CD62L (L-selectin) shedding for assessment of functional blockade of TNF alpha in anti-TNF treated IBD patients: clinical feasibility and perspectives (DOP060)

Background Tumor necrosis factor (TNF) inhibition is central to the therapy of inflammatory bowel diseases (IBD). However, loss of response (LOR) is frequent and additional tests to help decision making with costly anti-TNF Therapy are needed. Methods Consecutive IBD Patients receiving anti-TNF therapy (Infliximab (IFX) or Adalimumab (after IFX LOR) from Bern University Hospital were identified and followed prospectively. Patient whole blood was stimulated with a dose-titration of two triggers of TLR receptors human: TNF and LPS. Median fluorescence intensity of CD62L on the surface of granulocytes was quantified by surface staining with specific antibodies (CD33, CD62L) and flow cytometry and logistic curves to these data permits the calculation of EC50 or the half maximal effective concentration TNF concentration to induce shedding [1]. A shift in the concentration were CD62L shedding occurred was seen before and after the anti-TNF agent administraion which permits to predict the response to the drug. This predicted response was correlated to the clinical evolution of the patients in order to analyze the ability of this test to identify LOR to IFX. Results We collected prospective clinical data and blood samples, before and after anti-TNF agent administration, on 33 IBD patients, 25 Crohn's disease and 8 ulcerative colitis patients (45% females) between June 2012 and November 2013. The assay showed a functional blockade of IFX (PFR) for 22 patients (17 CD and 5 UC) whereas 11 (8 CD and 3 UC) had no functional response (NR) to IFX. Clinical characteristics (e.g. diagnosis, disease location, smoking status, BMI and number of infusions) were no significantly different between predicted PFR and NR. Among the 22 Patients with PRF, only 1 patient was a clinical non responder (LOR to IFX), based on clinical prospective evaluation by IBD gastroenterologists (PJ, AM), and among the 11 predicted NR, 3 had no clinical LOR. Sensitivity of this test was 95% and specificity 73% and AUC adjusted for age and gender was 0.81 (Figure 1). During follow up (median 10 mo, 3–15) 8 “hard” outcomes occured (3 medic. flares, 4 resections and 1 new fistula) 2 in the PFR and 6 in the NR group (25% vs. 75%; p < 0.01). Correlation with clinical response is presented in Figure 2. Figure 1. Figure 2. Correlation clinical response - log EC50 changes: 1 No, 2 partial, 3 complete clinical response. Conclusion CD62L (L-Selectin) shedding is the first validated test of functional blockade of TNF alpha in anti-TNF treated IBD patients and will be a useful tool to guide medical decision on the use of anti-TNF agents. Comparative studies with ATI and trough level of IFX are ongoing. 1. Nicola Patuto, Emma Slack, Frank Seibold and Andrew J. Macpherson, (2011), Quantitating Anti-TNF Functionality to Inform Dosing and Choice of Therapy, Gastroenterology, 140 (5, Suppl. I), S689.

Innate-like control of human iNKT cell autoreactivity via the hypervariable CDR3beta loop

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3beta, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3beta loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3beta in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist alpha-linked glycolipid antigen OCH and structurally different endogenous beta-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3beta sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3beta for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3beta dependent functional hierarchy of human iNKT cells.

Canine microglial cells: stereotypy in immunophenotype and specificity in function?

Microglial cells represent the endogenous immune system of the central nervous system (CNS). Upon pathological insults they reveal their immunological potential aimed at regaining homeostasis. These reactions have long been believed to follow a uniform and unspecific pattern which is irrespective to the underlying disease entity. Evidence is growing that this view seriously underrates microglial competence as the defenders of the CNS. In the present study, microglial cells of 47 dogs were examined ex vivo by means of flow cytometry. Ex vivo examination included immunophenotypic characterization using eight different surface markers and functional studies such as phagocytosis assay and the reactive oxygen species (ROS) generation test. The dogs were classified according to their histopathological diagnoses in disease categories (controls, canine distemper virus (CDV) induced demyelination, other diseases of the CNS) and results of microglial reaction profiles were compared. Immunophenotypic characterization generally revealed relative high conformity in the microglial disease response among the different groups, however the functional response was shown to be more specific. Dogs with intracranial inflammation and dogs with demyelination showed an enhanced phagocytosis, whereas a significant up-regulation of ROS generation was found in dogs with demyelination due to CDV infection. This strongly suggests a specific response of microglia to infection with CDV in the settings of our study and underlines the pivotal role of microglial ROS generation in the pathogenesis of demyelinating diseases, such as canine distemper.

Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro

BACKGROUND Platelet-rich concentrates are used as a source of growth factors to improve the healing process. The diverse preparation protocols and the gaps in knowledge of their biological properties complicate the interpretation of clinical results. QUESTIONS/PURPOSES In this study we aimed to (1) analyze the concentration and kinetics of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF), leukocyte- and platelet-rich plasma (L-PRP), and natural blood clot during in vitro culture; (2) investigate the migration of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as a functional response to the factors released; and (3) uncover correlations between individual growth factors with the initial platelet/leukocyte counts or the induced cell migration. METHODS L-PRF, L-PRP, and natural blood clot prepared from 11 donors were cultured in vitro for 28 days and media supernatants collected after 8 hours and 1, 3, 7, 14, and 28 days. Released transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), insulin growth factor (IGF-1), platelet-derived growth factor AB (PDGF-AB), and interleukin-1β (IL-1β) were measured in the supernatants with enzyme-linked immunosorbent assay. Migration of MSC and HUVEC induced by the supernatants was evaluated in Boyden chambers. RESULTS More TGF-ß1 was released (mean ± SD in pg/mL of blood) from L-PRF (37,796 ± 5492) compared with L-PRP (23,738 ± 6848; p < 0.001) and blood clot (3739 ± 4690; p < 0.001), whereas more VEGF and IL-1ß were released from blood clot (1933 ± 704 and 2053 ± 908, respectively) compared with both L-PRP (642 ± 208; p < 0.001 and 273 ± 386; p < 0.001, respectively) and L-PRF (852 ± 376; p < 0.001 and 65 ± 56, p < 0.001, respectively). No differences were observed in IGF-1 and PDGF-AB released from any of the concentrates. TGF-β1 release peaked at Day 7 in L-PRF and at 8 hours and Day 7 in L-PRP and 8 hours and Day 14 in blood clot. In all concentrates, main release of VEGF occurred between 3 and 7 days and of IL-1β between Days 1 and 7. IGF-1 and PDGF-AB were released until Day 1 in L-PRP and blood clot, in contrast to sustained release over the first 3 days in L-PRF. The strongest migration of MSC occurred in response to L-PRF, and more HUVEC migration was seen in L-PRF and blood clot compared with L-PRP. TGF-β1 correlated with initial platelet counts in L-PRF (Pearson r = 0.66, p = 0.0273) and initial leukocyte counts in L-PRP (Pearson r = 0.83, p = 0.0016). A positive correlation of IL-1β on migration of MSC and HUVEC was revealed (Pearson r = 0.16, p = 0.0208; Pearson r = 0.31, p < 0.001). CONCLUSIONS In comparison to L-PRP, L-PRF had higher amounts of released TGF-β1, a long-term release of growth factors, and stronger induction of cell migration. Future preclinical studies should confirm these data in a defined injury model. CLINICAL RELEVANCE By characterizing the biologic properties of different platelet concentrates in vitro, we may gain a better understanding of their clinical effects and develop guidelines for specific future applications.

Functional, structural and molecular plasticity of mammalian skeletal muscle in response to exercise stimuli

Biological systems have acquired effective adaptive strategies to cope with physiological challenges and to maximize biochemical processes under imposed constraints. Striated muscle tissue demonstrates a remarkable malleability and can adjust its metabolic and contractile makeup in response to alterations in functional demands. Activity-dependent muscle plasticity therefore represents a unique model to investigate the regulatory machinery underlying phenotypic adaptations in a fully differentiated tissue. Adjustments in form and function of mammalian muscle have so far been characterized at a descriptive level, and several major themes have evolved. These imply that mechanical, metabolic and neuronal perturbations in recruited muscle groups relay to the specific processes being activated by the complex physiological stimulus of exercise. The important relationship between the phenotypic stimuli and consequent muscular modifications is reflected by coordinated differences at the transcript level that match structural and functional adjustments in the new training steady state. Permanent alterations of gene expression thus represent a major strategy for the integration of phenotypic stimuli into remodeling of muscle makeup. A unifying theory on the molecular mechanism that connects the single exercise stimulus to the multi-faceted adjustments made after the repeated impact of the muscular stress remains elusive. Recently, master switches have been recognized that sense and transduce the individual physical and chemical perturbations induced by physiological challenges via signaling cascades to downstream gene expression events. Molecular observations on signaling systems also extend the long-known evidence for desensitization of the muscle response to endurance exercise after the repeated impact of the stimulus that occurs with training. Integrative approaches involving the manipulation of single factors and the systematic monitoring of downstream effects at multiple levels would appear to be the ultimate method for pinpointing the mechanism of muscle remodeling. The identification of the basic relationships underlying the malleability of muscle tissue is likely to be of relevance for our understanding of compensatory processes in other tissues, species and organisms.

Shifts in microbial communities do not explain the response of grassland ecosystem function to plant functional composition and rainfall change

Ecosystem functioning in grasslands is regulated by a range of biotic and abiotic factors, and the role of microbial communities in regulating ecosystem function has been the subject of much recent scrutiny. However, there are still knowledge gaps regarding the impacts of rainfall and vegetation change upon microbial communities and the implications of these changes for ecosystem functioning. We investigated this issue using data from an experimental mesotrophic grassland study in south-east England, which had been subjected to four years of rainfall and plant functional composition manipulations. Soil respiration, nitrogen and phosphorus stocks were measured, and the abundance and community structure of soil microbes were characterised using quantitative PCR and multiplex-TRFLP analysis, respectively. Bacterial community structure was strongly related to the plant functional composition treatments, but not the rainfall treatment. However, there was a strong effect of both rainfall change and plant functional group upon bacterial abundance. There was also a weak interactive effect of the two treatments upon fungal community structure, although fungal abundance was not affected by either treatment. Next, we used a statistical approach to assess whether treatment effects on ecosystem function were regulated by the microbial community. Our results revealed that ecosystem function was influenced by the experimental treatments, but was not related to associated changes to the microbial community. Overall, these results indicate that changes in fungal and bacterial community structure and abundance play a relatively minor role in determining grassland ecosystem function responses to precipitation and plant functional composition change, and that direct effects on soil physical and chemical properties and upon plant and microbial physiology may play a more important role.

Unfolded protein response suppresses CEBPA by induction of calreticulin in acute myeloid leukaemia

The unfolded protein response (UPR) is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). The role of the UPR during leukemogenesis is unknown so far. Here, we studied the induction of mediators of the UPR in leukaemic cells of AML patients. Increased expression of the spliced variant of the X-box binding protein 1 (XBP1s) was detected in 17.4% (16 of 92) of AML patients. Consistent with activated UPR, this group also had increased expression of ER-resident chaperones such as the 78 kD glucose-regulated protein (GRP78) and of calreticulin. Conditional expression of calreticulin in leukaemic U937 cells was found to increase calreticulin binding to the CEBPA mRNA thereby efficiently blocking translation of the myeloid key transcription factor CEBPA and ultimately affecting myeloid differentiation. Consequently, leukaemic cells from AML patients with activated UPR and thus increased calreticulin levels showed in fact suppressed CEBPA protein expression. We identified two functional ER stress response elements (ERSE) in the calreticulin promoter. The presence of NFY and ATF6, as well as an intact binding site for YY1 within these ERSE motifs were essential for mediating sensitivity to ER stress and activation of calreticulin. Thus, we propose a model of the UPR being activated in a considerable subset of AML patients through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation.

TRAIL signaling is mediated by DR4 in pancreatic tumor cells despite the expression of functional DR5

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and agonistic anti-DR4/TRAIL-R1 and anti-DR5/TRAIL-R2 antibodies are currently under clinical investigation for treatment of different malignancies. TRAIL activates DR4 and DR5 and thereby triggers apoptotic and non-apoptotic signaling pathways, but possible different roles of DR4 or DR5 in these responses has poorly been addressed so far. In the present work, we analyzed cell viability, DISC formation as well as IL-8 and NF-kappaB activation side by side in responses to TRAIL and agonistic antibodies against DR4 (mapatumumab) and against DR5 (lexatumumab) in pancreatic ductal adenocarcinoma cells. We found that all three reagents are able to activate cell death and pro-inflammatory signaling. Death-inducing signaling complex (DISC) analysis revealed that mapatumumab and lexatumumab induce formation of homocomplexes of either DR4 or DR5, whereas TRAIL additionally stimulated the formation of heterocomplexes of both receptors. Notably, blocking of receptors using DR4- and DR5-specific Fab fragments indicated that TRAIL exerted its function predominantly via DR4. Interestingly, inhibition of PKC by Goe6983 enabled DR5 to trigger apoptotic signaling in response to TRAIL and also strongly enhanced lexatumumab-mediated cell death. Our results suggest the existence of mechanisms that silence DR5 for TRAIL- but not for agonistic-antibody treatment.

Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

Perceived functional ability assessed with the spinal function sort: is it valid for European rehabilitation settings in patients with non-specific non-acute low back pain?

The aim of this study involving 170 patients suffering from non-specific low back pain was to test the validity of the spinal function sort (SFS) in a European rehabilitation setting. The SFS, a picture-based questionnaire, assesses perceived functional ability of work tasks involving the spine. All measurements were taken by a blinded research assistant; work status was assessed with questionnaires. Our study demonstrated a high internal consistency shown by a Cronbach's alpha of 0.98, reasonable evidence for unidimensionality, spearman correlations of >0.6 with work activities, and discriminating power for work status at 3 and 12 months by ROC curve analysis (area under curve = 0.760 (95% CI 0.689-0.822), respectively, 0.801 (95% CI 0.731-0.859). The standardised response mean within the two treatment groups was 0.18 and -0.31. As a result, we conclude that the perceived functional ability for work tasks can be validly assessed with the SFS in a European rehabilitation setting in patients with non-specific low back pain, and is predictive for future work status.

The transcriptional regulator megakaryoblastic leukemia-1 mediates serum response factor-independent activation of tenascin-C transcription by mechanical stress

The extracellular matrix protein tenascin-C (TNC) is up-regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain-induced TNC expression depends on RhoA-actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia-1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c-fos was abolished. Cyclic strain failed to induce TNC in MKL1-deficient but not in SRF-deficient fibroblasts, and strain-induced TNC expression strongly depended on the SAP domain of MKL1. Promoter-reporter and chromatin immunoprecipitation experiments unraveled a SAP-dependent, SRF-independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.

The link between visual exploration and neuronal activity: A multi-modal study combining eye tracking, functional magnetic resonance imaging and transcranial magnetic stimulation

In the present multi-modal study we aimed to investigate the role of visual exploration in relation to the neuronal activity and performance during visuospatial processing. To this end, event related functional magnetic resonance imaging er-fMRI was combined with simultaneous eye tracking recording and transcranial magnetic stimulation (TMS). Two groups of twenty healthy subjects each performed an angle discrimination task with different levels of difficulty during er-fMRI. The number of fixations as a measure of visual exploration effort was chosen to predict blood oxygen level-dependent (BOLD) signal changes using the general linear model (GLM). Without TMS, a positive linear relationship between the visual exploration effort and the BOLD signal was found in a bilateral fronto-parietal cortical network, indicating that these regions reflect the increased number of fixations and the higher brain activity due to higher task demands. Furthermore, the relationship found between the number of fixations and the performance demonstrates the relevance of visual exploration for visuospatial task solving. In the TMS group, offline theta bursts TMS (TBS) was applied over the right posterior parietal cortex (PPC) before the fMRI experiment started. Compared to controls, TBS led to a reduced correlation between visual exploration and BOLD signal change in regions of the fronto-parietal network of the right hemisphere, indicating a disruption of the network. In contrast, an increased correlation was found in regions of the left hemisphere, suggesting an intent to compensate functionality of the disturbed areas. TBS led to fewer fixations and faster response time while keeping accuracy at the same level, indicating that subjects explored more than actually needed.

The Lower Limb Functional Index: development and clinimetric properties

Background Existing lower-limb, region-specific, patient-reported outcome measures have clinimetric limitations, including limitations in psychometric characteristics (eg, lack of internal consistency, lack of responsiveness, measurement error) and the lack of reported practical and general characteristics. A new patient-reported outcome measure, the Lower Limb Functional Index (LLFI), was developed to address these limitations. Objective The purpose of this study was to overcome recognized deficiencies in existing lower-limb, region-specific, patient-reported outcome measures through: (1) development of a new lower-extremity outcome scale (ie, the LLFI) and (2) evaluation of the clinimetric properties of the LLFI using the Lower Extremity Functional Scale (LEFS) as a criterion measure. Design This was a prospective observational study. Methods The LLFI was developed in a 3-stage process of: (1) item generation, (2) item reduction with an expert panel, and (3) pilot field testing (n=18) for reliability, responsiveness, and sample size requirements for a larger study. The main study used a convenience sample (n=127) from 10 physical therapy clinics. Participants completed the LLFI and LEFS every 2 weeks for 6 weeks and then every 4 weeks until discharge. Data were used to assess the psychometric, practical, and general characteristics of the LLFI and the LEFS. The characteristics also were evaluated for overall performance using the Measurement of Outcome Measures and Bot clinimetric assessment scales. Results The LLFI and LEFS demonstrated a single-factor structure, comparable reliability (intraclass correlation coefficient [2,1]=.97), scale width, and high criterion validity (Pearson r=.88, with 95% confidence interval [CI]). Clinimetric performance was higher for the LLFI compared with the LEFS on the Measurement of Outcome Measures scale (96% and 95%, respectively) and the Bot scale (100% and 83%, respectively). The LLFI, compared with the LEFS, had improved responsiveness (standardized response mean=1.75 and 1.64, respectively), minimal detectable change with 90% CI (6.6% and 8.1%, respectively), and internal consistency (α=.91 and .95, respectively), as well as readability with reduced user error and completion and scoring times. Limitations Limitations of the study were that only participants recruited from outpatient physical therapy clinics were included and that no specific conditions or diagnostic subgroups were investigated. Conclusion The LLFI demonstrated sound clinimetric properties. There was lower response error, efficient completion and scoring, and improved responsiveness and overall performance compared with the LEFS. The LLFI is suitable for assessment of lower-limb function.

17Beta-estradiol affects the response of complement components and survival of rainbow trout (Oncorhynchus mykiss) challenged by bacterial infection

Research on the endocrine role of estrogens has focused on the reproductive system, while other potential target systems have been less studied. Here, we investigated the possible immunomodulating role of 17beta-estradiol (E2) using rainbow trout (Oncorhynchus mykiss) as a model. The aims of the study were to examine a) whether estrogens can modulate immune gene transcription levels, and b) whether this has functional implications for the resistance of trout towards pathogens. Trout were reared from fertilization until 6 months of age under (1) control conditions, (2) short-term E2-treatment (6-month-old juveniles were fed a diet containing 20 mg E2/kg for 2 weeks), or c) long-term E2-treatment (twice a 2-h-bath-exposure of trout embryos to 400 mug 17beta-estradiol (E2)/L, followed by rearing on the E2-spiked diet from start-feeding until 6 months of age). Analysis of plasma estrogen levels indicated that the internal estrogen concentrations of E2-exposed fish were within the physiological range and analysis of hepatic vitellogenin mRNA levels indicated that the E2 administration was effective in activating the endogenous estrogen receptor pathway. However, expression levels of the hepatic complement components C3-1, C3-3, and Factor H were not affected by E2-treatment. In a next step, 6-month-old juveniles were challenged with pathogenic bacteria (Yersinia ruckeri). In control fish, this bacterial infection resulted in significant up-regulation of the mRNA levels of hepatic complement genes (C3-1, C3-3, Factor B, Factor H), while E2-treated fish showed no or significantly lower up-regulation of the complement gene transcription levels. Apparently, the E2-treated trout had a lower capacity to activate their immune system to defend against the bacterial infection. This interpretation is corroborated by the finding that survival of E2-treated fish under bacterial challenge was significantly lower than in the control group. In conclusion, the results from this study suggest that estrogens are able to modulate immune parameters of trout with functional consequences on their ability to cope with pathogens.