ARTHRITIS & RHEUMATISMVol. 46, No. 8, August 2002, pp 2252–2264© 2002, American College of Rheumatology

LETTERS

DOI 10.1002/art.10345

Rituximab for the treatment of type II mixedcryoglobulinemia

To the Editor:Rituximab is a human/mouse chimeric monoclonal

antibody (IgG1�) that reacts specifically with CD20 antigen (atransmembrane protein present during different steps in thematuration of B lymphocytes, from early pre B to maturelymphocyte), inducing complement-dependent cytotoxicityand antibody-dependent cellular cytotoxicity. The marked andsystemic B cell depletion observed in non-Hodgkin’s lym-phoma patients treated with rituximab has recently led to useof this agent in autoimmune diseases, with the aim of inhibitingproduction of antibodies reacting against normal cells andtissues. Preliminary published results appeared encouraging,because rituximab showed activity in some cases of coldagglutinin disease (1–3), warm antibody hemolytic anemia (2),idiopathic thrombocytopenic purpura (4–6), paraproteinemicpolyneuropathy (7), and myasthenia gravis (8).

Type II mixed cryoglobulinemia (MC) is a systemicvasculitis strictly associated with hepatitis C virus (HCV)infection and is generally sustained by proliferation of non-neoplastic oligoclonal B cells (9,10). Based on the hypothesisthat rituximab could act on proliferating B cells and thus onsystemic autoimmune manifestations, we used the drug in 4patients with symptomatic type II MC who were unresponsiveto previous treatments. The main clinical features of thesepatients, before treatment with rituximab, are described below.

Patient 1 (previously described; see ref. 11) was a58-year-old man who had formerly been treated withinterferon-� (IFN�), cyclophosphamide, prednisone, danazol,and plasmapheresis. Forty-six months after the diagnosis oftype II MC, the patient still had diffuse purpura, arthralgias,fever, moderate anemia, and chronic renal failure.

Patient 2 was a 67-year-old woman previously treatedwith prednisone and plasmapheresis. One hundred eight

months after the diagnosis of type II MC, despite mildlypositive cryoglobulins and rheumatoid factor (RF), she hadpurpura, diffuse bullous lesions, and urticaria.

Patient 3 was a 68-year-old woman who was HCVnegative and had been unresponsive to plasmapheresis andprednisone. Five months after the diagnosis of type II MC, shepresented with diffuse purpura, ulcerative skin lesions on herlower limbs, and a severe, primarily sensorial polyneuropathy.

Patient 4, a 68-year-old woman, had previously beentreated with prednisone. Forty-eight months after the diagno-sis of type II MC, her clinical picture was characterized bydevelopment of a CD20� B cell non-Hodgkin’s lymphoma(small lymphocytic lymphoma according to the revisedEuropean-American lymphoma classification), with superficiallymphadenopathies and bone marrow involvement (�10% ofmarrow infiltrated), Coombs-negative hemolytic anemia, andgrade IV neutropenia (according to World Health Organiza-tion grading).

Rituximab was administered intravenously in a dose of375 mg/m2 on days 1, 8, 15, and 22. No other cytotoxic,immunosuppressive, or steroid drugs were allowed in associa-tion with use of rituximab. Response was evaluated by assess-ing the modifications of clinical signs, symptoms, and the mainimmunologic pattern.

Each patient’s clinical and laboratory features beforeand after rituximab treatment are summarized in Tables 1–4.Patient 4 had a complete remission of the clinical manifesta-tions and normalization of laboratory values. Patients 1, 2, and3 demonstrated good partial responses, as defined by �50%reduction in clinical and laboratory signs of disease. Theduration of response ranged from 3 to 13 months. Depletion ofCD20� B cells and reduction of serum IgG and RF levels wereobserved in all 4 patients. In patients 3 and 4, the disappear-ance of the serum monoclonal component IgM� was shown byimmunofixation. Normalization of complement levels was in-consistent, and disappearance of cryoglobulins was seen inpatients 1 (transient disappearance only) and 4.

Table 1. Clinical and laboratory features of patient 1 before and after rituximab treatment (375 mg/m2)*

Months afterRTX therapy

Main features of typeII MC

RF,units/ml

Cryo,mg/dl

IgM,gm/liter

IgA,gm/liter

IgG,gm/liter

C3,mg/dl

C4,mg/dl

CD20,�109/liter

CD19,�109/literPurpura Arthralgia

Baseline ��� 9 868 90 1.7 0.7 2.9 72 8 0.032 0.0321 � 2 159 �50 1.1 0.8 4.7 88 4 0.002 0.0022 � 2 71 Neg 0.8 0.6 3 80 4 0.002 0.0013 � 2 132 150 1 0.7 4.5 85 2 0.002 0.0014 �� 5 604 90 1.4 0.7 4.9 88 3 0.020 0.0205 �� 8 296 137 1.3 0.7 4.3 112 2 NA NA6 ��� 9 944 169 1.6 0.8 5.2 92 3 NA NA12, LF ��� 9 427 319 1.4 0.9 3 96 2 NA NA

* The patient was a 58-year-old, hepatitis C virus–positive man. Purpura was scored as follows: ��� � diffuse and persistent involvement of thetrunk and lower limbs; �� � diffuse and persistent involvement of the lower limbs; � � spare and fluctuating involvement of the lower limbs.Arthralgia was graded according to a patient-scored (range 0–10) visual analog scale. Rheumatoid factor (RF) normal range 0–14; C3 normal range90–180; C4 normal range 10–40. MC � type II mixed cryoglobulinemia; Cryo � cryoglobulins; RTX � rituximab; Neg � negative; NA � data notavailable; LF � last followup.

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In patient 1, the therapeutic regimen was interruptedafter the second infusion of rituximab because of the develop-ment of acute left-sided amaurosis with a documented throm-bosis of the retinal artery. A symmetric panniculitis at theelbows and knees, which spontaneously resolved after 1 month,was recorded for patient 2 on day 60. No other acute ordelayed relevant side effects or infectious complications oc-curred.

In this preliminary experience, rituximab treatmentproved effective in all 4 type II MC patients whose clinicalmanifestations had been unresponsive to conventional treat-ments. Clinical improvement was accompanied by improve-ment in disease-related laboratory values. In particular, thereduction in the levels of IgM and RF and the disappearanceof the monoclonal component of IgM� in 2 patients, inconjunction with the concomitant depletion of B cells from theperipheral blood, indicate that rituximab inhibited type IIMC–related B cell proliferation and pathogenic autoantibodyproduction. Rituximab therapy, although not targeted to thekey infectious trigger of type II MC, effectively treated mani-

festations of the disease, presumably by blocking the crucialpathobiologic events (RF and immunocomplex production)linked to the infection. This fact may be relevant in type II MCpatients, in whom HCV eradication may not be achievedbecause of inefficacy of or contraindications to treatment withantiviral agents.

Data for patients with non-Hodgkin’s lymphomas andautoimmune diseases indicate that use of rituximab is associ-ated with low toxicity and, in particular, absence of infectiouscomplications. This is important, because infection, the mostcommon cause of death in type II MC, is commonly associatedwith use of steroids and cytotoxic drugs. Administration ofrituximab was not associated with arterial or venous thrombo-sis in a large series of patients (12). However, the thromboticevent observed in patient 1 could have been related to factorspredisposing to type II MC (e.g., vasculitis, hyperviscosity).Thus, use of rituximab should be carefully supervised inpatients with type II MC, especially if additional risk factors forvascular events are present.

In conclusion, these preliminary results suggest that

Table 2. Clinical and laboratory features of patient 2 before and after rituximab treatment (375 mg/m2)*

Months afterRTX therapy

Main features of type II MCRF,

units/mlCryo,mg/dl

IgM,gm/liter

IgA,gm/liter

IgG,gm/liter

C3,mg/dl

C4,mg/dl

CD20,�109/liter

CD19,�109/literPurpura Urticaria Bullae

Baseline �� �� �� 42 �50 1 1.5 7 114 2 0.145 0.1201 0 0 0 48 �50 1 1.5 7.3 127 4 0.010 0.0052 0 0 0 51 �50 0.9 1.4 7.5 108 4 0.008 0.0043 0 0 0 20 �50 0.6 1.4 6.4 128 6 0.007 0.0014 0 0 0 64 �50 0.8 1.4 6.9 128 6 0.006 0.0015 0 0 0 50 �50 0.8 1.4 7.5 104 6 0.009 0.0096 � 0 0 40 �50 0.9 1.2 9.4 100 8 0.150 0.15013, LF 0 0 0 40 �50 0.9 2.2 9.4 98 4 NA NA

* The patient was a 67-year-old, hepatitis C virus–positive woman. Purpura, urticaria, and bullae were scored as follows: ��� � diffuse andpersistent involvement of the trunk and lower limbs; �� � diffuse and persistent involvement of the lower limbs; � � spare and fluctuatinginvolvement of the lower limbs. Rheumatoid factor (RF) normal range 0–14; C3 normal range 90–180; C4 normal range 10–40. See Table 1 fordefinitions.

Table 3. Clinical and laboratory features of patient 3 before and after rituximab treatment (375 mg/m2)*

Months afterRTX therapy

Main features of type II MCRF,

units/mlCryo,mg/dl

IgM,gm/liter

IgA,gm/liter

IgG,gm/liter

C3,mg/dl

C4,mg/dl

CD20,�109/liter

CD19,�109/literNeuropathy Purpura Skin ulcers

Baseline 8 ��� �� 79 1,500 14 1.6 7.4 107 2 0.053 0.0261 4 0 �� 34 1,200 10 1.2 3.4 179 6 0.007 0.0072 4 0 � 21 1,100 7 0.9 3.4 140 7 0.001 0.0023 4 0 � �15 3,900 5 0.8 3.4 143 10 0.007 0.0154 4 � 0 16 NA 7 0.9 4.8 145 10 0.020 0.0205, RTX � 4 4 �� 0 �15 NA 6 0.9 5 149 10 0.001 0.0016 4 �� 0 �15 1,613 2 0.9 2.7 168 11 NA NA7 4 �� 0 �15 1,421 1.7 0.9 1.9 154 11 0.001 0.0018 3 �� 0 �15 NA 0.4 0.9 3.3 167 14 0.010 0.0109 3 �� 0 47 2,924 2.3 0.9 3.3 155 13 0.056 0.05610 3 �� 0 40 1,324 2.4 1.1 3.3 134 12 NA NA11, LF 3 �� � �15 NA 2.9 1 3.2 148 10 NA NA

* The patient was a 68-year-old, hepatitis C virus–negative woman. Neuropathic symptoms (pain and paresthesias) were graded according to apatient-scored (range 0–10) visual analog scale. Purpura and ulcers were scored as follows: ��� � diffuse and persistent involvement of the trunkand lower limbs; �� � diffuse and persistent involvement of the lower limbs; � � spare and fluctuating involvement of the lower limbs. Rheumatoidfactor (RF) normal range 0–14; C3 normal range 90–180; C4 normal range 10–40. RTX � 4 � 4 infusions. See Table 1 for additional definitions.

LETTERS 2253

rituximab may represent an important alternative for thetreatment of patients with type II MC. Rituximab could be theideal replacement for corticosteroids and conventional cyto-toxic drugs, which increase the risk of infection and oncogenicevents. Moreover, rituximab could also be the ideal drug foruse in association with classic antiviral therapy (i.e., IFN�,ribavirin).

We wish to thank Roche Diagnostics S.p.A. (Monza, Italy) forkindly supplying rituximab and Professor Angelo Giumanini for editing.

Francesco Zaja, MDSalvatore De Vita, MDDomenico Russo, MDAngela MicheluttiRenato Fanin, MDGianfranco Ferraccioli, MDPoliclinico UniversitarioUdine, ItalyMichele Baccarani, MDPoliclinico S. OrsolaBologna, Italy

1. Berentsen S, Tjonnfjord GE, Gjertsen BT, Hammerstrom J,Langholm R, Sorbo JH, et al. Rituxan (rituximab) therapy forchronic cold agglutinin disease [abstract]. Blood 2000;96:3156a.

2. Lee E, Zamkoff KW, Gentile TC, Zimrin A. Rituxan in thetreatment of auto-immune haemolytic anemia (AIHA) [abstract].Blood 2000;96:2560a.

3. Zaja F, Russo D, Fuga G, Michelutti T, Sperotto A, Fanin R, et al.Rituximab in a case of cold agglutinin disease. Br J Haematol2001;115:232–3.

4. Perotta A, Sunnemberg JS, Ratanatharathorn V, Hook C, Attas L,Dawson D, et al. Rituxan in the treatment of chronic idiopathicthrombocytopenia purpura (ITP) [abstract]. Blood 1999;94:94a.

5. Ratanatharathorn V, Carson E, Reynolds C, Ayash LJ, Levine J,Yanik G, et al. Anti-CD20 chimeric monoclonal antibody treat-ment of refractory immune-mediated thrombocytopenia in a pa-tient with chronic graft-versus-host disease. Ann Intern Med2000;133:275–9.

6. Saleh MN, Gutheil J, Moore M, Feinberg B, Bunch P, Butler J, etal. A pilot study of anti-CD20 MoAb rituximab in patients withrefractory immune thrombocytopenic purpura (ITP) [abstract].Blood 2000;96:1086a.

7. Levine TD, Pestronk A. IgM antibody-related polyneuropathies:B-cell depletion chemotherapy using rituximab. Neurology 1999;52:1701–4.

8. Zaja F, Russo D, Fuga G, Perella G, Baccarani M. Rituximab formyasthenia gravis developing after bone marrow transplant. Neu-rology 2000;55:1062–3.

9. De Vita S, De Re V, Gasparotto D, Ballare M, Pivetta B,Ferraccioli G, et al. Oligoclonal non-neoplastic B cell expansion isthe key feature of type II mixed cryoglobulinemia: clinical andmolecular findings do not support a bone marrow pathologicdiagnosis of indolent B cell lymphoma. Arthritis Rheum 2000;43:94–102.

10. Langford CA. Treatment of polyarteritis nodosa, microscopicpolyangiitis, and Churg-Strauss syndrome: where do we stand?Arthritis Rheum 2001;44:508–12.

11. Zaja F, Russo D, Fuga G, Patriarca F, Ermacora A, Baccarani M.Rituximab for the treatment of type II mixed cryoglobulinemia.Haematologica 1999;84:1157–8.

12. McLaughlin P, Grillo-Lopez AJ, Link BK, Levy R, Czuczman MS,Williams ME, et al. Rituximab chimeric anti-CD20 monoclonalantibody therapy for relapsed indolent lymphoma: half of patientsrespond to a four-dose treatment program. J Clin Oncol 1998;16:2825–33.

DOI 10.1002/art.10351

Reply

To the Editor:The letter by Zaja and colleagues raises some impor-

tant points with regard to cryoglobulinemic vasculitis and itsmanagement. Since the discovery of HCV, it has becomeapparent that the vast majority of cases once identified asessential mixed cryoglobulinemia are associated with chronicHCV infection (1). Although the mechanisms involved in thedevelopment of cryoglobulinemic vasculitis continue to beactively investigated, current evidence suggests that in thesetting of HCV infection, stimulation of B cells leads to theproduction of IgM RF that complexes with HCV antigens andpolyclonal HCV-specific IgG. The deposition of these immunecomplexes in blood vessel walls triggers inflammation, result-ing in the clinical features of cryoglobulinemic vasculitis.

Treatment of HCV-related cryoglobulinemic vasculitishas primarily focused on elimination of HCV and on measuresthat interfere with the immune mechanisms responsible forblood vessel damage. For patients with HCV, combined treat-ment with IFN� and ribavirin has resulted in an overallsustained response rate of 40–45% (2,3). IFN� has beenshown to be beneficial in HCV-related cryoglobulinemic vas-culitis, with clinical response and reduction in the level of

Table 4. Clinical and laboratory features of patient 4 before and after rituximab treatment (375 mg/m2)*

Months afterRTX therapy

Main features of type II MCRF,

units/mlCryo,mg/dl

IgM,gm/liter

IgA,gm/liter

IgG,gm/liter

C3,mg/dl

C4,mg/dl

CD20,�109/liter

CD19,�109/literAHA, grade Neutropenia, grade

Baseline 1 IV 101 446 8 1.9 10 240 6 0.060 0.0431 0 0 40 293 5.1 1 8 133 10 0.006 0.0012 0 0 33 45 2.2 0.8 7 138 13 0.001 0.0023 0 0 21 Neg 2.1 0.8 8 134 19 0.006 0.0064 0 0 15 Neg 1.7 0.8 7.1 127 18 NA NA5, LF 0 0 15 Neg 1.7 0.8 7.1 119 20 0.020 0.020

* The patient was a 68-year-old, hepatitis C virus–positive woman. Anemia and neutropenia were graded according to World Health Organizationclassification. Rheumatoid factor (RF) normal range 0–14; C3 normal range 90–180; C4 normal range 10–40. AHA � autoimmune hemolyticanemia. See Table 1 for definitions.

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circulating cryoglobulins being related to suppression of vire-mia (4).

In the largest prospective, randomized, controlled trialto date, involving 65 patients with HCV-related cryo-globulinemic vasculitis, complete response was observed in53% of those who received IFN� plus methylprednisolone,53% who received IFN� alone, 17% who received methylpred-nisolone alone, and 7% who received no treatment (5). Al-though the rate of relapse following discontinuation of treat-ment was delayed in patients who received combined therapy,the addition of methylprednisolone to IFN� was found to be oflimited benefit overall. Long-term remission was observed in33% of patients treated with combined therapy, compared with25% of those treated with IFN� alone, and HCV RNA levelsincreased in almost 40% of methylprednisolone-treated pa-tients. This prior experience supports that IFN� plus ribavirinshould remain the first line of treatment for HCV-relatedcryoglobulinemic vasculitis, because it provides the best oppor-tunity for viral clearance and resultant improvement of dis-ease. The reality, however, is that �50% of patients may havea sustained response to this regimen, or patients may be unableto receive or tolerate IFN� plus ribavirin because of sideeffects. For such individuals, there is a great need to identifyother therapeutic options.

In their letter, Zaja and coworkers describe theirexperience with 4 patients with type II mixed cryoglobulinemiawho were treated with rituximab. Rituximab is a chimericmonoclonal antibody that binds to the CD20 antigen on Bcells, resulting in B cell death. Such a mechanism is intriguingin the setting of HCV-related cryoglobulinemic vasculitis,because it would theoretically deplete the population of B cellsthat are producing the pathogenic RF that is driving theimmune complex disease.

The experience of Zaja et al must be viewed with bothcaution and interest. Regarding safety, rituximab can beassociated with infusion reactions, and, although such reac-tions were not described, 1 patient had amaurosis and 1 hadpanniculitis, both of which were unclearly associated with themedication. The 4 individuals treated by Zaja and associateshad very diverse clinical pictures. Of the 3 with HCV-relatedcryoglobulinemic vasculitis, only 1 was previously treated withIFN� and ribavirin. One patient had a B cell non-Hodgkin’slymphoma, which is the primary indication for rituximab.Although a complete response of her hemolytic anemia andneutropenia was observed with treatment, these are not fea-tures of cryoglobulinemic vasculitis and were likely related tolymphoma. The 3 other patients, who had more characteristicmanifestations of cryoglobulinemic vasculitis, had a partialresponse.

Based upon this experience, it is difficult to draw anyconclusions regarding the safety or possible efficacy of ritux-imab in cryoglobulinemic vasculitis. However, the mechanismof action of rituximab does support the need for a standardizedstudy, which is currently being conducted at the NationalInstitutes of Health. Until such time when further informationbecomes available, management of cryoglobulinemic vasculitisshould continue to focus on identification of any underlyingdisease processes and, in the setting of HCV infection, provid-ing treatment when possible with IFN� and ribavirin.

Carol A. Langford, MD, MHSNational Institute of Allergy and Infectious DiseasesNational Institutes of HealthBethesda, MD

1. Agnello V, Chung RT, Kaplan LM. A role for hepatitis C virusinfection in type II cryoglobulinemia. N Engl J Med 1992;327:1490–5.

2. Poynard T, Marcellin P, Lee SS, Niederau C, Minuk GS, Ideo G, etal, International Hepatitis Interventional Therapy Group (IHIT).Randomised trial of interferon alpha2b plus ribavirin for 48 weeksor for 24 weeks versus interferon alpha2b plus placebo for 48 weeksfor treatment of chronic infection with hepatitis C virus. Lancet1998;352:1426–32.

3. McHutchison JG, Gordon SC, Schiff ER, Shiffman ML, Lee WM,Rustgi VK, et al, Hepatitis Interventional Therapy Group. Inter-feron alfa-2b alone or in combination with ribavirin as initialtreatment for chronic hepatitis C. N Engl J Med 1998;339:1485–92.

4. Misiani R, Bellavita P, Fenili D, Vicari O, Marchesi D, Sironi PL,et al. Interferon alfa-2a therapy in cryoglobulinemia associated withhepatitis C virus. N Engl J Med 1994;330:751–6.

5. Dammacco F, Sansonno D, Han JH, Shyamala V, Cornacchiulo V,Iacobelli AR, et al. Natural interferon-alpha versus its combinationwith 6-methyl-prednisolone in the therapy of type II mixed cryo-globulinemia: a long-term, randomized, controlled study. Blood1994;84:3336–43.

DOI 10.1002/art.10374

Anti–tumor necrosis factor therapy and Listeriamonocytogenes infection: report of two cases

To the Editor:The tumor necrosis factor (TNF)–blocking agents in-

fliximab and etanercept have recently been approved by theUS Food and Drug Administration and the respective Euro-pean licensing institutions for treatment of rheumatoid arthri-tis (RA) refractory to other medical therapy (1–3). Tolerabilityand therapeutic efficacy of both infliximab and etanercept aregood, and the number of patients treated with these drugs isincreasing steadily (4,5). Animal studies, however, have shownthat TNF is not only a key mediator of inflammation but alsoplays an essential role in the defense against infection (6). Micetreated with TNF-blocking agents became exquisitely sensitiveto infections with pathogens such as cryptosporidia, mycobac-teria, fungi, Leishmania, and Listeria, which require an intactTh1 response for recovery (7).

Increased susceptibility to such infections may also berelevant to humans receiving TNF-blocking therapy. In De-cember 2000, the manufacturers of infliximab reported that 33cases of active tuberculosis were potentially associated with useof this anti-TNF antibody. Moreover, 2 patients with Crohn’sdisease developed listeriosis and invasive pulmonary aspergil-losis, respectively, while receiving infliximab (8,9).

We now report 2 cases of Listeria monocytogenesinfection, 1 of which was fatal, in 2 patients who were receivinginfliximab together with other immunosuppressive disease-modifying antirheumatic drugs (DMARDs). Patient 1 was a60-year-old woman with a history of severe RA since 1984. Hertherapy included methotrexate, cyclosporin A, and repeated

LETTERS 2255

prednisolone pulses because of ongoing, highly active arthritis.In April 2000, she began receiving infliximab at a dosage of 10mg/kg every 4 weeks. RA activity was well controlled with thistherapy until September 2000 (14 days after receiving the sixthinfusion of infliximab), when she was admitted to the hospitalbecause of anemia, fever, and abdominal pain. A gastric ulcer,without evidence of acute hemorrhage, and acute cholecystitiswere diagnosed, together with a pulmonary infiltrate. Labora-tory tests revealed anemia and the following values: hemoglo-bin 6.2 gm/dl, white blood cell count (WBC) 4.2/nl with a leftshift in the differential, platelet count 70/nl, lactate dehydro-genase (LDH) 552 units/liter, aspartate aminotransferase(AST) 82 units/liter, alanine aminotransferase (ALT) 49 units/liter, creatinine 1.9 mg/dl, and C-reactive protein 254 mg/liter.

Cholecystectomy was performed, and the patient wasimmediately given antibiotic therapy with cefriaxone, metroni-dazole, and fluconazole. After surgery, she did not regainconsciousness appropriately and continued to require respira-tory support despite discontinuation of all sedative drugs. Theleukocyte count increased to 11.5/nl, and C-reactive proteindecreased slightly to 163 mg/dl. A cerebral computed tomo-graphy (CT) scan was performed and showed severe brainedema with small spots of subarachnoidal hemorrhage, whichwas initially interpreted as acute sinus vein thrombosis. Sub-sequently, the patient developed multi-organ failure and brain-stem herniation due to the massive brain edema and died 2days later, despite maximum supportive care. Unexpectedly,both a swab culture from the gallbladder obtained duringsurgery and a blood culture obtained 1 day later grew Lmonocytogenes.

Patient 2 was a 62-year-old woman with a diagnosis ofRA since 1992. Until July 2000, her therapy included metho-trexate together with cyclosporin A. Because of highly activearthritis, cyclosporin A was switched to infliximab, 200 mgintravenously, which was planned to be given at weeks 0, 2, 6,and 10 and at 8-week intervals thereafter. The arthritis im-proved after the first dose of infliximab. However, 14 days afterthe second dose, the patient developed cholecystitis and wasadmitted to the surgical department for cholecystectomy.

After surgery, she developed signs indicating an intra-cerebral process, including hemiparesis, aphasia, and fever. Acerebral CT scan showed a mass lesion (4 cm in diameter) inthe left frontal lobe, with circular contrast enhancement.Laboratory tests showed the following values: WBC 13.3/nl,C-reactive protein 126 mg/liter, AST 85 units/liter, ALT 48units/liter, and LDH 516 units/liter. Two days later, bloodcultures turned positive for L monocytogenes, and therapy withampicillin and gentamicin was started. Fever and the elevatedparameters of inflammation responded adequately, and it wasdecided that neurosurgical intervention would not be helpful.Antibiotic therapy was continued for a total of 18 weeks.Improvement of the neurologic symptoms was slow; however,repeated cerebral CT scans 5 months after the initial diagnosisrevealed a marked reduction in the size of the brain lesion,with no perifocal contrast enhancement.

The common features in these 2 cases of systemiclisteriosis are striking and include cholecystitis and involve-ment of the central nervous system (CNS), with meningoen-cephalitis and brain abscess, respectively. Several cases oflocalized Listeria infection, the majority in the form of mon-arthritis, have been reported in patients receiving immunosup-

pressive therapy, usually methotrexate (for review, see ref. 10).Pregnant women, patients receiving chronic immunosuppres-sive therapy, or organ transplant recipients (i.e., patients witha more or less pronounced T cell defect) are known to be atrisk for listeriosis. Both of our patients were severely immu-nocompromised, because they had received immunosuppres-sive disease-modifying therapy with methotrexate, cyclosporinA, and corticosteroids for an extended period of time beforeTNF-blocking therapy with infliximab was initiated. Bothpatients subsequently developed cholecystitis, bacteremia, andCNS infection from L monocytogenes. It should be noted thatneither patient had a major infection during long-term therapywith conventional immunosuppressive DMARDs (before in-fliximab treatment was started), and Listeria infections devel-oped in both patients shortly after initiation of TNF-blockingtherapy. Therefore, it can be hypothesized that the newlyadministered anti-TNF antibody therapy blocked the criticalstep required for appropriate defense against L monocytogenesinfection, resulting in systemic infection.

Increased susceptibility to Listeria in humans mighthave been predicted from in vivo experiments using infectiousagents in animals pretreated with TNF-blocking drugs. In theseexperiments, immunity against Listeria was shown to dependon appropriate secretion of TNF (7,11), most likely because inthis particular infection, TNF is essential for macrophageactivation (12,13).

The occurrence of severe infections associated withTNF-blocking drugs does not necessarily preclude their use,because these agents are clearly beneficial for patients withchronic inflammatory disease refractory to other therapies.Rather, we suggest routinely advising patients receiving TNF-blocking agents not to eat soft cheese or other nonpasteurizeddairy products, which are known to harbor Listeria (14). Allphysicians treating patients who receive TNF-blocking drugsshould be highly suspicious about atypical pathogens, includingListeria, once signs of infection occur in their patients. Antibi-otic susceptibility of Listeria is restricted to ampicillin, carba-penems, aminoglycosides, and trimethoprim-sulfamethoxazole.However, these agents are not used as first-line empirictherapy for fever in the immunocompromised host. Therefore,infection with Listeria must be aggressively sought and ruledout in such cases. Empiric antibiotic coverage for Listeria maybe appropriate in high-risk cases and should be mandatory ifsigns of CNS involvement are present.

T. Gluck, MDH.-J. Linde, MDJ. Scholmerich, MDU. Muller-Ladner, MDUniversity of RegensburgRegensburg, GermanyC. Fiehn, MDUniversity of HeidelbergHeidelberg, GermanyP. Bohland, MDDiakoniekrankenhaus MannheimMannheim, Germany

1. Maini R, St Clair EW, Breedveld F, Furst D, Kalden J, WeismanM, et al for the ATTRACT Study Group. Infliximab (chimericanti-tumour necrosis factor alpha monoclonal antibody) versus

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placebo in rheumatoid arthritis patients receiving concomitantmethotrexate: a randomised phase III trial. Lancet 1999;354:1932–9.

2. Weinblatt ME, Kremer JM, Bankhurst AD, Bulpitt KJ, Fleisch-mann RM, Fox RI, et al. A trial of etanercept, a recombinanttumor necrosis factor receptor: Fc fusion protein, in patients withrheumatoid arthritis receiving methotrexate. N Engl J Med 1999;340:253–9.

3. Feldman N, Maini RN. Anti-TNF therapy of rheumatoid arthritis:what have we learned? Annu Rev Immunol 2001;19:193–6.

4. Kremer JM. Combination therapy with biologic agents in rheuma-toid arthritis: perils and promise. Arthritis Rheum 1998;41:1548–51.

5. Markham A, Lamb HM. Infliximab: a review of its use in themanagement of rheumatoid arthritis. Drugs 2000;59:1341–59.

6. Echtenacher B, Falk W, Mannel DN, Krammer PH. Requirementof endogenous tumor necrosis factor/cachectin for recovery fromexperimental peritonitis. J Immunol 1990;145:3762–6.

7. Nakane A, Minagawa T, Kato K. Endogenous tumor necrosisfactor (cachectin) is essential to host resistance against Listeriamonocytogenes infection. Infect Immun 1988;56:2563–9.

8. Morelli J, Wilson FA. Does administration of infliximab increasesusceptibility to listeriosis? Am J Gastroenterol 2000;95:841–2.

9. Warris A, Bjorneklett A, Gaustad P. Invasive pulmonary aspergil-losis associated with infliximab therapy. N Engl J Med 2001;344:1099–100.

10. Polnau U, Braun MG, van der Boom H, Becker-Capeller D.Listerienarthritis bei chronischer polyarthritis unter low-dose-prednisolon- und methotrexat-therapie. Z Rheumatol 2001;60:41–6.

11. Nishikawa S, Miura T, Sasaki S, Nakane A. The protective role ofendogenous cytokines in host resistance against an intragastricinfection with Listeria monocytogenes in mice. FEMS ImmunolMed Microbiol 1996;31:291–8.

12. Roll JT, Young KM, Kurtz RS, Czuprynski CJ. Human rTNFalpha augments anti-bacterial resistance in mice: potentiation ofits effect by recombinant human rIL-1 alpha. Immunology 1990;69:316–22.

13. Kato K, Nakane A, Minagawa T, Kasai N, Yamamoto K, Sato N,et al. Human tumor necrosis factor increases the resistance againstListeria infection in mice. Med Microbiol Immunol 1989;178:337–46.

14. Rudol M, Scherer S. High incidence of Listeria monocytogenes inEuropean red smear cheese. Int J Food Microbiol 2001;63:91–8.

DOI 10.1002/art.10371

Reply

To the Editor:Dr. Gluck and colleagues should be commended for

their careful report of 2 cases of L monocytogenes infection inpatients with RA receiving infliximab. As use of new biotech-nology treatments expands, the medical community is likely tosee other reports of this kind (1).

The fact that their letter is based on an editorialauthored and published in 1998 (2) speaks to the need for acentralized collection system for reporting toxicities associatedwith the use of these new agents. One of the challenges facedby clinicians using new drugs is to increase awareness oftoxicity issues that inevitably emerge with expanded clinicaluse, while seeking to develop creative strategies to avoid thesenewly reported untoward events. Recent directives to performtargeted tuberculin skin testing in patients at risk (3) have thusbeen applied to the use of infliximab. The authors’ empiric

suggestion about counseling patients receiving TNF inhibitorsto limit potential exposure to Listeria by avoiding nonpasteur-ized dairy products makes some sense in this regard.

It is also possible that long-term intermittent use oftrimethoprim-sulfamethoxazole (TMP-SMX), often used forprophylaxis of Pneumocystis carinii pneumonia (4), might havethe additional benefit of avoidance of Listeria infections. It isunlikely that the effects of TMP-SMX would result in signifi-cant toxicity because this agent is now widely used withmethotrexate without additional toxicity when treating patientswith Wegener’s granulomatosis (5). Long-term observations inpatients receiving TNF inhibitors and TMP-SMX would beneeded in order to provide insight about whether this strategyis safe and effective. Because of the relative infrequency ofListeria infections, very large numbers of patients, as can befound only in large clinical data bases, will be needed toprovide these answers.

In the meantime, clinicians using biotechnology agentsmust remain vigilant for reports of opportunistic infections, asdescribed in the letter by Gluck et al, while being willing toincorporate new and creative strategies, when appropriate, toavoid them. The inhibition of TNF has resulted in significantgains in the fight against RA, and continued, expanded use ofthese agents is both likely and appropriate.

Joel M. Kremer, MDThe Center for RheumatologyAlbany, NY

1. Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J,Schwieterman WD, et al. Tuberculosis associated with infliximab, atumor necrosis factor alpha-neutralizing agent. N Engl J Med2001;345:1098–104.

2. Kremer JM. Combination therapy with biologic agents in rheuma-toid arthritis: perils and promise. Arthritis Rheum 1998;41:1548–51.

3. Cohn DL, O’Brien RJ. Targeted tuberculin testing and treatment oflatent tuberculosis infection. Am J Resp Crit Care Med 2000;161:S221–S247.

4. Ognibene FP, Shelhamer JH, Hoffman GS, Kerr GS, Reda D,Fauci AS, et al. Pneumocystis carinii pneumonia: a major compli-cation of immunosuppressive therapy in patients with Wegener’sgranulomatosis. Am J Respir Crit Care Med 1995;151:795–9.

5. Hoffman GS. Treatment of Wegener’s granulomatosis: time tochange the standard of care? Arthritis Rheum 1997;40:2099–104.

DOI 10.1002/art.10348

Bullous skin lesions following infliximab infusion in apatient with rheumatoid arthritis

To the Editor:Infliximab, a chimeric monoclonal antibody to tumor

necrosis factor � (TNF�), is a treatment option for patientswith rheumatoid arthritis (RA) (1). Infliximab has been asso-ciated with a variety of side effects, including hypersensitivityreactions, serum sickness–like reactions, and the developmentof a lupus-like syndrome with autoantibodies. However, to ourknowledge, development of bullous skin lesions in patientstreated with infliximab has not been reported.

Recently, a 72-year-old white man with RA presentedto our emergency department with severe pain and blisteringskin lesions. These symptoms occurred 1 day after the patientreceived his fourth dose of infliximab for the treatment of

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severe RA. Physical examination revealed huge (10–15 cm)flaccid bullae over the left elbow and right thigh that containedstraw-colored fluid (Figure 1).

Serologic testing revealed positive (2.0 units) antinu-clear antibodies (ANA) (�1.0 unit is considered negative), butantibodies to Sm, Ro, La, Jo-1, Scl-70, RNP, and double-stranded DNA (dsDNA) were not detected. The total comple-ment level was reduced (15 units/ml, normal range 30–75), aswas the level of C3 (54 mg/dl, normal range 75–175). Humanantichimeric antibodies (Prometheus Laboratories, San Diego,CA) were positive (2.57 �g/ml, reference �1.69), and chimericanti-TNF antibodies were also positive (2.25 �g/ml, reference�1.4).

Although the bullous skin lesions had some features incommon with pemphigus vulgaris and bullous pemphigoid,light microscopy and direct immunofluorescence of the skinbiopsy specimens revealed features that were not typical ofeither (e.g., discontinuous granular IgM deposition in thebasement membrane zone was seen).

Based on the temporal relationship between the inflix-imab infusion and development of the bullae, an infliximab-induced bullous skin reaction was diagnosed, and treatmentwas initiated with prednisone, 60 mg/day, with tapering to thepatient’s baseline prednisone dose over a 2-week period. Nofurther bullae developed after initiation of higher-dose pred-nisone, and the patient recovered completely.

The US Food and Drug Administration recently re-ported on the safety of TNF� antagonists (2). Infliximab rarelyhas been associated with cases of demyelinating disease,seizures, and intestinal perforation. Granulomatous infectionssuch as tuberculosis (3) and histoplasmosis have been reportedmore frequently; other reported infections include listeriosisand Pneumocystis carinii pneumonia.

Cutaneous adverse events have included rash, edema,urticaria, and rare cases of lupus-like illnesses and rashescompatible with subacute cutaneous and discoid lupus, associ-ated with increases in autoantibody titers (4). Low titers ofautoantibodies developed in �10% of patients, and drug-induced lupus developed in �1% in a long-term safety evalu-ation of infliximab (4). Antibodies to dsDNA of the IgM classare induced by infliximab in 5–7% of patients (5).

The safety of and adverse events associated withinfliximab have been reviewed by combining data from 453patients in 9 clinical trials (6). Human antichimeric antibodies(HACA) developed in 13% of patients with Crohn’s disease.Of note, patients receiving immunosuppressive treatment withmethotrexate, azathioprine, or 6-mercaptopurine were lesslikely to develop HACA responses. Infusion reactions werepositively correlated with the presence of HACA (6).

This report does not definitely show that infliximab wasthe cause of the patient’s bullous skin eruption. However, theclose temporal relationship between the infusion and theobserved reaction does signal a potential causal association.Bullous skin lesions have been described in patients with lupus,but the immunofluorescence studies in this case were nottypical of lupus (7). Our patient had developed positive ANAand HACA, but whether these played any role in the patho-genesis of the skin eruption is speculative. In addition, whilethe patient was receiving low-dose prednisone and hydroxy-chloroquine, he was not receiving methotrexate because ofhepatotoxicity caused by this drug. Lack of use of a potentimmunosuppressive medication in conjunction with infliximabmay have been a predisposing factor in this case.

Continued vigilance for similar adverse reactions isindicated. Whether concomitant therapy with agents such asmethotrexate must always be prescribed with infliximab is notknown, but it would seem prudent to pursue this therapywhenever possible and indicated.

Peter D. Kent, MDJohn M. Davis III, MDMark D. P. Davis, MDEric L. Matteson, MD, MPHMayo ClinicRochester, MN

1. Taylor PC. Anti-tumor necrosis factor therapies. Curr Opin Rheu-matol 2001;13:164–9.

2. Arthritis Advisory Committee. Safety update on the TNF-alphaantagonists. Open session of the Food and Drug AdministrationCenter for Biologics Evaluation and Research; 2001 Aug 17;Gaithersburg (MD). Online at http:www.fda.gov/ohrms/dockets/ac/01/transcripts/3779t2_01.pdf.

3. Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J,Schwieterman WD, et al. Tuberculosis associated with infliximab, atumor necrosis factor alpha-neutralizing agent. N Engl J Med2001;345:1098–104.

4. Shaible TF. Long-term safety of infliximab. Can J Gastroenterol2000;14 Suppl C:29C–32C.

5. Charles PJ, Smeenk RJ, De Jong J, Feldmann M, Maini RN.Assessment of antibodies to double-stranded DNA induced inrheumatoid arthritis patients following treatment with infliximab, amonoclonal antibody to tumor necrosis factor alpha: findings inopen-label and randomized placebo-controlled trials. ArthritisRheum 2000;43:2383–90.

6. Hanauer SB. Safety of infliximab in clinical trials. Aliment Phar-macol Ther 1999;13 Suppl 4:16–22.

7. Chan LS, Lapiere J, Chen M, Traczyk T, Mancini AJ, Paller AS, etal. Bullous systemic lupus erythematosus with autoantibodies rec-ognizing multiple skin basement membrane components, bullouspemphigoid antigen 1, laminin-5, laminin-6, and type VII collagen.Arch Dermatol 1999;135:569–73.

Figure 1. Large bulla on the right medial thigh, containing straw-colored fluid, with surrounding erythema.

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DOI 10.1002/art.10353

Reply

To the Editor:Infliximab-induced ANA and anti-dsDNA antibodies

of the IgM class, usually in low titer, are only rarely associatedwith a lupus-like illness (1). For example, from among a groupof 156 RA patients exposed to infliximab in clinical trials, wedocumented 1 who developed fever, a nonspecific rash, andpleuropericarditis and who, interestingly enough, developedboth IgG and IgM anti-dsDNA antibodies in high titer.Another patient, who developed a lupus-like, erythematousrash, was ANA positive but had no anti-dsDNA antibodies (2).

The striking reduction in complement in the patientwith the bullous eruption described by Kent et al wouldimplicate immune complexes in the pathogenesis of thoselesions. Whether the nuclear antigens complexed to ANAformed pathogenic immune complexes, as they might do inlupus, cannot be ascertained simply by observing an associationwith the occurrence of ANA. ANA may appear and disappearduring the course of infliximab therapy (1). The possibilitymust be considered that the pathogenic immune complexes inthis patient were composed of infliximab and anti-infliximabantibodies (also detected in the blood of this patient). Thispossibility is further supported by the close temporal relation-ship between the onset of rash and the fourth infusion ofinfliximab.

The speculation that coadministration of methotrexatemight have prevented the adverse reaction is supported by theobservation that the immunogenicity of infliximab is signifi-cantly reduced by combining methotrexate and infliximab (3).In contrast, the incidence of anti-dsDNA induction was notinhibited by methotrexate (1). The hypothesis that anti-infliximab antibodies played a part should be further investi-gated by seeking evidence of anti-infliximab antibodies inblood and deposition of infliximab in skin lesions by probingwith an antiidiotype antibody.

Ravinder N. Maini, MBBChir, FRCP, FMedSciPeter Charles, FIBMSKennedy Institute of Rheumatology DivisionImperial College of Science, Technology and MedicineLondon, UK

1. Charles PJ, Smeenk RJT, De Jong J, Feldmann M, Maini RN.Assessment of antibodies to double-stranded DNA induced inrheumatoid arthritis patients following treatment with infliximab, amonoclonal antibody to tumor necrosis factor �: findings in open-label and randomized placebo-controlled trials. Arthritis Rheum2000;43:2383–90.

2. Maini RN, St Clair EW, Breedveld F, Furst D, Kalden J, WeismanM, et al, ATTRACT Study Group. Infliximab (chimeric anti-tumour necrosis factor � monoclonal antibody) versus placebo inrheumatoid arthritis patients receiving concomitant methotrexate: arandomised phase III trial. Lancet 1999;354:1932–9.

3. Maini RN, Breedveld FC, Kalden JR, Smolen JS, Davis D, Mac-farlane JD, et al. Therapeutic efficacy of multiple intravenousinfusions of anti-tumor necrosis factor � monoclonal antibodycombined with low-dose weekly methotrexate in rheumatoid arthri-tis. Arthritis Rheum 1998;41:1552–63.

DOI 10.1002/art.10377

Synovial lymphocyte responses to microbiologicantigen stimulation indicate the etiology ofundifferentiated and reactive arthritis, and possibly ofrheumatoid arthritis: comment on the article bySchnarr et al

To the Editor:In the November 2001 issue of Arthritis & Rheumatism,

Schnarr et al (1) reported finding chlamydial DNA in thesynovial fluid of 9 of 52 patients with undifferentiated oligo-arthritis (UOA). They discussed the difficulty of attributing theetiology of UOA to the synovial DNA, commenting that,“Additional evidence supporting a bacterial etiology, suchas . . . bacteria-specific T cell responses, will be helpful.”

In 1980 (2), synovial mononuclear cells in Reiter’ssyndrome were found to respond to ureaplasmal and chlamyd-ial antigens. In 1985 (3), synovial lymphocytes from 8 patientswith enteric reactive arthritis were shown to respond to entericantigens, and in 12 patients with sexually transmitted reactivearthritis, they responded to either chlamydial or ureaplasmalantigens. In contrast, synovial lymphocytes from 14 patientswith rheumatoid arthritis showed no responses to these anti-gens. In addition, in all cases, peripheral blood lymphocytesusually showed minimal or no responses. Between 1989 and1994, these observations were confirmed in several countries(4–7).

In 1984 (8), synovial responses to chlamydia/ureaplasma and enteric antigens were studied in 31 patientswith unexplained inflammatory arthritis confined to the kneejoints, and comparisons between responses in rheumatoidarthritis and Reiter’s syndrome were made. In 10 of the 31cases of UOA, the synovial lymphocytes responded as if thepatients had Reiter’s syndrome.

Because synovial lymphocyte responses to microbio-logic antigen stimulation indicate the causes of undifferenti-ated and reactive arthritis, such reactivity can give significanceto the presence of microbiologic DNA or antigen in thesynovia of patients with arthritis of unknown etiology. Themere presence of these substances in the joint is not proof ofpathogenicity, but an association between a specific immunereaction within the joint and the same agent provides strongincriminating evidence for specific immunopathology. Summa-ries of the accumulated data from a 12-year study of synoviallymphocyte responses in 360 patients have provided docu-mented support for these viewpoints and have extended theobservations to the probable microbiologic etiology of rheu-matoid arthritis (9–12).

Denys K. Ford, MDVancouver, BC, Canada

1. Schnarr S, Putschky N, Jendro MC, Zeidler H, Hammer M,Kuipers JG, et al. Chlamydia and Borrelia DNA in synovial fluidof patients with early undifferentiated oligoarthritis: results of aprospective study. Arthritis Rheum 2001;44:2679–85.

2. Ford DK, da Roza DM, Shah P, Wenman WM. Cell-mediatedimmune responses of synovial mononuclear cells in Reiter’s syn-drome against ureaplasmal and chlamydial antigens. J Rheumatol1980;7:751–5.

3. Ford DK, da Roza DM, Schulzer M. Lymphocytes from the site of

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disease but not blood lymphocytes indicate the cause of arthritis.Ann Rheum Dis 1985;44:701–10.

4. Hermann E, Fleischer B, Mayet WJ, Poralla T, Meyer zumBuschenfelde KH. Response of synovial fluid T cell clones toYersinia enterocolitica in patients with reactive Yersinia arthritis.Clin Exp Immunol 1989;75:365–70.

5. Gaston JS, Life PF, Granfors K, Merilahti-Palo R, Bailey L,Consalvey S, et al. Synovial T lymphocyte recognition of organismsthat trigger reactive arthritis. Clin Exp Immunol 1989;76:348–53.

6. Sieper J, Kingsley G, Palacios-Boix A, Pitzalis C, Treharne J,Hughes R, et al. Synovial T lymphocyte-specific response toChlamydia trachomatis in Reiter’s disease. Arthritis Rheum 1991;34:588–98.

7. Horowitz H, Horowitz J, Taylor-Robinson D, Sukenik S, Apte RN,Bar-David J, et al. Ureaplasma urealyticum in Reiter’s syndrome.J Rheumatol 1994;21:877–82.

8. Ford DK, da Roza DM, Ward RH. Arthritis confined to kneejoints: synovial lymphocyte responses to microbial antigens corre-late with distribution of HLA. Arthritis Rheum 1984;27:1157–64.

9. Ford DK. Synovial lymphocytes can indicate specific microbiologiccauses of rheumatoid arthritis. Arthritis Rheum 1993;36:1350–2.

10. Ford DK, Schulzer M. Lymphocytes from the site of disease arefunctionally different from peripheral blood lymphocytes and maydemonstrate etiologically related antigen specificity. Immunol Lett1994;42:179–83.

11. Ford DK, Tingle A. Lymphocytes from site of disease indicateprobable microbiological etiology of “infective-immune” diseasessuch as rheumatoid arthritis. Infect Agents Dis 1996;5:223–30.

12. Ford DK. Understanding rheumatoid arthritis. J Rheumatol 1997;24:1464–6.

DOI 10.1002/art.10372

Reply

To the Editor:We thank Dr. Ford for his interest in our article. We

consider the presence of chlamydial DNA in the synovium tobe a valuable hint concerning the etiology of undifferentiatedarthritis, but we agree that this does not unequivocally provethe causative role of these bacteria in individual cases. Severalapproaches are possible to prove causality: first, demonstrationof viable and metabolically active bacteria at the site ofinflammation, as has been convincingly shown by Gerard et al(1); second, demonstration of antigen-specific host response tothe persisting bacteria; and third (the ultimate proof), cure ofthe arthritis by eradication of the bacteria.

With respect to these different approaches, we do notconsider results of synovial lymphocyte proliferation assays tobe sufficient proof of causality. In contrast to the literaturecited by Dr. Ford, Fendler et al describe low specificity ofsynovial lymphocyte proliferation assays due to cross-reactivityor other antigen-independent factors, such as the ratio of Tcells to macrophages or the grade of T cell activation (2).Consequently, they argue against use of proliferation assays fordiagnostic purposes. Wilkinson et al did not even find acorrelation between the presence of intraarticular chlamydialDNA and the antichlamydial synovial T cell response in 4patients with sexually acquired reactive arthritis and in 31patients with UOA (3). In our opinion, reduced T cell reactiv-ity in the presence of viable chlamydiae could be explained bythe phenomenon of chlamydia-infected macrophages inducingT cell apoptosis (4).

In summary, only a combination of techniques such aspolymerase chain reaction (PCR), reverse transcriptase PCR,specific readout systems for host response (e.g., flow

cytometry–based analysis of antigen-specific T cell responses),and, of course, ultimately the cure of arthritis by eradication ofthe persistent synovial infection can unequivocally prove thepathogenetic significance of intraarticular organisms.

Sebastian Schnarr, MDMichael C. Jendro, MDHenning Zeidler, MDJens G. Kuipers, MDHannover Medical SchoolHannover, Germany

1. Gerard HC, Branigan PJ, Schumacher HR Jr, Hudson AP. SynovialChlamydia trachomatis in patients with reactive arthritis/Reiter’ssyndrome are viable but show aberrant gene expression. J Rheu-matol 1998;25:724–42.

2. Fendler C, Wu P, Eggens U, Laitko S, Sorensen H, Diestler A, et al.Longitudinal investigation of bacterium-specific synovial prolifera-tion in reactive arthritis and Lyme arthritis. Br J Rheumatol1998;37:784–8.

3. Wilkinson NZ, Kingsley GH, Sieper J, Braun J, Ward ME. Lack ofcorrelation between the detection of Chlamydia trachomatis DNAin synovial fluid from patients with a range of rheumatic diseasesand the presence of an antichlamydial immune response. ArthritisRheum 1998;41:845–54.

4. Jendro MC, Deutsch T, Korber B, Kohler L, Kuipers JG, Krausse-Opatz B, et al. Infection of human monocyte-derived macrophageswith Chlamydia trachomatis induces apoptosis of T cells: a potentialmechanism for persistent infection. Infect Immun 2000;68:6704–11.

DOI 10.1002/art.10375

Criteria for TUNEL labeling in determining apoptosisin human osteoarthritis cartilage: comment on thearticle by Aigner et al

To the Editor:We read with great interest the article by Aigner and

colleagues (1) reporting apoptosis limited to the deep zone inosteoarthritic (OA) human cartilage. This result contrasts withthose of most of the published studies (2–7). The TUNELtechnique has limitations: it gives false-positive results, and thereaction may appear even in necrotic chondrocytes, as previ-ously reported (8,9). Although TUNEL is still a useful method,results must be interpreted very cautiously and require addi-tional support, including detection of other molecules of theapoptotic cascade, such as Fas and caspase 3 (the 17–22-kdfraction), and even the “ladder” DNA fragmentation sign.

The use of human cartilage also imposes certain re-strictions, because it is not always possible to ascertain thecomplete clinical history of the patient. Some patients mayhave received drugs that can substantially modify the results, asin the case of locally infiltrated glucocorticoids, which indeedstimulate apoptosis (10). The type of OA must also beconsidered. OA in elderly patients differs from traumatic OA,which can induce severe cartilage breakdown. The samplingmethod is another key factor. In a previous study (7), wedemonstrated that cartilage obtained by arthroscopy displayedmore TUNEL labeling compared with cartilage obtained byarthroplasty, suggesting that the procedure used for removingtissue might influence the results. In that study, very smalltissue samples were manipulated arthroscopically before fixa-

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tion. In contrast, using arthroplasty, entire condyles wereremoved and immediately fixed before the cartilage was di-vided into small portions to assess the TUNEL reaction. All ofthese factors must be evaluated when using human cartilage.

The occurrence of apoptosis in human OA cartilagehas been further supported by reports of Fas/FasL expression(3) and detection of the DNA ladder fragmentation sign (5).These observations strongly indicate that apoptosis might notbe as limited as suggested by Aigner et al. In view of thecurrent knowledge, it seems rather risky to assess apoptosisonly by TUNEL-labeled apoptotic bodies, because theTUNEL technique is associated with serious pitfalls, and theapoptotic bodies could easily be missed, as has been reported(9).

Using different experimental models and techniques inaddition to TUNEL, several authors (including our group)have described apoptosis within the entire depth of the carti-lage. As already reported, our rat OA model (11) showed thatboth the intensity and the arrangement of TUNEL nuclearlabeling varied according to both progression of damage (5–60days after surgery) and cartilage zone. In earlier stages ofdisease, the superficial chondrocytes displayed intense nuclearlabeling and changed morphologically from flattened torounded. These chondrocytes were not contracted, and theydid not display highly condensed chromatin. On the contrary,they showed an ultrastructural pattern characterized byrounded nuclei, a prominent rough endoplasmic reticulum,and frequent Golgi membranes (12). In contrast, chondrocytesin the middle and deep zones showed contraction and frag-mentation, and labeling was fuzzy or absent. In addition, whenwe compared nuclear TUNEL labeling of chondrocytes fromOA cartilage for all experiments, it was evident that thenumber of labeled nuclei and the intensity of labeling variedfrom abundant and intense (in the early stages) to scarce andfuzzy (in late stages).

These results, which are not possible to assess inhuman OA, made us assume that the intensity of TUNELlabeling is greater prior to the appearance of the presumedtypical morphologic pattern in apoptosis, and also that thelabeling seems to disappear according to the breakdown ofDNA. Therefore, combining research using human cartilagesamples with that involving an experimental model mightprovide a more integral view than that provided by a singleapproach. Confident use of TUNEL detection kits most cer-tainly requires adjustment of the limitations provided by themanufacturers and the use of accurate controls (1). However,it is important to consider that when the TUNEL technique isperformed outside of certain suggested parameters, false-negative results are possible.

Also worthy of discussion is the morphology of apo-ptosis in chondrocytes, which seems to differ from that de-scribed in lymphocytes. Regardless, in our experiments, themorphology in both human OA and the experimental OA inrats most certainly was not that of necrosis. Furthermore, someclustered chondrocytes displayed TUNEL labeling, and themorphology was different from the presumed morphology ofan apoptotic chondrocyte. It is possible that apoptotic death inchondrocytes might be diverse and different from that de-scribed in other cell types.

Regarding the article by Aigner et al, we are alsoconcerned because the authors credited our group with results

that are opposite to those that we reported (7). For instance,we described intense labeling in cartilage samples obtained byarthroscopy. In addition, we suggested that the difference inthe intensity of TUNEL labeling was apparently opposite to theapoptotic morphologic changes. It is highly unlikely that wewould establish a correlation between positive TUNEL label-ing and apoptotic morphology, because such a correlationwould probably be different from that expected, as docu-mented above. We believe that the authors might have inad-vertently misinterpreted our results while using them as amajor argument to sustain part of their hypothesis.

Cell death in OA cartilage is an intriguing subject thatis still not fully characterized and is worthy of clarification.More information regarding this issue might help shed light onthe pathogenesis of OA, which could in turn lead to develop-ment of new therapeutic strategies.

Juan B. Kouri, MD, PhDKarin Abbud-Lozoya, PhDCentro de Investigacion y de Estudios Avanzados

del Instituto Politecnico NacionalUniversidad Nacional Autonoma de MexicoMexico City, Mexico

1. Aigner T, Hemmel M, Neureiter D, Gebhard PM, Zeiler G,Kirchner T, et al. Apoptotic cell death is not a widespreadphenomenon in normal aging and osteoarthritis human articularknee cartilage: a study of proliferation, programmed cell death(apoptosis), and viability of chondrocytes in normal and osteoar-thritic human knee cartilage. Arthritis Rheum 2001;44:1304–12.

2. Blanco FJ, Guitian R, Vazquez-Martul E, de Toro FJ, Galdo F.Osteoarthritis chondrocytes die by apoptosis: a possible pathwayfor osteoarthritis pathology. Arthritis Rheum 1998;41:284–9.

3. Hashimoto S, Setareh M, Ochs RL, Lotz M. Fas/Fas ligandexpression and induction of apoptosis in chondrocytes. ArthritisRheum 1997;40:1749–55.

4. Hashimoto S, Ochs RL, Komiya S, Lotz M. Linkage of chondro-cyte apoptosis and cartilage degradation in human osteoarthritis.Arthritis Rheum 1998;41:1632–8.

5. Kim HA, Lee YJ, Seong SC, Choe KW, Song YW. Apoptoticchondrocyte death in human osteoarthritis. J Rheumatol 2000;27:455–62.

6. Kouri JB, Rosales-Encina JL, Chaudhuri PP, Luna J, Mena R.Apoptosis in human osteoarthritic cartilage: a microscopy report.Med Sci Res 1997;25:245–8.

7. Kouri JB, Aguilera JM, Reyes J, Abbud-Lozoya K, Gonzalez S.Apoptotic chondrocytes from osteoarthrotic human articular car-tilage and abnormal calcification of subchondral bone. J Rheuma-tol 2000;27:1005–19.

8. Gold R, Schmid M, Giegerich H, Breitschopf H, Hartung HP,Toyka KV, et al. Differentiation between cellular apoptosis andnecrosis by the combined use of in situ tailing and nick translationtechniques. Lab Invest 1994;71:219–25.

9. Walker JA, Quirke P. Viewing apoptosis through a ‘TUNEL’.J Pathol 2001;195:275–6.

10. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is asso-ciated with endogenous endonuclease activation. Nature 1980;284:555–6.

11. Abbud-Lozoya K, Kouri JB. A novel rat osteoarthrosis model toassess apoptosis and matrix degradation. Pathol Res Pract 2000;196:729–45.

12. Kouri-Flores JB, Abbud-Lozoya KA, Roja-Morales L. Kinetics ofthe ultrastructural changes from an osteoarthrosis rat model: awindow of comparison to the cellular mechanism of apoptosis inhuman chondrocytes. Ultrastruct Pathol 2002;26:33–40.

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DOI 10.1002/art.10346

Parvovirus B19 infection of cultured skin fibroblastsfrom systemic sclerosis patients: comment on thearticle by Ray et al

To the Editor:We read with interest the recent article by Ray and

coworkers reporting that human parvovirus B19 infection caninduce in vitro invasive properties in normal human synovialfibroblasts (1). Their study further supports previous observa-tions suggesting that B19 infection could be involved in thepathogenesis of rheumatoid arthritis and other rheumaticdisorders, including systemic sclerosis (SSc) (2–5). SSc is aconnective tissue disease of unknown etiology; its pathogenesisinvolves overproduction of collagen by altered fibroblasts andalterations of the microvasculature and immune system (6–7).Numerous genetic, environmental, and infectious agents havebeen proposed as possible etiologic factors (6–8).

We previously demonstrated the presence of parvovi-rus B19 infection in bone marrow and/or skin biopsy specimensfrom a significant number of unselected SSc patients (4–5).Bone marrow may represent a reservoir from which the viruscould spread to SSc target tissues (9). Given the role offibroblasts in scleroderma fibrotic lesions (6), we investigatedthe presence of parvovirus B19 DNA in cultured skin fibro-blasts from 8 unselected SSc patients (3 men, 5 women;mean � SD age 52 � 10 years, mean disease duration 10 � 9years). Patients were classified according to the AmericanCollege of Rheumatology (formerly the American Rheuma-tism Association) preliminary criteria (10) and were consecu-tively recruited after they had given informed consent to enterthe study.

Skin biopsy specimens obtained from the patient’sforearm were used for fibroblast and keratinocyte cultures.Skin samples were washed in a phosphate buffered saline(PBS) solution supplemented with antibiotics and cut into thinstrips, which were placed in petri dishes containing Dispase IIsolution (12.5 units/ml; Boehringer Mannheim, Indianapolis,IN) for 40–60 minutes. Sheets of epidermis were separatedfrom underlying dermis and incubated in a trypsin–EDTAsolution (0.25% trypsin/0.02% EDTA; Sigma-Aldrich, Milan,Italy) for 15 minutes to obtain a single-cell suspension ofkeratinocytes. Isolated dermis was cut into small pieces (2–3mm2), and fibroblasts were isolated by collagenase digestion(80 units/ml; Worthington Biochemical, Lakewood, NJ). Cellswere then seeded on 25-cm2 culture flasks containing a smallamount of culture medium (Dulbecco’s modified Eagle’s me-dium [DMEM]) supplemented with 10% fetal bovine serum(Sigma, St. Louis, MO), 4 mM L-glutamine (Sigma), andpenicillin/streptomycin ([100 units/ml]/[100 �g/ml]; Seromed,Berlin, Germany).

Keratinocyte cultures were obtained by using irradi-ated Swiss BALB/c 3T3 fibroblast feeder layers in culturemedium (DMEM/Ham’s F-12 [3:1]) with 10% FBS (Sigma), 4mM L-glutamine, penicillin/streptomycin (100 units/ml)/(100�g/ml), 0.4 mg/ml hydrocortisone succinate, 5 �g/ml insulin(Sigma), 5 �g/ml transferrin, 2 � 10�9M triiodo-L-thyronine,10�9M cholera toxin, and 24 �g/ml adenine.

After 6–7 days, preconfluent primary keratinocytes

and fibroblasts were dissociated into single cells by using a0.05% trypsin/0.02% EDTA PBS-buffered solution. Keratino-cyte cultures were first incubated with 0.02% EDTA solutionto remove the feeder layer. Human fibroblasts and keratino-cytes were expanded (split ratio 1:2; up to 6 passages whenpossible) and preserved in liquid nitrogen. For some samplesthe number of passages was limited because of bacterialcontamination. Because of the initial quality of tissue samples,keratinocyte isolation was unsuccessful in 3 cases.

Virologic studies included the detection of serumanti-B19 IgG and IgM antibodies using enzyme immunoassay(Biotrin, Dublin, Ireland) and B19 DNA in the serum, whole-skin biopsy, and cultured fibroblasts and keratinocytes bynested polymerase chain reaction (PCR) technique. In 5 of 8subjects, parvovirus B19 infection was also evaluated in bonemarrow biopsy samples. The target sequence for the amplifi-cation reaction was in the nonstructural region. The primers P1(1399–1422 nucleotides [nt]) and P6 (1682–1659 nt) were usedin the first reaction, whereas the primers P2 (1498–1525 nt)and P5 (1600–1576 nt) were employed as inner primers in thesecond reaction. The first and second reactions were per-formed for 35 cycles, each at 94°C for 45 seconds, 55°C for 60seconds, and 72°C for 90 seconds.

The main clinical, serologic, and virologic findings of 8SSc patients are summarized in Table 1. On the basis ofvirologic data, patients can be divided in 2 groups: 1) 4 subjectswith serum anti-B19 (IgG type), and parvovirus B19 infectionof the whole skin (in 3 of them B19 DNA was also found inbone marrow biopsy specimens) and 2) 4 B19-negative sub-jects.

The presence of B19 DNA was demonstrated in allpassages of fibroblast cultures in 4 of 4 patients with parvovirusB19 infection of the whole skin (Table 1). Comparable levels ofviral DNA, evaluated by semiquantitative PCR, were found inall passages of B19-positive fibroblast cultures. B19 DNA wasnever detected in the keratinocyte cultures. No significantclinical or serologic differences between B19-positive andB19-negative SSc patients were observed.

This preliminary study demonstrated that parvovirusB19 may infect skin fibroblasts of SSc patients. The pathologichallmark of SSc is fibrosis of the skin and various internalorgans (6–7). Scleroderma fibroblasts present an abnormallyactivated phenotype responsible for collagen overproduction(6); inflammatory cell-derived solution mediators might triggerthis fibroblast dysfunction. Alternatively, it could be a primaryevent caused by deep alterations in the regulatory pathwayscontrolling connective tissue gene expression. This lattermechanism is suggested by several studies showing that cul-tured scleroderma fibroblasts produce increased amounts oftype I collagen compared with fibroblasts from healthy subjects(6). Moreover, this hyperactivity is maintained for severalpassages in culture in the absence of potential extracellularactivating signals (6). The ability of parvovirus B19 to persis-tently infect scleroderma fibroblasts might be responsible forfibroblast alterations. This possible event is in keeping with thephenotype changes observed by Ray et al in normal humansynovial fibroblasts infected by parvovirus B19 in vitro (1).

SSc represents a spectrum of clinical and serologicvariants and is probably caused by a multifactorial process,including genetic and exogenous (toxic or infectious) factors

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(6–8). In this context, parvovirus B19 might be included amongthe potential causative agents of the disease.

Clodoveo Ferri, MDDilia Giuggioli, MDMarco Sebastiani, MDUniversity of PisaPisa, ItalySusi Panfilo, MDGiovanni Abatangelo, MDUniversity of PadovaPadova, ItalyKrystyna Zakrzewska, MDAlberta Azzi, MDUniversity of FlorenceFlorence, Italy

1. Ray NB, Nieva DRC, Seftor EA, Khalkhali-Ellis Z, NaidesSJ. Induction of an invasive phenotype by human parvovirus B19in normal human synovial fibroblasts. Arthritis Rheum 2001;44:1582–6.

2. Naides SJ. Rheumatic manifestations of parvovirus B19 infection.Rheum Dis Clin North Am 1998;24:375–401.

3. Kerr JR. Pathogenesis of human parvovirus B19 in rheumaticdisease. Ann Rheum Dis 2000;59:672–83.

4. Ferri C, Zakrzewska K, Longombardo G, Giuggioli D, Storino FA,Pasero G, et al. Parvovirus B19 infection of bone marrow insystemic sclerosis. Clin Exp Rehumatol 1999;17:718–20.

5. Ferri C, Zakrzewska K, Giuggioli D, Longombardo G, SebastianiM, Storino F, et al. Parvovirus B19 infection in the skin and bonemarrow of systemic sclerosis patients [abstract]. Arthritis Rheum2000;43 Suppl 9:315.

6. Jimenez SA, Hitraya E, Varga J. Pathogenesis of scleroderma:collagen [review]. Rheum Dis Clin North Am 1996;22:647–74.

7. White B. Immunopathogenesis of systemic sclerosis. Rheum DisClin North Am 1996;22:695–708.

8. Pandey JP, LeRoy EC. Human cytomegalovirus and the vascu-lopathies of autoimmune diseases (especially scleroderma), allo-graft rejection, and coronary restenosis. Arthritis Rheum 1998;41:10–5.

9. Cassinotti P, Burtonoboy G, Fopp M, Siegl G. Evidence forpersistence of human parvovirus B19 DNA in bone marrow. J MedVirol 1997;53:229–32.

10. Subcommittee for Scleroderma Criteria of the American Rheu-

matism Association Diagnostic and Therapeutic Criteria Commit-tee. Preliminary criteria for the classification of systemic sclerosis(scleroderma). Arthritis Rheum 1980;23:581–90.

DOI 10.1002/art.10352

Reply

To the Editor:The report by Ferri and colleagues offers a tantalizing

suggestion that parvovirus B19 plays a role in the pathogenesisof SSc. Using nested PCR methodology, the authors found B19DNA in cultured skin fibroblasts from 4 of 8 SSc patients, butnot in their serum. Although these results are promising, webelieve that it would be premature to conclude that parvovirusB19 is a causative agent in SSc, a caveat recognized by theauthors. Normal skin fibroblasts were not tested as a control.Soderlund et al demonstrated PCR positivity for B19 DNA inthe synovium of 13 (48%) of 27 healthy military recruitsundergoing arthroscopy for trauma (1). Like Ferri et al,Soderlund and colleagues reported serum anti-B19 IgG posi-tivity in all patients with B19 DNA–positive tests. It is possiblethat the parvovirus B19 detected in the cultured fibroblastsrepresents latent infection. Like many other DNA viruses,parvovirus B19 may prove to be capable of prolonged latency.The challenge in studying latent virus infection is in provingKoch’s postulates and demonstrating that the virus causesdisease rather than sojourns as an innocent passenger.

Confirming parvovirus B19 as an etiologic agent of SScwill require additional study. First, fibroblasts from a largernumber of patients with SSc will need to be tested for B19 inorder to strengthen the epidemiologic association between B19and disease. These patients should be compared with age- andsex-matched normal individuals recruited concurrently, to con-trol for geographic and temporal variations in community-acquired parvovirus B19 infection. In addition, fibroblastsfrom patients with skin disease other than SSc should be testedto eliminate the possibility that latent B19 is rendered detect-able in SSc patient skin fibroblasts due to nonspecific immunestimulation of viral replication. B19 in unstimulated normalskin would remain at background levels below the detection

Table 1. Demographic, clinical, serologic, and virologic findings in the SSc patients*

PatientAge/sex

Cutaneoussubset

Mainclinicalfeatures

Serumautoantibodies

Anti-B19antibodies

Parvovirus B19 DNA (PCR)

SerumBone

marrowWhole

skin

Culturedfibroblasts

(cultureno.)

Culturedkeratinocytes(culture no.)

1 42/F Diffuse L Anti–Scl-70 IgG� – � � � (3/3) � (2/2)2 59/F Limited E, U ACA IgG� – � � � (3/3) � (2/2)3 50/F Limited E, U ACA IgG� – � � � (6/6) ND4 56/M Diffuse L, H, E, U Anti–Scl-70, ANA IgG� – ND � � (6/6) ND5 70/M Limited L, H, E, U ACA – – ND – � (2/2) � (4/4)6 51/M Diffuse L, E, U ANA – – – – � (6/6) � (6/6)7 54/F Diffuse L, E, U ANA – – – – � (5/5) � (3/3)8 36/F Diffuse L, E, U Anti–Scl-70 – – ND – � (6/6) ND

* PCR � polymerase chain reaction; L � lung; ANA � antinuclear antibodies; E � esophagus; U � cutaneous ulcers; ACA � anticentromereantibodies; ND � not detected; H � heart involvement.

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threshold. Larger numbers of patients will also allow discern-ment of patterns of disease manifestation and serologic mark-ers uniquely associated with B19 infection.

Second, in situ hybridization of B19 DNA and immu-nocytochemical staining for B19 proteins and fibroblast phe-notype markers would allow confirmation that the B19 DNAdetected by the sensitive nested PCR is indeed in the culturedfibroblasts. Direct visualization and phenotyping of the in-fected cell would eliminate the possibility that the B19 signaldetected in the low-passage primary fibroblast cultures residesin a contaminating cell such as a blood-derived leukocyte.Similar visualization of B19 and phenotypic cell markers inintact skin biopsy specimens would offer histologic localizationof the infected cells. Parvovirus B19 has broad tissue tropismreflecting the broad distribution of its cell surface glycosphin-golipid receptors, a requirement for an etiologic agent of amultisystemic disease (2). The association would be furtherstrengthened by the presence of B19 in fibroblasts in SSc-involved internal organs but its absence in disease-free organs.

Third, a plausible and testable paradigm of B19 virus–cell interactions is required to explain disease induction. Ray etal demonstrated B19 induction of an invasive phenotype innormal human synovial fibroblasts, a finding of potentialsignificance in rheumatoid arthritis pathogenesis (3). Ferri etal suggested that parvovirus B19 induces a similar invasivephenotype in cutaneous fibroblasts in SSc, but this has notbeen directly demonstrated. The mechanism by which B19alters cellular function at the molecular level will need to be

described. Demonstrating parvovirus B19-induced up-regulation of collagen matrix deposition would support a rolefor B19 in SSc. Parvovirus B19 infection is widespread andcommon, while SSc remains relatively uncommon. Additionalhost and environmental factors that enable the hypothesizedB19 alterations would need to be identified.

Nancy B. Ray, PhDZhila Khalkhali-Ellis, PhDDaniel R. C. Nieva, BAElisabeth A. Seftor, BSUniversity of IowaIowa City, IAStanley J. Naides, MDMilton S. Hershey Medical Center

and Pennsylvania State UniversityHershey, PA

1. Soderlund M, von Essen R, Haapasaari J, Kiistala U, Kiviluoto O,Hedman K. Persistence of parvovirus B19 DNA in synovialmembranes of young patients with and without chronic arthro-pathy. Lancet 1997;349:1063–5.

2. Cooling LL, Koerner TA, Naides SJ. Multiple glycosphingolipidsdetermine the tissue tropism of parvovirus B19. J Infect Dis1995;172:1198–205.

3. Ray NB, Nieva DR, Seftor EA, Khalkhali-Ellis Z, Naides SJ. In-duction of an invasive phenotype by human parvovirus B19 innormal human synovial fibroblasts. Arthritis Rheum2001;44:1582–6.

DOI 10.1002/art.10475

On my knees (Instructions to Authors)

Oh Doctor, please don’t end this Trial,Be patient with your Patient’s Methods,Collect my Results, but withhold your Conclusions,Consider me not statistically yet significant,Don’t Table me as data figures,Figure me beyond the Figures,I am not figurative, I’m bone and flesh, soDon’t Abstract me, instead, instruct me, henceHave a Discussion on me but never a SummaryAnd, yes, please keep being my Reference.

Softly release my stiffness,Stiffly ease my softness,I’ll follow your steps, please try to healAnd watch my step, as well as heel,And guide me up the hillToward the right direction, since

The more affected are my knees,The more they need affection.

Dan Caspi, MDTel Aviv Medical CenterTel Aviv, Israel

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