Database of Optimized Proteomic Quantitative Methods for Human Drug Disposition-Related Proteins for Applications in Physiologically Based Pharmacokinetic Modeling.

The purpose of this study was to create an open access repository of validated liquid chromatography tandem mass spectrometry (LC-MS/MS) multiple reaction monitoring (MRM) methods for quantifying 284 important proteins associated with drug absorption, distribution, metabolism, and excretion (ADME). Various in silico and experimental approaches were used to select surrogate peptides and optimize instrument parameters for LC-MS/MS quantification of the selected proteins. The final methods were uploaded to an online public database (QPrOmics; www.qpromics.uw.edu/qpromics/assay/), which provides essential information for facile method development in triple quadrupole mass spectrometry (MS) instruments. To validate the utility of the methods, the differential tissue expression of 107 key ADME proteins was characterized in the tryptic digests of the pooled subcellular fractions of human liver, kidneys, intestines, and lungs. These methods and the data are critical for development of physiologically based pharmacokinetic (PBPK) models to predict xenobiotic disposition.

Ontogeny of Hepatic Drug Transporters as Quantified by LC-MS/MS Proteomics.

Protein expression of major hepatic uptake and efflux drug transporters in human pediatric (n = 69) and adult (n = 41) livers was quantified by liquid chromatography / tandem mass spectroscopy (LC-MS/MS). Transporter protein expression of OCT1, OATP1B3, P-gp, and MRP3 was age-dependent. Particularly, significant differences were observed in transporter expression (P < 0.05) between the following age groups: neonates vs. adults (OCT1, OATP1B3, P-gp), neonates or infants vs. adolescents and/or adults (OCT1, OATP1B3, and P-gp), infants vs. children (OATP1B3 and P-gp), and adolescents vs. adults (MRP3). OCT1 showed the largest increase, of almost 5-fold, in protein expression with age. Ontogenic expression of OATP1B1 was confounded by genotype and was revealed only in livers harboring SLCO1B1*1A/*1A. In livers >1 year, tissues harboring SLCO1B1*14/*1A showed 2.5-fold higher (P < 0.05) protein expression than SLCO1B1*15/*1A. Integration of these ontogeny data in physiologically based pharmacokinetic (PBPK) models will be a crucial step in predicting hepatic drug disposition in children.

A novel HLA-B allele, HLA-B*35:279, identified by sequencing-based typing in a Czech patient.

The identification of a novel HLA-B*35:279 allele in a Czech patient is described. This allele is identical to the B*35:03:01 variant except the G/A nucleotide exchange at position 652 of the HLA-B gene that corresponds to the amino acid substitution from valine to isoleucine in alpha 3 domain of the HLA-B antigen.

Sudden cardiac death thirty years ago and at present. The role of autonomic disturbances in acute myocardial infarction revisited.

The most common cause of sudden cardiac death is ventricular fibrillation (VF). In addition to the status, size and location of the ventricular focus, a major pathogenic mechanism triggering VF is autonomic dysbalance (disturbance). This term refers to a wide range of reflex changes in the ratio of sympathetic to vagal ventricular activation over time, occurring immediately after coronary artery occlusion at the onset of acute myocardial infarction (AMI). Another trigger of VF is autonomic disturbance due to emotional stress. Experimental and clinical research into autonomic disturbances associated with coronary artery occlusion and emotional stress was given considerable attention as early as some 30 years ago when researchers were already searching for ways of inhibiting autonomic disturbances using predominant sympathetic and vagal activation by beta-blockers (BB) and atropine, respectively. The aim of our paper is to compare results obtained 30 years ago with current status of experimental and clinical research into SCD prevention. Another aim is to identify questions that have remained unanswered to date; answers to these outstanding questions could help further reduce the risk of SCD.

Allo-SCT in children with high-risk leukemia using unmanipulated grafts from alternative donors.

Allogeneic HSCT is a curative treatment for high-risk leukemia. In Europe, approximately 15% of children have an HLA-matched sibling, but in 65-70% HLA allele-matched (9-10/10) unrelated donors (UD) can be identified. Transplantation using an HLA partially mismatched donor, unrelated cord blood or haploidentical family donor with graft manipulation is then considered with preference on the basis of local experience and/or availability. Here we evaluate the outcomes of 87 consecutive patients with leukemia transplanted with unmanipulated graft from matched or partially mismatched UD or cord blood (CB) at our institution between January 2001 and December 2007. Within the median follow-up of 30 months, the acute GVHD grade II was diagnosed in 70.9% patients; grades III-IV only in 4.6%. The overall incidence of chronic GVHD was 43.3% (extensive in 34.9%). The probability of 3-year EFS was 59.5% and that of 3-year overall survival was 66.9%. TRM at day +100 was 4.5%, and overall it was 13.8%. Fourteen patients (16.1%) died as a consequence of post-transplant leukemia relapse. We conclude that the prognosis of patients transplanted for leukemia using unmanipulated grafts from HLA-matched or partially mismatched UD or CB is comparable and satisfactory. TRM and relapse rate are lower than in the earlier period.

[HLA-DPB1 gene analysis in haematopoietic stem cell transplantations]. / Analýza HLA-DPB1 genu u transplantací hematopoetických kmenových bunek.

BACKGROUND: The HLA-DPB1 gene is probably one of HLA class II genes affecting the haematopoietic stem cell transplantation. The aim of the study was to analyse the HLA-DPB1 gene and its match/mismatch in patients transplanted from unrelated HSC donors. The PCR-SSP method was used for the typing of the HLA system. METHODS AND RESULTS: The cohort consisted of 201 pairs of patient/unrelated HSC donor. Match in HLA-A, -B, -Cw, -DRBI and -DQBI (e.g. 10/10 match) was found in 81 pairs. The HLA-DPBI was tested in them. 18 different HLA-DPBI alleles were identified in this cohort. Complete match (e.g.12/12) was detected in 3% of the 201 analysed pairs only. CONCLUSIONS: The probability of finding 12/12 matching unrelated HSC donor is limited due to the high percentage of mismatches and inaccessibility of previous HLA-DPB1 results.

[Significance of confirmatory testing of HLA system in unrelated haematopoietic stem cell donors]. / Význam konfirmacních testu HLA systému nepríbuzných dárcu hematopoetických kmenových bunek.

BACKGROUND: Haematopoietic stem cell transplantation is a standard curative therapy for some acquired haematological diseases and inherited metabolic and immunological disorders. The HLA compatibility in five loci (HLA class I -A, -B and -C and HLA class II -DRB1 and -DQB 1) of the donor/recipient pair is a prerequisite for the success of haematopoietic stem cell transplantation which represents a process of adoption donors immunity. METHODS AND RESULTS: HLA is the most polymorphic system in the human genome and this polymorphism is exactly detected by molecular genetics methods on DNA level only. In period of 2001-2004 we performed confirmatory testing of 366 unrelated haematopoietic stem cells donors from Czech and foreign registers for 256 patients. Only 16% of the donors completely matched the patients in all HLA loci. We detected HLA mismatches in the samples of 81% patient/donor pairs but these results were consonant with previous results from registers. 3% of confirmatory samples were discrepant with previous registry data. CONCLUSIONS: Despite of increasing number of available unrelated haematopoietic stem cell donors and the quality of registry HLA typing the possibility of finding the completely match donor is still limited.

[HLA-DRB1/DQA1/DQB1 alleles and haplotypes in Czech children with celiac sprue]. / HLA-DRB1/DQA1/DQB1 alely a haplotypy ceských deti s celiakální sprue.

BACKGROUND: The celiac disease (CD) is a multifactor disease resulting from a life time abnormal immune response to gluten accompanied by autoimmune characteristics, which can in sensitive individuals evoke small bowel mucosa morphologic changes. The genetically sensitive individual to CD has not been defined yet, it is obvious, however, that this illness is closely linked to the HLA class II genes. The objective of our study was to detect associations of HLA class II alleles and haplotypes DRB1/DQA1/DQB1 in Czech CD children. METHODS AND RESULTS: A group of Czech CD children diagnosed according to ESPGHAN criteria was genotyped HLA for alleles of DRB1/DQA1/DQB1 loci. Genotyping of the HLA-DRB1/DQB1 haplotypes proved statistically significant association CD with haplotypes and alleles of this genetic system. 92.9% of patients have in their HLA phenotype allele DQA1*0501 in either cis or trans configuration with the DQB1 allele *0201/*0202. The extended HLA haplotype DRB*0301/DQA1*0501/DRB1*0201 as well as the haplotype DRB1*0701/DQA1*0201/DQB1*0202, are presented in 63.6% or in 61.0% CD patients respectively. The individual HLA class II alleles DRB*0301, *0701, DQA1*0201, *0501, DQB1*0201, *0202 and the above mentioned HLA haplotypes inclusively provide genotypic frequencies significantly different from healthy Czech individuals (P < or = 0.06 +/- 0.001). These results support the opinion that the HLA molecule expressed on the cellular surface as a alpha beta heterodimer encoded by the DQA1*0501 and DQB1*0201/02 alleles in either cis or trans configuration is responsible for the primary sensitivity to this disease. We were, however, not able to find an association of various clinical forms of the CD with a certain HLA haplotype in the followed group. CONCLUSION: The CD patients have in comparison with healthy population significantly different frequency of HLA class II haplotypes. Though the finding of these alleles is not sufficient for an explicit confirmation of this diagnosis, the proof of this risky haplotype/s may notably contribute to it, namely in case of potential or latent forms of this disease.

Cytotoxic T lymphocyte precursor frequency analysis in the selection of HLA matched unrelated donors for hematopoietic stem cell transplantation: the correlation of CTLp frequency with HLA class I genotyping and aGVHD development.

The selection of human leukocyte antigen (HLA) compatible unrelated donors for hematopoietic stem cell transplantation (HSCT) is based on the direct genotyping of HLA class I and class II alleles (HLA-A, -B, -C, -DRB1, -DQB1 loci). The cellular test estimating the frequency of cytotoxic T lymphocyte precursors (CTLp) has been included into the selection procedure of unrelated donors to detect the class I alloreactivity and to predict acute graft versus host disease (aGVHD) occurrence and severity. The relationship between HLA-A, -B, -C high/medium resolution genotyping and CTLp activation was analysed in the cohort of 78 unrelated donor/patient pairs indicated for HSCT. The high frequency of CTLp (> 1:100,000) correlated significantly (p < or = 0.0002) with the incompatibilities in alleles of HLA-A, -B, -C loci. Nevertheless, the results of HLA-A, -B, -C genotyping and CTLp assay are not fully alternative, suggesting that the CTLp test gives its specific information. The high CTLp frequency (CTLpf) in 14/35 pairs fully matched by HLA class-I alleles genotyping could reflect the influence of another factors upon the CTLp activation. On the contrary, the low CTLp frequency values (< or = 1:100,000) found in 8/43 pairs with existing HLA class-I alleles incompatibilities could indicate the immunological permissivity of these particular mismatches. The clinical relevance of the CTLp test for aGVHD prediction has been also analysed. The relationship between CTLp activation in vitro and the incidence and severity of aGVHD was evaluated in 37 patients who underwent allogeneic HSCT. The severe form of aGVHD (grade III-IV) developed in 9 of 18 cases (50%) with the high pretransplant CTLpf value. The patients with the low CTLpf (n = 19) suffered from the severe form of aGVHD in 2 cases (10%) only, the remaining 17 patients from this group were without aGVHD symptoms or developed only the mild form of aGVHD (I-II). The relationship between CTLp results and the incidence and severity of aGVHD was found statistically significant (p < or = 0.01).

The relationship between HA-1 compatibility and the activation of helper T lymphocyte precursors.

The relationship between the compatibility in minor histocompatibility HA-1 antigen and the activation of helper (IL-2 producing) T lymphocyte precursors in vitro was studied in the group of 17 HLA-A2 positive HLA identical siblings. Although the number of pairs studied is still small, no correlation has been found between HA-1 compatibility and helper T lymphocyte precursors activation. The results presented here could suggest the possibility that the HTLp assay does not have to be a relevant parameter for the detection of HA-1 mismatches in HLA identical siblings.

[Variation in the R-R intervals on the electrocardiogram. A new diagnostic method in cardiology]. / Variabilita intervalu R-R elektrokardiogramu. Novejsí pomocná diagnostická metoda v kardiologii.

Periodic heart rate fluctuation depends on the oscillation of sympathetic and vagal activation of the heart. Periodic retardation and acceleration of heart rate related to respiration and to blood pressure changes can be registered on the ECG as the "variability of R-R intervals". Testing procedures of the variability of R-R intervals at rest, during deep breathing, daily activities, during exercise and other stress tests are described in the paper. For the evaluation of the R-R interval's variability, current statistical methods are used (e.g. mean with standard deviation, variation coefficient, mean beat to beat differences in R-R intervals etc.). Power spectral analysis in the variability of 200-600 successive R-R intervals commonly performed today uses either rapid Fourier transformation or the autoregulation model. The analysis shows high- and low frequency peaks corresponding to the rapid and slow oscillations in heart rate. Evaluation of the R-R interval variability, especially using power spectrum analysis, gave good results in testing drugs, e.g., beta blockers, calcium antagonists and antiarrhythmic drugs. Variability of R-R intervals is reduced in conditions affecting the cardiac autonomous nervous system such as diabetes. It is also decreased in patients with ischaemic heart disease and in those with cardiac failure of different aetiology. The decrease is not an expression of the disease itself: it shows an alteration in neurovegetative tonicity in the particular disease condition. The decreased variability of R-R intervals in patients with ischaemic heart disease has an important prognostic value. The predominance of the sympathetic over the depressed vagal activity signalizes an increased risk of sudden coronary death.

Dogs at high or low risk of ventricular fibrillation. An experimental study of acute myocardial ischaemia.

In the last 15 years, measurement of the ventricular fibrillation threshold (VFT) after ligation of the coronary artery in anaesthetised dogs has become our standard method for the evaluation of the stabilizing effect of antifibrillation drugs. Analysis of a group of 143 dogs revealed that in 75 animals the VFT 8 minutes after the ligation of the coronary artery dropped to less than 1 mA (high risk group), while in the remaining 68 dogs the decrease was smaller and not below 1 mA (low risk animals). The difference between the groups could be seen already before the ligation of the coronary artery. The high risk animals had a lower VFT and a higher heart rate. The groups also differed in the response to drugs administered 15 minutes after the ligation of the coronary artery. Metipranolol, a liposoluble beta blocker of the beta 1 and 2 cardiac receptors (Trimepranol Spofa 0.3 mg.kg-1 b.w.), increased greatly the VFT in both groups already 8 minutes after the injection of the drug and eliminated the difference between the groups. Flunitrazepam (Rohypnol Hoffmann-La Roche 0.25 mg.kg-1 b.w.) increased the VFT less than metipranolol and the difference between the groups disappeared only 30 minutes after its injection. Celiprolol (Selectol Linz Chemie 3.0 mg.kg-1 b.w.) blocking beta 1 and stimulating beta 2 receptors as well as trimecaine (sodium channel blocker, Mesocain Spofa 3.0 mg.kg-1 b.w.) led only to a small insignificant increase in the VFT and the difference between the groups of dogs remained.(ABSTRACT TRUNCATED AT 250 WORDS)

[The patient after heart transplantation]. / Obraz nemocného po srdecní transplantaci.

The data of the first 100 patients undergoing heart transplantation in the period between January 1984 and May 1993 were analyzed. Of this group, 57 patients are alive. Out of the total of 43 deaths, 14 patients died from graft failure within the first postoperative days, 6 died from surgical complications, 11 from infection, 10 deaths were due to accelerated coronary atherosclerosis, and 2 patients died from tumours. Early mortality rates (within 30 days since surgery) were 37% and 17% in patients operated on between 1984-88 and between 1989-93, respectively. The health condition of heart transplant recipients is affected by side effects of immunosuppressive therapy. Forty per cent of patients re-develop systemic hypertension within the first post-transplantation year. Five years after transplantation, hypertension is detected in 60% of patients. Elevated serum creatinine levels are present in 70% of patients by the end of the first post-transplantation year. In the ensuing period, there is no progression in renal function impairment, which does not require cyclosporin withdrawal and is not associated with the development of hypertension. In the first post-transplantation year, 45% of patients are markedly obese. All patients with overweight and obesity show markedly raised levels of serum cholesterol. Another undesirable effect (mainly due to corticosteroid therapy) is the development of ulcers in 16% of patients. Heart transplantation has become an established method at the Institute for Clinical and Experimental Medicine in Prague. Despite the above pitfalls, heart transplantation substantially prolongs the life of patients and dramatically alters the quality of their life.

[Directed sympathetic denervation of the heart--an actual therapeutic method or myth?]. / Cílená sympatická denervace srdce--aktuální lécebná metoda nebo legenda?

Aimed sympathetic denervation of the heart, i. e. bilateral removal of the stellate ganglion Th 1-Th 3 was used in Czechoslovakia since 1981 in eight patients who were followed up for prolonged periods. The patients were operated on account of angina at rest which caused deterioration of the quality of their lives. In case of extensive affection of the coronary arteries surgical treatment was not feasible and pharmacological treatment did not bring relief. At the time of a five-year check up 7 patients survived. All patients reported relief and their efficiency during ergometry improved. Four of the patients worked for several years. The authors discuss whether the above treatment is still justified.

[The effect of mechanical support of the heart on the extent of experimental myocardial necrosis. I]. / Vliv mechanické podpory srdce na rozsah experimentální nekrózy myokardu. I. cást.

The authors investigated the influence of mechanical cardiac support with a non-pulsating blood flow on the size of an ischaemic after ligature of the descendent branch of the left coronary artery in the dog. As compared with a control group of experimental animals where mechanical cardiac support was not used, the size of the necrotic focus of the heart muscle diminished by 50% (p less than 0.01). In some of the experimental animals the size of the necrotic focus did not change despite the use of a mechanical cardiac support. Analysis of haemodynamic parameters (assessed and calculated) revealed that for the fate of the necrotic focus of the myocardium the degree of relief (decompression) of the left ventricle is decisive. In complete relief the necrosis diminishes in size.

[Metabolic aspects of hypothermic extracorporeal circulation]. / K nekterým metabolickým aspektum hypotermního mimotelního obehu.

The submitted paper is devoted to some aspects of metabolic and immunity reactions of the organism to extracorporeal circulation. The authors focused attention in particular on a correlation of the whole-body oxygen consumption and the blood sugar level. Surprising findings comprise the rise of the whole-body oxygen consumption which occurs before the tissue temperature rises. As far as toxic oxygen radicals are concerned, their release is individual. It is, however, important that during the perfusion proper the activation of the immune system is suppressed. The course of the extracorporeal circulation was satisfactory from the clinical aspect; despite that it cannot be considered physiological, as also apparent from some biochemical findings.

Heart protection by cardioplegic solutions containing oxyhemoglobin pretreated by carbontetrachloride and freeze-drying with sucrose.

A technologically improved variant of native stroma-free oxyhemoglobin (SFH) pretreated by carbontetrachloride and freeze-drying with 240 mM sucrose were reconstituted in a properly diluted ionic solution to reach the final concentration of 66 g oxyhemoglobin/L, osmolality 280-320 m0sm and pH 7.4. Cardioplegia of isolated rat heart was induced and maintained by this solution without recirculation for 3 h at 20 degrees C prior to heterotopic allo-transplantation of the graft. Evaluation of the survival and performance of each graft after 24 h and extent of tissue necroses indicated that the given standardly produced SFH variant ensured reproducible heart preservation from ischemic and reperfusion injury similarly as did the renown crystalloid cardioplegic solution CUSTODIOL.

A comparison of the protective effect of a modified StTH solution and HTK-B on the energy and functional status of the isolated rat heart.

Using a model of the isolated beating rat heart, the authors compared the protective effect of St. Thomas Hospital cardioplegic solution enriched with glucose and mannitol (StTH-M) and Bretschneider solution (HTK-B). Results showed that, during 120-minute global ischaemia in cardioplegia, StTH-M was able to maintain levels of high-energy phosphates comparable with those found in a group of hearts perfused with HTK-B at 20 degrees C only when the temperature had been decreased to 12-15 degrees C. Under these conditions, repair of metabolic and functional parameters during post-ischaemic perfusion was also similar in both groups.

Analgosedation raises baroreflex sensitivity in acute myocardial ischaemia.

Acute local myocardial ischaemia is associated with a decrease in baroreflex sensitivity while electrical instability (vulnerability) of ventricles increases. Co-administration of a benzodiazepine and a potent analgesic (analgosedation) has been found to raise both baroreflex sensitivity and ventricular fibrillation threshold. Pharmacological modulation of neurovegetative ionization of the heart in the early phase of ischaemia is a promising method for preventing sudden coronary death.