HLA genotyping using the Illumina HLA TruSight next-generation sequencing kits: A comparison.

Illumina first introduced their TruSight human leucocyte antigen (HLA) next-generation sequencing (NGS) typing kit in 2015 and subsequently followed up with a new version in 2016. Here we report on our experience comparing the two versions of the Illumina HLA NGS kits.

HLA genotyping in the clinical laboratory: comparison of next-generation sequencing methods.

Implementation of human leukocyte antigen (HLA) genotyping by next-generation sequencing (NGS) in the clinical lab brings new challenges to the laboratories performing this testing. With the advent of commercially available HLA-NGS typing kits, labs must make numerous decisions concerning capital equipment and address labor considerations. Therefore, careful and unbiased evaluation of available methods is imperative. In this report, we compared our in-house developed HLA NGS typing with two commercially available kits from Illumina and Omixon using 10 International Histocompatibility Working Group (IHWG) and 36 clinical samples. Although all three methods employ long range polymerase chain reaction (PCR) and have been developed on the Illumina MiSeq platform, the methodologies for library preparation show significant variations. There was 100% typing concordance between all three methods at the first field when a HLA type could be assigned. Overall, HLA typing by NGS using in-house or commercially available methods is now feasible in clinical laboratories. However, technical variables such as hands-on time and indexing strategies are sufficiently different among these approaches to impact the workflow of the clinical laboratory.

Assessment of heat provocation tests on the human gingiva: the effect of periodontal disease and smoking.

UNLABELLED: Periodontal inflammation is associated with morphological changes in the blood vessels which may influence the regulation of gingival blood flow (GBF). Our aim was to adapt the heat provocation test to the human gingiva to assess vascular reactivity in periodontal inflammation. METHOD: GBF was recorded by Laser Doppler Flowmetry before and after heat provocation in healthy volunteers (n = 50). Heat was generated either by warm saline or a halogen lamp. The latter method was also utilized for a heat test in non-smoking and smoking patients with periodontal inflammation. The circulatory parameters were correlated to the inflammatory marker, i.e. gingival crevicular fluid (GCF) production measured by Periotron. RESULTS: Local application of heat caused a rapid, significant and transient increase in GBF regardless of the method used. The increase in the speed and not in the concentration of moving blood cells was responsible for increased GBF. Higher GCF values were correlated with increased peak flow, flux pulse amplitude and faster restoration of GBF after the test in non-smokers, but not in smokers. CONCLUSIONS: The heat test could be a valuable tool to check the vascular reactivity of gingival vessels. Moderate periodontal inflammation may facilitate gingival vascular responsiveness which can be suppressed by smoking.

Unusual spinal tuberculosis in an Avar Age skeleton (Csongrád-Felgyo, Ürmös-tanya, Hungary): A morphological and biomolecular study.

The paleopathological analysis of a well-preserved young adult female skeleton from the AD 7-8th century (Avar Age) in Hungary revealed multiple lytic lesions in all of the thoracic and lumbar vertebral bodies. The lesions were characterized by smooth marginal zones and space-occupying mass appearance. The considerable loss of spongy bone in the thoracolumbar vertebrae resulted in angular deformity and fusion, characteristic of the healing stage of TB. Osteolytic lesions were also observed on the vertebral processes, ribs and sternum. On the endocranial surface, abnormal blood vessel impressions were revealed, indicating some kind of meningitis. The X-ray and CT analysis of the affected bones detected abnormal structures and cystic zones of destruction. The lesions were however not always bordered by areas of increased density, which is typical in cystic TB. Vertebral remains were also subjected to biomolecular analysis in two different laboratories, which attested the presence of Mycobacterium tuberculosis complex (MTBC) DNA and supported the paleopathological diagnosis of TB. Spoligotyping analysis confirmed the presence of MTBC DNA and more specifically an infection caused by bacteria belonging to the M. tuberculosis lineage. This case study provides new data for the paleoepidemiology of TB in this geographical area and historical period, and draws attention to the great variability of TB lesions in the human skeleton.

Nutrient stress alters the glycosylation status of LGR5 resulting in reduced protein stability and membrane localisation in colorectal tumour cells: implications for targeting cancer stem cells.

BACKGROUND: LGR5 is an important marker of intestinal stem cells and performs its vital functions at the cell membrane. Despite the importance of LGR5 to both normal and cancer stem cell biology, it is not known how microenvironmental stress affects the expression and subcellular distribution of the protein. METHODS: Nutrient stress was induced through glucose starvation. Glycosylation status was assessed using endoglycosidase or tunicamycin treatment. Flow cytometry and confocal microscopy were used to assess subcellular distribution of LGR5. RESULTS: Glucose deprivation altered the glycosylation status of LGR5 resulting in reduced protein stability and cell surface expression. Furthermore, inhibiting LGR5 glycosylation resulted in depleted surface expression and reduced localisation in the cis-Golgi network. CONCLUSIONS: Nutrient stress within a tumour microenvironment has the capacity to alter LGR5 protein stability and membrane localisation through modulation of LGR5 glycosylation status. As LGR5 surface localisation is required for enhanced Wnt signalling, this is the first report to show a mechanism by which the microenvironment could affect LGR5 function.

Long-term hazards of neonatal blue-light phototherapy.

Blue-light phototherapy has been an essential therapeutic tool in the management of neonatal jaundice for decades. Rarely, it is accompanied by acute dermatological and systemic side-effects, but fortunately these are reversible and can be adequately and promptly treated in routine neonatal practice. In contrast, much less is known about the potential long-term side-effects of neonatal blue-light phototherapy (NBLP). Many of the data that are currently available on how NBLP influences melanocytic naevus (MN) development are controversial. The results of recent well-designed epidemiological surveys suggest that NBLP could well be a risk factor for MN formation, and highlight the need for additional in vivo and in vitro studies. NBLP is at present the mainstay of treatment for neonatal jaundice, but in the future greater consideration should be given to its long-term side-effects when phototherapy is indicated. It is relevant to emphasize the importance of appropriately restricted and adequate clinical guidelines, and strict monitoring of the management of hyperbilirubinaemia, in order to avoid the unnecessary overtreatment of newborn infants.

Removal of nonspecific binding proteins from cell and tissue extracts using 2-aminobenzimidazole-tethered affinity resin.

Cellular drug target identification through affinity chromatography is often hindered by the quantity of nonspecific binders, such as cytoskeletal and heat shock proteins. Thus, we prepared a 2-aminobenzimidazole-tethered depletion resin designed for removal of these proteins, and tested it on human lung carcinoma cell and rat tissue extracts. Column-bound proteins were identified by two-dimensional gel electrophoresis and MS. Among others, tubulins, actins and heat shock proteins were successfully depleted. Due to the reduction of these highly abundant proteins detection of potential drug targets is considerably facilitated in the pre-purified samples.

Influence of shear viscosity of quark-gluon plasma on elliptic flow in ultrarelativistic heavy-ion collisions.

We investigate the influence of a temperature-dependent shear viscosity over entropy density ratio η/s on the transverse momentum spectra and elliptic flow of hadrons in ultrarelativistic heavy-ion collisions. We find that the elliptic flow in √S(NN)=200 GeV Au+Au collisions at RHIC is dominated by the viscosity in the hadronic phase and in the phase transition region, but largely insensitive to the viscosity of the quark-gluon plasma (QGP). At the highest LHC energy, the elliptic flow becomes sensitive to the QGP viscosity and insensitive to the hadronic viscosity.

Translationally controlled tumour protein (TCTP) is a novel glucose-regulated protein that is important for survival of pancreatic beta cells.

AIMS/HYPOTHESIS: This study used proteomics and biochemical approaches to identify novel glucose-regulated proteins and to unveil their role in pancreatic beta cell function. Translationally controlled tumour protein (TCTP) was identified to be one such protein, and further investigations into its function and regulation were carried out. METHODS: Global protein profiling of beta cell homogenates following glucose stimulation was performed using two-dimensional gel electrophoresis. Proteins were identified by mass spectroscopy analysis. Immunoblotting was used to investigate alterations in TCTP protein levels in response to glucose stimulation or cell stress induced by palmitate. To investigate the biological function of TCTP, immunolocalisation, gene knockdown and overexpression of Tctp (also known as Tpt1) were performed. Apoptosis was measured in Tctp knockdown or Tctp-overexpressing cells. Glucose-stimulated insulin secretion was carried out in Tctp knockdown cells. RESULTS: TCTP was identified as a novel glucose-regulated protein, the level of which is increased at stimulatory glucose concentration. Glucose also induced TCTP dephosphorylation and its partial translocation to the mitochondria and the nucleus. TCTP protein levels were downregulated in response to cell stress induced by palmitate or thapsigargin treatments. Gene knockdown by small interfering RNA led to increased apoptosis, whereas overproduction of TCTP prevented palmitate-induced cell death. CONCLUSIONS/INTERPRETATION: Regulation of TCTP protein levels by glucose is likely to be an important cyto-protective mechanism for pancreatic beta cells against damage caused by hyperglycaemia. In contrast, high concentration of palmitate causes cell stress, reduction in TCTP levels and consequently reduced cell viability. Our results imply that TCTP levels influence the sensitivity of beta cells to apoptosis.

Relativistic shock waves in viscous gluon matter.

We solve the relativistic Riemann problem in viscous gluon matter employing a microscopic parton cascade. We demonstrate the transition from ideal to viscous shock waves by varying the shear viscosity to entropy density ratio eta/s from zero to infinity. We show that an eta/s ratio larger than 0.2 prevents the development of well-defined shock waves on time scales typical for ultrarelativistic heavy-ion collisions. Comparisons with viscous hydrodynamic calculations confirm our findings.

Appearance of hyperostosis frontalis interna in some osteoarcheological series from Hungary.

Hyperostosis frontalis interna (HFI) is a generalised pathological condition with an unknown etiology and variable clinical association. It is characterized by excess bone growth and manifested on the inner table of the frontal bone, occasionally extending onto the temporals, parietals and the occipital. The etiology of HFI is uncertain: it may be an unknown genetic predisposition, a common environmental exposure, or special metabolic diseases. The purpose of the present study is to report cases of HFI in some osteoarcheological series from Hungary and to emphasize the importance of the investigation of HFI in ancient populations. Twenty out of 803 adults with observable frontal bones exhibited HFI, ranging from early to mid-type, including 15 females and 5 males. Some overgrowths with edges were blending into the endocranial surface, and some were prominently protruding from the surface. Advanced cases of HFI (type C) were observed after age 40-60 years.

Modification of ionotropic glutamate receptor-mediated processes in the rat hippocampus following repeated, brief seizures.

The seizure-induced molecular and functional alterations of glutamatergic transmission in the hippocampus have been investigated. Daily repeated epileptic seizures were induced for 12 days by intraperitoneal administration of 4-aminopyridine (4-AP; 4.5 mg/kg) in adult Wistar rats. The seizure symptoms were evaluated on the Racine's scale. One day after the last injection, the brains were removed for in vitro electrophysiological experiments and immunohistochemical analysis. The glutamate receptor subunits NR1, NR2A, NR2B, GluR1, GluR1(flop), GluR2, and KA-2 were studied using the histoblotting method. The semi-quantitative analysis of subunit immunoreactivities in hippocampal layers was performed with densitometry. In the hippocampus, increase of GluR1, GluR1(flop) and NR2B immunostaining was observed in most of the areas and layers. The significant decrease of GluR2 staining intensity was observed in the CA1 and dentate gyrus. Calcium permeability of hippocampal neurons was tested by a cobalt uptake assay in hippocampal slices. The uptake of cobalt increased in the CA1 area and dentate gyrus, but not in the CA3 region following 4-AP treatment. Effects of AMPA and NMDA (N-methyl-d-aspartate) glutamate receptor antagonists (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) and D-APV respectively) were measured in hippocampal slices using extracellular recording. Analysis of the population spikes revealed the reduced effectiveness of the AMPA receptor antagonist GYKI 52466, while the effect of the NMDA receptor antagonist d-(2R)-amino-5-phosphonovaleric acid was similar to controls. The results demonstrated that repeated convulsions induced structural and functional changes in AMPA receptor-mediated transmission, while NMDA and kainate receptor systems displayed only alterations in receptor subunit composition.

Leflunomide and its metabolite A771726 are high affinity substrates of BCRP: implications for drug resistance.

BACKGROUND: Earlier publications have suggested a possible role for the efflux transporter breast cancer resistance protein (BCRP) in acquired resistance to disease-modifying antirheumatic drugs (DMARDs) such as leflunomide and its metabolite A771726 (teriflunomide). However, there is no direct evidence that BCRP interacts with these drugs. OBJECTIVES: To characterise the interaction between BCRP transporter and leflunomide and its active metabolite A771726, with emphasis on the nature of the interaction (substrate or inhibitor) and the kinetic characterisation of the interactions. METHODS: Different in vitro membrane-based methods (ATPase and vesicular transport assay) using BCRP-HAM-Sf9 membrane preparations and cellular assays (Hoechst assay and cytotoxicity assay) were performed on PLB985-BCRP and HEK293-BCRP cell lines overexpressing BCRP. RESULTS: In all assays used, an interaction between the investigated drugs and BCRP was detected. In the vesicular transport assay, both leflunomide and its metabolite inhibited BCRP-mediated methotrexate transport. Both compounds are likely substrates of BCRP as shown by the vanadate-sensitive ATPase assay. In line with the membrane assays, leflunomide and A771726 inhibited BCRP-mediated Hoechst efflux from PLB985-BCRP cells. In the cytotoxicity assay, overexpression of BCRP conferred 20.6-fold and 7.5-fold resistance to HEK293 cells against leflunomide and A771726, respectively. The resistance could be reversed by Ko134, a specific inhibitor of BCRP. CONCLUSION: Based on these results, BCRP could play an important role in the resistance to leflunomide and A771726 via interactions with these drugs. BCRP may also mediate drug-drug interactions when leflunomide is administered with other BCRP substrate drugs such as methotrexate.

ATG16L1 and IL23 receptor (IL23R) genes are associated with disease susceptibility in Hungarian CD patients.

BACKGROUND: North American and European genome-wide association scans have identified ATG16L1 and IL23R as novel inflammatory bowel disease (IBD) susceptibility genes and subsequent reports confirmed these findings in large independent populations. The aims of this study were to investigate the association and examine genotype-phenotype relationships in a Hungarian IBD cohort. METHODS: 415 unrelated IBD patients (CD: 266, age: 35.2+/-12.1 years, duration: 8.7+/-7.5 years and UC: 149, age: 44.4+/-15.4 years, duration: 10.7+/-8.9 years) and 149 healthy subjects were investigated. IL23R Arg381Gln (R381Q, rs11209026) and ATG16L1 Thr300Ala (T300A, rs2241880) polymorphisms were tested using LightCycler allele discrimination method. Detailed clinical phenotypes were determined by reviewing the medical charts. RESULTS: The association between IL23R rs11209026, ATG16L1 rs2241880 and CD was confirmed (OR(IL23R381Q): 0.38, 95% CI: 0.16-0.87; OR(ATG16L1300AA): 1.86, 95% CI: 1.04-3.40). No difference was found between patients with UC and either controls or CD. In CD, IL23R 381Gln heterozygosity was associated with inflammatory disease (70% vs. 34%, p=0.037), while disease restricted to the colon was more prevalent in patients with the ATG16L1 300Ala/Ala homozygosity (33.3% vs. 21.1%, p=0.036). In addition, carriage of the variant alleles did not predict response to steroids, infliximab or need for surgery. CONCLUSIONS: We confirmed that ATG16L1 and IL23R are susceptibility loci for CD in Hungarian CD patients. Further studies are needed to confirm the reported phenotype-genotype associations found in this study.

Cholesterol potentiates ABCG2 activity in a heterologous expression system: improved in vitro model to study function of human ABCG2.

ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by high-throughput systems using transfected insect and/or human cell lines. The determination of ABCG2-ATPase activity is one method to identify ABCG2 substrate and inhibitors. We demonstrate that the ATPase activities of the human ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ. Variation due to disparity in the glycosylation level of the protein had no effect on the transporter. The influence of cholesterol on ABCG2-ATPase activity was investigated because the lipid compositions of insect and human cells are largely different from each other. Differences in cholesterol content, shown by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes. Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells. MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. Our study indicates an essential role of membrane cholesterol for the function of ABCG2.

Inhibition of human primary melanoma cell proliferation by histamine is enhanced by interleukin-6.

BACKGROUND: Interleukin-6 (IL-6) is a bifunctional growth factor in malignant melanoma; its expression increases during the malignant progression of the disease. Histamine, detected in large amounts in normal and pathological proliferating tissues, is an important paracrine and autocrine regulator of normal and tumour cell proliferation as well. MATERIALS AND METHODS: We investigated the presence and function of IL-6 and histamine in the WM35 primary human melanoma cell line with respect to their direct role in cell proliferation and their regulatory interactions. RESULTS: IL-6 inhibited the proliferation of WM35 melanoma cells and increased significantly the expression of histidine decarboxylase as well as histamine production. It had dose-dependent effects on the proliferation: high concentration (10-5 M) was inhibitory through H1 histamine receptors while low histamine concentration acting on H2 receptors, with a simultaneous increase of cAMP, enhanced colony formation in the monolayer. Furthermore, IL-6 increased the H1- but decreased the H2-histamine receptor expression of the melanoma cells. On the other hand, histamine was locally synthesized by the WM35 melanoma cells. CONCLUSION: We suggest that the growth arrest induced by IL-6 is in part mediated by its dual action on histamine: a shift toward H1 receptor predominance and an elevation of locally produced histamine with prevalent action on the inhibitory response triggered through the H1 receptor. These findings suggest a local cross-talk between histamine and IL-6 in the regulation of melanoma growth.

One-generation reproduction toxicity study of Dithane M-45 (mancozeb) and lead acetate.

The reproductive toxicity of lead acetate and of a fungicide formulation (Dithane M-45) containing 80% mancozeb was studied on rats. Lead acetate was applied in the feed in the following dose groups: control, 1,000, 5,000 and 10,000 mg/kg of diet. The three treatment groups received, in addition to the above doses of lead acetate, 4,500 mg/kg Dithane M-45 in the diet. The method was based on the OECD Guideline for Testing of Chemicals No. 415 (1981). Clinical symptoms and mortality were not found in the parent generation. The body weight of female animals decreased significantly before the pregnancy period. This tendency was also seen in males after the combination treatment. At the two high dose levels a remarkable body weight increase was seen in the female animals during the lactation period. As a result of treatment, decreased body weight of offspring was measured during the lactation period. No gross pathological changes were seen. Histological examination showed general tubulonephrosis in the experimental animals. It can be established that the administration of Dithane M-45 did not enhance the reproductive toxicity of lead acetate.

Cimetidine and a tamoxifen derivate reduce tumour formation in SCID mice xenotransplanted with a human melanoma cell line.

Histamine is produced by many cells expressing histidine decarboxylase (HDC), the enzyme responsible for the synthesis of histamine. Since melanoma cells and tissue contain relatively large amounts of histamine, the functional significance of histamine was examined using specific antihistamines in vitro and in vivo in the human melanoma cell line HT168 and severe combined immunodeficiency (SCID) mice. It was shown that the H2 receptor antagonist cimetidine when combined with N, N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine-HCl (DPPE), a tamoxifen derivate, inhibits the proliferation of HT168 cells. Furthermore, it is suggested that there is a factor(s) that interferes with the exponential growth of HT168 cells xenografted to immunodeficient mice, and cimetidine and DPPE together significantly influence this factor(s). This combination of antihistamines also increases the survival of human melanoma-grafted mice. These changes are accompanied by enhanced infiltration of interferon-gamma- producing mouse macrophages into the tumour tissue. These findings suggest that two different mechanisms are probably acting concordantly: direct inhibition of tumour cell proliferation by the H2 receptor antagonists, and activation of the local immune response characterized by interferon-gamma production. These findings may help to elucidate the possibility of a rationally designed antihistamine strategy in melanoma therapy.