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.

GABA, GABA transporters, GABA(A) receptor subunits, and GAD mRNAs in the rat parabrachial and Kölliker-Fuse nuclei.

In the present study, we investigated the key molecules that determine gamma-aminobutyric acid (GABA)ergic signal transduction in the parabrachial/Kölliker-Fuse complex (PB/KF) by means of immunocytochemistry and in situ hybridization. Our data demonstrate a dense plexus of GABA-immunoreactive (-ir) varicosities throughout the nuclei of the PB and the KF. The number of neurons expressing GAD65 or GAD67 mRNA was fairly low in the PB, whereas caudally in the KF an accumulation of GAD-expressing neurons was observed. The GABA transporter-3 (GAT-3) was detected in all parts of the PB/KF, whereas immunolabeling for GAT1 was not observed. All nuclei of the PB and the KF exhibited immunoreactivity for the gamma2-, alpha2-, and alpha3-subunits of the GABA(A) receptor. Gamma2-ir was strong and similar in all PB/KF nuclei. In contrast, alpha2-labeling was particularly intense in the superior lateral PB, and alpha3-labeling was most prominent in the external lateral and external medial PB, compared with the remaining nuclei. With respect to the subcellular localization, we found gamma2-ir in cell bodies and higher order dendrites, whereas alpha2- and alpha3-ir was predominantly found in cell bodies. Immunolabeling for the beta2/3- and the alpha1-subunit was seen in cell bodies and presumed dendritic profiles. The staining intensity was strongest in the dorsal lateral PB. Most importantly, the external lateral PB and the waist area were totally devoid of beta2/3- and alpha1-ir. Our data suggest that neural processing in the PB/KF is under a strong GABAergic inhibition that is apparently mediated by different types of GABA(A) receptors in functionally different pathways through the PB/KF.

Co-localization of glutamate and homocysteic acid immunoreactivities in human photoreceptor terminals.

Consecutive semithin sections of human retinae were treated with antisera recognizing fixed homocysteic acid, glutamate or glutamine. Photoreceptor terminals displayed a co-localization of glutamate-like and homocysteic acid-like immunoreactivities. This was confirmed in the electron microscope by immunogold cytochemistry. A quantitative analysis of the immunogold labelling indicated that glutamate and homocysteic acid occurred at higher concentrations in the terminals than in outer parts of the receptor cells. No such gradient was found for glutamine immunoreactivity, which was concentrated in Müller cell processes. These processes were also labelled by the homocysteic acid antiserum, although less intensely than were the photoreceptor terminals. Control experiments suggested that the homocysteic acid antiserum visualized a pool of authentic homocysteic acid, although it could not be excluded that part of this pool had been generated by non-enzymatic oxidation of precursor molecules. Homocysteic acid immunoreactivity was also demonstrated in photoreceptor terminals of baboon. The present data indicate that primate photoreceptor terminals contain homocysteic acid in addition to glutamate and open up the possibility that homocysteic acid is released as a glutamate co-agonist at photoreceptor synapses.

Identification of neuronal plasma membrane microdomains that colocalize beta-amyloid and presenilin: implications for beta-amyloid precursor protein processing.

Alzheimer's disease (AD) is associated with the accumulation of extracellular deposits of the beta-amyloid protein (Abeta). Abeta is a result of misprocessing of the beta-amyloid precursor protein (APP). Gamma-secretase is involved in APP misprocessing and one hypothesis holds that this secretase is identical to PS1. We tested this hypothesis by determining whether PS is co-localised with Abeta in situ. Using confocal analyses and a sensitive immunogold procedure we show that PS and Abeta are co-localised within discrete microdomains of neuronal plasma membranes in AD patients and in aged dogs, an established model of human brain aging. Our data indicate that APP misprocessing occurs in discrete plasma membrane domains of neurons and provide evidence that PS1 is critically involved in Abeta formation.

Distribution of glutamate-like immunoreactivity in excitatory hippocampal pathways: a semiquantitative electron microscopic study in rats.

A semiquantitative electron microscopic immunocytochemical procedure was used to study the cellular and subcellular distribution of glutaraldehyde-fixed glutamate in rat hippocampal formation. Ultrathin plastic-embedded sections were incubated with a primary glutamate antiserum followed by a secondary antibody coupled to colloidal gold particles. A computer-assisted assessment of gold particle densities revealed that the axon terminals of all of the main excitatory pathways in the hippocampus were enriched with glutamate-like immunoreactivity relative to other tissue elements, including the parent cell bodies (granule and pyramidal cells). The different excitatory pathways showed slightly different labelling intensities: boutons in the termination zone of the lateral perforant path were covered by higher gold particle densities than boutons situated in the termination zones of the medial perforant path, the Schaffer collateral/commissural pathway and the hilar associational/commissural pathway. The mossy fibre terminals were significantly less enriched in immunoreactivity than terminals of the lateral perforant path and the Schaffer collateral/commissural pathway. Within the terminals, glutamate-like immunoreactivity was concentrated over synaptic vesicles and mitochondria. Terminals establishing symmetric junctions with cell bodies or dendritic stems displayed low particle densities, as did glial cell processes. These findings support the idea that glutamate is a major excitatory neurotransmitter in hippocampal excitatory synapses. Our observations are also in line with biochemical data pointing to the existence of a considerable neuronal and a smaller glial, metabolic pool of glutamate.

Cellular and subcellular redistribution of glutamate-, glutamine- and taurine-like immunoreactivities during forebrain ischemia: a semiquantitative electron microscopic study in rat hippocampus.

The effect of 20 min of ischemia on the cellular and subcellular distribution of glutamate, glutamine and taurine in the rat hippocampus was studied by means of an immunocytochemical procedure based on antisera raised against protein glutaraldehyde conjugates of the respective amino acids. Forebrain ischemia was induced by temporary occlusion of the common carotid arteries in rats with permanently occluded vertebral arteries. Within 90 s after removal of the carotid ligatures, the rats were perfused through the heart with a mixture of glutaraldehyde and paraformaldehyde. For semiquantitative electron microscopic analysis, ultrathin sections were incubated in a primary antiserum followed by a secondary antibody coupled to colloidal gold particles. The gold particle densities over different tissue compartments within the CA1 field and the mossy fiber zone of the hippocampus were determined by means of a specially designed computer program, and values from normal and ischemic animals were compared. It was found that in the astrocytes, the level of immunoreactivity for glutamine and taurine is unchanged or slightly decreased after ischemia, while that for glutamate is increased, particularly within the mitochondria (by about 100%). In contrast, pyramidal cell bodies display a reduced immunolabeling for all three amino acids following the ischemic episode. The results show that ischemia causes a redistribution of glutamate from neurons to glia. The observed increase in the glial immunolabeling for glutamate indicates that the capacity of the glial cells to metabolize glutamate is exceeded during ischemia. This glial response to ischemia has not previously been recognized and may play a role in the chain of events leading to "excitotoxic" cell death during or following an ischemic episode. The reduction of glutamate and taurine immunolabeling in neurons points to a possible amino acid efflux and is compatible with previous biochemical studies demonstrating an elevated extracellular level of these amino acids during ischemia.

Neuroactive amino acids in organotypic slice cultures of the rat hippocampus: an immunocytochemical study of the distribution of GABA, glutamate, glutamine and taurine.

Antisera raised against protein-glutaraldehyde-amino acid conjugates were used to study the light and electron microscopic distribution of GABA, glutamate, glutamine and taurine in organotypic slice cultures of rat hippocampi. In the stratum oriens and radiatum, glutamate-like immunoreactivity was particularly concentrated in nerve endings establishing asymmetric junctions with dendritic spines. Mossy fiber terminals in CA3 and the dentate hilus were also strongly labeled. A quantitative immunogold analysis of the glutamate-immunolabelled profiles showed a pattern that was highly reminiscent of that previously observed in perfusion-fixed hippocampi, including a correspondingly sparse labeling of glial processes and of presynaptic elements in symmetric synapses. GABA-like immunoreactivity was localized predominantly in interneurons and in presynaptic terminals contacting dendritic shafts and neuronal cell bodies, while immunoreactivities for glutamine and taurine were found mainly in astroglial cells and pyramidal cells, respectively. Our data indicate that the major intrinsic fiber systems of the cultured hippocampi have retained their normal transmitter phenotypes.

Ultrastructural evidence of fibrillar beta-amyloid associated with neuronal membranes in behaviorally characterized aged dog brains.

The aged dog brain accumulates beta-amyloid in the form of diffuse senile plaques, which provides a potentially useful in vivo model system for studying the events surrounding the deposition of beta-amyloid. We used postembedding immunocytochemistry at the electron microscopic level to determine the subcellular distribution of beta-amyloid 1-40 and beta-amyloid 1-42 peptides in the prefrontal and parietal cortex of behaviorally characterized dogs ranging in age from one to 17 years. Immunogold particles signaling beta-amyloid 1-42 occurred over intracellular and extracellular fibrils that were approximately 8 nm in width. Intracellular beta-amyloid 1-42 fibrils were found in close proximity to glial fibrillary acidic protein fibers within astrocytes, but only in cells with signs of plasma membrane disruption. Neuronal labeling of beta-amyloid 1-42 appears to be associated with the plasma membrane. Membrane-bound beta-amyloid 1-42 occurs in the form of fine fibrils that are embedded in the dendritic membrane and appear to project into the extracellular space as determined by quantitative analysis of the immunogold particle distribution. Bundles of beta-amyloid 1-42 were also closely associated and/or integrated with degenerating myelin sheaths of axons. In one dog that was impaired on several cognitive tasks, extensive beta-amyloid 1-42 deposition was associated with microvacuolar changes and vascular pathology. The present findings suggest that beta-amyloid 1-42 may be generated at the dendritic plasma membrane as well as in intracellular compartments. The close association between beta-amyloid 1-42 and destroyed myelin suggests one possible new mechanism by which beta-amyloid 1-42 induces neurodegeneration.

Expression of glial glutamate transporters GLT-1 and GLAST is unchanged in the hippocampus in fully kindled rats.

In situ hybridization techniques and quantitative western blotting were used to study the expression of the glial glutamate transporter GLT-1 and GLAST in the brains of normal (implanted, non-kindled) and fully kindled rats. Wistar rats were implanted with stimulating electrodes in the basolateral amygdala, and killed 28 days after the stimulated group had shown stage 5 seizures on five occasions. The brains were processed for in situ hybridization of messenger RNA for GLT-1 using 35S-labelled oligonucleotide probes or digoxigenin-labelled riboprobes. Paired (kindled and non-kindled) sections were used for qualitative and quantitative analyses. Image analysis of autoradiograms showed no change in expression of GLT-1 messenger RNA in any region of the hippocampus or in the cortex. An increase in expression of GLT-1 messenger RNA (expressed as percentage difference of control) was observed bilaterally in the striatum in kindled animals (16-21%, P<0.05). Nuclear emulsion-dipped sections showed predominant glial cell labelling in the hippocampus. Particle density analysis revealed reduced cell labelling in some kindled vs control pairs but overall there was no significant reduction in labelling in CA1. Equivalent results were found in CA1 using digoxigenin-labelled riboprobes. Quantitative immunoblotting also revealed no change in GLT-1 or GLAST transporter protein in the hippocampus of kindled animals. From these data we conclude that the enduring seizure susceptibility associated with the fully kindled state is unlikely to involve alterations in hippocampal GLT-1 messenger RNA or GLT-1 and GLAST transporter protein expression.

A quantitative electron microscopic immunocytochemical study of the distribution and synaptic handling of glutamate in rat hippocampus.

One of the major problems in glutamate immunocytochemistry has been the difficulty involved in separating immunocytochemical labelling due to metabolic glutamate from the labelling caused by transmitter glutamate. Another problem appears to be the accessibility of antigenic sites in conventional light microscopic preparations. In the present report, we have applied the primary glutamate antiserum onto ultrathin tissue sections, followed by the use of a colloidal gold detection system. The use of this postembedding immunogold procedure allows equal access of antibodies to all cellular compartments exposed at the section surface, allows quantitative assessment of the immunoreactivity, and affords a high resolution compatible with studies at the organelle level. When applied to slice preparations the immunogold procedure can be used to identify releasable pools of glutamate. These methodological advances have greatly increased the usefulness of glutamate immunocytochemistry as a tool to study putative glutamatergic terminals in the CNS.

Cloning and expression of a neuronal rat brain glutamate transporter.

Glutamate is the major excitatory transmitter in the mammalian central nervous system. Glutamate transporters, which keep the extracellular glutamate concentration low, are required both for normal brain function and for protecting neurons against harmful glutamatergic overstimulation. We have isolated the cDNA for a rat brain glutamate transporter (REAAC1) which has 90% amino acid and 86% nucleotide identity to the rabbit EAAC1. When REAAC1 was expressed in HeLa cells using a recombinant vaccinia-T7 virus expression system, a sodium dependent glutamate uptake was observed. The affinity of the carrier to various substrates was typical of brain "high affinity' glutamate uptake: threo-3-hydroxyaspartate, (R)-aspartate, (S)-glutamate and (S)-trans-pyrrolidine-2,4-dicarboxylic acid were strong inhibitors, but not (R)-glutamate or gamma-aminobutyrate. High resolution, non-radioactive in situ hybridization histochemistry in rat brain revealed the mRNA in several types of glutamatergic as well as non-glutamatergic neurons, but not in glial cells.

Molecular organization of cerebellar glutamate synapses.

The organization of key molecules at glutamatergic synapses in the rat cerebellar cortex as analyzed by high resolution immunocytochemical techniques using gold particles as markers. The distinct compartmentation of glutamate and glutamine was consistent with biochemical data indicating an active role of glia in the removal of released glutamate and in the supply of glutamine for de novo synthesis of transmitter glutamate. The presence in glial cells of two different glutamate transporters, GLT1 and GLAST, provided further support of this concept. Both transporters were selectively expressed in glial membranes and occurred at higher densities in glial processes surrounding parallel fiber synapses with spines than in glial processes associated with parallel fiber synapses with dendritic shafts. At the former type of synapse, gold particles signalling GLT1 and GLAST could be found within a few nanometers of the postsynaptic density. The rat cerebellum also contains a homologue (rEAAC1) of the glutamate transporter EAAC1, originally cloned from rabbit, mRNA encoding this transporter was restricted to neurons. The exact localization of the rEAAC1 transporter molecules at cerebellar synapses remains to be determined but immunocytochemical and physiological data from other laboratories suggest that they may be preferentially expressed in postsynaptic membranes. Gold particles representing immunoreactivity for the AMPA receptor subunits GluR2/3 were found along the entire mediolateral extent of the postsynaptic specialization of parallel fiber synapses and were rarely encountered at non-synaptic membranes. The present data show that molecules engaged in signalling at cerebellar glutamatergic synapses are precisely organized, consistent with the requirements for rapid signal transmission and efficient removal and recycling of transmitter.

Ischemic disruption of glutamate homeostasis in brain: quantitative immunocytochemical analyses.

More than 10 years ago, it was shown by microdialysis that the excitatory transmitter glutamate accumulates in the interstitial space of brain subjected to ischemic insult. This was one of the key observations leading to the formulation of the "glutamate hypothesis' of ischemic cell death. It is now assumed that even a transient glutamate overflow may set in motion a number of events that ultimately cause cell loss in vulnerable neuronal populations. The aim of the present review is to discuss the intracellular changes that underlie the dysregulation of extracellular glutamate during and after ischemia, with emphasis on data obtained by postembedding, electron microscopic immunogold cytochemistry. While the time resolution of this approach is necessarily limited, it can reveal, quantitatively and at a high level of spatial resolution, how the intracellular pools of glutamate and metabolically related amino acids are perturbed during and after an ischemic insult. Moreover, this can be done in animals whose extracellular amino acid levels are monitored by microdialysis, allowing a direct correlation of extra- and intracellular changes. Immunogold analyses of brains subjected to ischemia have identified dendrites and neuronal somata as likely sources of glutamate efflux, probably mediated by reversal of glutamate uptake. The vesicular glutamate pool has been found to be largely unchanged after 20 min of ischemia. Ischemia causes an increased glutamate content and an increased glutamate/glutamine ratio in glial cells, as revealed by double immunogold labelling. This argues against the idea that glial cells contribute to the extracellular overflow of glutamate in the ischemic brain.

Ultrastructural analyses of beta-amyloid in the aged dog brain: neuronal beta-amyloid is localized to the plasma membrane.

1. An electron microscopic study was undertaken to study beta-amyloid (Abeta) deposition and neuropathology in aged dogs. 2. A positive correlation between Abeta deposits and neuropathology was found in some dogs. Massive Abeta deposition was correlated to advanced lesions. 3. By use of immunocytochemistry Abeta fibers were identified within plaques, around vessels and in association with cell membranes.

Differential distribution of the glutamate transporters GLT1 and rEAAC1 in rat cerebral cortex and thalamus: an in situ hybridization analysis.

The distributions in rat cerebral cortex and thalamus of the mRNAs encoding the glutamate transporters GLT1 and rEAAC1 (a rat homologue of rabbit EAAC1) were investigated by nonautoradiographic in situ hybridization using digoxigenin-labelled riboprobes. The probe recognizing rEAAC1 mRNA labelled exclusively neurons while GLT1 mRNA was found in glia as well as in select neuronal populations. The neurons containing the GLT1 transcript exhibited a distribution that was different from, and at some sites complementary to, the distribution of neurons containing rEAAC1 mRNA. In the subiculum, neurons positive for GLT1 and rEAAC1 were found in the deep and superficial part of the cell layer, respectively, while in the parietal neocortex GLT1 predominated in layer VI and rEAAC1 in layer V. Very few neuronal populations, most notably cells in hippocampal subfields CA3 and CA4, and in layer II in the entorhinal cortex, appeared to be equipped with both transcripts. In the thalamus the GLT1 labelling predominated in the midline and intralaminar nuclei while rEAAC1 labelling was found throughout this structure. It was concluded that the cerebral cortex and thalamus show cellular, laminar, as well as regional heterogeneities in the expression of the two glutamate transporters.

Cytoprotective effects of growth factors: BDNF more potent than GDNF in an organotypic culture model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a preferential loss of dopaminergic (DAergic) neurons of the substantia nigra pars compacta (SNpc). Both glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) play key roles in maintaining the DAergic phenotype and exert a cytoprotective effect on these neurons in vivo and in vitro. However, controversy still exists regarding the relative potency of the two factors and the extent to which they act synergistically. In this study, we used a refined version of organotypic cultures as a model for PD. The neurotoxin 6-hydroxydopamine (6-OHDA) was applied unilaterally in slices of rat mesencephalon, allowing for internal controls and enabling a precise comparison between the two sides of the midbrain. We evaluated the cytoprotective and regenerative effects of BDNF, GDNF and the combination of these in terms of surviving tyrosine hydroxylase positive (TH+) cells and TH mRNA expression. Pre-, co-, or post-treatment with neurotrophic factors clearly protects DAergic neurons from cell death. Cell survival is particularly pronounced in cultures pre-treated with BDNF and is not further increased when BDNF is applied in combination with GDNF in equimolar dose. On the lesion side, surviving TH+ cells exposed to neurotrophic factors showed extensive sprouting, and BDNF treatment resulted in a two-fold increase in TH mRNA. Such effects were not seen in the absence of toxin exposure. Thus, we observed that BDNF induced an upregulation of the DAergic phenotype, which suggest a cytoprotective and regenerative effect.

Chronic fatigue is associated with impaired health-related quality of life in inflammatory bowel disease.

BACKGROUND: Fatigue is reported to reduce health-related quality of life (HRQOL) in chronic diseases. Studies on the importance of fatigue and its implications for the patient's HRQOL in inflammatory bowel disease (IBD) remain scarce and need to be explored. AIM: To investigate the influence of chronic fatigue on both generic and disease-specific HRQOL in IBD. METHODS: Patients in remission, with mild and moderate IBD completed the Fatigue Questionnaire, the Short-Form 36 (SF-36) and the Norwegian version of the Inflammatory Bowel Disease Questionnaire (N-IBDQ). In addition, demographic and clinical variables were obtained. RESULTS: In total, 140 patients were included; the mean age of patients with chronic fatigue was 44.2 years (s.d. = 15.8), that of nonfatigued was 44.7 years (s.d. = 16.0). Ulcerative colitis (UC)/Crohn's disease (CD) = 92/48. Chronic fatigue was associated, after controlling for covariates, with a reduction of HRQOL scores in 6/8 SF-36 dimensions in UC and 5/8 dimensions in CD. In N-IBDQ, chronic fatigue was associated with a reduction of HRQOL in four subdimensions and total score in CD and all dimensions in UC. CONCLUSIONS: Fatigue is associated with reduction of HRQOL scores in IBD. The physical HRQOL domains are particularly affected. The impact of fatigue on disability, sick leave, school and work attendance has to be studied further.

Beta-amyloid causes downregulation of calcineurin in neurons through induction of oxidative stress.

Calcineurin is an abundant cytosolic protein that is implicated in the modulation of glutamate release. Here we show that the expression level of this enzyme is reduced in primary neuronal cultures treated with beta-amyloid. Parallel experiments in ETNA cell lines expressing SOD1 suggested that the effect of beta-amyloid on calcineurin expression is mediated by oxidative stress. The relevance of the in vitro experiments was assessed by analysis of tissue from patients with Alzheimer's disease (AD) and tissue from two strains of transgenic mice that mimic aspects of AD. The tissue from the AD brains displayed a pronounced downregulation of calcineurin immunoreactivity in profiles that were negative for glial fibrillary acidic protein (GFAP). In the hippocampus of the transgenic animals (which were analyzed in an early stage of the disease) the downregulation of calcineurin was restricted to mossy fiber terminals. A downregulation of the presynaptic pool of calcineurin may contribute to the dysregulation of glutamate release that is considered a hallmark of AD.

[New aspects of the pathogenesis of ischemic brain damage. Possible involvement of excitatory amino acids]. / Nye synspunkter på patogenesen av ischemisk hjerneskade. Eksitatoriske aminosyrer kan vaere involvert.

Glutamate, a major excitatory transmitter substance, is neurotoxic at high concentrations. Brain dialysis experiments have demonstrated an extracellular overflow of glutamate during ischemia, and there is good evidence from several animal models that glutamate antagonists offer partial protection against the development of ischemic cell degeneration. These and other experimental data indicate that glutamate may be involved in the pathogenesis of ischemic brain damage. Quantitative immunocytochemical investigations carried out in the authors' laboratory suggest that ischemia is associated with loss of glutamate from nerve cell bodies, and reduced ability of the glial cells to metabolize glutamate. We discuss possibilities of new therapeutic strategies.