Design of an artificial phage-display library based on a new scaffold improved for average stability of the randomized proteins.

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the µM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site.

Daclatasvir and Sofosbuvir With or Without Ribavirin in Liver Transplant Recipients: A Single-Center Real-World Study.

BACKGROUND: Treatment with direct-acting antiviral drugs in interferon-free regimens is currently recommended for viral hepatitis C recurrence after liver transplantation. There are limited data regarding its results in this population, and no optimal treatment scheme has yet been singled out. METHODS: We report our real-world results in liver transplant (LT) recipients. All patients were hepatitis C virus (HCV) monoinfected and completed a 12-week treatment course, followed 12 weeks later by HCV polymerase chain reaction testing with 12 IU/mL sensibility. Liver fibrosis was graded with the use of biopsies taken <12 months before treatment and stratified as early (0-1) or moderate to advanced (2-4) according to the Metavir score. RESULTS: Median postoperative time was 5.2 years. Genotype 3 was found in 66.7% of the sample. The following regimens were prescribed: daclatasvir-sofosbuvir with (n = 11) or without (n = 28) ribavirin. Genotypes 1 and 3 were evenly distributed between the regimens. Sustained virologic response (SVR) was obtained in 24 out of 28 patients (85.7%) who received daclatasvir-sofosbuvir and in all patients (100%) who received daclatasvir-sofosbuvir-ribavirin (global SVR 89.7%). All patients that failed treatment had genotype 3 HCV. Fibrosis was evaluated in 79.5% of the sample: 48.4% had early and 51.6% had moderate to advanced fibrosis, for which ribavirin was more commonly prescribed (P = .001). CONCLUSIONS: The SVR rate in our LT recipients was similar to that previously reported in the literature. The addition of ribavirin to DAA treatment appears to be justified in this population.

Overexpression of CD45RA isoforms in carriers of the C77G mutation leads to hyporeactivity of CD4+CD25highFoxp3+ regulatory T cells.

Disorders in regulatory T-cell (T(reg)) function can result in the breakdown of immunological self-tolerance. Thus, the identification of mechanisms controlling the activity of T(reg) is of great relevance. We used T(reg) from individuals carrying the C77G polymorphism as models to study the role of CD45 molecules in humans. C77G prevents splicing of CD45 exon A thereby leading to an aberrant expression pattern of CD45 isoforms in affected individuals. Resting and in vitro expanded/activated CD4(+)CD25(high)Foxp3(+) T(reg) from carriers of C77G strongly expressed CD45RA isoforms whereas these isoforms were almost absent in cells from individuals with wild-type CD45. C77G T(reg) showed diminished upregulation of activation markers, lower phosphorylation of p56(lck)(Y505) and a reduced proliferative potential when stimulated with anti-TcR or anti-TcR plus CD28 mAb suggesting decreased responsiveness to activating stimuli. In addition, the capacity to suppress proliferation of conventional CD4(+) T cells was impaired in C77G T(reg). Furthermore, microarray studies revealed distinct gene expression patterns in T(reg) from C77G carriers. These data suggest that the changes in CD45 isoform combination resulting from the C77G mutation alter the responsiveness of T(reg) to TcR-mediated signaling. Targeting CD45 isoform expression might be a useful approach to modulate T(reg) function.

Comparison Between IGL-1 and HTK Preservation Solutions in Deceased Donor Liver Transplantation.

The effectiveness of liver preservation solutions remains in evidence. Cold ischemia time, steatosis, expanded criterion donors, operational cost, and survival represent important roles in its success. In a prospective cohort study between August 2009 and April 2014, 178 patients were allocated into an Institut Georges Lopez - 1 (IGL-1) solution group (63.5%) or histidine-tryptophan-ketoglutarate (HTK) group (36.5%). There were no differences among recipient's characteristics including age, skin color, gender, Model for End-stage Liver Disease score, acute rejection, cholestasis, and reperfusion syndrome incidences. Also, donors, age average, skin color, donor risk index, time in intensive care unit, hemodynamic variables, infections, and steatosis incidences were similar. The average cold ischemia time was 494 minutes in the IGL-1 group and 489 minutes in the HTK group (P = .77). Alanine aminotransferase and aspartate aminotransferase serum levels on the first postoperative day were 707 and 1185 mg/dL, respectively, with IGL-1 and 1298 and 2291 mg/dL, respectively, with HTK (P = .016) and similar at day 15 (P > .88). The incidence of delayed graft function was 4.5% with IGL-1 and 4.6% with HTK (P = .90). The incidence primary nonfunction was 2.7% with IGL-1 and 3.1% with HTK (P = .71). The incidence of perioperative death was 11.5% with IGL-1 and 13.8% with HTK (P = .94). The survival in 30 months was 86% in IGL-1 group and 82% in HTK group (P = .66). Both preservation solutions are efficient to liver transplantations with deceased donors. Major prospective trials are necessary to evaluate each preservation solution's particularities. The preservation solution availability in each transplantation center must guide its use at the present moment.

Poster - Thur Eve - 70: Quantification of tumour dose enhancement at kilo-voltage energies due to the presence of gold nanoparticles during radiation therapy: EGSnrcMP Monte Carlo study.

One of the greatest challenges in radiation therapy is the ability to deliver a lethal dose of radiation to a tumour while sparing the surrounding normal tissues. In theory, the dose delivered to a tumour during photon-based radiation therapy can be enhanced by loading high atomic number (Z) materials into the tumour, which results in greater photoelectric absorption and hence increased photoelectron fluence within the tumour than in surrounding tissues. The EGSnrcMP Monte Carlo code, together with DOSXYZnrc, a three-dimensional voxel dose calculation module has been used to study the macroscopic dose enhancement factor (MDEF) in a tumour infused with gold nanoparticles at the kilo-voltage energies. We observed that gold nanoparticles infused in a tumour irradiated with kilo-voltage energies has the potential to enhance the tumour dose by a factor ranging from 0.25 to about 5 depending on the mean energy of the beam and the concentration of gold nanoparticles in the tumour. The increase in dose can be attributed to the significant increase in the photoelectron fluence within the tumour loaded with gold particles during the irradiation. Future studies will involve the characterization of the MDEF at megavoltage energies.

Poster - Thur Eve - 66: A planning comparison between RapidArc and intensity modulated radiotherapy for head and neck cancer.

Volumetric modulated arc therapy (VMAT) has recently been used to improve the dose distribution and efficiency of treatment delivery over the standard intensity-modulated radiotherapy (IMRT) technique. This study compares the dosimetry between RapidArc plan and standard IMRT plan for head and neck cancer. Three head and neck patients treated clinically with sliding window intensity-modulated radiotherapy (IMRT) technique at Grand River Regional Cancer Center were selected randomly and re-planned using RapidArc technique with 6 MV photon beams generated by a Varian 21EX linac with 120-leaf multileaf collimator. Three dose prescriptions were used to deliver 70 Gy, 63 Gy and 58.1 Gy to the regions of the primary tumors, intermediate-risk nodes and low-risk nodal level, respectively, in 35 fractions. Dosimetric comparison based on the dose-volume histogram, target coverage, organ at risk (OAR) dose sparing were studied between the RapidArc plan and IMRT plan. RapidArc technique from Varian Medical Systems showed superior target coverage, better OAR sparing, fewer monitor units per fraction with less treatment time over IMRT technique for head and neck cancers. The average homogeneity index, defined as the difference between the percentage dose covering 5% and 95% of the PTV, is 9.5 for RapidArc plan and 10.5 for IMRT plan. All RapidArc plans met the dose objectives for the primary OAR: spinal cord, brainstem, brain etc. Both parotid mean dose and D50% are lower for RapidArc plan than those of the IMRT plan. The technique is currently being used clinically at our cancer center.

Randomized clinical assay for hepatic grafts preservation with University of Wisconsin or histidine-tryptophan-ketoglutarate solutions in liver transplantation.

University of Wisconsin (UW) solution has been the standard for preservation of liver transplantation grafts since 1989. However, some studies demonstrated that histidine-tryptophan-ketoglutarate (HTK) solution is also effective. The purpose of this study was to compare the efficacy of both solutions in liver transplantation. From January 2003 to August 2004 the livers of deceased donors were randomized into HTK and UW groups. The 102 studied patients included 65 (63.7%) in the UW group and 37 (36.3%) in the HTK group. Sex, race, hemodynamic state, use of adrenergic drugs, and presence of steatosis in the donor were similarly distributed in the two groups (P > .05). The mean age of the donors was 38.1 years (SD +/-14.4) in the UW group and 44.6 years (SD +/-14.2) in the HTK cohort (P = .036). Sex, race, age, etiology of the cirrhosis, retransplant, acute liver failure, portal thrombosis, and Child-Pugh and MELD scores in the recipients were similarly distributed in the two recipient samples (P > .05). Among 89 patients who completed 4 months of follow-up, the HTK group included eight cases (25.8%) of biliary complications versus five cases (8.6%) in the UW group (P = .033; OR = 2.0 95% CI = 1.2-3.5). The incidence of graft dysfunction was 2.8% in the HTK group and 9.4% in the UW group (P = .15). In conclusion, UW and HTK solutions were equally effective for the preservation of the hepatic graft. The routine use of HTK solution can reduce the costs of liver transplantation.

Normalized data for the estimation of fetal radiation dose from radiotherapy of the breast.

There can be several reasons why a pregnant patient may receive a radiological examination. It could have been a planned exposure, or the exposure might have resulted from an emergency when a thorough evaluation of pregnancy was impractical. Sometimes the pregnancy was unsuspected at the time of the examination and, with younger women being diagnosed with breast cancer, the likelihood of this will increase in radiotherapy departments. Whatever the reason, when presented with a pregnant patient who has received a radiological examination involving ionizing radiation, the dose to the fetus should be assessed based on the patient's treatment plan. However, a major source of uncertainty in the estimation of fetal absorbed dose is the influence of fetal size and position as these change with gestational age. Consequently, dose to the fetus is related to gestational age. Various studies of fetal dose during pregnancy have appeared in the literature. Whilst these papers contain many useful data for estimating fetal dose, they usually contain limited data regarding the depth and size of the fetus within the maternal uterus. We have investigated doses to the fetus from radiation therapy of the breast of a pregnant patient using an anthropomorphic phantom. Normalized data for estimating fetal doses that takes into account the fetal size (gestational age: 8-20 weeks post-conception) and depth within the maternal abdomen (4-16 cm) for different treatment techniques have been provided. The data indicate that fetal dose is dependent on both depth within the maternal abdomen and gestational age, and hence these factors should always be considered when estimating fetal dose. The data show that fetal dose can be underestimated up to about 10% or overestimated up to about 30% if the dose to the uterus is assumed instead of the actual fetal dose. It can also be underestimated up to about 23% or overestimated up to about 12% if a mean depth of 9 cm is assumed, instead of using the actual depth of the fetus within the maternal abdomen. Multi-segments sMLC technique showed consistently lower fetal doses compared with all the wedged plans employed.

Oxidative stress, hepatocellular integrity, and hepatic function after initial reperfusion in human hepatic transplantation.

BACKGROUND: The mechanisms underlying liver graft dysfunction are not completely defined, although much of the injury derives from oxidative stress in organ reperfusion. The antioxidant glutathione in its reduced form (GSH) is an important agent to detoxify oxygen species after reperfusion. However, this effect might be limited by low concentrations at the end of cold storage. The objective of this study was to evaluate GSH and glutathione oxidized (GSSG) hepatic levels pre- and postreperfusion and correlate with hepatocellular injury and liver function in the 5 subsequent days after transplantation. METHODS: Liver biopsies were taken immediately before implant and 2 hours after venous reperfusion in 34 grafts, determining GSH, GSSG levels, and GSSG/GSH ratio. Aminotransferases (ALT, AST) and PT were measured for 5 days. RESULTS: There was a strong decrease in GSH concentration (P <.0001), increase of GSSG levels (P <.01), and increase of the GSSG/GSH ratio (P <.0001). No correlations were found between GSH, GSSG, or GSH/GSSH levels and AST, ALT, and PT. CONCLUSION: Glutathione levels showed significant changes after 2 hours of reperfusion, due to intense oxidative stress. Therapies to replenish GSH should be considered as a protective measure to avoid liver graft dysfunction after transplantation.

A stepwise mapping approach for localization and ablation of ectopic right, left, and septal atrial foci using electroanatomic mapping.

AIMS: We describe a new strategic stepwise mapping approach for fast and accurate identification and ablation of ectopic atrial foci using an electroanatomic mapping system. METHODS AND RESULTS: Mapping procedures started with the acquisition of four points at the superior/septal part of the tricuspid annulus. According to this activation sequence, maps were continued towards the right atrial free wall if relatively early activation was shown at the superior part of the initial map or towards the triangle of Koch and, if necessary, to the left atrium, in cases of relatively early activation at the septum. High density mapping and detailed electrogram analysis only of the target area allowed identification and ablation of 34 foci in 30 of the 32 studied consecutive patients. A small number of mapping points were sufficient within a procedure time of 90 +/- 41 min for right 148 +/- 68 min and for left sided foci and a total fluoroscopy time of 9.6 +/- 7.2 min and 24.8 +/- 16.4 min respectively. Sixteen foci were located at the right free wall, eight at the left free wall, and 10 at the right or left side of the septum. CONCLUSION: Strategic electroanatomic mapping with fast identification of the area of tachycardia origin and high density mapping only of this target area allowed fast and successful localization and ablation of right and left free wall and septal ectopic atrial foci.

Electromagnetic versus fluoroscopic mapping of the inferior isthmus for ablation of typical atrial flutter: A prospective randomized study.

BACKGROUND: Radiofrequency catheter ablation within the tricuspid annulus-inferior caval vein isthmus can cure typical atrial flutter. The target for ablation, nonetheless, is relatively wide, and standard ablation procedures may require significant exposure to radiation. METHODS AND RESULTS: A total of 50 patients (mean age, 58+/-11 years) with typical atrial flutter were prospectively randomized to receive isthmus ablation using conventional fluoroscopy for catheter navigation (group I, n=24) or electromagnetic mapping (group II, n=26). Complete bidirectional isthmus block was verified with double potential mapping. If complete isthmus block could not be achieved after 20 radiofrequency pulses or 25 minutes of fluoroscopy, the patients were switched to the other group. Eight patients from group I (33%) but only 1 patient from group II (4%) were switched. Overall, complete isthmus block was achieved in 47 of 50 patients (94%). The overall fluoroscopy time, including the placement of the diagnostic catheters, was 22.0+/-6.3 minutes in group I and 3.9+/-1.5 minutes in group II (P:<0.0001). The fluoroscopy time needed for isthmus mapping was 17.7+/-6.5 minutes in group I and 0.2+/-0.3 minutes in group II (P:<0.0001). CONCLUSIONS: Electromagnetic mapping during the induction of linear lesions for the ablation of atrial flutter permitted a highly significant reduction in exposure to fluoroscopy while maintaining high efficacy, and it allowed the time required for fluoroscopy to be reduced to levels anticipated for diagnostic electrophysiological studies.

Serum albumin distribution in early treated anorexia nervosa.

We investigated the effects of malnutrition and refeeding on albumin distribution and metabolism in patients undergoing treatment for anorexia nervosa. Using autologous 125I-labelled albumin, we measured the fractional catabolic rate and calculated the relative sizes of the plasma and extravascular albumin pools in 6 female anorexia nervosa subjects and 6 matched controls. We were unable to demonstrate any differences in either the catabolic rate of albumin (fractional or absolute) or in serum albumin concentration between anorexia nervosa and control subjects. There was a large expansion of the extravascular albumin pool in the anorexia nervosa subjects--36% when expressed in relation to body weight. We conclude that, at the time of study, there were no effects of anorexia nervosa on albumin catabolism in these subjects. However, the condition and its treatment are associated with a significant relative expansion of the extravascular albumin pool. This contrasts to some extent with previous work, which suggested that in protein depletion the plasma albumin pool is maintained at the expense of the extravascular albumin pool. The expansion of the extravascular albumin pool is possibly related to the relative excess of interstitial fluid seen in starvation and in the initial phases of refeeding.

Development of an enzyme-linked immunosorbent assay for human metallothionein-1 in plasma and urine.

The development of a sensitive enzyme-linked immunosorbent assay (ELISA) for human metallothionein-1 is reported. Metallothionein was purified from postmortem human liver and used to raise high-titer antibodies in rabbits. The assay was specific for human metallothionein-1 (MT-1), and there was no significant cross-reaction with human metallothionein-2. The detection limit (sensitivity) of the assay was 5 ng/ml, and the added MT-1 could be fully recovered from plasma and urine. The normal reference range for MT-1 was 32 +/- 16 ng/ml in plasma and 10 +/- 6 ng MT-1 per micromole of creatinine in random samples of urine. No significant differences were found between the values for males and females. The concentration of MT-1 was greatly increased between 24 and 48 hours after surgery, indicating that the protein behaves like an acute phase reactant in human subjects.

The effects of experimental malnutrition on albumin metabolism and distribution in rabbits.

In order to determine the extent to which the concentration of albumin in plasma is maintained at the expense of the extravascular pool during protein-energy malnutrition, the rates of exchange between albumin in plasma (IA) and the extravascular pool (EA) and consequently the distribution of albumin between intravascular and extravascular pools (expressed as EA:IA) were measured in protein-energy-depleted and control rabbits. The fractional rates of synthesis (FSR) and catabolism (FCR), the concentration of albumin and the plasma volume (PV) were also measured. In animals in which protein-energy intake was reduced by 58% the concentration of albumin in plasma remained unchanged, whereas FCR decreased by 38% and FSR by 30%. No significant changes in EA:IA or PV were found. We conclude that albumin concentration during protein-energy depletion is not maintained at the expense of extravascular albumin, but by parallel changes in the rates of catabolism and synthesis.

Albumin catabolic rate and protein-energy depletion.

Severe malnutrition was induced in New Zealand White rabbits by restricting dietary nitrogen and energy to 9.1 and 57.4% of normal intake, respectively. Albumin distribution and metabolism were then determined in these and in control animals. The fractional catabolic rate was reduced by 33.7% in the depleted animals, but there were no changes in the other distribution rate constants. In particular, we were unable to demonstrate a significant change in the relative distribution of albumin between the intravascular and extravascular spaces. In addition, albumin concentration in the depleted group only fell toward the end of the experiment. We conclude that, with acute severe malnutrition, plasma albumin concentration is maintained until late and is therefore not a good nutritional marker. Also, maintenance of intravascular albumin concentration is not at the expense of extravascular albumin.