Search

Objectives: Patients who are referred to home health care after an acute care hospitalization may not receive home health care, resulting in incomplete home health referrals. This study examines the prevalence of incomplete referrals to home health, defined as not receiving home health care within 7 days after an initial hospital discharge, and investigates the relationship between home health referral completion and patient outcomes., Design: Retrospective cohort study., Setting and Participants: Medicare beneficiaries who are discharged from short-term acute care hospitals between October 2015 and December 2016 with a discharge status code on the hospital claim indicating home health care., Methods: Patient characteristics and outcomes were compared between Medicare beneficiaries with complete and incomplete home health referrals after hospital discharge. The outcomes included mortality, readmission rate, and total spending over a 1-year episode following hospitalization. These outcomes were risk-adjusted using patient demographic, socioeconomic, clinical characteristic, hospital characteristic, and state fixed effects., Results: Approximately 29% of the 724,700 hospitalizations in the analytic dataset had incomplete home health referrals after discharge. The rate of incomplete home health referrals varied among clinical conditions, ranging from 17% among joint/musculoskeletal patients and 38% among digestive/endocrine patients. Risk-adjusted 1-year mortality and readmission rates were 1.4 and 2.4 percentage points lower and total spending was $1053 higher among patients with complete home health referrals as compared with those with incomplete home health referrals after hospital discharge., Conclusions and Implications: The analysis revealed that almost 1 in 3 patients discharged from a hospital with a discharge status of home health does not receive home health care. In addition, complete home health referrals are associated with lower mortality and readmission rates and higher spending. As home health care utilization increases, policymakers should pay attention to the tradeoff between quality and cost when implementing alternative policies and payment models., (Copyright © 2020 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.)

Objectives: This study aimed to demonstrate the effect of treating titanium-implant surfaces with plasma from two different sources on wettability and initial single-cell adhesion of human osteoblasts and to investigate whether aging affects treatment outcomes., Methods: Titanium disks with sandblasted and acid-etched (SLA) surfaces were treated with atmospheric pressure plasma (APP) and low-pressure plasma (LPP). For wetting behavior of the specimens after plasma treatment, the water contact angle was measured. The single-cell detachment force and amount of work of detachment of human osteoblasts were determined with single-cell force spectroscopy (SCFS). To evaluate the aging effect in APP-treated specimens, SCFS was conducted 10 and 60 min after treatment., Results: Significantly higher hydrophilicity was observed in the APP and LPP treatment groups than in the control group, but no significant difference was observed between the APP and LPP groups. No significant difference in cell-detachment force or work of detachment was observed, and there were no significant differences according to the conditioning mechanisms and storage time., Significance: Conditioning of the titanium surfaces with APP or LPP was not a significant influencing factor in the initial adhesion of the osteoblasts., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The aim of this in vitro study was to identify changes in gene expression of proinflammatory cytokines in human whole blood after contact with titanium implant surfaces after plasma treatment., Materials and Methods: Grade 4 titanium dental implants were conditioned with low-pressure plasma (LPP) and atmospheric-pressure plasma (APP) and submerged in human whole blood in vitro. Unconditioned implants and blood samples without implants served as control and negative control groups, respectively. Sampling was performed at 1, 8, and 24 h. Changes in mRNA expression levels of interleukin 1-beta (IL1-β) and tumor necrosis factor-alpha (TNF-α) were assessed using RT-qPCR., Results: In the control group, significant increases in IL1-β and TNF-α expression were observed. Significant decreases in the expression of IL1-β and TNF-α were identified in blood with implants after plasma treatment., Conclusion: Differences in gene expression of proinflammatory cytokines after incubation of plasma-conditioned titanium implants can be assessed using human whole blood. The results of the present study indicate that plasma treatment (APP and LPP) of titanium dental implants leads to downregulation of proinflammatory cytokine gene expression, which might be beneficial in early osseointegration., (Copyright © 2019 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Purpose: Successful implant therapy is based on fast, safe, and predictable osseointegration. Several surface modifications have been introduced to improve the bone-to-implant interaction. This in vivo study evaluates the impact of plasma surface conditioning on early wound healing and osseointegration., Materials and Methods: A total of 16 dental implants with a sand-blasted and acid-etched surface were conditioned with cold atmospheric plasma prior to insertion in the frontal bone of four miniature pigs. Sequential fluorescence labeling was administered to label bone metabolism, and after 8 weeks, bone blocks were harvested for radiological, histological, and histomorphometrical evaluation., Results: The plasma conditioning had no impact on the morphology of the implant surface. The bone-to-implant contact ratio was 90.4% and 86.5%, the interthread bone density 72.5% and 63.4%, and the periimplant bone density 60.5% and 61.1%, in the plasma conditioned group and control group, respectively. Concentric bands of fluorescence enrichment indicated a chronological and homogenous mineralization of newly formed bone. No unwanted periimplant side effects were detected., Conclusion: The increased parameters for osseointegration in this in vivo study merit further investigation in prospective clinical trials., (Copyright © 2018 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

In two recent studies of Spanish individuals, gender was suggested as a factor that contributes to human eye colour variation. However, gender did not improve the predictive accuracy on blue, intermediate and brown eye colours when gender was included in the IrisPlex model. In this study, we investigate the role of gender as a factor that contributes to eye colour variation and suggest that the gender effect on eye colour is population specific. A total of 230 Italian individuals were typed for the six IrisPlex SNPs (rs12913832, rs1800407, rs12896399, rs1393350, rs16891982 and rs12203592). A quantitative eye colour score (Pixel Index of the Eye: PIE-score) was calculated based on digital eye images using the custom made DIAT software. The results were compared with those of Danish and Swedish population samples. As expected, we found HERC2 rs12913832 as the main predictor of human eye colour independently of ancestry. Furthermore, we found gender to be significantly associated with quantitative eye colour measurements in the Italian population sample. We found that the association was statistically significant only among Italian individuals typed as heterozygote GA for HERC2 rs12913832. Interestingly, we did not observe the same association in the Danish and Swedish population. This indicated that the gender effect on eye colour is population specific. We estimated the effect of gender on quantitative eye colour in the Italian population sample to be 4.9%. Among gender and the IrisPlex SNPs, gender ranked as the second most important predictor of human eye colour variation in Italians after HERC2 rs12913832. We, furthermore, tested the five lower ranked IrisPlex predictors, and evaluated all possible 3(6) (729) genotype combinations of the IrisPlex assay and their corresponding predictive values using the IrisPlex prediction model [4]. The results suggested that maximum three (rs12913832, rs1800407, rs16891982) of the six IrisPlex SNPs are useful in practical forensic genetic casework., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

We analysed the effects of all-trans retinoic acid (ATRA) on proliferation and changes in the global proteome of the nullipotent human embryonal carcinoma cell line 2102Ep and the pluripotent cell line NTERA2 cl.D1 (NT2). Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of proteins of the retinoid pathway. We established a proteome map of the germ cell tumor (GCT) cell line NT2 showing neuronal differentiation under ATRA treatment for 7days. Using bioinformatic analyses, we identified functional groups of altered proteins and potentially involved pathways, of which changes to the organization of the cytoskeleton and anti-apoptotic effects were the most prominent. Changes observed in the expression of factors involved in the retinoid pathway under ATRA, namely an upregulation of CRBP and CRABP2, were also reflected in GCT tissues of different histologies, providing further insight into factors involved in the differentiation of these pluripotent tumors., Biological Significance: Treatment of NT2 germ cell tumor cells with all-trans retinoic acid (ATRA) is a model to investigate differentiation. We analysed differentially expressed proteins by 2D-PAGE and mass spectrometry and provide a proteome map of NT2 cells under 7days of ATRA. By bioinformatic analyses, functional groups of proteins and involved pathways like changes to the cytoskeleton and anti-apoptotic effects were identified. Factors involved in the retinoid pathway, in particular upregulation of CRBP, CRABP1 and CRABP2, also showed differential expression in tumors with different histological subtypes, which provides insight into gene regulation under induced and spontaneous differentiation in germ cell tumors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this study, we present a new objective method for measuring the eye colour on a continuous scale that allows researchers to associate genetic markers with different shades of eye colour. With the use of the custom designed software Digital Iris Analysis Tool (DIAT), the iris was automatically identified and extracted from high resolution digital images. DIAT was made user friendly with a graphical user interface. The software counted the number of blue and brown pixels in the iris image and calculated a Pixel Index of the Eye (PIE-score) that described the eye colour quantitatively. The PIE-score ranged from -1 to 1 (brown to blue). The software eliminated the need for user based interpretation and qualitative eye colour categories. In 94% (570) of 605 analyzed eye images, the iris region was successfully extracted and a PIE-score was calculated. A very high correlation between the PIE-score and the human perception of eye colour was observed. The correlations between the PIE-scores and the six IrisPlex SNPs (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, TYR rs1393350, SLC45A2 rs16891982 and IRF4 rs12203592) were analyzed in 570 individuals. Significant differences (p<10(-6)) in the PIE-scores of the individuals typed as HERC2 rs12913832 G (PIE=0.99) and rs12913832 GA (PIE=-0.71) or A (PIE=-0.87) were observed. We adjusted for the effect of HERC2 rs12913832 and showed that the quantitative PIE-scores were significantly associated with SNPs with minor effects (OCA2 rs1800407, SLC24A4 rs12896399 and TYR rs1393350) on the eye colour. We evaluated the two published prediction models for eye colour (IrisPlex [1] and Snipper[2]) and compared the predictions with the PIE-scores. We found good concordance with the prediction from individuals typed as HERC2 rs12913832 G. However, both methods had difficulties in categorizing individuals typed as HERC2 rs12913832 GA because of the large variation in eye colour in HERC2 rs12913832 GA individuals. With the use of the DIAT software and the PIE-score, it will be possible to automatically compare the iris colour of large numbers of iris images obtained by different studies and to perform large meta-studies that may reveal loci with small effects on the eye colour., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objectives: The aim of this preliminary prospective study was to evaluate the clinical outcome, the oral health-related quality of life (OHRQoL), and the subjective chewing ability of patients with mandibular complete dentures retained by a single implant placed in the mandible midline., Methods: Patients wearing complete dentures were treated with a single implant in the mandible, followed by relining of the dentures and incorporation of ball attachments for implant retention. Implant outcome, prosthodontic maintenance, subjective chewing ability, and the oral health impact profile of the patients were assessed at baseline and at four weeks after connecting the denture and implant., Results: Eleven patients were enrolled in this investigation, and the mean observation period was 43.4 months (minimum period: 35, maximum period: 52 months). No implants were lost during observation period, but four dentures needed repair because of the fracture of the denture base in the midline area. A significant improvement was observed in the OHRQoL of the patients after the attachment of the mandibular dentures with a single midline implant. Furthermore, the subjective chewing ability of the patients was significantly improved after implant connection., Conclusions: Within the limitations of this preliminary prospective clinical study, single implant-supported mandibular overdentures were a successful treatment option for older edentulous patients who showed improvements in their OHRQoL and chewing ability., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We have conducted a meta-analysis of the clinical effects of morphine and hydromorphone to compare their benefit in analgesia. Embase and Medline were searched with an end-date of June 2009 for randomized, controlled trials or observational studies that addressed comparative analgesic and side-effects or particular side-effects. Two researchers independently identified included studies and extracted the data. Estimates of opioid effects were combined by using a random-effects model. Meta-analysis of eight studies suggested that hydromorphone (494 patients) provides slightly better (P=0.012) clinical analgesia than morphine (510 patients). The effect-size was small (Cohen's d=0.266) and disappeared when one study was removed, although the advantage of hydromorphone was more evident in studies of better quality (Jadad's rating). Side-effects were similar, for example, nausea (P=0.383, nine studies, 456 patients receiving hydromorphone and 460 morphine); vomiting (P=0.306, six studies, 246 patients receiving hydromorphone and 239 morphine); or itching (P=0.249, eight studies, 405 patients receiving hydromorphone, 410 morphine). This suggests some advantage of hydromorphone over morphine for analgesia. Additional potential clinical pharmacological advantages with regard to side-effects, such as safety in renal failure or during acute analgesia titration, are based on limited evidence and require substantiation by further studies.

The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to study the adhesion of platelets to immobilized FKN under physiologic flow conditions. Platelet adhesion to von Willebrand factor (VWF) was increased in the presence of FKN at 600 inverse seconds. Additional platelet adhesion to FKN coimmobilized with VWF was dependent on the FKN receptor CX3CR1 and activation of glycoprotein (GP) IIb/IIIa. The number of platelets rolling on VWF was likewise enhanced in the presence of FKN. The enhancement of rolling on FKN and VWF was insensitive to anti-CX3CR1 antibody but was fully inhibited by neutralizing GPIbα function. The extracellular domain of GPIbα was covalently coupled to fluorescent microspheres, and microsphere binding was significantly higher in the presence of FKN. Platelet adhesion to activated endothelium in vitro and to intact human arteries was substantially increased in an FKN-dependent manner. These data demonstrate that endothelial expressed FKN activates platelets via its cognate receptor CX3CR1, whereas platelet adhesion is predominantly mediated by GPIbα and independent of CX3CR1.

Unlabelled: The synthetic arginine-derived direct thrombin inhibitor argatroban is an attractive anticoagulant for percutaneous coronary intervention (PCI), because of its rapid onset and offset, and its hepatic elimination. Argatroban was approved for PCI in patients with heparin-induced thrombocytopenia (HIT). However, there are limited data about argatroban in non-HIT patients. The objective of this open-label, multiple-dose, controlled study was to examine the safety and efficacy of argatroban in patients undergoing elective PCI., Methods and Results: Of 140 patients randomized to three argatroban dose groups (ARG250, ARG300, and ARG350 with 250, 300, or 350 μg/kg bolus, followed by 15, 20, or 25 μg/kg/min infusion) and one unfractionated heparin (UFH) group (70-100 IU/kg bolus), 138 patients were analyzed. Argatroban dose-dependently prolonged activated clotting time (ACT) with more patients reaching the minimum target ACT after the initial bolus injection (ARG250: 86.1%, ARG300: 89.5%, and ARG350: 96.8%) compared to 45.5% in UFH (p<0.001). The patient proportion who did not require additional bolus injections to start PCI was significantly higher in argatroban than in UFH (p ≤ 0.002). Consequently, the time to start of PCI was shortened in argatroban groups. Composite incidences of death, myocardial infarction, and urgent revascularization until day 30 were not significantly different between the groups (ARG250: 2.8%, ARG300: 0.0%, ARG350: 3.2% vs. UFH: 3.0%). Major bleeding was observed only in UFH (3.0%), while minor bleeding occurred in ARG350 (3.2%) and UFH (6.1%, n.s.)., Conclusion: Argatroban dose-dependently increases coagulation parameters and, compared to UFH, demonstrates a superior predictable anticoagulant effect in patients undergoing elective PCI., (Copyright © 2010. Published by Elsevier Ireland Ltd.)

Aim: The main goal of this prospective clinical study was to evaluate the outcome of inlay-retained fixed dental prostheses (FDPs) made from heat-pressed lithium-disilicate glass-ceramic., Methods: Forty-five FDPs were placed in 42 patients (21 women, mean age 36.1 years and 21 men, mean age 42.0 years). The FDPs replaced 4 premolars and 19 molars in the maxilla and 4 premolars and 18 molars in the mandible. Preparations were performed in accordance with general principles for ceramic inlay restorations. Five of the 45 FDPs were hybrid-retained restorations, i.e. one abutment tooth with an inlay retainer and one with a full crown retainer. All FDPs were pressed in one piece using lithium-disilicate ceramic (IPS e.max Press, Ivoclar Vivadent). The minimum dimensions for the proximal connector were 4mm in height and 4mm in width (16 mm(2)) with a minimum occlusal ceramic thickness of 1.5mm. The surfaces of the inlay retainer were conditioned by etching with hydrofluoric acid 5% and silane application. Standard adhesive luting techniques were performed using a dentin adhesive (Syntac Classic, Ivoclar Vivadent) and a resin composite (Variolink II, Ivoclar Vivadent). Clinical follow-up examinations were performed annually., Results: The mean observation periods were 70 months (minimum 4, maximum 123 months). Twenty-seven FDPs (60%) failed during the observation period and had to be replaced. The Kaplan-Meier survival rate for inlay-retained FDPs was 57% after 5 years and 38% after 8 years, while for hybrid-retained FDPs it was 100% after 5 and 60% after 8 years., Conclusions: Inlay-retained FDPs made from lithium-disilicate ceramic present a high clinical failure rate and therefore cannot be recommended., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Selective inhibitors of cyclooxygenase-2 (COX-2) called coxibs, are effective anti-inflammatory and analgesic drugs. Recently, these drugs were associated with an increased risk for myocardial infarction and atherothrombotic events. The hypothesis of thromboxane-prostacyclin imbalance has been preferred to explain these unwanted effects., Methods: We studied the effects of 14 days intake of rofecoxib (25 mg q.d.), celecoxib (200 mg b.i.d.), naproxen (500 mg b.i.d.) and placebo in a randomized, blinded, placebo-controlled study in young healthy volunteers (median age 25-30 years, each group n = 10). We assessed prostanoid metabolite excretion (PGE-M, TXB(2), 6-keto-PGF(1alpha), 11-dehydro-TXB(2), 2,3-dinor-TXB(2), and dinor-6-keto-PGF(1alpha)), the expression of platelet activation markers (CD62P, PAC-1, fibrinogen), platelet-leukocyte formation, the endogenous thrombin potential, platelet cAMP content and plasma thrombomodulin level., Results: Naproxen suppressed biosynthesis of PGE-M, prostacyclin metabolites and thromboxane metabolites and thrombomodulin levels. In contrast, both coxibs had an inhibitory effect only on PGE-M, 6-keto-PGF(1alpha), and on dinor-6-keto-PGF(1alpha), whereas TXB(2), 2,3-dinor-TXB(2) and 11-dehydro-TXB(2) excretion were unaffected. None of the coxibs exerted significant effects on the expression of platelet activation markers, cAMP generation, platelet-leukocyte formation, or on thrombomodulin plasma levels. Interestingly, platelet TXB(2) release during aggregation was enhanced after coxib treatment following arachidonic acid or collagen stimulation., Conclusion: In young healthy volunteers coxibs inhibit systemic PGE(2) and PGI(2) synthesis. Platelet function and expression of platelet aggregation markers are not affected; however, coxibs can stimulate TXB(2) release from activated platelets. Combined decrease in vasodilatory PGE(2) and PGI(2) together with increased TXA(2) in proaggregatory conditions may contribute to coxib side effects.

Background: Angiotensin-converting enzyme inhibitors (ACEIs) have been shown to lower hematocrit and erythropoietin (EPO), but a direct link between angiotensin II (Ang II) and EPO in humans has not been shown., Methods: Placebo or Ang II was infused for six hours in nine healthy male volunteers with and without blockade of the Ang II subtype 1 receptor (AT1R). EPO concentrations were measured 3, 6, 12, and 24 hours after the start of the infusion., Results: Ang II raised the mean arterial pressure by about 20 mm Hg. Consistent with the known diurnal variation, EPO levels rose significantly (P < or = 0.02) during the day in all groups. During Ang II infusion, EPO levels rose to significantly higher levels after 6 and 12 hours compared with placebo [9.9 +/- 3.5 vs. 7.2 +/- 3.1 mU/mL (3 h, P = NS); 16.9 +/- 4.5 vs. 8.8 +/- 3.7 mU/mL (6 h, P = 0.01); 17.0 +/- 8.6 vs. 11.1 +/- 4.7 mU/mL (12 h, P = 0.01)] and returned to baseline after 24 hours (7.9 +/- 3.8 vs. 10.6 +/- 8.6 mU/mL, P = NS). With AT1R blockade, blood pressure remained normal during Ang II infusion, and EPO levels were never significantly different from placebo [6.8 +/- 4.8, 10.5 +/- 5.6, 13.1 +/- 9.0, and 12.4 +/- 10.1 mU/mL at 3, 6, 12, and 24 h after infusion, respectively, P = NS]., Conclusions: Ang II increases EPO levels in humans. This increase requires the participation of AT1R.

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

Whiplash injury, common after a motor vehicle crash, has a variable prognosis that is difficult to predict. To assess the role of various factors on this prognosis, we assembled a historical cohort of 3014 individuals who sustained a whiplash injury resulting from a motor vehicle crash in the Province of Quebec, Canada, in 1987 and were followed for 6 years. The data were obtained from the computerized databases created by the province's universal automobile insurance plan and police accident reports. The recovery time from whiplash, as measured by duration of compensation, was the primary outcome. Socio-demographic and crash-related factors measured at the time of the crash were investigated. The median recovery time for the cohort was 31 days, with 22% recovering within a week and 3% still not recovered after 1 year. For the 1551 subjects with a whiplash injury only, the socio-demographic factors that were found to be independently associated with a slower recovery from whiplash in this cohort are female gender, older age, having dependents, and not having full-time employment. The significant crash-related factors are occupancy in a truck or bus, being a passenger in the vehicle, colliding with a moving object, and being in a head-on or perpendicular collision. We classified the subjects according to a prediction score ranging from 0 to 11, devised from these factors. Subjects with a score of 0 to 2, that is those who had at most two risk factors present, had the fastest median recovery time of 19 days compared with 71 days for subjects who had a score of 6 or more. We conclude that several sociodemographic and crash-related factors are independently associated with a slow and costly recovery from whiplash injury. They are easily measurable at the time of the crash and combined so as to be simply incorporated in intervention programs aimed at early identification and management of whiplash patients with a poor prognosis.

Background: The chromosomal translocation t(11;14)(q13;q32) is the hallmark of mantle cell lymphoma (MCL) in which it can be detected cytogenetically in about 75% of cases. The t(11;14) translocation juxtaposes the bcl-1 locus in chromosome band 11q13 next to the IgH locus in chromosome band 14q32 and, thus, leads to deregulation of the cell cycle regulatory protein cyclin D1, which is encoded by the CCND1 gene localized at the telomeric border of the bcl-1-locus. MCL has the worst prognosis of all low-grade non-Hodgkin's lymphomas (NHL). In some instances, however, histopathologic differentiation between MCL and other low-grade B-cell NHL is difficult. Therefore, detection of the t(11;14) translocation is of essential diagnostic value for the risk-adjusted management of patients with MCL. Unfortunately, chromosome analyses are frequently hampered by the low yield and quality of tumor metaphases. As the 11q13 breakpoints are scattered over a region of more than 120 kb the application of molecular genetic techniques is also limited., Patients and Methods: We established an interphase fluorescence in situ hybridization (FISH) approach for the detection of the t(11;14) translocation by use of a cosmid probe hybridizing to the IgH constant region and a YAC spanning the bcl-1 region. Cells containing a t(11;14) translocation show a colocalisation of the signals for IgH and bcl-1. Eight control samples and 15 MCL specimens were investigated., Results: According to our control studies, samples containing more than 10% of cells with this signal constellation can be diagnosed as carrying a clonal t(11;14) translocation. All eleven MCL found to carry the t(11;14) translocation by chromosome analysis were positive in our FISH assay. Additionally, two of four MCL lacking a clonal t(11;14) translocation by chromosome analysis were shown to carry this aberration in 14% and 37% of interphase nuclei. Southern blot data indicate that our FISH assay reliably detects the t(11;14) translocation irrespective of the location of the breakpoints within the bcl-1 region., Conclusions: The described interphase FISH assay provides a reliable and routinely applicable tool for diagnosis of the t(11;14) translocation.

The translocation t(8;14)(q24;q32) is the characteristic chromosomal aberration of Burkitt's-type lymphomas and leukemias (BLs). On the molecular level, the t(8;14) juxtaposes the c-myc gene in 8q24 next to the IgH locus in 14q32, resulting in overexpression of the transcription factor c-Myc. The detection of a t(8;14) is a major aim in the diagnostic process of all patients with high-grade B-cell lymphomas because treatment strategies differ between BL and other high-grade lymphomas. As chromosome analyses are sometimes hampered by the low yield or poor quality of metaphase spreads and as the application of molecular genetic techniques is limited by the distribution of the 8q24 breakpoints over a region of about some hundred kilobases, we set out to establish an interphase fluorescence in situ hybridization (FISH) assay for the detection of the t(8;14). A cosmid probe hybridizing to the IgH constant region in 14q32 was combined with a differently labeled probe of pooled cosmid clones spanning the c-myc locus in 8q24. Interphase nuclei lacking a t(8;14) show two separated signals corresponding to each probe, whereas interphase nuclei carrying a t(8;14) display a split of the c-myc probe and a colocalization of at least one of the splitted signals with the IgH probe. Based on the results of extensive control studies, the cutoff level for this stringent (type I) criteria was set at 2%. Additionally, colocalization of at least one c-myc signal with one IgH signal alone (without signal split for the c-myc probe) was used as a less stringent (type II) criteria with a cutoff limit of 11%. Nine BLs and one Burkitt-like lymphoma were investigated by this approach. Cytogenetically, all tumors contained a translocation t(8;14)(q24;q32) except for one BL, in which cytogenetic analysis had failed. In interphase FISH, all lymphomas and leukemias met the less stringent criteria for the diagnosis of the t(8;14). Additionally, in all tumors but the Burkitt-like lymphoma, a t(8;14) could be diagnosed according to the stringent criteria. The percentage of cells found to harbor the t(8;14) by FISH ranged from 4.3% to 100%. Comparison of cytogenetic and FISH results revealed a significantly lower percentage of t(8;14)+ interphase nuclei than metaphase cells (P = .004). In conclusion, the described FISH assay provides a feasible and sensitive tool for the routine detection of the translocation t(8;14) in interphase cells which might also offer new insights into the biology of high-grade B-cell lymphomas.

Cytokine-induced expression of adhesion molecules on leukocytes and endothelial cells (EC) is a crucial point in the process of organ transplant rejection. It has been shown that protein kinase C (PKC) is involved in this activation process. Verapamil and other calcium channel blockers seem to possess immunosuppressive qualities in vivo and in vitro; some authors suggested that this is due to PKC- or calmodulin-antagonism. Thus our objectives were to further investigate the second-messenger systems involved in the stimulation of EC and to analyze whether the beneficial influence of calcium channel blockers on the outcome of transplantation is due to impaired expression of adhesion molecules on EC. Our results, obtained in an in vitro model using human umbilical vein EC, show that IL-1-induced expression of intercellular adhesion molecule-1 (ICAM-1) is in part mediated by PKC and that parallel activation of calmodulin is required. Expression of ICAM-1 was reduced to 38.5% by PKC-inhibitor H7 and to 77.2% by calmodulin-inhibitor W7. In addition, data on the intracellular events in TNF-alpha-induced expression of vascular cell adhesion molecule-1 (VCAM-1) is presented, showing that both PKC and, to a higher extent, calmodulin, are involved in this process. Expression of VCAM-1 was reduced to 63.7% by H7 and to 27.7% by W7. IL-1-induced expression of endothelial leukocyte adhesion molecule-1 (ELAM-1) is PKC-dependent but insensitive to blocking of calmodulin. Though activation of adhesion molecule expression utilizes PKC and/or calmodulin as second-messenger pathways the investigated calcium channel blockers verapamil (R- and S-enantiomers), diltiazem and Ro 40-5967 failed to inhibit adhesion molecule expression. Surprisingly, higher concentrations of verapamil (> 12.5 micrograms/ml) or Ro 40-5967 (5 micrograms/ml) significantly enhanced IL-1-induced expression of ELAM-1. ICAM-1-expression was also enhanced by verapamil, but not by Ro 40-5967 or diltiazem. This enhancement was only seen if verapamil was added maximally one hour after the cytokine stimulus indicating that transcriptional modulation is responsible for the observed effects. Our findings indicate that calcium channel blockers have an immunomodulating effect independent of adhesion molecule expression.

The effect of N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, on the energy metabolism in isolated hepatocytes was investigated. Incubation of cells with NAPQI (400 microM) resulted in an immediate uptake into the mitochondria, followed by both reduction and glutathione conjugation of the quinone imine. These reactions were extremely rapid and were associated with depletion of the mitochondrial ATP content (greater than 80% depletion after 1 min exposure). The loss of ATP was accompanied by increases in ADP and AMP, as well as NADP. No effect on mitochondrial NAD was observed during this initial phase. Similar alterations were produced by NAPQI in the cytosolic compartment. Furthermore, incubation of hepatocytes with NAPQI inhibited oxygen consumption by nearly 90% within 10 s. In parallel to these biochemical changes, there was marked bleb formation on the surface of the hepatocytes, which was found to precede cell death (trypan blue uptake). In conclusion, our results demonstrate that during exposure of hepatocytes to NAPQI, dramatic changes in cellular energy metabolism occur. These biochemical alterations may be caused by a rapid decrease in mitochondrial function, and they may play an important role in the initiation of NAPQI-induced cytotoxicity.

In this paper we describe an anaerobic titrator made virtually from glass with a small amount of high vacuum epoxy mounted directly to a quartz EPR tube. A complete titration may be carried out with as little as 600 microliters of sample. This cell features the anaerobic manipulation of an electrochemically poised solution from an electrochemical pouch to an EPR tube. The cell uses a gold foil working electrode and Ag/AgCl reference and counter electrodes. The reference and counter electrodes are isolated from the sample by leached Vycor glass. In the work reported here, we used this cell to determine the equilibrium redox potential of methyl viologen in an EPR titration. With methyl viologen as an indicator we found that the cell has a residual oxygen level of 1.5 microM with a leak rate of 0.005 nmol/min. After moving the solution into the EPR tube, freezing, performing EPR, and thawing, the potential of the methyl viologen solution drifted only 2 mV. During the titration, the poised potentials were stable, drifting only 1 mV/min. Formal potentials as low as -630 mV in a vitamin B12-type protein have been determined with this cell (S. R. Harder, W.-P. Lu, B. A. Feinberg, and S. W. Ragsdale (1989) Biochemistry, in press).