Ultrasonographic evaluation of parathyroid hyperplasia.

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Nephrology Dialysis Transplantation

Letters and replies Ultrasonographic evaluation of parathyroid hyperplasia We appreciate the supportive letter from Pavlovic et al. [1] for our paper [2 ] on the value of parathyroid sonography in secondary hyperparathyroidism. In response to the letter, we would like to offer our reply to the question they raised, ‘Is gland size sensitive enough to differentiate nodular and diffuse hyperplasia?’ This is an important question because even small nodules are composed of monoclonally proliferating cells [3 ] with a lower concentration of vitamin D receptor [4 ], thus resistant to medical therapy including calcitriol pulse therapy. Recent histological data in surgically removed parathyroid glands have clearly revealed that more than 90% of the glands heavier than 0.5 g were nodular hyperplasia [5]. This weight roughly corresponds to 0.5 cm3 in volume, or 1 cm in diameter. Thus we have sufficient rationale to set this volume as the critical size to predict parathyroid responsiveness to calcitriol therapy [6 ]. However, one problem which remains is that even smaller glands may be nodular hyperplasia. Actually, about 50% of glands between 0.25 and 0.5 g were nodular hyperplasia [5 ]. In such patients, positive blood supply detected by Doppler flow mapping suggests vigorous proliferation of parathyroid cells even in small glands. Power Doppler imaging, which displays the integrated power of Doppler signals, is very sensitive to detect even lower blood supply and thus is especially useful for this purpose. In percutaneous ethanol injection therapy (PEIT ) of para-

thyroid hyperplasia, detection of blood supply is also helpful to optimize the site of ethanol injection, to confirm the complete destruction of the tissues, and to detect the relapse of cell growth. By routinely using Doppler flow mapping, we have recently developed a new protocol of repeated intensive PEIT of parathyroid ( RIPP), in which all glands larger than 0.5 cm3 are destroyed within 1 week with minimum quantities of ethanol [7 ]. In addition, the latest device for high-resolution ultrasonography may be able to visualize suspected nodules in enlarged parathyroid glands (Figure 1). Although comparison studies between sonographic and histological findings need to be done, it may become possible to identify nodules in parathyroid glands by ultrasonography in near future. First Department of Internal Medicine University of Tokyo School of Medicine Tokyo Division of Endocrinology and Metabolism Showa General Hospital Kodaira and Tokai University School of Medicine Isehara Japan

M Fukagawa M Kitaoka K Kurokawa

1. Pavlovic D, Brzac HT, Cala S, Jankovic. The value of parathyroid sonography. Nephrol Dial Transplant 1997; 12: 367 2. Fukagawa M, Kitaoka M, Kurokawa K. Ultrasonographic intervention of parathyroid hyperplasia in chronic dialysis patients: a theoretical approach. Nephrol Dial Transplant 1996; 11 [Suppl 3]: 125–129 3. Tominaga Y, Kohara S, Namii Y et al. 1996 Clonal analysis of nodular hyperplasia in renal hyperparathyroidism. World J Surg 1996; 20: 744–752 4. Fukuda N, Tanaka H, Tominaga Y, Fukagawa M, Kurokawa K, Seino Y. Decreased 1,25-dihydroxyvitamin D receptor density is 3 associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest 1993; 92: 1436–1443 5. Tominaga Y, Takagi H. Histology, pathophysiology and indications for surgical treatment of renal hyperparathyroidism. Semin Surg Oncol (in press) 6. Fukagawa M, Kitaoka M, Yi H et al. Serial evaluation of parathyroid size by ultrasonography is another useful marker for the long-term prognosis of calcitriol pulse therapy in chronic dialysis patients. Nephron 1994; 68: 221–228 7. Fukagawa M, Kitaoka M, Kurokawa K. A new and safer protocol for percutaneous ethanol injection therapy ( PEIT ) of severe parathyroid hyperplasia (abstract). J Am Soc Nephrol 1996; 7: 1813

Reply

Fig. 1. Multiple nodular lesions within the left lower gland, which were suspected nodular hyperplasia of the parathyroid.

Based on the experience of our Japanese colleagues as well as ourselves there is no doubt that sonography is essential in the management of secondary hyperparathyroidism [1,2]. Today there is less problem in detecting the enlarged parathyroid glands, than in how to differentiate diffuse and nodular hyperplasia, i.e. more severe hyperparathyroidism. Tominaga et al. has documented that even in glands weighting

© 1997 European Renal Association–European Dialysis and Transplant Association

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between 0.1 and 0.5 g nodular hyperplasia could be found [ 3]. Therefore the gland weight is not sensitive enough to differentiate between diffuse and nodular hyperplasia. Could it be done by Doppler flow mapping? In our opinion more data are required to confirm the sensitivity and specificity of Doppler flow mapping in differentiating between these two forms of parathyroid-gland hyperplasia. Despite that, as we have already stated, we believe that new diagnostic methods, particularly high-resolution ultrasonography, probably power Doppler imaging, and fine-needle aspiration biopsy would teach us more about the pathological changes in parathyroid glands in primary and secondary hyperparathyroidism [ 4]. The advances in our knowledge may have a great impact in the decision of how to treat our patients. Sveti Duh General Hospital Zagreb Clinical Hospital University of Zagreb Croatia

D. Pavlovic´ H. Tomic´ Brzac ˇ ala S. C N. Jankovicˇ

1. Fukagawa M, Kitaoka M, Kurokawa K. Ultrasonographic intervention of parathyroid hyperplasia in chronic dialysis patients: a theoretical approach. Nephrol Dial Transplant 1996; 11 [Suppl 3]: 125–129 ˇ ala S, Jankovicˇ N. The value of 2. Pavlovic´ D, Tomic´ Brzac H, C parathyroid sonography in secondary hyperparathyroidism. Nephrol Dial Transplant 1997; 12: 367 3. Tominaga Y, Tanaka Y, Sato K, Nagasaka T, Takagi H. Histopathology, pathophysiology, and indications for surgical treatment of renal hyperparathyroidism. Semin Surg Oncol 1997; 13: 78–86 4. Pavlovic´ D, Tomic´ Brzac H. Unusual changes in parathyroid glands in patients with chronic renal failure. J Clin Endocrinol Metab 1997; 82: 703–704

Sulphonamides in vasculitides: which mechanism? Sir, Several reports describe beneficial effects of sulphonamides, mostly in association with trimethoprim, in diseases with circulating antineutrophil cytoplasmic autoantibodies directed against proteinase 3, or myeloperoxidase such as Wegener’s granulomatosis and related vasculitides. Prolonged medication with co-trimoxazole tends to decrease the incidence of recurrences in Wegener’s granulomatosis once disease has been brought into remission by immunosuppressive treatment. In addition, co-trimoxazole may be adequate for limited stages of the disease [ 1,2]. Several lines of evidence suggest a pathophysiological role for micro-organisms, especially Staphylococcus aureus, in triggering diseases activity in Wegener’s granulomatosis. It is therefore accepted that the beneficial of sulphonamides is related to their antimicrobial activity, as recently reviewed in this journal [2]. The antileprosy drug dapsone, a sulphonamide, is well known for its use in a variety of cutaneous leukocytoclastic vasculitides such as Scho¨nlein–Henoch syndrome [3,4 ]. Anecdotal, recently summarized communications indicate that Scho¨nlein–Henoch syndrome may respond dramatically to this agent [ 4]. Sulphonamides appear to control leukocytoclastic vasculitis rather than cure the underlying condition [ 4]. The mechanism of action of sulphonamides in leukocytoclastic vasculitis, which is largely speculative, includes both inhibition of the process by which neutrophils leave the circulation and migrate to lesional sites as well as the prevention of the tissue destruction normally caused by the neutrophils [4 ].

The observations in patients with Scho¨nlein–Henoch syndrome suggest that the beneficial of sulphonamides in vasculitides with circulating antineutrophil cytoplasmic autoantibodies is caused at least in part by a direct antivasculitic effect. Division of Nephrology Department of Pediatrics University of Bern Inselspital Bern Switzerland

M. G. Bianchetti A. C. Truttmann

1. De Remee RA. Empiricism and Wegener’s granulomatosis. N Engl J Med 1996; 335: 54–55 2. Stegerman CA, Cohen Tervaert JW, Kallenberg CGM. Co-trimoxazole and Wegener’s granulomatosis: more than a coincidence? Nephrol Dial Transplant 1997; 12: 652–655 3. Bernstein JE, Lorincz AL. Sulfonamides and sulfones in dermatologic therapy. Int J Dermatol 1981; 20: 81–82 4. Hoffbrand BI. Dapsone in Henoch–Schonlein purpura—worth a trial. Postgrad Med J 1991; 67: 961–962

Reply Sir, Drs Bianchetti and Truttmann correctly point out that the beneficial effects of co-trimoxazole in Wegener’s granulomatosis may be unrelated to its antimicrobial effect. First, as suggested in their letter, sulphamethoxazole, the sulphonamide component of co-trimoxazole, may act on neutrophils and macrophages by interfering with the myeloperoxidase– hydrogen peroxide–halogen system and diminish the generation of damaging products [1 ]. However, the scavenging potential of co-trimoxazole in vitro is much lower than of diafenylsulfon and sulphapyridine, drugs that have been reported to be effective in leukocytoclastic and Henoch–Scho¨nlein vasculitis [2 ]. Furthermore, sulphamethoxazole hydroxylamine, an in vivo encountered metabolite of sulphamethoxazole, has been shown to interfere with in vitro lymphocyte activation, probably by affecting intracellular esterase activity [3 ]. Lastly, co-trimoxazole, by its antagonism of folic acid metabolism, may have effects on the proliferation of immune competent cells thereby exerting immunosuppressive effects. It should be noted that in vitro the proliferation of mammalian cells is not affected by antimicrobially effective concentrations [4 ]. In addition, we were unable to demonstrate a significant effect of 24 months treatment with co-trimoxazole on cANCA titres in patients with Wegener’s granulomatosis [5 ]. So although some in vitro data suggest anti-inflammatory or immunosuppressive effects of co-trimoxazole, we have no proof that these effects are contributing to the therapeutic effect seen in Wegener’s granulomatosis. The same, however, holds true for the proposed pathophysiological role for Staphylococcus aureus in Wegener’s granulomatosis and the question whether the beneficial effect of co-trimoxazole in this disease is due to its antimicrobial activity [6 ]. Department of Internal Medicine Division of Nephrology and Clinical Immunology University Hospital Groningen Hanzeplein Groningen The Netherlands

C. A. Stegeman J. W. Cohen Tervaert C. G. M. Kallenberg

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1. Roberts DE, Curd JG. Sulfonamides as antiinflammatory agents in the treatment of Wegener’s granulomatosis. Arthritis Rheum 1990; 33: 1590–1593 2. Anderson R, Grabow G, Dosthuizen R, Theron A, van Rensburg AJ. Effects of sulfamethoxazole and trimethoprim on human neutrophil and lymphocyte functions in vitro: in vivo effects of co-trimoxazole. Antimicrob Agents Chemother 1980; 17: 322–326 3. Leeder JS, Nakhooda A, Spielberg SP, Dosch H-M. Cellular toxicity of sulfamethoxazole reactive metabolites. II. Inhibition of natural killer activity in human peripheral blood mononuclear cells. Biochem Pharmacol 1991; 41: 575–583 4. Burchall JJ. Mechanism of action of trimethoprim/ sulfamethoxazole, II. J Infect Dis 1973; 128 [suppl ]: 437–441 5. Stegeman CA, Cohen Tervaert JW, de Jong PE, Kallenberg CGM, for the Dutch Co-trimoxazole Wegener Study Group. Trimethoprim-sulfamethoxazole for the prevention of relapses of Wegener’s granulomatosis. N Engl J Med 1996; 335: 16–20 6. Stegeman CA, Cohen Tervaert JW, Kallenberg CGM. Co-trimoxazole and Wegener’s granulomatosis: more than a coincidence. Nephrol Dial Transplant 1997; 12: 652–655

Calcifying panniculitis or ‘simple’ inflammation? Biopsy is better than a bone scan Sir, Calciphylaxis, a nephrological mystery wrapped in an enigma, should always make a good case report. So it was with expectation that I read the case report by Koch Nagueira and colleagues [ 1]. This was immediately of interest to me, because here was a putative case of calciphylaxis induced by LMW heparin, within months of another report, in this journal, of the very same material apparently being responsible for resolution of calciphylaxis [2,3]. Calciphylaxis, like glomerulonephritis, is best diagnosed by histopathology, where the characteristic calcific obliterative arteriolopathy with tissue necrosis can be identified. There is a wide differential diagnosis. Cases without histology are suspect, unless contextually unimpeachable. Calciphylaxis is very, very unusual indeed in children—barely a handful of cases in the literature can be scrutinized. The problem I have with this report is twofold. First, subcutaneous heparin can cause a variety of skin lesions, including inflammation and necrosis, that have nothing at all to do with calciphylaxis [4], and second the naı¨ve assumption that a technetium 99m-labelled bone scan can unequivocally diagnose ‘calcification’. This scan technique, while indeed useful in the examination of the osseus skeletion, has a wellknown ability to detect sites of acute inflammation. Perhaps the best example is its use in myocardial infarction, where in dogs myocardial uptake is seen 12 h after coronary ligation, and in man 48–72 hours after clinical onset [5 ]. This is not due to calcification, but the presence of complexed calcium in the soft tissues. Naturally, with cell injury and death, intracellular calcium homeostasis is deranged, and it is not surprising that a calciotropic agent should localize to the region of injury. I think that the authors have described a skin reaction to subcutaneous LWM heparin, which they have shown very elegantly with the use of a bone-scanning technique adept at detecting acute inflammation. Without any histology, and with the unusually favourable outcome, I can’t accept this case as calciphylaxis. Trafford Dept. of Renal Medicine Royal Sussex County Hospital Brighton, UK

2.

3. 4. 5.

Calcifying panniculitis in a child after renal transplantation. Nephrol Dial Transplant 1997; 12: 216–218 Peres-Mijares R, Guzman-Zamadio JL, Payan-Lopez J, Rodriguez-Fernandez A, Gomez-Fernandez P, Almaraz-Jimenez. Calciphylaxis in a haemodialysis patient: functional protein S deficiency? Nephrol Dial Transplant 1996; 11: 1856–1859 Goldsmith DJA. Calciphylaxis in a haemodialysis patient: functional protein S deficiency? Nephrol Dial Transplant 1997; 12: 1082–1083 Bircher AJ. The differential diagnosis of heparin-induced skin lesions. Br J Haematol 1993; 85: 837 Parkey RW, Broute FJ, Buja LM, Stokely EN, Willerson JT. Myocardial infarct imaging with Tc99m-phosphate. Semin Nucl Med, 1977; 7: 15–28

Reply With interest we read the comments from Dr Goldsmith about the case recently reported by us in this journal [1]. In reply, we do agree with his main argument that biopsy is better than a bone scan with respect to the diagnosis of calciphylaxis. This was stated in the published report [1 ] and we assume that the diagnosis without a biopsy is presumptive at best. Nevertheless the hypothesis that the spots detected by technetium 99m scan could represent an inflammation process without true calcification cannot be retained since a plain radiography revealed subcutaneous calcifications at the same places where the spots had been detected ( Figure 1). As stated in the report, we confirm that the clinical picture fits with the original description of experimental calciphylaxis [2]. But, again in accordance with Dr Goldsmith, the fortunate favourable outcome was very unexpected and our main motivation to report the case was exactly that the clinical course was unusual.

D.J.A. Goldsmith

1. Koch Nagueira PC, Giuliani C, Rey N, Said M-H, Cochat P.

Fig. 1. Subcutaneous calcifications in the arm.

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Unite´ de Ne´phrologie Pe´diatrique Hoˆpital Edouard Herriot Lyon France


P. C. Koch Nogueira P. Cochat

1. Koch Nogueira PC, Giuliani C, Rey N, Saı¨d M-H, Cochat P. Calcifying panniculitis in a child after renal transplantation. Nephrol Dial Transplant 1997; 12: 216–218 2. Selye H, Nielsen K. Histogenesis of experimental cutaneous calcinosis. Acta Morphol Acad Sci Hung 1961; 10: 327–336

Interleukin-6 production by endothelial cells: effect of corticosteroids Sir, Interleukin 6 ( IL6) is a cytokine involved in B and T lymphocyte activation during inflammation [1], produced by several cell lines [1], including endothelial cells ( EC ) [ 2,3]. Its role in vasculitic disorders has been postulated [ 4–6 ], but not yet clarified. Corticosteroids have been claimed to reduce IL6 serum levels in some inflammatory disorders [ 7,8] and the expression of its receptor is upregulated by dexamethasone [1]. We studied the effect of corticosteroids on the endothelial production of IL6. Human EC, isolated from umbilical veins as previously described [9 ], were cultured in 6-well plates in M199 medium with penicillin 100 U/ml, streptomycin 50 U/ml, hepes 1%, fetal calf serum 20%, heparin 50 U/ml, and endothelial cell growth factor ( ECGF) (Boehringer Mannheim) 50 mg/ml. Subconfluent cellular monolayers were stimulated with lipopolysaccharide ( LPS) 1 mg/ml in the same culture medium without heparin and ECGF, in the presence or absence of increasing doses (0, 25, 50, 125, 250, 500 mg/ml ) of methylprednisolone (MP). Peripheral blood lymphocytes (PBL) isolated from healthy donors [ 10] at concentration of 106/ml were identically treated. Cellular supernatants were harvested after 18 h and IL6 was detected with ELISA (Predicta Interleukin-6 Kit, Genzyme diagnostics, Cambridge, MA). LPS determined an increase in IL6 concentration in MP-free PBL supernatants (from 0.16 to 0.41 pg/ml ) and in MP-free EC supernatants (from 0.27 to 0.54 pg/ml ). In MP- and LPS-treated samples, inhibition of IL6 production was observed, which was notable in PBL cultures ( MP-free sample, 0.41 pg/ml; MP 500 mg/ml, 0.17 pg/ml; MP 250 mg/ml, 0.26 pg/ml; MP 125 mg/ml, 0.18 pg/ml; MP 50 mg/ml, 0.25 pg/ml; MP 25 mg/ml, 0.28 pg/ml ), and less evident in EC cultures (MP-free sample, 0.54 pg/ml; MP 500 mg/ml, 0.44 pg/ml; MP 250 mg/ml, 0.47 pg/ml; MP 125 mg/ml, 0.54 pg/ml; MP 50 mg/ml, 0.49 pg/ml; MP 25 mg/ml, 0.48 pg/ml ) ( Figure 1). The anti-inflammatory properties of corticosteroid, at least in part, may be mediated through inhibition of IL6 production. In our experiments this effect was marked in lymphocyte culture, and less evident in EC samples. EC play an important pathogenetic role in vasculitis diseases, being involved in activation and modulation of the inflammatory process, through release of different mediators. It is a common notion that clinical activity of vasculitides is insufficiently controlled by corticosteroids alone, since only the addition of immunosuppressive treatment dramatically improved prognosis of these patients [11]. Thus, our experimental data could find a direct clinical counterpart, as we may speculate that corticosteroid inad-

Fig. 1. IL-6 production by EC and PBL stimulated by LPS and MP EC: endothelial cells; PBL: peripheral blood lymphocytes; LPS: lipopolysaccharide; MP: methylprednisolone; IL-6: interleukin 6.

equacy may be due partly to incomplete inhibition of IL6 production by EC. Institute of Nephrology and Department of Clinical Physiopathology University of Turin Italy

C. Rollino S. Borsa G. Bellone E. Menegatti F. Quarello G. Emanuelli G. Piccoli

1. Choi I, Kang HS, Yang Y, Pyun KH. IL6 induces hepatic inflammation and collagen synthesis in vivo. Clin Exp Immunol 1994; 95: 530–535 2. Xin X, Cai Y, Matsumoto K, Agui T. Endothelin-induced interleukin-6 production by rat aortic cells. Endocrinology 1995; 136: 132–137 3. Rafii S, Shapiro P, Pettengell R. Human bone marrow microvascular endothelial cells support long-term proliferation and differentiation of myeloid and megakaryocytic progenitors. Blood 1995; 86: 3353–3363 4. Weyand CM, Hicock KC, Hunder GG, Goronzy JJ. Tissue cytokine patterns in patients with polymialgia rheumatica and giant cell arteritis. Ann Intern Med 1993; 121: 484–491 5. Wagner AD, Goronzy JJ, Weyand CM. Functional profile of tissue infiltrating and circulating CD68+ cells in giant cell arteritis. J Clin Invest 1994; 94: 1134–1140 6. Yokoyama A, Kohno N, Fujino S, Inoue Y, Hiwada K. IgG and IgM rheumatoid factor levels parallel interleukin-6 during the vasculitic phase in a patient with Churg–Strauss syndrome. Intern Med 1995; 34: 646–648 7. Teoh KH, Bradley CA, Gauldie J, Burrows H. Steroid inhibition of cytokine-mediated vasodilation after warm heat surgery. Circulation 1995; 92: S11347–11353 8. van den Brick HR, van Wijk MJ, Geertzen RG, Bijlsma JW. Influence of corticosteroid pulse therapy on the serum levels of interleukin 2 receptor, interleukin 6 and interleukin 8 in patients with rheumatoid arthritis. J Rheumatol 1994; 21: 430–434 9. Rollino C, Borsa S, Bellone G, Piccoli G, Emanuelli G. False positive results with MTT assay. J Immunol Methods 1995; 185: 141–143 10. Boyum A. Separation of blood leucocytes, granulocytes and lymphocytes. Tissue Antigens 1974; 4: 269–274 11. Fauci AS, Haynes BF, Katz P, Wolff SM. Wegener’s granulomatosis: prospective clinical and therapeutic experience with 85 patients for 21 years. Ann Intern Med 1983; 98: 76–85


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ANCA in Behçet’s disease* Behçet’s disease ( BD) is a multisystem inflammatory disorder of unknown aetiology. It is characterized by recurrent oral and genital ulcers and uveitis. The disease is also associated both to thrombophlebitis and arthritis in addition to systemic manifestations resulting from small- and large-vessel vasculitis. The microscopy of recurrent aphthae shows an inflammatory reaction with aggregation of lymphocytes and monocytes and an intense infiltration of polymorphonuclear leukocytes with small-vessel necrosis, especially at mucosal and cutaneous sites [1]. Antineutrophil cytoplasmic antibodies (ANCA) are recognized in patients with necrotizing glomerulonephritis. Recent investigations have shown that ANCA are present in vasculitides. There are two main different patterns of ANCA: c and p (cytoplasmic and perinuclear) respectively. While c-ANCA are highly specific serological markers for active Wegener’s granulomatosis, p-ANCA have been detected in primary and secondary vasculitis [ 2]. Some anecdotal case reports of BD with positive ANCA were reported [3–6 ]. In Tunisia, as in the other Mediterranean countries, Behçet’s disease is common. To further appreciate the role of polymorphonuclear leukocytes in this disease we reviewed our large series of 300 patients and looked for the presence of ANCA in a group of patients with active and inactive Behçet’s disease. Forty-six Tunisian BD patients, 40 men and 6 women, were studied. The mean age was 37±7 years. All of them fulfilled the ISG criteria [7] with recurrent aphthous ulcers or scarring in 40 (86%), uveitis or retinitis in 26 ( 56%), cutaneous vasculitis in 27 (58%), synovitis in 20 ( 43%), meningoencephalitis in 2 ( 4%), and positive pathergy test in 20 (43%). Disease duration was 8±5 years. Renal function and urine analyses were normal. The disease was active in 16 patients, ( 35%) and inactive in 30 (65%). A total of 46 serum samples from these 46 consecutive patients with BD were tested for the presence of ANCA. All sera were collected at the time of the patient’s follow-up or during hospitalization, at different stages of disease evolution, and were kept at −70°C until use. All tests were controlled at Laboratory of Reference for France, Necker Hospital, according to indirect immunofluorescence standard tech-

nique, using smears containing human neutrophils, also some lymphocytes and eosinophilic granulocytes, in order to distinguish antinuclear antibodies from true ANCA, and sera diluted at 1/20 [8 ]. ELISA was also employed using as antigens the proteinase 3 of the granulocytes cytoplasm (PR3) and the myeloperoxidase ( MPO), a protein of the polymorphonuclear cytoplasm [9]. None of the 46 sera from patients with active and inactive BD had detectable ANCA. Only one patient had a weak perinuclear positivity corresponding to antinuclear antibodies (identical fixation in neutrophils, eosinophils, and lymphocytes). When tested by anti-MPO ELISA, no anti-MPO antibodies were detected. BD is a multisystem disease characterized by oral and genital ulceration associated with systemic manifestations due to large- and small-vessel vasculitis. Lesions are characterized histologically by acute inflammation and an intense infiltration of polymorphonuclear leukocytes with smallvessel necrosis, especially at mucosal and cutaneous sites [10]. BD is heterogeneous and besides the conventional triad, additional features of this syndrome include synovitis, cutaneous vasculitis resembling erythema nodosum, meningoencephalitis, large-artery aneurysms, and thrombophlebitis. The exact cause is unclear, and there is no universally accepted diagnostic test. However, numerous immunological abnormalities in patients with BD have been detected [11]. These include circulating antibodies to human mucosal cells and lymphocytotoxicity to oral epithelial cells [ 12]. Moreover, ANCA were found in some anecdotal reports of BD [3–6 ]. When stated, the clinical findings from these patients included the presence of crescentic glomerulonephritis, suggesting an overlap necrotizing angiitis syndrome. Five isolated cases of BD ( Table 1) with such atypical features with an associated renal involvement (crescentic glomerulonephritis) and vasculitic skin lesions were reported [3–6 ]. More interestingly, ANCA were sought in some series of 129 active and inactive BD patients and were found to be positive to a titre of 1/10 in only one patient [13–15 ]. The ANCA immunofluorescence specificity was not reported. It seems that ANCA have no role in the aetiopathogenesis of BD per se, unless there is another associated vasculitic process. No ANCA are detected in BD. Thus, although many agreed that the pathological hallmark of the BD is vasculitis, this disease is not mediated by autoantibodies directed MPO and PR3 as in systemic vasculitis.

Table 1. Previous and current case reports of ANCA in Behç et’s disease Authors

ANCA (+)/total cases

Atypical BD features

ANCA-IF/specificity

Hamza and Meyer 1990 [13 ] Vaiopoulos et al. 1993 [ 15]

1/28 3/29

Yang et al. 1993 [ 3]

1/1

— p-ANCA/PR3MPO p-ANCA/PR3 p-ANCA/PR3 c-ANCA/NS

Khan et al. 1994 [4 ] Baleva et al. 1994 [ 5] Ohta et al. 1994 [ 6 ] Aydintug et al. 1994 Present series 1997

1/1 2/2 1/1 0/72 0/46

none none ( 1) thrombosis ( 1) retinal vasculitis ( 1) crescentic GN Vasculitic skin lesions renal vasculitis NS crescentic GN none none

NS: Not stated. GN: Glomerulonephritis. * Parts of this work were presented at the 33rd EDTA–ERA Congress, Amsterdam, 18–21 June 1996.

c-ANCA/NS c-ANCA/NS c-ANCA/NS — —

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We conclude that, contrary to previous anecdotal reports about positive ANCA in BD, our study strongly suggests that ANCA cannot be proposed as a contributing antibody or diagnostic test in BD. Departments of Nephrology and Internal Medicine, H. Chaker Hospital, Sfax, Tunisia Department of Immunology, Faculty of Medicine, Sfax, Tunisia Department of Nephrology, Necker Hospital, Paris, France

M. Ben Hmida J. Hachicha N. Kaddour H. Makni F. Z. Aydel N. Chakroun Z. Bahloul H. Ayadi L. H. Noe¨l A. Jarraya

1. Behçet’s disease. Lancet 1989; 1: 761–762 2. Kallenberg, CGM, Brouwer E, Weening JJ, Tervaert JW. Antineutrophil cytoplasmic antibodies, current diagnostic and pathophysiological potential. Kidney Int 1994; 46: 1–15 3. Yang CW, Park IS, Kim SY et al. Antineutrophil cytoplasmic autoantibody associated vasculitis and renal failure in Behç et disease. Nephrol Dial Transplant 1993; 8: 871–873 4. Khan IH, Catto GRD, MacLeod AM. Antineutrophil cytoplasmic autoantibody associated vasculitis and renal failure in Behçet’s disease. Nephrol Dial Transplant 1994; 9: 332 5. Baleva M, Kolarov Z, Nikolov K. Antineutrophil cytoplasmic autoantibody in two patients with Behç et’s disease. Nephrol Dial Transplant 1994; 9: 876 6. Ohta S, Yokoyama H, Matsuda I et al. A case of ANCAassociated rapidly progressive glomerulonephritis with oral aphtha and erythema nodosum. Nippon Jinzo Gakkai Shi 1994; 36: 1184–1190 7. International Study Group for Behçet’s disease. Criteria for diagnosis of Behç et’s disease. Lancet 1990; 1: 1078–1080 8. Wiik A. Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. Acta Pathol & Microbiol Immunol Scand 1989; 97: 12–13 9. Geffriaud-Ricouard C, Noe¨l LH, Chauveau D, Houhou S, Gru¨nfled JP, Lesavre P. Clinical spectrum associated with ANCA of defined antigen specificities in 98 selected patients. Clin Nephrol 1993; 39: 125–136 10. O’Duffy JD. Behçet’s syndrome. N Engl J Med 1990; 322: 326–328 11. Lehner T. Behç et’s syndrome and autoimmunity. Br Med J 1967; 1: 465–466 12. Rogers RS, Sams W, Shorter RG. Lymphocytotoxicity in recurrent aphthous stomatitis: lymphocytotoxicity for oral epithelial cells in recurrent aphthous stomatitis and Behçet’s syndrome. Arch Dermatol 1974; 109: 361–362 13. Hamza M, Meyer O. Negative antineutrophil cytoplasmic autoantibody in Behçet’s disease. Ann Rheum Dis 1990; 49: 817 14. Aydintug AO, Tokgoz G, D’Cruz DP et al. Antibody to endothelial cells in patients with Behçet’s disease. Clin Immunol Immunopathol 1993; 67: 157–162 15. Vaiopoulos G, Hatzinikolaou P, Tsiroyanni A et al. Antineutrophil cytoplasmic autoantibodies in Adamantiadis– Behçet’s disease. Br J Rheumatol 1994; 33: 406–407

Anti-b -glycoprotein I and anti-prothrombin antibodies 2 in haemodialysis patients Sir, Vascular access thrombosis ( VAT ) is a major source of morbidity in haemodialysis patients. Technical problems related to surgery and diminished flow through the vascular access caused by stenosis or by perdialytic or postdialytic hypotension largely contribute to thrombosis. Other problems with haemostasis that may predispose to hypercoagulability also may contribute. Many studies have reported a

high prevalence of anticardiolipin antibodies in haemodialysis patients [1,2]. Nevertheless, studies on the role of anticardiolipin antibodies (aCLA) on vascular access thrombosis have given conflicting results [1–3 ]. The significance of antiphospholipid antibodies as a marker for recurrent thrombosis is decreased by their extreme heterogeneity, not taken into account by usual laboratory tests (anticardiolipin antibodies and lupus anticoagulant). Recent studies have shown that some aCLA might not bind directly to cardiolipin but rather to plasma proteins with affinity for anionic phospholipids, namely b 2 glycoprotein I (b -GPI) or prothrombin [4 ]. Anti-b -GPI 2 2 have been detected in autoimmune diseases but not in infections. Conversely, aCLA found in patients with syphilis do not seem to require this cofactor, nor do syphilis patients develop an antiphospholipid syndrome (APS ), thus suggesting that pathogenicity of aCLA may depend on their need of b -GPI for reactivity [5]. Moreover the association 2 of thromboses with anti-b -GPI in patients with SLE was 2 found to be stronger than to aCLA, and anti-b -GPI are 2 also frequently found in patients with primary antiphospholipid syndrome [6 ]. The purpose of this study was to determine whether the presence of greater than normal titre of aCLA, anti-b -GPI and anti-prothrombin antibodies in patients 2 on haemodialysis is related to recurrent vascular access thrombosis. Forty-five haemodialysed patients were tested for the presence of anticardiolipin, anti b -GPI and anti-prothrombin 2 antibodies. IgG ACA were assayed with ELISA procedure and considered negative when 23. Anti b -GPI and anti-prothrombin antibodies were assayed 2 with ELISA. Twelve patients (26.6%) exhibited aCLA. Two of them (16.6%) exhibited both anti-b -GPI and anti2 prothrombin antibodies. Primary renal disease was APS in one of them. None of the aCLA-negative patients had anticofactor antibodies. Age, gender, diabetes, type of vascular access, duration of haemodialysis, and primary renal disease were not different in patients with and without aCLA. Patients with aCLA had thrombosed their vascular access at a rate of 0.53/patient/year whereas patients without aCLA had thrombosed at a rate of 0.39/patient/year (P=NS). Mean aCLA titre was 19.5±7.2 IU in patients without recurrent VAT ( 0 or 1 in thrombosis) and 20.7±8.3 in patients with recurrent VAT (>1 thrombosis) (P=NS ). None of the two patients with anti-cofactor antibodies experienced vascular access thrombosis. Nevertheless one of them received oral anticoagulant because of APS. In conclusion, we found no association between the presence of aCLA and the prevalence of vascular access thrombosis in haemodialysis patients. Haemodialysis-associated aCLA seem to interact directly with phospholipids. The absence of both anti-b -GPI and anti-prothrombin antibodies 2 may explain the irrelevance of aCLA in the pathogenesis of vascular access thrombosis in haemodialysis patients. Department of Nephrology and Renal Transplantation Hopital Saint Jacques Besançon, France

D. Ducloux A. Florea J.-M. Rebibou M. Jamali J.-M. Chalopin

1. Gronhagen-Riska C, Teppo AM, Helentera A, Honkanen E, Julkunen. Raised concentration of antibodies to cardiolipin in patients receiving dialysis. Br Med J 1990; 300: 1696–1697 2. Garcia-Martin F, De Arriba G, Carrascosa T, Moldenhauer F, Martin-Escobar E, Val J, Saiz F. Anticardiolipin antibodies and lupus anticoagulant in end-stage renal disease. Nephrol Dial Transplant 1991; 6: 543–547

Nephrol Dial Transplant ( 1997) 12: 2467 3. Prakash R, Miller CC, Suki WN. Anticardiolipin antibody in patients on maintenance hemodialysis and its association with recurrent arteriovenous graft thrombosis. Am J Kidney Dis 1995; 26: 347–352 4. Galli M, Comfurius P, Maassen C. Anticardiolipin antibodies directed not to cardiolipin but to plasma protein factor. Lancet 1990; 335: 1544–1547 5. Matsuura E, Igarashi Y, Fujimoto M. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 1990; 336: 177–178 6. Viard JP, Amoura Z, Bach JF. Association of antib2-glycoprotein I antibodies with lupus-type circulating anticoagulant and thrombosis in systemic lupus erythematosus. Am J Med 1992; 93: 223–229

Hepatitis G virus infection in haemodialysis patients Sir, The recent discovery of a new blood-transmitted RNA virus belonging to the Flaviviridae family and called hepatitis G Virus (HGV ) [1 ], induced studies to establish the epidemiological and clinical role of HGV virus in high-risk groups such as drug addicts and transfused or haemodialysis patients. The epidemiological data available so far show a significant discrepancy of HGV prevalence among haemodialysed patients. In fact the prevalence ranges from 3.1% reported by Japanese authors [2] to 57.5% reported by French authors [ 3]. We studied 78 patients (45 males and 33 females) who were undergoing maintenance haemodialysis at the Nephrology Unit of our Department. Characteristics of patients are reported in Table 1. Patient sera were tested for HGV-RNA and HCV-RNA. Informed consent was obtained from each patient. HGV-RNA analysis was performed by nested and semi-nested polymerase chain reaction using primers derived from the NS3/helicase and 5∞ non-coding regions respectively. HCV-RNA analysis was performed by nested PCR using primers derived from the 5∞ non-coding region. Two of our 78 patients (2.6%) proved HGV-RNA positive. No HBV and/or HCV coinfection was found in these patients. Both patients had previously been transfused. ALT values were normal and no clinical signs of liver disease were present. HGV infection prevalence was low in our haemodialysed patients ( 2.6%). This prevalence is similar to what was reported by Japanese authors [2] but quite different compared to what was reported by other authors of our geographic area [3–5]. This finding would therefore exclude a racial distribution of HGV infection. It is difficult to explain the significant differences found in the published series. Methodological factors in HGV detection do not explain these discrepancies, because in our work we amplified both 5∞UTR and NS3 regions, increasing the sensitivity of HGV detection. HGV infection is particularly prevalent in all polytransfused patients. Thus we think that the history of transfusions in haemodialysed patients may play an important role in transmitting HGV. The percentage of transfused patients is relatively low ( 23/78, 29.5%) in our Table 1. Characteristics of patients Patients (n) 78 Sex ( M/F ) 45/33 Age (M±SD) 62±14 (range 17–90) Duration of haemodialysis (M±SD) 52±51 months (range 1–273) History of transfusion 23/78 HBsAg+ve 3/78 Anti-HCV+ve 22/78

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series and it is significantly lower than that reported by other authors [3] whose papers indicate a higher HGV prevalence. This data may support the low prevalence of HGV infection we observed. Moreover we would like to point out that 19 of the 23 transfused patients underwent transfusion after testing for HCV had begun. All these patients were antiHCV and HCV-RNA negative. This finding might explain the low prevalence of HGV considering the significant presence of HCV-HGV coinfection in polytransfused patients. These observations suggest that the history of transfusion may influence the HGV prevalence. Our HGV patients had no clinical and/or biochemical signs of liver disease. In particular ALT values were persistently normal. On the basis of these data liver biopsy was not performed. Our experience shows that HGV infection probably has a poor epidemiological and clinical impact in haemodialysed patients. Further studies are needed to determine the natural history of the infection. Department of Internal Medicine Gastroenterology and Nephrology Units Institute of Biology and Genetics University of Genova Italy

A. Picciotto N. Campo N. Sinelli R. Brizzolara G. Poggi G. Gurreri R. Russo S. Saffioti D. A. Coviello G. Celle

1. Simons JN, Leary TP, Dawson GJ et al. Isolation of novel viruslike sequences associated with human hepatitis. Nat Med 1995; 1: 564–569 2. Masuko K, Mitsui T, Iwano K et al. Infection with hepatitis GB virus C in patients on maintenance hemodialysis. N Engl J Med 1996; 334: 1485–1490 3. De Lamballerie X, Charrel RN, Dussol B. Hepatitis GB virus C in patients on hemodialysis. N Engl J Med 1996; 334: 1549 4. Sampietro M, Badalamenti S, Lunghi G et al. Hepatitis GB Virus C. N Engl J Med 1996; 335: 1392 5. Sampietro M, Badalamenti S, Graziani G et al. Hepatitis G virus infection in hemodialysis patients. Kidney Int 1997; 51: 348–352

Mycophenolate mofetil toxicity in an anorexic kidney transplant patient treated with sulphinpirazone Sir, Mycophenolate mofetil (MMF ) is receiving increasing attention by nephrologists for its efficiency in preventing rejection and in the treatment of ongoing rejection [1–3]. MMF may also exert a preventive effect on the development and progression of proliferative arteriolopathy, a critical pathological lesion in chronic rejection [4], and it has been hypothesized that MMF may have a ‘steroid-sparing’ effect [1 ]. However, clinicians should be alert for side-effects and drug interactions not encountered in the clinical trials. In our outpatient clinic we are now following some carriers of kidney grafts in which long-lasting chronic immunosuppression has been modified from azathioprine to MMF. Filler and Ehrich [ 3] recently described a young patient with severe MMF toxicity and suggested that monitoring mycophenolic acid (MFA) concentrations may be necessary. In keeping with their observation, we would like to describe an adult patient who developed severe gastrointestinal symptoms, life-threatening hyponatraemia, and acute-on-chronic renal failure after starting MMF treatment.

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Case report. A 50-year-old woman was admitted with severe gastrointestinal symptoms and acute-on-chronic renal failure of the transplanted kidney. The patient was suffering from adult polycystic kidney disease and 8 years previously had received a cadaveric kidney graft treated by standard triple therapy (methylprednisolone 6 mg, cyclosporin A 150 mg, azathioprine 75 mg). She was anorexic, and her body-weight was 50 kg, height 165 cm. Her creatinine clearance was 21 ml/min (serum creatinine and urea 2.1 and 80 mg/dl respectively) and a recent kidney biopsy was suggestive of chronic rejection with intimal fibrosis. A month before the present admission, her azathioprine had been substituted by MMF 1 g b.d. and sulphinpirazone ( Enturen 400 mg) had been added due to high uric acid level. About a week after, the patient started to vomit 2–4 times daily with pyrosis and abdominal pain. A week before the admission, she contacted her GP who prescribed glucose and amino acid i.v. Four days before the admission she presented with aqueous diarrhoea 5–6 times daily. On admission, she looked extremely dehydrated. Body weight was 45 kg. There was no evidence of either viral or bacterial infection. Yersinia, salmonella and shigella were negative in the stools. EGDS showed no specific lesions. ECG was unmodified. Leukopenia was not present. Serum creatinine was 3 mg/dl, urea 136 mEq/l, uric acid 13.6 mg/dl, sodium 113 mEq/l, potassium 3.5 mEq/l, chloride 88 mEq/l, pH 7.35, HCO 16.2 mEq/l, PCO 29.3 mmol/l. 3 2 MMF and sulphinpirazone were withdrawn, azathioprine re-instituted, and the patient was treated with heavy saline and electrolyte infusion. Subsequently the patient stopped vomiting, epigastralgias persisted for 4 days and the aqueous diarrhoea improved after 5 days and finally ceased after 10 days. At discharge, serum creatinine and urea were respectively 2.3 and 80 mg/dl. Comment. In this patient we did not perform pharmacodynamic or pharmacokinetic monitoring of MMF [2 ]. However, it seems likely that gastroenteric symptoms were related to MMF intoxications since 3 weeks of constant vomiting disappeared after stopping MMF administration. On the other hand, long-lasting diarrhoea following MMF toxicity has been described by other authors [3 ]. The question is why our patient developed severe gastroenteric symptoms on the recommended MMF dose for GFR