Analysis of Drug/Plasma Protein Interactions by Means of Asymmetrical Flow Field-Flow Fractionation

Purpose. The applicability of Asymmetrical Flow Field-Flow Fractionation (Asymmetrical Flow FFF) as an alternative tool to examine the distribution of a lipophilic drug (N-Benzoyl-staurosporine) within human plasma protein fractions was investigated with respect to high separation speed and loss of material on surfaces due to adsorption. Methods. Field-Flow Fractionation is defined as a group of pseudo-chromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicochemical properties. This method was used to separate human plasma in its protein fractions. The drug distribution in the fractions was investigated by monitoring the fractionated eluate for drug content by fluorescence spectroscopy. Results. Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), ₂-macroglobulin and low density lipoprotein (LDL) fractions in less than ten minutes. Calibration of the system and identification of the individual fractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composition on the absolute recovery of N-Benzoyl-staurosporine and fluorescein-isothio-cyanate-albumin (FITC-albumin) was found to be quite significant. Both factors were optimized during the course of the investigations. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA. Conclusions. If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorption at the separation membrane, Asymmetrical Flow FFF shows high recoveries and fast separation of human plasma proteins, and can be a reliable tool to characterize drug / plasma protein interactions. For analytical purposes it has the potential to rival established technologies like ultracentrifugation in terms of ease-of-use, precision, and separation time.     

Detection of antibodies against pancreatic islet cells in clinical practice

Development of diabetes mellitus caused by pancreatic beta-cell destruction of autoimmune origin is the result of a long lasting process. The most easily examinable feature of this stage is the occurrence of the islet cell antibodies. The sera which are positive for islet cell cytoplasmic antibodies (ICA), examined by indirect immunofluorescence, contain a mixture of antibodies. The glutamic acid decarbocylase (GAD), the tyrosin phosphatase (IA2), the insulin, and the GM2-1 glycolipid can be the targets of these antibodies. One can routinely examine the ICA, the GADA, the IA2 antibodies. The detection of antibodies against insulin (IAA) and GM-2-1 glycolipid is not invented in the routine laboratory work. The aim of the authors was the evaluation of clinical significance of occurrence of islet cell antibodies: one hundred and eighteen nondiabetic children an adult human being without known diabetic first degree relatives and 366 type 1 diabetic children and adult patients served as controls. The authors evaluated the predictive value of the different islet cell antibodies to the development of type 1 diabetes mellitus in 596 nondiabetic children with type 1 diabetic first degree relatives. The authors looked for markers of beta-cell destruction among sera of 320 diabetics manifested after 30 years of age with at least half a year of non-insulin-dependency and in the sera of 68 females suffered from gestational diabetes after 0-14 years of the index pregnancy. Finally the authors report 7 cases in which the examination of islet cell antibodies helped the diagnosis and classification of diabetes mellitus. Indirect immunofluorescence method was used for the detection of ICA, radioimmunoassay for that of GADA and IA2 antibodies. There was no positive reaction for ICA and GADA in the nondiabetic population without diabetic first degree relatives. Among the freshly diagnosed type 1 diabetic children 39% were positive for only ICA, 44% for only GADA and 80% for any antibodies. Among the freshly manifested type 1 diabetic adults ICA positivity only was observed in 21%, GADA positivity only in 7.1% and 93% for any antibodies. From the 595 nondiabetic children with type 1 diabetic first degree relatives 23 were positive for ICA, from whom 5 became diabetic during a two years observation period. These diabetic children had multiplex autoantibodies besides ICA. One child from this group, who was negative for ICA became diabetic, too. Among type 2 diabetic patients 13% were positive for ICA alone, 17% were positive for GADA alone and 27% were positive for any antibodies. The insulin dependency manifested in a short time was associated with antibody positivity. Among the gestational diabetics 10 were found positive for ICA. From them, 7 were type 1 diabetics, and 3 were type 2 diabetics at the time of the detection of antibodies. The authors suggest the need of determination of islet cell antibodies in the group of nondiabetic first degree relatives of type 1 diabetic patients (ICA, GADA, IA2 and IAA), in the group of non-insulin-dependent diabetics (ICA and GADA) as a screening for later insulin dependency, and in gestational diabetes after delivery (ICA) as screening for type 1 diabetes mellitus.     

Effect of nalbuphine on the motility of the sphincter of Oddi in patients with suspected sphincter of Oddi dysfunction

BACKGROUND: Nalbuphine is an ideal supplementary analgesic drug for midazolam-induced conscious sedation during operative endoscopy because it has no cardiovascular effect and only a moderate depressive effect on respiration. However, no data are available as to whether nalbuphine is suitable as an analgesic drug during endoscopic sphincter of Oddi manometry. The aim of the present study was to investigate the effect of nalbuphine on the sphincter of Oddi motility in patients with a suspected sphincter of Oddi dysfunction. METHODS: Seventeen patients who were suspected clinically to have SOD after cholecystectomy were prospectively investigated. Five mg of midazolam was administered intravenously before the procedure to induce conscious sedation. After approximately 5 minutes of stationary sphincter of Oddi manometry recording (baseline), either 10 mg of nalbuphine or saline solution (placebo) was administered intravenously in random fashion and pressure was recorded for a further 5 minutes. Maximum sphincter of Oddi basal pressure and average phasic contraction amplitude and frequency were measured before and after the infusion of the drug or saline solution. RESULTS: Nalbuphine administration effectively enhanced the sedation obtained with midazolam without any adverse effect. When the sphincter of Oddi manometric periods before and after the administration of nalbuphine versus placebo were compared, there was a significantly increased basal sphincter of Oddi pressure only in the nalbuphine group: respectively, 49 (18) and 77 (29) mm Hg (p = 0.003) versus 51 (24) and 49 (23) mm Hg (p = 0.9). The phasic contraction amplitude did not change in response to nalbuphine, but the phasic contraction frequency increased significantly, from 5 (3) to 8 (4) per minute (p = 0.04). CONCLUSIONS: Nalbuphine has a stimulatory effect on sphincter of Oddi motility in patients with a suspected sphincter of Oddi dysfunction. Nalbuphine should not be used as premedication before endoscopic ERCP if sphincter of Oddi manometry is to be performed.     

Sex determination in skeletal remains from the medieval Eastern Adriatic coast – discriminant function analysis of humeri

Aim

To investigate the usefulness of humerus measurement for sex determination in a sample of medieval skeletons from the Eastern Adriatic Coast. Additional aim was to compare the results with contemporary female population.

Methods

Five humerus measurements (maximum length, epicondylar width, maximum vertical diameter of the head, maximum and minimum diameter of the humerus at midshaft) for 80 male and 35 female medieval and 19 female contemporary humeri were recorded. Only sufficiently preserved skeletons and those with no obvious pathological or traumatic changes that could affect the measurements were included. For ten samples, analysis of DNA was performed in order to determine sex using amelogenin.

Results

The initial comparison of men and women indicated significant differences in all five measures (P < 0.001). Discriminant function for sex determination indicated that as much as 85% of cases could be properly categorized, with better results in men (86%) than women (80%). Furthermore, the comparison of the medieval and contemporary women did not show significant difference in any of the measured features. Sex results obtained by anthropological and DNA analysis matched in all 10 cases.

Conclusion

The results indicate that humerus measurement in Croatian medieval population may be sufficient to determine the sex of the skeleton. Furthermore, it seems that secular changes have not substantially affected contemporary population, suggesting that the results of this study are transferable to contemporary population as well.Once the skeletal remains are uncovered, anthropologists initially aim to reconstruct the biological profile of the person, which includes sex, age, and height estimation. During the reconstruction process, numerous issues may arise, including bone fragmentation and poor preservation of skeletal remains, coupled with the complexity of human skeleton (1,2). Sex determination is one of the first and basic steps of assessing the biological profile. Although the analysis of DNA is the most reliable method for sex determination (3), it is also the most expensive and time consuming method, which can also be hindered by local conditions. This may especially be true in cases of poor preservation of the remains, inhibitors effects, or a small amount of extracted DNA from the sample.In absence of DNA results, skeletal remains can be used to infer subject’s sex via two methods, morphological and anthropometric. The morphological approach is based on the examination of the bones that show the strongest sexual dimorphism, principally the skull and the pelvis (4). However, this method is not always reliable, especially if the skull is fragmented or incomplete. Age can also affect the results, especially in elderly women, in which morphological characteristics of the skull tend to resemble those of men (5). Although morphological methods are very important for a preliminary sex assessment, they additionally rely on the experience of the examiner and are therefore rather subjective and unreliable.The second approach is based on anthropometric analysis, which relies on the bone measurements. The main analytic approach is based on discriminant function analysis, which attempts to classify subjects into each of the sexes, by using either one or more bones (6). This kind of analysis is a very important quantitative method (7) for sex determination as it reduces the subjectivity of the examiner (2,4,8). So far, only a few such studies have been published using Croatian bone samples. These include medieval and contemporary femurs and tibias (8-11) and medieval and contemporary mandibles and teeth (6,12). Such studies are important, since clear differences were observed in different populations (13-15), making this a locally-specific feature that requires the development of regional standards, applicable for local population (16).Besides already used femurs and tibias, humerus is another long bone from the body that is presumably informative for sex determination. This idea was initially derived from the empirical investigations of the skeletal remains, and further supported by the previously reported sexual dimorphism of humeri (7,17), even in cases of severe bone fragmentation (18). Furthermore, such location-specific results may be of interest in modern forensics as well, since observed changes in the skeleton marked predominantly by the increase in height (19), appear to be proportional, with no indication of sexual dimorphism in ancient and modern samples (20). Therefore, the aim of this study was to investigate the possibility to determine the sex of the subject based on anthropometric analysis of humeri measures.     

The HLA 8.1 ancestral haplotype is strongly linked to the C allele of -429T>C promoter polymorphism of receptor of the advanced glycation endproduct (RAGE) gene. Haplotype-independent association of the -429C allele with high hemoglobinA1C levels in diabetic patients

Previously we reported on strong linkage disequilibrium (LD) between the mono-S-C4B-RCCX module (mono-S) and the TNF2 allele (both known constituents of the 8.1 ancestral haplotype (8.1 AH)) in two Caucasian populations. The gene for the receptor of advanced glycation endproducts (RAGE) is encoded between the RCCX module and the HLA class II genes in the central MHC region. In order to assess the relationship between the promoter polymorphisms of the RAGE gene and the 8.1 AH, we performed a family study in eight informative families affected with type 1 diabetes mellitus; haplotypes of a RAGE promoter SNP (-429T>C) with the HLA-DQ2, -DR-3(17) and TNF2 alleles, as well as the mono-S genotype were determined. A similar analysis was performed in 82 unrelated patients with type 1 diabetes mellitus, and in unrelated healthy individuals of three different Caucasian populations (Hungarians, Ohioian females, Icelandics). In the diabetic patients clinical correlations were also investigated. Out of the 32 paternal and maternal chromosome 6 from the eight families, 15 different MHC haplotypes were found. Haplotypes containing at least three of the known constituents of the 8.1 AH (HLA-DQ2, -DR17, mono-S, TNF2) were always linked to the RAGE -429C allele. The RAGE -429C allele exhibited highly significant (p<0.0001) LD coefficients to known constituents of the 8.1 AH both in healthy persons and patients with type 1 diabetes. In the group of patients with diabetes we found significantly (p=0.013) higher maximal hemoglobinA1C concentration in the carriers of the RAGE -429C allele, this trait, however was not linked to the 8.1 AH. Our present findings indicate that the RAGE -429C allele can be considered as a candidate member of the 8.1 AH. The results also reveal a spectrum of recombinant MHC haplotypes in addition to the conserved ancestral haplotypes.     

Network meta-analysis of prophylactic pancreatic stents and non-steroidal anti-inflammatory drugs in the prevention of moderate-to-severe post-ERCP pancreatitis

BackgroundThere is an ongoing debate that non-steroidal anti-inflammatory drugs (NSAID) or prophylactic pancreatic stents (PPS) are more beneficial in preventing post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP). In our present network meta-analysis, we aimed to compare PPSs to rectal NSAIDs in the prevention of moderate and severe PEP in average- and high-risk patients.MethodsWe performed a systematic search for randomized controlled trials (RCT) from MEDLINE (via PubMed), Embase and Cochrane Central databases. RCTs using prophylactic rectal NSAIDs or PPSs in patients subjected to ERCP at average- and high-risk population were included. The main outcome was moderate and severe PEP defined by the Cotton criteria. Pairwise Bayesian network meta-analysis was performed, and interventions were ranked based on surface under cumulative ranking (SUCRA) values.ResultsSeven NSAID RCTs (2593 patients), and 2 PPS RCTs (265 patients) in the average-risk, while 5 NSAID RCTs (1703 patients), and 8 PPS RCTs (974 patients) in the high-risk group were included in the final analysis. Compared to placebo, only PPS placement reduced the risk of moderate and severe PEP in both patient groups (average-risk: RR = 0.07, 95% CI [0.002–0.58], high-risk: RR = 0.20, 95% CI [0.051–0.56]) significantly. Rectal NSAID also reduced the risk, but this effect was not significant (average-risk: RR = 0.58, 95% CI [0.22–1.3], high-risk: RR = 0.58, 95% CI [0.18–2.3]). Based on SUCRA, PPS placement was ranked as the best preventive method.ConclusionProphylactic pancreatic stent placement but not rectal NSAID seems to prevent moderate-to-severe PEP better both, in average- and high-risk patients.     

Increased Neutrophil-to-Lymphocyte Ratio is Associated with Unfavorable Functional Outcome in Acute Ischemic Stroke

Neurocritical Care - Inflammatory response is the hallmark of secondary brain injury in stroke patients. Neutrophil-to-lymphocyte ratio (NLR) emerged as a marker for functional outcome in several...     

Cardiovascular autonomic dysfunction in diabetes mellitus.

The aim was to assess cardiovascular autonomic dysfunction in children and adolescents with diabetes mellitus. A total of 110 children and adolescents with type 1 (insulin dependent) diabetes (aged 6 to 18 years) and 130 non-diabetic controls were studied. Resting heart rate, heart rate variation to deep breathing, heart rate response to standing from a lying position, fall in systolic blood pressure on standing, and rise in diastolic blood pressure during sustained handgrip were measured. A reference range of results was obtained in the controls. Diabetic children had significantly increased resting heart rate [92.4 (SEM 2.5) v 84.2 (2.2) beats/min], decreased deep breathing heart rate variation [25.3 (0.9) v 32.8 (0.6) beats/min], and lower standing/lying heart rate ratio [1.23 (0.04) v 1.31 (0.03)] compared with controls. 46 diabetic children (42%) had at least one abnormal autonomic test result. Of these, 20 (15%) had only one abnormal test and 26 (24%) had two or more abnormal tests. Using multiple logistic regression analysis, longer diabetes duration and worse long term metabolic control were independently predictive of cardiovascular autonomic dysfunction as the dependent variable [adjusted OR (95% CI): 2.9 (1.1-5.9) and 3.3 (1.2-6.4), respectively]. Cardiovascular autonomic dysfunction is not rare in children with diabetes. Efforts should be made to maintain the best metabolic control to prevent or delay these complications.