Alfred D. Steinberg

Methylprednisolone and Cyclophosphamide, Alone or in Combination, in Patients with Lupus Nephritis: A Randomized, Controlled Trial

Therapy for patients with life-threatening systemic lupus erythematosus has included high doses of corticosteroids and cytotoxic or cytostatic drugs [1-20]. Cyclophosphamide, given in intermittent intravenous boluses, has been widely used to treat renal [1-68, 15, 21] and central nervous system disease [2, 3, 6, 7, 19-21], but this therapy is sometimes withheld in the hope that disease might be controlled with corticosteroids or other immunosuppressive drugs. Moreover, some patients do not respond adequately to therapy with intermittent boluses of cyclophosphamide, and these patients might benefit from more intensive therapy. In a previous study [3], monthly administration of methylprednisolone (1.0 g/m2 body surface area) was less effective than bolus therapy with cyclophosphamide. However, the limited duration of the methylprednisolone regimen [6 months] might have been insufficient to treat lupus nephritis. To address this concern, we evaluated patients receiving methylprednisolone once a month for 1 year; additional boluses were given as needed to control disease. We compared these patients with patients receiving our standard therapy: intermittent boluses of cyclophosphamide. A group of patients randomly assigned to receive both cyclophosphamide and methylprednisolone was also included for three major reasons: 1) some patients with lupus nephritis respond inadequately to boluses of cyclophosphamide, 2) anecdotal experience had suggested that cyclophosphamide therapy might be more effective for all patients when given with substantial doses of corticosteroids, and 3) animal studies had shown the advantage of combined chemotherapy for lupus nephritis [22, 23]. Our study design was modified from previous designs so that therapy could be intensified for patients with refractory or relapsing disease. Methods Patient Selection We enrolled 82 patients with lupus nephritis into this randomized, parallel study at the Clinical Center of the National Institutes of Health (Bethesda, Maryland) between 1986 and 1990. To enter the study, patients had to have both glomerulonephritis and a diagnosis of systemic lupus erythematosus [24]. Glomerulonephritis was defined as a sediment on two or more urinalyses that showed either 10 or more erythrocytes per high-power field or erythrocyte or leukocyte casts (without evidence of infection) or both, plus histologic evidence of active proliferative lupus glomerulonephritis on a renal biopsy specimen obtained within 3 months of study entry (provided that a biopsy could be done safely). Scores for renal histologic activity and chronicity were assessed as reported elsewhere [25]. All eligible patients were invited to participate. Exclusion criteria were 1) receipt of cytotoxic drug treatment for more than 2 weeks during the 6 weeks before study entry or receipt of cyclophosphamide therapy for more than 10 weeks at any time; 2) receipt of pulse therapy with corticosteroids during the 6 weeks before study entry; 3) need [at the time of study entry] for oral corticosteroids in dosages greater than 0.5 mg of a prednisone equivalent per kilogram of body weight per day to control extrarenal disease; 4) active or chronic infection; 5) pregnancy; 6) the presence of only one kidney; 7) insulin-dependent diabetes mellitus; and 8) allergy to methylprednisolone or cyclophosphamide. Study Design The protocol that we used was approved by the NIDDK/NIAMS (National Institute of Diabetes and Digestive and Kidney Diseases/National Institute of Arthritis and Musculoskeletal and Skin Diseases) Institutional Review Board [86-AR-0189]. After giving signed, written informed consent, patients were randomly assigned to one of three treatment groups by drawing from a masked card sequence arranged from a table of random numbers. Each group received one of the following regimens: 1) intravenous methylprednisolone [1 g/m2 body surface area], given as boluses over 60 minutes on 3 consecutive days followed by at least 12 consecutive monthly single infusions; 2) intravenous cyclophosphamide, given as boluses once a month for 6 consecutive months and then once every 3 months for at least 2 more years; and 3) the combination of these two regimens. After a patient completed 1 year of study, a decision about whether therapy would be modified was made on the basis of the patients renal status at that time (Figure 1). In patients receiving methylprednisolone, therapy was discontinued if urine studies showed that renal remission had occurred. Renal remission was defined as the presence of fewer than 10 dysmorphic erythrocytes per high-power field, the absence of cellular casts, and excretion of less than 1 g of protein per day. If a renal remission was not evident, the patient continued to receive methylprednisolone every month for 6 more months. After the additional 6 months, if renal remission was still not evident, the patient received treatment for another 6 months. Therapy with methylprednisolone was limited to a maximum of 36 monthly boluses. Figure 1. Treatment regimens and decision pathways used in this clinical trial for lupus nephritis. At 1 year, patients who had been receiving cyclophosphamide alone or in combination with methylprednisolone continued to receive or began to receive cyclophosphamide alone, once every 3 months, if the results of urine studies were substantially improved. Substantial improvement was defined as a reduction of at least 50% in 1) the number of dysmorphic erythrocytes seen in urine samples, 2) the number of cellular casts, and 3) proteinuria, without a mmol of the serum creatinine level. Quarterly administration of cyclophosphamide was continued for 2 years after renal remission occurred, after which time therapy was stopped. After the first year of the study, patients in any treatment group who were no longer receiving monthly therapy but who had evidence of the reactivation of glomerular disease had their originally assigned regimens reinstituted as if they were beginning therapy from enrollment. Reactivation of glomerular disease was defined as new active nephritis with an increase of at least 50% (relative to the lowest reproducible values obtained during the study) in at least two of the following: number of dysmorphic erythrocytes ( 10 per high-power field), number of cellular casts, proteinuria ( 1 g of protein per day), or serum creatinine level. One year after the reinstitution of therapy, patients were again evaluated for evidence of active glomerulonephritis (as described above). As before, patients could be withdrawn from therapy, could restart treatment, or could continue to receive cyclophosphamide every 3 months. Patients could restart therapy no more than twice; if therapy failed more than three times, patients were declared to be nonresponders. Treatment and Follow-up Cyclophosphamide was infused for 60 minutes at an initial dose of 0.75 g/m2 body surface area. If the leukocyte nadir was greater than 3000 cells/mm3, the cyclophosphamide dose was increased by 25%, to a maximum of 1 g/m2 body surface area. The dose was reduced by 25% for leukocyte counts less than 1500 cells/mm3. Patients with a creatinine clearance of less than 30 mL/min received an initial dose of 0.5 g/m2 body surface area, and subsequent doses were adjusted on the basis of the lowest leukocyte count. Patients treated with cyclophosphamide were hydrated, and diuretics were used to maintain neutral fluid balance. Thiethylperazine, 10 mg, with 25 mg of diphenhydramine or 0.25 mg of lorazepam, was administered orally or intravenously every 6 hours for nausea. After the middle of 1990, patients were treated in a day hospital setting, where they received intravenous saline, 200 mL per hour for 10 hours. Mesna (2-mercaptoethanesulfonate), at 20% of the cyclophosphamide dose, was infused intravenously for 10 minutes before cyclophosphamide was administered and every 3 hours thereafter, for a total of four doses. Ondansetron, 8 mg, was given every 4 hours beginning 4 hours after infusion of cyclophosphamide, for a total of three doses. Dexamethasone, 10 mg, was given 4 hours after administration of cyclophosphamide [26]. Patients were instructed to continue oral hydration after discharge from the day hospital to maintain a dilute and frequent diuresis for at least 24 hours after infusion of cyclophosphamide. All patients were initially given oral prednisone, 0.5 mg/kg per day for 4 weeks. The prednisone dose was then tapered by 5 mg every other day each week to the minimal dose required to control extrarenal disease or 0.25 mg/kg every other day, whichever was greater. For severe extrarenal flares of lupus, patients were permitted to receive prednisone, 1.0 mg/kg per day for 2 weeks. Blood pressure was closely monitored and was maintained within 110 to 130/70 to 85 mm Hg with antihypertensive therapy. The intervals at which patients were followed were dictated by the activity of lupus and nephritis. In general, all patients were seen monthly during the first year of the study and every 3 months thereafter. At each study visit, patients were questioned about and examined for adverse events. Outcome Measures The primary study outcome was the response to the study drugs as defined by 1) the percentage of patients who achieved renal remission, 2) the number of nonresponders (nonresponse was defined as 10 erythrocytes per high-power field, cellular casts, proteinuria [>1 g of protein per day], and doubling of the serum creatinine level), and 3) the percentage of adverse events. The outcome data, with the exception of data on adverse events, were collected in a blinded manner on 1 May 1995, 5 years after the last patient was enrolled in the study. Secondary outcome measures were renal failure that required dialysis (end-stage renal disease), stable doubling of the serum creatinine level, and number of renal relapses (renal relapse was defined as a reactivation of renal disease after 6 or more months of remissio

Dietary enrichment with the polyunsaturated fatty acid eicosapentaenoic acid prevents proteinuria and prolongs survival in NZB x NZW F1 mice.

Prostaglandins and related compounds are active mediators of inflammation, but data concerning their role in the pathogenesis of the glomerulonephritis of New Zealand Black x New Zealand White (NZB x NZW) F1 mice are conflicting. Dietary eicosapentaenoic acid (EPA, C20:5), a fatty acid analogue of arachidonic acid (C20:4), has been shown to impair platelet aggregation in humans, apparently through inhibition of the synthesis of prostaglandins and thromboxanes from arachidonic acid. We report here the effects of a diet high in EPA on the development of renal disease and survival in female NZB x NZW F1 mice. Animals from 4--5 wk of age were fed diets containing 25% lipid, supplied either as beef tallow or menhaden oil, with fatty acid analysis of less than 0.05 and 14.4% EPA, respectively. In the first experiment, by 13.5 mo of age, mice on the beef tallow diet had all (9/9) developed proteinuria and the majority (6/9) had died, with renal histologic examination revealing severe glomerulonephritis. In contrast, none of 10 menhaden oil-fed animals had developed proteinuria, and all were alive at this time (P less than 0.005 for both proteinuria and survival). In a second experiment using 50 mice in each dietary group, 56% of the beef tallow group vs. none of the menhaden oil group had developed proteinuria at 9 mo of age (P less than 0.005). Native DNA binding at 6 mo of age was 23.9 +/- 14.7 vs. 10.1 +/- 9.7% in the beef and menhaden oil groups, respectively (P less than 0.01). Weights were similar in all groups, and there was no evidence of essential fatty acid deficiency in any group. These results demonstrate that a diet high in EPA protects NZB x NZW F1 mice from the development of glomerulonephritis.

B-lymphocyte alloantigens associated with systemic lupus erythematosus.

We examined B-lymphocyte alloantigens in 41 patients with systemic lupus erythematosus and 184 controls, using a panel of 47 pregnancy serums, and compared reaction frequencies of individual serums. One serum, la-715, reacted with B lymphocytes from 75.6 per cent of patients and 14.1 per cent of controls (Pc less than 0.005, relative risk 18.8). Twenty-eight of the patients were also typed with a panel of HLA-D-related serums from the Seventh International Histocompatibility Workshop, HLA-DRw types assigned, and compared to 490 Workshop controls. Both HLA-DRw2 (57.1 per cent vs. 26.4 per cent, Pc less than 0.004) and HLA-DRw3 (46.4 per cent vs. 22.2 per cent, Pc less than 0.03) were increased in systemic lupus erythematosus. This study demonstrates that select B-lymphocyte alloantigens, which are controlled by genes in the major histocompatibility complex, are present in increased frequency in systemic lupus erythematosus.

The Pathogenesis of Autoimmunity in New Zealand Black Mice

This paper will review the immune disease of New Zealand Black (NZB) mice and their F1 hybrids produced by mating with New Zealand White mice (NZB/NZW). It will attempt to highlight important recent observations made in many laboratories around the world without exhaustively reviewing all papers written on the subject. Previous reviews have emphasized pathological changes and disease descriptions in New Zealand mice (Howie and Helyer, 1968; Helyer and Howie, 1963 a). The present effort will stress the immunological abnormalities of these mice. An attempt will be made to relate these immunological abnormalities with genetic and viral factors known to be important in their disease processes. The authors and their co-workers have studied approximately 20 000 NZB and NZB/NZW mice with regard to natural history, immunology, pathogenesis of disease and therapy. They will draw upon this experience when possible to fill in details not available in published reports.

Cerebrospinal fluid antibodies to neuronal cells: association with neuropsychiatric manifestations of systemic lupus erythematosus.

The validity of the hypothesis that some of the neuropsychiatric manifestations of systemic lupus erythematosus (SLE) are mediated by the direct effects of antibody binding to neuronal cell membranes is dependent on the demonstration of antineuronal activity within the central nervous system of patients with active central nervous system disease. Using a radiolabelled staphylococcal protein A assay, we tested cerebrospinal fluid from 27 patients with SLE and central nervous system manifestations, and cerebrospinal fluid from 18 additional patients with SLE but free of central nervous system disease for antibody reactive with the cultured human neuronal cell line SK-N-SH. Cerebrospinal fluid from 20 of 27 patients with active lupus central nervous system disease had increased immunoglobulin G (IgG) antineuronal activity compared with cerebrospinal fluid from two of 18 patients with SLE without central nervous system disease. Ninety percent of the patients with psychosis, organic brain syndrome or generalized seizures had increased IgG antineuronal activity as compared with only 25 percent of the patients who presented with hemiparesis or with chorea/hemiballismus. Antineuronal activity per microgram of IgG was concentrated eightfold in the cerebrospinal fluid of patients with active central nervous system disease as compared with the serum activity. Patients with or without active central nervous system disease did not differ significantly in the amount of serum antineuronal binding activity. The results are consistent with the hypothesis that the more diffuse central nervous system manifestations of SLE are a direct result of the interaction of antibody with neuronal cell membranes.

Alterations in immunoregulatory T cell subsets in active systemic lupus erythematosus.

To determine whether imbalance among subsets of human T cells exists in patients with systemic lupus erythematosus (SLE), we analyzed peripheral blood lymphocytes in SLE patients during active and inactive stages of disease. For this analysis, we used monoclonal antibodies to the surface antigens of inducer (T4) and suppressor (T5/T8) T cell subsets, as well as a common T cell antigen (T3). In contrast to normal and inactive SLE patients, the percentage of T3+ cells was reduced in all active SLE patients. More importantly, there was a selective decrease in T5+/T8+ suppressor T cells in 12 of 14 active patients, including 1 of 2 patients with drug-induced SLE. Serial analysis of three SLE patients showed a significant correlation between the presence of T5+/T8+ subset and clinical disease activity in all patients. We conclude that aberrations in suppressor T cell subsets are an important correlate of disease in patients with SLE.

Systemic Lupus Erythematosus: Evolving Concepts

Abstract Systemic lupus erythematosus, a disease of unknown cause and protean manifestations, continues to excite substantial investigational interest. These papers bring together recent advances i...

A defect of immunoregulatory T cell subsets in systemic lupus erythematosus patients demonstrated with anti-2H4 antibody.

The cell surface phenotype of peripheral blood lymphocytes (PBL) of systemic lupus erythematosus (SLE) patients was characterized with the anti-2H4 monoclonal antibody that defines the human suppressor inducer subset. The T4+2H4+ population of cells has been shown to be critical for the activation of T8+ suppressor cells. Patients with SLE has a markedly decreased percentage of T4+2H4+ cells (13 +/- 2%) in their PBL compared with normal controls (21 +/- 1%) (P less than 0.001). This reduction was greatest in patients with active SLE, especially those with renal disease. Serial analysis of patients with SLE and renal disease showed a correlation between percent positive circulating T4+2H4+ cells and disease activity. Moreover, there was a significant correlation between a low percentage of T4+2H4+ cells and decreased suppressor-inducer function in autologous mixed lymphocyte reaction-activated T4+ cells from SLE patients. Thus, a deficiency exists in SLE patients with active renal disease in the T4+2H4+ suppressor-inducer T cell subset.

Effect of Treatment on the Evolution of Renal Abnormalities in Lupus Nephritis

We retrospectively studied the evolution of histopathologic features in successive renal biopsies in patients with lupus nephritis, to evaluate the effects of various treatment regimens. Repeat renal biopsies had been performed in 62 patients after more than 18 months of observation (median interval, 44 months) in randomized therapeutic trials comparing prednisone with cytotoxic drugs. Renal histopathologic features were graded individually, and a composite score reflecting the number and severity of irreversible lesions was defined as a chronicity index. The chronicity index for patients treated with conventional high-dose prednisone increased linearly with the interval between biopsies, whereas the index in the group receiving cytotoxic-drug treatments did not increase over time. After statistical adjustment for important prognostic factors (age and initial chronicity index) identified by multiple linear regression, the difference in the slopes between the group receiving prednisone and the group receiving cytotoxic drugs was significant (P less than 0.0001). We conclude that cytotoxic-drug treatment reduces the likelihood of progressive renal scarring in lupus nephritis.

Defective EBV-Specific Suppressor T-Cell Function in Rheumatoid Arthritis

Several lines of evidence, including high antibody titers to Epstein-Barr virus (EBV)-associated antigens and rapid transformation of B cells into lymphoblastoid cells lines, suggest an association between EBV and rheumatoid arthritis. When lymphocytes from normal immune donors were infected with EBV in culture, they produced an exponentially increasing number of immunoglobulin-secreting cells for eight to 10 days. Thereafter, there was a marked late suppression of their response, mediated by immunoregulatory T cells; by 12 days in culture, this suppression averaged 90 per cent. Lymphocytes from 20 EBV-immune patients with rheumatoid arthritis also responded with increasing production of immunoglobulin-secreting cells, but the late suppression expected in immune donors was absent. Tests of several other T-cell functions in these patients gave normal results, suggesting a more restricted defect in suppressor-T cell function relating specifically to EBV. Since EBV persists in host B cells and thus represents a potential stimulus for immunoglobulin production, this persistence, along with a specific regulatory T-cell defect, may contribute to many of the immune abnormalities underlying rheumatoid arthritis.