L-mimosine increases the production of vascular endothelial growth factor in human tooth slice organ culture model.

AIM To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model. METHODOLOGY Human teeth were sectioned transversely into 600-μm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48 h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test. RESULTS Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P < 0.05). This effect on VEGF was reduced by echinomycin (P < 0.01). Changes in normalized IL-6 and IL-8 levels upon treatment with L-mimosine did not reach the level of significance (P > 0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P < 0.05) and IL-8 levels (P < 0.05). CONCLUSIONS The prolyl hydroxylase inhibitor L-mimosine increased VEGF production via HIF-1 alpha in the tooth slice organ culture model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration.

Automatic multi-resolution shape modeling of multi-organ structures.

Point Distribution Models (PDM) are among the most popular shape description techniques and their usefulness has been demonstrated in a wide variety of medical imaging applications. However, to adequately characterize the underlying modeled population it is essential to have a representative number of training samples, which is not always possible. This problem is especially relevant as the complexity of the modeled structure increases, being the modeling of ensembles of multiple 3D organs one of the most challenging cases. In this paper, we introduce a new GEneralized Multi-resolution PDM (GEM-PDM) in the context of multi-organ analysis able to efficiently characterize the different inter-object relations, as well as the particular locality of each object separately. Importantly, unlike previous approaches, the configuration of the algorithm is automated thanks to a new agglomerative landmark clustering method proposed here, which equally allows us to identify smaller anatomically significant regions within organs. The significant advantage of the GEM-PDM method over two previous approaches (PDM and hierarchical PDM) in terms of shape modeling accuracy and robustness to noise, has been successfully verified for two different databases of sets of multiple organs: six subcortical brain structures, and seven abdominal organs. Finally, we propose the integration of the new shape modeling framework into an active shape-model-based segmentation algorithm. The resulting algorithm, named GEMA, provides a better overall performance than the two classical approaches tested, ASM, and hierarchical ASM, when applied to the segmentation of 3D brain MRI.

Duration-dependent influence of dynamic torsion on the intervertebral disc: an intact disc organ culture study.

PURPOSE Mechanical loading is an important parameter that alters the homeostasis of the intervertebral disc (IVD). Studies have demonstrated the role of compression in altering the cellular metabolism, anabolic and catabolic events of the disc, but little is known how complex loading such as torsion-compression affects the IVD cell metabolism and matrix homeostasis. Studying how the duration of torsion affects disc matrix turnover could provide guidelines to prevent overuse injury to the disc and suggest possible beneficial effect of torsion. The aim of the study was to evaluate the biological response of the IVD to different durations of torsional loading. METHODS Intact bovine caudal IVD were isolated for organ culture in a bioreactor. Different daily durations of torsion were applied over 7 days at a physiological magnitude (±2°) in combination with 0.2 MPa compression, at a frequency of 1 Hz. RESULTS Nucleus pulpous (NP) cell viability and total disc volume decreased with 8 h of torsion-compression per day. Gene expression analysis suggested a down-regulated MMP13 with increased time of torsion. 1 and 4 h per day torsion-compression tended to increase the glycosaminoglycans/hydroxyproline ratio in the NP tissue group. CONCLUSIONS Our result suggests that load duration thresholds exist in both torsion and compression with an optimal load duration capable of promoting matrix synthesis and overloading can be harmful to disc cells. Future research is required to evaluate the specific mechanisms for these observed effects.

Time Course of Antibiotic and Antifungal Concentrations in Corneal Organ Culture.

PURPOSE Contamination with bacteria and/or fungi is a serious complication in organ-cultured corneas. Hence, antibiotic and antifungal agents are added to the culture medium. The concentration of different antimicrobial and antifungal additives to the media over time has so far not been investigated in detail and is the aim of this study. METHODS Nine human fresh corneoscleral discs were stored in corneal culture medium consisting of 2% fetal bovine serum and minimal essential medium. In addition, the culture medium contained 1200 μg/mL penicillin G, 25 μg/mL amphotericin B, 120 μg/mL streptomycin, and 100 μg/mL voriconazole. The concentration of amphotericin B used was 10 times higher than in clinical routine to facilitate its detection. The cultures were kept at 37°C for 28 days. At days 0, 7, 14, 21, and 28, samples of the culture medium were harvested for analysis of antimicrobial concentrations by liquid chromatography and electrospray ionization tandem mass spectrometry. RESULTS During corneal storage, the concentration of all antibiotics and antifungal agents declined significantly. By day 28, penicillin G was reduced to 14% of the original concentration. Amphotericin B and streptomycin retained approximately 60% of the original concentration to the end of the experiment and voriconazole maintained stable concentrations after an initial decline to approximately 80% at 7 days. CONCLUSIONS Throughout the entire storage period, the concentrations of penicillin G, streptomycin, and voriconazole exceeded the minimum inhibitory concentrations of all common contaminants, obviating the need for a change of the medium for antimicrobial reasons. Based on the minimum inhibitory concentrations and our findings, the initial concentration of amphotericin B should be raised to 5 μg/mL.

CRTC2 polymorphism as a risk factor for the incidence of metabolic syndrome in patients with solid organ transplantation.

Metabolic syndrome after transplantation is a major concern following solid organ transplantation (SOT). The CREB-regulated transcription co-activator 2 (CRTC2) regulates glucose metabolism. The effect of CRTC2 polymorphisms on new-onset diabetes after transplantation (NODAT) was investigated in a discovery sample of SOT recipients (n1=197). Positive results were tested for replication in two samples from the Swiss Transplant Cohort Study (STCS, n2=1294 and n3=759). Obesity and other metabolic traits were also tested. Associations with metabolic traits in population-based samples (n4=46'186, n5=123'865, n6>100,000) were finally analyzed. In the discovery sample, CRTC2 rs8450-AA genotype was associated with NODAT, fasting blood glucose and body mass index (Pcorrected<0.05). CRTC2 rs8450-AA genotype was associated with NODAT in the second STCS replication sample (odd ratio (OR)=2.01, P=0.04). In the combined STCS replication samples, the effect of rs8450-AA genotype on NODAT was observed in patients having received SOT from a deceased donor and treated with tacrolimus (n=395, OR=2.08, P=0.02) and in non-kidney transplant recipients (OR=2.09, P=0.02). Moreover, rs8450-AA genotype was associated with overweight or obesity (n=1215, OR=1.56, P=0.02), new-onset hyperlipidemia (n=1007, OR=1.76, P=0.007), and lower high-density lipoprotein-cholesterol (n=1214, β=-0.08, P=0.001). In the population-based samples, a proxy of rs8450G>A was significantly associated with several metabolic abnormalities. CRTC2 rs8450G>A appears to have an important role in the high prevalence of metabolic traits observed in patients with SOT. A weak association with metabolic traits was also observed in the population-based samples.The Pharmacogenomics Journal advance online publication, 8 December 2015; doi:10.1038/tpj.2015.82.

Risk Behavior and Reciprocity of Organ Donation Attitudes in Young Men

Background Lack of donor organs remains a major obstacle in organ transplantation. Our aim was to evaluate (1) the association between engaging in high-risk recreational activities and attitudes toward organ donation and (2) the degree of reciprocity between organ acceptance and donation willingness in young men. Methods A 17-item, close-ended survey was offered to male conscripts ages 18 to 26 years in all Swiss military conscription centers. Predictors of organ donation attitudes were assessed in bivariate analyses and multiple logistic regression. Reciprocity of the intentions to accept and to donate organs was assessed by means of donor card status. Results In 1559 responses analyzed, neither motorcycling nor practicing extreme sports reached significant association with donor card holder status. Family communication about organ donation, student, or academic profession and living in a Latin linguistic region were predictors of positive organ donation attitudes, whereas residence in a German-speaking region and practicing any religion predicted reluctance. Significantly more respondents were willing to accept than to donate organs, especially among those without family communication concerning organ donation. Conclusions For the first time, it was shown that high-risk recreational activities do not influence organ donation attitudes. Second, a considerable discrepancy in organ donation reciprocity was identified. We propose that increasing this reciprocity could eventually increase organ donation rates.

Waddlia chondrophila induces systemic infection, organ pathology, and elicits Th1-associated humoral immunity in a murine model of genital infection

Waddlia chondrophila is a known bovine abortigenic Chlamydia-related bacterium that has been associated with adverse pregnancy outcomes in human. However, there is a lack of knowledge regarding how W. chondrophila infection spreads, its ability to elicit an immune response and induce pathology. A murine model of genital infection was developed to investigate the pathogenicity and immune response associated with a W. chondrophila infection. Genital inoculation of the bacterial agent resulted in a dose-dependent infection that spread to lumbar lymph nodes and successively to spleen and liver. Bacterial-induced pathology peaked on day 14, characterized by leukocyte infiltration (uterine horn, liver, and spleen), necrosis (liver) and extramedullary hematopoiesis (spleen). Immunohistochemistry demonstrated the presence of a large number of W. chondrophila in the spleen on day 14. Robust IgG titers were detected by day 14 and remained high until day 52. IgG isotypes consisted of high IgG2a, moderate IgG3 and no detectable IgG1, indicating a Th1-associated immune response. This study provides the first evidence that W. chondrophila genital infection is capable of inducing a systemic infection that spreads to major organs, induces uterus, spleen, and liver pathology and elicits a Th1-skewed humoral response. This new animal model will help our understanding of the mechanisms related to intracellular bacteria-induced miscarriages, the most frequent complication of pregnancy that affects one in four women.

A novel organ-slice culturing system to simulate meniscal repair: Proof of concept using a synovium-based pool of meniscoprogenitor cells.

Meniscal injuries can occur secondary to trauma or be instigated by the changes in knee-joint function that are associated with aging, osteo- and rheumatoid arthritis, disturbances in gait and obesity. Sixty per cent of persons over 50 years of age manifest signs of meniscal pathology. The surgical and arthroscopic measures that are currently implemented to treat meniscal deficiencies bring only transient relief from pain and effect but a temporary improvement in joint function. Although tissue-engineering-based approaches to meniscal repair are now being pursued, an appropriate in-vitro model has not been conceived. The aim of this study was to develop an organ-slice culturing system to simulate the repair of human meniscal lesions in vitro. The model consists of a ring of bovine meniscus enclosing a chamber that represents the defect and reproduces its sequestered physiological microenvironment. The defect, which is closed with a porous membrane, is filled with fragments of synovial tissue, as a source of meniscoprogenitor cells, and a fibrin-embedded, calcium-phosphate-entrapped depot of the meniscogenic agents BMP-2 and TGF-ß1. After culturing for 2 to 6 weeks, the constructs were evaluated histochemically and histomorphometrically, as well as immunohistochemically for the apoptotic marker caspase 3 and collagen types I and II. Under the defined conditions, the fragments of synovium underwent differentiation into meniscal tissue, which bonded with the parent meniscal wall. Both the parent and the neoformed meniscal tissue survived the duration of the culturing period without significant cell losses. The concept on which the in-vitro system is based was thus validated. This article is protected by copyright. All rights reserved.

Intravital and whole-organ imaging reveals capture of melanoma-derived antigen by lymph node subcapsular macrophages leading to widespread deposition on follicular dendritic cells.

Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy.

Organ inflammation in porcine Escherichia coli sepsis is markedly attenuated by combined inhibition of C5 and CD14.

Sepsis is an infection-induced systemic inflammatory syndrome, potentially causing organ failure. We previously showed attenuating effects on inflammation, thrombogenicity and haemodynamics by inhibiting the Toll-like receptor co-factor CD14 and complement factor C5 in a porcine Escherichia coli-induced sepsis model. The present study explored the effect on organ inflammation in these pigs. Tissue samples were examined from the combined treatment group (n = 8), the positive (n = 8) and negative (n = 6) control groups after 4h of sepsis. Inflammatory biomarkers were measured using ELISA, multiplex and qPCR analysis. Combined inhibition of C5 and CD14 markedly attenuated IL-1β by 31-66% (P < 0.05) and IL-6 by 54-96% (P < 0.01) in liver, kidney, lung and spleen; IL-8 by 65-100% in kidney, lung, spleen, and heart (P < 0.05) and MCP-1 by 46-69% in liver, kidney, spleen and heart (P < 0.05). Combined inhibition significantly attenuated tissue factor mRNA upregulation in spleen (P < 0.05) and IP-10 mRNA upregulation in four out of five organs. Finally, C5aR mRNA downregulation was prevented in heart and kidney (P < 0.05). Combined inhibition of C5 and CD14 thus markedly attenuated inflammatory responses in all organs examined. The anti-inflammatory effects observed in lung and heart may explain the delayed haemodynamic disturbances observed in septic pigs receiving combined inhibition of C5 and CD14.

Fulminant hepatic and multiple organ failure following acute viral tonsillitis: a case report.

BACKGROUND Pyogenic tonsillitis may often be observed in the general Western population. In severe cases, it may require antibiotic treatment or even hospitalization and often a prompt clinical response will be noted. Here we present an unusual case of progressive multiple organ failure including fulminant liver failure following acute tonsillitis initially mistaken for "classic" pyogenic (that is bacterial) tonsillitis. CASE PRESENTATION A 68-year-old previously healthy white man was referred with suspicion of pyogenic angina. After tonsillectomy, he developed acute liver failure and consecutive multiple organ failure including acute hemodynamic, pulmonary and dialysis-dependent renal failure. Immunohistopathological analysis of his tonsils and liver as well as serum polymerase chain reaction analyses revealed herpes simplex virus-2 to be the causative pathogen. Treatment included high-dose acyclovir and multiorgan supportive intensive care therapy. His final outcome was favorable. CONCLUSIONS Fulminant herpes simplex virus-2-induced multiple organ failure is rarely observed in the Western hemisphere and should be considered a potential diagnosis in patients with tonsillitis and multiple organ failure including acute liver failure. From a clinical perspective, it seems important to note that fulminant herpes simplex virus-2 infection may masquerade as "routine" bacterial severe sepsis/septic shock. This persevering condition should be diagnosed early and treated goal-oriented in order to gain control of this life-threatening condition.

Compression Loading Induced Cellular Stress Response of Intervertebral Disc Cells in Organ Culture

Introduction: Mechanical stress is often associated to interverterbal disc (IVD) degeneration and the effect of mechanical loading on IVD has been studied and reviewed.1,2 Previously, expression of heat shock proteins, HSP70 and HSP27 has been found in pathological discs.3 However, there is no direct evidence on whether IVD cells respond to the mechanical loading by expression of HSPs. The objective of this study is to investigate the stress response of IVD cells during compressive loading in an organ culture. Materials and Methods: Fresh adult bovine caudal discs were cultured with compressive loading applied at physiological range. Effect of loading type (static and dynamic) and repeated loading (2 hours per day for 2 days) were studied. Nucleus pulposus (NP) and annulus fibrosus (AF) of the IVD were retrieved at different time points: right after loading and right after resting. Positive control discs were heat shocked (43°C). Cell activity was assessed and expression of stress response genes (HSP70 and HSF1) and matrix remodeling genes (ACAN, COL2, COL1, ADAMTS4, MMP3 and MMP13) were studied. Results: Cell activity was maintained in all groups. Both NP and AF expressed high level of HSP70 in heat shock groups, confirming their expression in response to stress. In NP, expression of HSP70 was up-regulated after static loading and dynamic loading with higher fold change was observed after static loading. During repeated loading, HSP70 appeared to be upregulated right after loading and decreased after resting. Such trend was not observed in AF and HSF1 levels. Expressions of matrix remodeling genes did not change significantly with loading except ADAMTS4 decreased in AF during static loading. Conclusion: This study demonstrated that NP cells upregulate expression of HSP70 in response to loading induced stress without changing cell activity and matrix remodeling significantly. Acknowledgments: This project was funded by AO Spine (AOSPN) (grant number: SRN_2011_14) and a fellowship exchange award by AO Spine Scientific Research Network (SRN).

Nocardia infection in solid organ transplant recipients: a multicenter European case-control study.

BACKGROUND  Nocardiosis is a rare, life-threatening opportunistic infection, affecting 0.04% to 3.5% of patients after solid organ transplantation (SOT). The aim of this study was to identify risk factors for Nocardia infection after SOT and to describe the presentation of nocardiosis in these patients. METHODS  We performed a retrospective case-control study of adult patients diagnosed with nocardiosis after SOT between 2000 and 2014 in 36 European (France, Belgium, Switzerland, Netherlands, Spain) centers. Two control subjects per case were matched by institution, transplant date and transplanted organ. A multivariable analysis was performed using conditional logistic regression to identify risk factors for nocardiosis. RESULTS  One hundred and seventeen cases of nocardiosis and 234 control patients were included. Nocardiosis occurred at a median of 17.5 [range 2-244] months after transplantation. In multivariable analysis, high calcineurin inhibitor trough levels in the month before diagnosis (OR=6.11 [2.58-14.51]), use of tacrolimus (OR=2.65 [1.17-6.00]) and corticosteroid dose (OR=1.12 [1.03-1.22]) at the time of diagnosis, patient age (OR=1.04 [1.02-1.07]) and length of stay in intensive care unit after SOT (OR=1.04 [1.00-1.09]) were independently associated with development of nocardiosis; low-dose cotrimoxazole prophylaxis was not found to prevent nocardiosis. Nocardia farcinica was more frequently associated with brain, skin and subcutaneous tissue infections than were other Nocardia species. Among the 30 cases with central nervous system nocardiosis, 13 (43.3%) had no neurological symptoms. CONCLUSIONS  We identified five risk factors for nocardiosis after SOT. Low-dose cotrimoxazole was not found to prevent Nocardia infection. These findings may help improve management of transplant recipients.