A Mechano-Activated Cell Reporter System as a Proxy for Flow-Dependent Endothelial Atheroprotection.

The vascular endothelium plays a critical role in the health and disease of the cardiovascular system. Importantly, biomechanical stimuli generated by blood flow and sensed by the endothelium constitute important local inputs that are translated into transcriptional programs and functional endothelial phenotypes. Pulsatile, laminar flow, characteristic of regions in the vasculature that are resistant to atherosclerosis, evokes an atheroprotective endothelial phenotype. This atheroprotective phenotype is integrated by the transcription factor Kruppel-like factor-2 (KLF2), and therefore the expression of KLF2 can be used as a proxy for endothelial atheroprotection. Here, we report the generation and characterization of a cellular KLF2 reporter system, based on green fluorescence protein (GFP) expression driven by the human KLF2 promoter. This reporter is induced selectively by an atheroprotective shear stress waveform in human endothelial cells, is regulated by endogenous signaling events, and is activated by the pharmacological inducer of KLF2, simvastatin, in a dose-dependent manner. This reporter system can now be used to probe KLF2 signaling and for the discovery of a novel chemical-biological space capable of acting as the "pharmacomimetics of atheroprotective flow" on the vascular endothelium.

Hemodynamic Control of Endothelial Cell Fates in Development.

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. From the onset of blood flow, the embryonic vasculature is continuously exposed to a variety of hemodynamic forces. These biomechanical stimuli are key determinants of vascular cell specification and remodeling and the establishment of vascular homeostasis. In recent years, major advances have been made in our understanding of mechano-activated signaling networks that control both spatiotemporal and structural aspects of vascular development. It has become apparent that a major site for mechanotransduction is situated at the interface of blood and the vessel wall and that this process is controlled by the vascular endothelium. In this review, we discuss the hemodynamic control of endothelial cell fates, focusing on arterial-venous specification, lymphatic development, and the endothelial-to-hematopoietic transition, and present some recent insights into the mechano-activated pathways driving these cell fate decisions in the developing embryo.

Frailty and Transplantation.

Consequences of aging are gaining clinical relevance. In transplantation, aging and immunosenescence impact treatment and outcomes. The impact of aging, however, will critically depend on distinguishing healthy, chronological aging from biological aging that may result into frailty. Approximately 15% of individuals older than 65 years are frail, and it is expected that this condition will gain more clinical relevance with an expected increase to greater than 20% over the next 5 years. Clearly, frailty impacts various general aspects of health care and organ transplantation in particular including patient selection, waitlist management and treatment after transplantation. In general, frailty has been characterized by a compromised physiological reserve and an augmented vulnerability. In comparison to healthy aging, inflammatory markers and cytokines are increased in frail older adults. Thus, modifications of the immune response, in addition to physical limitations and changes of metabolism, are likely to impact outcomes after transplantation. Here, we provide a risk assessment of frailty at the time of transplant evaluation and review effects on outcomes and recovery after transplantation. Moreover, we summarize our current understanding of the pathophysiology of frailty and consequences on immune responses and metabolism.

Immunosenescence in renal transplantation: a changing balance of innate and adaptive immunity.

PURPOSE OF REVIEW: With global demographic changes and an overall improved healthcare, more older end-stage renal disease (ESRD) patients receive kidney transplants. At the same time, organs from older donors are utilized more frequently. Those developments have and will continue to impact allocation, immunosuppression and efforts improving organ quality. RECENT FINDINGS: Findings mainly outside the field of transplantation have provided insights into mechanisms that drive immunosenescence and immunogenicity, thus providing a rationale for an age-adapted immunosuppression and relevant clinical trials in the elderly. With fewer rejections in the elderly, alloimmune responses appear to be characterized by a decline in effectiveness and an augmented unspecific immune response. SUMMARY: Immunosenescence displays broad and ambivalent effects in elderly transplant recipients. Those changes appear to compensate a decline in allospecific effectiveness by a shift towards an augmented unspecific immune response. Immunosuppression needs to target those age-specific changes to optimize outcomes in elderly transplant recipients.

Ischemia/reperfusion Injury and its Consequences on Immunity and Inflammation.

Ischemia/reperfusion injury (IRI), an inherent component of transplantation, affects organ quality and transplant outcomes. Although the complexity of the pathophysiology is recognized, detailed mechanisms remain unclear, and strategies preventing the consequences of IRI have been challenging. Of critical significance appears the link between IRI, the initiation of innate immune responses, and the (potential) augmentation of adaptive immunity. An improved understanding of those complex mechanisms and interactions may pave the way for more effective treatment strategies.

Mechanisms and consequences of injury and repair in older organ transplants.

Donor organ scarcity remains a significant clinical challenge in transplantation. Older organs, increasingly utilized to meet the growing demand for donor organs, have been linked to inferior transplant outcomes. Susceptibility to organ injury, reduced repair capacity, and increased immunogenicity are interrelated and impacted by physiological and pathological aging processes. Insights into the underlying mechanisms are needed to develop age-specific interventional strategies with regards to organ preservation, immunosuppression, and allocation. In this overview, we summarize current knowledge of injury and repair mechanisms and the effects of aging relevant to transplantation.

Effect of primary versus revisional Roux-en-Y gastric bypass: inferior weight loss of revisional surgery after gastric banding.

BACKGROUND: Laparoscopic adjustable gastric banding is a popular and effective restrictive bariatric procedure. However, with longer follow-up, it has become clear that a considerable number of patients require revisional surgery, of which Roux-en-Y gastric bypass (RYGB) is the most commonly performed procedure. Studies that compared the outcomes of primary RYGB and revisional RYGB have not been conclusive. Our objective was to determine whether significant differences exist in the 1-year outcomes between primary RYGB (prim-RYGB) and revisional RYGB after laparoscopic adjustable gastric banding (rev-RYGB) at a major training hospital in The Netherlands. METHODS: All prim-RYGB and rev-RYGB procedures performed from 2007 to 2009 were analyzed. Data were collected regarding weight loss, hospitalization, operative time, postoperative complications, and co-morbidities. RESULTS: A total of 292 RYGB procedures were performed: 66 rev-RYGB and 226 prim-RYGB procedures. The operative time was significantly shorter in the prim-RYGB group (136.6±37.5 versus 167.5±40.6 min; P<.0001). No significant differences were found in hospitalization time (4.4±1.7 versus 4.9±2.4 d; P= .063) or complication rate (14.7% versus 15.2%; P=.962). No deaths occurred in either group. The number of patients with resolved diabetes and hypertension did not differ between the 2 groups (50.1% versus 23.1%; P=.116; and 40.7% versus 25.0%; P=.384, respectively). Weight loss was significantly greater in the prim-RYGB group in terms of excess weight loss (71.6%±20.8% versus 48.4%±26.8%; P<.0001), body mass index reduction (13.0±3.8 versus 10.2±5.6 kg/m(2); P<.0001), absolute weight loss (37.4±11.5 versus 29.3±17.2 kg; P=.001), and percentage of weight loss (29.7%±8% versus 21.7%±11.5%; P<.0001). CONCLUSION: rev-RYGB is a safe procedure with outcomes similar to those of prim-RYGB in terms of complication rate, hospitalization time, and effect on co-morbidity. Weight loss, however, was significantly less after rev-RYGB than after prim-RYGB.

Spectrum of ATM gene mutations in a hospital-based series of unselected breast cancer patients.

Blood relatives of patients with the inherited disease ataxia telangiectasia (A-T) have an increased susceptibility for breast cancer. We therefore looked for sequence alterations of the ATM gene in a large hospital-based series of unselected breast cancer patients. The whole ATM coding sequence was analyzed in genomic DNA samples from a core group of 192 consecutive breast cancer cases to define the spectrum of ATM gene mutations. Common sequence alterations were then screened in the whole series of 1000 breast cancer patients and in 500 random individuals. In the core group, 21 distinct sequence alterations were identified throughout the ATM coding region, and 1 common splicing mutation was uncovered in intron 10. Almost half of the breast cancer patients (46%) were heterozygotes for 1 of 16 different amino acid substitutions, and three patients (1.6%) carried a truncating mutation. These data indicate that approximately 1 in 50 German breast cancer patients is heterozygous for an A-T-causing mutation. In our extended series, the most common A-T mutation 1066-6T-->G was disclosed in 7 of 1000 (0.7%) breast cancer patients. Transcript analyses indicated that the loss of exon 11 in the ATM mRNA was the pathogenic consequence of this splicing mutation, which produced a <10% of full-length ATM mRNA and ATM protein in a homozygous A-T patient. We also found an excess of rare missense substitutions in the breast cancer cohort compared with random individuals (7.9% versus 5.3% of alleles; odds ratio = 1.6; P < 0.01). One missense substitution, S707P in exon 15, was two times more frequent in breast cancer patients (odds ratio = 2.4; 95% confidence interval, 1.0-5.8) and five times more frequent in patients with bilateral disease than in random individuals (P < 0.001). We conclude that a large variety of distinct ATM mutations and variants exist among breast cancer patients, some of which can contribute to the etiology and progression of the malignancy. Screening for frequent A-T mutations such as the 1066-6-->G splice site substitution can be effective to prospectively identify A-T heterozygotes in an unselected cancer patient population.

Mutagen sensitivity of human lymphoblastoid cells with a BRCA1 mutation in comparison to ataxia telangiectasia heterozygote cells.

Our previous results indicated a close relationship between the presence of a BRCA1 mutation in lymphocytes and hypersensitivity for the induction of micronuclei by gamma irradiation and hydrogen peroxide (H(2)O(2)). Comparative investigations with the comet assay (single-cell gel electrophoresis) suggested a normal rate of damage removal and pointed to a disturbed fidelity of DNA repair as a direct or indirect consequence of a BRCA1 mutation. We now wanted to see whether similar results could be obtained with lymphoblastoid cell lines (LCLs) and whether such permanent cells are suitable as a model for the investigation of mechanisms involved in mutagen sensitivity. Our results show that LCLs with a BRCA1 mutation are also hypersensitive to the chromosome-damaging effects of gamma irradiation or H(2)O(2), as revealed by the micronucleus test. Interestingly, LCLs heterozygous for an ataxia telangiectasia (AT) mutation have similar characteristics as BRCA1 cells with respect to the induction and repair of DNA damage induced by either gamma irradiation or H(2)O(2). However, caffeine enhanced the induction of micronuclei by gamma irradiation only in normal and heterozygous AT cells but not in BRCA1 cells, thus indicating a difference in the pathways leading to mutagen sensitivity in cells with a BRCA1 or an AT mutation. Our results suggest that caffeine could be useful in discriminating AT heterozygotes from carriers of a BRCA1 mutation, as well as BRCA1 mutation carriers from normal individuals.

Characterization of ATM gene mutations in 66 ataxia telangiectasia families.

Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. The gene mutated in AT, designated the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. In this study, we investigated the mutational spectrum of the ATM gene in a cohort of AT patients living in Germany. We amplified and sequenced all 66 exons and the flanking untranslated regions from genomic DNA of 66 unrelated AT patients. We identified 46 different ATM mutations and 26 sequence polymorphisms and variants scattered throughout the gene. A total of 34 mutations have not been described in other populations. Seven mutations occurred in more than one family, but none of these accounted for more than five alleles in our patient group. The majority of the mutations were truncating, confirming that the absence of full-length ATM protein is the most common molecular basis of AT. Transcript analyses demonstrated single exon skipping as the consequence of most splice site substitutions, but a more complex pattern was observed for two mutations. Immunoblot studies of cell lines carrying ATM missense substitutions or in-frame deletions detected residual ATM protein in four cases. One of these mutations, a valine deletion proximal to the kinase domain, resulted in ATM protein levels >20% of normal in an AT lymphoblastoid cell line. In summary, our results survey and characterize a plethora of variations in the ATM gene identified by exon scanning sequencing and indicate a high diversity of mutations giving rise to AT in a non-isolated population.

Operations on the colon without preoperative oral antibiotic therapy.

The ideal preparation of the intestine prior to elective colonic resection has been a controversial subject. With the development of new and more effective antibiotics, many modalities of intestinal preparation preoperatively have been used in an effort to reduce the number of infectious complications. Mechanical cleansing of the intestine, when accompanied by perioperative parenteral antibiotics, is adequate preparation prior to resection of the intestine. One hundred consecutive operations upon the colon by one surgeon were the subject of this retrospective study. Approximately 70 per cent of the operations were for carcinoma and the remainder for inflammatory disease. Preparation consisted of the administration of cathartics and saline solution enemas combined with a liquid diet for 48 hours prior to operation. Perioperative parenteral antibiotic therapy was given for 48 to 72 hours or as long as intravenous fluids were administered. All anastomoses were the open, two layer type, using nonabsorbable suture material. All abdominal incisions were closed primarily. There were no operative deaths, anastomotic leaks, intra-abdominal abscesses or fistulas. There were six instances of wound infection. Our results with this type of management compare favorably with those of other surgeons using various modes of preoperative oral antibiotic therapy and suggest that oral preoperative antibiotic therapy may not be necessary in elective operations on the colon.