SAFER ACELLULAR AND WHOLE-CELL PERTUSSIS VACCINES

1427 n Dr CROSS OFFICE 2 ELEMENTARY SCHOOL 3 JUNIOR COLLEGE OF THE KEYS

MANAGEMENT OF NON-CONVULSIVE STATUS EPILEPTICUS

4 NURSLING HOME

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5 HOSPITAL 6 ANIMAL SHELTER 7 COUNTY OFFICES 8Dr DEANS OFFICE 9 WASTE PILE

SiR,—Your April 25 editorial on non-convulsive status epilepticus states that treatment of absence status "is by intravenous diazepam, clonazepam, or acetazolamide". Whilst endorsing the choice of the former two drugs, but perhaps in reverse order of preference, we were surprised that you recommend acetazolamide. Although this drug has been reported to be sometimes effective against absence seizures, 13 its usefulness is limited by the rapid development of tolerancewhich is most likely to be due to an adaptive increase in carbonic anhydrase production.s With high doses or prolonged medication, drowsiness and paraesthesias may occur, and since teratogenic effects have been demonstrated in animals acetazolamide should not be administered during pregnancy.’ In our experience absence status responds rapidly to intravenous clonazepam or diazepam, oral sodium valproate or clonazepam being the long-term prophylactic drugs of choice.6

Key West and Stock Island, Florida. Both veterinarians accept that the other’s observations about CDV are valid, and they attribute the higher rate of distemper on Stock Island to the following: Stock Island is poorer than Key West, with many trailer homes; stray and wild dogs are more common (26 wild ones were trapped in 1986); the vaccination rate was probably lower than Key West’s in the early 1970s; dogs on Stock Island may also have been more poorly nourished and have had a higher incidence of infections; before 1978 the many dogs destroyed, because of distemper, at the animal shelter on Stock Island were put on a waste

pile nearby (figure).

We could not reconcile these veterinary data with the information on the MS epidemic in the Key West area until the results of a case-controlled epidemiological study were reported in the Nov 10, 1985, issue of the Miami Herald. This study, done jointly with an epidemiologist at the University of South Florida, found that 15 of the 26 patients with MS lived or worked on Stock Island, and another 4 lived on nearby Key Haven. Places of employment included Florida Keys Hospital, Florida Keys Community College, a nursing home, county offices, and the elementary school, all on Stock Island (figure). Thus there is evidence, albeit anecdotal, for an extraordinarily high rate of CDV in dogs on Stock Island and parts of Key West from 1969 until the mid-1970s, and this CDV outbreak has been associated with an extraordinary clustering of MS, with onset since 1975, among people who lived or worked on Stock Island. We are not saying that CDV caused MS in the Key West/Stock Island area but merely point out that the apparent CDV and MS findings may be consistent with the CDV/MS hypothesis. However, if MS is transmitted from dog to man by CDV, we would expect the incidence of MS in the Key West area to fall over the next 10 years, provided that the incidence of CDV infection remains low. VA Medical Center, East Orange, and Department of Neurosciences, New Jersey Medical School, Newark, New Jersey 07103, USA

S. D. COOK B. BLUMBERG P. C. DOWLING

Stock Island, Florida

W. DEANS

Key West, Florida

R. CROSS

DC, Withum DG, MacLeod CL, Whiteside ME. tropical island of multiple sclerosis. Lancet 1985; ii: 618. Cook SD, Dowling PC. Multiple sclerosis and viruses: an overview. Neurology 1980;

1. Sheremata

Unusual

2.

WA,

Poskanzer

occurrence on a

30: 80-91. 3. Cook SD, Dowling PC, Russell WC. Multiple sclerosis and canine distemper. Lancet 1978; i: 605-06. 4. Cook SD, Dowling PC, Norman J, Jablon S. Multiple sclerosis and canine distemper in Iceland. Lancet 1979; i: 380-81. 5. Cook SD, Gudmundsson G, Benedikz J, Dowling PC. Multiple sclerosis and distemper in Iceland: 1966-1978. Acta Neurol Scand 1980; 61: 244-51 6. Cook SD, Cromarty JI, Tapp W, et al. Declining incidence of multiple sclerosis in the Orkney Islands Neurology 1985; 34: 545-51 7. Pryse-Phillips WEM. The incidence and prevalence of multiple sclerosis in Newfoundland and Labrador, 1960-1984. Ann Neurol 1986; 20: 323-28. 8. Cook SD, Dowling PC. Distemper and multiple sclerosis in Sitka, Alaska. Ann Neurol 1982; 11: 192-94.

Department of Neurology, Royal Perth Hospital, Perth, Western Australia 6001

GRAEME J. HANKEY EDWARD G. STEWART-WYNNE

a carbonic anhydrase inhibitor, its use in epilepsy Neurology 1954, 4: 863-68 2. Lombroso CT, Davidson DT, Grossi-Bianchi ML. Further evaluation of acetazolamide in the treatment of epilepsy. JAMA 1956; 160: 268-72. 3. Chao DHC, Plumb RL. Diamox in epilepsy a critical review of 178 cases J Pediatr 1961; 58: 211-18 4. Weiner IM, Mudge GH. Diuretics and other agents employed in the mobilization of oedema fluid In Gilman AG, Goodman LS, Rall TW, Murad FM, eds. The pharmacological basis of therapeutics, 5th ed New York: Macmillan, 1985: 890 5. Koch A, Woodbury DM. Effects of carbonic anhydrase inhibition on brain excitability. J Pharmacol Exp Ther 1958; 122: 335-42. 6. Dunne JW, Summers QA, Stewart-Wynne EG Non-convulsive status epilepticus: a prospective study in an adult general hospital. Quart J Med 1987; 236: 117-26.

1 Merlis S. Diamox:

SAFER ACELLULAR AND WHOLE-CELL PERTUSSIS VACCINES SIR,-ln many parts ot the world whole-cell pertussis vaccines will be the only vaccines available for several years yet. It is therefore disturbing that Ashworth et aP have identified active pertussis toxin (lymphocytosis/leucocytosis-promoting factor, LPF; histaminesensitising factor, HSF) in detoxified whole-cell vaccines. This should not, perhaps, cause surprise since the ability of whole-cell vaccines to induce lymphocytosis in children and to sensitise mice to histamine is well known. Nevertheless, those with long experience with whole-cell pertussis vaccines do not seem to have considered the possibility that these properties may be related to the rare central-nervous-system reactions and encephalopathies thought by some to be associated with whole-cell vaccines. Sato and colleagues2.3 were the first to identify LPF as a mouse-protective antigen and to recommend its use in an acellular vaccine. The favoured formulation contains both LPF and fimbrial haemagglutinin (an antigen thought to be involved in the attachment of Bordetella pertussis to the ciliated cells of the tracheal epithelium). When treated with formalin2 or glutaraldehyde/ lysine,’ LPF loses some or all of its LPF/HSF activity; activity returns when the formalin-treated, but not the glutaraldehydelysine-treated, preparation is dialysed.2,s These observations are analogous to the detoxification ("toxoiding") of purified diphtheria toxin. The acellular vaccines may still be histamine-sensitising6 and be associated with CNS reactions and encephalopathies.7 Since the LPF in these vaccines is detoxified with formalin3 this suggests the possibility of reversal. Toxoiding of LPF may not be straightforward. Following the recognition of reversal of purified diphtheria toxoid and its apparent precipitation by lack of aminoacids or other suitable nitrogenous compounds in the toxoiding menstruum, Linggood et al8 showed that aminoacids are not all equally effective in preventing reversal. Lysine was chosen for diphtheria toxin because it prevents reversal, gives a much more stable toxoid, and enhances its antigenicity; glycine, on the other hand, prevents reversal but reduces stability and antigenicity. The use of glutaraldehyde and lysine with LPF may fortuitously have been effective, but a more detailed study involving the additional variables mentioned seems to be warranted.

1428 Detoxification of diphtheria toxin is a two-stage process, the first stage being irreversible and completed within 3 days of exposure to formalin while the second stage takes several weeks and appears to be reversible. Shukuda9 has described a reduction in the LPF toxicity of an acellular pertussis vaccine from 240 to 0.16 units/ml after exposure to 0-4% formalin for 7 days. Since Ashworth et aP found that a single dose of whole-cell vaccine may contain up to 0-68 g LPF, of which as much as 0-28 g may be active or non-detoxified, there is the disturbing possibility that some LPF in acellular vaccines may in time revert to the active form since residual formalin, which might help to prevent reversal is required by regulation to be removed from the final vaccine

product. Our experiments (unpublished)

have shown that when wholecell vaccine is treated to induce reversal to toxin, no active LPF is detected in the Chinese hamster ovary cell test. Does this mean that the active LPF within the formalin-killed bacterial cells is undetectable even by a highly sensitive method--or is inactive LPF somehow activated in vivo? It seems clear that absence of reversal of LPF must be tested for in acellular vaccines. For whole-cell vaccines it is equally necessary to try to assess the importance of their active LPF component. Institut Armand-Frappier, University of Quebec, Laval des Rapides, Quebec, Canada H7N 4Z3

JACK CAMERON

1. Ashworth

LAE, Robinson A, Irons LI, Morgan CP, Isaacs D. Antigens m whooping cough vaccine and antibody levels induced by vaccination of children. Lancet 1983;

ii: 878-81. 2. Sato Y, Arai H, Suzuki K. Leukocytosis-promoting factor of Bordetella pertussis III: Its identity with protective antigen. Infect Immun 1974; 9: 801-10. 3. Sato Y, Kimura M, Fukumi H. Development of a pertussis component vaccine in Japan. Lancet 1984; i: 122-26. 4. Munoz JJ, Arai H, Cole RL Mouse-protecting and histamine-sensitizing activities of pertussigen and fimbnal haemagglutinin from Bordetella pertussis. Infect Immun

1981; 32: 243-50. 5. Munoz JJ, Arai H. Studies on crystalline pertussigen. In: Robbins JB, Hill JC, Sadoff JC, eds. Seminars in infectious disease. New York: Thieme-Stratton, 1982. 6. Iwasa S, Ishida S, Asakawa S, Akama K. A curious histamine-sensitizing activity shown by the newly-developed Japanese acellular pertussis vaccine. Develop Biol Standard 1985; 61: 453-60. 7. Kimura M, Hikino N. Results with a new DTP vaccine in Japan. Develop Biol

Standard 1985; 61: 545-61.

Linggood FV, Stevens MF, Fulthorpe AJ, Woiwod AJ, Pope CG. The toxoiding of purified diphtheria toxin. Br J Expt Pathol 1963; 44: 177-88. 9. Shukuda Y. In: Workshop on acellular pertussis vaccines. US Department of Health and Human Services, Washington, DC: US Public Health Service, 1986: 24. 8.

TRANSIENT IMPAIRMENT OF RENAL FUNCTION AFTER GENERALISED SEIZURES

SiR,—It is difficult to evaluate the reported change in renal patients after generalised seizures on the information presented by Dr Nielsen and colleagues (May 2, p 1043). The validity of creatinine clearance as an indicator of changing glomerular filtration rate (GFR) has been questioned. The analytical variation of creatinine is at least 10%1 and day-to-day intra-individual variation has been reported to be as high as 27 %.’2 Our table shows creatinine clearance measured on four highly motivated healthy volunteers over a 3 day period while on a constant, meat-free diet. If we accept that a significant alteration of GFR occurs only when creatinine clearance changes by more than 25%, only five of the patients studied by Nielsen et al show a true change in GFR with time. The change in median clearance of 30 ml/min (and in patient E of 56 ml/min) cannot easily be reconciled with the statement that plasma creatinine was normal in all patients throughout the study. To account for this variation in clearance, marked changes must function in

have occurred in plasma and/or 24 h urine creatinine excretion and these are not provided. Faulty urine collection is the greatest source of error in creatinine clearance measurement. A mild increase in plasma myoglobin was detected in only eight patients and increased creatine kinase (CK) in only five. Myoglobin is cleared rapidly from plasma.3 The levels quoted are unlikely to have caused renal damage without an associated factor being present.4 We have reviewed one hundred cases of rhabdomyolysis, including eleven patients with generalised seizures (mean CK 11030, range 1000-51500 U/1) and have found that renal impairment, as judged by serum creatinine level, occurs only when rhabdomyolysis accompanies an insult known to cause renal damage, such as hypoxia, hypotension, ischaemia, or dehydration. Since generalised seizures are common, it is important to establish the true prevalence of renal impairment in this condition.

Department of Biochemistry, Royal Infirmary,

Glasgow G4 0SF

W. D. FRASER M. D. GARDNER J. O’DONNELL F. J. DRYBURGH

Payne RB. Creatmine clearance: a redundant clinical investigation. Ann Clin Biochem 1986; 23: 243-50. 2. Brochner-Mortensen J, Rodbro P. Selection of routine method for determination of glomerular filtration rate in adult patients. Scand J Clin Lab Invest 1976; 36: 35-43. 3. Koskelo P, Kekki M, Wager O. Kinetic behaviour of 131I-labelled myoglobin in human beings. Clin Chim Acta 1967; 17: 339-47. 4. Stein JH, Lifschitz MD, Barnes LD. Current concepts on the pathophysiology of acute renal failure. Am J Physiol 1978; 234: 171-81. 1.

HIV ENVELOPE-CD4 INTERACTION NOT INHIBITED

BY SYNTHETIC OCTAPEPTIDES

SiR,—The exterior envelope glycoprotein (gpl20) of human

immunodeficiency virus (HIV) binds specifically to a receptor protein (CD4) present at high concentration on the surface of helper T lymphocytes. Both entry of the virus into the cell and cell killing are dependent upon gpl20-CD4 binding. Definition of the region of gp 120 that interacts with the CD4 molecule is critical to attempts to interfere with binding. Pert and colleagues’ have reported that several octapeptides derived from the gp120 of HIV competitively inhibit this binding and that even at concentrations of 1 nmol/1 the octapeptides inhibited HIV replication. The peptides used by Pert et al correspond in sequence to a highly variable region of gpl20. Even on optimal alignment not one of the eight aminoacids was conserved in all fifteen HIV strains sequenced, some of which contained only one or two out of the eight aminoacid residues in this region. Besides residue changes there were also gaps. This heterogeneity, in a region proposed’ to be critical for CD4 binding, is unexpected. We have tested directly the ability of octapeptides to inhibit gpl20-CD4 interaction. We used Jurkat-tatm and Raji-tatm cell lines;2 SupTl CD4positive lymphocytes (obtained from Dr James Hoxie); C8166 cells established by HTLV-1 transformation;3 and H9 cells infected with the HIV (HTL V -11m supplied by Dr Robert C. Gallo). Jurkat-a:,,, and Raji-tatm were transfected with 10 ug caesium chloride purified plasmid DNA. Syncytia in Jurkat-tatm cells were scored.4 Raji-tat"i cells 72 h post-transfection were centrifuged, washed twice with phosphate-buffered saline (PBS) and mixed with a three-fold excess of twice washed SupTl cells. Either a 1:400 dilution of OKT4 or OKT4A monoclonal antibodies or peptide T or dT suspended in PBS was added. The mixture was returned to a 37°C COz incubator for 6 h and syncytia were counted. Peptides T (Ala-Ser-Thr-ThrThr-Asn-Tyr-Thr) and dT (dAla form) more than 95 % pure were obtained from Peninsula Laboratories.

CREATININE STUDIES IN FOUR HEALTHY VOLUNTEERS OVER THREE DAYS