The effect of adalimumab on key drivers in the pathogenesis of psoriasis.

BACKGROUND: The use of recently introduced biologics targeting specific immune mechanisms has identified crucial steps in the pathogenesis of psoriasis. Studying the dynamics of changes of these target mechanisms in sequential skin biopsies during treatment with biologics may reveal potential biomarkers. Correlation between clinical parameters and the expression of specific genes during treatments may identify markers indicative of treatment response. OBJECTIVES: This observational open-label study aimed to provide an overview of important cell biological changes in lesional skin during treatment with adalimumab, and their relationship to clinical improvement. METHODS: Ten patients with moderate-to-severe plaque psoriasis were included and treated with adalimumab for 16 weeks. At baseline, and after 10 days and 16 weeks of treatment clinical scores were assessed and biopsies were taken to examine gene expression at the mRNA and protein level. RESULTS: The expression of marker genes for innate immunity, and epidermal differentiation and proliferation was rapidly restored to normal levels, whereas genes of the adaptive immune system showed a delayed decrease. The static and dynamic course of CD1a+ Langerhans cells and Ki67+ nuclei showed a significant strong correlation to the Psoriasis Area and Severity Index score. No correlation between interleukin-17 expression and clinical scores was found. CONCLUSIONS: The innate immune system is affected during adalimumab treatment well before the changes in the adaptive immune system become apparent. We may speculate that the addition of a treatment with an early effect on adaptive immunity to adalimumab may result in superior effectiveness compared with monotherapies.

Balance of Treg vs. T-helper cells in the transition from symptomless to lesional psoriatic skin.

BACKGROUND: In the pathogenesis of psoriasis, proinflammatory T cells are strongly involved in the inflammatory process, where regulatory T-cell (Treg) function is impaired. OBJECTIVES: As effective Treg function is associated with a numerical balance between Treg and effector T cells, we wondered whether Treg/T-helper cell ratios may be associated with certain stages of the inflammatory process. We opted for the margin zone model as a dynamic approach. METHODS: From nine patients with chronic plaque psoriasis, 3-mm punch biopsies were obtained from the centre and margin of the lesion, perilesional skin and distant uninvolved skin. Skin biopsies of 10 healthy volunteers were included as a control. Samples were analysed using immunohistochemistry and immunofluorescence. RESULTS: In the transition from symptomless to lesional skin, a significant increase of CD3+, CD4+ and Foxp3+ cells was found. In seven of nine patients the ratio of Treg (Foxp3+) vs. CD4+ T cells was higher in the distant uninvolved skin than in the perilesional and lesional skin. Interestingly, the Foxp3/CD4 ratio in the distant uninvolved skin was even higher than in the skin of healthy controls. Notably, we found that most of the interleukin (IL)-17 expression was not related to CD4+ cells, but to mast cells. CONCLUSIONS: The relatively high Foxp3/CD4 ratio in symptomless skin of patients with psoriasis suggests an active immune controlling mechanism distant from the psoriatic plaque. In the margin and centre of the plaque the ratio appears skewed towards effector cells associated with inflammation. IL-17, an important driver of the psoriatic process, is mostly related to mast cells, and only sporadically to T cells.

Gap junctional remodeling in relation to stabilization of atrial fibrillation in the goat.

OBJECTIVE: It has been postulated that high atrial rate induced changes at the level of the gap junctions ('gap junctional remodeling', i.e. changes in distribution, intercellular orientation and expression of gap junction proteins), could be part of the vicious circle of electrophysiologic and structural changes leading to sustained atrial fibrillation (AF). To obtain experimental evidence in favour of such a postulate the timing of this remodeling process was studied in relation to the development of sustained AF in a goat model. METHODS AND RESULTS: Thin sections from the left (LAA) and right atrial appendage (RAA) from goats in sinus rhythm (SR) or AF, induced through programmed endocardial burst pacing for time periods between 0 and 16 weeks, were immunolabeled with antibodies against connexin(Cx)40 and Cx43 and analysed by immunofluorescence and confocal laser scanning microscopy. During SR the distribution pattern for Cx43 was completely homogeneous (LAA and RAA) and for Cx40 mostly homogeneous (LAA: all five goats, RAA: three out of five goats). The distribution pattern for Cx43 remained stable during AF, while the Cx40 distribution pattern became increasingly heterogeneous, both in the LAA and RAA, with increasing duration of pacing. This increase in heterogeneity in Cx40 distribution correlated (Spearman rank order) with an increase in stability of AF and the occurrence of structural changes (myolysis) in atrial myocytes. The Cx40/Cx43 immunofluorescence signal ratio in both the LAA and RAA appeared to be significantly lower in AF (1-16 weeks) as compared to SR (0 weeks); going from 0 to 16 weeks average ratios decreased 54.5% (n=5; P=0.026) in the LAA and 35.8 (n=5; P=0.034) in the RAA. Western blot analyses revealed similar decreases in the total Cx40/Cx43 protein ratio, on average 50.0% (n=5; P=0.008) and 47.8% (n=5; P=0.02) in the LAA and RAA, respectively. No changes were measured in the levels of Cx40 or Cx43 mRNA, as was assessed through RT-PCR. CONCLUSION: The time course of changes in the distribution and content of Cx40 gap junctions as observed during endocardial burst pacing of the goat atrium suggests that Cx40 gap junctional remodeling might be involved in the pathogenesis of sustained atrial fibrillation.

Genomic organization of the rat connexin40 gene: identical transcription start sites in heart and lung.

OBJECTIVES: The gap junction protein connexin(Cx)40 is developmentally and tissue-specifically expressed. How Cx40 expression is regulated is unknown. We therefore set out to characterize the 5'-untranslated end of both the Cx40 gene and mRNA from different tissues and ages and to identify the Cx40 promoter region. METHODS: The PCR method 5'-RACE was used to amplify the 5'-end of rat Cx40 mRNAs. Genomic rat Cx40 clones were isolated from a lambda EMBL3 library. The promoter sequence was isolated by long distance PCR. The transcription start site was identified by primer extension and RNase protection assays. RESULTS: Comparison of Cx40 genomic DNA and mRNA sequences revealed that the Cx40 gene contains a small untranslated exon, exon I, which is separated from the coding sequences by an intron of at least 5.5 Kb. The untranslated 5'-end of Cx40 mRNA sequences from adult rat lung, neonatal and adult rat heart and the rat aortic smooth muscle cell line A7r5 were identical. While the same transcription start site was found for the Cx40 mRNAs from different tissues and ages, and amount of Cx40 mRNA differed between tissues as follows: A7r5 cells > neonatal lung > adult lung > or = neonatal atrium > neonatal ventricle; Cx40 mRNA from adult atrium and ventricle was not readily detected by primer extension and RNase protection analyses. The genomic sequence upstream of the transcription start site contains multiple consensus binding sites for transcription factors putatively responsible for spatio-temporal control of Cx40 gene expression. CONCLUSIONS: Similar to other connexin genes, the Cx40 gene contains two exons. The same exon I sequence is present in all tissues and developmental stages examined and the relative amounts of Cx40 mRNA in these compare well with published data. Together our data suggest that tissue-specific and developmentally regulated expression of the Cx40 gene is controlled within the same promoter region by mechanisms that have yet to be detailed.

Altered pattern of connexin40 distribution in persistent atrial fibrillation in the goat.

INTRODUCTION: Since altered expression of gap junction proteins (connexins) in diseased myocardial tissue may lead to abnormal electrical coupling between cardiomyocytes and hence contribute to arrhythmogenesis, the expression of connexin(Cx)40 and Cx43 was studied in atrial appendage from goats in sinus rhythm (SR) and persistent atrial fibrillation (AF). METHODS AND RESULTS: Biopsies were taken from the left and right atrial appendages from goats in SR or after pacing-induced persistent AF. Analyses of Cx40 and Cx43 mRNA and protein levels, using quantitative (competitive) polymerase chain reaction and western blotting, respectively, revealed no significant changes in the overall expression of Cx40 and Cx43 as a result of persistent AF. At the cellular level, immunohistochemistry and confocal laser scanning microscopy showed a homogeneous distribution of either connexin in atrial sections taken during SR. After induction of AF, the distribution of Cx43 gap junctions was unchanged whereas the Cx40 pattern showed marked inhomogeneities with small areas (0.15 to 0.6 mm in diameter, 25% of section surface area) of low-density Cx40 located between larger areas of normal (unchanged) Cx40 density. Activation mapping (244 electrodes, spatial resolution 2.25 mm) of the right atrial wall did not reveal changes in atrial conduction velocity. CONCLUSION: Pacing-induced persistent AF in the goat gave rise to changes in the spatial organization of Cx40 gap junctions. Although the overall conduction velocity appeared not to have changed, microheterogeneities in conduction due to the local redistribution of Cx40 gap junctions might have contributed to the initiation and maintenance of AF.

Cell cycle-regulated degradation of Xenopus cyclin B2 requires binding to p34cdc2.

The protein kinase activity of the cell cycle regulator p34cdc2 is inactivated when the mitotic cyclin to which it is bound is degraded. The amino (N)-terminus of mitotic cyclins includes a conserved "destruction box" sequence that is essential for degradation. Although the N-terminus of sea urchin cyclin B confer cell cycle-regulated degradation to a fusion protein, a truncated protein containing only the N-terminus of Xenopus cyclin B2, including the destruction box, is stable under conditions where full length molecules are degraded. In an attempt to identify regions of cyclin B2, other than the destruction box, involved in degradation, the stability of proteins encoded by C-terminal deletion mutants of cyclin B2 was examined in Xenopus egg extracts. Truncated cyclin with only the first 90 amino acids was stable, but other C-terminal deletions lacking between 14 and 187 amino acids were unstable and were degraded by a mechanism that was neither cell cycle regulated nor dependent upon the destruction box. None of the C-terminal deletion mutants bound p34cdc2. To investigate whether the binding of p34cdc2 is required for cell cycle-regulated degradation, the behavior of proteins encoded by a series of full length Xenopus cyclin B2 cDNA with point mutations in conserved amino acids in the p34cdc2-binding domain was examined. All of the point mutants failed to form stable complexes with p34cdc, and their degradation was markedly reduced compared to wild-type cyclin. Similar results were obtained when the mutant cyclins were synthesized in reticulocyte lysates and when cyclin mRNA was translated directly in a Xenopus egg extract. These results indicate that mutations that interfere with p34cdc2 binding also interfere with cyclin destruction, suggesting that p34cdc2 binding is required for the cell cycle-regulated destruction of Xenopus cyclin B2.

Mitotic arrest caused by the amino terminus of Xenopus cyclin B2.

Progression through mitosis requires the inactivation of the protein kinase activity of the p34cdc2-cyclin complex by a mechanism involving the degradation of cyclin. We have examined the stability in Xenopus egg extracts of radiolabeled Xenopus or sea urchin B-type cyclins synthesized in reticulocyte lysates. Xenopus cyclin B2 and sea urchin cyclin B were stable in metaphase extracts from unfertilized eggs but were specifically degraded following addition of Ca2+ to the extracts. The degradation of either cyclin was inhibited by the addition of an excess of unlabeled Xenopus cyclin B2 but not by the addition of a number of control proteins. A truncated protein containing only the amino terminus of Xenopus cyclin B2, including sequences known to be essential for cyclin degradation in other species, also inhibited cyclin degradation, even though the truncated protein was stable in extracts following Ca2+ addition. The addition of the truncated protein did not stimulate histone H1 kinase activity in extracts but prevented the loss of H1 kinase activity that normally follows Ca2+ addition to metaphase extracts. When the amino-terminal fragment was added to extracts capable of several cell cycles in vitro, progression through the first mitosis was inhibited and elevated histone H1 kinase activity was maintained. These results indicate that although the amino terminus of cyclin does not contain all of the information necessary for cyclin destruction, it is capable of interacting with components of the cyclin destruction pathway and thereby preventing the degradation of full-length cyclins.

Induction of DNA replication in mammalian G1 nuclei by a heat-inactivible component from permeable S phase cells; an assay system.

The in vitro initiation of DNA replication was studied in permeable mammalian cells by a newly developed procedure. Pairs of monolayer cultures, one synchronized in G1 and the other in S phase, were incubated in a sandwich with assay solution, containing Triton X-100 for permeabilization and [3H]TTP as a tracer. After 1.5 h DNA synthesis was shown to be induced in 36 to 81% of the G1 nuclei. The inducing capacity of the S phase cultures was diminished by at least 50% after a 10 min exposure to 60 degrees C prior to incubation. The suitability application of this in vitro system for testing components that might effect the initiation of DNA replication is shown in an assay with G1 cultures where the addition of up to 1 mM Ap4A led to an increase of DNA synthesizing cells from 4 to 15%.

Evidence for contamination of origin DNA isolated after an in vivo treatment of mammalian cells with 4,5',8-trimethylpsoralen.

We have studied the effect of in vivo treatment with trioxsalen on DNA replication in mammalian cells. In vitro cultured bovine liver cells were exposed to two or four cycles of treatment with 45 microM trioxsalen followed by irradiation with long-wave ultraviolet light. Thymidine incorporation was reduced by about 95% during the first hour after a double treatment. A large proportion of the label was released in alkaline sucrose gradients as a low molecular weight fraction (average length about 500 nucleotides) which was supposed to consist of replication origins containing DNA fragments. From the relative quantities of this DNA obtained at different times of the S phase we concluded that it contains a considerable but not precisely determinable proportion of non-origin DNA. We also find that the fraction is contaminated by a large excess of non-replicating bulk DNA.

Protein composition of the chromosomal scaffold and interphase nuclear matrix.

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.

In vitro DNA replication in association with the nuclear matrix of permeable mammalian cells.

DNA replication has been studied in in vitro cultured bovine liver cells permeabilized in 0.02% Triton X-100. The Km for TTP was 20 microM. The initial incorporation rate at 10 microM TTP concentration was about 12% of the in vivo synthesis and declined very strongly within 1 h. A similar decline of the incorporation rate was found at 0.12 microM TTP concentration. DNAase I digestion of DNA-matrix complexes obtained from isolated nuclei in 2 M NaCl revealed that newly replicated DNA was preferentially bound to the nuclear matrix. A similar digestion with S1 nuclease caused a selective release of short duplexes of Okazaki fragments with the complementary parental strand. The results show that in vivo replication continues in permeabilized cells in an almost unchanged way, except for a gradual decline of its rate which is mainly due to inactivation of one or more essential components.

Attachment of origins of replication to the nuclear matrix and the chromosomal scaffold.

We have investigated the attachment of DNA to the nuclear matrix and chromosomal scaffold in synchronized bovine liver cells. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the subsequent G1 phase and with a residual protein structure from dehistonized chromosomes during mitosis. On the other hand label incorporated during mid or late S phase was about equally distributed over the DNA molecule after a chase into the G1 phase. These results suggest that DNA is attached to the nuclear matrix and chromosome scaffolds by the origins of replication.

An unusually long non-coding region in rat lens alpha-crystallin messenger RNA.

Most of the mRNA sequence coding for the alpha A2 chain of the ocular lens protein alpha-crystallin from rat, has been determined by sequencing cloned DNA copies of this mRNA. The 892-base pair cDNA sequence encompasses all but 52 N-terminal amino acids of the alpha A2 chain. It lacks the sequence characteristic for the 22 extra amino acids inserted in the alpha A2 -like chain, named alpha AIns. A stretch of 583 nuceotides, representing more than 50% of the entire mRNA sequence, is located 3' wards of the alpha A2 coding sequence. It contains the characteristic AAUAAA signal involved in poly(A) -addition and represents an unexpectedly long non-coding region. Examination of the total cytoplasmic poly(A) RNA of rat lens by filter-hybridization and subsequent translation of the electrophoretically separated mRNA fractions shows that the alpha A2 chain is encoded by mRNA species which are distinct from the alpha AIns encoding mRNA. No evidence is obtained for an extensive size heterogeneity in the 3' untranslated regions of these two different rat lens mRNAs.