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.

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.

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.

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.

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.

Experience with a commercial preparation of 125I-labelled human albumin for study of albumin metabolism.

Evaluation of a commercial preparation of (125)I-labelled albumin for use in the study of albumin metabolism is described. In eight subjects with normal albumin metabolism the proportion of the dose of radioiodide excreted was stable throughout a period of 17 days, indicating that there was no excessive denaturation of the iodinated albumin. Characteristics of albumin metabolism-pool sizes, catabolic rate, etc-were in agreement with currently accepted normal values. It is concluded that this preparation of iodinated albumin is suitable for metabolic use.

Chemical and kinetic study of the lipoproteins in abetalipoproteinaemic plasma.

Abetalipoproteinaemic plasma lipoproteins were fractionated by molecular sieve chromatography into two classes on the basis of size. Each class had the same chemical and immunochemical composition and seemed to be interconvertible in vitro, presumably as a result of aggregation/disaggregation. The low levels of circulating apolipoprotein A-I found in abetalipoproteinaemic subjects have been shown by kinetic analysis to result from reduced synthesis of the apoprotein and not from increased catabolism or redistribution between vascular and extravascular compartments.

Early time course of the acute phase protein response in man.

The rate at which the acute phase protein response occurred after both major and minor surgery was explored. Increases in the plasma concentration of C-reactive protein (CRP), alpha-1-acid glycoprotein (alpha 1 AG) and fibrinogen were not detected until 6-8 h after the initial incision. The peak concentration of CRP occurred at 48 h and that of fibrinogen at 96 h; alpha 1 AG concentrations rose rapidly until 48 h followed by little change until about 120 h. Although there was widespread variation in the concentrations of individual proteins in patients, severity of injury did not seem to have a significant effect on the time course of the change. Plasma cortisol concentration and the total white blood cell count (WBC) reached their peaks before the acute phase proteins, cortisol at 6 h and WBC at 12 h.

Early plasma protein and mineral changes after surgery: a two stage process.

Sequential changes in albumin, transferrin, alpha 1-acid glycoprotein, C reactive protein, fibrinogen, copper, iron, and zinc in plasma up to 24 h after hysterectomy were measured. No increases in the concentrations of the acute phase proteins alpha 1-acid glycoprotein, C reactive protein, and fibrinogen were observed until 6 h after the skin incision. These increases were preceded by significant falls at 2-4 h, and this was shown also by albumin, transferrin, iron, zinc, and copper. The ratios of iron and zinc to their binding proteins, transferrin and albumin, did not decrease until 4-6 h and their concentrations remained low for at least 24 h. These patterns suggest that at least two mechanisms operate after trauma. The early fall in the concentrations of the proteins in plasma is consistent with a prompt increase in microvascular permeability. The later decrease in binding of the metals iron and zinc to their transport proteins and the increase in concentrations of the acute phase proteins could be initiated by a common mediator.

The protein metabolic response to short-term starvation in man.

Plasma protein concentrations, in particular the serum albumin concentration, are often quoted as indicators of nutritional state in surgical patients. Injury also affects the circulating concentrations of these proteins. Twenty students were subjected to a 3-day total fast and the concentration of plasma proteins, generally affected by trauma, determined before and after the fast. The only protein to show any negative response was retinal binding protein which fell by 16% during the starvation period (p < 0.001). Albumin, the protein often used as a nutritional index, demonstrated a significant (p < 0.001) increase during this period. This was not related to changes in the hydration state of the subjects.

The effect of environmental temperature on protein and energy changes following burn injury in the rat.

The metabolic response to a standard burn injury in rats (25% of body surface area) was investigated at environmental temperature 20 degrees C and 30 degrees C. With an intake of 15 g diet (20% w/w protein) per day, burned rats at 20 degrees C were found to be in negative energy balance mainly due to increased insensible (evaporative) losses, and they lost weight. Fat was the main endogenous source of energy, although protein was also catabolised from both carcass and pelt. At 30 degrees C, insensible losses of burned rats were still high, but they had lower sensible (mainly radiative) losses leading to a positive energy balance and a gain in weight. This consisted mainly of carcass water, fat and protein. Urine catecholamines were higher in burned rats at 20 degrees C than at 30 degrees C and correlated with heat loss, urine nitrogen and urine 3-methylhistidine, supporting the hypothesis that catecholamines play a central role in mediating the protein and energy changes following burn injury. For the first 4-6 days, urine nitrogen and 3-methylhistidine excretion increased in rats at 30 degrees C, suggesting that this early part of the metabolic response is obligatory. These findings support the use of raised environmental temperatures to reduce the metabolic response to burns.

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.

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.

Simplified analytical methods for the measurement of the synthesis rate of plasma proteins in vivo by the [14C]carbonate method.

We describe a method for obtaining the specific activity of 14C in urea, essential in the measurement of the synthesis rate of a plasma protein in vivo, which is simpler than the original procedure. The principle is the measurement of 14CO2 and NH4+ separately, after incubation with urease. A simple alteration gives samples of 13CO2 for mass spectrometry. The 'recoveries' of 14C and 13C in urea were invariably between 90 and 96% and the CV was 3%.

Temperatures used in the determination of enzyme activity in clinical biochemistry laboratories in Britain: results of a survey.

Information on the temperature used in the determination of enzyme activity in clinical biochemistry laboratories in Britain was obtained by circulating a questionnaire to 400 laboratories in Britain. Replies were analysed from 321 laboratories (80%). A large majority of laboratories use 37 degrees C to determine enzyme activity: 99% use this temperature for amylase, 88% for alkaline phosphatase, and 81% for aspartate and alanine amino-transferases. A greater proportion of laboratories with large workloads use 37 degrees C than do those with smaller workloads. The majority of laboratories use equipment with incubation temperatures that are selectable within the laboratory. When there is evidence of advantage in using a particular temperature most laboratories use this temperature. Almost one-quarter of the laboratories replying participate in a local agreement to use 37 degrees to determine enzyme activity. About one-third of laboratories consider it impracticable to change to 30 degrees C, and of those replying to the question on whether they were willing to change to 30 degrees C, 49% indicated that they were not willing to do so.

Drift correction--a comparative evaluation of some alternatives.

Three drift correction methods are described. The results obtained from computer simulation of these methods and their application to raw data obtained from a survey of multichannel continuous-flow analysers are discussed, with particular reference to the effects of each method on analytical precision and accuracy, and on between-analyser variation. A geometrically derived method of drift correction was found to be the most satisfactory.

Optimal conditions for the estimation of ammonium by the Berthelot reaction.

A technique for optimising reagent concentrations on the AutoAnalyzer has been applied to the estimation of ammonia by the Berthelot reaction in the determination of urea and organic nitrogen. Comparison of the use of phenol and salicylate revealed that the optimum concentration of the latter is about four times that of the former. The optimum concentration of hypochlorite is five times greater with salicylate than with phenol, and for the catalyst, sodium nitroprusside, the factor is two. The precision obtained with the different methods is similar.

A limited protocol for assessment of within-batch performance of multichannel analysers.

An experimental protocol is described to evaluate rapidly the performance of biochemical analysers. A trial in which this protocol was applied to 15 multichannel continuous-flow analysers is described and the results are discussed. Some consideration is given to the effects of applying a commonly-used drift correction procedure to the raw data, and values of within-batch standard deviation are proposed which may be useful in assessing performance.

Variations in mechanical sampling: their causes and effects.

Three characteristics of a sample of AutoAnalyzer Sampler II modules have been measured: eccentricity of the cam, eccentricity of the spindle driving the cam, and the lengths of the chords of the cam lobes. Imprecision in timing may make a significant contribution to analytical error.