European multicentre experience of staged hybrid atrial fibrillation ablation for the treatment of persistent and longstanding persistent atrial fibrillation.

The management of non-paroxysmal atrial fibrillation (AF) remains controversial. We examined the efficacy and safety of the 2 stage Hybrid AF ablation approach by analysing the largest series of this technique reported so far. METHODS: The approach aims to electrically isolate the left atrial posterior wall incorporating the pulmonary veins ('box-set'pattern). An initial video-assisted thoracoscopic (VATS) epicardial ablation is followed after a minimum of 8 weeks by endocardial radiofrequency catheter ablation. RESULTS: Of 175 patients from 4 European cardiothoracic centers, who underwent the surgical (COBRA Fusion, AtriCure Inc) 1st stage ablation, 166 went on to complete 2nd stage catheter ablation. At median follow up of 18 months post 2nd stage procedure 93/166 (56%) had remained free of AF or atrial tachycardia (AT) recurrence off antiarrhythmic drugs. 110/175 62.9% were in sinus rhythm off all antiarrhythmic drugs at last clinic follow-up (132/175 75.4% including those on antiarrhythmic drugs). 18 patients (10.8%) underwent a further re-do ablation (mean of 1.1 ablations per patient) 105/166 (63%) remained free of AF/AT recurrence off antiarrhythmic drugs following last ablation procedure.Latterly, ILRs have been implanted in patients (n = 56); 60% have remained fully arrhythmia free and 80% have shown AF burden < 5% at a median 14 months follow-up [IQR: 13.5 (8-21.5)]. Only 10.9% have reverted to persistent AF. 5 patients (2.9%) had a perioperative stroke and 4 patients (2.3%) exhibited persistent weakness of the right hemidiaphragm following stage 1 VATS epicardial ablation. One patient died following stroke (overall mortality 0.6%). CONCLUSIONS: In patients with non-paroxysmal AF with unfavourable characteristics for catheter ablation, the staged hybrid approach results in acceptable levels of freedom from recurrent atrial arrhythmia, however, complication rates are higher than with catheter ablation alone.

Older recipient age is paradoxically associated with a lower incidence of chronic GVHD in thymoglobulin recipients: a retrospective study exploring risk factors for GVHD in allogeneic transplantation with thymoglobulin GVHD prophylaxis.

Thymoglobulin (TG) given with conditioning for allogeneic haematopoietic SCT (alloHSCT) is effective in reducing the risk of acute and chronic GVHD (cGVHD). Whether conventional risk factors for GVHD apply to TG-conditioned alloHSCT is unknown. We retrospectively studied 356 adults from three centres who received TG 4.5 mg/kg prior to alloHSCT for haematologic malignancy. Donors were unrelated in 64%. At 3 years, OS was 61% (95% confidence interval (CI) 55-67%), cumulative incidence of relapse was 28% (95% CI 23-33%) and non-relapse mortality was 19% (14-24%). The cumulative incidences of grade 2-4, and grade 3-4 acute GVHD were 23% (95% CI 19-28%) and 10% (95% CI 6-13%), respectively. The cumulative incidence of cGVHD requiring systemic immunosuppression (cGVHD-IS) at 3 years was 32% (95% CI 27-37%). On multivariate analysis, counterintuitively, recipient age over 40 was associated with a significantly decreased risk of cGVHD-IS (P=0.001). We report for the first time a paradoxical association of older age with reduced cGVHD in TG recipients, and conclude that traditional risk factors for GVHD may behave differently in the context of pre-transplant TG.

Quantitative classification of tumor cell morphological changes on selectively functionalized biochips.

Proteomics information of cancer has shown that abnormalities at the levels of growth factors, receptors, intracellular mediators and transcription factors play major role in the disease progression. We report a directly quantifiable approach to measure tumor cell behavior on functionalized chips. The chips were functionalized with aptamer molecules that were selective against epidermal growth factor receptor (EGFR), a commonly overexpressed cancer biomarker. The chip-bound aptamer selectively isolated tumor cells from cell mixture samples. The isolated cells were thus bound to the chip surface. However, some normal cells also got captured on the surface. The selectivity and sensitivity of tumor isolation changed when the surface of the chip was chemically treated to create nanoscale texture. The captured cancer cells showed distinctly different behavior on the surface of the chip than that for the normal cells. The behavior quantification can serve as a novel modality to detect cancer cells from simple samples like blood, saliva or urine.

IL15 levels on day 7 after hematopoietic cell transplantation predict chronic GVHD.

Chronic GVHD (cGVHD) is an important complication of allogeneic hematopoietic cell transplantation (HCT). As preemptive therapy might be efficacious if administered early post transplant, we set out to determine whether cGVHD can be predicted from the serum level of a biomarker on day 7 or 28. In a discovery cohort of 153 HCT recipients conditioned with BU, fludarabine and rabbit antithymocyte globulin (ATG), we determined serum levels of B-cell-activating factor, vascular endothelial growth factor, soluble TNF-α receptor 1, soluble IL2 receptor α, IL5, IL6, IL7, IL15, γ-glutamyl transpeptidase, cholinesterase, total protein, urea and ATG. Patients with low levels of IL15 (<30.6 ng/L) on day 7 had 2.7-fold higher likelihood of developing significant cGVHD (needing systemic immunosuppressive therapy) than patients with higher IL15 levels (P<0.001). This was validated in a validation cohort of 105 similarly-treated patients; those with low IL15 levels had 3.7-fold higher likelihood of developing significant cGVHD (P=0.001). Low IL15 was not associated with relapse; it trended to be associated with acute GVHD and was associated with low infection rates. In conclusion, low IL15 levels on day 7 are predictive of cGVHD, and thus could be useful in guiding preemptive therapy.

Differential regulation of microRNA expression in irradiated and bystander cells.

The ionizing radiation (IR) induces a variety of biological effects in irradiated cells. Additionally, the irradiated cells communicate with unirradiated cells and induce changes in them through a phenomenon termed as the bystander effect. The nature of the bystander effect signal and how it impacts unirradiated cells remains to be discovered. Examination of molecular changes in bystander cells due to signals from irradiated cells could lead to the identification of the pathways underlying the bystander effect. To gain insight into the molecular pathways affected by the transmission of signal from irradiated cells to bystander cells, we monitored the microRNA (miRNA) transcriptional changes. miRNAs control gene expression at the posttranscriptional level. In previous studies from our laboratory the modulation of miRNA in irradiated human cells were identified. In the present work human lymphoblasts TK6 cells in a medium exchanged bystander effect model system were used to analyze miRNA expression alterations by employing the real time RT-PCR technology. The relative expression of several miRNAs involved in RAS, c-MYC and BCL2 gene regulation were examined. The let-7 family of miRNAs was upregulated in irradiated cells but most of these miRNAs remained repressed in bystander cells. The miR-17-3p, miR-19b, and miR-18a were upregulated in irradiated cells but were repressed in the bystander cells. The miR-17-5p, miR-142-3p, miR-142-5p, and miR-19a were induced only for a short time in bystander cells. The miR-15a, miR-16, miR-143, miR-145, miR-155, and miR21 were upregulated in irradiated TK6 cells. While the expression of miR-15a, miR-16, miR-155, and miR-21 was repressed, the miR-143 and miR-145 expression was induced in bystander cells. These results indicate the involvement of miRNA modulation in irradiated and bystander cells.

Influence of comorbidities on transplant outcomes in patients aged 50 years or more after myeloablative conditioning incorporating fludarabine, BU and ATG.

Non-myeloablative (MA) and reduced intensity allo-SCT regimens are offered to older patients and/or those with comorbidities because the morbidity and mortality attributable to fully MA conditioning is thought to be unacceptably high. A total of 207 patients aged 50-66 years were treated between 1999 and 2008 with SCT after MA conditioning with fludarabine 50 mg/m(2) daily × 5 and i.v. BU 3.2 mg/kg daily × 4.90 (43%) had additional TBI 200 cGy × 2. GVHD prophylaxis was CsA, MTX and thymoglobulin (4.5 mg/kg total dose). As defined by the hematopoietic cell transplantation co-morbidity index (HCT-CI) scoring system 117 (57%) pts scored 0 and 90 (43%) 1. At 5 years OS was 39 vs 54% (P=0.008), disease-free survival 38 vs 49% (P=0.03), TRM 39 vs 19% (P=0.003) and relapse 36 vs 39% (P=ns) in those with scores of 0 and 1, respectively. Multivariate analysis confirmed the influence of HCT-CI scores on TRM (subhazard ratios=2.29; 95% confidence interval=1.29-4.08; P=0.005). We conclude that comorbidities as assessed by the HCT-CI do influence TRM with this regimen but that age alone should not be an indication to prefer a less intense protocol.

Expression of the oxidative base excision repair enzymes is not induced in TK6 human lymphoblastoid cells after low doses of ionizing radiation.

Most of the DNA damage produced by ionizing radiation is repaired by the base excision repair (BER) pathway. To determine whether the BER genes were up-regulated by low doses of ionizing radiation, we investigated their expression in TK6 human lymphoblastoid cells by measuring mRNA levels using real-time quantitative PCR. No induction at the transcriptional level of any of the base excision repair genes, NTH1 (NTHL1), OGG1, NEIL1, NEIL2, NEIL3, APE1, POLB, or accessory protein genes, LIG3, XRCC1 or XPG, was found at gamma-radiation doses ranging from 1 cGy to 2 Gy in a 24-h period. As has been measured in other cell lines, a dose-dependent induction of CDKN1A (WAF1) mRNA levels was observed in TK6 cells in the dose range of 0.5 to 2.0 Gy. We also examined BER enzyme activity on 8-oxoguanine-, dihydrouracil- and furan-containing oligonucleotide substrates and found no increase in extracts of TK6 cells after gamma-ray doses of 0.5-2.0 Gy. These data were corroborated by Western blot analysis of APE1 and NTH1, suggesting that the BER enzymes are also not up-regulated at the post-transcriptional level after ionizing radiation exposure.

Head injuries: a prospective observational study evaluating the potential impact of the Galasko report on Accident and Emergency departments.

In June 1999, the Galasko report of the Royal College of Surgeons of England recommended that in the next 5 years, the Accident and Emergency (A&E) departments should admit and supervise head-injured patients for up to 48 h. A prospective observational study was carried out for a 6 weeks period at the A&E department of Birmingham Heartland's Hospital to identify the potential impact of implementation of the Galasko report. The cost implications of this perceived additional workload were considered.Of the 786 head-injured patients seen during study period, 665 (85%) were discharged home directly from the A&E department. Of the remaining 121 patients, who were hospitalized, 76 (63%) were admitted to the A&E observation ward (AEOW) and 19 (16%) patients were admitted to a paediatric ward. All of these patients were discharged home within 24 h. The remaining 21% patients were admitted to other specialities and had prolonged stays in hospital. All of the 9% of the patients admitted under orthopaedics would have been admitted to the AEOW if the Galasko recommendations were implemented leading to an extra 22 bed days over the 6 weeks study period. The estimated annual cost of admission alone for these patients would be pound 38,200. Our study has demonstrated an expected additional workload and cost implications on a single A&E department.

Unusual cardiac injury following blunt chest trauma.

Cardiac injury following blunt chest trauma is known to occur, but traumatic rupture of ventricular septum is a rare injury, especially following blunt chest trauma. A case of a 20-year-old male is presented who fell on his back from a 9th-floor window and was resuscitated for 3 hours to no avail. Post-mortem examination confirmed a fracture of the pelvis, pulmonary contusion and rupture of ventricular septum of the heart.

Response of base excision repair enzymes to complex DNA lesions.

There is now increasing evidence that ionizing radiation generates complex DNA damage, i.e. two or more lesions--single-strand breaks or modified nucleosides--located within one to two helical turns on the same strand or on opposite strands. Double-strand breaks are the most readily recognizable clustered lesions, but they may constitute a relatively minor fraction of the total. It is anticipated that clustered lesions may play a significant role in cellular response to ionizing radiation since they may present a major challenge to the DNA repair machinery. The degree of lesion complexity increases with increasing LET. This has potential implications for space travel because of exposure to high-LET cosmic radiation. It is therefore critical that we begin to understand the consequences of such damaged sites, including their influence on DNA repair enzymes. This paper presents a short review of our current knowledge of the action of purified DNA repair enzymes belonging to the base excision repair pathway, including DNA glycosylases and apurinic/apyrimidinic endonucleases, on model complex lesions.

Paradoxical effects of trichostatin A: inhibition of NF-Y-associated histone acetyltransferase activity, phosphorylation of hGCN5 and downregulation of cyclin A and B1 mRNA.

Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), is widely used to study the role of histone acetylation in gene expression, since genes that use histone acetylation as a means of regulating expression may be up regulated when TSA is added. In this study, however, we show that TSA has an unexpected paradoxical effect leading to inhibition of NF-Y-associated histone acetyl transferase (HAT) activity and phosphorylation of the HAT, hGCN5. TSA treatment of cells resulted in diminished levels of NF-Y-associated HAT activity without changes in NF-Y(A) amount. hGCN5 is one of the HATs known to associate with NF-Y. The association of hGCN5 with NF-Y was not altered by TSA treatment. The enzymatic activity of hGCN5 is known to be inhibited by phosphorylation. TSA treatment of Hela cells resulted in phosphorylation of hGCN5. Exposure of the NF-Y immunoprecipitates from TSA-treated cells to a phosphatase resulted in enhanced HAT activity. We have also shown that the mRNA levels of several genes, cyclin B1 and cyclin A, are downregulated by TSA; these effects do not require protein synthesis and the downregulation of cyclin B1 by TSA occurs through transcription. These results suggest that TSA can have contradictory effects, on one hand stimulating HAT activity in general by inhibition of HDACs, but also resulting in inhibition of NF-Y-associated HAT activity and phosphorylation of hGCN5.

The effects of heparin and extracorporeal circulation on platelet counts and platelet microaggregation during cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass is associated with platelet activation and reduced platelet counts. Platelet activation may artifactually lower platelet counts by causing aggregation. In vivo platelet activation may increase existent platelet microaggregation ex vivo. We studied platelet counts and existent platelet microaggregation at different stages of cardiopulmonary bypass. METHODS: Twenty-one patients were studied before and after heparinization (300 U. kg(-1)) and at the end of cardiopulmonary bypass. Unaggregated (or single) platelets were counted in hirudin-anticoagulated blood, and total platelets were counted in ethylenediaminetetraacetic acid-anticoagulated blood. RESULTS: The total platelet count, 198 +/- 61 x 10(9). L(-1), was unaffected by heparin and stayed at 197 +/- 60 x 10(9). L(-1) (P =.7) but fell during extracorporeal circulation; the hemodilution-corrected count was 163 +/- 52 x 10(9). L(-1) (P =.0004). Heparinization reduced the unaggregated platelet count from (mean +/- 1 SD) 178 +/- 62 x 10(9). L(-1) to 155 +/- 60 x 10(9). L(-1) (P =.0001). Extracorporeal circulation had little additional effect. The hemodilution-corrected count was 142 +/- 48 x 10(9). L(-1) (P =.6). CONCLUSIONS: Heparinization caused platelet activation and increased existent platelet microaggregation ex vivo. During extracorporeal circulation, there was a reduction in total platelets that was greater than could be explained by hemodilution alone, but the unaggregated platelet count did not change significantly when corrected for hemodilution. Furthermore, the increased platelet microaggregation observed after heparinization was no longer evident after this loss. These findings suggest that during extracorporeal circulation, the platelets that formed into microaggregates after heparinization were lost from the circulation in preference to single platelets.

Quantitative detection of platelet aggregates in whole blood without fixation.

Platelet counting detects lesser degrees of platelet aggregation than conventional aggregometry. In order to prevent progressive platelet aggregation or disaggregation after sampling it is customary to fix blood samples. However fixation may introduce other artefacts. We first compared stability of platelet counts in EDTA-, citrate- and r-hirudin-anticoagulated blood from healthy volunteers. Second, the stability of platelet counts in unfixed EDTA- and hirudin-anticoagulated blood was compared with glutaraldehyde-fixed blood in the same anticoagulants. Third, the effect of in vivo heparin administration on platelet counts in EDTA- and hirudin-anticoagulated blood was studied. Platelet counts within 2 h of collection were significantly higher in EDTA- than in hirudin- or citrate-anticoagulated blood (P = 0.002 vs. hirudin and P = 0.001 vs. citrate). Twenty-four hour counts in hirudin and EDTA were unchanged (P = 0.3 and P = 0.2, respectively, vs. earlier counts). Counts in citrate increased significantly (P = 0.007; n = 10). Platelet counts in fixed blood did not differ significantly from those in unfixed blood. Heparin administered for cardiopulmonary bypass reduced platelet counts in hirudin-anticoagulated blood from (mean +/- 1 standard deviation) 180 +/- 45 to 162 +/- 30 x 10(9) l-1 (P = 0.01; n = 14), without significantly lowering counts with EDTA-anticoagulation, consistent with increased platelet aggregation. Hirudin and EDTA provided stable platelet counts, suggesting that fixation is unnecessary.

Porcine versus pericardial bioprostheses: eleven-year follow up of a prospective randomized trial.

BACKGROUND AND AIM OF THE STUDY: There is renewed interest in the pericardial heart valve as an alternative to the porcine bioprosthesis. The long-term results of a randomized trial comparing a second-generation pericardial valve against a well-tested porcine bioprosthesis are presented. Seven-year follow up has been reported previously. Production of the Bioflo pericardial prosthesis used in this trial was discontinued due to fears related to bovine spongiform encephalopathy. METHODS: Between February 1987 and March 1990, 170 patients undergoing aortic (AVR) or mitral (MVR) valve replacement were assigned randomly to receive either the Bioflo pericardial bioprosthesis or the Carpentier-Edwards (CE) supra-annular porcine bioprosthesis. Eighty-five patients received 93 Bioflo valves (46 AVR, 31 MVR, eight AVR+MVR), and the remaining 85 received 99 CE valves (48 AVR, 23 MVR, 14 AVR+MVR). Mean patient age was 61.0 years (range: 38-77 years) for the Bioflo group and 62.1 years (range: 41-77 years) for the CE group. Current follow up is 100% complete and totals 1,391 patient-years; mean +/- SD follow up was 8.2 +/- 3.4 years (maximum 12.2 years). RESULTS: The operative mortality rate was 4.12%. There were 70 patients still at risk at 11 years (31 Bioflo, 39 CE); of these, 91.4% were in NYHA classes I/II. No significant difference in survival or valve-related complications was seen between the groups. Mean (+/- SEM) survival at 11 years was 41.4 +/- 6.8% in the Bioflo group and 55.3 +/- 6.8% in the CE group (p = 0.15). There were 16 valve-related deaths (nine in the Bioflo group, seven in the CE group). At 11 years, freedom from valve-related mortality was 89.5 +/- 3.9% for the Bioflo group and 91.0 +/- 3.5% for the CE group (p = 0.4). Valve position had no impact on survival. At 11 years, freedom from structural valve deterioration was 83.9 +/- 5.4% and 87.5 +/- 4.2% in the Bioflo and CE groups, respectively (p = 0.9). CONCLUSION: Over the 11-year period of follow up, clinical performance of the Bioflo pericardial valve was comparable with that of the Carpentier-Edwards supra-annular porcine bioprosthesis. No difference was apparent between the two valve types when implanted in either the aortic or the mitral position.

Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease.

The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure.

Relative affinities of poly(ADP-ribose) polymerase and DNA-dependent protein kinase for DNA strand interruptions.

Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are important nuclear enzymes that cooperate to minimize genomic damage caused by DNA strand interruptions. DNA strand interruptions trigger the ADP-ribosylation activity and phosphorylation activity of PARP and DNA-PK respectively. In order to understand the relationship of PARP and DNA-PK with respect to DNA binding required for their activation, we analyzed the kinetics of the reactions and determined the apparent dissociation constants (Kd app) of the enzymes for DNA strand interruptions. PARP has a high binding affinity for blunt ends of DNA (Kd app=116 pM) and 3' single-base overhangs (Kd app=332 pM) in comparison to long overhangs (Kd app=2.6-5.0 nM). Nicks are good activators of PARP although the affinity of PARP for nicks (Kd app=467 pM) is 4-fold less than that for blunt ends. The Kd app of DNA-PK for 3' single-base overhangs, blunt ends and long overhangs is 704 pM, 1.3 nM and 1.4-2.2 nM respectively. These results demonstrate that (1) PARP, when compared to DNA-PK, has a greater preference for blunt ends and 3' single-base overhangs but a weaker preference for long overhangs, and (2) nicks are effective in attracting and activating PARP. The possible implications of the preferences of PARP and DNA-PK for DNA strand interruptions in vivo are discussed.

Removal by human apurinic/apyrimidinic endonuclease 1 (Ape 1) and Escherichia coli exonuclease III of 3'-phosphoglycolates from DNA treated with neocarzinostatin, calicheamicin, and gamma-radiation.

DNA strand breaks with terminal 3'-phosphoglycolate groups are produced by agents that can abstract the hydrogen atom from the 4'-carbon of DNA deoxyribose groups. Included among these agents are gamma-radiation (via the OH radical) and enediyne compounds, such as neocarzinostatin and calicheamicin. However, while the majority of radiation-induced phosphoglycolates are found at single-strand breaks, most of the phosphoglycolates generated by these two enediynes are found at bistranded lesions, including double-strand breaks. Using a 32P-post-labelling assay, we have compared the enzyme-catalyzed removal of phosphoglycolates induced by each of these agents. Both human apurinic/apyrimidinic endonuclease 1 (Ape 1) and its Escherichia coli homolog exonuclease III rapidly removed over 80% of phosphoglycolates from gamma-irradiated DNA, although there appeared to be a small resistant subpopulation. The neocarzinostatin-induced phosphoglycolates were removed more slowly, though not to completion, while the calicheamicin-induced phosphoglycolates were extremely refractory to both enzymes. These data suggest that unless other enzymes are capable of acting upon the phosphoglycolate termini at enediyne-induced double-strand breaks, such termini will be resistant to end rejoining repair pathways.