Serum golimumab concentration and anti-drug antibodies are associated with treatment response and drug survival in patients with inflammatory joint diseases: data from the NOR-DMARD study.

Objectives: This study aimed to identify the therapeutic target concentration and frequency of anti-drug antibodies (ADAbs) in golimumab-treated patients with inflammatory joint disease (IJD).Method: Associations between golimumab concentration, ADAbs, and treatment response were examined in 91 patients with IJD [41 axial spondyloarthritis (axSpA), 20 rheumatoid arthritis (RA), and 30 psoriatic arthritis (PsA)] included in the NOR-DMARD study. Treatment response was defined by Ankylosing Spondylitis Disease Activity Score (ASDAS) clinically important improvement in axSpA, European League Against Rheumatism (EULAR) good/moderate response in RA, and improvement of ≥ 50% in modified Disease Activity index for PSoriatic Arthritis (DAPSA) (28 swollen/tender joint counts) in PsA. Serum drug concentrations and ADAbs were analysed using automated in-house assays.Results: At inclusion, 42% were biological disease-modifying anti-rheumatic drug naïve and 42% used concomitant synthetic disease-modifying anti-rheumatic drug. The median golimumab concentration was 2.2 (interquartile range 1.0-3.5) mg/L. The proportions of responders after 3 months among patients with golimumab concentration < 1.0, 1.0-3.9, and ≥ 4.0 mg/L were 19%, 49%, and 74%, respectively. A higher rate of treatment discontinuation was seen in patients with serum golimumab concentration < 1.0 compared to ≥ 1.0 mg/L (hazard ratio 3.3, 95% confidence interval 1.8-6.0, p < 0.05). ADAbs were detected in 6%, and were associated with lower drug concentrations and both reduced treatment response and drug survival.Conclusions: Golimumab concentrations ≥ 1.0 mg/L were associated with improved treatment response and better drug survival, although some patients may benefit from higher concentrations. This study suggests a rationale for dosing guided by therapeutic drug monitoring in golimumab-treated patients with IJD. The results should be confirmed in larger studies including trough samples, and the efficacy of such a strategy must be examined in randomized controlled trials.

Long-term efficacy and safety of biosimilar infliximab (CT-P13) after switching from originator infliximab: open-label extension of the NOR-SWITCH trial.

BACKGROUND AND OBJECTIVES: The 52-week, randomized, double-blind, noninferiority, government-funded NOR-SWITCH trial demonstrated that switching from infliximab originator to less expensive biosimilar CT-P13 was not inferior to continued treatment with infliximab originator. The NOR-SWITCH extension trial aimed to assess efficacy, safety and immunogenicity in patients on CT-P13 throughout the 78-week study period (maintenance group) versus patients switched to CT-P13 at week 52 (switch group). The primary outcome was disease worsening during follow-up based on disease-specific composite measures. METHODS: Patients were recruited from 24 Norwegian hospitals, 380 of 438 patients who completed the main study: 197 in the maintenance group and 183 in the switch group. In the full analysis set, 127 (33%) had Crohn's disease, 80 (21%) ulcerative colitis, 67 (18%) spondyloarthritis, 55 (15%) rheumatoid arthritis, 20 (5%) psoriatic arthritis and 31 (8%) chronic plaque psoriasis. RESULTS: Baseline characteristics were similar in the two groups at the time of switching (week 52). Disease worsening occurred in 32 (16.8%) patients in the maintenance group vs. 20 (11.6%) in the switch group (per-protocol set). Adjusted risk difference was 5.9% (95% CI -1.1 to 12.9). Frequency of adverse events, anti-drug antibodies, changes in generic disease variables and disease-specific composite measures were comparable between arms. The study was inadequately powered to detect noninferiority within individual diseases. CONCLUSION: The NOR-SWITCH extension showed no difference in safety and efficacy between patients who maintained CT-P13 and patients who switched from originator infliximab to CT-P13, supporting that switching from originator infliximab to CT-P13 is safe and efficacious.

Assays for Infliximab Drug Levels and Antibodies: A Matter of Scales and Categories.

Immunogenicity is a frequent cause of secondary non-response to tumour necrosis factor (TNF) inhibitors. Drug level measurement and detection of antidrug antibodies have been shown to be cost effective and clinically relevant, and a large number of assays are available for these purposes. It is, however, difficult to compare assays and translate results into clinical meaningful information due to different methodological approaches and a lack of assay standardization. We have analysed infliximab drug levels and antidrug antibodies in 107 patient samples using enzyme-linked immunoassays (ELISA), immunofluorometric assays (IFMA) and reporter-gene assays (RGA). The RGA gave the lowest results for drug levels, whereas the IFMA detected the highest number of antidrug antibody positive sera. Applying individualized therapeutic ranges to each assay resulted in agreement among all three assays in 74% of samples for drug levels and 98% of samples for antidrug antibodies. We found that TNF inhibitor monitoring assays measure on different scales and that the agreement between quantitative results is limited. However, interassay differences can partially be overcome by assay-individualized translations of quantities into categories, which also is necessary for a meaningful clinical application. Our data demonstrate that assays should not be used interchangeably and that direct comparison of quantitative drug levels obtained with different assays should be avoided.

Long term performance of porous platinum coated neural electrodes.

Over the last several years, there has been a growing interest in neural implants for the study and diagnostics of neurological disorders as well as for the symptomatic treatment of central nervous system related diseases. One of the major challenges is the trade-off between small electrode sizes for high selectivity between single neurons and large electrode-tissue interface areas for excellent stimulation and recording properties. This paper presents an approach of increasing the real surface area of the electrodes by creating a surface microstructure. Two major novelties let this work stand out from existing approaches which mainly make use of porous coatings such as platinum black or iridium oxide, or Poly(3,4-ethylenedioxythiophene) (PEDOT). Roughening is carried out by a dry etching process on the silicon electrode core before being coated by a sputtered platinum layer, eliminating complicated deposition processes as for the materials described above. The technology is compatible with any commonly used coating material. In addition, the surface roughening is compatible with high aspect ratio penetrating electrode arrays such as the well-established Utah electrode array, whose unique geometry presents a challenge in the surface modification of active electrode sites. The dry etching process is well characterized and yields a high controllability of pore size and depth. This paper confirms the superior electrochemical properties including impedance, charge injection capacity, and charge storage capacity of surface engineered electrode arrays compared to conventional arrays over a period of 12 weeks. Furthermore, mechanical stability of the modified electrodes was tested by implantation in the brain of a recently deceased rat. In conclusion, the larger interface surface of the electrodes does not only decrease the impedance which should lead to enhanced Signal to noise ratio (SNR) for recording purposes, but also yields higher charge injection capacities, which improve the stimulation characteristics of the implants.

Reusable High Aspect Ratio 3-D Nickel Shadow Mask.

Shadow Mask technology has been used over the years for resistless patterning and to pattern on unconventional surfaces, fragile substrate and biomaterial. In this work, we are presenting a novel method to fabricate high aspect ratio (15:1) three-dimensional (3D) Nickel (Ni) shadow mask with vertical pattern length and width of 1.2 mm and 40 µm respectively. The Ni shadow mask is 1.5 mm tall and 100 µm wide at the base. The aspect ratio of the shadow mask is 15. Ni shadow mask is mechanically robust and hence easy to handle. It is also reusable and used to pattern the sidewalls of unconventional and complex 3D geometries such as microneedles or neural electrodes (such as the Utah array). The standard Utah array has 100 active sites at the tip of the shaft. Using the proposed high aspect ratio Ni shadow mask, the Utah array can accommodate 300 active sites, 200 of which will be along and around the shaft. The robust Ni shadow mask is fabricated using laser patterning and electroplating techniques. The use of Ni 3D shadow mask will lower the fabrication cost, complexity and time for patterning out-of-plane structures.

Identification of a lymphokine that stimulates eosinophil differentiation in vitro. Its relationship to interleukin 3, and functional properties of eosinophils produced in cultures.

Factors stimulating eosinophil differentiation in vitro have been studied by means of a liquid bone marrow culture system in which the number of eosinophils is estimated directly by morphology or indirectly by assay for eosinophil peroxidase. The results show that eosinophil colonies are not formed in agar, emphasizing the importance of the liquid culture system. Three types of evidence identify a novel lymphokine, eosinophil-differentiating factor (EDF). (a) Coordinate analysis of lymphokine activity in media conditioned by a panel of parasite antigen and another panel of alloantigen-reactive T cell clones indicates that EDF is distinct from interleukin 2 (IL-2), IL-3, and bone marrow proliferation activity (BMPA). (b) A T hybrid (NIMP-TH1) produces EDF but no IL-2, IL-3, interferon, or colony-stimulating factor. (c) Gel filtration of conditioned media (CM) indicates that NIMP-TH1 and a T clone (NIMP-T2) produce EDF (Mr 46,000). NIMP-T2 also produced IL-3 (Mr 26,000) but this was easily separated from EDF. IL-3 is also shown to have eosinophil differentiation activity (EDA) but this represents a very small proportion of the EDA in T2-CM. Fractionation of WEHI-3-CM indicates that EDA from this source has a similar elution profile to IL-3 (Mr 35-36,000). Furthermore, a comparison of the relative activities in purified IL-3 and WEHI-3-CM indicates that all the EDA can be attributed to the IL-3 in the latter. EDF is shown to stimulate production of eosinophils in long-term bone marrow cultures; the kinetics of eosinophil production suggests that EDF is acting on committed precursors in the bone marrow. The transient nature of eosinophil production suggests that precursors from multipotential stem cells are not produced. The eosinophils produced in these cultures are morphologically normal and functional in that they lysed sheep red blood cells coated with IgG1, IgG2a, and IgG2b, but not with IgM, IgA, or IgE. In addition, they were capable of adhering to and killing Schistosoma mansoni schistosomula.

Murine eosinophil differentiation factor. An eosinophil-specific colony-stimulating factor with activity for human cells.

A purified murine lymphokine, eosinophil differentiation factor (EDF), was found to be a selective stimulus for the clonal proliferation and differentiation of murine eosinophil progenitor cells, establishing it as the murine eosinophil colony-stimulating factor (Eo-CSF). EDF was also active on human eosinophil progenitors and mature blood eosinophils, but had no effect on neutrophil or macrophage precursor cells, nor on blood neutrophils. In culture of human bone marrow cells, EDF stimulated equal numbers and equal sizes of eosinophil colonies to develop when compared with human placental conditioned medium, a source of human CSFs, suggesting that all responsive progenitor cells were stimulated. Clone transfer experiments and the linear relationship between number of bone marrow cells plated and colonies produced confirmed that the action of EDF was directly on eosinophil progenitor cells. EDF increased the capacity of human blood eosinophils, but not neutrophils, to kill antibody-coated tumor cells and to phagocytose serum-opsonized yeast cells. This functional activation was associated with the enhanced expression of functional antigens (GFA-1, GFA-2, and the receptor for C3bi) on eosinophils. The possession by EDF (Eo-CSF) of all the properties expected of a human eosinophil CSF raises the possibility that a human analog of this molecule exists, and is involved in the regulation of production and function of human eosinophils in vivo.

Characterization of monoclonal antibodies directed against squamous cell carcinoma antigens: report of the second TD-10 workshop.

Eight monoclonal antibodies directed against Squamous Cell Carcinoma Antigens (A1 and A2) were collected and evaluated by three working groups. Recombinant antigens, fusion proteins and native antigens from normal tissue were used to evaluate antibody specificity. Five antibodies reacted with both A1 and A2. Two of these antibodies (K123 and K131) showed related binding characteristics, whereas SCC140, K182 and SCC111 demonstrated unique epitope specificity and were not related to the reference antibodies included (F1H3, F2H7 and SCC107). SCC111 reacted particularly well with antigen on Western blot, indicating that the epitope was partly hidden when the antigen was in solution. Two antibodies (SCC103 and SCC109) reacted only with A2 and the fusion protein A1/A2, indicating that they recognized an A2 epitope in exon 8. The A2-specific antibodies are unique in their binding to A2 and are different from the reference antibodies included (SCC104 and K122). SCC103 is probably the best A2-specific antibody available. One antibody, K136, was A1-specific and is related to reference antibody K135. The new antibodies can be used to establish immunometric assays for specific measurement of A1, A2 or both A1 and A2 together.

Quantitation of 6-thioguanine residues in peripheral blood leukocyte DNA obtained from patients receiving 6-mercaptopurine-based maintenance therapy.

The antimetabolite 6-mercaptopurine is widely utilized in maintenance therapy for childhood acute lymphoblastic leukemia. Following p.o. administration, this prodrug undergoes extensive biotransformation, resulting in the generation of a plethora of metabolites including 2'-deoxy-6-thioguanosine triphosphate. Incorporation of 6-thioguanine (6-TG) bases into DNA is generally considered to be central to thiopurine-mediated cytotoxicity. We have developed a novel precolumn derivatization HPLC technique for quantifying 6-TG base accumulation into leukocyte DNA obtained from acute lymphoblastic leukemia patients receiving 6-mercaptopurine maintenance therapy. The method is based on enzymatic degradation of DNA to 2'-deoxyribonucleosides and the derivatization of released 2'-deoxy-6-thioguanosine with a thiol-reactive reagent containing a 7-amino-4-methylcoumarin-3-acetic acid fluorophore. The 2'-deoxy-6-thioguanosine-7-amino-4-methylcoumarin-3-acetic acid adduct is resolved by reversed-phase HPLC and quantified fluorometrically. Assay response is linear from 15 pmol to 60 fmol 6-TG bases/microgram DNA with a limit of quantitation corresponding to the incorporation of 1 6-TG residue per 50,000 bases. In a small cohort of acute lymphoblastic leukemia patients receiving p.o. 6-mercaptopurine-based maintenance therapy, significant interindividual variation in the accumulation of 6-TG bases into leukocyte DNA was revealed. The determined levels of drug base incorporation ranged from 95 to 710 fmol 6-TG bases/microgram DNA (6-TG base:nucleotide ratio 1:32,000 to 1:4,000). The assay may provide a novel methodology for pharmacological monitoring of thiopurine therapy either in the routine clinical setting or during studies of alternative routes of drug delivery.

Protective effects of tumor necrosis factor on murine hematopoiesis during cycle-specific cytotoxic chemotherapy.

Tumor necrosis factor (TNF) is a pleiotropic cytokine which exerts a wide range of effects when administered in vivo. Using a murine model, we have investigated the effect of pretreatment with 1 microgram (2.6 x 10(4) units) per mouse of recombinant murine TNF-alpha on hematopoietic recovery following administration of cyclophosphamide, 5-fluorouracil, methotrexate, or vinblastine. TNF pretreatment results in enhanced regeneration of circulating neutrophils and hematopoietic progenitors, as measured by in vivo and in vitro assays, in animals given cycle-specific chemotherapeutic agents. The results may suggest that TNF affects cycle kinetics in hematopoietic progenitor cell populations, thus making these cells less prone to the cytocidal effects of the chemotherapeutic agents. As myeloablation is a frequent and often critical side effect following cancer treatment, these findings may have clinical implications.

Restoring motor control and sensory feedback in people with upper extremity amputations using arrays of 96 microelectrodes implanted in the median and ulnar nerves.

OBJECTIVE: An important goal of neuroprosthetic research is to establish bidirectional communication between the user and new prosthetic limbs that are capable of controlling >20 different movements. One strategy for achieving this goal is to interface the prosthetic limb directly with efferent and afferent fibres in the peripheral nervous system using an array of intrafascicular microelectrodes. This approach would provide access to a large number of independent neural pathways for controlling high degree-of-freedom prosthetic limbs, as well as evoking multiple-complex sensory percepts. APPROACH: Utah Slanted Electrode Arrays (USEAs, 96 recording/stimulating electrodes) were implanted for 30 days into the median (Subject 1-M, 31 years post-amputation) or ulnar (Subject 2-U, 1.5 years post-amputation) nerves of two amputees. Neural activity was recorded during intended movements of the subject's phantom fingers and a linear Kalman filter was used to decode the neural data. Microelectrode stimulation of varying amplitudes and frequencies was delivered via single or multiple electrodes to investigate the number, size and quality of sensory percepts that could be evoked. Device performance over time was assessed by measuring: electrode impedances, signal-to-noise ratios (SNRs), stimulation thresholds, number and stability of evoked percepts. MAIN RESULTS: The subjects were able to proportionally, control individual fingers of a virtual robotic hand, with 13 different movements decoded offline (r = 0.48) and two movements decoded online. Electrical stimulation across one USEA evoked >80 sensory percepts. Varying the stimulation parameters modulated percept quality. Devices remained intrafascicularly implanted for the duration of the study with no significant changes in the SNRs or percept thresholds. SIGNIFICANCE: This study demonstrated that an array of 96 microelectrodes can be implanted into the human peripheral nervous system for up to 1 month durations. Such an array could provide intuitive control of a virtual prosthetic hand with broad sensory feedback.

Successful pregnancy in a diabetic patient treated with continuous ambulatory peritoneal dialysis.

In this article we describe the successful management of pregnancy and delivery in a 26-yr-old patient with advanced diabetic nephropathy and chronic renal failure. Targets for control of blood urea and hemoglobin were achieved with the aid of continuous ambulatory peritoneal dialysis (CAPD). Peritoneal dialysis did not interfere with normal recovery from cesarean section. With CAPD, successful pregnancy is now possible in this group of patients, among whom fetal loss would otherwise be high.

Induction of a reversible block in murine CFU-C differentiation by exposure to nitrous oxide.

Patients subjected to prolonged exposure to nitrous oxide (N2O) often develop megaloblastic bone marrow changes. This toxicity is due to the N2O-mediated inactivation of cobalamin-dependent enzymes with resultant perturbations in cell metabolism. The effect of N2O on the behavior of murine colony-forming units-cytokine (CFU-C) in vitro was studied by incubating granulocyte/macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cultures for 7 days in an atmosphere of either 5% CO2 in air or 50% N2O/5% CO2 in air. Exposure of bone marrow cells in agarose to N2O resulted in an approximately 50% reduction in colony formation when compared with cultures incubated in air. In contrast, when residual CFU-C numbers were determined in bone marrow liquid cultures after 7 days of incubation in the presence of GM-CSF, exposure to N2O was found to dramatically enhance CFU-C recovery. Since these liquid cultures contain a strong differentiation inducer, and are unable to support CFU-C generation, the enhancement of CFU-C recovery in N2O-exposed cultures appears to be related to its ability to induce a reversible block in CFU-C differentiation. The reversible block in CFU-C maturation seen in vitro parallels clinical observations where a rapid hematologic recovery is seen in N2O-exposed patients treated with hydroxycobalamin. These observations would suggest that N2O is not markedly cytotoxic to CFU-C and that its action is, at least in part, cytostatic in nature.

Effect of methotrexate on murine bone marrow cells in vitro: evidence of a reversible antiproliferative action.

Methotrexate (MTX) acts by inducing cellular depletion of reduced folates, which ultimately leads to an inhibition of DNA synthesis. Like many anticancer drugs, this antimetabolite has little selectivity for tumor cells, and its effectiveness is limited by toxicity to normal tissues, particularly gastrointestinal epithelium and bone marrow. Previous studies have shown that MTX inhibits colony formation of the hematopoietic progenitor cells (CFU-C) in vitro. Whether this effect is due to a cytotoxic or a cytostatic mechanism has not been resolved. The present study was undertaken to eludicate the mechanism by which MTX inhibits CFU-C formation. Bone marrow cells in agarose cultures supplemented with recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) were incubated for 7 days in the presence or absence of MTX. Exposure to 33 nM to 1 microM MTX reduced colony formation by more than 80% when compared to control cultures. When bone marrow suspension cultures supplemented with rmGM-CSF were incubated for 5 days in the presence or absence of MTX, exposure to 10 nM to 1 microM MTX resulted in a 60 to 80% reduction in cell numbers when compared to untreated cultures. Residual CFU-C numbers were determined in the same cultures by replating into agarose. Exposure to 10 nM MTX was found to enhance CFU-C recovery three-fold as compared to controls and cultures exposed to higher MTX concentrations. Addition of 10 microM of the reduced folate leucovorin (LV; 5-formyl-tetrahydrofolate) prevented CFU-C accumulation in the presence of 10 nM MTX. The kinetics of LV rescue of CFU-C, pre-exposed to 100 nM MTX, were investigated in clonogenic assays. The addition of 1 microM LV to semisolid bone marrow cultures preincubated with 100 nM MTX for up to 8 days completely abolished the inhibition of colony formation seen with 100 nM MTX alone. When the dose range of MTX was expanded from 33 nM to 3.3 microM, we found that administration of 10 microM LV on day 5 rescued the hematopoietic progenitors from MTX inhibition in all groups. These observations suggest that MTX is not cytotoxic to hematopoietic progenitors over its entire dose range but that it can induce a reversible block in the proliferation and differentiation of cells in the progenitor compartment.

Influence of interleukins 3,5, and 6 on the growth of eosinophil progenitors in highly enriched human bone marrow in the absence of serum.

Purified preparations of natural murine interleukin (IL)-5 and recombinant human IL-1 alpha, IL-3, and IL-6 were evaluated alone, and in combination, for effects in vitro on colony formation by eosinophil progenitors (eosinophil colony-forming units, CFU-Eos) in either nonadherent low-density T-lymphocyte depleted (NALT-) or fluorescence-activated cell sorted NALT-HLA-DR+ CD34 (My10) cells from normal human bone marrow, in the absence and presence of serum. Interleukins were assessed on CFU-Eos plated directly in agar, or on CFU-Eos placed in suspension culture for 7 days prior to plating in agar in the presence of IL-5. Eosinophil colonies were identified in situ by staining with Luxol fast blue. IL-3 and IL-5 acted in agar culture as direct stimulators of CFU-Eos, using highly purified cells in the HLA-DR+ My10 fraction of cells, the specificity for CFU-Eos being greatest for IL-5 and most demonstrable in the absence of serum. The IL-3 and IL-5 direct stimulating effects on CFU-Eos were additive. IL-1 alpha and IL-6 had little or no effect, alone or in combination with IL-3 and IL-5; however, IL-3, IL-5, and IL-6 each enhanced the number of IL-5-responsive CFU-Eos found after suspension culture compared to control medium, with the individual effects being additive when the molecules were combined. These results demonstrate that both IL-3 and IL-5, alone and in combination have direct-acting effects on CFU-Eos, with the greatest specificity for stimulation of pure Eos colonies and clusters residing in IL-5, and that IL-3, IL-5, and IL-6, alone and in combination, may play a role in the survival of CFU-Eos.

Effect of acute oral beta adrenergic blockade on muscle blood flow in man.

We have determined the effect of beta blocking drugs in man to ascertain whether cardioselectivity or intrinsic sympathomimetic activity influence muscle blood flow response to exercise. Exercise muscle blood flow was determined using a miniaturised lightweight cadmium telluride detector attached to the skin surface. Mean exercise muscle blood flow for the six subjects, during a constant work load, did not differ significantly either for predosing values on the six separate study days or at 2, 4 and 6 h after dosing with placebo. All the beta blocking agents caused a reduction in exercise muscle blood flow. Propranolol caused significant reduction in muscle blood flow at 2 h only. Metoprolol and atenolol produced the greatest and most prolonged effect and oxprenolol the smallest effect on exercise muscle blood flow. Intrinsic sympathomimetic activity may protect against reduction in exercise muscle blood flow in man.

Assessment of cadmium telluride detector system for measurement of muscle blood flow during dynamic exercise in man.

We have used a miniature cadmium telluride detector in the assessment of muscle blood flow during dynamic exercise in man following local injection of 133Xenon. The results obtained were compared with those using a conventional sodium iodide detector. There was no significant difference between the results obtained with the two detectors. The reproducibility of results was greater with cadmium telluride than with sodium iodide. The cadmium telluride system has the advantage of being small, lightweight and portable and enables measurements to be made during dynamic exercise.

Detection of eosinophils using an eosinophil peroxidase assay. Its use as an assay for eosinophil differentiation factors.

A colorimetric assay for peroxidase has been applied to the detection of eosinophils in bone marrow cultures and tissue cell suspensions. The substrate solution consists of 0.1 mM o-phenylenediamine in 0.05 M Tris-HCl buffer pH 8.0 containing 0.1% Triton and 1 mM hydrogen peroxide. The method is shown to be an easy and reproducible method of detecting eosinophils, with insignificant interference from neutrophils.