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.

Fast and efficient synthesis of Zorro-LNA type 3'-5'-5'-3' oligonucleotide conjugates via parallel in situ stepwise conjugation.

Zorro-LNA is a new class of therapeutic anti-gene oligonucleotides (ONs) capable of invading supercoiled DNA. The synthesis of single stranded Zorro-LNA is typically complex and laborious, requiring reverse phosphoramidites and a chemical linker connecting the two separate ON arms. Here, a simplified synthesis strategy based on 'click chemistry' is presented with a high potential for screening Zorro-LNA ONs directed against new anti-gene targets. Four different Zorro type 3'-5' 5'-3' constructs were synthesized via parallel in situ Cu(i) [3 + 2] catalysed cycloaddition. They were prepared from commercially obtained ONs functionalized on solid support (one ON with the azide and the other ON with the activated triple bond linker N-propynoylamino)-p-toluic acid (PATA)) and after cleavage from resin, they were conjugated in solution. Our report shows the benefit of combining different approaches when developing anti-gene ONs, (1) the ability for rapid and robust screening of potential targets and (2) refining the hits with more anti-gene optimized constructs. We present as well the first report showing double-strand invasion (DSI) efficiency of two combined Zorro-LNAs.

Celiac disease and risk of fracture in adults--a review.

Patients with celiac disease (CD) have low bone mineral density. Evidence of increased fracture risk in these patients is conflicting, and the indication for bone mineral density screening of all adult CD patients is debated. Our aim was to review current published data on fractures in CD. Cross-sectional cohort studies and one case study were identified by searching Medline and Embase. Although the identified studies are heterogeneous and difficult to compare, the overall findings indicate a positive association between CD and risk of fracture. Adult patients with CD should be considered for bone densitometry in order to estimate fracture risk.

Density of CD163+ CD11c+ dendritic cells increases and CD103+ dendritic cells decreases in the coeliac lesion.

Coeliac disease is a chronic inflammation of the intestinal mucosa controlled by gluten-specific T cells restricted by disease-associated HLA-DQ molecules. We have previously reported that mucosal CD11c(+) dendritic cells (DCs) are responsible for activation of gluten-reactive T cells within the coeliac lesion. In mice, intestinal CD11c(+) DCs comprise several functionally distinct subsets. Here, we report that HLA-DQ(+) antigen-presenting cells (APCs) in normal human duodenal mucosa can be divided into four subsets with striking similarities to those described in mice: CD163(+) CD11c(-) macrophages (74%), and CD11c(+) cells expressing either CD163 (7%), CD103 (11%) or CD1c (13%). CD103(+) and CD1c(+) DCs belonged to partly overlapping populations, whereas CD163(+) CD11c(+) APCs appeared to be a distinct population. In the coeliac lesion, we found increased density of CD163(+) CD11c(+) APCs, whereas the density of CD103(+) and CD1c(+) DCs was decreased, suggesting that distinct subpopulations of APCs in coeliac disease may exert different functions in the pathogenesis.

Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease.

The action of tissue Transglutaminase (TGase) on specific protein-bound glutamine residues plays a critical role in numerous biological processes. Here we provide evidence for a new role of this enzyme in the common, HLA-DQ2 (and DQ8) associated enteropathy, celiac disease (CD). The intestinal inflammation in CD is precipitated by exposure to wheat gliadin in the diet and is associated with increased mucosal activity of TGase. This enzyme has also been identified as the main target for CD-associated anti-endomysium autoantibodies, and is known to accept gliadin as one of its few substrates. We have examined the possibility that TGase could be involved in modulating the reactivity of gliadin specific T cells. This could establish a link between previous reports of the role of TGase in CD and the prevailing view of CD as a T-cell mediated disorder. We found a specific effect of TGase on T-cell recognition of gliadin. This effect was limited to gliadin-specific T cells isolated from intestinal CD lesions. We demonstrate that TGase mediates its effect through an ordered and specific deamidation of gliadins. This deamidation creates an epitope that binds efficiently to DQ2 and is recognized by gut-derived T cells. Generation of epitopes by enzymatic modification is a new mechanism that may be relevant for breaking of tolerance and initiation of autoimmune disease.

Gliadin-specific, HLA-DQ(alpha 1*0501,beta 1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients.

Celiac disease (CD) is most probably an immunological disease, precipitated in susceptible individuals by ingestion of wheat gliadin and related proteins from other cereals. The disease shows a strong human HLA association predominantly to the cis or trans encoded HLA-DQ(alpha 1*0501,beta 1*0201) (DQ2) heterodimer. T cell recognition of gliadin presented by this DQ heterodimer may thus be of immunopathogenic importance in CD. We therefore challenged small intestinal biopsies from adult CD patients on a gluten-free diet in vitro with gluten (containing both gliadin and other wheat proteins), and isolated activated CD25+ T cells. Polyclonal T cell lines and a panel of T cell clones recognizing gluten were established. They recognized the gliadin moiety of gluten, but not proteins from other cereals. Inhibition studies with anti-HLA antibodies demonstrated predominant antigen presentation by HLA-DQ molecules. The main antigen-presenting molecule was established to be the CD-associated DQ(alpha 1*0501, beta 1*0201) heterodimer. The gluten-reactive T cell clones were CD4+, CD8-, and carried diverse combinations of T cell receptor (TCR) V alpha and V beta chains. The findings suggest preferential mucosal presentation of gluten-derived peptides by HLA-DQ(alpha 1*0501, beta 1*0201) in CD, which may explain the HLA association.

The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase.

The great majority of patients that are intolerant of wheat gluten protein due to celiac disease (CD) are human histocompatibility leukocyte antigen (HLA)-DQ2(+), and the remaining few normally express HLA-DQ8. These two class II molecules are chiefly responsible for the presentation of gluten peptides to the gluten-specific T cells that are found only in the gut of CD patients but not of controls. Interestingly, tissue transglutaminase (tTG)-mediated deamidation of gliadin plays an important role in recognition of this food antigen by intestinal T cells. Here we have used recombinant antigens to demonstrate that the intestinal T cell response to alpha-gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides that overlap by a seven-residue fragment of gliadin. We show that tTG converts a glutamine residue within this fragment into glutamic acid and that this process is critical for T cell recognition. Gluten-specific T cell lines from 16 different adult patients all responded to one or both of these deamidated peptides, indicating that these epitopes are highly relevant to disease pathology. Binding studies showed that the deamidated peptides displayed an increased affinity for DQ2, a molecule known to preferentially bind peptides containing negatively charged residues. Interestingly, the modified glutamine is accommodated in different pockets of DQ2 for the different epitopes. These results suggest modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide-MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.

Fatty acid-spermine conjugates as DNA carriers for nonviral in vivo gene delivery.

The lack of efficient in vivo gene delivery is a well-known shortcoming of nonviral delivery vectors, in particular of chemical vectors. We developed a series of novel nonviral carriers for plasmid-based in vivo gene delivery. This new transport device is based on the assembly of DNA plasmids with synthetic derivatives of naturally occurring molecules-fatty acid-spermine conjugates (or lipospermines). We tested the ability of these fatty acid conjugates to interact with plasmid DNA (pDNA) and found that they formed DNA nanocomplexes, which are protected from DNase I degradation. This protection was shown to directly correlate with the length of the aliphatic component. However, this increase in the length of the hydrocarbon chain resulted in increased toxicity. The cationic lipids used for transfection typically have a C(16) and C(18) hydrocarbon chain. Interestingly, toxicity studies, together with further characterization studies, suggested that the two most suitable candidates for in vivo delivery are those with the shortest hydrocarbon chain, butanoyl- and decanoylspermine. Morphological characterization of DNA nanocomplexes resulting from these lipospermines showed the formation of a homogenous population, with the diameter ranging approximately from 40 to 200 nm. Butanoylspermine was found to be the most promising carrier from this series, resulting in a significantly increased gene expression, in relation to naked plasmid, in both tissues herein targeted (dermis and M. tibialis anterior). Thus, we established a correlation between the in vitro properties of the ensuing DNA nanocarriers and their efficient in vivo gene expression.

Transcriptional profiling reveals monocyte-related macrophages phenotypically resembling DC in human intestine.

The tissue dendritic cell (DC) compartment is heterogeneous, and the ontogeny and functional specialization of human tissue conventional DC (cDC) subsets and their relationship with monocytes is unresolved. Here we identify monocyte-related CSF1R+Flt3- antigen presenting cells (APCs) that constitute about half of the cells classically defined as SIRPα+ DCs in the steady-state human small intestine. CSF1R+Flt3- APCs express calprotectin and very low levels of CD14, are transcriptionally related to monocyte-derived cells, and accumulate during inflammation. CSF1R+Flt3- APCs show typical macrophage characteristics functionally distinct from their Flt3+ cDC counterparts: under steady-state conditions they excel at antigen uptake, have a lower migratory potential, and are inefficient activators of naïve T cells. These results have important implications for the understanding of the ontogenetic and functional heterogeneity within human tissue DCs and their relation to the monocyte lineage.

Diet adherence and gluten exposure in coeliac disease and self-reported non-coeliac gluten sensitivity.

BACKGROUND/OBJECTIVES: Adherence to gluten-free diet in self reported non-coeliac gluten sensitive subjects is scarcely researched. Objectives of the study were to compare dietary adherence in coeliac disease (CD) subjects and in non-coeliac gluten sensitive (NCGS) subjects, and to estimate gluten exposure based on weighed food records and analysis of gluten content in selected food items. SUBJECTS/METHODS: Twenty-three subjects with biopsy verified CD on a gluten-free diet and 34 HLA-DQ2+ NCGS subjects on a self-instituted gluten-free diet were enrolled. The latter group was under investigation of CD. Dietary adherence was assessed by frequency questionnaire and structured forms supplied by weighed food records. For the analyses of food samples, the sandwich R5-ELISA, Ridascreen® Gliadin competitive method was used. RESULTS: There was no difference in dietary adherence between CD and NCGS subjects (83% vs 68%, p = 0.21). NCGS subjects were mainly self-educated in gluten-free diet compared to CD subjects (91% and 39%, respectively, p < 0.001). In non-adherent subjects, there was no difference in gluten exposure between CD and NCGS (10 vs 138 mg/day, p = 0.83). There was no difference in BMR-factor between CD and NCGS subjects, or between adherent and non-adherent subjects. CONCLUSIONS: Both CD and NCGS subjects were largely adherent, and adherence did not differ between the groups. Gluten exposure varied greatly, and some CD and NCGS subjects reached gluten intake above 500 mg/day, which might have considerable health effects on the individual, especially in case of coeliac disease.

Immunization with HIV-1 envelope T20-encoding DNA vaccines elicits cross-clade neutralizing antibody responses.

BACKGROUND: Genetic immunization is expected to induce the expression of antigens in a native form. The encoded peptide epitopes are presented on endogenous MHC molecules, mimicking antigen presentation during a viral infection. We have explored the potential of enfuvirtide (T20), a short HIV peptide with antiviral properties, to enhance immune response to HIV antigens. To generate an expression vector, the T20 sequence was cloned into a conventional plasmid, the novel minicircle construct, and a replicon plasmid. In addition, 3 conventional plasmids that express the envelope of HIV-1 subtypes A, B and C and contain T20 in their gp41 sequences were also tested. RESULTS: All combinations induced HIV-specific antibodies and cellular responses. The addition of T20 as a peptide and as an expression cassette in the 3 DNA vectors enhanced antibody responses. The highest anti-HIV-1 Env titers were obtained by the replicon T20 construct. This demonstrates that besides its known antiviral activity, T20 promotes immune responses. We also confirm that the combination of slightly divergent antigens improves immune responses. CONCLUSIONS: The antiretroviral T20 HIV-1 sequence can be used as an immunogen to elicit binding and neutralizing antibodies against HIV-1. These, or similarly modified gp41 genes/peptides, can be used as priming or boosting components for induction of broadly neutralizing anti-HIV antibodies. Future comparative studies will reveal the optimal mode of T20 administration.

Responsive population dynamics and wide seeding into the duodenal lamina propria of transglutaminase-2-specific plasma cells in celiac disease.

A hallmark of celiac disease is autoantibodies to transglutaminase 2 (TG2). By visualizing TG2-specific antibodies by antigen staining of affected gut tissue, we identified TG2-specific plasma cells in the lamina propria as well as antibodies in the subepithelial layer, inside the epithelium, and at the brush border. The frequency of TG2-specific plasma cells were found not to correlate with serum antibody titers, suggesting that antibody production at other sites may contribute to serum antibody levels. Upon commencement of a gluten-free diet, the frequency of TG2-specific plasma cells in the lesion dropped dramatically within 6 months, yet some cells remained. The frequency of TG2-specific plasma cells in the celiac lesion is thus dynamically regulated in response to gluten exposure. Laser microdissection of plasma cell patches, followed by antibody gene sequencing, demonstrated that clonal cells were seeded in distinct areas of the mucosa. This was confirmed by immunoglobulin heavy chain repertoire analysis of plasma cells isolated from individual biopsies of two untreated patients, both for TG2-specific and non-TG2-specific cells. Our results shed new light on the processes underlying the B-cell response in celiac disease, and the approach of staining for antigen-specific antibodies should be applicable to other antibody-mediated diseases.

Altered gut microbiota profile in common variable immunodeficiency associates with levels of lipopolysaccharide and markers of systemic immune activation.

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency characterized by low immunoglobulin (Ig)G and IgA, and/or IgM. In addition to bacterial infections, a large subgroup has noninfectious inflammatory and autoimmune complications. We performed 16S ribosomal RNA-based profiling of stool samples in 44 CVID patients, 45 patients with inflammatory bowel disease (disease controls), and 263 healthy controls. We measured plasma lipopolysaccharide (LPS) and markers of immune cell activation (i.e., soluble (s) CD14 and sCD25) in an expanded cohort of 104 patients with CVID and in 30 healthy controls. We found a large shift in the microbiota of CVID patients characterized by a reduced within-individual bacterial diversity (alpha diversity, P<0.001) without obvious associations to antibiotics use. Plasma levels of both LPS (P=0.001) and sCD25 (P<0.0001) were elevated in CVID, correlating negatively with alpha diversity and positively with a dysbiosis index calculated from the taxonomic profile. Low alpha diversity and high dysbiosis index, LPS, and immune markers were most pronounced in the subgroup with inflammatory and autoimmune complications. Low level of IgA was associated with decreased alpha diversity, but not independently from sCD25 and LPS. Our findings suggest a link between immunodeficiency, systemic immune activation, LPS, and altered gut microbiota.

T-cell receptor beta usage by 35 different antigen-specific T-cell clones restricted by HLA-Dw4 or -Dw14.1.

We studied whether antigen-specific T cells being restricted by the very similar HLA-Dw4 and/or -Dw14.1 molecules might demonstrate homogeneities in parts of their TCR. TCCs were generated from three individuals who were all HLA-Dw4/Dw14.1 heterozygous. Thirty-five TCCs specific for PPD or TT and restricted by HLA-Dw4 and/or -Dw14.1 were selected for TCR beta gene sequencing. We found that 19 different V beta genes from 13 V beta families were expressed by these TCCs. Thus, it seems that many different TCRV beta genes may be used by TCCs restricted by these HLA molecules. For PPD-specific TCCs, a possible biased usage of V beta 8, as well as possible preferential usage of a CDR3 motif, were found.

Antigen-specific T cells restricted by HLA-DQw8: importance of residue 57 of the DQ beta chain.

CD4+ T-lymphocyte clones reactive with antigen from herpes simplex virus type 1 were established from a DQw8+ donor. One T-lymphocyte clone was able to recognize HSV antigen only when presented by antigen presenting cells expressing DQw8, and only HLA-DQ-specific mAbs could inhibit the response. Using antigen presenting cells from an extensive panel of donors whose HLA-DQ molecules and genes had been established, it could be demonstrated that alanine at residue 57 of the DQ beta chain played a critical role in presentation of herpes simplex virus--derived antigen(s) to T cells. The results are interesting, since the amino acids present at residue 57 of the DQ beta chains seems to play an important role in determining susceptibility to develop or protection against insulin-dependent diabetes mellitus.

T cells from the small intestinal mucosa of a DR4, DQ7/DR4, DQ8 celiac disease patient preferentially recognize gliadin when presented by DQ8.

CD is an immunologic disease of the small intestine which is precipitated by ingestion of wheat gliadin. Most patients carry the HLA-DQ (alpha 1*0501, beta 1*0201) (DQ2) heterodimer. We recently reported that a preponderance of gliadin-specific T cells from the small intestinal mucosa of DQ2-positive CD patients were restricted by this DQ heterodimer. However, a small percentage of CD patients do not carry this DQ heterodimer, and most of them instead carry DQ (alpha 1*0301, beta 1*0302) (DQ8). Here we report that a majority of gliadin-specific T cells from the small intestinal mucosa of a DR4,DQ7/DR4,DQ8 heterozygous CD patient are restricted by DQ8. Thus, preferential presentation of gliadin-derived peptides to T cells by the CD-associated DQ2 and DQ8 molecules may be an initial and important immunopathogenic step in CD.

T cells recognizing an HLA-DQ alpha beta heterodimer encoded in cis by the DR4DQw4 haplotype and in trans by DR4DQw8/DRw8DQw4 heterozygous cells.

We generated alloreactive DQ-specific CD4+ T-lymphocyte clones. One of these (TLC HH58) was only restimulated with cells having the DR4DQw4 haplotype or cells being DR4DQw8/DRw8DQw4 heterozygous. The former cells carry the DQA1*0301 and DQB1*0401 alleles in cis position while the latter cells carry DQA1*0301 and DQB1*0402 alleles (DQB1*0402 is identical to DQB1*0401 except for codon 23) in trans position. Thus, very similar DQ alpha beta heterodimers are encoded by these genes in both cis and trans positions, which are recognized by the same T cells.

HLA-DQ2-restricted T-cell recognition of gluten-derived peptides in celiac disease. Influence of amino acid substitutions in the membrane distal domain of DQ beta 1*0201.

CD is precipitated in susceptible individuals by ingestion of wheat gluten. The disease is strongly associated to the HLA-DQ(alpha 1*0501, beta 1*0201) (DQ2) heterodimer, where both the DQ alpha and DQ beta chains are required for susceptibility. We have recently shown that gluten-specific CD4+ T cells from the small intestinal mucosa of CD patients are predominantly restricted by the CD-associated HLA-DQ(alpha 1*0501, beta 1*0201) heterodimer. Here we report studies on the influence of aa substitutions in the DQ beta 1*0201 chain on DQ2-restricted T-cell recognition of gluten antigens. A B-LCL expressing the DQ(alpha 1*501, beta 1*0301) heterodimer was transfected with the DQB1*0201 gene, or with DQB1*0201 genes altered by site-directed mutagenesis. Surface expression of the wild-type or mutated DQ(alpha 1*0501, beta 1*0201) heterodimers was observed in the transfectants. Seven DQ2-restricted, gluten-specific TCCs were then investigated with respect to their ability to recognize antigen presented by the transfectants. All TCCs were sensitive to one or more of the aa substitutions induced but showed different response patterns. The results demonstrate that single aa substitutions of the DQ beta 1*0201 chain at positions in the peptide-binding cleft of DQ(alpha 1*0501, beta 1*0201) may affect binding of gluten-derived peptides and/or interfere with T-cell recognition. Because all seven TCCs studied were differently affected, they probably differ with respect to glutenpeptide and/or DQ(alpha 1*0501, beta 1*0201) restriction fine specificity.

T cells recognize a peptide derived from alpha-gliadin presented by the celiac disease-associated HLA-DQ (alpha 1*0501, beta 1*0201) heterodimer.

CD is unique among the HLA-associated diseases since (a) the disease-promoting agent (gliadin) is known and (b) the disease is precipitated mainly in individuals carrying a particular cis- or trans-encoded HLA-DQ heterodimer; i.e., DQ(alpha 1*0501, beta 1*0201). Further, a preponderance of gliadin-specific T cells derived from the small intestinal mucosa of CD patients are restricted by this DQ heterodimer. T-cell recognition of gliadin peptides presented by the DQ(alpha 1*0501, beta 1*0201) heterodimer may thus be of importance in CD. Here we report that a T-cell clone from a patient with CD recognizes a synthetic alpha-gliadin peptide, when presented by the cis- or trans-encoded CD-associated DQ(alpha 1*0501, beta 1*0201) heterodimer. The minimal peptide recognized by the T-cell clone corresponds to residues 31-47 of alpha-gliadin, which is included in the part of alpha-gliadin previously shown to have disease-promoting activity. When testing analogue peptides derived from other alpha-gliadin sequences, one peptide differing by one amino acid was recognized by the T-cell clone, whereas the other peptide differing by two amino acids was not recognized. Our findings demonstrate that the CD-associated DQ(alpha 1*0501, beta 1*0201) heterodimer may serve as an antigen-presenting molecule to T cells for certain gliadin peptides.