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Background: The complement system is involved in pathogenesis of cardiovascular disease, and might play a role in accelerated atherogenesis in spondylarthropathies (SpA). Hence, we examined complement activation in SpA, and its relationship to antirheumatic treatment, inflammatory and cardiovascular markers., Methods: From PSARA, a prospective observational study, we examined 51 SpA patients (31 psoriatic arthritis (PsA), and 20 ankylosing spondylitis (AS)), starting tumor necrosis factor (TNF) inhibitor alone (n = 25), combined with methotrexate (MTX) (n = 10), or MTX monotherapy (n = 16). Complement activation was determined by the soluble terminal complement complex (sC5b-9), inflammation by erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), and endothelial function by finger plethysmography (Endopat) at baseline, after 6 weeks and 6 months of treatment., Results: SpA patients had sC5b-9 levels at (PsA) or above (AS) the upper limit of the estimated reference range. Median sC5b-9 levels decreased significantly from baseline to 6 weeks, with no significant difference between the AS and PsA group. Notably, a significant reduction in sC5b-9 was observed after administration of TNF inhibitor ± MTX, whereas no significant changes were observed in patients treated with MTX alone. Between 6 weeks and 6 months, sC5b-9 remained stable across all subgroups. Reduction in sC5b-9 was independently related to decreased ESR and CRP, and to increased high density cholesterol and total cholesterol. Reduction in sC5b-9 from baseline to 6 weeks was associated with improved EF in age and gender adjusted analyses., Conclusion: TNF-inhibition, but not MTX monotherapy, led to rapid and sustained reduction of complement activation in SpA. Thus, the observed decrease in cardiovascular morbidity in patients treated with TNF-inhibitors might be partly due to its beneficial effect on complement., Trial Registration: Clinical Trials (NCT00902005), retrospectively registered on the 14th of May 2009., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient's eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function., Methods: Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed., Results: Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = -0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin β1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups., Conclusion: For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient., Competing Interests: The authors have declared that no competing interests exist.

Objectives: The reason for increased cardiovascular risk in inflammatory arthritis (IA) is unclear. Interestingly, selenium-deficiency is suspected to contribute to the development of cardiovascular disease (CVD) in the general population. Although the reference range of serum selenium (s-selenium) is 50-120 μg/L, there are indications that levels up to 85 μg/L might not be sufficient for optimal cardioprotection. Our aim was to examine s-selenium levels in rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS), to evaluate the effect of anti-rheumatic treatment on s-selenium levels, and to assess relationships between s-selenium levels and clinical and laboratory parameters including markers of disease activity and CVD risk., Methods: We examined 64 patients with RA, 40 with PsA and 26 with AS starting with methotrexate (MTX) monotherapy or anti-tumor necrosis factor therapy (anti-TNF) with or without methotrexate (anti-TNF ± MTX) due to active disease. S-selenium, inflammatory biomarkers, endothelial function (EF) and other variables were examined at baseline and after 6 weeks and 6 months of treatment., Results: In the total IA group, s-selenium increased within 6 weeks of anti-rheumatic treatment, and thereafter the levels remained stable until the end of the 6 months follow-up period. There were no significant differences in s-selenium changes between the three diagnostic groups and between the two treatment regimens. Changes in s-selenium were negatively related to changes in C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), but there were no significant relationships to any other of the examined risk parameters for CVD including EF., Conclusion: IA patients had s-selenium within the reference range, but below the level that might be necessary for optimal CVD protection. Anti-rheumatic treatment had a relatively rapid and sustained effect on s-selenium levels. The increase in s-selenium was related to reduction in inflammatory activity. In theory, anti-rheumatic drugs might improve s-selenium levels through inhibition of pro-inflammatory processes or through other mechanisms. Although we have not revealed any significant relationships between s-selenium and CVD risk parameters, the role of suboptimal s-selenium levels in pathogenesis of premature CVD in IA cannot be ruled out., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Background: Vitamin D has an important role in the immune system, and has been linked to rheumatoid arthritis (RA) and coronary artery disease (CAD). The exact mechanisms by which vitamin D is involved in these processes are still unclear. Therefore, we wanted to search for differences in expression of genes involved in the vitamin D receptor (VDR) activation pathway and genes that are known to alter upon vitamin D stimulation, in the aortic adventitia of CAD patients with and without RA., Methods: Affymetrix microarray was used to determine gene expression profile in surgical specimens from the adventitia of the ascending aorta of CAD patients with RA (n = 8) and without RA (n = 8) from the Feiring Heart Biopsy Study., Results: We identified three vitamin D associated genes that were differentially expressed between RA and non-RA patients: Growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A) (FC = 1.47; p = 0.006), Nuclear Receptor Co-repressor 1 (NCOR1) (FC = 1,21; p = 0.005) and paraoxonases 2 (PON2) (FC = -1.37; p = 0.01). High expression of GADD45A in RA tissues was confirmed by real-time qRT-PCR. GADD45A expression correlated with plasma levels of 1,25(OH)2D3 (rs = 0.69; p = 0.003)., Conclusions: Microarray analyses revealed higher expression of GADD45A and NCOR1; and lower expression of PON2 in the aortic adventitia of RA than non-RA patients. Further studies are needed to elucidate if and how GADD45A, NCOR1 and PON2 are involved in the development of accelerated atherosclerosis in RA. In theory, some of these factors might have proatherogenic effects whereas others might reflect an underlying vascular pathology promoting atherogenesis (such as vascular stress)., Competing Interests: The authors have declared that no competing interests exist.

Limbal stem cell deficiency can be treated with transplantation of cultured human limbal epithelial cells (LEC). It can be advantageous to produce LEC in centralized labs and thereafter ship them to eye clinics. The present study used transport simulations of LEC to determine if vigorous shaking during transport altered the viability, morphology and phenotype during a 4 day-long storage of LEC with a previously described serum-free storage method. Inserts with LEC cultured on amniotic membranes were sutured to caps inside air-tight containers with generous amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM). The containers were distributed among the following testing conditions: 6 hours with full containers, 36 hours with full containers, 36 hours with container three quarters full of medium, and 36 hours with container full of medium containing a shear-protecting agent (Pluronic-F68). Compared to stored, but non-transported controls, no statistically significant changes in viability and immunohistochemical staining were observed. The epithelial sheets remained intact. However, an air-liquid interface in the containers reduced the number of desmosomes and hemi-desmosomes compared to the controls. In conclusion, cultured LEC sheets appear to endure vigorous shaking for at least 36 hours if the container is full.

The advancement of human retinal pigment epithelial cell (hRPE) replacement therapy is partly dependent on optimization of cell culture, cell preservation, and storage medium. This study was undertaken to search for a suitable storage temperature and storage medium for hRPE. hRPE monolayer sheets were cultured under standard conditions at 37°C and then randomized for storage at six temperatures (4, 16, 20, 24, 28, and 37°C) for 7 days. After revealing a suitable storage temperature, hRPE sheets were subsequently stored with and without the silk protein sericin added to the storage medium. Live/dead assay, light microscopy, pH, and phenotypic expression of various proteins were used to assess cell cultures stored at different temperatures. After 7 days of storage, hRPE morphology was best preserved at 4°C. Addition of sericin to the storage medium maintained the characteristic morphology of the preserved cells, and improved pigmentation and levels of pigmentation-related proteins in the cultured hRPE sheets following a 7-day storage period at 4°C.

Background: Inflammatory arthritis (IA), including rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA), leads to increased cardiovascular disease occurrence probably due to atherosclerosis. One of the first stages in atherogenesis is endothelial dysfunction (ED). Therefore, we aimed to compare endothelial function (EF) in patients with IA, and to examine the effects of methotrexate (MTX) monotherapy and antitumor necrosis factor (anti-TNF) treatment with or without MTX comedication (anti-TNF ± MTX) on EF., Methods: From the PSARA observational study, all patients with RA (n = 64), PsA (n = 29), and AS (n = 20) were evaluated for EF. In patients with ED at baseline (n = 40), we evaluated changes in the Reactive Hyperemic Index (RHI) after 6 weeks and 6 months of antirheumatic therapy., Results: In IA patients with ED, RHI significantly improved after 6 weeks (p < 0.001) and 6 months (p < 0.001) of treatment, independent of changes in disease activity parameters. After 6 months, RHI had improved more in the MTX group than in the anti-TNF ± MTX group, and the difference remained statistically significant after adjustments for potential confounders. Among patients with active RA, AS, and PsA, those with AS appeared to have the worst endothelial function, although they were the youngest., Conclusion: Treatment with MTX and anti-TNF ± MTX was associated with a relatively fast improvement of EF in IA patients with ED, independent of change in disease activity. Therefore, modes of action other than the anti-inflammatory effect may contribute to the EF improvement. After 6 months, the EF improvement was more pronounced in the MTX group than in the anti-TNF ± MTX group., Trial Registration: Clinicaltrials, NCT00902005 . Registered on 13 May 2009.

Objectives: Vitamin D modulates inflammation, and this may explain the observed associations between vitamin D status and disorders driven by systemic inflammation, such as coronary artery disease (CAD) and inflammatory rheumatic diseases (IRDs). The aims of this study were to assess vitamin D status in patients with CAD alone and in patients with CAD and IRD, and to explore potential associations between vitamin D status and the presence of mononuclear cell infiltrates (MCIs) in the aortic adventitia of these patients., Method: Plasma levels of 25-hydroxyvitamin D 3 [(25(OH)D 3 ] were determined by radioimmunoassay and 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] by enzyme immunoassay in the 121 patients from the Feiring Heart Biopsy Study (FHBS) who had available histology data on adventitial MCIs; 53 of these had CAD alone and 68 had CAD and IRD., Results: In the crude analysis, vitamin D levels were similar in CAD patients with and without IRD. After adjustment for potential confounders, IRD was associated with an increase of 8.8 nmol/L [95% confidence interval (CI) 1.0-16.6; p = 0.027] in 25(OH)D 3 and an increase of 18.8 pmol/L (95% CI 4.3-33.3; p = 0.012) in 1,25(OH) 2 D 3 , while MCIs in the aortic adventitia were associated with lower levels of 1,25(OH) 2 D 3 (β = -18.8, 95% CI -33.6 to -4.0; p = 0.014)., Conclusions: IRD was associated with higher levels of both 25(OH)D 3 and 1,25(OH) 2 D 3 . These findings argue against the hypothesis that patients with high systemic inflammatory burden (CAD+IRD) should have lower vitamin D levels than those with less inflammation (CAD only). Of note, when controlled for potential confounders, low 1,25(OH) 2 D 3 levels were associated with adventitial aortic inflammation.

Purpose: Inflammatory rheumatic diseases (IRD) are associated with accelerated coronary artery disease (CAD), which may result from both systemic and vascular wall inflammation. There are indications that complement may be involved in the pathogenesis of CAD in Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA). This study aimed to evaluate the associations between circulating complement and complement activation products with mononuclear cell infiltrates (MCI, surrogate marker of vascular inflammation) in the aortic media and adventitia in IRDCAD and non-IRDCAD patients undergoing coronary artery bypass grafting (CABG). Furthermore, we compared complement activation product deposition patterns in rare aorta adventitial and medial biopsies from SLE, RA and non-IRD patients., Methods: We examined plasma C3 (p-C3) and terminal complement complexes (p-TCC) in 28 IRDCAD (SLE = 3; RA = 25), 52 non-IRDCAD patients, and 32 IRDNo CAD (RA = 32) from the Feiring Heart Biopsy Study. Aortic biopsies taken from the CAD only patients during CABG were previously evaluated for adventitial MCIs. The rare aortic biopsies from 3 SLE, 3 RA and 3 non-IRDCAD were assessed for the presence of C3 and C3d using immunohistochemistry., Results: IRDCAD patients had higher p-TCC than non-IRDCAD or IRDNo CAD patients (p<0.0001), but a similar p-C3 level (p = 0.42). Circulating C3 was associated with IRD duration (ρ, p-value: 0.46, 0.03). In multiple logistic regression analysis, IRD remained significantly related to the presence and size of MCI (p<0.05). C3 was present in all tissue samples. C3d was detected in the media of all patients and only in the adventitia of IRD patients (diffuse in all SLE and focal in one RA)., Conclusion: The independent association of IRD status with MCI and the observed C3d deposition supports the unique relationship between rheumatic disease, and, in particular, SLE with the complement system. Exaggerated systemic and vascular complement activation may accelerate CVD, serve as a CVD biomarker, and represent a target for new therapies.

Background: Pentraxin 3 is proposed to be a marker of inflammation and cardiovascular risk, but its role in inflammatory rheumatic diseases (IRDs) is still uncertain. Therefore, we wanted to examine if anti-rheumatic treatment reduced serum PTX3 (s-PTX3) levels in IRDs, and if s-PTX3 levels were related to other markers of inflammation and to endothelial function (EF)., Methods: We examined s-PTX3, EF and established inflammatory biomarkers in 114 IRD patients from the PSARA study before and after 6 weeks and 6 months of treatment with methotrexate (MTX) or anti-tumor necrosis factor alpha (anti-TNF) therapy with or without MTX co-medication., Results: s-PTX3 levels in all IRD diagnoses were above the upper limit of the reference range. In contrast to established inflammatory markers, in particular CRP and ESR, s-PTX3 levels did not change significantly after 6 weeks and 6 months of anti-rheumatic therapy. There was no difference in change in s-PTX3 levels from baseline to 6 weeks and 6 months between MTX monotherapy and anti-TNF regimens. CRP, ESR and EF were not related to changes in s-PTX3 neither in crude nor adjusted analyses., Conclusion: IRD patients have increased s-PTX3 levels, which, in contrast to other inflammatory markers, do not seem to improve within 6 months of therapy with MTX and/or anti-TNF. Thus, s-PTX3 might reflect a persisting immune process, even a causal factor of inflammation, not inhibited by the standard anti-rheumatic treatment. Furthermore, even though s-PTX3 is thought to be a strong predictor of cardiovascular prognosis, it was not related to EF.

Purpose: The development of a suitable storage method for retinal pigment epithelium (RPE) is necessary in the establishment of future RPE replacement therapy, and storage temperature has proven to be pivotal for cell survival. ARPE-19, a widely used model for RPE, has been shown to yield the greatest number of viable cells when stored at 16°C compared to other storage temperatures. In this study, we analyze the gene expression profile of cultured ARPE-19 cells after seven days of storage at different temperatures in an effort to predict the gene-level consequences of storage of RPE transplants., Materials and Methods: ARPE-19 cells were cultured until confluence and then stored in minimum essential medium at 4°C, 16°C, and 37°C for seven days. The total RNA was isolated and the gene expression profile was determined using DNA microarrays. The Results were validated using qPCR., Results: Principal component and hierarchical clustering analyses show that the gene expression profiles of cell cultures stored at different temperatures cluster into separate groups. Cultures stored at 4°C cluster closest to the control cultures that were not stored and display the least change in gene expression after storage (157 differentially expressed genes). Cultures stored at 16°C and 37°C display a much larger change in differential gene expression (1787 and 1357 differentially expressed genes, respectively). At 16°C, the expression of several genes with proposed tumor suppressor functions was markedly increased. Changes in regulation of several known signaling pathways and of oxidative stress markers were discovered at both 16°C and 37°C, and activation of the angiogenesis marker vascular endothelial growth factor (VEGF) was discovered at 37°C. There was no evidence of the activation of inflammatory processes in stored cell cultures., Conclusion: ARPE-19 cultures stored at 16°C show the greatest propensity to modulate their gene expression profile in a manner that supports cell survival during storage.

Ingestion of the Agaricus blazei Murill-based mushroom extract AndoSan ™ has been shown in randomized placebo-controlled studies to improve symptoms in Crohn's disease (CD) and ulcerative colitis (UC) and also fatigue and quality of life in the latter patients. The aim was to examine whether this clinical impact of AndoSan ™ intake could be explained by influence on foremost pro-inflammatory cytokines in the patients. Fifty patients with symptomatic UC and CD were randomized and blinded for oral daily intake of AndoSan ™ or placebo. Blood samples taken before (visit 1) and after 21 days' (visit 3) consumption were analysed for cytokines IL-1ß, IL-2, IL-4-8, IL-10, IL-12-13, IL-17, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1ß and TNF-α. Baseline cytokine levels were similar in CD and UC. In CD, cytokine levels at visit 1 versus visit 3 were unaltered within the AndoSan ™ and the placebo groups. Only IL-2 was significantly reduced at visit 3 in the Andosan ™ compared with the placebo group. However, when combining IL-1ß, IL-6 and G-CSF in the patients with CD, the cytokine levels were significantly lower in the AndoSan TM - versus the placebo group, visit 3. In UC, levels of IL-2, IL-5 and MIP-1ß were reduced within the AndoSan ™ group. IL-5 was also reduced at visit 3 compared with placebo. Generally, the effect on reduction in systemic cytokine levels by consumption of AndoSan ™ was limited and supported only marginally anti-inflammatory effects in these patients. Therefore, other explanations behind the clinical anti-inflammatory effects than the contribution of cytokines seem more pertinent, including anti-allergic and antioxidant activities., (© 2016 The Foundation for the Scandinavian Journal of Immunology.)

Background: Ingestion of AndoSanTM, based on the mushroom Agaricus blazei Murill, has previously shown an anti-inflammatory effect through reduction of pro-inflammatory cytokines in healthy individuals and patients with Crohn's disease (CD). In this randomized single-blinded placebo-controlled study we examined whether intake of AndoSanTM also resulted in clinical effects., Methods and Findings: 50 patients with symptomatic CD were randomized for oral daily consumption of AndoSanTM or placebo for a 21-day experimental period, in this per-protocol study. Patients reported validated scores for symptoms, fatigue and health related quality of life (HRQoL) at days 0, 14 and 21. Fecal calprotectin and general blood parameters were also analyzed. In the AndoSanTM group (n = 25) symptoms improved from baseline (day 0) to days 14 and 21, with respective mean scores (95% CI) of 5.52 (4.64-6.40), 4.48 (3.69-5.27) and 4.08 (3.22-4.94) (p<0,001). We found significant improvements in symptom score for both genders in the AndoSanTM group, and no significant changes in the placebo (n = 25) group. There were however no significant differences between the groups (p = 0.106), although a marginal effect in symptom score for men (p = 0.054). There were comparable improvements in physical, mental and total fatigue for both groups. HRQoL versus baseline were at day 21 improved for bodily pain and vitality in the AndoSanTM group and for vitality and social functioning in the placebo group. No crucial changes in general blood samples and fecal calprotectin were detected., Conclusions: The results from this single-blinded randomized clinical trial shows significant improvement on symptoms, for both genders, in the AndoSanTM group, but no significant differences between the study groups. The results on fatigue, HRQoL, fecal calprotectin and blood samples were quite similar compared with placebo. The patients did not report any harms or unintended effects of AndoSanTM. CD patients with mild to moderate symptoms may have beneficiary effects of AndoSanTM as a safe supplement in addition to conventional medication., Trial Registration: ClinicalTrials.gov NCT01496053.

Phenotype of cultured ocular epithelial transplants has been shown to affect clinical success rates following transplantation to the cornea. The purpose of this study was to evaluate the relationship between cell nucleus morphometry and phenotype in three types of cultured epithelial cells. This study provides knowledge for the development of a non-invasive method of determining the phenotype of cultured epithelium before transplantation. Cultured human conjunctival epithelial cells (HCjE), human epidermal keratinocytes (HEK), and human retinal pigment epithelial cells (HRPE) were analyzed by quantitative immunofluorescence. Assessments of nucleus morphometry and nucleus-to-cytoplasm ratio (N/C ratio) were performed using ImageJ. Spearman's correlation coefficient was employed for statistical analysis. Levels of the proliferation marker PCNA in HCjE, HEK, and HRPE correlated positively with nuclear area. Nuclear area correlated significantly with levels of the undifferentiated cell marker ABCG2 in HCjE. Bmi1 levels, but not p63α levels, correlated significantly with nuclear area in HEK. The N/C ratio did not correlate significantly with any of the immunomarkers in HCjE (ABCG2, CK7, and PCNA) and HRPE (PCNA). In HEK, however, the N/C ratio was negatively correlated with levels of the undifferentiated cell marker CK14 and positively correlated with Bmi1 expression. The size of the nuclear area correlated positively with proliferation markers in all three epithelia. Morphometric indicators of phenotype in cultured epithelia can be identified using ImageJ. Conversely, the N/C ratio did not show a uniform relationship with phenotype in HCjE, HEK, or HRPE. N/C ratio therefore, may not be a useful morphometric marker for in vitro assessment of phenotype in these three epithelia.

Purpose: Cultured epidermal cell sheets (CECS) are used in the treatment of large area burns to the body and have potential to treat limbal stem cell deficiency (LSCD) as shown in animal studies. Despite widespread use, storage options for CECS are limited. Short-term storage allows flexibility in scheduling surgery, quality control and improved transportation to clinics worldwide. Recent evidence points to the phenotype of cultured epithelial cells as a critical predictor of post-operative success following transplantation of CECS in burns and in transplantation of cultured epithelial cells in patients with LSCD. This study, therefore assessed the effect of a range of temperatures, spanning 4-37 °C, on the phenotype of CECS stored over a 2-week period in a xenobiotic-free system., Materials and Methods: Progenitor cell (p63, ΔNp63α and ABCG2) and differentiation (C/EBPδ and CK10) associated marker expression was assessed using immunocytochemistry. Immunohistochemistry staining of normal skin for the markers p63, ABCG2 and C/EBPδ was also carried out. Assessment of progenitor cell side population (SP) was performed using JC1 dye by flow cytometry., Results: P63 expression remained relatively constant throughout the temperature range but was significantly lower compared to control between 20 and 28 °C (p < 0.05). High C/EBPδ together with low p63 suggested more differentiation beginning at 20 °C and above. Lower CK10 and C/EBPδ expression most similar to control was seen at 12 °C. The percentage of ABCG2 positive cells was most similar to control between 8 and 24 °C. Between 4 and 24 °C, the SP fluctuated, but was not significantly different compared to control. Results were supported by staining patterns indicating differentiation status associated with markers in normal skin sections., Conclusions: Lower storage temperatures, and in particular 12 °C, merit further investigation as optimal storage temperature for maintenance of undifferentiated phenotype in CECS.

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Background: Ingestion of AndoSan™, based on the mushroom Agaricus blazei Murill, has previously been shown to exhibit anti-inflammatory effects because of reduction of pro-inflammatory cytokines in healthy individuals and patients with ulcerative colitis. In this randomized single-blinded placebo controlled study we examined whether intake of AndoSan™ also resulted in clinical effects., Methods and Findings: 50 patients with symptomatic ulcerative colitis were block-randomized and blinded for oral daily intake of AndoSan™ or placebo for the 21 days' experimental period. The patients reported scores for symptoms, fatigue and health related quality of life (HRQoL) at days 0, 14 and 21. Fecal calprotectin and general blood parameters were also analyzed. In the AndoSan™ group (n = 24) symptoms improved from baseline (day 0) to days 14 and 21, with respective mean scores (95% CI) of 5.88 (4.92-6.83), 4.71 (3.90-5.52) (p = 0.002) and 4.50 (3.70-5.30) (p = 0.001). Corresponding improved mean scores (±SD) for total fatigue were 16.6 (5.59), 14.1 (4.50) (p = 0.001) and 15.1 (4.09) (p = 0.023). These scores in the placebo group (n = 26) were not improved. When comparing the two study groups using mixed model statistics, we found significant better scores for the AndoSan™-patients. HRQoL for dimensions bodily pain, vitality, social functioning and mental health improved in the AndoSan™ group. There were no alterations in general blood samples and fecal calprotectin., Conclusions: Beneficiary effects on symptoms, fatigue and HRQoL from AndoSan™ consumption were demonstrated in this per-protocol study, supporting its use as a supplement to conventional medication for patients with mild to moderate symptoms from ulcerative colitis. The patients did not report any harms or unintended effects of AndoSan™ in this study., Trial Registration: ClinicalTrials.gov NCT01496053.

The open window theory indicates altered immunity 3 to 72 hours after exercise. The J-curve describes the risk of illness in response to exercise. The aim of this study was to examine the secretion of proinflammatory and anti-inflammatory cytokines before and after long-term strenuous exercise. Fourteen marathon and 16 half-marathon runners and 10 military cadets participating in a military ranger-training course were recruited to this study. Within-subject design was used measuring levels of plasma cytokines before, during, and after exercise. Plasma cytokines were measured using Luminex multiplex technology and ELISA. Comparing pre/post plasma levels both the marathon- and the half-marathon runners showed heavily increased levels of IL-6, IL-10, and IL-8 (P < 0.001). LPS stimulation among the half-marathon runners decreased the postrace levels of IL-6, IL-1b, and TNFα by 45%, 24%, and 43%, respectively (P < 0.01). During the ranger training course the spontaneous and LPS-stimulated levels of IL-6, IL-8, IL-10, IL-1b, and TNFα changed in a similar fashion as in the half-marathon runners although the fluctuations were smaller. Our study supports the open window and the J-curve theory; the immune system is more activated and the subjects are more threatened to infectious pathogens after intensive physical activity and in the period after exercise.

Purpose/aims: To assess the effect of storage temperature on the viability, phenotype, metabolism, and morphology of cultured human oral keratinocytes (HOK)., Materials and Methods: Cultured HOK cells were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium (MEM) at nine temperatures in approximately 4 °C increments from 4 °C to 37 °C for seven days. Cells were characterized for viability by calcein fluorescence, phenotype retention by immunocytochemistry, metabolic parameters (pH, glucose, lactate, and O2) within the storage medium by blood gas analysis, and morphology by scanning electron microscopy and light microscopy., Results: Relative to the cultured, but non-stored control cells, a high percentage of viable cells were retained only in the 12 °C and 16 °C storage groups (85% ± 13% and 68% ± 10%, respectively). Expression of ABCG2, Bmi1, C/EBPδ, PCNA, cytokeratin 18, and caspase-3 were preserved after storage in the 5 groups between 4 °C and 20 °C, compared to the non-stored control. Glucose, pH and pO2 in the storage medium declined, whereas lactate increased with increasing storage temperature. Morphology was best preserved following storage of the three groups between 12 °C, 16 °C, and 20 °C., Conclusion: We conclude that storage temperatures of 12 °C and 16 °C were optimal for maintenance of cell viability, phenotype, and morphology of cultured HOK. The storage method described in the present study may be applicable for other cell types and tissues; thus its significance may extend beyond HOK and the field of ophthalmology.

Aim/purpose of the Study: To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets., Materials and Methods: Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3)., Results: The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03)., Conclusion: Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days.

Purpose: To evaluate the effect of storage temperature on the morphology, viability, cell number and metabolism of cultured human conjunctival epithelial cells (HCjEs)., Materials and Methods: Three-day cultured HCjEs were stored at nine different temperatures between 4 °C and 37 °C for four and seven days. Phenotype was assessed by immunofluorescence microscopy, morphology by scanning electron microscopy, viability and cell number by a microplate fluorometer and glucose metabolism by a blood gas analyzer., Results: Cultured cells not subjected to storage expressed the conjunctival cytokeratins 7 and 19 and the proliferation marker proliferating cell nuclear antigen. Cell morphology was best maintained following four-day storage between 12 °C and 28 °C and following 12 °C storage after seven days. Assessed by propidium iodide uptake, the percentage of viable cells after four-day storage was maintained only between 12 °C and 28 °C, whereas it had decreased in all other groups (p < 0.05; n = 4). After seven days this percentage was maintained in the 12 °C group, but it had decreased in all other groups, compared to the control (p < 0.05; n = 4). The total number of cells remaining in the cultures after four-day storage, compared to the control, had declined in all groups (p < 0.05; n = 4), except 12 °C and 20 °C groups. Following seven days this number had decreased in all groups (p < 0.01; n = 4), except 12 °C storage. Four-day storage at 12 °C demonstrated superior preservation of the number of calcein-stained viable cells (p < 0.05) and the least accumulation of ethidium homodimer 1-stained dead cells (p < 0.001), compared to storage at 4 °C and 24 °C (n = 6). The total metabolism of glucose to lactate after four-day storage was higher in the 24 °C group compared to 4 °C and 12 °C groups, as well as the control (p < 0.001; n = 3)., Conclusions: Storage at 12 °C appears optimal for preserving the morphology, viability and total cell number in stored HCjE cultures. The superior cell preservation at 12 °C may be related to temperature-associated effects on cell metabolism.

Purpose. Replacement of the diseased retinal pigment epithelium (RPE) with cells capable of performing the specialized functions of the RPE is the aim of cell replacement therapy for treatment of macular degenerative diseases. A storage method for RPE is likely to become a prerequisite for the establishment of such treatment. Herein, we analyze the effect of storage temperature on key functions of cultured RPE cells. Methods. Cultured ARPE-19 cells were stored in Minimum Essential Medium at 4°C, 16°C, and 37°C for seven days. Total RNA was isolated and the gene expression profile was determined using DNA microarrays. Comparison of the microarray expression values with qRT-PCR analysis of selected genes validated the results. Results. Expression levels of several key genes involved in phagocytosis, pigment synthesis, the visual cycle, adherens, and tight junctions, and glucose and ion transport were maintained close to control levels in cultures stored at 4°C and 16°C. Cultures stored at 37°C displayed regulational changes in a larger subset of genes related to phagocytosis, adherens, and tight junctions. Conclusion. RPE cultures stored at 4°C and 16°C for one week are capable of maintaining the expression levels of genes important for key RPE functions close to control levels.

Introduction: There is increasing evidence that retinal pigment epithelium (RPE) can be used to treat age-related macular degeneration, one of the leading causes of blindness worldwide. However, the best way to store RPE to enable worldwide distribution is unknown. We investigated the effects of supplementing our previously published storage method with seven additives, attempting to improve the number of viable adult retinal pigment epithelial (ARPE)-19 cells after storage., Materials and Methods: ARPE-19 cells were cultured on multiwell plates before being stored for 1 week at 16 °C. Unsupplemented Minimal Essential Medium (MEM) (control) and a total of seven individual additives (DADLE ([D-Ala(2), D-Leu(5)]-encephalin), capsazepine, docosahexaenoic acid (DHA), resveratrol, quercetin, simvastatin and sulforaphane) at three to four concentrations in MEM were tested. The individual effect of each additive on cell viability was analyzed with a microplate fluorometer. Cell phenotype was investigated by both microplate fluorometer and epifluorescence microscopy, and morphology by scanning electron microscopy., Results: Supplementation of the storage medium with DADLE, capsazepine, DHA or resveratrol significantly increased the number of viable cells by 86.1% ± 41.9%, 67.9% ± 24.7%, 36.5% ± 10.3% and 21.1% ± 6.4%, respectively, compared to cells stored in unsupplemented MEM. DHA and resveratrol significantly reduced caspase-3 expression, while expression of RPE65 was maintained across groups., Conclusion: The number of viable ARPE-19 cells can be increased by the addition of DADLE, capsazepine, DHA or resveratrol to the storage medium without perturbing apoptosis or differentiation.

Bradykinin (BK) receptor-2 (B2R) and β2-adrenergic receptor (β2AR) have been shown to form heterodimers in vitro. However, in vivo proofs of the functional effects of B2R-β2AR heterodimerisation are missing. Both BK and adrenergic stimulation are known inducers of tPA release. Our goal was to demonstrate the existence of B2R-β2AR heterodimerisation in myocardium and to define its functional effect on cardiac release of tPA in vivo. We further investigated the effects of a non-selective β-blocker on this receptor interplay. To investigate functional effects of B2R-β2AR heterodimerisation (i. e. BK transactivation of β2AR) in vivo, we induced serial electrical stimulation of cardiac sympathetic nerves (SS) in normal pigs that underwent concomitant BK infusion. Both SS and BK alone induced increases in cardiac tPA release. Importantly, despite B2R desensitisation, simultaneous BK infusion and SS (BK+SS) was characterised by 2.3 ± 0.3-fold enhanced tPA release compared to SS alone. When β-blockade (propranolol) was introduced prior to BK+SS, tPA release was inhibited. A persistent B2R-β2AR heterodimer was confirmed in BK-stimulated and non-stimulated left ventricular myocardium by immunoprecipitation studies and under non-reducing gel conditions. All together, these results strongly suggest BK transactivation of β2AR leading to enhanced β2AR-mediated release of tPA. Importantly, non-selective β-blockade inhibits both SS-induced release of tPA and the functional effects of B2R-β2AR heterodimerisation in vivo, which may have important clinical implications.

Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic-free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.

The current study investigates whether microRNA (miRNA) regulators of epithelial-mesenchymal transition (EMT), tissue fibrosis, and angiogenesis are differentially expressed in human primary pterygium. Genome-wide miRNA and mRNA expression profiling of paired pterygium and normal conjunctiva was performed in the context of conventional excision of pterygium with autotransplantation of conjunctiva (n = 8). Quantitative real time polymerase chain reaction (qRT-PCR) was used to validate the expression of key molecules previously detected by microarray. In pterygium, 25 miRNAs and 31 mRNAs were significantly differentially expressed by more than two-fold compared to normal conjunctiva. 14 miRNAs were up-regulated (miR-1246, -486, -451, -3172, -3175, -1308, -1972, -143, -211, -665, -1973, -18a, 143, and -663b), whereas 11 were down-regulated (miR-675, -200b-star, -200a-star, -29b, -200b, -210, -141, -31, -200a, -934, and -375). Unsupervised hierarchical cluster analysis demonstrated that members of the miR-200 family were coexpressed and down-regulated in pterygium. The molecular and cellular functions that were most significant to the miRNA data sets were cellular development, cellular growth and proliferation, and cellular movement. qRT-PCR confirmed the expression of 15 of the 16 genes tested and revealed that miR-429 was down-regulated by more than two-fold in pterygium. The concerted down-regulation of four members from both clusters of the miR-200 family (miR-200a/-200b/-429 and miR-200c/-141), which are known to regulate EMT, and up-regulation of the predicted target and mesenchymal marker fibronectin (FN1), suggest that EMT could potentially play a role in the pathogenesis of pterygium and might constitute promising new targets for therapeutic intervention in pterygium., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objectives: To examine the effect of methotrexate (MTX) with or without tumor necrosis factor alpha (TNF-α)-inhibitors on serum lipoprotein(a) (s-Lp(a)), and to explore a possible relationship between s-Lp(a) and endothelial function (EF) in terms of serum levels of adhesion molecules and reactive hyperaemic index (RHI) in patients with rheumatoid arthritis (RA)., Methods: Serum levels of Lp(a), endothelial adhesion molecules, RHI and inflammatory markers were studied in 64 RA patients, starting with either MTX (n=34) or MTX+TNF-α-inhibitor treatment (n=30) at baseline and after 6 weeks and 6 months., Results: Compared to baseline values, s-Lp(a) was significantly reduced after 6 weeks (p=0.001) and 6 months (p=0.001) in RA patients treated with MTX, and after 6 weeks (p=0.001) in the MTX+TNF-α-inhibitor group. A non-significant reduction was found after 6 months (p=0.102) in the MTX+TNFα-inhibitor group. Serum E-selectin (s-E-selectin) was significantly reduced in both RA treatment groups at both control points. S-Lp(a) correlated positively with s-E-selectin at baseline (p=0.004), and change in s-E-selectin correlated with the change in s-Lp(a) during follow-up (p6weeks= 0.008, p 6months=0.009). No association was found between s-Lp(a) and the other adhesion molecules and RHI., Conclusions: MTX or MTX combined with a TNFα-inhibitor appears to significantly reduce Lp(a). This finding indicate that s-Lp(a) might be related to systemic inflammation, or that the examined drugs might reduce s-Lp(a) by other mechanisms. Anti-inflammatory treatment might be a novel therapeutic option to decrease s-Lp(a). The associations between s-E-selectin and s-Lp(a) suggest an interaction between these factors, or a common cause.

The transplantation of cultured limbal epithelial cells (LEC) has since its first application in 1997 emerged as a promising technique for treating limbal stem cell deficiency. The culture methods hitherto used vary with respect to preparation of the harvested tissue, choice of culture medium, culture time, culture substrates, and supplementary techniques. In this chapter, we describe a procedure for establishing human LEC cultures using a feeder-free explant culture technique with human amniotic membrane (AM) as the culture substrate.

Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7% ± 9.8%; P < 0.01 compared to 4°C, 8°C, and 24°C-37°C; P < 0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.

Myocardial ischemia induces cardiac tissue plasminogen activator (tPA) release, declining by repeated periods of ischemia. However, the mechanisms and cellular sources are unknown. Sympathetic nerve stimulation (SS) and bradykinin (BK), an endogenous inducer of endothelial tPA release, may play roles, potentially involving different sources or mechanisms revealed by different release patterns. Therefore, we compared the cardiac tPA release patterns during repeated coronary BK infusions and SS, both with an ensuing period of local myocardial ischemia/reperfusion (I/R). Nine pigs were subjected to four periods of coronary BK infusion (4 min) and another nine animals to four periods of SS (4 min). Finally, 10 min of I/R was induced in both groups. The single-peaked BK-induced tPA release declined toward baseline by repeated infusions, but tPA release reappeared during I/R. In contrast, total tPA release during repeated SS and subsequent I/R was more stable, and SS-induced total tPA and norepinephrine (NE) releases were strongly correlated. Surprisingly, the instantaneous SS-induced tPA release was biphasic with a stable first peak, and a second peak declining toward baseline by repeated stimulations. The fluctuations in cardiac release of plasminogen activator inhibitor-1 and the endogenous BK inhibitor angiotensin-converting enzyme, could not explain the diverging tPA release patterns. Different tPA release patterns were demonstrated during SS and BK stimulation, as well as diverging responses to repeated stimulations and subsequent I/R. This study demonstrates strong association between tPA and NE during SS and possibly two different sources or mechanisms for SS-induced tPA release.

Background: Oral intake (60 ml daily) over 12 days in eight healthy volunteers of an immunostimulatory extract based on the medicinal mushroom Agaricus blazei Murill (AbM (AndoSan™)), reduced the monocyte and granulocyte release of mainly proinflammatory cytokines in vivo, suggesting an anti-inflammatory effect. In this foremost in vivo study, the aim was to examine the effect of such AndoSan™ consumption on the expression of adhesion molecules CD11b, CD11c and CD62L and production of reactive oxygen species (ROS) in leukocytes., Methodology/principal Findings: As shown by flow cytometry, there was a significant increase of CD62L expression on monocytes and granulocytes from before (day 0) compared with 12 days after daily AndoSan™ consumption. However, only minor alterations and no clear trend in the expression of CD11b and CD11c were detected. Intracellular ROS (mainly superoxide ion) were significantly reduced in these cells from days 0 to 12., Conclusions/significance: These results support that oral intake of AndoSan™ exhibits an anti-inflammatory effect in humans in vivo.

Objective: To examine whether serum level of cartilage oligomeric matrix protein (S-COMP) is related to methotrexate (MTX) or to MTX and tumor necrosis factor-α (TNF-α) combination treatment for rheumatoid arthritis (RA); and to investigate whether S-COMP is related to cardiovascular risk factors including endothelial dysfunction and level of anticitrullinated protein antibodies (ACPA) in patients with RA., Methods: Clinical and laboratory measures, including S-COMP and reactive hyperemic index (RHI), were examined in 55 consecutive patients with RA starting with either MTX (n = 34) or MTX and anti-TNF-α treatment (n = 21) at baseline, and after 6 weeks and 6 months., Results: S-COMP was similar in the 2 treatment regimens during followup. We found a positive relationship between S-COMP at baseline and the use of disease-modifying antirheumatic drugs the last year preceding the study (p = 0.001), and a negative relation to current use of systemic glucocorticosteroids (p = 0.044). The nonsignificant change in S-COMP between baseline and the 6-month followup was positively and independently related to change in ACPA level (p = 0.009). There was no significant association between RHI and level of S-COMP at baseline., Conclusion: The cartilage turnover marker S-COMP did not change significantly after 6 months' treatment with MTX with or without a TNF-α inhibitor in patients with RA. The positive association between S-COMP and ACPA suggests that these factors might interact, and could both be contributors to an unknown link between inflammation and cartilage destruction in patients with RA. S-COMP was not related to endothelial function in patients with RA, or to other cardiovascular risk factors studied. Clinical Trials registration number NCT00902005.

The effect of a single time exposure of SLS to the buccal mucosa of mice was compared to one application of the hapten OXA (oxazolone), evaluated by routine histology, immunohistochemistry and ELISA quantifications of cytokines. The SLS concentrations (2%, 4% and 8%) resulted in epithelial surface necrosis at 1-6 h, after 2-6 h accumulation of intra-epithelial neutrophils and at 24 h the main inflammatory cells were mononuclear. Increased concentrations of SLS gave more severe damage. CD4(+) T cells were found at 6 h and increased slightly up to 24 h and were most frequently seen at the lowest SLS dose. The CD8(+) T cells were kept at a low number during the whole 24 h observation period, but increased proportionally to the CD4(+) T cells. One application of 1% OXA did not raise the number of cells of either phenotype (2-24 h). Neither IL-2 nor IFN-γ demonstrated increased levels during the week of observation at any concentration of SLS, contrary to one application of OXA which caused increased IL-2 levels both at the local application site and in the regional and distant lymph nodes. Regardless of SLS concentration, a minor increase in regional lymph node weight was observed 8-12 h after substance application, quickly to subside whilst one OXA application gave a maximal weight increase at 48-72 h. We conclude that oral mucosa irritant SLS reactions gave early surface necrosis and neutrophil infiltrations and later mononuclear cell infiltrations dominated by CD4(+) T cells. The cytokines IL-2 and IFN-γ and lymphocyte proliferation in the regional lymph nodes was not observed after SLS application, contrary to hapten application., (Copyright © 2011. Published by Elsevier Ltd.)

Background: Recovery and survival of transfused platelets (PLTs) are usually assessed by radioisotope labeling methods for evaluation of transfusion efficacy and new progress in the processing of PLT concentrates. Alternative, nonradioactive methods are warranted., Study Design and Methods: A multicolor flow cytometry method was developed for simultaneous studies of recovery, survival, and function of transfused PLTs. Eight consecutive patients undergoing allogeneic stem cell transplantation (TX) were transfused with apheresis PLTs of nonself human leukocyte antigen (HLA) Class I types, and HLA Class I discrepancy between donor and recipient was used to identify transfused PLTs. Hematologic status and HLA Class I surface expression were analyzed immediately before transfusion, 1 and 6 hours after transfusion, and daily during the subsequent week. PLT activation was assessed by surface expression of CD63, CD62P, or CD42a, before and after stimulation with thrombin receptor agonist peptide., Results: PLT recovery was 43, 41, and 31% for fresh (5-72 hr old) and 30, 27, and 17% for stored (73-148 hr old) PLTs, after 1, 6, and 15 to 28 hours, respectively. Survival of fresh versus stored PLTs were 160 and 105 hours, respectively. Spontaneous PLT activation and residual activation potential were almost equal for fresh and stored PLTs. PLT engraftment was detected between Day 7 and Day 9, which was significantly earlier than first sign of neutrophil engraftment (Days 11-19; p=0.01)., Conclusion: Flow cytometry is an attractive alternative to radiolabeling of PLTs for simultaneous studies of survival, recovery, and function of transfused PLTs and early detection of PLT engraftment after allogeneic stem cell TX., (© 2011 American Association of Blood Banks.)

Background: Early intramedullary nailing (IMN) of long bone fractures in severely injured patients has been evaluated as beneficial, but has also been associated with increased inflammation, multi organ failure (MOF) and morbidity. This study was initiated to evaluate the impact of primary femoral IMN on coagulation-, fibrinolysis-, inflammatory- and cardiopulmonary responses in polytraumatized patients., Methods: Twelve adult polytraumatized patients with femoral shaft fractures were included. Serial blood samples were collected to evaluate coagulation-, fibrinolytic-, and cytokine activation in arterial blood. A flow-directed pulmonary artery (PA) catheter was inserted prior to IMN. Cardiopulmonary function parameters were recorded peri- and postoperatively. The clinical course of the patients and complications were monitored and recorded daily., Results: Mean Injury Severity Score (ISS) was 31 ± 2.6. No procedure-related effect of the primary IMN on coagulation- and fibrinolysis activation was evident. Tumor necrosis factor alpha (TNF-α) increased significantly from 6 hours post procedure to peak levels on the third postoperative day. Interleukin-6 (IL-6) increased from the first to the third postoperative day. Interleukin-10 (IL-10) peaked on the first postoperative day. A procedure-related transient hemodynamic response was observed on indexed pulmonary vascular resistance (PVRI) two hours post procedure. 11/12 patients developed systemic inflammatory response syndrome (SIRS), 7/12 pneumonia, 3/12 acute lung injury (ALI), 3/12 adult respiratory distress syndrome (ARDS), 3/12 sepsis, 0/12 wound infection., Conclusion: In the polytraumatized patients with femoral shaft fractures operated with primary IMN we observed a substantial response related to the initial trauma. We could not demonstrate any major additional IMN-related impact on the inflammatory responses or on the cardiopulmonary function parameters. These results have to be interpreted carefully due to the relatively few patients included., Trial Registration: ClinicalTrials.gov: NCT00981877.

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23°C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23°C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell-cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03±0.38 microvilli/μm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69±0.54 microvilli/μm; P=0.98 and 0.89±1.0 microvilli/μm; P=0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07±1.0 microvilli/μm; P=0.47 and 0.07±0.07 microvilli/μm; P=0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4±0.3 cell layers, as opposed to 4.0±0.9 cell layers; P=0.89 after 4 days of HEPES-MEM storage and 2.8±0.6 cell layers; P=0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7±0.2 cell layers; P=0.46 and 3.4±0.4 cell layers; P=0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4%±3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1%±4.5%; P=0.99 and 85.1%±13.7%; P=0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7%±15.2%; P=0.97 and 79.8%±15.7%; P=0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23°C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Introduction: Operations in trauma patients represent a second insult and the extent of the surgical procedures influences the magnitude of the inflammatory response. Our hypothesis was that a reamer-irrigator-aspirator (RIA) system would cause a lesser inflammatory response than traditional reaming (TR)., Materials and Methods: Coagulation, fibrinolysis and cytokine responses were studied in Norwegian landrace pigs during and after intramedullary nailing (IMN) with two different reaming systems using ELISA and chromogenic peptide substrate assays. The TR (n=8) and the RIA (n=7) reaming systems were compared to a control group (n=7). The animals were followed for 72 h. Arterial, mixed venous and femoral vein blood were withdrawn simultaneously peroperatively and until 2 h after the nail was inserted for demonstration of local, pulmonary and systemic activation of the cascade systems. At 6 h, 24 h, 48 h and 72 h postoperatively arterial blood samples were withdrawn., Results: Significantly procedure-related increased levels were found for thrombin-antithrombin (TAT) and tissue plasminogen activator (t-PA) in the TR group and TAT in the RIA group. The local and the pulmonary activation of coagulation and fibrinolysis were more pronounced in the TR than in the RIA group, the difference reached significance for plasminogen activator inhibitor-1 (PAI-1) (arterial blood). The cytokine response, mainly represented by IL-6 increase, was more pronounced in the TR than the RIA group, and was significant for IL-6 in femoral vein blood. The arterial levels of IL-6 exceeded the mixed venous levels indicating an additional pulmonary activation of IL-6. Two animals in the TR group, who died of pulmonary embolism (PE) prior to planned study end point, had a more pronounced response compared to the rest of the TR group., Conclusion: A procedure-related coagulation and fibrinolytic response was demonstrated in both reaming groups, with more pronounced response in the TR than in the RIA group. Elevated levels of cytokines were demonstrated related to reaming and nailing, with significantly higher IL-6 levels in the TR than in the RIA group., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Elevated plasma levels of vascular inflammatory markers have been reported in patients with peripheral arterial disease (PAD). We assessed the effect of supervised exercise training (ET) on vascular inflammation, hypothesizing that ET reduces plasma levels of the endothelial adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-I (VCAM-I). Twenty-nine patients with PAD underwent a supervised ET program for 8 weeks. Before and after ET, walking distances (pain-free, PWD; maximal, MWD) were determined by a standard treadmill test. Plasma levels of E-selectin and ICAM-I were significantly reduced (E-selectin: 45.5-40.4 ng/mL, P = .013); ICAM-I: 342.0-298.0 ng/mL, P = .016). VCAM-1 levels were unchanged. Walking distances increased significantly (PWD: median 77-150 m, P < .001; MWD: median 306-535 m, P < .001). In conclusion, 8 weeks of ET in patients with PAD reduces plasma levels of the specific endothelium-derived inflammatory markers E-selectin and ICAM-I.

An immunomodulatory extract (AndoSan™) based on the medicinal mushroom Agaricus blazei Murill (AbM) has shown to reduce blood cytokine levels in healthy volunteers after 12 days' ingestion, pointing to an anti-inflammatory effect. The aim was to study whether AndoSan™ had similar effects on cytokines in patients with ulcerative colitis (UC) and Crohn's disease (CD). Calprotectin, a marker for inflammatory bowel disease (IBD), was also measured. Patients with CD (n = 11) and with UC (n = 10) consumed 60 ml/day of AndoSan™. Patient blood plasma was harvested before and after 6 h LPS (1 ng/ml) stimulation ex vivo. Plasma and faecal calprotectin levels were analysed using ELISA and 17 cytokines [IL-2, IFN-γ, IL-12 (Th1), IL-4, IL-5, IL-13 (Th2), IL-7, IL-17, IL-1β, IL-6, TNF-α, IL-8, MIP-1β, MCP-1, G-CSF, GM-CSF and IL-10] by multiplex assay. After 12 days' ingestion of AndoSan™, baseline plasma cytokine levels in UC was reduced for MCP-1 (40%) and in LPS-stimulated blood for MIP-1β (78%), IL-6 (44%), IL-1β (41%), IL-8 (30%), G-CSF (29%), MCP-1 (18%) and GM-CSF (17%). There were corresponding reductions in CD: IL-2 (100%), IL-17 (55%) and IL-8 (29%) and for IL-1β (35%), MIP-1β (30%), MCP-1 (22%), IL-8 (18%), IL-17 (17%) and G-CSF (14%), respectively. Baseline concentrations for the 17 cytokines in the UC and CD patient groups were largely similar. Faecal calprotectin was reduced in the UC group. Ingestion of an AbM-based medicinal mushroom by patients with IBD resulted in interesting anti-inflammatory effects as demonstrated by declined levels of pathogenic cytokines in blood and calprotectin in faeces., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4(-/-) mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4(-/-)-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased syndecan-4 levels with decreased pS179 which was associated with increased NFAT activation. In conclusion, our data show that syndecan-4 is essential for compensatory hypertrophy in the pressure overloaded heart. Specifically, syndecan-4 regulates stretch-induced activation of the calcineurin-NFAT pathway in cardiomyocytes. Thus, our data suggest that manipulation of syndecan-4 may provide an option for therapeutic modulation of calcineurin-NFAT signaling.

The medicinal mushroom Agaricus blazei Murill from the Brazilian rain forest has been used in traditional medicine and as health food for the prevention of a range of diseases, including infection, allergy, and cancer. Other scientists and we have examined whether there is scientific evidence behind such postulations. Agaricus blazei M is rich in the immunomodulating polysaccharides, β-glucans, and has been shown to have antitumor, anti-infection, and antiallergic/-asthmatic properties in mouse models, in addition to anti-inflammatory effects in inflammatory bowel disease patients. These effects are mediated through the mushroom's stimulation of innate immune cells, such as monocytes, NK cells, and dendritic cells, and the amelioration of a skewed Th1/Th2 balance and inflammation.

Introduction: Intramedullary orthopaedic procedures may increase the intramedullary pressure (IMP) and thereby cause intravasation of bone marrow contents. In recent studies by the authors the reamer-irrigator-aspirator (RIA) has been demonstrated to reduce IMP and coagulation-, fibrinolysis- and cytokine responses, but did not prove any significant difference in cardiopulmonary function parameters or numbers of emboli when compared to a traditional reaming (TR) system. The correlations between IMP increase, regardless type of reamer, and inflammatory- and coagulation responses, pulmonary embolization, and cardiopulmonary alterations have, however, not previously been analyzed in this material. Our hypothesis was that a lower IMP would result in reduced occurrence of pulmonary embolization, reduced inflammatory-and coagulation responses, as well as reduced cardiopulmonary alterations., Materials and Methods: Twenty-eight young Norwegian landrace pigs were exposed to femoral intramedullary reaming, with either the TR (n = 10) or the RIA (n = 10) system, or used as controls (n = 8). IMP was recorded during reaming and nailing. Serial blood samples for demonstration of coagulation-, fibrinolysis-, and cytokine activation were withdrawn peroperatively and until 72 hours post nail insertion. Circulatory and pulmonary effects were monitored peroperatively and until two hours postoperatively. The animals were sacrificed 72 hours post nail insertion and lung tissue biopsies were harvested and examined for lung emboli., Results and Conclusions: A strong correlation between increased IMP and increased coagulation-and cytokine responses was found. The number of emboli was not significantly correlated to IMP, but was strongly correlated to changes in the coagulation- and cytokine responses. No clinical relevant correlations were observed between increased IMP or numbers of lung emboli and changes in hemodynamic- or pulmonary function parameters. A correlation between coagulation activation and cytokine activation was observed. This study confirms the connection between increased IMP, increased coagulation activation and the magnitude of pulmonary emboli in a model evaluating the effects of intramedullary reaming of intact pig femora. In this model, the lowering of IMP during reaming, as obtained with RIA, reduced the magnitude of and the effects of bone marrow extravasation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Intramedullary reaming and nailing increases intramedullary pressure. This may cause intravasation of bone marrow contents, leading to bone marrow embolization and altered cardiopulmonary function. Possible beneficial effects of attenuation of the intramedullary pressure increase by the use of a reamer-irrigator-aspirator (RIA) system were studied with the hypothesis that the RIA technique would cause lower numbers of pulmonary embolisms (PEs) and lesser cardiopulmonary affection than traditional reaming (TR)., Material and Methods: Intramedullary reaming and nailing was performed in intact femora of young Norwegian landrace pigs using either a standard intramedullary nailing technique (n = 8) or a RIA technique (n = 7). The hemodynamic and pulmonary effects were investigated during the reaming and nailing procedure and for 2 hours postoperatively. The animals were killed after 72 hours, and the lung/carcass weight ratio and the numbers of PEs were investigated., Results and Conclusion: The pattern of the procedure-related hemodynamic and pulmonary effects did not differ significantly between the RIA and the TR groups. The RIA group had lower numbers (ns) of embolisms per square centimeter lung area than the TR group. After reaming with the TR device, two animals died of PEs, the first postoperative day. The patients with femoral shaft fracture and additional cardiopulmonary injury or preexisting reduced cardiopulmonary function, however, need special attention, and the use of RIA may, in these cases, represent a better operative alternative with a lesser operative burden.

Using ELISA, we have quantified the levels of IL-2 and IFN-gamma in the oral mucosa, ear skin and regional and distant lymph nodes in an experimental murine model of contact sensitivity (CS), induced by the hapten oxazolone (OXA). Compared to normal conditions, the levels of IL-2 peaked early (4-6 h) after hapten exposure in the hapten-exposed tissues analysed both during the first hapten exposure (sensitization) and the second (elicitation) phase, thereafter quickly to subside. The oral mucosa displayed maximal 24-fold increase in IL-2 levels after sensitization and 39-fold increase after elicitation. Respective figures for ear skin were x27 and x35 and for regional lymph nodes x8 and x9, respectively. The distant lymph nodes displayed only minor cytokine increases at any time. IFN-gamma-levels did not increase after sensitization with OXA. An increase in IFN-gamma was seen after the second exposure, peaking at 8-24 h, thereafter quickly subsiding. The oral mucosa IFN-gamma increased x14 after elicitation, the ear skin x8 and regional lymph nodes x37. The weight of the four regional lymph nodes increased from 10 to 38 mg, and the total number of cells in these lymph nodes was increased x11, peaking 48 h after the elicitation. We conclude that in CS reactions, tissue levels of IL-2 increased in buccal mucosa, ear skin and in regional lymph nodes after hapten exposure and re-exposure, IFN-gamma appeared only after re-exposure to the hapten. The increased weight of the regional lymph nodes was mainly attributed to cell proliferation. The common ectodermal origin and the similarity of the CS reactions on skin and in buccal mucosa indicate that these tissues share common immunological patterns of Th1 cell reactivity, at least in dealing with haptens like OXA.

Purpose: To investigate whether human limbal epithelial cells (HLEC) that have been cultured on intact or de-epithelialized amniotic membranes (AMs) demonstrate differences in morphology after 1 week of eye bank storage., Methods: HLEC were cultured from limbal explants for 3 weeks on intact AM and AM deprived of the amniotic epithelial cells by incubation with 0.02% ethylene diamine tetra acetic acid followed by mechanical scraping. The HLEC cultures were stored for 1 week in a closed container in a serum-based medium at 23°C. The surface morphology was assessed using scanning electron microscopy, and a quantitative comparison of desmosome and hemidesmosome numbers was performed using transmission electron microscopy., Results: Although most superficial epithelial cells were closely attached to each other, with tightly opposed cell junctions and distinct cell borders, there was evidence of some cell separation in HLEC that had been cultured on intact and denuded AM after 1 week of storage. In both experimental groups, the epithelia were well stratified, consisting of basal column-shaped cells, suprabasal cuboid wing cells, and flat squamous superficial cells, but dilated intercellular spaces were observed. The total number of desmosomes per micron was 1.39 ± 0.77 in HLEC cultured on intact AM versus 0.98 ± 0.45 in HLEC expanded on denuded AM (P = 0.76). The total number of hemidesmosomes per micron in HLEC cultured on intact AM and denuded AM was 0.87 ± 0.34 and 0.78 ± 0.31, respectively (P = 0.70)., Conclusions: Denuding of AM does not improve the structural integrity of cultured HLEC after eye bank storage.

The edible mushroom Agaricus blazei Murill (AbM), which has been used in traditional medicine against a range of diseases and possess immunomodulating properties, probably due to its high content of beta-glucans. Others and we have demonstrated stimulatory effects of extracts of this mushroom on different immune cells. Dendritic cells are major directors of immune function. We wanted to examine the effect of AbM stimulation on signal substance release from monocyte-derived dendritic cells (MDDC). After 6d incubation with IL-4 and GM-CSF, the cells were true MDDC. Then the cells were further incubated with up to 10% of the AbM-based extract, AndoSan, LPS (0.5 microg/ml) or PBS control. We found that the AbM extract promoted dose-dependent increased levels of IL-8, G-CSF, TNFalpha, IL-1beta, IL-6 and MIP-1beta, in that order. The synthesis of IL-2, IL-8 and IFNgamma were similar for the AbM extract and LPS. However, AndoSan induced a 10- to 2-fold higher production than did LPS of G-CSF, TNFalpha and IL-1beta, respectively. AbM did not induce increased synthesis of Th2 or anti-inflammatory cytokines or the Th1 cytokine IL-12. We conclude that stimulation of MDDC with an AbM-based extract resulted in increased production of proinflammatory, chemotactic and some Th1-type cytokines in vitro., (2009. Published by Elsevier Ltd.)

Purpose: To investigate whether human limbal epithelial cells (HLECs) derived from various regions of the limbus exhibit differences in gene expression and epithelial characteristics., Methods: HLECs were derived from explants taken from the superior, nasal, inferior, and temporal limbus and cultured for 21 days. Whole genome transcript profiling was performed with a gene microarray. The microarray results were validated by using RT-PCR. Epithelial morphology was studied with light microscopy and transmission electron microscopy, and phenotype was evaluated by immunohistochemistry., Results: Epithelial outgrowth was present in most cultures of superior origin (88%) in contrast to cultures of temporal origin (38%). The epithelial thickness and number of cell layers were significantly greater in cultures of superior origin than in cultures from inferior and temporal areas. TRIM36, OSR2, and RHOU, which are involved in morphogenesis, were significantly differentially expressed in the superior region, compared with the other regions. Proposed limbal stem cell, progenitor, and differentiation markers were not differentially expressed. The uniform gene expression of ocular surface markers correlated with homogeneous immunostaining of corresponding protein markers in HLEC cultures from all regions, demonstrating an undifferentiated phenotype (p63(+), DeltaNp63alpha(+), ABCG2(+), K19(+), vimentin(+), integrin beta1(+), nestin(-), K3(-), K5(+), and E-cadherin(+))., Conclusions: No major transcriptional or phenotypic differences were observed in cultured HLECs derived from different regions of the limbus. However, explants of superior origin demonstrated the highest outgrowth success rate and generated epithelia with greater epithelial thickness and number of cell layers, which may prove useful for transplantation purposes.

Aim: Bilirubin overload caused by haemolysis of transfused blood and breakdown of extravasated blood constitutes an important causative factor of jaundice in trauma patients. Intravenous infusions of large amounts of unconjugated bilirubin (UCB) block biliary phospholipid secretion and produce canalicular membrane lesions in pigs, putatively because of enhanced cytotoxicity of bile. Severe intrahepatic cholestasis is the functional end result. The aim of the study was to investigate whether bilirubin ditaurate (BDT) overload, which also inhibits biliary phospholipid secretion, also induces intrahepatic cholestasis., Methods: Six pigs were infused with 2.8 g of BDT for 150 min. Six control pigs were infused with albumin sham solution for 150 min. Bile samples were analysed for bile acid- and phospholipid-secretion rates. Bile and serum samples were analysed for bilirubin concentration. Liver biopsies were obtained for scanning electron microscopic studies (SEM)., Results: Biliary bile acid secretion fell by 7.7% and biliary phospholipid secretion rate fell by 88% after BDT infusion. Thus, infusion of BDT for 150 min did not cause intrahepatic cholestasis. SEM showed some variability in the size of canalicular membrane microvilli, but no evidence of gross destruction., Conclusion: BDT overload markedly lowers biliary phospholipid secretion. In contrast to UCB, BDT does not induce canalicular membrane damage nor cause intrahepatic cholestasis. Sustained, marked inhibition of phospholipid secretion does therefore not adequately explain UCB-induced cholestasis. Accumulation of UCB in the canalicular membrane may be the important factor in the pathogenesis of canalicular membrane lesions and intrahepatic cholestasis during UCB overload.

Background/aims: To assess sterility of cultured human limbal epithelial cells (HLEC) and to investigate the viability, morphology and phenotype of cultured HLEC following 2 and 3 weeks of organ culture storage., Methods: HLEC cultured on amniotic membranes were stored in organ culture medium in a closed container at 23 degrees C. Sterility of storage media was tested using a Bactec 9240 blood culture instrument (Becton Dickinson, Maryland) for incubation and periodic reading. Viability was analysed by calcein-acetoxymethyl ester/ethidium homodimer-1 assay, morphology by light microscopy and cellular phenotype by immunohistochemistry., Results: No microbial contamination was observed after 1 week's storage. Viability of cultured HLEC was 87.9 (SD 6.4)% and 52.7 (13.1)% after 2 and 3 weeks of storage, respectively, compared with 98.8 (2.6)% before storage (p<0.001). The multilayered structure was preserved in 70% of cultures following 2 weeks of storage but lost after 3 weeks. A less differentiated phenotype was maintained., Conclusion: This study is the first to verify the sterility of HLEC cultures prior to transplantation. Although a slight decrease in viability was observed following 2 weeks of storage, the HLEC sheets remain acceptable, whereas 3 week's storage was unsatisfactory.