The efficacy and safety of rituximab in a chart review study of 15 patients with systemic lupus erythematosus.

In contrast to randomised clinical trials, open-label studies have suggested that B cell depletion by a course of rituximab is associated with a significant clinical benefit. Our aim was to assess the safety and efficacy of rituximab in 15 refractory lupus patients, particularly those with more than one course of therapy. Disease activity was measured by the classic British Isles Lupus Assessment Group (BILAG) index, anti-DNA antibodies and complement levels. We assessed immunoglobulin levels, functional antibodies and serious adverse events. The mean patient age ± SD was 37.9 ± 7.2 years and mean disease duration was 8.5 ± 3.3 years; 46% were Afro-Caribbean, 27% South Asian, 20% Caucasian and 7% others. Twelve patients responded by 6 months; six avoided major flare for >1 year. Complete absence of disease activity (BILAG D/E) lasted for 5.5 (SD 3.8) months and 4.8 (SD 3.6) months after the first (n = 15) and second (n = 9) rituximab course, respectively. The mean 6-month reduction in daily prednisolone was 10.4 (SD 11.4) mg/day and 10.7 (SD 9.3) mg/day from baseline after the first and second course, respectively. Patients with low C3/C4 normalised their C3 by 6 months. Most patients with raised anti-dsDNA normalised after rituximab courses. Serious adverse events only occurred after more than four courses of rituximab. Rituximab was safe and efficacious for treating patients with refractory systemic lupus erythematosus (SLE) and was associated with significant steroid reduction, but more than four courses of rituximab was associated with an increased risk of serious infection in two patients.

Bilateral retinal vasculitis in a patient with systemic lupus erythematosus and its remission with rituximab therapy.

Severe retinal vasculitis is a rare, but potentially blinding, complication of patients with systemic lupus erythematosus (SLE). We describe here the first reported case of treating severe bilateral SLE-associated retinal vasculitis with the anti-CD20 monoclonal antibody rituximab, a drug which has established its role in rheumatoid arthritis and has shown promise in case series for the treatment of severe SLE that is unresponsive to other therapies. This case suggests that rituximab-induced B-cell depletion may provide an important new therapeutic option for refractory cases of this devastating ocular complication.

Effects of rituximab on resistant SLE disease including lung involvement.

We present a retrospective review of 11 patients with refractory systemic lupus erythematosus (SLE) treated with rituximab after failing corticosteroids and at least one other immunosuppressive drug. We measured clinical response using the Classic British Isles Lupus Assessment Group (BILAG) index, serum complement and reduction in maintenance prednisolone dose. B cells were measured using flow cytometry, and lung function testing was used to assess severe pulmonary disease (three patients). The median patient age was 42 years (range, 25-64) with median disease duration 6 years (range, 2-12). In all, 10 of 11 patients responded initially, with median global BILAG reduction of 7.5 at 6 months (P = 0.007), with loss of all A and B scores by 7 months. Rituximab treatment was associated with normalisation of complement (C3 P = 0.008, C4 P = 0.018) and reduction in steroid requirement, median reduction 15 mg/day (P = 0.036). In 9 of 10 patients who responded, all other immunosuppressants were stopped. There was no significant difference in anti-dsDNA antibody titres in these responders, but they were negative or had low titres at baseline. B-cell depletion continued for median 4 months (range, 2-9), and disease flare occurred at a median 6.6 months (range, 1.5-23) and was preceded by B-cell recovery in all but two patients. Rituximab was beneficial in refractory SLE including severe neurological and cardiorespiratory disease by inducing disease remission, allowing withdrawal of other agents and reduction in steroid requirement. Rituximab appeared to stabilise and possibly improve progressive lung disease.

BILAG-2004 index captures systemic lupus erythematosus disease activity better than SLEDAI-2000.

OBJECTIVE: To assess the reliability of Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2000 index in routine practice and its ability to capture disease activity as compared with the British Isles Lupus Assessment Group (BILAG)-2004 index. METHODS: Patients with systemic lupus erythematosus from 11 centres were assessed separately by two raters in routine practice. Disease activity was assessed using the BILAG-2004 and SLEDAI-2000 indices. The level of agreement for items was used to assess the reliability of SLEDAI-2000. The ability to detect disease activity was assessed by determining the number of patients with a high activity on BILAG-2004 (overall score A or B) but low SLEDAI-2000 score (<6) and number of patients with low activity on BILAG-2004 (overall score C, D or E) but high SLEDAI-2000 score (>or=6). Treatment of these patients was analysed, and the increase in treatment was used as the gold standard for active disease. RESULTS: 93 patients (90.3% women, 69.9% Caucasian) were studied: mean age was 43.8 years, mean disease duration 10 years. There were 43 patients (46.2%) with a difference in SLEDAI-2000 score between the two raters and this difference was >or=4 in 19 patients (20.4%). Agreement for each of the items in SLEDAI-2000 was between 81.7 and 100%. 35 patients (37.6%) had high activity on BILAG-2004 but a low SLEDAI-2000 score, of which 48.6% had treatment increased. There were only five patients (5.4%) with low activity on BILAG-2004 but a high SLEDAI-2000 score. CONCLUSIONS: SLEDAI-2000 is a reliable index to assess systemic lupus erythematosus disease activity but it is less able than the BILAG-2004 index to detect active disease requiring increased treatment.

BILAG 2004. Development and initial validation of an updated version of the British Isles Lupus Assessment Group's disease activity index for patients with systemic lupus erythematosus.

OBJECTIVE: To devise a more discriminating version of the British Isles Lupus Assessment Group (BILAG) disease activity index and to show that it is reliable. METHODS: A nominal consensus approach was undertaken by members of BILAG to update and improve the BILAG lupus disease activity index. The index has been revised following intense consultations over a 1-yr period. It has been assessed in two real-patient exercises. These involved patients with diverse clinical features of SLE, including gastrointestinal, hepatic and ophthalmic problems, which the earlier versions of the index did not fully take into account. Reliability in terms of the ability to differentiate patients was assessed by calculating intraclass correlation coefficients. The level of agreement between physicians was determined by calculating the ratio of estimates of the standard error (SE) attributable to the physicians to the SE attributable to the patients. RESULTS: Good reliability and high levels of physician agreement were observed in one or both exercises in the constitutional, mucocutaneous, neurological, cardiorespiratory, renal, ophthalmic and haematological systems. In contrast, the musculoskeletal system did not score as well, although providing more clear-cut glossary definitions should greatly improve the situation. CONCLUSIONS: Some significant changes in the BILAG disease activity index to assess patients with SLE are proposed. The process of demonstrating validity and reliability has started with these two exercises assessing real patients. Further validation studies are under way. BILAG 2004 is likely to be valuable in clinical trials assessing new therapies for the treatment of SLE, as it provides a more comprehensive system-based disease activity measure than has been available previously.

Prevalence and predictors of fragility fractures in systemic lupus erythematosus.

OBJECTIVE: To establish the prevalence of reduced bone mineral density (BMD) and fractures, and risk factors for fractures, in a cross sectional study of a large cohort of patients with systemic lupus erythematosus (SLE). METHODS: All SLE patients willing to take part in the study had bone densitometry in 1999/2000 and completed a questionnaire on risk factors for osteoporosis and on drugs used. Accumulated damage was scored using the SLICC/ACR damage index (SDI). Only fractures occurring since the onset of SLE and unrelated to trauma were included, and the SDI score was modified to exclude osteoporotic fractures. Statistical analysis was by chi(2) test, Fisher's exact test, and binary logistic regression. RESULTS: 242 patients were studied, median age 39.9 years (range 18 to 80), median disease duration 7.0 years (range 0 to 42). Of these, 123 (50.8%) had reduced BMD (T score <-1.0) and 25 (10.3%) were in the osteoporotic range (T score <-2.5). Fragility fractures had occurred in 22 patients (9.1%) since diagnosis of SLE. Of these, two (9.1%) had normal BMD and 20 (90.9%) had reduced BMD, while seven (31.8%) were within the osteoporotic range. Non-Afro-Caribbean race and exposure to prednisolone >10 mg daily were significantly associated with reduced BMD, while age and menopause were associated with osteoporosis. The risk factors for fractures were reduced BMD and age. CONCLUSIONS: Reduced BMD, osteoporosis, and fragility fractures appear to be prevalent in patients with SLE. Steroids were not an independent risk factor for fractures, although their effect could be mediated through reduced bone mineral density.

EULAR randomised controlled trial of pulse cyclophosphamide and methylprednisolone versus continuous cyclophosphamide and prednisolone followed by azathioprine and prednisolone in lupus nephritis.

OBJECTIVE: To compare the efficacy and side effects of intermittent pulse cyclophosphamide plus methylprednisolone with continuous oral cyclophosphamide plus prednisolone, followed by azathioprine, in patients with proliferative glomerulonephritis caused by systemic lupus erythematosus (SLE). METHODS: A multicentre randomised controlled trial was conducted between June 1992 and May 1996 involving eight European centres. All patients satisfied the American College of Rheumatology criteria for SLE and had biopsy proven proliferative lupus nephritis. All received corticosteroids in addition to cytotoxic drugs, as defined in the protocol, for two years. The trial was terminated after four years as recruitment was disappointing. RESULTS: 32 SLE patients with lupus nephritis were recruited: 16 were randomised to intermittent pulse cyclophosphamide and 16 to continuous cyclophosphamide plus azathioprine. Mean duration of follow up was 3.7 years in the continuous group (range 0 to 5.6) and 3.3 years in the pulse group (range 0.25 to 6). Three patients were excluded from the pulse therapy group as they were later found to have pure mesangial glomerulonephritis. Two patients in the continuous therapy group developed end stage renal failure requiring dialysis, but none in the intermittent pulse therapy (p = 0.488; NS). There were similar numbers of side effects and withdrawals from treatment in both groups. There were three deaths: two in the intermittent pulse therapy group and one in the continuous therapy group. CONCLUSIONS: There was no statistically significant difference in efficacy and side effects between the two regimens. Infectious complications occurred commonly, so careful monitoring is required during treatment.

The prevalence of patients with rheumatoid arthritis in the West Midlands fulfilling the BSR criteria for anti-tumour necrosis factor therapy: an out-patient study.

OBJECTIVES: There are currently two anti-tumour necrosis factor (anti-TNF) therapies licensed for treatment of rheumatoid arthritis (RA). A British Society for Rheumatology (BSR) working party defined criteria for patients that would be suitable for such treatment. The aim of this study was to determine the prevalence of these patients attending rheumatology out-patient departments across the West Midlands. METHODS: Data were collected over a 2-week period in adult out-patient departments of 12 centres. A questionnaire was completed at each patient review. Disease activity scores (DAS-28) were recorded for those who had failed methotrexate treatment and at least one other disease-modifying anti-rheumatic drug (DMARD) in the absence of contraindications to anti-TNF therapy. Information was also collected on the number of DMARDs failed and the use of steroid therapy. RESULTS: A total of 1441 patients with RA were assessed; 177 (12.3%) patients had failed methotrexate and at least one other DMARD. Of these, 19 had contraindications to the use of anti-TNF therapy. In the remaining 158 patients (11%), 80 (5.6%) had a DAS-28 score of >5.1, thus fulfilling BSR criteria for use of anti-TNF therapy. Those with a DAS-28 score of < or = 5.1 were significantly more likely to have been taking steroids compared with those with a DAS-28 score >5.1 (68.2 and 49.3%, respectively, P=0.024). CONCLUSIONS: Of patients with RA attending adult rheumatology out-patient clinics in the West Midlands, 5.6% would meet BSR criteria for use of anti-TNF therapy. Eligibility may be affected by steroid use.

Association of damage with autoantibody profile, age, race, sex and disease duration in systemic lupus erythematosus.

OBJECTIVE: To determine if there is any association between autoantibody profile and damage in a cohort of patients with systemic lupus erythematosus (SLE). METHODS: A prospective cohort of SLE patients attending two SLE clinics in Birmingham was analysed. All patients fulfilled ARA criteria for SLE. Detailed clinical and serological information was recorded at each visit. Damage according to the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SLICC/ACR DI) was recorded 6-monthly and the last score in the year 2000 or prior to death was used in the analysis. Univariate analysis was performed with the chi(2) test, Fisher's exact test or univariate analysis of variance. Multivariate analysis was done with binary logistic regression. RESULTS: A total of 348 patients (326 females) were studied, comprising 208 Caucasians, 65 Afro-Caribbeans, 59 Asians, four Orientals and 12 others. There were 32 (9.2%) deaths and 156 (44.8%) patients had damage recorded during follow-up. The presence of damage showed no significant association with race, sex or anti-cardiolipin, anti-Ro, anti-La, anti-Sm, anti-RNP and anti-dsDNA antibodies. Only age, disease duration and other antibodies to extractable nuclear antigens (ENA) were found to be associated with the presence of damage. When individual organ damage was analysed, the only significant associations were of anti-Ro with ocular damage and of other anti-ENA antibodies (anti-Scl-70 and/or anti-Jo-1) with premature gonadal failure. Other autoantibodies were not predictive of damage in individual organs. CONCLUSIONS: Although autoantibodies are useful in diagnosis and predicting disease activity in SLE, they do not appear to be useful in predicting damage in SLE.

Dendritic cell-specific MHC class II transactivator contains a caspase recruitment domain that confers potent transactivation activity.

The MHC class II transactivator (CIITA) is a critical transcription factor that regulates genes involved in antigen presentation function. At least three functional forms of CIITA gene products are transcribed from three different promoters. The CIITA gene expressed in dendritic cells (DC-CIITA) has a unique first exon encoding an extended N-terminal region of CIITA. Here, we show that the N terminus of DC-CIITA has high homology to a caspase recruitment domain (CARD) found in components of apoptosis and nuclear factor-kappaB signaling pathways. However, DC-CIITA does not regulate cell death, nor does it induce nuclear factor-kappaB activity. Instead, DC-CIITA is transcriptionally a more potent activator of the MHC class II gene than the form expressed in B cells. A single amino acid substitution in the CARD of DC-CIITA, predicted to disrupt CARD-CARD interactions, diminished the transactivation potential of DC-CIITA. These results indicate that the CARD in the context of CIITA serves as a regulatory domain for transcriptional activity and may function to selectively enhance MHC class II gene expression in dendritic cells.

The evolution of ribonucleotide reduction revisited.

Ribonucleotide reductases (RNRs) catalyze the conversion of both purine and pyrimidine nucleotides to deoxynucleotides in all organisms and provide all the monomeric precursors essential for both DNA replication and repair. RNRs have been divided into three classes on the basis of their unique metallo-cofactors. The exquisitely controlled free radical chemistry used by all RNRs, and the commonality of the structures of the subunits where the nucleotide reduction process occurs, together provide compelling evidence for the importance of chemistry in the divergent evolution of RNRs from a common progenitor.

Effective antigen-specific immunotherapy in the marmoset model of multiple sclerosis.

Mature T cells initially respond to Ag by activation and expansion, but high and repeated doses of Ag cause programmed cell death and can suppress T cell-mediated diseases in rodents. We evaluated repeated systemic Ag administration in a marmoset model of experimental allergic encephalomyelitis that closely resembles the human disease multiple sclerosis. We found that treatment with MP4, a chimeric, recombinant polypeptide containing human myelin basic protein and human proteolipid protein epitopes, prevented clinical symptoms and did not exacerbate disease. CNS lesions were also reduced as assessed in vivo by magnetic resonance imaging. Thus, specific Ag-directed therapy can be effective and nontoxic in primates.

Molecular cloning, expression, and activity of zebrafish semaphorin Z1a.

Semaphorins/collapsins are a large family of secreted and cell surface molecules that are thought to guide growth cones to their targets. Although some members are clearly repulsive to specific growth cones in vitro, the in vivo role of many of these molecules in vertebrate embryos is still unclear. As a first step towards clarifying the in vivo role of semaphorins/collapsins, we analyzed semaZ1a in the simple and well-characterized zebrafish embryo. SemaZ1a is a secreted molecule that is highly homologous to Sema III/D/collapsin-1, and it can collapse chick dorsal root ganglion growth cones in vitro. It is expressed in highly specific patterns within the developing embryo, which suggests that it influences outgrowth by a variety of growth cones including those of the posterior lateral line ganglion. Consistent with this hypothesis, the peripherally extending growth cones of posterior lateral line neurons retract and partially collapse during normal outgrowth.

Analysis of a Zebrafish semaphorin reveals potential functions in vivo.

The semaphorin/collapsin gene family is a large and diverse family encoding both secreted and transmembrane proteins, some of which are thought to act as repulsive axon guidance molecules. However, the function of most semaphorins is still unknown. We have cloned and characterized several semaphorins in the zebrafish in order to assess their in vivo function. Zebrafish semaZ2 is expressed in a dynamic and restricted pattern during the period of axon outgrowth that indicates potential roles in the guidance of several axon pathways. Analysis of mutant zebrafish with reduced semaZ2 expression reveals axon pathfinding errors that implicate SemaZ2 in normal guidance.

Molecular cloning and expression of two novel zebrafish semaphorins.

The large, conserved semaphorin/collapsin gene family encodes putative axon guidance molecules. We describe the cloning and expression of two n ovel zebrafish semaphorins that represent an increase in the size and diversity of the family. These semaphorins are expressed in unique and dynamic patterns during development.

Zebrafish semaphorin Z1a collapses specific growth cones and alters their pathway in vivo.

The semaphorin/collapsin gene family encodes secreted and transmembrane proteins several of which can repulse growth cones. Although the in vitro activity of Semaphorin III/D/Collapsin 1 is clear, recent analyses of two different strains of semaphorin III/D/collapsin 1 knockout mice have generated conflicting findings. In order to clarify the in vivo action of this molecule, we analyzed sema Z1a, a zebrafish homolog of semaphorin III/D/collapsin 1. The expression pattern of sema Z1a suggested that it delimited the pathway of the growth cones of a specific set of sensory neurons, the posterior ganglion of the lateral line, in zebrafish. To examine the in vivo action of this molecule, we analyzed (1) the pathways followed by lateral line growth cones in mutants in which the expression of sema Z1a is altered in an interesting way, (2) response of lateral line growth cones to exogenous Sema Z1a in living embryos, and (3) the pathway followed by lateral line growth cones when Sema Z1a is misexpressed by cells along their normal route. The results suggest that a repulsive action of Sema Z1a helps guide the growth cones of the lateral line along their normal pathway.

Carboplatin-induced cell death in model prostate cancer systems.

BACKGROUND: Many chemotherapeutic agents are believed to kill cancer cells by inflicting cellular damage which triggers the cell to enter apoptosis (programmed cell death). We investigated the means by which carboplatin induces cell death in three model cancer systems: the human prostate carcinoma cell lines PC-3 and LNCaP and the human cervical carcinoma cell line HeLa. MATERIALS AND METHODS: Drug cytotoxicity, cell cycle effects, bcl-2 deactivation, and multiple markers for apoptosis were utilized to examine carboplatin activity within these cell lines. RESULTS: In HeLa cells, carboplatin appears to induce an S-phase block followed by apoptosis. In contrast, PC-3 and LNCaP cells show no cell cycle phase block and die from necrosis rather than apoptosis. The effects of carboplatin contrast sharply with the effects of paclitaxel, which induces an M-phase block and apoptosis in all three cell lines. CONCLUSIONS: These results show that PC-3 and LNCaP cells are relatively resistant to carboplatin and suggest two causes of resistance: bypassing the cell cycle checkpoints which serve as points of entry into apoptosis, and incomplete execution of the effector mechanisms of apoptosis. Carboplatin resistance in the prostate cancer cell lines fits into the developing scheme of apoptosis-necrosis and raises valuable questions about the root causes of cancer resistance to chemotherapeutic agents.

The effect of castanospermine on the metastatic properties of prostate cancer cells.

BACKGROUND: Most deaths from prostate cancer result from the metastatic spread of the disease. Castanospermine has been shown to inhibit tumor growth and metastasis in mouse and rat models. We hypothesized that castanospermine might inhibit metastasis in the Dunning model of rat prostate adenocarcinoma by interfering with the metastatic properties of tumor cells. MATERIALS AND METHODS: We examined the cytotoxicity of castanospermine toward the metastatic MAT-LyLu and nonmetastatic AT. 1 cell lines and its effects on cell motility and adhesion to endothelial cells. We assessed castanospermine's effects on in vivo metastasis in Copenhagen rats. RESULTS: Castanospermine was not cytotoxic toward the MAT-LyLu and AT. 1 cell lines at concentrations through 10 micrograms/mL, nor did it significantly affect cell motility, adhesion to endothelial cells, or in vivo metastasis. CONCLUSIONS: Within the Dunning model, castanospermine did not appear to significantly affect cell characteristics related to metastatic potential.

Regulation of interleukin-6 expression in the lymphoma cell line OCI-LY3.

Previously we described a cell line OCI-LY3 derived from a patient with non-Hodgkin's lymphoma. The cell line produced interleukin-6 (IL-6) mRNA and protein and demonstrated an autocrine pattern of growth for IL-6. Southern blot analysis of the IL-6 gene did not reveal any rearrangement. To determine whether the production of IL-6 by OCI-LY3 was due to subtle changes in the promoter of IL-6 or due to the expression of trans-acting factors chloramphenicol acetyltransferase (CAT) reporter constructs containing from -1,180 to +13 to -112 to +13 of a normal IL-6 gene were electroporated into the cell line. When these constructs are transferred into unstimulated fibroblasts, no CAT activity is seen; however, CAT activity is induced when the cells are stimulated with either IL-1 alpha, lipopolysaccharide (LPS), or cyclic adenosine monophosphate (cAMP) analogues. When the cell line OCI-LY3 was transfected with these constructs, CAT activity was observed; it was not necessary to stimulate the cells with exogenous factors to observe this activity. No CAT activity was observed in a second lymphoma cell line, OCI-LY13.1, that does not produce IL-6. These results suggest that the constitutive production of IL-6 by the cell line OCI-LY3 is due to the presence of trans-acting factors that stimulate the expression of IL-6 and not due to a cis-acting mutation of the IL-6 promoter.