Efficacy and safety of subcutaneous tabalumab in patients with systemic lupus erythematosus: results from ILLUMINATE-1, a 52-week, phase III, multicentre, randomised, double-blind, placebo-controlled study.

OBJECTIVES: Evaluate efficacy and safety of tabalumab, a human IgG4 monoclonal antibody that binds and neutralises membrane and soluble B-cell activating factor (BAFF) versus placebo plus standard of care (SoC) in patients with systemic lupus erythematosus (SLE). METHODS: This phase III, 52-week study randomised 1164 patients with moderate-to-severe SLE (Safety of Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index ≥6 at baseline). Patients received SoC plus subcutaneous injections of tabalumab or placebo, starting with a loading dose (240 mg) at week 0 and followed by 120 mg every two weeks (120 Q2W, n=387), 120 mg every four weeks (120 Q4W, n=389) or placebo Q2W (n=388). PRIMARY ENDPOINT: proportion of patients achieving SLE Responder Index 5 (SRI-5) response at week 52. RESULTS: Similar proportions of patients in each group achieved SRI-5 response at week 52 (120 Q2W: 31.8%; 120 Q4W: 35.2% and placebo: 29.3%). Key secondary endpoints were not met. In a sensitivity analysis not excluding patients who decreased antimalarials or immunosuppressants, SRI-5 response was achieved with 120 Q4W (37.0% vs 29.8% placebo; p=0.021), but not 120 Q2W (34.1%; p=0.171). Significant reductions in anti-dsDNA antibodies, increases in C3 and C4, and reductions in total B cells and immunoglobulins were observed with tabalumab. No differences were observed between treatment groups in percentage of deaths (120 Q2W: 0.8%; 120 Q4W: 0.5%; placebo: 0.5%), serious adverse events (AEs) (range 11.1-14.4%) or treatment-emergent AEs (range 81.1-82.3%). CONCLUSIONS: Tabalumab had biological activity-changes in anti-dsDNA, complement, B cells and immunoglobulins-consistent with BAFF pathway inhibition. Key clinical efficacy endpoints did not achieve statistical significance. Safety profiles were similar with tabalumab and placebo. TRIAL REGISTRATION NUMBER: NCT01196091.

Diagnosis and risk stratification in patients with anti-RNP autoimmunity.

INTRODUCTION: Anti-RNP autoantibodies occur either in mixed connective tissue disease (MCTD) (with a frequently favorable prognosis), or in systemic lupus erythematosus (SLE) cases with aggressive major organ disease. It is uncertain how to assess for the risk of severe disease in anti-RNP + patients. METHODS: Following institutional review board-approved protocols, clinical data and blood were collected from patients with known or suspected anti-RNP autoimmunity and normal controls in a cohort study. Samples were screened for parameters of immune activation. Groups were compared based on clinical diagnoses, disease classification criteria, disease activity and specific end-organ clinical manifestations. RESULTS: Ninety-seven per cent of patients satisfying Alarcon-Segovia MCTD criteria also met Systemic Lupus International Collaborating Clinic (SLICC) SLE criteria, while 47% of the anti-RNP + SLE patients also met MCTD criteria. Among SLICC SLE patients, MCTD criteria were associated with reduced rates of renal disease (odds ratio (OR) 4.3, 95% confidence interval (CI) 1.3-14.0), increased rates of Raynaud's phenomenon (OR 3.5, 95% CI 1.3-9.5) and increased serum B-cell maturation antigen, transmembrane activator and CAML interactor and TNFα levels. Circulating immune markers and markers of type I interferon activation were not effective at distinguishing clinical subgroups. CONCLUSIONS: Among anti-RNP patients, the question of MCTD versus SLE is not either/or: most MCTD patients also have lupus. MCTD classification criteria (but not a broad set of immune markers) distinguish a subset of SLE patients at reduced risk for renal disease.

Seed train development for the fermentation of bagasse from sweet sorghum and sugarcane using a simplified fermentation process.

A process was developed for seed culture expansion (3.6 million-fold) using 5% of the hemicellulose hydrolysate from dilute acid pretreatment as the sole organic nutrient and source of sugar. Hydrolysate used for seed growth was neutralized with ammonia and combined with 1.0mM sodium metabisulfite immediately before inoculation. This seed protocol was tested with phosphoric acid pretreated sugarcane and sweet sorghum bagasse using a simplified process with co-fermentation of fiber, pentoses, and hexoses in a single vessel (SScF). A 6h liquefaction (L) step improved mixing prior to inoculation. Fermentations (L+SScF process) were completed in 72 h with high yields (>80 gal/US ton). Ethanol titers for this L+SScF process ranged from 24 g/L to 32 g/L, and were limited by the bagasse concentration (10% dry matter).

Injection of air into the headspace improves fermentation of phosphoric acid pretreated sugarcane bagasse by Escherichia coli MM170.

Microaeration (injecting air into the headspace) improved the fermentation of hemicellulose hydrolysates obtained from the phosphoric acid pretreatment of sugarcane bagasse at 170°C for 10 min. In addition, with 10% slurries of phosphoric acid pretreated bagasse (180°C, 10 min), air injection into the headspace promoted xylose utilization and increased ethanol yields from 0.16 to 0.20 g ethanol/g bagasse dry weight using a liquefaction plus simultaneous saccharification and co-fermentation process (L+SScF). This process was scaled up to 80 L using slurries of acid pretreated bagasse (96 h incubation; 0.6L of air/min into the headspace) with ethanol yields of 312-347 L (82-92 gal) per tone (dry matter), corresponding to 0.25 and 0.27 g/g bagasse (dry weight). Injection of small amounts of air into the headspace may provide a convenient alternative to subsurface sparging that avoids problems of foaming, sparger hygiene, flotation of particulates, and phase separation.

Effect of reduced sulfur compounds on the fermentation of phosphoric acid pretreated sugarcane bagasse by ethanologenic Escherichia coli.

The addition of reduced sulfur compounds (thiosulfate, cysteine, sodium hydrosulfite, and sodium metabisulfite) increased growth and fermentation of dilute acid hydrolysate of sugarcane bagasse by ethanologenic Escherichia coli (strains LY180, EMFR9, and MM160). With sodium metabisulfite (0.5mM), toxicity was sufficiently reduced that slurries of pretreated biomass (10% dry weight including fiber and solubles) could be fermented by E. coli strain MM160 without solid-liquid separation or cleanup of sugars. A 6-h liquefaction step was added to improve mixing. Sodium metabisulfite also caused spectral changes at wavelengths corresponding to furfural and soluble products from lignin. Glucose and cellobiose were rapidly metabolized. Xylose utilization was improved by sodium metabisulfite but remained incomplete after 144 h. The overall ethanol yield for this liquefaction plus simultaneous saccharification and co-fermentation process was 0.20 g ethanol/g bagasse dry weight, 250 L/tonne (61 gal/US ton).

Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160.

Hexose and pentose sugars from phosphoric acid pretreated sugarcane bagasse were co-fermented to ethanol in a single vessel (SScF), eliminating process steps for solid-liquid separation and sugar cleanup. An initial liquefaction step (L) with cellulase was included to improve mixing and saccharification (L+SScF), analogous to a corn ethanol process. Fermentation was enabled by the development of a hydrolysate-resistant mutant of Escherichia coli LY180, designated MM160. Strain MM160 was more resistant than the parent to inhibitors (furfural, 5-hydroxymethylfurfural, and acetate) formed during pretreatment. Bagasse slurries containing 10% and 14% dry weight (fiber plus solubles) were tested using pretreatment temperatures of 160-190°C (1% phosphoric acid, 10 min). Enzymatic saccharification and inhibitor production both increased with pretreatment temperature. The highest titer (30 g/L ethanol) and yield (0.21 g ethanol/g bagasse dry weight) were obtained after incubation for 122 h using 14% dry weight slurries of pretreated bagasse (180°C).

T cells in the pathogenesis of systemic lupus erythematosus.

The role of T cells in the pathogenesis of systemic lupus erythematosus (SLE) is reviewed with a focus on autoantigen-specific T cells in SLE. The initial clue to a role for T cells in SLE was histopathologic studies demonstrating extensive infiltration of T cells at the sites of inflammation. Later studies, showing association between HLA polymorphisms and specific autoantibodies, directly implicated a role for T cells in autoantibody production. More recently, we and others have identified and characterized autoantigen-specific T cells in SLE. We review these studies on the role of autoantigen-specific T cells in SLE and present new findings on the molecular characterization of T cell immunity to Sm-B, Sm-D and U1-70kD small nuclear ribonucleoprotein (snRNP) autoantigens.

Structural analysis of TCRalpha and beta chains from human T-Cell clones specific for small nuclear ribonucleoprotein polypeptides Sm-D, Sm-B and U1-70 kDa: TCR complementarity determining region 3 usage appears highly conserved.

Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are systemic autoimmune diseases that are characterized by the presence of autoantibodies reactive with U small nuclear RNP (snRNP) autoantigens. Both B and T cells are important in the pathogenesis of the disease, and T- and B-cell immunity against snRNP polypeptides have been shown to be linked in vivo. Currently, several alternative hypotheses for the pathogenesis of these diseases have been proposed. These include loss of tolerance, modified self-antigens, molecular mimicry and nondirected immune activation. To help distinguish between the various models of disease pathogenesis, we have characterized the T-cell receptor (TCR) CDR3 from a large panel of well-characterized human T-cell clones and lines specific for individual snRNP polypeptides. The results presented here reveal highly restricted TCR usage across patients by the snRNP-reactive T cells based on the deduced amino acid sequence of the CDR3 loop. These data support the hypothesis that T-cell responses against self antigens in SLE and MCTD are antigen driven and that there are a limited number of T-cell epitopes present on the snRNP autoantigens.

T cell epitope mapping of the Smith antigen reveals that highly conserved Smith antigen motifs are the dominant target of T cell immunity in systemic lupus erythematosus.

B cell and T cell immunity to the Smith Ag (Sm) is a characteristic feature of systemic lupus erythematosus (SLE). We have shown that T cell immunity against Sm can be detected in SLE patients, and that T and B cell immunity against Sm are linked in vivo. TCR usage by Sm-reactive T cells is highly restricted and characteristic of an Ag-driven immune response. Sm is a well-characterized complex Ag consisting of proteins B1, B2, D1, D2, D3, E, F, and G. A unique feature of all Sm proteins is the presence of homologous motifs, Sm motif 1 and Sm motif 2. We used limiting dilution cloning and synthetic peptide Ags to characterize the human T cell immune response against Sm in seven SLE patients. We sought to determine the precise antigenic peptides recognized, the common features of antigenic structure recognized, and the evolution of the T cell response against Sm. We found there was a highly restricted set of Sm self-peptides recognized by T cells, with three epitopes on Sm-B and two epitopes on Sm-D. We found that T cell immunity against Sm-B and Sm-D was encoded within the highly conserved Sm motif 1 and Sm motif 2, and that immunity against these epitopes appeared stable. The present study supports the concept that T cell immunity to Sm is an Ag-driven immune response directed against a highly restricted set of self-peptides, encoded within Sm motif 1 and Sm motif 2, that is shared among all Sm proteins.

The appearance of U1 RNP antibody specificities in sequential autoimmune human antisera follows a characteristic order that implicates the U1-70 kd and B'/B proteins as predominant U1 RNP immunogens.

OBJECTIVE: To observe the order of development of anti-U1 RNP peptide antibodies in humans. METHODS: Immunoblots against Jurkat cell lysates were performed on 5,882 serum samples from 3,668 patients referred on clinical grounds for RNP antibody testing to a reference laboratory between 1989 and 1999. In patients from whom multiple samples were drawn, we determined the order in which IgG antibodies to the U1 RNP peptides A, B'/B, C, D, and 70 kd appeared. RESULTS: One hundred sixty-three patients with serial samples were identified in whom antibodies to at least one U1 RNP peptide initially were not present but later appeared. The first RNP antibodies to appear were most often directed against the 70 kd and B'/B peptides (P < 0.01). Antibodies to the A and C peptides usually developed after other RNP peptide antibodies, and antibodies to D often emerged only after immunity to multiple other U1 RNP proteins had appeared. B'/B, but not 70 kd, was a frequent early target of spreading after initial immunity to other RNP peptides. CONCLUSION: Orderly patterns of emergence of U1 RNP peptide antibodies appear to exist in humans. Two peptides, 70 kd and B'/B, show characteristics of early immunogens in the development of human RNP immunity.

Mixed connective tissue disease.

A defining feature of mixed connective tissue disease (MCTD) is the presence of antibodies against the U1-ribonucleoprotein (RNP) complex, but other autoantibodies in MCTD have recently been described. Research has also further elucidated the immune responses directed against U1-RNP in humans and in murine models of disease. Hypotheses implicating modified self-antigens and/or infectious agents in the pathogenesis of MCTD have been advanced. Links between the immunologic and clinical phenomena in MCTD are emerging. Longitudinal study of patients with MCTD highlights the impact of pulmonary hypertension on disease outcome.

Autoantibody recognition of distinctly modified forms of the U1-70-kd antigen is associated with different clinical disease manifestations.

OBJECTIVE: To examine whether autoantibody recognition of modified forms of the U1-70-kd RNP antigen correlates with manifestations of rheumatic disease. METHODS: Blinded to clinical disease manifestations, sera from 27 rheumatic disease patients with U1-70-kd antibodies were used to immunoblot control, apoptotic, and oxidatively modified HeLa cell lysates. Using densitometry, recognition of antigen fragments was quantitated. The presence or absence of 1) lupus skin disease and 2) Raynaud's phenomenon (RP) was determined for each patient by chart review. The ability of patient sera to recognize the different fragments was compared for patients with and without skin disease and with and without RP. RESULTS: Patients with lupus skin disease had higher recognition of apoptotic U1-70 kd than did patients without skin disease (mean +/- SD fragment recognition index [FRI] 1.35 +/- 0.57 versus 0.95 +/- 0.25; P < 0.024, by Student's t-test). Patients with RP had higher recognition of oxidatively modified U1-70 kd than did patients without RP (mean +/- SD FRI 0.95 +/- 0.80 versus 0.24 +/- 0.22; P < 0.048). CONCLUSION: Recognition of apoptotically and oxidatively modified forms of the U1-70-kd autoantigen are associated with distinct clinical rheumatic disease manifestations. This finding provides in vivo evidence for the hypothesis that immune recognition of modified forms of self antigens may be relevant to the pathogenesis of systemic rheumatic diseases. Understanding the antigenic modifications to which patients react may help predict the expression of rheumatic syndromes.

Immunization with a bacterial ATP-binding cassette transporter fragment suppresses autoimmunity and prolongs survival in MRL/lpr lupus-prone mice.

The present study was undertaken to better define the role of the U1 70 kDa antigen in a spontaneous murine model of systemic lupus erythematosus (SLE) by testing whether immunization with the U1 70 kDa polypeptide could alter the production of antibodies against U1 70 kDa or against other small nuclear ribonucleoproteins (snRNP), modify disease expression or alter survival. We found that, while immunization with a U1 70 kDa derived fusion protein (70 KFP) tended to delay the development of anti-snRNP antibodies in the sera of MRL/lpr mice, it had no effect on autoimmune-mediated renal disease or survival. Unexpectedly, it was found that MRL/lpr mice immunized with a 367 amino acid fragment of a bacterial ATP-binding cassette transporter, MFP, had prolonged survival compared to saline injection or U1 70 kDa immunization and that this was associated with a delay in the onset of SLE-like proliferative glomerulonephritis. This is the first study, to our knowledge, in which a bacterial ATP-binding cassette transporter was shown to be beneficial in treating a murine model of SLE. We report that MFP significantly prolonged longevity in the MRL/lpr murine model of SLE compared to saline injection or 70 KFP immunization and that improved survival was associated with a delay in the onset of SLE-like glomerulonephritis.

Comparison of 18S and ITS-1 rDNA sequences of selected geographic isolates of Myxobolus cerebralis.

Myxobolus cerebralis, the myxosporean parasite-causing salmonid whirling disease, was first reported among rainbow trout (Oncorhynchus mykiss) in Germany in 1903. The parasite was reported for the first time in North America in 1958 among hatchery-reared trout in the eastern USA, presumably arriving with frozen trout shipments from Europe. A comparison of 18S and ITS-1 ribosomal DNA sequences was conducted to identify potential strain differences between selected geographic isolates of this parasite from Europe and North America. Only fourteen of 1700 base pairs were different in the 18S rRNA gene from isolates obtained from California and West Virginia in the USA, and the Federal German Republic. No evidence for strain differences was obtained from ITS-1 sequences that were found to be identical among all parasite isolates. This finding is consistent with the hypothesis that the parasite was recently introduced to the USA from Europe.

Induction of primary human CD8+ T lymphocyte responses in vitro using dendritic cells.

The ability of two different human professional APCs, specifically macrophages (Mphi) and dendritic cells (DC), to stimulate primary responses in human CD8+ T lymphocytes was examined using both allogeneic and Ag-pulsed autologous APCs. CTL responses in CD8+ T lymphocytes isolated from HIV-uninfected donors were evaluated against six different HIV epitopes that are restricted by four different HLA alleles using autologous human PBMC-derived Mphi and DCs for primary stimulation. In a side-by-side experiment, immature DCs, but not Mphi, were able to prime a CTL response against the B14-restricted p24gag 298-306 epitope; mature DCs were also able to prime a response against this epitope. In addition, DCs were capable of priming CD8+ CTL responses against the B8-restricted p24gag 259-267 epitope. In contrast, Mphi were unable to prime strong CTL responses against other epitopes. Since the Ag-specific cytotoxic responses required subsequent rounds of restimulation before they could be detected, the ability of the allogeneic Mphi and DCs to directly prime CD8+ T lymphocyte responses without subsequent restimulation was examined. Similar to the aforementioned peptide-specific results, DCs were more efficient than Mphi in priming both allogeneic proliferative and cytotoxic responses in human CD8+ T lymphocytes. Collectively, these results promote an enhanced status for DCs in the primary stimulation of human CD8+ T lymphocytes.

Analysis of T cell receptors specific for U1-70kD small nuclear ribonucleoprotein autoantigen: the alpha chain complementarity determining region three is highly conserved among connective tissue disease patients.

The U1-70kD autoantigen is a major target of B cell responses in patients with connective tissue diseases (CTD). T cell responses are important in the pathogenesis of CTD, however little is known about autoantigen-specific T cells in these diseases. We have recently proven that U1-70kD-reactive human T cells exist. To further characterize these autoreactive T cells, U1-70kD-reactive T cell clones have been generated from patients with CTD using either a recombinant fusion protein or synthetic peptides spanning the U1-70kD polypeptide. T cell receptors (TCR) isolated from the U1-70kD-reactive T cell clones were sequenced and the third complementarity-determining region (CDR3) compared to determine if a common motif was present. mAb blocking of antigen-induced proliferation was done to determine the HLA restriction element used in recognition of the U1-70kD autoantigen by T cells. The results presented here indicate that TCRAV CDR3 usage is highly restricted among U1-70kD autoantigen-specific human T cells clones derived from CTD patients with distinctive structural features. Furthermore, the recognition of the U1-70kD autoantigen occurs in the context of HLA-DR.

Long-term outcome in mixed connective tissue disease: longitudinal clinical and serologic findings.

OBJECTIVE: To determine the long-term clinical and immunologic outcomes in a well-characterized cohort of 47 patients with mixed connective tissue disease (MCTD), including reactivity with U small nuclear RNP (snRNP) polypeptides. METHODS: Patients were followed up over a period of 3-29 years with immunogenetic and systematic clinical and serologic analysis. Sera were analyzed for reactivity with snRNP polypeptides U1-70 kd, A, C, B/B', and D, for anti-U1 RNA, and for anticardiolipin antibodies (aCL). RESULTS: The typical core clinical features of MCTD tended to develop over time; features of inflammation as well as Raynaud's phenomenon and esophageal hypomotility diminished, while pulmonary hypertension, pulmonary dysfunction, and central nervous system disease persisted, following treatment. A favorable outcome was observed in 62% of patients; 38% had continued active disease or had died, with death associated with pulmonary hypertension and aCL. All patients had autoantibodies to the U1-70 kd polypeptide of snRNP, and most were positive for anti-U1 RNA. An orderly progression of intramolecular spreading of autoantibody reactivity against snRNP polypeptides was observed, as was the novel finding of "epitope contraction" followed by disappearance of anti-snRNP autoantibodies during prolonged remission. CONCLUSION: These patients demonstrated the typical immunogenetic, clinical, and serologic findings of MCTD, and the condition rarely evolved into systemic lupus erythematosus or systemic sclerosis. The majority of patients had favorable outcomes, with pulmonary hypertension being the most frequent disease-associated cause of death. Intramolecular spreading of autoantibody reactivity against snRNP polypeptides was observed, followed by "epitope contraction" and ultimate disappearance of anti-snRNP autoantibodies during prolonged disease remission.

T cell receptor beta-chain third complementarity-determining region gene usage is highly restricted among Sm-B autoantigen-specific human T cell clones derived from patients with connective tissue disease.

OBJECTIVE: To determine the structure of T cell receptors (TCR) used by Sm-B-reactive human T cell clones, to map T cell epitopes on the Sm-B autoantigen, and to determine the HLA restriction element used in the recognition of Sm-B by T cells. METHODS: Sm-B-reactive T cell clones were generated from patients with connective tissue disease by using either a recombinant fusion protein or synthetic peptides. The TCR structure was defined with the use of polymerase chain reaction and DNA sequencing. Synthetic peptides were used to map T cell epitopes on Sm-B. HLA restriction element usage was defined by using monoclonal antibody blocking. RESULTS: Usage of the TCR third complementarity-determining region (CDR3) was highly restricted among Sm-B autoantigen-specific human T cell clones. Only amino acids 48-96 of the Sm-B2 autoantigen were recognized by T cells, and this occurred in the context of HLA-DR. CONCLUSION: TCR CDR3 gene usage is highly conserved by Sm-B autoantigen-specific T cell clones, and this appears to be related to the recognition of a limited number of T cell epitopes on the Sm-B autoantigen presented in the context of HLA-DR.