Cultured Human Retinal Pigment Epithelial (hRPE) Sheets: A Search for Suitable Storage Conditions.

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.

Nucleus Morphometry in Cultured Epithelial Cells Correlates with Phenotype.

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.

miRNA and mRNA expression profiling identifies members of the miR-200 family as potential regulators of epithelial-mesenchymal transition in pterygium.

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.

Serum levels of lipoprotein(a) and E-selectin are reduced in rheumatoid arthritis patients treated with methotrexate or methotrexate in combination with TNF-α-inhibitor.

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.

Recovery, survival, and function of transfused platelets and detection of platelet engraftment after allogeneic stem cell transplantation.

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.

Effect of AndoSan™ on expression of adhesion molecules and production of reactive oxygen species in human monocytes and granulocytes in vivo.

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.

Cardiopulmonary response to reamed intramedullary nailing of the femur comparing traditional reaming with a one-step reamer-irrigator-aspirator reaming system: an experimental study in pigs.

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.

Tumor necrosis factor inhibitors are associated with reduced complement activation in spondylarthropathies: An observational study.

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.

Regeneration with proliferation of the endothelium of cultured human donor corneas with extended postmortem time.

PURPOSE: To examine the endothelium of donor corneas with extended postmortem time for survival and reparative mechanisms in an eye bank organ culture storage system. METHODS: We obtained 14 pairs of donor corneas with a postmortem time ranging from 29 to 163 hours. One cornea of a pair was immediately fixed for the study of structural changes postmortem and to serve as a control. The second was stored in organ culture for 3 days and thereafter fixed to be studied for reparative processes. Examination was done with light microscopy and scanning electron microscopy. Immunohistochemical staining with antibodies against proliferating cell nuclear antigen, Ki-67, and n-cadherin was performed to examine for cell proliferation and to characterize the cells. RESULTS: The control corneas showed increasing endothelial cell damage with increasing postmortem time. After 5-7 days postmortem, most cells were structurally damaged. After 3 days in organ culture, all corneas acquired an endothelial covering of the posterior surface, with cells, suggesting proliferation in both scanning preparations and in cross-sections. Positive endothelial cell staining with proliferating cell nuclear antigen was found in all cultured corneas. Ki-67 staining of the endothelium was found in 9 of the cultured corneas. CONCLUSIONS: The study showed survival of the corneal endothelium up to 7 days postmortem, and accordingly, the potential clinical use of donor corneas with extended postmortem time. Our results furthermore suggest that repair of the endothelium in donor corneas during organ culture storage occurs also by proliferation and not only by migration and enlargement of existing cells. If we uncover the mechanisms regulating cell proliferation in corneal endothelium, it should be possible to develop better storage methods of corneal transplants to improve quality and supply.

Effect of anti-rheumatic treatment on selenium levels in inflammatory arthritis.

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 ±â€¯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.

Differential expression of vitamin D associated genes in the aorta of coronary artery disease patients with and without rheumatoid arthritis.

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).

Effects of organ culture and Optisol-GS storage on structural integrity, phenotypes, and apoptosis in cultured corneal epithelium.

PURPOSE: A previous report has described the use of eye bank storage of cultured human limbal epithelial cells (HLECs) to provide a reliable source of tissue for treating limbal stem cell deficiency. In the present study, conventional organ culture (OC) storage and Optisol-GS (Bausch & Lomb, Irvine, CA) storage of cultured HLECs were compared. METHODS: Three-week HLEC cultures were either organ cultured at 31 degrees C or 23 degrees C or stored in Optisol-GS at 5 degrees C in a closed container for 1 week. Morphology was studied by light microscopy and transmission electron microscopy, and phenotypic characterization was assessed by immunohistochemistry. Apoptosis was evaluated by real-time RT-PCR microarray analysis, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). RESULTS: The ultrastructure was preserved at 23 degrees C, while storage at 31 degrees C and 5 degrees C was associated with enlarged intercellular spaces, separation of desmosomes, and detachment of epithelial cells. Cultured HLECs remained undifferentiated in all storage conditions. The expression of the antiapoptotic gene BCL2 was prominently upregulated in storage at 23 degrees C and 5 degrees C. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 in 23 degrees C and 5 degrees C storage conditions suggests a reduction in nuclear factor-kappaB activity. No significant increase in cleaved caspase-3 and TUNEL staining was observed in response to eye bank storage, and the labeling indices of cleaved caspase-3 (range, 0.0%-4.7%) and TUNEL (range, 0.0%-7.8%) were low. CONCLUSIONS: These data indicate that OC storage of cultured HLECs at ambient temperature is superior to OC storage at 31 degrees C and Optisol-GS storage at 5 degrees C and that apoptosis is minimal after eye bank storage of cultured HLECs.

Effect of an extract of the mushroom Agaricus blazei Murill on expression of adhesion molecules and production of reactive oxygen species in monocytes and granulocytes in human whole blood ex vivo.

We have reported that an extract of the edible officinal mushroom Agaricus blazei Murill (AbM) stimulates synthesis of pro-inflammatory cytokines in human monocytes and vein endothelial cells in vitro and reduces the extent of lethal septicemia in mice with bacterial peritonitis. In the present study on human monocytes and granulocytes in whole blood ex vivo, we studied the dynamic changes of cell adhesion molecules (CD11b, CD62L) and the production of reactive oxygen species (ROS) after stimulation with AbM. The presence of AbM resulted in a similarly increased expression of CD11b in monocytes and granulocytes, although at a lower AbM concentration in monocytes (0.5%) than in granulocytes (2%). Furthermore, there was an AbM-mediated decrease in CD62L expression mirroring the effect on CD11b expression regarding magnitude and dose response. The intracellular production of ROS increased slightly but significantly in granulocytes, but not in monocytes stimulated with AbM. The results suggested that the major effect of AbM on monocytes and granulocytes was the upregulation of CD11b expression, thereby increasing both the phagocytic potential and the ablility to induce diapedesis into inflammatory foci. The rich beta-glucan content of AbM could play a crucial role in this immune response.

Decreased levels of asymmetric dimethylarginine during acute hyperinsulinemia.

Endothelial dysfunction is reflected by an impaired nitric oxide (NO)-mediated vasodilatation. Insulin resistance may be linked to endothelial dysfunction by several mechanisms, including disturbances in signaling pathways common to both insulin action and NO production. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, may contribute to endothelial dysfunction, and elevated ADMA levels have been associated with both insulin levels and the degree of insulin resistance. The direct link between insulin and ADMA, however, has not yet been established. In the present study, we aimed to investigate the effects of acute hyperinsulinemia on circulating ADMA and L-arginine levels and on forearm blood flow (FBF). Male volunteers, aged 21 to 24 years, with borderline hypertension were included in the study. The participants underwent a 90-minute hyperinsulinemic isoglycemic glucose clamp with insulin levels at the postprandial levels (n=20) or a saline infusion (control) (n=9). Fasting blood samples were drawn at baseline and after 90 minutes. Insulin infusion was accompanied by a reduction in ADMA (0.78 to 0.68 micromol/L, P<.01), which was significantly different (P=.001) from the increase seen in the saline control group (0.69 to 0.79 micromol/L, P<.05). The same profile was obtained for L-arginine with a significantly more pronounced decrease (P<.001) in the insulin clamp group (74 to 61 micromol/L, P<.001) than in the saline control group (59 to 57 micromol/L, P=.95). The FBF level and nitrate/nitrite (NOx) levels were not affected by any of the clamp procedures. Short-term administration of insulin was accompanied by a decrease in both ADMA and L-arginine levels, with no change in FBF, in our population of young men with borderline hypertension. The possible influence of insulin on ADMA levels in a chronic state of insulin resistance can, however, not be deduced from the present investigation.

A novel method for preserving cultured limbal epithelial cells.

AIM: To investigate organ culture preservation of cultured limbal epithelial cells in order to enhance the availability of tissue-engineered epithelia that are used to treat patients with limbal stem cell deficiency. METHODS: Limbal epithelial cells were cultured for 3 weeks on intact amniotic membrane fastened to a polyester membrane carrier. The cultured epithelia were stored for 1 week at 23 degrees C in organ culture medium. The preserved epithelia were then examined using a colorimetric cell viability assay, light microscopy and immunohistochemistry. RESULTS: The viability of the preserved epithelia was 84% (20%), and no statistically significant difference was found compared with non-preserved epithelia. In general, the cell borders were maintained, the nuclei showed no sign of degeneration, and the original layered structure was preserved. Mild intercellular oedema was occasionally observed. Expression of p63, K19 and vimentin was maintained. CONCLUSIONS: Cultured limbal epithelial cells can be preserved in organ culture medium for 1 week at room temperature, while maintaining the original layered structure and undifferentiated phenotype.

LeuCAM and reactive oxygen species during long-term exercise combined with sleep and energy deficiency.

INTRODUCTION: During the Norwegian military ranger-training course, cadets are exposed to prolonged physical exercise combined with sleep-, energy-, and food deficiency. The open-window postexercise hypothesis indicates that after hard physical activity, there is an increased risk of contracting infectious diseases. PURPOSE: The purpose of the present study was to determine leukocyte reactive oxygen species (ROS) levels, total antioxidant status (TAS), leukocyte expression of the cell adhesion molecules CD62L and CD11b, and plasma levels of soluble adhesion molecule L-selectin before, during, and in the recovery phase of a military ranger-training course. METHODS: Ten cadets from the Norwegian Military Academy were recruited to the study. Flow cytometry was used to study the intracellular levels of ROS in leukocytes (basally, as well as after in vitro stimulation with phorbol myristate acetate (PMA)), applying the probes dihydroethidium (DHE) and dihydrorhodamine 123 (DHR) and the leukocyte expression of adhesion molecules CD62L and CD11b. ELISA was used to assess the plasma levels of soluble L-selectin, and TAS in plasma was measured using the ABTS+ reduction assay kit. RESULTS: The basal levels of ROS as well as PMA-stimulated ROS in leukocytes declined gradually during the ranger-training course, being lowest on the last day (P < 0.05). The level of TAS increased (P < 0.01) during the course. A striking decrease (P < 0.001) was observed in leukocyte CD62L expression and was sustained even after 3 d of recovery. The leukocyte expression of CD11b remained unchanged. CONCLUSION: The ranger-training course leads to a partial exhaustion of the leukocyte ROS-generating machinery and to a nearly total extinguishing of leukocyte CD62L expression. These changes may support the open-window hypothesis indicating reduced ability to combat microbial invasions before total restitution.

Exposure, lung function decline and systemic inflammatory response in asphalt workers.

OBJECTIVES: The aim of this study was to determine the association between exposures in asphalt work and changes in lung function, blood concentrations of interleukin-6 (IL-6), micro-C-reactive protein, and fibrinogen among asphalt workers during a work season. METHODS: Blood samples from all asphalt workers (N=140) in Norway's largest road construction and maintenance company were taken in April-May 2005 and again in September-October 2005. Spirometric tests of the asphalt workers and a reference group (heavy construction workers, N=126) were carried out before the asphalt season, and the asphalt workers were tested again at the end of the season. Exposure to total dust, oil mist, polycyclic aromatic hydrocarbons, and gases was measured by personal samplers during the asphalt season. RESULTS: The asphalt workers had a significantly a lower forced expiratory volume in 1 second (FEV(1)) and forced expiratory flow rate of 50% of the forced vital capacity than the reference group at the beginning of the season. The asphalt workers were divided according to their exposure into two groups, asphalt pavers (N=81) and asphalt plant operators and truck drivers (N=54). The screedmen, a group of the asphalt pavers, had a statistically significant lower FVC and FEV(1) after one season of asphalt work than all of the other asphalt workers (P<0.05). The mean plasma concentration of IL-6 increased among the asphalt pavers from 1.55 pg/ml before the season to 2.67 pg/ml at the season's end (P=0.04, adjusted for current smoking). CONCLUSIONS: Exposure in asphalt paving may enhance the risk of lung function decline.

Cardiac accumulation of bone marrow mononuclear progenitor cells after intracoronary or intravenous injection in pigs subjected to acute myocardial infarction with subsequent reperfusion.

OBJECTIVE: The purpose of the present study was to compare the efficacy of intracoronary and intravenous injection of autologous progenitor cells for homing to the acutely infarcted but reperfused myocardium in pigs. METHODS: Myocardial infarction was induced in 11 anesthetized pigs by 60-min balloon inflation in the mid LAD. After balloon deflation, reperfusion was verified and autologous CD31(+) progenitor cells, or bone marrow mononuclear cells, labeled with PKH67, were injected either intracoronarily (n=6) or intravenously (n=3). By autopsy, 4-5 days after induction of infarction, tissue from the heart and other organs was obtained for fluorescence microscopy. RESULTS: In the heart, PKH(+) cells were detected throughout the reperfused infarcted myocardium, and the number of PKH(+) cells was significantly higher after intracoronary than after intravenous injection (3.2+/-0.55 vs. 0.33+/-0.17 cells/high-power field/10(6) cells injected, P=.01). Few PKH(+) cells were detected in the spleen, lung, mesenteric lymph node, and bone marrow. In an additional animal with a coil placed in the mid LAD, progenitor cells were not detected in the infarcted myocardium or in the normal myocardium. CONCLUSION: Autologous mononuclear and CD31(+) cells from bone marrow accumulated in the infarcted myocardium when injected intracoronarily or intravenously after established reperfusion, and the accumulation of cells was significantly greater after intracoronary injection than after intravenous injection. Accumulation of PKH(+) cells did not appear in the normal myocardium or in the nonreperfused infarcted myocardium. PKH(+) cells were detected in spleen, lung, and bone marrow but to a lesser degree than in the infarcted myocardium.

Impact of Storage Temperature on the Expression of Cell Survival Genes in Cultured ARPE-19 Cells.

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.

Plasma complement and vascular complement deposition in patients with coronary artery disease with and without inflammatory rheumatic diseases.

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.