Peripheral blood marker of residual acute leukemia after hematopoietic cell transplantation using multi-plex digital droplet PCR.

Background: Relapse remains the primary cause of death after hematopoietic cell transplantation (HCT) for acute leukemia. The ability to identify minimal/measurable residual disease (MRD) via the blood could identify patients earlier when immunologic interventions may be more successful. We evaluated a new test that could quantify blood tumor mRNA as leukemia MRD surveillance using droplet digital PCR (ddPCR). Methods: The multiplex ddPCR assay was developed using tumor cell lines positive for the tumor associated antigens (TAA: WT1, PRAME, BIRC5), with homeostatic ABL1. On IRB-approved protocols, RNA was isolated from mononuclear cells from acute leukemia patients after HCT (n = 31 subjects; n = 91 specimens) and healthy donors (n = 20). ddPCR simultaneously quantitated mRNA expression of WT1, PRAME, BIRC5, and ABL1 and the TAA/ABL1 blood ratio was measured in patients with and without active leukemia after HCT. Results: Tumor cell lines confirmed quantitation of TAAs. In patients with active acute leukemia after HCT (MRD+ or relapse; n=19), the blood levels of WT1/ABL1, PRAME/ABL1, and BIRC5/ABL1 exceeded healthy donors (p<0.0001, p=0.0286, and p=0.0064 respectively). Active disease status was associated with TAA positivity (1+ TAA vs 0 TAA) with an odds ratio=10.67, (p=0.0070, 95% confidence interval 1.91 - 59.62). The area under the curve is 0.7544. Changes in ddPCR correlated with disease response captured on standard of care tests, accurately denoting positive or negative disease burden in 15/16 (95%). Of patients with MRD+ or relapsed leukemia after HCT, 84% were positive for at least one TAA/ABL1 in the peripheral blood. In summary, we have developed a new method for blood MRD monitoring of leukemia after HCT and present preliminary data that the TAA/ABL1 ratio may may serve as a novel surrogate biomarker for relapse of acute leukemia after HCT.

Comparison of predictions of developmental toxicity for compounds of solvent data set.

We have considered a series of 235 compounds technically classified as solvents. Chemically, they belong to different classes. Their potential developmental toxicity was evaluated using two models available on platform VEGA HUB; model CAESAR and the Developmental/ Reproductive Toxicity library (PG) model. Models provide beside the prediction of developmental toxicity additional information on similar compounds from models' training sets. In the report, first, we compare the predictions. Second, the sets of similar compounds have been used to implement the clustering scheme. The Kohonen artificial neural network method has been applied as a clustering method. The clusters obtained have been discussed for both models.

Thickness dependent photocurrent spectra and current-voltage characteristics of P3HT:PCBM photovoltaic devices.

P3HT:PCBM based photovoltaic devices with different active layer thicknesses (ALTs) were examined in photodetector and solar cell operation modes. The photodetector photocurrent spectra and solar cell current density-voltage characteristics were measured. All experimental results were reproduced by the unique drift-diffusion model which excludes the optical interference and allows the parameters of photogeneration, transport, and recombination to be ALT dependent. The active layer optical characterization indicated a thickness dependence of optical parameters too. A conclusion was drawn that the P3HT:PCBM film thickness and morphology are strongly correlated which leads to a non-monotonic change of film parameters with its thickness.

Comparison of Demographic, Epidemiological, Immunological, and Clinical Characteristics of Patients with HIV Mono-infection Versus Patients Co-infected with HCV or/and HBV: A Serbian Cohort Study.

OBJECTIVE: The study aimed to correlate the status of hepatitis C (HCV) and hepatitis B virus (HBV) co-infection in patients with human immunodeficiency virus (HIV) infection with clinical and demographic data prior to starting highly active antiretroviral therapy (HAART) and assess the impact of HCV and HBV co-infection on the natural history of HIV infection. PATIENTS AND METHODS: The study involved a total of 836 treatment-naive patients with available serological status for HBV and HCV at the point of therapy initiation. Patients were stratified into four groups: HIV mono-infection, HIV/HCV, HIV/HBV, and HIV/HCV/HBV co-infection. Demographic, epidemiological, immunological and clinical characteristics were analyzed in order to assess the possible impact of HCV and HBV co-infection on HIV - related immunodeficiency and progression to AIDS. RESULTS: The prevalence of HCV and HBV co-infection in our cohort was 25.7% and 6.3%, respectively. Triple HIV/HCV/HBV infection was recorded in 1.7% of the patients. In comparison with those co-infected with HCV, patients with HIV mono-infection had lower levels of serum liver enzymes activity and higher CD4 cell counts, and were less likely to have CD4 cell counts below100 cells/µL and clinical AIDS, with OR 0.556 and 0.561, respectively. No difference in the development of advanced immunodeficiency and/or AIDS was recorded between patients with HIV monoinfection and those co-infected with HBV, or both HCV/HBV. CONCLUSION: HIV/HCV co-infection was found to be more prevalent than HIV/HBV co-infection in a Serbian cohort. Co-infection with HCV was related to more profound immunodeficiency prior to therapy initiation, reflecting a possible unfavorable impact of HCV on the natural history of HIV infection.

Extensive depolarization and lack of recovery of leech Retzius neurons caused by 2,4 diaminobutyric acid.

In this paper we present, for the first time, a detailed account of electrophysiological effects of 2,4-diaminobutyric acid (2,4-DABA). 2,4-DABA is a neurotoxic non-protein amino acid produced by Cyanobacteria with a possible link to neurodegenerative disorders in animals and humans. Intracellular recordings were performed on Retzius nerve cells of the leech Haemopis sanguisuga using glass microelectrodes filled with 3 mol/L KCl. Our results show that 2,4-DABA is an excitatory amino acid, causing membrane depolarization in a concentration-dependent manner. The most prominent depolarizations of 39.63±2.22 mV and 47.05±4.33 mV, induced by 5×10-3 and 10-2 mol/L 2,4-DABA respectively, are several times larger than maximal depolarizations induced by either Glutamate, Aspartate, ß-N-methylamino-alanine (BMAA) or ß-N-oxalylamino-alanine (BOAA) on our model. These 2,4-DABA induced depolarizations evolve through two distinct stages, which is a novel phenomenon in electrical cell activity upon application of an excitatory amino acid, at least on our model. Involvement of two separate mechanisms, suggested by the two stage phenomenon, is discussed in the paper. We also provide evidence that 2,4-DABA induces irreversible functional disturbances in neurons in a concentration-dependent manner, since only half of the cells recovered normal electrical activity after application of 5×10-3 mol/L 2,4-DABA, and none recovered after application of 10-2 mol/L 2,4-DABA. Effects of both L-2,4-DABA and DL-2,4-DABA were tested and are not significantly different.

Prenatal Diagnosis of a Fetal Epidural Hematoma.

A fetal epidural hematoma is a rare finding during the prenatal period, with different etiologies such as maternal trauma, infections, or maternal use of specific medications such as warfarin. Both ultrasonography and magnetic resonance imaging (MRI) have been used successfully to evaluate the fetal central nervous system. Although these methods are also useful in detecting and evaluating a fetal epidural hematoma, brain function of the neonate cannot be exactly predicted on the basis of the prenatal findings. According to the literature data to date, this is the first reported case of prenatally detected fetal epidural hematoma of unknown etiology with a good outcome.

Failure to detect viral RNA in bat samples collected in the Balkan region.

Bats represent a known reservoir of emerging viruses, yet no molecular data are found about the occurrence of zoonotic viruses in bats in the Balkans. The aim of this study was to determine the presence of paramyxo- and hanta-viruses in bats, examined by PCR in 95 deceased bats, that were collected in Serbia and Montenegro, during the period 2002 to 2009. All samples tested positive for beta-actin mRNA, confirming successful RNA isolation and amplification. However, no sample tested positive for virus specific RNA. Our findings might reflect tissue degradation in carcass samples and do not exclude bats as potential viral reservoir in the surveyed geographic area.

Primary resistance to integrase strand-transfer inhibitors in Europe.

OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

Genetic detection of Dobrava-Belgrade hantavirus in the edible dormouse (Glis glis) in central Serbia.

Hantaviruses are endemic in the Balkans, particularly in Serbia, where sporadic cases and/or outbreaks of hantaviral human disease have been reported repeatedly, and evidenced serologically. Here, we present genetic detection of Dobrava-Belgrade virus (DOBV) hantaviral sequences in wild rodents trapped in central Serbia. All the animals were pre-screened serologically by indirect immunofluorescence (IF) test and only those with a positive finding of hantaviral antigens were further tested by polymerase chain reaction. Of the total of 104 trapped animals, 20 were found to be IF positive and of those three were positive for hantaviral RNA: one Microtus arvalis for Tula virus, and one each of Apodemus agrarius and Glis glis for DOBV. Phylogenetic analysis of the obtained sequences implies putative DOBV spillover infection of A. agrarius and G. glis from Apodemus flavicollis. However, future investigations should help to identify the most common natural host and geographical distribution of DOBV in its reservoir hosts in Serbia.

Genetic analysis of Dobrava-Belgrade virus from western Serbia--a newly detected focus in the Balkan Peninsula.

Dobrava-Belgrade virus (DOBV) is a hantavirus species that causes the most severe form of haemorrhagic fever with renal syndrome (HFRS) in Europe. DOBV has been detected in three Apodemus rodents: A. flavicollis, A. agrarius and A. ponticus. These emerging viruses appear throughout the Balkan Peninsula including Serbia as its central part. In this study, we examined the seroprevalence, molecular epidemiology and phylogenetics of DOBV from A. flavicollis captured at six Serbian localities. Furthermore, we applied microsatellite typing of host animal genome to analyse the role of host kinship in DOBV animal transmission. The overall IgG seropositivity rate over 3 years (2008-2010) was 11.9% (22/185). All seropositive samples were subjected to RT-PCR and DNA sequencing for S and L genome segments (pos. 291-1079 nt and 2999-3316 nt, respectively). DOBV was genetically detected in three samples from mountain Tara in western Serbia, a newly detected DOBV focus in the Balkans. No sequence data from human cases from Serbia are available for the studied period. However, collected DOBV isolates in this work phylogenetically clustered together with isolates from Serbian human cases dating from 2002, with 1.9% nucleotide divergence. We determined the level of kinship between seropositive and seronegative animal groups and found no significant difference, suggesting that horizontal virus transmission in the studied population was the same within and among the hatches. Our findings are the first genetic detection of DOBV in rodents in Serbia. We confirm wide and continuous hantavirus presence in the examined parts of the Balkans, underlying the necessity of continual monitoring of hantavirus circulation in A. flavicollis.

Typing of macrolide resistant group A streptococci by random amplified polymorphic DNA analysis.

OBJECTIVE: Several studies of group A streptococci (GAS) have revealed that a small number of dominant resistant clones might be responsible for the spread of Streptococcus (S.) pyogenes resistance to macrolides. We aimed to determine the genetic diversity of macrolide resistant group A streptococci (MRGAS), isolated from patients with pharyngitis in Serbia. MATERIALS AND METHODS: The clonal relationships among 76 MRGAS isolates collected during 2008 were studied using two molecular typing methods: emm typing and random amplified polymorphic DNA (RAPD) analysis. Isolates that share the same emm type and RAPD pattern were considered to belong to the same clone. RESULTS: Out of 7 distinct emm types identified, the 3 most frequently occurring overall were emm12, emm75 and emm77 (> 90% of isolates). Although as many as 26 different RAPD patterns were found among the isolates studied, two clones with emm12 and emm77 accounted 32 out of 76 (42%) isolates. CONCLUSIONS: The results indicate a polyclonal spread of erythromycin-resistant Streptococcus pyogenes in our country. Furthermore, predominance of two clones, particularly among emm12 and emm77 strains indicates that erythromycin-resistant GAS of the same clonal origin are widely distributed in Serbia.

Neurobehavior in fetal life.

Assessment fetal behavior gave a promising opportunity to understand the hidden function of the developmental pathway of the fetal central nervous system. After the assessment of normal neurobehavioral development by four-dimensional (4D) ultrasound, attempts have been made to identify functional characteristics of the fetus that predict a range of subsequent developmental dysfunction. These attempts resulted in producing the Kurjak Antenatal Neurodevelopmental Test (KANET). Assessment of fetal behavior by 4D ultrasound and application of KANET scoring test has been recently published in several journals and summarized results are presented in this review.

HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries.

BACKGROUND: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. METHODS: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. RESULTS: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs. CONCLUSION: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Relation between serum urea and mortality of hemodialysis patients.

BACKGROUND: It is well known that serum urea concentration is not a good predictor of mortality in hemodialysis patients. On the other hand, urea kinetic modeling has been very successfully used to measure dialysis dose by the Kt/V index, which was found to be a good predictor of mortality. Could there be a relation between urea and mortality, but in some more complex way? METHODS: This is a post-hoc analysis of a single center observation study that included 242 patients and an 11-year observation period. Mortality rates between the quartiles of serum urea levels were examined by a 2 x 4 table with the chi(2) test. Both univariate and multivariate survival analyses were performed with standard and segmented extended Cox regression. RESULTS: The relation between mean urea in the baseline period and mortality showed an irregular U-shaped curve. The lowest mortality was observed in the third quartile (28 to 31 mmol/L). The relation between mean urea in the whole observation period and mortality was a J-shaped curve. The lowest mortality was in the second quartile (25-27 mmol/L). Urea was not a predictor of mortality in the whole cohort, but low-urea (binary) and high-urea (binary) were independent predictors of mortality in the corresponding models using standard or extended Cox regression. CONCLUSION: This study revealed a complex relationship between urea and mortality in hemodialysis patients. Patients with low or high urea levels exhibited higher mortality than those with medium levels, while both low and high levels of urea were independent predictors of all-cause mortality.

Could depression be a new branch of MIA syndrome?

The aims of the present study were to determine the prevalence of depression in our dialysis patients, to detect the most powerful variables associated with depression, and to determine the role of depression in prediction of mortality. The prospective follow-up study of 128 patients (77 HD and 51 CAPD, 65 male, aged 53.8 +/- 13.5 years, dialysis duration 64.7 +/- 64.8 months) was carried out over 36 months. Depression by the Beck Depression Inventory-BDI-II score, laboratory parameters (hemoglobin, serum albumin and creatinine concentration), immunological status (cytokines and hsCRP), comorbidity by Index of Physical Impairment (IPI) and adequacy of dialysis by Kt/V were monitored. The overall prevalence of depression in the dialysis patients (BDI score > or = 14) was 45.3%, and 28.2%, respectively, for moderate and severe depression (BDI > or = 20). The most powerful variable associated with depression was IL-6, but associations with albumin, hemoglobin, creatinine and IPI score were also found. During the follow-up period 36 patients died, 7 patients left the cohort and 2 patients were transplanted. If IPI score was not included in the multivariate Cox analysis, the BDI score remained one of the best predictors of mortality along with albumin. In conclusion, because of the close association of depression with inflammation, malnutrition, and cardiovascular mortality, it could be speculated that depression is one branch of the MIA (malnutrition, inflammation, atherosclerosis) syndrome.

Hemodialysis modality, percentage of body fat, and patient survival.

BACKGROUND: A number of studies have reported lower mortality of overweight hemodialysis patients. This post hoc analysis of an observational prospective single-center study was aimed at elucidating whether both being overweight and surviving longer could result from changes in the hemodialysis modality. METHODS: The study included a cohort of 242 patients who were gradually switched from cuprophane membrane and acetate dialysis to polysulfone (including high-flux) membranes and bicarbonate dialysis. The analysis involved 12 months of baseline data obtained during the first calendar year after the patients entered the study (1994-2001) and repeated measurements for up to 132 months of follow-up (until 2004). Anthropometric measurements were made during the winter season and the percentage of body fat (%fat) was calculated from triceps, biceps, subscapular, and suprailiac skinfolds (K/DOQI guidelines).Kt/V, normalized protein catabolic rate, and cardiovascular comorbidity were also determined and laboratory analyses undertaken. RESULTS: Significant correlations were found between %fat and bicarbonate dialysate as well as polysulfone membrane and high-flux membrane. The linear mixed model showed dependence of %fat on polysulfone and high-flux membrane (p<0.01) Multivariate Cox regression (time-dependent covariates) found %fat to be an independent factor for longer survival, in addition to polysulfone and high-flux membranes. CONCLUSION: Changes in hemodialysis modality were followed by both higher body fat percentage and patient survival. The reverse epidemiology of overweight patients might be at least partially the result of the influence of nonnutritional factors, such as a change in hemodialysis modality (introducing biocompatible high-flux and low-flux membranes and bicarbonate dialysis).

Gorlin-Goltz syndrome.

Gorlin-Goltz syndrome, also known as basal cell nevus syndrome, is an uncommon, autosomal dominant inherited disorder, which is characterized by numerous basal cell carcinomas, maxillary keratocysts, and musculoskeletal malformations. Occasionally, it is associated with aggressive basal cell carcinomas and internal malignancies. Early diagnosis and treatment are essential, as well as genetic counseling. A patient with characteristic symptoms of nevoid basal cell carcinomas and a review of the literature are presented.