175

remaining after most of the bacteria have been cleared. Either way it doubtful that antibiotics would be of benefit in most cases of Crohn’s disease. seems

We thank J. B. You for preparation of primers. Funded by grant from the World Health Organisation to T. S. B. Y.

Department of Biochemistry, Chang Gung Medical College, Taipei, Republic of China

SARAH W. P. WU CHIA C. PAO

Department of Pathology, UCSM, San Francisco

JULIA CHAN*

Department of Pathology, School of Medicine, University of California, San Francisco, California 94143, USA

T. S. BENEDICT YEN

*Present address.

Pathology Associates, Los Gatos, California.

SJ, McFadden JJ, Hermon-Taylor J. Mycobacteria and Crohn’s disease. Gut 1988; 29: 1017-19. 2. Chiodoni RJ, van Kruiningen HJ, Thayer WR, Coutu JA. Spheroplastic phase of mycobacteria isolated from patients with Crohn’s disease. J Clin Microbiol 1986; 24: 357-63. 3 Collins J, Beaman B, Arthur M, Gitnick G. Isolation of mycobacteria from intestinal tissues. Gastroenterology 1986; 90: 1377. 4. Tytgat GNJ, Mulder CJJ. The etiology of Crohn’s disease. Int J Colorect Dis 1986; 1: 188-92 5. McFadden JJ, Butcher PD, Chiodini R, Hermon-Taylor J. Crohn’s disease-isolated mycobacteria are identical Mycobacterium paratuberculosis as determined by DNA probes that distinguish between mycobacterial species. Clin Microbiol 1987; 25: 796-801. 6. Butcher PD, McFadden JJ, Hermon-Taylor J. Investigation of mycobacteria in Crohn’s disease tissue by Southern blotting and DNA hybridization with cloned mycobacterial genomic DNA probes from a Crohn’s disease isolated mycobacteria. Gut 1988; 29: 1222-28. 7. Pao CC, Yen TSB, You JB, et al. Detection and identification of mycobacterium tuberculosis by DNA amplification method. J Clin Microbiol 1990; 28: 1877-80. 8. Seto E, Yen TSB. Detection of cytomegalovirus infection using DNA isolated from paraffin-embedded tissues and dot hybridization. Am J Pathol 1987; 127: 409-13. 9 Chehab FF, Xiao X, Kan YW, Yen TSB. Detection of cytomegalovirus in paraffin-embedded tissue specimens with the polymerase chain reaction. Mod Pathol 1989; 2: 75-78. 1. Hampson

bcl-2 expression in

lymphomas

SIR,-Dr Pezzella and colleagues (Dec 15, p 1510) report heterogeneous expression of bcl-2 protein in follicular lymphomas which is not dependent on the presence of a t(14;18) translocation. We have used the same monoclonal antibody to bcl-2 protein to examine six cases of low-grade B-cell lymphoma of mucosaassociated lymphoid tissue (MALT) in which colonisation of reactive follicles was prominent. This occurrence results in neoplastic (ie, Ig light chain restricted) follicles with the phenotype of MALT lymphoma (CD 10-, KB61 +) rather than follicular lymphoma (CD 10 +, KB61-) (figure). Monoclonality and absence of the t(14,18) translocation has been shown to be characteristic of MALT lymphomaand was confirmed in these cases by Southern blotting with DNA probes specific for the immunoglobulin heavy chain joining region and for the bcl-2 major and minor breakpoints. In all six

cases tumour

cells outside the colonised follicles

expressed bcl-2 protein while those within the germinal centres were negative (figure). Staining with Ki-67 showed a very high rate of proliferation of the tumour cells within the colonised follicles, similar to that seen in non-neoplastic follicle centres and in striking contrast to the low proliferation rate in the diffusely infiltrating bcl-2 protein positive tumour cells. This finding suggests a close association between the rate of cell proliferation and failure to express bcl-2 protein, and would explain why all small (resting) lymphocytes express the protein whereas follicle centres, which normally have a very high proliferation rate, do not. Unlike normal reactive follicles and colonised follicles in MALT lymphoma the follicles in follicular lymphoma have a low proliferation rate and, therefore, might be expected to express bcl-2 protein. Although bcl-2 protein is consistently absent in normal proliferating follicle centre cells,s,6 at the transcriptional level increased bcl-2 mRNA has been found in mitogen-stimulated B and T lymphocytesExpression of the bcl-2 gene may be regulated at the transcriptional level by both stimulatory and inhibitory factors and at the post-transcriptional level by a labile repressor which

low-grade B-cell gastric lymphoma of MALT. Stained (A) for kappa light chain, (B) for lambda light chain, (C) with CD10, (D) with KB61, (E) with anti-bc/-2 protein, and (F) with Ki67. 0 Follicle shows lambda light cham restriction and is negative with CD10 and positive with KB61 bcl-2 protein is expressed by MALT lymphoma cells diffusely infiltrating the mucosa but the follicle is largely negative. Ki67 staining shows numerous proliferating cells m follicle. (Immunoalkaline phosphatase ) Follicle from

could be inefficient

mitogen induced.’ It has also been suggested that synthesis of bcl-2 protein may result from a number of

proposed translation "false start" sites at the 5’ end of the mRNA.5,8 Finally, diminished bcl-2 protein expression in rapidly proliferating lymphocytes may simply reflect failure to store the protein. The expression of bcl-2 protein by MALT lymphomas supplements Pezzella and colleagues’ finding that bcl-2 protein is not specific for the i(14d8) translocauon. Aberrant loss of

176

in the tumour cells that colonise follicles is a further feature that distinguishes MALT lymphomas from other low-

expression

grade B-cell lymphomas. P. G. ISAACSON A. C. WOTHERSPOON T. C. DISS L. X. PAN

Department of Histopathology, UCMSM,

University College London, London WC1E 6JJ, UK 1 Isaacson

PG, Wotherspoon AC, Diss TC, Pan LX. Follicular colonization in B cell lymphoma of mucosa associated lymphoid tissue. J Pathol 1990, 161: 340a (abstr) 2. Isaacson PG, Dogan A, Price SK, Spencer J Immunoproliferative small intestinal disease: an immunohistochemical study. Am J Surg Pathol 1989; 13: 1023-33. 3. Pan L, Diss TC. Cunningham D, Isaacson PG. The bcl-2 gene in primary B cell lymphomas of mucosa associated lymphoid tissue (MALT). Am J Pathol 1989;

diseases if one component of microbiological evidence is not available. The publication bias that would result from this policy would be a matter of concern. The answer to the question about "exposure to various factors" is given in the last paragraph of the results section. Any variation in measurement technique, which includes having more than one interviewer, can produce bias. We decided at the outset of this study that any bias from these sources would have little effect. None of the 95% confidence intervals (CI) for the odds ratio for the dose-response relation in table n enclose unity. The 95% CI (Fisher’s exact test) are as follows: Odds ratio (95% Cl) Days per week

135: 7-11. 4. Hall PA, Richards MA, Gregory WM, D’Ardenne AJ, Lister TA, Stansfeld AG. The prognostic value of Ki67 immunostaining in non-Hodgkin’s lymphoma J Pathol

1988, 154: 223-35 Ngan BY, Chen-Levy Z, Weiss LM, Warnke RA, Cleary ML Expression in non-Hodgkin’s lymphoma of the bcl-2 protein associated with the t(14;18) chromosomal translocation. N Engl J Med 1988; 318: 1638-44. 6. Pezzella F, Tse AGD, Cordell JL, Pulford KAF, Mason DY. Expression of the bcl-2 oncogene protein is not specific for the 14;18 chromosomal translocation. Am J Pathol 1990; 137: 225-32. 7. Reed JC, Tsujimoto Y, Alpers JD, Croce CM, Nowell PC. Regulation of bcl-2 proto-oncogene expression during normal human lymphocytic proliferation

5

8.

Science 1987, 236: 1295-99. Tsujimoto Y, Croce CM. Analysis of the structure, transcripts, and protein products of the bcl-2, the gene involved in human follicular lymphoma. Proc Natl Acad Sci USA 1986; 83: 5214-18

Birds, milk, and campylobacter SIR,-Dr Southern and colleagues (Dec 8, p 1425) report no microbiological evidence of milk contamination. Moreover, we are not given details of the exposure to "various factors" asked about at interview. Cases and controls were interviewed either at home or by How many in each group were interviewed in the two and could a bias have been introduced by this method? The ways, dose-response subgroups are small. What are the confidence intervals for the odds ratios in table n? Southern et al admit that "the weak protective association with handling of other dogs may be a chance finding", and this may well apply to the milk-bottle attacks also. Bradford Hill’ has pointed out eight attributes that increase the likelihood of a causal relation, of which temporality is the only indispensable condition. Campylobacter infections are rising at a time when more milk is being delivered in cartons. Southern and colleagues also cite strength of association and dose-response effect, yet only 3 cases witnessed birds attacking milk bottles. A weak association between bird attack and campylobacter infection may have been shown, but these data provide insufficient evidence to claim that method of milk deliverv will eliminate the risk.

telephone.

ANN BISSET LESLEY MACDONALD JON P. CRESSWELL SUSAN MACPHEE K. M. VENKAT NARAYAN

Department of Public Health Medicine, Grampian Health Board, Aberdeen AB9 8QP, UK

1. Bradford Hill A. The environment and disease: association

Med

or causation.

The X2 test for trend is probably more appropriate in this circumstance. Chance can explain any epidemiological finding, but our results are consistent with our hypothesis and chance is unlikely to be the explanation for all of them. Laboratory isolates of campylobacter infections are increasmg and several explanations for this change exist. I do not see how an increase in delivery of cartoned milk relates to the hypothesis under study. We have investigated a new mode of transmission that we believe accounts for a large part of the spring rise in C jejuni infection. All current evidence points to this mode being a spring effect only, which does not take place at other times of the year. Our data strongly support this hypothesis; were such a study conducted outside the spring peak then the associations would probably not even exist. Huntingdon Health Authority, Huntingdon PE18 6SE, UK

J. P. SOUTHERN

SIR,-Dr Southern and colleagues attribute the transmission of Campylobacter infections to milk-bottle attacks by birds, though they admit that few people witness the attacks. Obstetricians are more likely to be up and about at the time of milk deliveries and I have seen cats, my own included, remove milk-bottle tops with their teeth and drink the first 2 cm or so from the bottle. The authors think that cats and dogs in the household may help scare birds away. May I suggest that the converse is true in the Bridgend area and that cats are a more likely source of Campylobacter than birds. Princess of Wales

Hospital, Bridgend CF31 1RO, UK

R. P. BALFOUR

SIR,-Dr Southern and colleagues suggest that a change to delivery of milk in cartons may reduce doorstep attacks on milk by birds. I have learned, at the cost of several litres of milk, that cartons afford no protection against magpies. It is the milk they are after, not the bottle caps. "Bag-end", Tonroe, Oranmore,

Co Galway, Ireland

MARTIN G. CORMICAN

Proc R Soc

1965, 58: 295-300

Tuberculous pericarditis confirmed by DNA

amplification *** This letter has been shown follows.-ED. L.

to

Dr

Southern, whose reply

SiR,—The lack of microbiological data neither weakens nor strengthens our epidemiological evidence. We were late in conducting this experiment and so only had three attacked bottles for examination. However, C jejuni has been isolated elsewhere from attacked milk bottles and a reference to that point was cited in our paper. It would be difficult to detect C jejuni in milk that had caused human illness since a long time elapses between infection and laboratory isolation from a faecal specimen. Direct evidence could be secured through challenge tests, but this method is probably unethical. Dr Bisset and colleagues imply that it is incorrect to publish results of epidemiological studies into infectious

StR,—Tuberculous pericarditis is usually diagnosed clinically and may subsequently be confirmed by culture of the pericardial fluid in 25-59% of cases.’ We have used a nested polymerase chain reaction (PCR)Z with two pairs of primers within the IS6110 sequenceto amplify DNA from Mycobacterium tuberculosis from the pericardial fluid of a patient with a presumed tuberculous effusion. The procedure took less than two days. IS6110 is present in multiple copies in the M tuberculosis complex but not in related bacteria. A 52-year-old woman presented after 4 weeks of right-sided hypochondrial pain, 2 weeks of fever and night sweats, and 1 week of chest pain and a dry cough. Her temperature was 37 5° C and her pulse was regular at 90 per min. A chest radiograph revealed a large