Liver preservation with machine perfusion and a newly developed cell-free oxygen carrier solution under subnormothermic conditions.

We describe a new preservation modality combining machine perfusion (MP) at subnormothermic conditions(21 °C) with a new hemoglobin-based oxygen carrier (HBOC) solution. MP (n=6) was compared to cold static preservation (CSP; n=6) in porcine orthotopic liver transplants after 9 h of cold ischemia and 5-day follow-up. Recipients' peripheral blood, serial liver biopsies, preservation solutions and bile specimens were collected before, during and after liver preservation. Clinical laboratorial and histological analyses were performed in addition to mitochondrial functional assays, transcriptomic, metabolomic and inflammatory inflammatory mediator analyses. Compared with CSP, MP animals had: (1) significantly higher survival (100%vs. 33%; p<0.05); (2) superior graft function (p<0.05);(3) eight times higher hepatic O2 delivery than O2 consumption (0.78 mL O2/g/h vs. 0.096 mL O2/g/h) during MP; and (4) significantly greater bile production (MP=378.5 ± 179.7; CS=151.6 ± 116.85). MP downregulated interferon (IFN)-α and IFN-γ in liver tissue. MP allografts cleared lactate, produced urea, sustained gluconeogenesis and produced hydrophilic bile after reperfusion. Enhanced oxygenation under subnormothermic conditions triggers regenerative and cell protective responses resulting in improved allograft function. MP at 21 °C with the HBOC solution significantly improves liver preservation compared to CSP.

The CUPDAY Study: prebiotic-probiotic milk product in 1-3-year-old children attending childcare centres.

AIM: The aim of the study was to evaluate the efficacy of a milk product containing probiotics and prebiotics (CUPDAY Milk) on the incidence of diarrhoea in children attending daycare centres. METHODS: The study was undertaken in a randomized controlled trial with 496 children aged 1-3 years attending 29 childcare centres in Perth, Australia. The endpoint for the study was the number of days in which children were recorded as having four or more stools. The diarrhoeal rates were analyzed by Poisson regression using 'intention to treat' (all children) and 'reduced' (children enrolled for more than 10 days) data sets. RESULTS: There was no difference in demographic characteristics between the case and control groups. In the 'intention to treat' analysis, a total of 41 745 child-days were included in the study. The adjusted risk ratio for those consuming the 'Cupday' milk drink was 0.82 (95% CI 0.73-0.94) for the intention to treat sample (n = 496) and 0.80 (95% CI 0.70-0.91) for the reduced sample (n = 315). The children consuming the 'Cupday' drink had a 20% reduction in the number of days experiencing four or more stools per day. CONCLUSION: A milk containing probiotics and prebiotics reduced the number of days children attending child care who had four or more stools by 20%.

The altered fluid distribution in obesity may reflect plasma hypertonicity.

OBJECTIVE: This study explored whether the increased extracellular relative to intracellular fluid (ECF/ICF) ratio in obesity might reflect osmotic effects of elevated plasma solute concentrations. DESIGN: Cross-sectional, epidemiological survey. SETTING AND SUBJECTS: The present analysis used nationally representative data from the Third National Health and Nutrition Examination Survey on community-dwelling adults (aged 40-59 years) in the US without evidence of glucose dysregulation or chronic disease (n=1285). INTERVENTION: Body mass index (BMI) was estimated from measured height and weight. Total body reactance, an index of body fluid distribution, was determined by bioelectrical impedance analysis. Plasma tonicity (the cumulative index of osmotically effective plasma solute) was estimated from plasma glucose, sodium and potassium. Sex-specific relative odds of lower reactance (or=295 mmol/l) associated with overweight (25or=30) were estimated using logistic regression models that controlled for sociodemographic variables, smoking, leisure-time physical activity, total energy intake, serum creatinine, plasma insulin and glucose. Multinomial logistic regression models tested for associations between weight status and specific serum solute. RESULTS: Independent of covariates, in men and women, overweight and obesity were associated with increased odds of lower reactance and hypertonicity. Overweight and obese individuals with lower reactance had significantly higher serum sodium than normal weight individuals. CONCLUSIONS: Elevated plasma solute concentrations are associated with obesity in free-living adults. Physicians and researchers should be alert to a possible link between hypertonicity and obesity. SPONSORSHIP: Grants from the NIH, Nestle Waters.

Effects of usual nutrient intake and vitamin D status on markers of bone turnover in Swiss adolescents.

OBJECTIVE: To evaluate the effects of nutrient intake and vitamin D status on markers of type I collagen formation and degradation in adolescent boys and girls. DESIGN: Cross-sectional study. SETTING: Canton of Vaud, West Switzerland. SUBJECTS: A total of 92 boys and 104 girls, aged 11-16 y. Data were collected on height, weight, pubertal status (self-assessment of Tanner stage), nutrient intake (3-day dietary record) and fasting serum concentration of 25-hydroxyvitamin D (25OHD), and markers of collagen formation (P1NP) and degradation (serum C-terminal telopeptides: S-CTX). RESULTS: Tanner stage was a significant determinant of P1NP in boys and girls and S-CTX in girls. Of the nutrients examined, only the ratio of calcium to phosphorus (Ca/P) was positively associated with P1NP in boys, after adjustment for pubertal status. 25OHD decreased significantly at each Tanner stage in boys. Overall, 15% of boys and 17% of girls were identified as being vitamin D insufficient (serum 25OHD <30 nmol/l), with the highest proportion of insufficiency at Tanner stage 4-5 (29%) in boys and at Tanner stage 3 (24%) in girls. A significant association was not found between 25OHD and either bone turnover marker, nor was 25OHD insufficiency associated with higher concentrations of the bone turnover markers. CONCLUSIONS: The marked effects of puberty on bone metabolism may have obscured any possible effects of diet and vitamin D status on markers of bone metabolism. The mechanistic basis for the positive association between dietary Ca/P ratio and P1NP in boys is not clear and may be attributable to a higher Ca intake per se, a critical balance between Ca and P intake or higher dairy product consumption. A higher incidence of vitamin D insufficiency in older adolescents may reflect a more sedentary lifestyle or increased utilisation of 25OHD, and suggests that further research is needed to define their requirements. SPONSORSHIP: Nestec Ltd and The Swiss Foundation for Research in Osteoporosis.

Effect of bite tray impression technique on relocation accuracy in frameless stereotactic radiotherapy.

A previously developed method for achieving patient relocation in fractionated stereotactic radiotherapy (attachment of an infrared fiducial system to a bite tray) relies on the integrity of a bite tray system that incorporates moulding to the patient's upper dentition. Reproducible and accurate patient positioning requires stability of the bite tray and mould during the full treatment process, both during the time the bite tray is inserted in the patient's mouth, and between separate bite tray insertions. The optimum construction method for a stable reproducible tray has not been sufficiently investigated. We undertook a study to identify factors which might influence the integrity of the hard palate bite tray system. Reprosil Fast Set Putty was used to construct 3 impression conditions; teeth only; teeth and alveolar sulcus; and teeth, alveolar sulcus, and the hard palate. Reproducibility was assessed by volunteers inserting the impressions multiple times and recording the locations of 8 standard reference points. Our results showed the optimal impression technique (i.e., the one that led to the smallest ranges in positional and rotational errors) was that which incorporated the teeth, alveolar sulcus, and hard palate.

Effect of training location on students' clinical skills.

The clinical skills of second-year medical students trained in a hospital-based setting were compared with those of students trained in a community setting using an objective structured clinical exam. No statistically significant difference was found.

Selenium absorption and retention from a selenite- or selenate-fortified milk-based formula in men measured by a stable-isotope technique.

The present study was designed to determine the apparent absorption and retention of the inorganic Se compounds SeO3(2-) and SeO4(2-), which are commonly used for Se fortification of clinical nutrition products and infant formulas. Ten healthy men were fed a milk-based formula labelled with 40 microg Se as 74SeO3(2-) or 76SeO4(2-) on two consecutive days using a randomised crossover design. Se stable-isotope analysis of 9 d complete collections of urine and faeces was used to calculate apparent Se absorption and retention. Se retention from 74SeO3(2-) (41.0 (SD 8.4) %) and from 76SeO4(2-) (46.0 (SD 7.9) %) was not significantly different (P > 0.05). However, Se absorption was significantly higher from SeO4(2-) than from SeO3(2-) (91.3 (SD 1.4) % v. 50.2 (SD 7.8) %, P < 0.05). Urinary excretion of the administered dose was 9.2 (SD 1.8) % for 74SeO3(2-) and 45.3 (SD 8.2) % for 76SeO4(2-) (P < 0.05). Urinary Se excretion kinetics differed significantly for the two Se compounds; 90 % of the total urinary Se was excreted after 121 h for 74SeO32- and after 40 h for 76SeO42- These results suggest that although Se absorption and urinary excretion differ for SeO3(2-) and SeO4(2-), both Se compounds are equally well retained when administered at a relatively low dose (40 microg Se). The nutritional impact of Se fortification of foods would thus be expected to be similar when SeO4(2-) or SeO3(2-) are used.

Iron bioavailability in infants from an infant cereal fortified with ferric pyrophosphate or ferrous fumarate.

BACKGROUND: Infant cereals are commonly fortified with insoluble iron compounds with low relative bioavailability, such as ferric pyrophosphate, because of organoleptic changes that occur after addition of water-soluble iron sources. OBJECTIVE: Our objective was to compare iron bioavailability from ferric pyrophosphate with an alternative iron source that is soluble in dilute acid, ferrous fumarate, and to evaluate the influence of ascorbic acid on iron bioavailability from ferrous fumarate in infants. DESIGN: Iron bioavailability was measured as the incorporation of stable iron isotopes into erythrocytes 14 d after administration of labeled test meals (25 g dry wheat and soy infant cereal, 100 g water, and 2.5 mg Fe as [57Fe]ferric pyrophosphate or [57Fe]ferrous fumarate). Ascorbic acid was added to all test meals (25 mg in study 1 or 25 or 50 mg in study 2). Infants were fed each test meal on 4 consecutive days under standardized conditions. The 2 different test meals within each study were administered 2 wk apart in a crossover design. RESULTS: Geometric mean iron bioavailability was significantly higher from [57Fe]ferrous fumarate than from [57Fe]ferric pyrophosphate [4.1% (range: 1.7-14.7%) compared with 1.3% (range: 0. 7-2.7%); n = 8, P = 0.008]. In this study, doubling the ascorbic acid content did not further enhance iron bioavailability; the geometric means (range) were 3.4% (1.9-6.6%) and 4.2% (1.2-18.7%) for the test meals with 25 and 50 mg ascorbic acid added, respectively (n = 9). CONCLUSION: Iron bioavailability from iron-fortified infant cereals can be improved by using an iron compound with high relative bioavailability and by ensuring adequate ascorbic acid content of the product.

Oxygen delivery during retrograde cerebral perfusion in humans.

UNLABELLED: Retrograde cerebral perfusion (RCP) potentially delivers metabolic substrate to the brain during surgery using hypothermic circulatory arrest (HCA). Serial measurements of O2 extraction ratio (OER), PCO2, and pH from the RCP inflow and outflow were used to determine the time course for O2 delivery in 28 adults undergoing aortic reconstruction using HCA with RCP. HCA was instituted after systemic cooling on cardiopulmonary bypass for 3 min after the electroencephalogram became isoelectric. RCP with oxygenated blood at 10 degrees C was administered at an internal jugular venous pressure of 20-25 mm Hg. Serial analyses of blood oxygen, carbon dioxide, pH, and hemoglobin concentration were made in samples from the RCP inflow (superior vena cava) and outflow (innominate and left carotid arteries) at different times after institution of RCP. Nineteen patients had no strokes, five patients had preoperative strokes, and four patients had intraoperative strokes. In the group of patients without strokes, HCA with RCP was initiated at a mean nasopharyngeal temperature of 14.3 degrees C with mean RCP flow rate of 220 mL/min, which lasted 19-70 min. OER increased over time to a maximal detected value of 0.66 and increased to 0.5 of its maximal detected value 15 min after initiation of HCA. The RCP inflow-outflow gradient for PCO2 (slope 0.73 mm Hg/min; P < 0.001) and pH (slope 0.007 U/min; P < 0.001) changed linearly over time after initiation of HCA. In the group of patients with preoperative or intraoperative strokes, the OER and the RCP inflow-outflow gradient for PCO2 changed significantly more slowly over time after HCA compared with the group of patients without strokes. During RCP, continued CO2 production and increased O2 extraction over time across the cerebral vascular bed suggest the presence of viable, but possibly ischemic tissue. Reduced cerebral metabolism in infarcted brain regions may explain the decreased rate of O2 extraction during RCP in patients with strokes. IMPLICATIONS: Examining the time course of oxygen extraction, carbon dioxide production, and pH changes from the retrograde cerebral perfusate provided a means to assess metabolic activity during hypothermic circulatory arrest.

Physiologic study of pressure point techniques used in the martial arts.

BACKGROUND: Study physiologic changes occurring during "knockouts" produced by application of pressure point techniques during martial arts demonstrations. EXPERIMENTAL DESIGN: prospective analysis of physiologic variables during and immediately following an acute event. SETTING: martial arts demonstration carried out at a medical center hospital. SUBJECTS: 12 normal volunteers participating in a martial arts demonstration. INTERVENTIONS: application of various pressure point techniques that have been observed to produce states of unresponsiveness in volunteers. MEASURES: continuous ECG and video/EEG monitoring with measurements of blood pressure and oxygen saturation. Qualitative analysis of EEG and ECG recordings and quantitative comparison of heart rate, blood pressure, and oxygen saturation measurements before during and after the period of induced unconsciousness. RESULTS: No significant changes in blood pressure, oxygen saturation, cardiac rate or rhythm, or electroencephalogram are noted during the knockouts produced by application of pressure point techniques. There was only variable inability for subjects to remember words spoken to them during the episode of apparent unresponsiveness. CONCLUSIONS: The mechanism for the state of unresponsiveness produced by application of pressure point techniques is not related to a significant cardiac or pulmonary process. There is no evidence of reduced cerebral blood flow during this time or of other dangerous physiologic changes. The exact mechanism for this phenomenon remains uncertain.

Determination of selenium stable isotopes by gas chromatography-mass spectrometry with negative chemical ionisation.

A gas chromatography mass spectrometric method using negative chemical ionisation was developed for the determination of stable isotopes of selenium for evaluation of selenium absorption and retention from foods in humans. The method involves an acid digestion to convert all selenium into selenite, which subsequently reacts with 4-nitro-o-phenylene-diamine to form a volatile piazselenole. The piazselenole, after extraction into an organic solvent, was analysed for its isotopic selenium composition by gas chromatography mass spectrometry. Negative chemical ionisation is reported for the first time for the determination of selenium stable isotopes and its analytical characteristics were compared to those of electron impact mass spectrometric ionisation, classically used for the determination of selenium. The negative chemical ionisation technique allowed accurate determination of total selenium by isotope dilution and of selenium isotope ratios in biological samples. The repeatability for total selenium and for stable isotope ratios was good (R.S.D.< or =10%) within the range of 50 to 250 ng selenium. The detection limit for the investigated selenium isotopes was approximately 1 pg (signal to noise ratio at 3). The applicability of the developed stable isotope methodology was demonstrated by the determination of the selenium absorption and retention from foods in a pilot study using one human adult.

Importance of a conserved TCR J alpha-encoded tyrosine for T cell recognition of an HLA B27/peptide complex.

Human HLA B27-restricted cytotoxic T lymphocytes (CTL) specific for the influenza A epitope NP383-391 use similar TCR alpha and beta chains, with two closely related J alpha segments used by six of nine CTL clones from three unrelated donors (Bowness et al., Eur J. Immunol. 1993. 23: 1417-1421). The role of TCR complementarity-determining region (CDR)3alpha residues 93 and 100-102 was examined by site-directed mutagenesis, following expression of the TCR alpha and beta extracellular domains from one clone as a TCR zeta fusion heterodimer in rat basophil leukemia (RBL) cells. For the first time we have measured direct binding of tetrameric HLA B*2705/NP383-391 complexes to transfected TCR. Independently peptide-pulsed antigen-presenting cells (APC) were used to induce TCR-mediated degranulation of RBL transfectants. Our results show a key role for the conserved TCRalpha CDR3 J alpha-encoded residue Y102 in recognition of HLA B27/NP383-391. Thus the Y102D mutation abolished both tetramer binding and degranulation in the presence of peptide-pulsed APC. Even the Y102F mutation, differing only by a single hydroxyl group from the native TCR, abolished detectable degranulation. Further mutations F93A and S100R also abolished recognition. Interestingly, the N101A mutation recognized HLA B27/NP in functional assays despite having significantly reduced tetramer binding, a finding consistent with "kinetic editing" models of T cell activation. Modeling of the GRb TCR CDR3alpha loop suggests that residue Y102 contacts the HLA B*2705 alpha1 helix. It is thus possible that selection of germ-line TCRAJ-encoded residues at position 102 may be MHC driven.

Stuttering: a brief review.

The etiology of stuttering is controversial. The prevailing theories point to measurable neurophysical dysfunctions that disrupt the precise timing required to produce speech. Stuttering is a common disorder that usually resolves by adulthood. Almost 80 percent of children who stutter recover fluency by the age of 16 years. Mild stuttering is self-limited, but more severe stuttering requires speech therapy which is the mainstay of treatment. Delayed auditory feedback and computer-assisted training are currently used to help slow down speech and control other speech mechanisms. Pharmacologic therapy is seldom used, although haloperidol has been somewhat effective.

Relationships between cerebral blood flow velocities and arterial pressures during intra-aortic counterpulsation.

OBJECTIVE: To determine the effects of intra-aortic counterpulsation (IABP) on cerebral blood flow velocity. DESIGN: Prospective self-controlled study. SETTING: University hospital surgical intensive care unit. PARTICIPANTS: Nineteen cardiac surgical patients requiring perioperative IABP assistance. INTERVENTIONS: Simultaneous recording of transcranial Doppler middle cerebral artery blood flow velocity and arterial pressure in response to changes in the magnitude of augmentation and trigger ratio. MEASUREMENTS AND MAIN RESULTS: Instantaneous cerebral blood flow velocities correlated with arterial pressures during IABP (r = 0.650) at different magnitudes of augmentation and trigger ratios. The increase in arterial pressure during balloon inflation was associated with an increase in cerebral blood flow velocity, and the decrease in arterial pressure in response to balloon deflation was associated with a decrease in cerebral blood flow velocity that was dependent on the magnitude of augmentation. Different magnitudes of augmentation or trigger ratios had no effect on peak systolic cerebral blood flow velocity, mean cerebral blood flow velocity, mean arterial pressure, or the mean velocity-to-pressure ratio. Instantaneous cerebral blood flow velocity to arterial pressure ratios were lowest in response to balloon deflation at the time of pre-ejection. CONCLUSIONS: IABP modified the phasic profile of cerebral blood flow to reflect the arterial pressure waveform without affecting mean cerebral blood flow velocity. Peak systolic cerebral blood flow velocity was maintained in augmented beats despite the decreased systolic arterial pressure associated with afterload reduction. The acute decrease in cerebral blood flow velocity at pre-ejection was balanced by increased cerebral blood flow velocity during balloon inflation in diastole.

Effects of irradiation on the expression of major histocompatibility complex class I antigen and adhesion costimulation molecules ICAM-1 in human cervical cancer.

PURPOSE: We initiated studies to analyze the effects of high doses of gamma irradiation on the surface antigen expression of MHC Class I, Class II, and ICAM-1 on human cervical carcinoma cell lines. METHODS AND MATERIALS: The expression of surface antigens (MHC Class I, Class II, and ICAM-1) was evaluated by FACS analysis on two cervical cell lines at different time points, following their exposure to high doses of gamma irradiation (i.e., 25.00, 50.00, and 100.00 Gy). RESULTS: The CaSki and SiHa cervical cancer cells we analyzed in this study expressed variable levels of MHC Class I and ICAM-1 antigens, while Class II surface antigens were not detectable. Whereas irradiation doses of 25.00 Gy were not sufficient to totally block cell replication in both cell lines, exposure to 50.00 or 100.00 Gy was able to completely inhibit cell replication. Range doses from 25.00 to 100.00 Gy significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation but were unable to induce neoexpression of antigens previously not expressed by these cells (i.e., MHC Class II). Importantly, such upregulation was shown to be dose dependent, with higher radiation doses associated with increased antigen expression. Moreover, when the kinetic of this upregulation was studied after 2 and 6 days after irradiation, it was shown to be persistent and lasted until all the cells died. CONCLUSIONS: These findings may partially explain the increased immunogenicity of tumor cells following irradiation and may suggest enhanced immune recognition in tumor tissue in patients receiving radiation therapy.