Dorothy E. Scott

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

Smallpox vaccine–induced antibodies are necessary and sufficient for protection against monkeypox virus

Vaccination with live vaccinia virus affords long-lasting protection against variola virus, the agent of smallpox. Its mode of protection in humans, however, has not been clearly defined. Here we report that vaccinia-specific B-cell responses are essential for protection of macaques from monkeypox virus, a variola virus ortholog. Antibody-mediated depletion of B cells, but not CD4+ or CD8+ T cells, abrogated vaccine-induced protection from a lethal intravenous challenge with monkeypox virus. In addition, passive transfer of human vaccinia-neutralizing antibodies protected nonimmunized macaques from severe disease. Thus, vaccines able to induce long-lasting protective antibody responses may constitute realistic alternatives to the currently available smallpox vaccine (Dryvax).

Inhibitory Effects of B Cells on Antitumor Immunity

B-cell functions in antitumor immunity are not well understood. In this study, we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag), D5 mouse melanoma, or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments, spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors), IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells.

Distinct roles for B7-1 (CD-80) and B7-2 (CD-86) in the initiation of experimental allergic encephalomyelitis.

The activation and differentiation of T cells require both antigen/MHC recognition and costimulatory signals. The present studies examined the role of B7-1 (CD80) and B7-2 (CD86) costimulation in the prototypic autoimmune disorder, experimental allergic encephalomyelitis (EAE). In adoptively transferred EAE, in vitro activation of myelin basic protein (MBP)-specific lymph node cells was inhibited by the combination of anti-CD80 plus anti-CD86, but not individually. However, in actively induced disease, one injection of anti-CD80 significantly reduced disease, while anti-CD86 exacerbated disease. Interestingly, one injection of CTLA-4Ig suppressed disease, while multiple injections resulted in enhanced disease. Thus, the costimulation provided by B7-1 molecules appears to be important for the development of encephalitogenic T cells. The enhanced disease caused by multiple injections of CTLA-4Ig or a single injection of anti-CD86 suggests an inhibitory function for CD86 interaction with its counterreceptors CD28 and CTLA-4 in EAE. Alternatively, these results are consistent with an essential timing requirement for the coordinated interaction of B7 and CD28 family receptors, and that disruption of this critical timing can have opposing results on the outcome of an immune response.

Immunity and protection against Brucella abortus

Brucella abortus is an intracellular pathogen that causes disease in cattle and in humans. The response against B. abortus involves the whole gamut of the immune system, from innate to adaptive immunity resulting from stimulation of antigen-presenting cells, NK cells, CD4(+) and CD8(+) T cells, and B cells.

Severe Eczema Vaccinatum in a Household Contact of a Smallpox Vaccinee

BACKGROUND We report the first confirmed case of eczema vaccinatum in the United States related to smallpox vaccination since routine vaccination was discontinued in 1972. A 28-month-old child with refractory atopic dermatitis developed eczema vaccinatum after exposure to his father, a member of the US military who had recently received smallpox vaccine. The father had a history of inactive eczema but reportedly reacted normally to the vaccine. The childs mother also developed contact vaccinia infection. METHODS Treatment of the child included vaccinia immune globulin administered intravenously, used for the first time in a pediatric patient; cidofovir, never previously used for human vaccinia infection; and ST-246, an investigational agent being studied for the treatment of orthopoxvirus infection. Serological response to vaccinia virus and viral DNA levels, correlated with clinical events, were utilized to monitor the course of disease and to guide therapy. Burn patient-type management was required, including skin grafts. RESULTS The child was discharged from the hospital after 48 days and has recovered with no apparent systemic sequelae or significant scarring. CONCLUSION This case illustrates the need for careful screening prior to administration of smallpox vaccine and awareness by clinicians of the ongoing vaccination program and the potential risk for severe adverse events related to vaccinia virus.

IL-12 Induction by a Th1-Inducing Adjuvant In Vivo: Dendritic Cell Subsets and Regulation by IL-10

IL-12 induction is critical for immune responses against many viruses and intracellular bacterial pathogens. Recent studies suggest that IL-12-secreting dendritic cells (DC) are potent Th1-inducing APC. However, controversy exists concerning the function of DC subsets. Murine studies have suggested that CD8+ DC preferentially induce Th1 responses, whereas CD8− DC induce Th2 development; in this model, different DC subsets prime different responses. Alternatively, the propensity of DC subsets to prime a Th1 response could depend upon the type of initial stimulus. We used a prototypic Th1-inducing adjuvant, heat-killed Brucella abortus (HKBA) to assess stimulation of DC subsets, relationship between Ag burden and IL-12 production, and down-regulation of DC subset IL-12 production by IL-10. In this study, we show that DC were sole producers of IL-12, although most HKBA uptake was by splenic macrophages and granulocytes. More CD8− than CD8+ DC produced IL-12 after HKBA challenge, whereas only CD8+ DC produced IL-12 after injection of another Th1-promoting microbial substance, soluble Toxoplasma gondii Ags. Studies in IL-10-deficient mice revealed that IL-10 down-regulates frequency and duration of IL-12 production by both DC subsets. In the absence of IL-10, IL-12 expression is enabled in CD11clow cells, but not in macrophages or granulocytes. These findings support the concept of DC as the major IL-12 producers in spleens, but challenge the notion that CD8+ and CD8− DC are destined to selectively induce Th1 or Th2 responses, respectively. Thus, the nature of the stimulating substance is important in determining which DC subsets are activated to produce IL-12.

Therapeutic potential of phosphodiesterase type 4 inhibition in chronic autoimmune demyelinating disease

It was recently demonstrated that selective phosphodiesterase type 4 (PDE4) inhibition suppresses the clinical manifestations of acute experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), and inhibits the production of tumor necrosis factor-alpha (TNF-alpha), a pathogenetically central cytokine. Since the most common presentation of MS in humans is a relapsing-remitting course, we investigated the therapeutic potential of PDE4 inhibition in the relapsing-remitting EAE model of the SJL mouse. Administration of rolipram, the prototypic PDE4 inhibitor, reduced the clinical signs of EAE during both the initial episode of disease and subsequent relapses. In parallel, there was marked reduction of demyelination and also less inflammation throughout the central nervous system (CNS) of rolipram-treated animals. Gene expression of proinflammatory cytokines in the CNS was reduced in most of the rolipram-treated animals. Additional experiments demonstrated that PDE4 inhibition acted principally by inhibiting the secretion of Th1 cytokines, however, the encephalitogenic potential of myelin basic protein-specific T cells was not impaired. Our findings suggest that PDE4 inhibitors are a promising cytokine-directed therapy in chronic demyelinating disease.

Immunoglobulin G3 from Polyclonal Human Immunodeficiency Virus (HIV) Immune Globulin Is More Potent than Other Subclasses in Neutralizing HIV Type 1

ABSTRACT Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab′)2 fragments or with papain to generate Fab fragments. IgG3 F(ab′)2 fragments were still more efficient in neutralization than F(ab′)2 of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.

Opposing effects of CTLA4-Ig and anti-CD80 (B7-1) plus anti-CD86 (B7-2) on experimental allergic encephalomyelitis.

The roles of the B7 receptors, CD80 and CD86, during actively induced experimental allergic encephalomyelitis were examined with specific monoclonal antibodies and CTLA4-Ig. Injection of CTLA4-Ig on day 2 post-immunization resulted in decreased incidence and severity of resultant disease. Anti-CD80 injection on day 2 blocked development of the first disease episode. Subsequent relapses were unaffected. In contrast, injection of anti-CD86 alone had no effect. Surprisingly, combined anti-CD80 + anti-CD86 monoclonal antibody injection on day 2 resulted in marked exacerbation of disease. Examination of cytokine production in the draining lymph node cells demonstrated a reduction in both interferon (IFN)-gamma and interleukin (IL)-2 producing cells, but a dramatic increase in tumor necrosis factor (TNF)-alpha secretion in animals receiving both monoclonal antibodies. These results suggest distinct roles for CD80 and CD86 in the initiation of EAE, resulting in the diverse clinical outcomes observed in this model of EAE.