Secukinumab improves hand, foot and nail lesions in moderate-to-severe plaque psoriasis: subanalysis of a randomized, double-blind, placebo-controlled, regimen-finding phase 2 trial.

BACKGROUND: Psoriasis affecting sites such as the hands, feet and nails can be particularly difficult to treat. There are limited data on the efficacy of biological agents to treat these specific localizations. OBJECTIVE: This analysis of a phase 2 regimen-finding study evaluated the efficacy of secukinumab in subjects with moderate-to-severe psoriasis and non-pustular involvement of the hands, feet and/or nails. METHODS: Subjects were randomized (1 : 2 : 2 : 1) to one of three subcutaneous secukinumab 150-mg induction regimens [Single (Week 0), Monthly (Weeks 0, 4, 8), Early (Weeks 0, 1, 2, 4)] or placebo. In the subgroup (n = 131) with hand and/or foot psoriasis [baseline 5-point hand/foot Investigator's Global Assessment (IGA) score ≥2], efficacy was assessed as percentage of subjects achieving an IGA response [a score of 0 (clear) or 1 (minimal) and an improvement of ≥2 points on the 5-point hand/foot scale vs. baseline] at Week 12. In the subgroup (n = 304) with fingernail psoriasis (baseline composite score ≥1), efficacy was assessed as mean percentage change from baseline to Week 12 in a composite score. RESULTS: At Week 12, a markedly higher percentage of subjects with hand and/or foot psoriasis achieved an IGA response with the Early regimen vs. placebo (54.3% vs. 19.2%, P = 0.005). The composite fingernail score improved with the Early and Monthly regimens, but worsened with placebo [percentage mean change from baseline (SE): -19.1% (6.12) and -10.6% (7.06) vs. 14.4% (11.92); P = 0.010 vs. placebo for Early, P = 0.027 for Monthly). Secukinumab was well tolerated. CONCLUSION: Secukinumab demonstrated a beneficial effect on psoriasis of the hands/feet/nails in this short-term assessment.

Secukinumab induction and maintenance therapy in moderate-to-severe plaque psoriasis: a randomized, double-blind, placebo-controlled, phase II regimen-finding study.

BACKGROUND: Interleukin (IL)-17A has major proinflammatory activity in psoriatic lesional skin. OBJECTIVES: To assess the efficacy and safety of secukinumab, a fully human IgG1κ monoclonal anti-IL-17A antibody, in moderate-to-severe plaque psoriasis in a phase II regimen-finding study. METHODS: A total of 404 patients were randomized to subcutaneous placebo (n = 67) or one of three secukinumab 150 mg induction regimens: single (week 0; n = 66), early (weeks 0, 1, 2, 4; n = 133) and monthly (weeks 0, 4, 8; n = 138 patients). The primary outcome was ≥ 75% improvement from baseline Psoriasis Area and Severity Index score (PASI 75) at week 12. PASI 75 responders from active treatment arms at week 12 were rerandomized to either a fixed-interval (secukinumab 150 mg at weeks 12 and 24; n = 65) or a treatment-at-start-of-relapse maintenance regimen (secukinumab 150 mg at visits at which a start of relapse was observed; n = 67). RESULTS: At week 12, early and monthly induction regimens resulted in higher PASI 75 response rates vs. placebo (54·5% and 42·0% vs. 1·5%; P < 0·001 for both). Among PASI 75 responders at week 12 entering the maintenance period, PASI 75 and PASI 90 achievement at least once from week 20 to week 28 was superior with the fixed-interval regimen [85% (n = 55) and 58% (n = 38), respectively] vs. the start-of-relapse regimen [67% (n = 45), P = 0·020, and 21% (n = 14), respectively]. Fifteen weeks after last study drug administration, < 10% of patients in the fixed-interval and start-of-relapse groups experienced a start of relapse. No immunogenicity was observed, and no injection-site reactions were reported. Reported cases of neutropenia were mild-to-moderate (≤ grade 2); none was associated with clinically significant adverse events or resulted in study discontinuation. Due to the brief duration of the safety assessment, no firm conclusions can be drawn regarding long-term safety. CONCLUSIONS: Secukinumab shows efficacy for induction and maintenance treatment of moderate-to-severe plaque psoriasis.

How to make ends meet in V(D)J recombination.

In most vertebrate species analyzed so far, the diversity of soluble or membrane-bound antigen-receptors expressed by B and T lymphocytes is generated by V(D)J recombination. During this process, the coding regions for the variable domains of antigen-receptors are created by the joining of subexons that are randomly selected from arrays of tandemly repeated V, D (sometimes) and J gene segments. This involves the site-specific cleavage of chromosomal DNA by the lymphocyte-specific recombination-activating gene (RAG)-1/2 proteins, which appear to have originated from an ancient transposable element. The DNA double-strand breaks created by RAG proteins are subsequently processed and rejoined by components of the nonhomologous DNA end-joining pathway, which is conserved in all eukaryotic organisms - from unicellular yeast up to highly complex mammalian species.

Normal V(D)J recombination in cells from patients with Nijmegen breakage syndrome.

The majority of antigen receptor diversity in mammals is generated by V(D)J recombination. During this process DNA double strand breaks are introduced at recombination signals by lymphoid specific RAG1/2 proteins generating blunt ended signal ends and hairpinned coding ends. Rejoining of all DNA ends requires ubiquitously expressed DNA repair proteins, such as Ku70/86 and DNA ligase IV/XRCC4. In addition, the formation of coding joints depends on the function of the scid gene encoding the catalytic subunit of DNA-dependent protein kinase, DNA-PK(CS), that is somehow required for processing of coding end hairpins. Recently, it was shown that purified RAG1/2 proteins can cleave DNA hairpins in vitro, but the same activity was also described for a protein complex of the DNA repair proteins Nbs1/Mre11/Rad50. This leaves the possibility that either protein complex might be involved in coding end processing in V(D)J recombination. We have therefore analyzed V(D)J recombination in cells from patients with Nijmegen breakage syndrome, carrying a mutation in the nbs1 gene. We find that V(D)J recombination frequencies and the quality of signal and coding joining are comparable to wild-type controls, as analyzed by a cellular V(D)J recombination assay. In addition, we did not detect significant differences in CDR3 sequences of endogenous Ig lambdaL and kappaL chain gene loci cloned from peripheral blood lymphocytes of an NBS patient and of healthy individuals. These findings suggest that the Nbs1/Mre11/Rad50 complex is not involved in coding end processing of V(D)J recombination.

A comparative analysis of the phosphorylation and biochemical properties of wild type and host range variant DNA binding proteins of human adenovirus 5.

Specific mutation of the DNA binding protein (DBP) of human adenovirus types 2 and 5 can extend the host range of these viruses to simian cells. These mutations replace histidine at position 130 in the highly phosphorylated N-terminal domain of DBP with a potentially phophorylatable tyrosine residue. To investigate the possibility that alternative phosphorylation might contribute to the functional differences between wild type (wt) and host range (hr) DBP molecules, radiolabeled proteins were compared by partial proteolysis and tyrosine phosphorylation was analyzed. These studies confirmed the previous tentative assignment of a chymotrypsin-sensitive site at position 121 of DBP. No host range-specific tyrosine phosphorylation was detected, and no gross difference in the extent of phosphorylation between wt and hr DBP was observed. However, the cleaved N-terminal domains of wt and hr DBP exhibited different sensitivities to further chymotryptic digestion in vitro and different fragmentation patterns, suggesting that they might have different conformations. Such a difference could underlie the differing ability of these proteins to support Ad replication in simian cells.

Monoclonal antibodies that recognize the native human thyrotropin receptor.

Monoclonal antibodies have been produced that recognize the native human thyrotropin receptor by using a sensitive screening protocol based on flow cytofluorimetry combined with recombinant eukaryotic cells expressing high levels of the full-length functional receptor. The more standard screening method of ELISA preferentially selected antibodies that only reacted with the denatured receptor. Mice were immunized with recombinant receptor produced in either eukaryotic or prokaryotic systems; after screening and cloning, three stable hybridoma lines were established. An IgM antibody (7B5) produced in response to the eukaryotic material recognized only the native receptor (by flow cytofluorimetry) and did not react with denatured material on ELISA or immunoblotting, suggesting that its epitope is conformational. In contrast, two IgG1 antibodies (2C11 and 3B12) produced in response to the prokaryotic material recognized both native and denatured receptor (by flow cytofluorimetry, immunoprecipitation and immunoblotting). The use of different recombinant constructs in the immunoblotting procedure allowed the epitopes for both the IgG1 antibodies to be assigned to the region 125-369. None of the antibodies stimulated production of cAMP by recombinant cells expressing the full-length functional receptor, but one of the IgG1 antibodies (2C11) did inhibit binding of radiolabelled thyrotropin to these same cells. These antibodies, and others that can now be produced with this screening protocol, will help define the relationship between structure and function of this important receptor.

Production of antibodies to the human thyrotropin receptor and their use in characterising eukaryotically expressed functional receptor.

The structure of the human thyrotropin receptor expressed as a recombinant protein in eukaryotic cells was investigated by immunochemical and functional means using two types of polyclonal rabbit antisera: one raised against the large N-terminal extracellular region (residues 1-415) expressed in E. coli and the other raised against a synthetic peptide (residues 313-330). Both types of antisera gave similar results, with the former being more effective. As expected from the lack of conformation of the immunogens, the antisera worked well in immunoblotting. Less predictably, the antisera also recognised the functional receptor in its native state (detected by flow cytofluorimetry and immunoprecipitation), and inhibited the binding of thyrotropin. Thus the region 313-330 is on the outside of the receptor molecule and falls within, or close to, the binding site of thyrotropin. None of the antisera stimulated cAMP production, showing that this is a very special property, largely restricted to certain human autoantibodies. The antisera were used to immunoprecipitate radioiodinated proteins from Chinese hamster ovary cell (CHO) lines expressing recombinant receptor. The most abundant and reproducible cell-surface molecule that correlated with the presence of full-length functional receptor was a glycopolypeptide of approximately 100 kDa, of which 15 kDa is attributable to carbohydrate, in good agreement with the size predicted for the polypeptide from the cDNA sequence. Three other molecular species were also variably detected at the cell surface: 55 kDa, 180 kDa and large molecular weight material at the top of the polyacrylamide gel.(ABSTRACT TRUNCATED AT 250 WORDS)

Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model.

Intercellular junctions are fundamental to the interactions between cells. By means of these junctions, the activities of the individual cells that make up tissues are co-ordinated, enabling each tissue system to function as an integrated whole. In this review, the work of the authors on one specific type of junction--the cardiac gap junction--is presented as a case model to illustrate how the application of a range of microscopical methods, as part of a multidisciplinary approach, can help extend our understanding of cell junctions and their functions. In the heart, gap junctions form the low-resistance pathways for rapid impulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. Gap junctions also form pathways for direct intercellular communication, a function of particular importance for morphogenetic signalling during development. The work discussed demonstrates some of the applications of techniques in electron microscopy, immunocytochemistry and confocal scanning laser microscopy to the understanding of the structural basis of the function of gap junctions in the normal adult heart, the developing heart and the diseased heart. Freeze-fracture electron microscopy of heart tissue prepared by rapid freezing techniques, in which excision-related structural damage to the cells is minimized or avoided, makes it possible to deduce the structure of the functioning gap junction in vivo. Gap junctions in hearts that are beating normally in the living animal until the very instant of freezing consist of connexons (transmembrane channels) organized in a quasi-crystalline arrangement, not a 'random' arrangement as proposed in the original hypothesis on the structural correlates of gap junction function. Alterations in connexon arrangement occur in response to ischaemia and hypoxia, though the relationship of these to gap-junctional permeability is indirect. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera to synthetic peptides matching portions of the sequence of connexin43, the major gap-junctional protein reported in the heart, were raised. The specificity of the antisera was confirmed by dot blotting, Western blotting and by immunogold labelling of isolated gap junctions. One antiserum (that raised to residues 131-142) was found to be particularly effective as a cytochemical probe. An immunofluorescence labelling procedure for use with confocal scanning laser microscopy was developed to enable the three-dimensional precision mapping of gap junctions through thick slices of cardiac tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the extracellular region of the human thyrotrophin receptor expressed as a recombinant protein.

DNA encoding the N-terminal 415 residues of the human thyrotrophin receptor (predicted to code for the large extracellular region) was introduced into Chinese hamster ovary (CHO) cells using the glutamine synthetase/cytomegalovirus amplifiable expression system, and into E. coli using the pGEX-3X expression vector. Substantial quantities of insoluble fusion protein product resulted from bacterial expression; by Western blot analysis, this was shown to be reactive with anti-receptor antibodies raised against a peptide corresponding to residues 313-330. Immunoreactivity was not retained by the solubilized protein. In eukaryotic expression, several successful CHO transfectants were observed and one (ExG2) was characterized thoroughly. Using agarose-bound Concanavalin A, a glycoprotein with an M(r) of approximately 60,000 was detected in a detergent extract of metabolically labelled ExG2 cells, agreeing with the predicted molecular size of 45,000, plus carbohydrate. The same protein could also be detected by immunoprecipitation using the experimental anti-peptide antisera and also sera from patients with Graves' disease. The protein was immunoreactive in Western blot analyses of ExG2 cells using the experimental antisera but not the pathological sera, supporting the view that linear sequences are not sufficient for autoantibody binding. These are the first studies in which visualization of eukaryotically expressed recombinant receptor by such immunological techniques has been possible, presumably because of the higher expression of the glutamine synthetase system. Surprisingly, the recombinant protein was retained within the cells rather than being secreted. The recombinant protein was very effective at absorbing the adenylate cyclase-stimulating activity of the sera from patients with Graves' disease, but not that of thyrotrophin. This suggests that the large N-terminal extracellular region contains epitopes for stimulatory autoantibodies, but that high affinity thyrotrophin binding requires additional components.

Characterization of the glutamine synthetase amplifiable eukaryotic expression system applied to an integral membrane protein--the human thyrotropin receptor.

An amplifiable eukaryotic expression system, based upon glutamine synthetase, has been applied to the production of a complex integral membrane glycoprotein, the human receptor for the polypeptide hormone thyrotropin (TSH). Production of recombinant protein was achieved in chinese hamster ovary (CHO) cells at levels at least 10-fold higher than has been achieved in any other system. After amplification of the inserted gene, the gene copy number was found to be increased in most (but not all) subclones in the range of 3- to 50-fold; mRNA levels of the individual cell lines broadly followed their gene copy number. The level of protein production (measured both functionally and structurally, by radioligand binding and cytofluorimetry, respectively) also reflected these increases in DNA and RNA, but appeared to be limited to a maximum value which we conclude is the maximum that the cells can tolerate without impairing their viability. The receptor is efficiently coupled to adenylate cyclase (22-45 pM TSH producing a 50% response), although the coupling mechanism appeared to be saturated at higher receptor numbers. The high level of expression has allowed, for the first time, the detection of recombinant TSH receptor by immunochemical means. This expression system should prove very useful, not only in facilitating characterization of the TSH receptor, but also for the production of many other integral membrane proteins in their native form.

Nucleotide sequence and chromosomal assignment of a cDNA encoding the large isoform of human glutamate decarboxylase.

Glutamic acid decarboxylase (GAD) catalyses the conversion of L-glutamic acid to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Two forms of human GAD, GAD65 and GAD67, are encoded by two separate genes. A full length human GAD67 cDNA has been isolated from a human frontal cortex cDNA library and the nucleotide sequence determined. The GAD67 gene has been mapped to chromosome 2 using the polymerase chain reaction to amplify specifically the human sequence in rodent/human somatic cell hybrid DNA. This confirms that human GAD67 is not syntenic with the smaller GAD isoform GAD65 which has been assigned to chromosome 10. Production of polyclonal antiserum to a baculovirus-expressed GAD67 enabled immunocytological detection of GAD in the rat brain.

The use of the amplifiable high-expression vector pEE14 to study the interactions of autoantibodies with recombinant human thyrotrophin receptor.

In order to produce significant quantities of the human thyrotrophin (TSH) receptor we have investigated the use of two eukaryotic high expression systems. DNA encoding the receptor was obtained by the polymerase chain reaction (PCR) applied to thyroid cDNA. Receptor DNA was inserted into the baculovirus system; despite high mRNA levels there was little or no demonstrable protein production. However, using a novel amplifiable glutamine synthetase system, clones of transfected Chinese hamster ovary (CHO) cells expressed a high affinity TSH receptor (KD 0.225 +/- 0.046 nM, Bmax 20,000-45,000 sites/cell for individual clones). This was coupled to adenylate cyclase as measured by a TSH-stimulatable increase in extracellular cyclic AMP (cAMP), a detectable response being noted at 1 microU/ml TSH with half-maximal at around 25-50 microU/ml. The high expression allowed detection of both TSH binding inhibition and adenylate cyclase stimulation by autoantibodies in unfractionated sera from patients with Graves' disease.

Cardiac myocyte gap junctions: evidence for a major connexon protein with an apparent relative molecular mass of 70,000.

It is widely accepted that there is a family of gap junction connexon proteins, their distribution appearing to vary with tissue type and species. In cardiac tissues the major junctional channel component identified is a 43K (K = 10(3) Mr) polypeptide. Using a gap junction isolation protocol in which low temperatures are maintained, and which is detergent-free, we have identified a second gap junction-related protein in cardiac tissues with an apparent relative molecular mass of 70,000. Antibodies raised to three synthetic peptides matching portions of the 43K gap junction protein cDNA sequence cross-react with the 70K protein, but biochemical studies indicate that these proteins are distinct from one another. The structures that contain the 70K protein are susceptible to fragmentation at warm temperatures, and by electron microscopy this can be correlated with loss of 'minidomains' within the junctional plaque. Using a gap junction enriched-fraction prepared from purified isolated adult myocytes we show that both the 43K and 70K gap junction proteins are present in ventricular cardiac myocytes. In such preparations, and those from whole heart, the 70K protein appears to be the major gap junction-related protein present.

Cardiac gap junctions in rat ventricle: localization using site-directed antibodies and laser scanning confocal microscopy.

Antibodies were raised to three synthetic peptides, each constructed to match a different cytoplasmic region of the 43kD protein of the cardiac gap junction. These antibodies have all been used for immunohistochemical localization of gap junctions in the rat ventricle. Each is shown to have different staining characteristics which provide supporting evidence for the model proposed for the configuration of the gap junction protein within the membrane. Laser scanning confocal microscopy has confirmed that the localization of ventricular myocyte gap junctions is solely within intercalated disks. This technique is demonstrated to provide a greater overview of the three dimensional arrangement of the junctions than other microscopical techniques. Larger gap junctions occur typically at the periphery of the disk, with smaller junctions situated in the central zone. The analysis of the number and distribution of gap junctions over large volumes of myocardial tissue (three-dimensional tomography) is now feasible with this approach, and may be used to delineate conduction pathways.

Analysis of the rat liver gap junction protein: clarification of anomalies in its molecular size.

The major gap junction polypeptide in most tissues has an apparent molecular mass of 27 kDa with a 47 kDa dimer present in junction-enriched fractions. However, a 54 kDa protein recognized by gap junction-specific antibodies has been reported and a complementary DNA (cDNA) sequence for both human and rat liver gap junctions codes for a 32 kDa protein. In this paper we show that these are all forms of the same gap junction protein that can be observed on SDS-polyacrylamide gels simply by varying the concentration of acrylamide in the gels. A 64 kDa dimer is also obtainable. Antibodies to the gap junction protein or to a synthetic peptide constructed to match the rat liver gap junction amino-terminal sequence recognize all of these forms. Under some conditions a 54 kDa dimer is 'preferred', explaining the presence of this species in whole tissue homogenate Western blots. These results clarify several controversies and indicate that the protein forming the gap junction channel probably undergoes no major post-translational modification as the cDNA sequence codes for a protein of molecular mass 32 kDa and this protein species and its 64 kDa dimer are demonstrable on SDS-polyacrylamide gels under appropriate conditions.