OSTEOPENIA IN VERY LOW BIRTHWEIGHT INFANTS

1399 SEROLOGICAL EVIDENCE AGAINST ROLE FOR CANINE DISTEMPER VIRUS IN PATHOGENESIS OF PAGET’S DISEASE OF BONE

SIR,-Ultrastructural and immunohistochemical evidence supports a role for a slow viral infection in the pathogenesis of Paget’s disease of bone. The viruses implicated have been in the

paramyxovirus family-namely, measles virus and respiratory syncytial virus (RSV).1,2 Another paramyxovirus, and one with significant cross-reactivity with measles virus, is canine distemper (CDV) .3,1 O’Driscoll and Andersons have reported that past dog ownership was significantly more frequent in patients with Paget’s disease than in controls, although others have disputed the validity of that fmding.6.7 We have looked for IgM and IgG antibodies to CDV in the serum of 18 adult patients with Paget’s disease of bone diagnosed on radiographic appearances with an otherwise unexplained increase in serum alkaline phosphatase and in 18 controls with no evidence of Paget’s disease, nQ bone symptoms, and normal serum alkaline phosphatase activity. A solid phase immunoradiometric assay was used. (The general method is described elsewhere8 and further details may be had from R. J. H.) RESULTS (IN COUNTS/MIN) OF IMMUNORADIOMETRIC ASSAYS FOR virus

ANTIBODIES REACTIVE WITH CDV IN PATIENTS WITH PAGET’S DISEASE OF BONE

The amount of IgG or IgM antibody reactive with CDV in normal human sera was similar to that in patients with Paget’s disease (table). The patient with the highest level of IgG antibody (15 900 cpm) was the oldest patient in the series and has had severe polyostotic Paget’s disease for many years. Other patients with severe, longstanding disease, however, had very low values, and there was nearly complete overlap between the patient and control groups. The lack of a significant difference in IgG or IgM CDV antibodies between controls and patients with Paget’s disease can be explained in several ways: CDV has no role in Paget’s disease.-The only evidence for such a role is a study demonstrating increased frequency of prior dog ownership in Paget’s patients and the close antigenic relation between CDV and the other paramyxoviruses. The location of the virus within osteoclasts prevents the virus from being antigenic.-However, the accelerated remodelling of pagetoid bone should lead to continuous release of antigen from its intracellular location as osteoclasts die. Viraemia may be of a sufficient level to complex all available antibodies. A pre-existing inability to raise a sufficient antibody titre might predispose to chronic viral infection. Low-level CDV antibodies in serum of Paget’s patients may be masked by cross-reactivity with measles virus atitibodv.--CDV antibodies react with most of the polypeptides of measles virus.3 However, we did not attempt to adsorb measles antibody because it has proved difficult to determine which antibodies have been adsorbed.9,1O Paget’s disease may be a slow virus infection caused by a mutant, defective, or recombinant form of virus.-Such a virus may not express the same antigenic determinants as the conventional virus. We thank Inez

Campbell for secretarial

assistance.

Supported by

the

Veterans Administration. VA Medical Center, Houston, Texas 77211, USA, and Baylor College of Medicine, Houston

RICHARD J. HAMILL ROBERT E. BAUGHN LAWRENCE E. MALLETTE DANIEL M. MUSHER

Microbiological Associates, Inc, Bethesda, Maryland

DAVID B. WILSON

BG, Singer FR Nuclear inclusions in Paget’s disease of the bone. Science 1976; 194: 201-02. 2 Mills BG, Singer FR, Weiner LP, Suffin SC, Stabile E, Hoist P. Evidence for both respiratory syncytial virus and measles virus antigens in the osteoclasts of patients with Paget’s disease of bone. Clin Orthop 1984; 183: 303 - 11 3. Stephensen JR, ter Meulen V. Antigenic relationship between measles and canine distemper viruses: comparison of immune response m animals and humans to individual polyspecific polypeptides. Proc Natl Acad Sci USA 1979; 76: 6601-05. 4. Hall WW, Lamb RA, Choppin PW. The polypeptides of canine distemper virus synthesis in infected cells and relatedness to the polypeptides of other morbilliviruses. Virology 1980; 100: 433-49 5 O’Driscoll JB, Anderson DC Past pets and Paget’s disease. Lancet 1985; ii 919-21. 6 Barker KJP, Detheridge FM. Dogs and Paget’s disease. Lancet 1985; ii: 1245. 7 Pippard ED, ed The aetiology of Paget’s disease of bone MRC scientific report no 5. Southampton Environmental Epidemiology Unit, Southampton General Hospital, 1984 11-14 8 Baughn RE, Musher DM. Radioimmoassays for the detection of antibodies to treponemal polypeptide antigens in serum. J Clin Microbiol 1985, 21: 922-29. 9. Appel MJ, Glickman LT, Raines CS, Tourtellottee WW. Canine viruses and multiple sclerosis. Neurology 1981, 31: 944-49. 10 Arnadottin T. Measles and canine distemper virus antibodies in patients with multiple sclerosis determined by radioimmunoassay. Acta Neurol Scand 1980, 62: 81-89. 1 Mills

OSTEOPENIA IN VERY LOW BIRTHWEIGHT INFANTS

SIR,—Dr McIntosh and colleagues comment (Oct 25, p 981) that factors other than deficient mineral intake may contribute to osteopenia in preterm, very low bithweight infants. They relate these predominantly to milk feeding. However, mineral balance requires regulated output as well as adequate input, and the ability of the renal tubule to regulate mineral output and the renal tubule’s sensitivity to drugs should be considered. Mineral depletion by urinary losses in the first two weeks of life as a result of renal tubule immaturity may not be reflected by osteopenia until some weeks later, when linear growth makes demands on mineral stores. We describe two babies in whom frusemide, a calciuric drug, was given in the first weeks of life to "cover" a top-up blood transfusion. This preceded a fall in serum calcium (Ca), a rise in urinary phosphate (P04) and calcium, and an increase in serum alkaline phosphatase (AP). The AP increase began at the time of the disturbance but took 2-3 weeks to reach a peak, reflecting progressive changes in bone turnover. This disturbance in mineral metabolism happened before any significant postnatal growth, suggesting an effect of frusemide on the tubule persisting after the diuretic effect had ceased. A 780 g baby, born at 27 weeks’ gestation, was ventilated from birth for respiratory distress syndrome. Fluid intake was glucoseelectrolyte solution only until day 5 when nasogastric feeding with expressed breast milk was introduced. Parenteral feeding was given from day 5 to day 14. The baby was extubated after 11 days and

regained birthweight at 21 days. A top-up blood transfusion was given on day 9, covered by 2 mg frusemide because of a heart murmur. The serum AP, which had been unchanged at 600-800 IU/1, then increased to 1350 IU/1 within 24 h and to 1650 IU/1 within 2 days, remaining at that level for 5 weeks despite calciferol and mineral supplements. There were

radiological changes. Urinary mineral excretion, on spot urine samples, was expressed as Ca or P04 ratios to urinary creatinine (Cr) (mmol Ca or P04/mmol creatinine.) The ratios changed from Ca/Cr of19 and a P04/Cr of 015 on day 7 to Ca/Cr of 2 54 and a P04/Cr of 8-28 on day 15; these ratios remained high for several weeks. Although no changes in bone density were detected on X ray the AP values are compatible with those we have found in clinically significant bone disease. A growth-retarded twin baby was delivered at 28 weeks’ gestation weighing 620 g. He was ventilated from birth for respiratory distress syndrome. Initial fluid intake was glucose-electrolyte solution. Expressed breast milk was introduced from day 3. Calciferol supplements were given at 400 IU/kg daily from day 7; these were increased to 1300 IU/kg at 5 weeks. Parenteral feeding was given from day 10 for 1 month. Birthweight was regained after 21 days. A top-up transfusion was given on day 5 and then again on day 9, this time with frusemide cover (03 mg). A significant ductus arteriosus developed on day 12 and two further doses of frusemide were given on days 12 (0 25 mg) and 13 (03 mg) before ligation of the duct. no

1400 AP was less than 600 IU/1 until day 15, after which it rose to 2500 1 U/I by 1 month of age. Urinary P04 excretion changed from undetectable at day 8 to P04iCr 10on day 15. Urinary calcium excretion increased from Ca/Cr 3-26 on day 8 to 8.31 on day 10 and 4-20 on day 15. The serum calcium fell from 2-23 to 1-86 mmol/1 over the same period. fhis hypocalcaemia was corrected between days 14 and 16. These mineral outputs persisted over the subsequent month with a Ca/Cr of 5 63 and a PO4/Cr of 7-15 at 5 weeks of age-this despite radiological evidence of progressive bone demineralisation from 4 weeks of age. At 6 weeks fractures of one humerus and four ribs occurred without major trauma. Preterm, very low birth weight infants require a large positive calcium and phosphate balance to maintain the bone growth that would normally have taken place in utero. Anything which disturbs this balance can seriously affect bone metabolism. These two cases illustrate disturbances of balance in the first 2 weeks of life which were followed by persistent loss of calcium and phosphate in the urine. In one case this led to radiologically detectable osteopenia and fractures. In both cases administration of frusemide preceded the: biochemically detected disturbances in mineral balance. In such infants where dietary mineral intake is low, early administration of frusemide, a known calciuric agent, may produce long-term effects on mineral loss and precipitate frank metabc’Uc bone disease. Birmingham Maternity Hospital, Queen Elizabeth Medical Centre, Birmingham B15 21 G

M. E. I.MORGAN S. E. EVANS

This study confirms the earlier findings’ that London patients with AS do not have antibodies against Y enterocolitica 0:3. It is probable that a variety of microorganisms are connected with development of AS, and that their epidemiology and ecology is

geographically different. These studies were supported by grants from the Sigrid Jusehus Foundation and US Public Health Service (no 5 R01 AM33311-02).

Departments of Medicine and Medical Microbiology, Turku University, Turku, Finland

AULI TOIVANEN TOM H. STAHLBERG KAISA GRANFORS

Department of Biochemistry, King’s College, London; and Bloomsbury Rheumatology Unit, University College and Middlesex Hospitals, London WIN 8AA

ALAN EBRINGER

1. Kinsella TD, Fritzler ML, McNeil

2.

DJ. Ankylosing spondylitis, a disease m search of microbes. J Rheumatol 1983; 10: 2. Geczy AF, Prendergast JK, Sullivan JS, Upfold LI, Edmonds JP, Bashir HV. Possible role of entenc organisms in the pathogenesis of the seronegative arthropathies. In: Ziff M, Cohen SB, eds. Advances m inflammation research. New

York: Raven Press, 1985: 129-37. Bames M, Ptaszynska T Spondyloarthritis, uveitis, HLA-B27 and Klebsiella. Immunol Rev 1985; 86: 101-16. 4. Agner E. Natural course of raised Yersinia enterocolitica antibody titer m an unselected adult population followed during one year. Dan Med Bull 1983; 30: 100. 5. Granfors K, Isomaki H, von Essen R, Maatela J, Kalliomaki JL, Toivanen A Yersinia antibodies m inflammatory joint diseases. Clin Exp Rheumatol 1983; 1: 215-18. 6. van Bohemen CG, Nabbe AJJM, Goei The HS, Dekker-Sayes AJ, Zanen HC. Antibodies to Enterobacteriaceae in ankylosing spondylitis. Scand J Rheumatol 3.

Ebringer A,

1986; 15: 143-47. Ebringer A, Panayi G, Ebringer R, James DCO. HLA-B27 and the immune response to enterobacterial antigens in ankylosing spondylitis. Clin Exp Immunol 1984; 55: 74-80. 8. Towbin H, Staehelin T, Gordon J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-54. 9. Ståhlberg TH, Granfors K, Toivanen A. Immunoblot analysis of human IgM, IgG and IgA responses to plasmid-encoded antigens of Yersinia enterocolitica serovar 03. J Med Microbiol (in press) 10. Granfors K Measurement of immunoglobulin M (IgM), IgG, and IgA antibodies against Yersinia enter ocolitica by enzyme-linked immunosorbent assay: Persistence of serum antibodies during disease. J Clin Med Microbiol 1979, 9: 336—41 7. Trull A,

ABSENCE OF ANTIBODIES TO YERSINIA ENTEROCOLITICA IN PATIENTS WITH ANKYLOSING SPONDYLITIS IN LONDON

SiR,—Bacteria of the family Enterobacteriaceae are thought to be involved in the pathogenesis of ankylosing spondylitis (AS).’ Patients with AS have an increased frequency of Klebsiella in faecal cultures and an increased level of serum antibodies to klebsiella.’ Also increased antibody levels against Yersirria enterocolitica 0:3 havebeen observed in AS in Scandinaviass and the Netherlands,6 but not in London.’ To exclude the possibility that the negative findings in the London patients are not due to technical errors we have done a double-blind study on sera from 12 patients with inactive AS (London), 12 with active AS (London), and 10 with rheumatoid arthritis (London), from 10 blood donors (London), and from 4 patients with yersinia-triggered reactive arthritis (Turku). The sera were analysed using sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) followed by immunoblotting.8 For this, growth of Yersinia enterocolitica 0:3 (strain 4147) was pelleted, treated with SDS and 2mercaptoethanol, and separated by electrophoresis on a vertical SDS-polyacrylamide gradient gel (10-125%). The separated components in the gel were electrotransferred immediately to nitrocellulose sheets, and non-specific binding sites were blocked by incubation in phosphate-buffered saline containing 10% horse serum (HS-PBS) at 4°C overnight. After washing, the sheets were cut into vertical strips and reacted with a 1:100 dilution of sera from the patients in HS-PBS at 4°C overnight. The strips were washed and incubated for 3 h at room temperature with peroxidase-labelled rabbit antihuman IgG diluted 1:1000 in HS-PBS. After washing, the strips were developed with 4-chloro- I -naphthol.’ Only the 4 Finnish sera contained antibodies against about twenty different bacterial components, with molecular weights 15-89 kD. A few of the other sera gave very weak reactions with some components, the immunoblotting pattern being completely different from that obtained with the Finnish sera. This faint reactivity must be due to cross-reactions between Y enterocolitica 0:3 and other microorganisms. The results were confirmed by enzyme immunoassay’° for IgM, IgG, and IgA class antibodies against Y enterocolitica 0:3, with the same resulis. o- iiv the 41;-_:>Î1 s

oeing pù:O;.>-LlB

e.

GERMS AND JOINTS SiR,—There is widespread interest in gut involvement in seronegative arthropathies.1 There is an association between sterile seronegative spondyloarthropathies (eg, ankylosing spondylitis, Reiter’s syndrome, and reactive arthritis), the HLA-B27 antigen, and enterobacteria (eg, Klebsiella, Shigella, and Yersinia). Two theories have been proposed to explain this association: molecular mimicry2 and cell surface receptors.3 Molecular mimicry invokes similarity between B27-like antigenic epitopes on enterobacteria and B27 self antigens while, in the second theory, expression of cell surface receptors for enterobacterial antigens by B27-positive host cells follows transfection with enterobacterial plasmid DNA. Both theories suggest that the immune response to enterobacteria also affects host tissues. However, the two theories appear incompatible and neither explains why the joint is the primary site of disease. We propose a combined theory which could be tested in B27-associated reactive arthritis (ReA), an acute disease. In ReA, the chronically high immunoglobulin concentrations in serum’and synovial fluids may reflect interference in communication between joint-related lymphocyte subpopulations. 1,6 Associations of enterobacterial antigens (perhaps B27-like) with HLA class-1 receptors (eg, B27)’ may disrupt T-suppressor cell and B-cell interaction without inducing intrinsic receptor activity in these cells. It is known that the immune response to streptococcal antigen is controlled by suppressor T-cells8 although other factors-eg, disease resistance or susceptibility genes9-may be involved. Our combined theory could be tested by investigating the interaction of partially purified enterobacterial antigens, including B27-like antigens, with isolated lymphocyte subpopulations obtained from joints of ReA patients. Ankylosing spondylitis (AS), a chronic disease, may not directly involve enterobacterial antigens. Enterobacteria with freely accessible B27-like antigenic epitopes on their cell surface have been