANLEY CRYZ2
`
`Center for Vaccine Development, Division of Geographic Medicine,
`Department of Medicine, University of Maryland School of
`Medicine, Baltimore, Maryland, USA;' and Swiss Serum and
`Vaccine Institute, Berne, Switzerland
`
`Summary
`
`The genes encoding the A (toxic) subunit
`of cholera toxin were deleted from
`pathogenic Vibrio cholerae 01 strain 569B by recombinant
`techniques, leaving intact production of immunogenic,
`non-toxic B subunit. The resultant strain, CVD 103,
`evaluated for safety, immunogenicity, and efficacy as a live
`oral vaccine, was highly attenuated and elicited strong
`antibacterial and antitoxic immune responses; a single dose
`significantly protected volunteers against challenge with
`pathogenic V cholerae 01 of either serotype or biotype. A
`further derivative, CVD 103-HgR, which has an Hg"-
`resistance gene to differentiate it from wild-type vibrios, was
`also well-tolerated, immunogenic, and protective;
`moreover, faecal excretion of this derivative was
`significantly lower than that of CVD 103, which should
`minimise environmental spread of the vaccine. CVD
`103-HgR is a candidate for expanded clinical trials in
`endemic areas.
`
`Introduction
`CHOLERA is characterised clinically by voluminous
`diarrhoea and dehydration and epidemiologically by
`explosive outbreaks. Parenteral killed whole-cell cholera
`vaccines usually protect older people for only a few months
`and do not protect many young children at all.' The World
`Health Organisation has therefore made the development of
`
`improved cholera vaccines a priority.2 To achieve the
`greatest public health effect in less developed countries, the
`new vaccine will have to be safe, easy to administer,
`inexpensive, and highly protective after just a single dose.
`Our efforts to develop a new cholera vaccine have been
`guided by five observations. Cholera in volunteers confers
`substantial protection (for at least 3 years) against
`rechallenge with pathogenic Vibrio cholerae 01 of either the
`homologous or heterologous serotype." Cholera toxin
`(enterotoxin) causes the severe purging of cholera gravis; as
`little as 5µg purified toxin by mouth can elicit this
`syndrome.8 Antibacterial immunity is protective even
`without antitoxic immunity.3'9 There is no agreement on
`the critical antigens causing antibacterial imrnunity.8 The
`presence of serum vibriocidal antibody correlates with
`immunity from natural infection or whole-cell vaccines,"
`apparently by indicating the existence of intestinal secretory
`IgA antibodies against critical vibrio surface antigens."-"
`We used recombinant DNA methods to attenuate
`pathogenic V cholerae 01 strains known to cause cholera and
`to elicit immunity in volunteers. By site-directed
`mutagenesis, the genes encoding the cholera holotoxin or
`the A (enzymically active, toxic) subunit of cholera toxin
`were precisely deleted."'" This deletion leaves intact the
`genes encoding the antigenic but non-toxic B subunit, as
`well as all surface antigens of the vibrio that interact with the
`human intestinal immune system. The first recombinant
`vaccines of this variety tested in man, including A — B —
`JBK 70 and A — B + CVD 101, were highly attenuated
`compared with their pathogenic parents.9 Nevertheless,
`they caused mild diarrhoea in half the recipients.9 One
`hypothesis to explain the residual reactions provoked by the
`first genetically engineered candidate vaccine strains was
`that accessory toxins elaborated by V cholerae 01 were
`responsible. If so, strains lacking the accessory toxins might
`produce minimally reactogenic, yet protective, live oral
`vaccines. Specific toxins thought to provoke reactions were
`the El Tor haemolysin/cytotoxin" and a Shiga-like
`cytotoxin.'8 Deletion of the genes encoding the El Tor
`haemolysin/cytotoxin from JBK 70 and CVD 101 did not
`sufficiently reduce their capacity to provoke reactions.9
`8609 © The Lancet Ltd, 1988
`
`404AFFvaii
`
`Ex. 1047 - Page 2
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`The Lancet Saturday 27 August 1988
`
`4,
`
`SAFETY, IMMUNOGENICITY, AND EFFICACY
`OF RECOMBINANT LIVE ORAL CHOLERA
`VACCINES, CVD 103 AND CVD 103-HgR
`
`MYRON M. LEVINE'
`DEIRDRE HERRINGTON'
`GENEVIEVE LOSONSKY1
`BEN TALL'
`
`JAMES B. KAPER1
`JULIAN KETLEY1
`CAROL 0. TACKET1
`STANLEY CRYZ2
`
`Center for Vaccine Development, Division of Geographic Medicine,
`Department of Medicine, University of Maryland School of
`Medicine, Baltimore, Maryland, USA;1 and Swiss Serum and
`Vaccine Institute, Berne, Switzerland2
`
`Summary
`
`The genes encoding the A (toxic) subunit
`of cholera toxin were deleted from
`pathogenic Vibrio cholerae 01 strain 569B by recombinant
`techniques, leaving intact production of immunogenic,
`non-toxic B subunit. The resultant strain, CVD 103,
`evaluated for safety, immunogenicity, and efficacy as a live
`oral vaccine, was highly attenuated and elicited strong
`antibacterial and antitoxic immune responses; a single dose
`significantly protected volunteers against challenge with
`pathogenic V cholerae 01 of either serotype or biotype. A
`further derivative, CVD 103-HgR, which has an Hg"-
`resistance gene to differentiate it from wild-type vibrios, was
`also well-tolerated, immunogenic, and protective;
`moreover, faecal excretion of this derivative was
`significantly lower than that of CVD 103, which should
`minimise environmental spread of the vaccine. CVD
`103-HgR is a candidate for expanded clinical trials in
`endemic areas.
`
`Introduction
`CHOLERA is characterised clinically by voluminous
`diarrhoea and dehydration and epidemiologically by
`explosive outbreaks. Parenteral killed whole-cell cholera
`vaccines usually protect older people for only a few months
`and do not protect many young children at all.' The World
`Health Organisation has therefore made the development of
`
`improved cholera vaccines a priority' To achieve the
`greatest public health effect in less developed countries, the
`new vaccine will have to be safe, easy to administer,
`inexpensive, and highly protective after just a single dose.
`Our efforts to develop a new cholera vaccine have been
`guided by five observations. Cholera in volunteers confers
`substantial protection (for at least 3 years) against
`rechallenge with pathogenic Vibrio cholerae 01 of either the
`homologous or heterologous serotype.3-7 Cholera toxin
`(enterotoxin) causes the severe purging of cholera gravis; as
`little as 5 1..tg purified toxin by mouth can elicit this
`syndrome.8 Antibacterial immunity is protective even
`without antitoxic immunity .3-7'8 There is no agreement on
`the critical antigens causing antibacterial immunity.8 The
`presence of serum vibriocidal antibody correlates with
`immunity from natural infection or whole-cell vaccines,10
`apparently by indicating the existence of intestinal secretory
`IgA antibodies against critical vibrio surface antigens."-14
`We used recombinant DNA methods to attenuate
`pathogenic V cholerae 01 strains known to cause cholera and
`to elicit immunity in volunteers. By site-directed
`mutagenesis, the genes encoding the cholera holotoxin or
`the A (enzymically active, toxic) subunit of cholera toxin
`were precisely deleted.15.16 This deletion leaves intact the
`genes encoding the antigenic but non-toxic B subunit, as
`well as all surface antigens of the vibrio that interact with the
`human intestinal immune system. The first recombinant
`vaccines of this variety tested in man, including A — B —
`JBK 70 and A — B + CVD 101, were highly attenuated
`compared with their pathogenic parents.9 Nevertheless,
`they caused mild diarrhoea in half the recipients .8 One
`hypothesis to explain the residual reactions provoked by the
`first genetically engineered candidate vaccine strains was
`that accessory toxins elaborated by V cholerae 01 were
`responsible. If so, strains lacking the accessory toxins might
`produce minimally reactogenic, yet protective, live oral
`vaccines. Specific toxins thought to provoke reactions were
`the El Tor haemolysin/cytotoxin17 and a Shiga-like
`cytotoxin.'s Deletion of the genes encoding the El Tor
`haemolysin/cytotoxin from JBK 70 and CVD 101 did not
`sufficiently reduce their capacity to provoke reactions.8
`8609 © The Lancet Ltd, 1988
`
`Ex. 1047 - Page 3
`
`(cid:9)
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`478
`
`THE LANCET, AUGUST 27, 1988
`
`SYNOVIAL FLUID T CELL REACTIVITY
`AGAINST 65 1(1) HEAT SHOCK PROTEIN OF
`MYCOBACTERIA IN EARLY CHRONIC
`ARTHRITIS
`
`CEES G. SCHAAR'
`PIETER C. M. RES' (cid:9)
`FERDINAND C. EREEDVELD2 WILLEM VAN EDEN3
`IRUN R. COHEN5
`JAN D. A. VAN EMBDEN4 (cid:9)
`RENE R. P. DE VRIES'
`
`Department of lmmunohaematology and Blood Bank, University
`Hospital, Leiden;' Department of Rheumatology, University
`Hospital, Leiden,2 Department of Immunology, Veterinary Faculty,
`State University of Utrecht;' Laboratory of Bacteriology, National
`Institute of Public Health and Environmental Hygiene, Bilthoven,
`The Netherlands,4 and Department of Cell Biology, Weizmann
`Institute of Science, Rehovot, Israel'
`
`Summary (cid:9)
`
`The in vitro proliferative response against a
`recombinant 65 kD Mycobacterium bovis
`protein that has 100% homology with the 65 kD protein of
`M tuberculosis was tested in synovial fluid and peripheral
`blood mononuclear cells from patients with rheumatoid
`arthritis (RA) and other types of chronic arthritis. An
`acetone precipitate (AP) of M tuberculosis, and a purified
`protein derivative (PPD) of M tuberculosis were also tested.
`Responsiveness of synovial fluid lymphocytes to the
`mycobacterial antigens was found both in patients with RA
`and in patients with other forms of chronic inflammatory
`arthritis, but not among controls. T cell reactivity against
`mycobacterial antigens was nearly always higher in synovial
`fluid than in peripheral blood in those patients who showed
`reactivity. A significant association was found between
`responsiveness of synovial T cells to the 65 kD protein and
`AP, but no relation between responsiveness to the 65 kD
`protein and PPD. Both the number of 65 kD protein
`responders and the mean proliferative response of synovial
`T cells to the 65 kD protein were inversely correlated with
`duration of joint inflammation. Thus, a 65 kD-protein-
`specific reactivity of synovial T cells, mainly present in an
`early stage of joint inflammation, may be responsible for
`triggering chronic arthritis.
`
`Introduction
`ARTHRITIS can be induced in a genetically susceptible
`strain of Lewis rats by immunisation with Freund's
`complete adjuvant (heat-killed Mycobacterium tuberculosis in
`oil). From lymph node cells of such an immunised rat a T
`cell clone which could induce adjuvant arthritis (AA) after
`transfer to irradiated recipient rats was isolated.' This T cell
`clone (A2b) showed cross-reactivity for an epitope present
`on a M tuberculosis protein and a proteoglycan-associated
`molecule. The M tuberculosis protein epitope is contained
`within a peptide composed of the aminoacids at position
`180-188 of a 65 kD mycobacterial heat shock protein.2
`Another T cell clone (A2c) was shown to induce resistance to
`AA, and clone A2c probably recognised the same epitope on
`the 65 kD protein as clone A2b.3
`Susceptibility to rheumatoid arthritis (RA) is genetically
`controlled and associated with histocompatibility antigen
`DR4. In Spanish leprosy patients who had intradermal skin
`tests with several mycobacterial antigens, DR4-positive
`patients showed a stronger specific response to M
`tuberculosis than non-DR4 patients.4 Therefore, an Ir-gene
`controlling the T cell response to mycobacterial antigen or
`antigens could explain the association between RA and
`DR4.
`
`Synovial fluid mononuclear cells from patients with RA
`for less than 1 yr showed a high proliferative response
`against an acetone-precipitable (AP) fraction of M
`tuberculosis.5 In these patients, mononuclear cells from
`peripheral blood showed almost no reaction against Al'.
`However, in patients with known RA for 1-10 yr a high
`AP-specific proliferation was measured in peripheral blood,
`suggesting a primary activation and expansion of AP-
`specific T cells from the affected joints.5
`We have now investigated whether synovial fluid and
`peripheral blood mononuclear cells of patients with RA
`show reactivity to the 65 kD protein and to AP and PPD
`preparations of mycobacteria. Patients with other forms of
`chronic arthritis, gout, and osteoarthritis were also
`examined.
`
`Patients and Methods
`
`32 patients with chronic inflammatory arthritis were studied at
`the department of rheumatology, Leiden University Hospital.
`Synovial fluid was aspirated from joints as part of their routine
`treatment, and patients volunteered to give blood on the same day.
`22 patients had rheumatoid arthritis, 14 of whom were seropositive
`for rheumatoid factor; 3 had psoriatic arthritis, 1 of whom was tested
`twice; 1 had ankylosing spondylitis; 2 had reactive arthritis; and 4
`had unclassified chronic arthritis, 1 of whom was tested twice. 5
`patients without chronic inflammatory arthritis were studied as
`controls: 3 with osteoarthritis, 1 with gout, and 1 with dialysis
`arthropathy.
`The patients with chronic inflammatory arthritis had a mean age
`of 49.2 yr (range 18-84), 12 were female, 20 were male, and they had
`had arthritis for an average of 9.7 yr (range 4 mo-29 yr). Most of
`these patients were treated with non-steroidal anti-inflammatory
`drugs and disease modifying drugs (eg, gold, D-penicillamine, or
`antimalarials). 2 patients were treated with low doses of prednisone
`and 1 with azathioprine. The control patients had a mean age of 62.8
`yr (range 53-68). 2 were male, 3 female, and they had had joint
`disease for an average of 2.7 yr (range 7 mo-6 yr). These patients
`were only treated with non-steroidal anti-inflammatory drugs.
`The acetone-precipitate (AP) of M tuberculosis was prepared as
`previously described,' as was recombinant BCG 65 kD protein.'
`PPD was purchased from Statens Laboratory, Copenhagen. The
`proliferation assay was performed in Greiner flat bottom 96-well
`microtitre plates. Cells were grown in triplicate at a concentration of
`10' cells per well in culture medium in the presence or absence of 20
`µg/ml AP, 1 1.1g/m1 65 kD protein, and 1:100 dilution of PPD.
`(Culture medium consisted of Iscove's Modified Dulbecco's
`Medium supplemented with 58 µg/ml glutamine, 100 pg/m1
`penicillin, 100 µg/ml streptomycin, and 10% type AB human
`serum.) Cells were cultured for 6 d, the last 16 h in the presence of
`tritiated thymidine. The cells were then harvested and the
`incorporation of thymidine into DNA was measured in a liquid
`scintillation counter. The lymphocyte response was expressed as a
`stimulation index (SI): the ratio of mean counts per minute (cpm) in
`the presence of antigen divided by the mean cpm without antigen.
`
`Results
`The accompanying figure (a) shows the stimulation
`indices (SI) of the proliferative responses to the 65 kD
`protein of mononuclear cells from synovial fluid (SF-MNC)
`and peripheral blood (PB-MNC) from 31 patients tested.
`For most patients, reactivity to the 65 kD protein was higher
`in synovial fluid than in peripheral blood. The mean
`AP-specific and PPD-specific proliferative responses were
`also increased in synovial fluid (data not shown).
`The table shows the relation between responsiveness .of
`synovial mononuclear cells to the 65 kD protein (arbitrarily
`defined as SI 3), and to AP (A), and PPD (B) in 28
`patients. A significant relation between a proliferative
`response to the 65 kD protein and AP (A) was found,
`
`Ex. 1047 - Page 4
`
`

`

`THE LANCET, AUGUST 27, 1988
`
`SI
`
`SI
`
`18
`
`16
`
`14
`
`12 •
`
`10 •
`
`8 •
`
`6 -
`
`4 -
`
`2 -
`
`SF-MNC
`
`PB-MNC
`
`>3 years
`-5. 3 years (cid:9)
`chronic arthritis
`
`Proliferative responses to the 65 kD protein.
`a) Expressed as stimulation indices (SI) of synovial fluid (SF) and
`peripheral blood (PB) mononuclear cells (MNC) from patients with chronic
`arthritis. One patient was tested twice; the response of another patient is not
`included because no PB-MNC were available.
`b) SI of SF-MNC from joints inflamed for 0-3 yr, or > 3 yr. Responses of
`4 patients are included in fig b but not in the table because of insufficient
`synovial lymphocytes to test PPD reactivity. 2 patients were tested twice; both
`responses are included in the figure.
`
`whereas the proliferative response to 65 kD and PPD was
`not related (B). A correlation was found between AP and
`PPD proliferative responses (data not shown; p = 0.01). 8 of
`11 responders to the 65 kD protein also gave a positive
`response to AP. Responsiveness of synovial T cells to the 65
`kD protein was found in all types of chronic arthritis
`patients: in 6 of 22 RA patients, and 7 of 10 patients with
`other forms of chronic inflammatory arthritis. Anti-
`mycobacterial T cell responsiveness did not appear to be
`associated with HLA-DR4: 7 out of 15 DR4-positive
`patients were 65 kD protein responders and the same
`number were reactive to AP, and similar proportions were
`observed in DR4-negative individuals.
`We looked for a correlation between T cell reactivity
`against the mycobacterial antigens and various clinical data
`(eg, presence or absence of rheumatoid factors, age, time
`since onset of arthritis, and time since onset of inflammation
`
`RELATION BETWEEN RESPONSIVENESS OF SYNOVIAL
`MONONUCLEAR CELLS TO 65 kD PROTEIN AND AP (A) AND PPD (B)
`RESPECTIVELY
`
`(A) (cid:9)
`
`65 kD
`
`8 (cid:9)
`3 (cid:9)
`
`5
`12
`
`p =0.03
`
`PPD
`
`(B)
`
`65 kD
`
`5 (cid:9)
`6 (cid:9)
`
`5
`12
`
`p=0.32
`
`479
`
`in the joint aspirated: these periods could be the same, but in
`some cases the onset of inflammation in the joint involved
`was years after the time the diagnosis was made). The only
`significant relation found was between responsiveness of
`synovial mononuclear cells to the 65 kD antigen and a recent
`( (cid:9) 3 yr) onset of disease activity in that joint (fig b). The
`mean 65 kD-specific response and the percentage of 65 kD
`responders (SI 3) were higher in the aspirates from joints
`with the more recent onset of arthritis (p <0.05). In one
`patient, who was tested at 2 and 4 yr after onset of
`inflammation of his knee joint, the reactivity to the 65 kD
`antigen that was observed at the first investigation had
`disappeared at the second. In 3 patients with osteoarthritis, 1
`with gout, and 1 with dialysis arthropathy, no
`responsiveness to 65 kD protein was detected, even though 3
`of these patients had suffered from joint disease for less than
`3 yr.
`Most of the AP responders who did not respond to the 65
`kD protein had had inflammation in the joint for more than
`3 yr. The relation between AP and 65 kD protein responses
`for individual patients is therefore only apparent for patients
`with a more recent onset of inflammation (p AP-65
`kD 0.01), and not in patients with longer lasting
`inflammation (p AP-65 kD = 0•40). Response to AP was
`associated with a more recent onset of joint inflammation,
`but this association was less striking than the one found for
`65 kD. No relation was detected between duration of
`inflammation and PPD reactivity.
`
`Discussion
`Earlier studies suggested that T cell reactivity against 65
`kD protein might be involved in the pathogenesis of RA,
`and we therefore tested the 65 kD-protein-reactivity of
`synovial fluid and peripheral blood mononuclear cells from
`patients with RA and other forms of chronic arthritis. We
`also tested reactivity to AP and to PPD to compare our
`results with those of Holoshitz et al.5 Reactivity to the 65 kD
`protein, when present, was most often higher in synovial
`fluid than in peripheral blood, but responsiveness to the 65
`kD protein did not seem to be specific for RA patients and
`was found among all types of chronic inflammatory arthritis.
`However, this reactivity was seen almost exclusively in those
`patients with no more than 3 yr of inflammation of the joint
`from which the synovial fluid was aspirated, however long a
`period had elapsed since chronic arthritis was diagnosed
`because of involvement of other joints (in some cases, over
`10 years). No reactivity against the 65 kD protein, AP, or
`PPD was found in the synovial fluid or peripheral blood of
`control patients. Synovial T cell reactivity against the 65 kD
`protein, mainly in an early stage of joint inflammation,
`suggests a causal role for the 65 10 protein in T
`cell-mediated chronic arthritis.
`Chronic inflammatory diseases, characterised by
`infiltrations of mononuclear cells, may be caused by T
`lymphocytes that cross-react with an exogenous and an
`endogenous antigen. T cells that start the inflammatory
`process will be present at the site of inflammation at an early
`stage. Our results suggest that a proliferative response of
`synovial T cells to the 65 kD protein may be a trigger of
`chronic arthritis. T cell reactivity to the 65 kD protein, and
`to two other mycobacterial antigen preparations (AP and
`PPD), was higher in synovial fluid than it was in peripheral
`blood in almost all patients. We found a significant
`association between responsiveness of synovial lymphocytes
`to the 65 10 protein and to AP, but not to PPD, despite a
`
`Ex. 1047 - Page 5
`
`

`

`480
`
`THE LANCET, AUGUST 27, 1988
`
`correlation between AP and PPD reactivity. A similar
`antigen specificity was observed for the relation between
`synovial T cell reactivity and a relatively recent ( 3 years)
`onset of inflammation in the joint tested, which was
`significant for the 65 kD protein, failed to reach significance
`for AP, and was not present for PPD. Only a minority of the
`patients who had had inflammation of the joint concerned
`for longer than 3 yr show 65 kD antigen responsiveness of
`synovial T cells: perhaps the response to the 65 kD protein
`has been suppressed. AP probably contains the 65 kD
`protein (the rat T cell clone Alb recognised AP as well as the
`65 kD protein) and this would explain our finding that
`almost all the 65 kD protein-responders were AP-
`responders. A number of AP-responders do not show
`responsiveness to the 65 10 protein, and so reactivity to
`other components of AP may occur. Some DR4-positive
`patients were non-responders to 65 kD protein and AP.
`This was surprising, because Ottenhoff and co-workers'
`showed that DR4-positive Spanish leprosy patients had a
`stronger DTH reaction to M tuberculosis than DR4-negative
`leprosy patients. However, Palacios-Boix et al' observed a
`high DR4-associated responsiveness to a 4710 protein, but
`not to the 65 kD protein of M tuberculosis, and so DR4 could
`be associated with reactivity to the 47 kD protein.
`Some caution seems warranted in assigning the antigen(s)
`recognised by synovial-fluid-derived T cells to the
`mycobacterial 65 kD protein and not to E coli contaminants
`also present in the recombinant 65 kD preparation used by
`us.6 However, this assignment is not only the most logical
`but also a particularly interesting one, because the 65 kD
`protein appears to be one of the "heat-shock" or "stress"
`proteins, produced by bacteria during growth at high
`temperature. The stress proteins have been highly
`conserved during the course of evolution and homology has
`been detected between prokaryotic and eukaryotic stress
`proteins.8 Bacteria inside phagocytic cells are "under stress"
`and may well produce increased levels of stress proteins,
`such as the 65 kD protein, which might be presented to local
`T cells.' A T cell epitope on the 65 kD protein of
`mycobacteria might be a common epitope present on stress
`proteins in many different bacterial strains. If so, this could
`explain why adjuvant arthritis can be induced by many
`bacterial species in addition to mycobacteria. Synovial fluid
`T cell-reactivity against the 6510 protein could be triggered
`by other bacterial or even non-bacterial agents through
`cross-reactivity between the 65 kD stress protein and joint
`components.
`We thank Jelle Thole for the 65 kD recombinant BCG protein; John
`Haanen and Marleen Harteveld for performing part of the experiments;
`Dienne Elferink and Anneke Jansson for help with the isolation of cells; and
`Ieke Schreuder and colleagues for HLA typing. We are grateful to Prof A.
`Cats, Harry Marcusse, and several colleagues from the Rheumatology
`Department for providing clinical material and data and helpful comments.
`This work was supported by the Dutch League against Rheumatism.
`Correspondence should be addressed to R. R. P. de V., Department of
`Immunohaematology and Blood Bank, Building 1, E3-Q, Academisch
`Ziekenhuis, Postbus 9600, 2300 RC Leiden, The Netherlands.
`
`REFERENCES
`
`I. Holoshitz J, Naparstek Y, Ben-Nun A, Cohen IR. Lines of T lymphocytes induce or
`vaccinate against autoirnmune arthritis. Science 1983; 219: 56-58.
`2. Van Eden