A20/TNFAIP3 heterozygosity predisposes to behavioral symptoms in a mouse model for neuropsychiatric lupus.

Background: Neuropsychiatric lupus (NPSLE) refers to the neurological and psychiatric manifestations that are commonly observed in patients with systemic lupus erythematosus (SLE). An important question regarding the pathogenesis of NPSLE is whether the symptoms are caused primarily by CNS-intrinsic mechanisms or develop as a consequence of systemic autoimmunity. Currently used spontaneous mouse models for SLE have already contributed significantly to unraveling how systemic immunity affects the CNS. However, they are less suited when interested in CNS primary mechanisms. In addition, none of these models are based on genes that are associated with SLE. In this study, we evaluate the influence of A20, a well-known susceptibility locus for SLE, on behavior and CNS-associated changes in inflammatory markers. Furthermore, given the importance of environmental triggers for disease onset and progression, the influence of an acute immunological challenge was evaluated. Methods: Female and male A20 heterozygous mice (A20+/-) and wildtype littermates were tested in an extensive behavioral battery. This was done at the age of 10±2weeks and 24 â€‹± â€‹2 weeks to evaluate the impact of aging. To investigate the contribution of an acute immunological challenge, LPS was injected intracerebroventricularly at the age of 10±2weeks followed by behavioral analysis. Underlying molecular mechanisms were evaluated in gene expression assays on hippocampus and cortex. White blood cell count and blood-brain barrier permeability were analyzed to determine whether peripheral inflammation is a relevant factor. Results: A20 heterozygosity predisposes to cognitive symptoms that were observed at the age of 10 â€‹± â€‹2 weeks and 24 â€‹± â€‹2 weeks. Young A20+/- males and females showed a subtle cognitive phenotype (10±2weeks) with distinct neuroinflammatory phenotypes. Aging was associated with clear neuroinflammation in female A20+/- mice only. The genetic predisposition in combination with an environmental stimulus exacerbates the behavioral impairments related to anxiety, cognitive dysfunction and sensorimotor gating. This was predominantly observed in females. Furthermore, signs of neuroinflammation were solely observed in female A20+/- mice. All above observations were made in the absence of peripheral inflammation and of changes in blood-brain barrier permeability, thus consistent with the CNS-primary hypothesis. Conclusions: We show that A20 heterozygosity is a predisposing factor for NPSLE. Further mechanistic insight and possible therapeutic interventions can be studied in this mouse model that recapitulates several key hallmarks of the disease.

Treatment Steps, Surgery, and Hospitalization Rates During the First Year of Follow-up in Patients with Inflammatory Bowel Diseases from the 2011 ECCO-Epicom Inception Cohort.

BACKGROUND AND AIMS: The ECCO-EpiCom study investigates the differences in the incidence and therapeutic management of inflammatory bowel diseases [IBD] between Eastern and Western Europe. The aim of this study was to analyse the differences in the disease phenotype, medical therapy, surgery, and hospitalization rates in the ECCO-EpiCom 2011 inception cohort during the first year after diagnosis. METHODS: Nine Western, five Eastern European centres and one Australian centre with 258 Crohn's disease [CD], 380 ulcerative colitis [UC] and 71 IBD unclassified [IBDU] patients [female/male: 326/383; mean age at diagnosis: 40.9 years, SD: 17.3 years] participated. Patients' data were registered and entered in the web-based ECCO-EpiCom database [www.epicom-ecco.eu]. RESULTS: In CD, 36 [19%] Western Europe/Australian and 6 [9%] Eastern European patients received biological therapy [p = 0.04], but the immunosuppressive [IS] use was equal and high in these regions [Eastern Europe vs Western Europe/Australia: 53% vs 45%; p = 0.27]. Surgery was performed in 17 [24%] CD patients in Eastern Europe and 13 [7%] in Western Europe/Australia [p < 0.001, pLogRank = 0.001]. Of CD patients from Eastern Europe, 24 [34%] were hospitalized, and 39 [21%] from Western Europe/Australia, [p = 0.02, pLogRank = 0.01]. In UC, exposure to biologicals and colectomy rates were low and hospitalization rates did not differ between these regions during the 1-year follow-up period [16% vs 16%; p = 0.93]. CONCLUSIONS: During the first year after diagnosis, surgery and hospitalization rates were significantly higher in CD patients in Eastern Europe compared with Western Europe/Australia, whereas significantly more CD patients were treated with biologicals in the Western Europe/Australian centres.

Incidence and initial disease course of inflammatory bowel diseases in 2011 in Europe and Australia: results of the 2011 ECCO-EpiCom inception cohort.

BACKGROUND AND AIMS: The aim of the present study was to validate the IBD (inflammatory bowel diseases) incidence reported in the 2010 ECCO-EpiCom (European Crohn's and Colitis Organization-Epidemiological Committee) inception cohort by including a second independent inception cohort from participating centers in 2011 and an Australian center to investigate whether there is a difference in the incidence of IBD between Eastern and Western European countries and Australia. METHODS: Fourteen centers from 5 Eastern and 9 Western European countries and one center from Australia participated in the ECCO-EpiCom 2011 inception cohort. Patients' data regarding disease type, socio-demographic factors, extraintestinal manifestations and therapy were entered into the Web-based EpiCom database, www.ecco-epicom.eu. RESULTS: A total of 711 adult patients were diagnosed during the inclusion year 2011, 178 (25%) from Eastern, 461 (65%) from Western Europe and 72 (10%) from Australia; 259 (37%) patients were diagnosed with Crohn's disease, 380 (53%) with ulcerative colitis and 72 (10%) with IBD unclassified. The mean annual incidence rate for IBD was 11.3/100,000 in Eastern Europe, 14.0/100,000 in Western Europe and 30.3/100,000 in Australia. Significantly more patients were diagnosed with complicated disease at diagnosis in Eastern Europe compared to Western Europe (43% vs. 27%, p=0.02). CONCLUSION: Incidence rates, disease phenotype and initial treatment characteristics in the 2011 ECCO-EpiCom cohort were not significantly different from that reported in the 2010 cohort.

Health care and patients' education in a European inflammatory bowel disease inception cohort: an ECCO-EpiCom study.

BACKGROUND AND AIMS: The EpiCom study and inception cohort was initiated in 2010 in 31 centers from 14 Western and 8 Eastern European countries, covering a 10.1million person background population. Our aim was to investigate whether there is a difference between Eastern and Western Europe in health care and education of patients with inflammatory bowel disease (IBD). METHODS: A quality of care (QoC) questionnaire was developed in the EpiCom group consisting of 16 questions covering 5 items: time interval between the onset of symptoms and diagnosis, information, education, empathy and access to health care providers. RESULTS: Of 1,515 patients, 947 (217 east/730 west) answered the QoC questionnaire. Only 23% of all patients had knowledge about IBD before diagnosis. In Eastern Europe, significantly more patients searched out information about IBD themselves (77% vs. 68%, p<0.05), the main source was the Internet (92% vs. 88% p=0.23). In Western Europe, significantly more patients were educated by nurses (19% vs. 1%, p<0.05), while in Eastern Europe, gastroenterologists were easier to contact (80% vs. 68%, p<0.05). CONCLUSION: Health care differed significantly between Eastern and Western Europe in all items, but satisfaction rates were high in both geographic regions. Because of the low awareness and the rising incidence of IBD, general information should be the focus of patient organizations and medical societies. In Western Europe IBD nurses play a very important role in reducing the burden of patient management.

Improved spatial learning is associated with increased hippocampal but not prefrontal long-term potentiation in mGluR4 knockout mice.

Although much information about metabotropic glutamate receptors (mGluRs) and their role in normal and pathologic brain function has been accumulated during the last decades, the role of group III mGluRs is still scarcely documented. Here, we examined mGluR4 knockout mice for types of behavior and synaptic plasticity that depend on either the hippocampus or the prefrontal cortex (PFC). We found improved spatial short- and long-term memory in the radial arm maze, which was accompanied by enhanced long-term potentiation (LTP) in hippocampal CA1 region. In contrast, LTP in the PFC was unchanged when compared with wild-type controls. Changes in paired-pulse facilitation that became overt in the presence of the GABAA antagonist picrotoxin indicated a function of mGluR4 in maintaining the excitation/inhibition balance, which is of crucial importance for information processing in the brain and the deterioration of these processes in neuropsychological disorders such as autism, epilepsy and schizophrenia.

LPA5 receptor plays a role in pain sensitivity, emotional exploration and reversal learning.

Lysophosphatidic acid (LPA) is a bioactive lipid acting on the nervous system through at least 6 different G protein-coupled receptors. In this study, we examined mice lacking the LPA5 receptor using an extensive battery of behavioral tests. LPA5-deficient mice showed decreased pain sensitivity in tail withdrawal, faster recovery in one inflammatory pain procedure (complete Freund's adjuvant-induced inflammation) and attenuated responses under specific neuropathic pain conditions. Notably, deletion of LPA5 also induced nocturnal hyperactivity and reduced anxiety in the mutant mice. Several exploratory tasks revealed signs of reduced anxiety in LPA5 knockout mice including increased visits to the arena center and reduced thigmotaxis in the open field, and more open arm entries in the elevated plus maze. Finally, LPA5 knockout mice also displayed marked reduction in social exploration, although several other tests indicated that these mice were able to respond normally to environmental stimuli. While learning and memory performance was not impaired in LPA5-deficient mice, we found differences, e.g., targeted swim strategy and reversal learning, as well as scheduled appetitive conditioning that might indicate differential motivational behavior. These results imply that LPA5 might be involved in both nociception and mechanisms of pain hypersensitivity, as well as in anxiety-related and motivational behaviors. These observations further support the proposed involvement of LPA signaling in psychopathology.

Early clinical remission and normalisation of CRP are the strongest predictors of efficacy, mucosal healing and dose escalation during the first year of adalimumab therapy in Crohn's disease.

BACKGROUND: Adalimumab is a fully human monoclonal antibody targeting tumour necrosis factor with proven efficacy in the treatment of Crohn's disease (CD). AIM: To investigate the predictors of medium-term clinical efficacy and mucosal healing during adalimumab therapy, in patients with CD, in specialised centres approved for biological therapy in Hungary. METHODS: Data capture of the 201 CD patients was standardised and prospective (male/female: 112/89, median age: 33.0 years, duration: 8 years). Previous infliximab therapy had been administered in 48% of patients, concomitant steroids in 41%, azathioprine in 69% and combined therapy in 27% of patients. RESULTS: Overall clinical response and remission rates at 24 weeks were 78% and 52%, respectively; at 52 weeks were 69% and 44%, respectively. Endoscopic improvement and healing were achieved in 43% and 24% of patients. In a logistic regression model, clinical efficacy and CRP at week 12, need for combined immunosuppression at induction, shorter disease duration and smoking were identified as independent predictors for 12-month clinical outcome, whereas CRP at week 12, clinical remission at week 24, inflammatory parameters and nonsmoking were associated to endoscopic improvement/healing. Intensification to weekly dosing was needed in 16% of patients. Parallel azathioprine therapy and clinical remission at week 12 were inversely associated with dose escalation. CONCLUSIONS: Clinical efficacy and normalised CRP at week 12 (early deep clinical remission) are associated with medium-term clinical efficacy and mucosal healing during adalimumab therapy, whereas need for combined immunosuppression at induction and smoking status are predictors for non-response. Parallel azathioprine therapy may decrease the probability for dose escalation.

Validation of thin layer and high performance thin layer chromatographic methods.

Analytical validation is a key requirement to asses and to prove a method's reliability and suitability for an intended use. Planar chromatographic procedures are used in different applications ranging from simple screening tests to sophisticated instrumental quantitative assays of analytes in complex matrices. This paper intends to give guidance on how to adopt international accepted formal requirements and guidelines for validation of these different TLC/HPTLC procedures. In addition, some selected parameters for robustness testing and for on going quality assurance of analytical performance based on control charts are reported.

Impurity profiling of pharmaceuticals by thin-layer chromatography.

Although there is a tendency in current pharmacopoeias for favouring HPLC, thin-layer chromatography (TLC) is still a very popular and frequently used analytical method in the pharmaceutical industry. This paper highlights the possibilities of this method in the different areas of pharmaceutical analysis like in-process and intermediate control, illustrated by impurity testing of active ingredients and final products, as well as its application in pharmaceutical research and development, based on some examples reported mainly in the last five years.

Validation and quality assurance of planar chromatographic procedures in pharmaceutical analysis.

Within the process of the International Conference on Harmonization (ICH), 2 guidelines were released containing a standardized terminology, a verified model of requirements for the validation of analytical procedures, and some guidance in the practical aspects of conducting validation studies in pharmaceutical analysis. For planar chromatographic procedures, which may be used at different levels either in qualitative identity testing, assays, semiquantitative limit tests, or quantitative determination of impurities, this paper tries to transfer these formal requirements into practical approaches for validation. Basic acceptance criteria for evaluation of validation experiments based on practical experience are proposed. In addition, selected parameters for robustness testing of given procedures and quality assurance of quantitative planar chromatographic testing by control charts is described.

Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex.

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide-major histocompatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen.These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

Cleaning validation procedure eased by using overpressured layer chromatography.

In the manufacturing plants of many pharmaceutical companies the reaction apparatus is suitable to produce different active pharmaceutical ingredients. After completing the production of a compound the equipment should be cleaned in order to avoid the cross contamination in the next lot of the other products. In the authors' laboratory several chromatographic methods were introduced to measure the amount of the residual substances remaining on the surface of the apparatus after the cleaning procedure. A sensitive and fairly rapid overpressured layer chromatographic (OPLC) procedure--suitable to separate and control five steroid hormone compounds (allylestrenol, estradiol, ethynodiol diacetate, levonorgestel, norethisterone) produced in the same equipment at different times--was developed and validated.

Influence of intensive physical training on urinary nitrate elimination and plasma endothelin-1 levels in patients with congestive heart failure.

BACKGROUND: Congestive heart failure (CHF) is associated with increased peripheral vascular resistance. Exercise-induced shear stress may release endothelial relaxing factors, such as nitric oxide (NO), and inhibit the production of vasoconstrictors such as endothelin-1 (ET-1) thereby modulating vascular tone. We examined the effect of intensive training on ET-1 plasma concentrations and NO-metabolite elimination in patients with CHF after acute myocardial infarction. METHODS: Seventeen patients with CHF after a myocardial infarction were randomized to an exercise group (n = 9), who performed physical training for 8 weeks, or a control group (n = 8) who received usual care. A physical examination, pulmonary function test, and a maximum exercise test were performed, and 24-hour urinary nitrate elimination and ET-1 in plasma were determined before and at the end of the study period. RESULTS: Maximal oxygen uptake remained unchanged in controls (17.9 +/- 1.4 to 18.1 +/- 1.5 mL/(kg min) but increased in the exercise group (from 20.4 +/- 0.75 to 26.7 +/- 1.4 mL/(kg min). After 8 weeks the urinary nitrate elimination in controls was significantly decreased (1.25 +/- 0.20 to 1.03 +/- 0.22 mmol/24 hours; P < 0.001), while it was unchanged in the exercise group (1.26 +/- 0.23 to 1.39 +/- 0.28; P = 0.71). Plasma ET-1 levels did not change after 8 weeks (7.87 +/- 0.62 versus 7.57 +/- 0.75 and 7.13 +/- 0.6 versus 7.35 +/- 0.7 pg/mL for control and exercise groups, respectively). CONCLUSION: In patients with CHF after acute myocardial infarction nitrate elimination decreases over the subsequent 2 months. This trend was reversed by training. Because nitrate elimination mirrors endogenous NO production, these results suggest that training may positively influence endothelial vasodilator function.

Point mutations in the beta chain CDR3 can alter the T cell receptor recognition pattern on an MHC class I/peptide complex over a broad interface area.

To study how the T cell receptor interacts with its cognate ligand, the MHC/peptide complex, we used site directed mutagenesis to generate single point mutants that alter amino acids in the CDR3beta loop of a H-2Kb restricted TCR (N30.7) specific for an immunodominant peptide N52-N59 (VSV8) derived from the vesicular stomatitis virus nucleocapsid. The effect of each mutation on antigen recognition was analyzed using wild type H-2Kb and VSV8 peptide, as well as H-2Kb and VSV8 variants carrying single replacements at residues known to be exposed to the TCR. These analyses revealed that point mutations at some positions in the CDR3beta loop abrogated recognition entirely, while mutations at other CDR3beta positions caused an altered pattern of antigen recognition over a broad area on the MHC/peptide surface. This area included the N-terminus of the peptide, as well as residues of the MHC alpha1 and alpha2 helices flanking this region. Assuming that the N30 TCR docks on the MHC/peptide with an orientation similar to that recently observed in two different TCR-MHC/peptide crystal structures, our findings would suggest that single amino acid alterations within CDR3beta can affect the interaction of the TCR with an MHC surface region distal from the predicted CDR3beta-Kb/VSV8 interface. Such unique recognition capabilities are generated with minimal alterations in the CDR3 loops of the TCR. These observations suggest the hypothesis that extensive changes in the recognition pattern due to small perturbations in the CDR3 structure appears to be a structural strategy for generating a highly diversified TCR repertoire with specificity for a wide variety of antigens.

An in vitro study of the dynamic features of the major histocompatibility complex class I complex relevant to its role as a versatile peptide-receptive molecule.

The major histocompatibility complex class I complex consists of a heavy chain and a light chain (beta2-microglobulin, beta2m), which assemble with a short endogenously derived peptide in the endoplasmic reticulum. The class I peptide can be directly exchanged, either at the cell surface or, as recently described, in vesicles of the endocytic compartments, thus allowing exogenous peptides to enter the class I presentation pathway. To probe the interactions between the components of the class I molecule, we analyzed the exchange of peptide and beta2m by using purified, recombinant H2-Kb/peptide complexes in a cell-free in vitro system. The exchange of competitor peptide was primarily dependent on the off-rate of the original peptide in the class I binding groove. Peptide exchange was not enhanced by the presence of exogenous beta2m, as exchange occurred to the same extent in its absence. Thus, the exchange of peptide and beta2m are independent events. The exchange rate of beta2m also was not affected by the dissociation rates of the original peptides. Furthermore, peptides could substantially exchange into class I molecules over a pH range of 5.5 to 7.5, conditions prevalent in certain endocytic compartments. We conclude that the dynamic properties of the components of class I molecules explain its function as a highly peptide-receptive molecule. The major histocompatibility complex class I can readily receive peptides independent of the presence of exogenous beta2m, even at a low pH. Such properties are relevant to class I peptide acquisition, which can occur at the cell surface, as well as in specialized endosomes.

[The effect of prolonged acenocoumarol therapy on bone density]. / A tartós acenocumarol kezelés hatása a csonttömegre.

The effect of chronic cumarin treatment on bone mineral content was investigated. Bone mineral density was determined by double photon densitometry (Lunar DPXL). The density data (mean +/- SE) of 45 cardiac patients (age: 57.0 = +/- 6.3 y, body mass index: 26.7 +/- 3.8 kp/m2, cardiac stadium score, according to New York Heart Association: 2-3), had been treated by acenocumarol at least for 2 years (duration of treatment: 75.0 +/- 52 months), were compared to the values of 45 age, body mass index, cardiac status matched patients not treated by anticoagulant. The density values of L2-L4 lumbar regions were lower in the treated group (1.041 +/- 0.17 vs. controlls: 1.13 +/- 0.15 g/cm2, p < 0.05), while no differences in ultradistal ulnar and radial regions were detected. No correlation between bone mineral density and the length, or the dose of the cumarin treatment were observed. This observation suggests the importance of the regular bone densitometry control of cumarin treated patient.

The role of self peptides in the allogeneic cross-reactivity of CTLs.

This study presents data relevant to understanding the molecular and structural basis for the cross-reactivity of many CTLs on multiple MHC targets. Five anti-H-2Kb alloreactive CTL clones derived from B6.C-H-2bm1 (bm1), B6.C-H-2bm8 (bm8), and B6.C-H-2bm11 (bm11) mice and a Sendai virus-specific H-2Kb-restricted CTL clone were studied. Self peptides extracted from Kb molecules were fractionated by HPLC and tested for their ability to be recognized on RMA/s (H-2b) cells by those clones. For each alloreactive clone, a single dominant peptide peak was found to sensitize target cells. In addition to recognizing peptides presented by the Kb molecule, the five alloreactive clones and the one Sendai virus-specific clone all showed cross-reactivities on a panel of Kbm mutant cells in a peptide-dependent manner. Two CTL clones, one alloreactive and one virus specific, cross-recognized Kbm targets by each responding to a unique self peptide in the context of the mutant MHC molecules. Our data underscore the prevalent idea that TCR-alpha beta have an inherent structural capability to react with several peptide/MHC structural patterns other than the original peptide/MHC pattern that might have been used to select that TCR. The high incidence of cross-reactivity seems to reflect a feature of the mechanism of positive selection in the thymus and the need for T cells in the repertoire to have an expanded capability for responding to a wide variety of foreign Ags.

Generation of HLA-B55 restricted T lymphocyte mediated cytotoxicity against autologous LCL.

The lymphocytes of one HLA-A11 positive individual (A1, A11, B49, B55) were stimulated in vitro with the autologous EBV transformed lymphoblastoid cell line (LCL). The culture contained HLA-A1, A11 and B55 restricted, LCL selective cytotoxic T cells (CTLs). In the T cell culture stimulated four times, the lysis of A11 and B55 carrying targets suggested that the subsets of the two latter CTL types had similar size. After further stimulations the B55 restricted CTLs were enriched in the culture. Earlier results suggested that in HLA-A11 positive individuals the A11-restricted LCL-selective CTL subset dominates. The sensitivity of a target panel including Burkitt lymphoma (BL) lines suggested that the peptide presented by the B55 molecule differs in A and B type EBV strain carrying cells.

Cytotoxic susceptibility and defective MHC class I expression do not correlate with mutation of p53 in human carcinomas.

Twenty-nine samples of ex vivo ovarian and lung carcinomas were investigated for the relationship between the presence of mutated protein 53 (mp53) and cytotoxic susceptibility. Unaltered expression of MHC class I alleles was required for the cytotoxic susceptibility of tumour cells to the autologous ex vivo blood lymphocytes, i.e. all 4 sensitive tumours belonged to the group of 11 tumours without defect in MHC class I expression. In contrast, the susceptibility did not correlate with the presence of mp53, i.e. cases with mp53 were randomly distributed between the sensitive and resistant tumours (2/4 and 10/17 respectively). There was no correlation either between the p53 mutation and down-regulation of MHC class I alleles. The results suggest that in these tumours the mutated p53 is not the source of immunogenic peptides and that the lack of recognition of the tumours with mp53 is not caused by a defect in the expression of MHC class I molecules.