Orosomucoid (alpha1-acid glycoprotein) plasma concentration and genetic variants: effects on human immunodeficiency virus protease inhibitor clearance and cellular accumulation.

BACKGROUND AND OBJECTIVE: Protease inhibitors are highly bound to orosomucoid (ORM) (alpha1-acid glycoprotein), an acute-phase plasma protein encoded by 2 polymorphic genes, which may modulate their disposition. Our objective was to determine the influence of ORM concentration and phenotype on indinavir, lopinavir, and nelfinavir apparent clearance (CL(app)) and cellular accumulation. Efavirenz, mainly bound to albumin, was included as a control drug. METHODS: Plasma and cells samples were collected from 434 human immunodeficiency virus-infected patients. Total plasma and cellular drug concentrations and ORM concentrations and phenotypes were determined. RESULTS: Indinavir CL(app) was strongly influenced by ORM concentration (n = 36) (r2 = 0.47 [P = .00004]), particularly in the presence of ritonavir (r2 = 0.54 [P = .004]). Lopinavir CL(app) was weakly influenced by ORM concentration (n = 81) (r2 = 0.18 [P = .0001]). For both drugs, the ORM1 S variant concentration mainly explained this influence (r2 = 0.55 [P = .00004] and r2 = 0.23 [P = .0002], respectively). Indinavir CL(app) was significantly higher in F1F1 individuals than in F1S and SS patients (41.3, 23.4, and 10.3 L/h [P = .0004] without ritonavir and 21.1, 13.2, and 10.1 L/h [P = .05] with ritonavir, respectively). Lopinavir cellular exposure was not influenced by ORM abundance and phenotype. Finally, ORM concentration or phenotype did not influence nelfinavir (n = 153) or efavirenz (n = 198) pharmacokinetics. CONCLUSION: ORM concentration and phenotype modulate indinavir pharmacokinetics and, to a lesser extent, lopinavir pharmacokinetics but without influencing their cellular exposure. This confounding influence of ORM should be taken into account for appropriate interpretation of therapeutic drug monitoring results. Further studies are needed to investigate whether the measure of unbound drug plasma concentration gives more meaningful information than total drug concentration for indinavir and lopinavir.

Ceftriaxone--bilirubin-albumin interactions in the neonate: an in vivo study.

The in vivo bilirubin-albumin binding interaction of ceftriaxone (CRO) was investigated in 14 non-jaundiced newborns, aged 33-42 weeks of gestation, during the first few days of life after they had reached stable clinical condition. CRO (50 mg/kg) was infused intravenously over 30 min. The competitive binding effect of CRO on the bilirubin-albumin complex was estimated by determining the reserve albumin concentration (RAC) at baseline, at the end of CRO infusion, and at 15 and 60 min thereafter. Immediately after the end of drug administration, RAC decreased from 91.9 (+/- 25.1) mumol/l to 38.6 (+/- 10.1) mumol/l (P = 0.0001). At the same time the plasma bilirubin toxicity index (PBTI) increased from 0.64 (+/- 0.40) before drug infusion to 0.96 (+/- 0.44) thereafter (P = 0.0001). The highest displacement factor (DF) was calculated to be 2.8 (+/- 0.6) at the end of drug infusion. Average total serum bilirubin concentrations decreased from a baseline value of 59.6 (+/- 27.0) mumol/l to 55.2 (+/- 27.1) mumol/l (P = 0.026). Sixty minutes after the end of CRO infusion, RAC was 58.3 (+/- 21.7) mumol/l, PBTI regained baseline, but DF was still 1.9 (+/- 0.2). No adverse events were recorded. Our results demonstrate significant competitive interaction of CRO with bilirubin-albumin binding in vivo. Thus, ceftriaxone should not be given to the neonate at risk of developing bilirubin encephalopathy.

Systematic review of population pharmacokinetic analyses of imatinib and relationships with treatment outcomes

Several population pharmacokinetic (PPK) analyses of the anticancer drug imatinib have been performed to investigate different patient populations and covariate effects. The present analysis offers a systematic qualitative and quantitative summary and comparison of those. Its primary objective was to provide useful information for evaluating the expectedness of imatinib plasma concentration measurements in the frame of therapeutic drug monitoring. The secondary objective was to review clinically important concentration-effect relationships to provide help in evaluating the potential suitability of plasma concentration values. Nine PPK models describing total imatinib plasma concentration were identified. Parameter estimates were standardized to common covariate values whenever possible. Predicted median exposure (Cmin) was derived by simulations and ranged between models from 555 to 1388 ng/mL (grand median: 870 ng/mL and interquartile "reference" range: 520-1390 ng/mL). Covariates of potential clinical importance (up to 30% change in pharmacokinetic predicted by at least 1 model) included body weight, albumin, α1 acid glycoprotein, and white blood cell count. Various other covariates were included but were statistically not significant or seemed clinically less important or physiologically controversial. Concentration-response relationships had more importance below the average reference range and concentration-toxicity relationships above. Therapeutic drug monitoring-guided dosage adjustment seems justified for imatinib, but a formal predictive therapeutic range remains difficult to propose in the absence of prospective target concentration intervention trials. To evaluate the expectedness of a drug concentration measurement in practice, this review allows comparison of the measurement either to the average reference range or to a specific range accounting for individual patient characteristics. For future research, external PPK model validation or meta-model development should be considered.

Quality of care and survival of haemodialysed patients in western Switzerland.

BACKGROUND: Many factors affect survival in haemodialysis (HD) patients. Our aim was to study whether quality of clinical care may affect survival in this population, when adjusted for demographic characteristics and co-morbidities. METHODS: We studied survival in 553 patients treated by chronic HD during March 2001 in 21 dialysis facilities in western Switzerland. Indicators of quality of care were established for anaemia control, calcium and phosphate product, serum albumin, pre-dialysis blood pressure (BP), type of vascular access and dialysis adequacy (spKt/V) and their baseline values were related to 3-year survival. The modified Charlson co-morbidity index (including age) and transplantation status were also considered as a predictor of survival. RESULTS: Three-year survival was obtained for 96% of the patients; 39% (211/541) of these patients had died. The 3-year survival was 50, 62 and 69%, respectively, in patients who had 0-2, 3 and >or=4 fulfilled indicators of quality of care (test for linear trend, P < 0.001). In a Cox multivariate analysis model, the absence of transplantation, a higher modified Charlson's score, decreased fulfilment of indicators of good clinical care and low pre-dialysis systolic BP were independent predictors of death. CONCLUSION: Good clinical care improves survival in HD patients, even after adjustment for availability of transplantation and co-morbidities.

T helper 1 (Th1) and Th2 characteristics start to develop during T cell priming and are associated with an immediate ability to induce immunoglobulin class switching.

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cgamma2a and Cgamma1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon gamma in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone. The addition of killed Bordetella pertussis to the hapten-protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten-protein can proceed in a node where there is substantial Th1 activity.

ANIBAL, stable isotope-based quantitative proteomics by aniline and benzoic acid labeling of amino and carboxylic groups

Identification and relative quantification of hundreds to thousands of proteins within complex biological samples have become realistic with the emergence of stable isotope labeling in combination with high throughput mass spectrometry. However, all current chemical approaches target a single amino acid functionality (most often lysine or cysteine) despite the fact that addressing two or more amino acid side chains would drastically increase quantifiable information as shown by in silico analysis in this study. Although the combination of existing approaches, e.g. ICAT with isotope-coded protein labeling, is analytically feasible, it implies high costs, and the combined application of two different chemistries (kits) may not be straightforward. Therefore, we describe here the development and validation of a new stable isotope-based quantitative proteomics approach, termed aniline benzoic acid labeling (ANIBAL), using a twin chemistry approach targeting two frequent amino acid functionalities, the carboxylic and amino groups. Two simple and inexpensive reagents, aniline and benzoic acid, in their (12)C and (13)C form with convenient mass peak spacing (6 Da) and without chromatographic discrimination or modification in fragmentation behavior, are used to modify carboxylic and amino groups at the protein level, resulting in an identical peptide bond-linked benzoyl modification for both reactions. The ANIBAL chemistry is simple and straightforward and is the first method that uses a (13)C-reagent for a general stable isotope labeling approach of carboxylic groups. In silico as well as in vitro analyses clearly revealed the increase in available quantifiable information using such a twin approach. ANIBAL was validated by means of model peptides and proteins with regard to the quality of the chemistry as well as the ionization behavior of the derivatized peptides. A milk fraction was used for dynamic range assessment of protein quantification, and a bacterial lysate was used for the evaluation of relative protein quantification in a complex sample in two different biological states

Identification of secretory IgA, free secretory piece and serum IgA in the ovine and caprine species.

Caprine and ovine IgA were identified by cross-reaction with anti-human and anti-bovine IgA sera in colostrum, mature milk, saliva, urine and serum. Secretory component (SC) was shown in the free form and associated with polymeric serum IgA in secretions. Mean molecular weights were determined for the IgA and the free secretory components. The high IgA content of saliva suggested that it was a major secretory immunoglobulin in these species. Traces of secretory IgA were also found in normal sera but most of the serum IgA had no secretory determinant. Secretory IgA, serum IgA and free secretory component were purified. Levels of the sheep and goat immunoglobulins were measured in various fluids.

Exudative mineral losses after serious burns: a clue to the alterations of magnesium and phosphate metabolism.

Hypomagnesemia and hypophosphatemia are frequent after severe burns; however, increased urinary excretion does not sufficiently explain the magnitude of the mineral depletion. We measured the mineral content of cutaneous exudates during the first week after injury. Sixteen patients aged 34 +/- 9 y (mean +/- SD) with thermal burns were studied prospectively and divided in 3 groups according to the extent of their burn injury and the presence or absence of mineral supplements: group 1 (n = 5), burns covering 26 +/- 5% of body surface; group 2 (n = 6), burns covering 41 +/- 10%; and group 3 (n = 5), burns covering 42 +/- 6% with prescription of magnesium and phosphate supplements. Cutaneous exudates were extracted from the textiles (surgical drapes, dressings, sheets, etc) surrounding the patients from day 1 to day 7 after injury. Mean magnesium serum concentrations decreased below reference ranges in 12 patients between days 1 and 4 and normalized thereafter. Phosphate, normal on day 0, was low during the first week. Albumin concentrations, normal on day 0, decreased and remained low. Urinary magnesium and phosphate excretion were within reference ranges and not larger in group 3. Mean daily cutaneous losses were 16 mmol Mg/d and 11 mmol P/d (largest in group 2). Exudative magnesium losses were correlated with burn severity (r = 0.709, P = 0.003). Cutaneous magnesium losses were nearly four times larger than urinary losses whereas cutaneous phosphate losses were smaller than urinary phosphate losses. Mean daily losses of both magnesium and phosphate were more than the recommended dietary allowances. Exudative losses combined with urinary losses largely explained the increased mineral requirements after burn injury.

Origin-related, environmental, sex, and age determinants of immunocompetence, susceptibility to ectoparasites, and disease symptoms in the barn owl

Knowledge of the role of origin-related, environmental, sex, and age factors on host defence mechanisms is important to understand variation in parasite intensity. Because alternative components of parasite defence may be differently sensitive to various factors, they may not necessarily covary. Many components should therefore be considered to tackle the evolution of host-parasite interactions. In a population of barn owls (Tyto alba), we investigated the role of origin-related, environmental (i.e. year, season, nest of rearing, and body condition), sex, and age factors on 12 traits linked to immune responses [humoral immune responses towards sheep red blood cells (SRBC), human serum albumin (HSA) and toxoid toxin TT, T-cell mediated immune response towards the mitogen phytohemagglutinin (PHA)], susceptibility to ectoparasites (number and fecundity of Carnus haemapterus, number of Ixodes ricinus), and disease symptoms (size of the bursa of Fabricius and spleen, proportion of proteins that are immunoglobulins, haematocrit and blood concentration in leucocytes). Cross-fostering experiments allowed us to detect a heritable component of variation in only four out of nine immune and parasitic parameters (i.e. SRBC- and HSA-responses, haematocrit, and number of C. haemapterus). However, because nestlings were not always cross-fostered just after hatching, the finding that 44% of the immune and parasitic parameters were heritable is probably an overestimation. These experiments also showed that five out of these nine parameters were sensitive to the nest environment (i.e. SRBC- and PHA-responses, number of C. haemapterus, haematocrit and blood concentration in leucocytes). Female nestlings were more infested by the blood-sucking fly C. haemapterus than their male nestmates, and their blood was less concentrated in leucocytes. The effect of year, season, age (i.e. reflecting the degree of maturation of the immune system), brood size, position in the within-brood age hierarchy, and body mass strongly differed between the 12 parameters. Different components of host defence mechanisms are therefore not equally heritable and sensitive to environmental, sex, and age factors, potentially explaining why most of these components did not covary.

Physicochemical and biological characterization of single-walled and double-walled carbon nanotubes in biological media

To study the toxicity of nanoparticles under relevant conditions, it is important to reproducibly disperse nanoparticles in biological media in in vitro and in vivo studies. Here, single-walled nanotubes (SWNTs) and double-walled nanotubes (DWNTs) were physicochemically and biologically characterized when dispersed in phosphate-buffered saline (PBS) and bovine serum albumin (BSA). BSA-SWNT/DWNT interaction resulted in a reduction of aggregation and an increase in particle stabilization. Based on the protein sequence coverage and protein binding results, DWNTs exhibited higher protein binding than SWNTs. SWNT and DWNT suspensions in the presence of BSA increased interleukin-6 (IL-6) levels and reduced tumor necrosis factor-alpha (TNF-α) levels in A549 cells as compared to corresponding samples in the absence of BSA. We next determined the effects of SWNTs and DWNTs on pulmonary protein modification using bronchoalveolar lavage fluid (BALF) as a surrogate collected form BALB/c mice. The BALF proteins bound to SWNTs (13 proteins) and DWNTs (11 proteins), suggesting that these proteins were associated with blood coagulation pathways. Lastly, we demonstrated the importance of physicochemical and biological alterations of SWNTs and DWNTs when dispersed in biological media, since protein binding may result in the misinterpretation of in vitro results and the activation of protein-regulated biological responses.

Cerebral and extracerebral cholesterol metabolism and CSF markers of Alzheimer's disease.

The disturbances of the cholesterol synthesis and metabolism described in Alzheimer's disease (AD) may be both a consequence of the neurodegenerative process and a contributor to the pathogenesis. These putative relationships and their underlying mechanisms are not well understood. The aim of this study was to evaluate the relationship between the cerebral and extracerebral cholesterol synthesis and metabolism, and the AD pathology as reflected by CSF markers in humans. We evaluated the relationships between the plasma and the cerebrospinal fluid (CSF) concentrations of cholesterol, the cholesterol precursors lanosterol, lathosterol and desmosterol, and the cholesterol elimination products 24S-hydroxycholesterol and 27-hydroxycholesterol, and the CSF markers for AD pathology Aβ1-42 and p-tau181 in 86 subjects with normal cognition and in 107 AD patients. CSF desmosterol, cholesterol and 24S-hydroxycholesterol in the AD group, and CSF 24S-hydroxycholesterol in the control group correlated with the p-tau181 levels. Neither CSF nor plasma concentrations of the included compounds correlated with the CSF Aβ1-42 levels. In multivariate regression tests including age, gender, albumin ratio, number of the APOEε4 alleles, and diagnosis, p-tau181 levels independently predicted the CSF desmosterol, cholesterol and 24S-hydroxycholesterol concentrations. The associations remained significant for CSF cholesterol and 24S-hydroxycholesterol when analyses were separately performed in the AD group. The results suggest that alterations of CNS cholesterol de novo genesis and metabolism are related to neurodegeneration and in particular to the cerebral accumulation of phosphorylated tau.

Inhibition of glutathione efflux in the perfused rat liver and isolated hepatocytes by organic anions and bilirubin. Kinetics, sidedness, and molecular forms.

Using isolated, in situ, single-pass perfused rat livers, incubations of freshly isolated hepatocytes, and sinusoidal membrane-enriched vesicles, we and others have shown the saturability of transport (efflux) of hepatic glutathione (GSH). These observations have implicated a carrier mechanism. Our present studies were designed to provide further evidence in support of a carrier mechanism for hepatic GSH efflux by demonstrating competition by liver-specific ligands which are taken up by hepatocytes. Perfusing livers with different substances, we found that: (a) sulfobromophthalein-GSH (BSP-GSH) had a dose-dependent and fully reversible inhibitory effect on GSH efflux, while GSH alone did not have any effect; (b) taurocholate had no inhibitory effect; (c) all of the organic anions studied, i.e., BSP, rose bengal, indocyanine green, and unconjugated bilirubin (UCB), manifested potent, dose-dependent inhibitory effects, with absence of toxic effects and complete reversibility of inhibition in the case of UCB. The inhibitory effects of UCB could be overcome partially by raising (CoCl2-induced) hepatic GSH concentration. Because of the physiological importance of UCB, we conducted a detailed study of its inhibitory kinetics in the isolated hepatocyte model in the range of circulating concentrations of UCB. Studies with Cl- -free media, to inhibit the uptake of UCB by hepatocytes, showed that the inhibition of GSH efflux by UCB is apparently from inside the cell. This point was confirmed by showing that the inhibition is overcome only when bilirubin-loaded cells are cleared of bilirubin (incubation with 5% bovine serum albumin). Using Gunn rat hepatocytes and purified bilirubin mono- and diglucuronides, we found that both UCB and glucuronide forms of bilirubin inhibit GSH efflux in a dose-dependent manner. We conclude that the organic anions, although taken up by a mechanism independent of GSH, may competitively inhibit the carrier for GSH efflux from inside the hepatocyte.

Hypercalcaemia and acute renal failure after major burns: An under-diagnosed condition.

BACKGROUND: Hypercalcaemia has been shown to occur in about 20% of patients with major burns requiring prolonged intensive care unit (ICU) treatment, and it may be associated with renal failure. Having observed the early onset of hypercalcaemia, the study aimed to determine the frequency and timing of this condition in a European patient cohort. METHODS: A retrospective cohort study on a prospectively collected, computerised database of the 225 burn-injury ICU admissions between 2001 and 2007 was undertaken. The inclusion criteria included: burns &gt;20% of the body surface area (BSA) or in-hospital stay &gt;20 days. Hypercalcaemia was defined as an ionised plasma calcium (Ca(2+)) concentration &gt;1.32 mmol l(-1) (or total corrected calcium=[Ca]c&gt;2.55 mmol l(-1)). Four emblematic cases are reported in this article. RESULTS: A total of 73 patients met the inclusion criteria (age: 13-88 years, burns: 12-85% BSA): of these, 22 (30%) developed hypercalcaemia. The median time to the first hypercalcaemia value was 21 days. Only 11 patients had both high Ca(2+) and elevated [Ca]c (which remained normal in others). The risk factors of the disorder were burned surface (p=0.017) and immobilisation (fluidised bed use: p&lt;0.05, duration: p=0.02) followed by burned BSA. Acute renal failure tended to be more frequent in hypercalcaemic patients (five (23%) vs. three (6%): p=0.11), while mortality was not increased. The disorder resolved with hydration and mobilisation in most cases: pamidronate was successful in three cases that were most severe. CONCLUSION: Hypercalcaemia and associated acute renal failure occur more frequently and earlier than previously reported. Determining the ionised Ca rather than the total Ca with albumin correction enables earlier detection of hypercalcaemia. Bisphosphonates are an effective treatment option in controlling severe hypercalcaemia and preventing bone loss.

A new measurement of energy expenditure and physical activity in patients treated by maintenance hemodialysis

Purpose: The accurate estimation of total energy expenditure (TEE) is essential to allow the provision of nutritional requirements in patients treated by maintenance hemodialysis (MHD). The measurement of TEE and resting energy expenditure (REE) by direct or indirect calorimetry and doubly labeled water are complicated, timeconsuming and cumbersome in this population. Recently, a new system called SenseWear® armband (SWA) was developed to assess TEE, physical activity and REE. This device works by measurements of body acceleration in two axes, heat production and steps counts. REE measured by indirect calorimetry and SWA are well correlated. The aim of this study was to determine TEE, physical activity and REE on patients on MHD using this new device. Methods and materials: Daily TEE, REE, step count, activity time, intensity of activity and lying time were determined for 7 consecutive days in unselected stable patients on MHD and sex, age and weightmatched healthy controls (HC). Patients with malnutrition, cancer, use of immunosuppressive drugs, hypoalbumemia <35 g/L and those hospitalized in the last 3 months, were excluded. For MHD patients, separate analyses were conducted in dialysis and non-dialysis days. Relevant parameters known to affect REE, such as BMI, albumin, pre-albumin, hemoglobin, Kt/V, CRP, bicarbonate, PTH, TSH, were recorded. Results: Thirty patients on MHD and 30 HC were included. In MHD patients, there were 20 men and 10 women. Age was 60,13 years ± 14.97 (mean ± SD), BMI was 25.77 kg/m² ± 4.73 and body weight was 74.65 kg ± 16.16. There were no significant differences between the two groups. TEE was lower in MHD patients compared to HC (28.79 ± 5.51 SD versus 32.91 ± 5.75 SD kcal/kg/day; p <0.01). Activity time was significantly lower in patients on MHD (101.3 ± 12.6SD versus 50.7 ± 9.4 SD min; p = 0.0021). Energy expenditure during the time of activity was significantly lower in MHD patients. MHD patients walked 4543 ± 643 SD vs 8537 ± 744 SD steps per day (p <0.0001). Age was negatively correlated with TEE (r = -0.70) and intensity of activity (r = -0.61) in HC, but not in patients on MHD. TEE showed no difference between dialysis and non-dialysis days (29.92 ± 2.03 SD versus 28.44 ± 1.90 SD kcal/kg/day; p = NS), reflecting a lack of difference in activity (number of steps, time of physical activity) and REE. This finding was observed in MHD patients both older and younger than 60 years. However, age stratification appeared to have an influence on TEE, regardless of dialysis day, (29.92 ± 2.07 SD kcal/kg/day for <60 years-old versus 27.41 ± 1.04 SD kcal/kg/day for ≥60 years old), although failing to reach statistical significance. Conclusion: Using SWA, we have shown that stable patients on MHD have a lower TEE than matched HC. On average, a TEE of 28.79 kcal/kg/day, partially affected by age, was measured. This finding gives support to the clinical impression that it is difficult and probably unnecessary to provide an energy amount of 30-35 kcal/kg/day, as proposed by international guidelines for this population. In addition, we documented for the first time that MHD patients exert a reduced physical activity as compared to HC. There were surprisingly no differences in TEE, REE and physical activity parameters between dialysis and non-dialysis days. This observation might be due to the fact that patients on MHD produce a physical effort to reach the dialysis centre. Age per se did not influence physical activity in MHD patients, contrary to HC, reflecting the impact of co-morbidities on physical activity in this group of patients.