Blood Fractions Research Articles

Bloodless Autologous Hematopoeitic Stem Cell Transplant: An Alternative for Jehovah's Witnesses and Others Who Prefer to Go Bloodless

Topic Significance & Study Purpose/Background/Rationale Every year people are diagnosed with Non-Hodgkin's Lymphoma (NHL) and Multiple Myeloma (MM). As an alternative treatment modality, they may seek to have a Hematopoietic Stem Cell Transplant (HSCT), which usually requires blood product transfusions. This poses a challenge when treating Jehovah's Witnesses, who normally refuse whole blood products based on religious beliefs; however, many will accept minor blood fractions and recombinant products, such as erythropoietin and granulocyte colony stimulating factors (G-CSF). Methods, Intervention, & Analysis In this study, five Jehovah's Witnesses were enrolled in the Bloodless Care Program and went through a series of pretransplant testing. Testing included an echocardiogram, electrocardiogram, and stress test to evaluate possible underlying coronary artery disease and cardiac amyloidosis. Once they qualified for a transplant, a conditioning regimen was determined and they were prepared for apheresis. Before apheresis, the patient must have hemoglobin of 11.0 g/dL and platelet count of 100 K/mm3; if not, erythropoietin and intravenous iron were given to achieve that goal. Patients were then prepped with G-CSF and Plerixafor for stem cell collection. The goal for collection was a minimum of 4 million CD34+ cells per kilogram. Post collection, there was a two week window given prior to admission for the bloodless autologous HSCT. The conditioning regimen varied on their diagnosis. For the four MM patients, the regimen was Melphalan 140mg/m2; for the one NHL patient, the regimen was Carmustine, Etoposide, and Cytoxan (BECy). When the platelet count fell below 30 K/mm3 post-transplant, Amicar was administered. Minimal phlebotomy and pediatric tubes were recommended to reduce iatrogenic anemia. Findings & Interpretation The Average length of stay was 16.7 days. After transplant, the median nadir hemoglobin was 9.15 g/dL, while the platelets were 5.5 K/mm3. One patient was readmitted within 30 days after transplant for fever during engraftment. One patient relapsed and received a second bloodless autologous HSCT 9 months later. There were no mortalities. Discussion & Implications Utilizing minor fractions, blood conservation modalities, and appropriate pre-transplant workup, resulted in safely performing bloodless autologous HSCT for Jehovah's Witness; therefore, proving this can be an alternative treatment for those who prefer to go bloodless.

Estimation of diffusion, perfusion and fractional volumes using a multi-compartment relaxation-compensated intravoxel incoherent motion (IVIM) signal model

Compartmental diffusion MRI models that account for intravoxel incoherent motion (IVIM) of blood perfusion allow for estimation of the fractional volume of the microvascular compartment. Conventional IVIM models are known to be biased by not accounting for partial volume effects caused by free water and cerebrospinal fluid (CSF), or for tissue-dependent relaxation effects. In this work, a three-compartment model (tissue, free water and blood) that includes relaxation terms is introduced. To estimate the model parameters, in vivo human data were collected with multiple echo times (TE), inversion times (TI) and b-values, which allowed a direct relaxation estimate alongside estimation of perfusion, diffusion and fractional volume parameters. Compared to conventional two-compartment models (with and without relaxation compensation), the three-compartment model showed less effects of CSF contamination. The proposed model yielded significantly different volume fractions of blood and tissue compared to the non-relaxation-compensated model, as well as to the conventional two-compartment model, suggesting that previously reported parameter ranges, using models that do not account for relaxation, should be reconsidered.

Reduced Intensity Conditioned Bone Marrow Transplant with Post-Transplant Cyclophosphamide and Donor-Derived Mesenchymal Stromal Cell Infusions for Recessive Dystrophic Epidermolysis Bullosa

Introduction: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe, life-limiting systemic genodermatosis, characterized by COL7A1 mutation(s) yielding inadequate type VII collagen to maintain the integrity of the cutaneous basement and mucosal membranes. The mainstay of therapy for RDEB is supportive care to minimize the daily morbidity of blistering/scarring, pain/pruritis, systemic inflammation, and high metabolic demand. Leveraging the pluripotency of bone marrow cells, investigations of BMT as a systemic therapy for RDEB showed promise in pre-clinical murine (Tolar J, et al., Blood 2009, 113(5): 1167-74) as well as early phase human clinical trials utilizing fully myeloablative conditioning (Wagner J, et al., NEJM 2010, 363(7): 629-39). However, regimen-rated toxicity limited dampened enthusiasm for the clinical approach.Methods: In an effort to modulate disease activity with decreased toxicity, we investigated the safety of and preliminary responses to BMT with a reduced-intensity conditioning regimen (rabbit anti-thymocyte globulin 2.5 mg/kg with a methylprednisolone taper, cyclophosphamide 28 mg/kg, fludarabine 150 mg/m2, and low dose total body irradiation 300-400 cGy). Graft-versus-host disease (GvHD) prophylaxis included post-transplant cyclophosphamide (PTCy, 50 mg/kg recipient weight/dose on days +3 and 4) and mycophenylate mofetil from day +5 to 35. All except HLA-matched related donor recipients received tacrolimus from day +5 until tapered at day +100. Immunomodulatory donor-derived mesenchymal stromal cells (MSCs, 2 x 106 cells/kg recipient weight/dose) were infused at 60, 100 and 180 days post-BMT. Skin biopsies, medical photography, and dynamic assessments of RDEB disease activity by providers and parents were completed at baseline, day +100, +180 and 1 year post-BMT.Results: Ten RDEB patients were transplanted at a median age of 9.9 years (range 1.8 to 22.1), with a median follow-up of 16 months (1 year evaluations available for only 4 of 10). Donors were haploidentical related (n=6), HLA-matched related (n=3), and HLA-matched unrelated (n=1). BMT complications included graft failure in 3 patients (2 elected to pursue a 2nd PTCy BMT), veno-occlusive disease in 2, posterior reversible encephalopathy in 1, and chronic GvHD in 1, the latter deceased. Infectious complications in the first 100 days were limited to 3 viremia, 7 bacteremia, and 0 fungemia episodes. No serious adverse events were observed with MSC infusions. In the 9 ultimately engrafted patients, median donor chimerism at day +180 was 100% in both myeloid and lymphoid fractions of peripheral blood and 27% in skin. Skin biopsies at day +180 show stable (n=7) to improved (n=2) type VII collagen protein expression by immunofluorescence (IF) and gain of anchoring fibril components by transmission electron microscopy in 4 of 9 patients. Early clinical response included a trend toward reduced body surface area of blisters/erosions from a median of 45.5% at baseline to 27.5% at day +100 (p=0.05), with parental measures indicating stable quality of life. Select medical photography and skin biopsy results are shown for Patient 1 [IF: 40x merged dapi (blue) and C7 collagen antibody (red); immunoelectron microscopy with C7 collagen-directed immunostain (black)].Conclusions: BMT using reduced-intensity conditioning, PTCy and donor-derived MSC infusion for RDEB was largely well tolerated with low rates of GvHD and death from regimen-related toxicity in 1 of 10 patients undergoing 12 total BMTs. Early follow-up suggests this treatment modulates RDEB disease activity but requires longer follow-up for evaluation of efficacy. Importantly, the PTCy BMT platform provides a means of attaining immunotolerance for future donor-derived cellular grafts. [Display omitted] DisclosuresWagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding.

Obtaining High-Quality Blood Specimens for Downstream Applications: A Review of Current Knowledge and Best Practices.

Blood is a biological fluid that contains multiple blood fraction and cellular components. High-quality blood specimens are essential prerequisites for various downstream applications such as molecular epidemiology studies, genomics, and proteomics studies. Currently, protocols and research publications concerning the collection, handling, preservation, and stability of blood or blood fractions are constantly emerging. Moreover, standardized guidelines are a requirement for biorepositories to tightly control preanalytical variables originating from these procedures and obtain high-quality blood specimen for downstream analyses. In this review article, we summarize the best practices and fit-for-purpose protocols regarding blood collection, processing, storage, and stability. In addition, we present some typical quality biomarkers, which could be used to evaluate the integrity of blood specimens.

CD133 expression in circulating hematopoietic progenitor cells.

Circulating hematopoietic progenitors (HPCs) are involved in inflammatory diseases such as atherosclerosis, the immune response to cancer, and disorders of the hematopoietic system. HPC characterization by flow cytometry typically utilizes CD34 in combination with other cell surface markers to identify cell populations that give rise to specific hematopoietic lineages. CD133, also known as prominin-1, is a cell surface protein found in HPCs that has a similar but not interchangeable expression pattern with CD34 for characterization of HPC populations. Our goal was to determine the distribution of CD133 expression within HPC sub-types amongst circulating CD34+ cells. The quantity of different HPC populations within the CD34+ CD133+ and CD34+ CD133- fractions of venous blood obtained from healthy human subjects was measured using multicolor flow cytometry. The majority of circulating CD34+ cells is CD133-. CD133+ CD34+ cells express low levels of CD38, contain cell populations bearing cell surface markers of hematopoietic stem cells, multipotent progenitors, and multilymphoid progenitors, and are largely devoid of CD38 expressing lineage specified progenitors. These findings clarify the composition of circulating CD133+ CD34+ cell types in adult human subjects. © 2018 International Clinical Cytometry Society.

Detection of activated neutrophils by reactive oxygen species production using a hematology analyzer

Neutrophils are recruited to infection sites and kill bacteria by phagocytosis and reactive oxygen species (ROS) production. It has been reported that vacuoles are present in neutrophils that produce ROS and are present in large numbers in blood smears of patients with bacterial infections. The leukocyte differentiation function on the Sysmex automated hematology analyzer classifies leukocytes by flow cytometry. Particularly, side-scattered light is known to reflect the quantity of organelles.This study investigated the possibility of detecting vacuoles or invagination of cell membrane in neutrophils producing ROS using a hematology analyzer.Whole blood and polymorphonuclear (PMN) cell fractions were activated with phorbol myristate acetate (PMA) or formylmethionylleucylphenylalanine (fMLP) and analyzed using the Sysmex XE-2100 automated hematology analyzer. PMN fractions were morphologically analyzed with a confocal laser scanning microscope (CLSM), electron microscope (EM), and general-purpose conventional flow cytometer.In the white blood cell differentiation scattergram obtained in this analysis, a new cluster separate from the original neutrophil cluster appeared in the eosinophil area in an area of higher side-scattering (SSC) intensity. Flow cytometry analysis of the PMN fractions revealed that the cells in this new cluster were CD16b- and APF-positive, indicating that the cells were activated neutrophils that produced ROS. CLSM and EM findings revealed that ROS production occurred in the cytoplasm and that the activated neutrophils contained some vacuole-like structures of vacuoles or invagination of cell membrane.Vacuole-like Sstructures were found within the cytoplasm of neutrophils producing ROS. These neutrophils were detected as an independent cluster in the eosinophil area with higher SSC intensity than that shown by neutrophils in the traditional cluster on the white blood cell differentiation scattergram, likely because the vacuole-like structures increased the SSC intensity.

Exogenous phytase and xylanase exhibit opposing effects on real-time gizzard pH in broiler chickens

ABSTRACT1. The current study was conducted to evaluate the influence of high phytase doses and xylanase, individually and in combination, on performance, blood inositol and real-time gastric pH in broilers fed wheat-based diets.2. In a 42-d experiment, a total of 576 male Ross 308 broiler chicks were allocated to 4 dietary treatments. Treatments consisted of a 2 × 2 factorial arrangement, with 500 or 2500 FTU/kg phytase and 0 or 16 000 BXU/kg xylanase, fed in two phases (starter 0–21; grower 21–42 d). Heidelberg pH capsules were administered to 8 birds from each treatment group, pre- and post-diet phase change, with readings captured over a 5.5-h period.3. At 21 and 42 d, birds fed 500 FTU/kg phytase without xylanase had on average 127 and 223 g lower weight gain than all other treatments, respectively (P < 0.05). At 42 d, body weight-corrected feed conversion ratio (bwcFCR) was reduced (P < 0.05) by supplementing 2500 FTU/kg phytase or xylanase, with the combination giving a 12 point reduction in bwcFCR compared to birds fed 500 FTU/kg phytase without xylanase. Inositol content of plasma was twice that of the erythrocyte (P < 0.001), with 2500 FTU/kg phytase tending to increase (P = 0.07) inositol content in both blood fractions.4. Across all treatments, capsule readings ranged from pH 0.54 to 4.84 in the gizzard of broilers. Addition of 2500 FTU/kg phytase to the grower diet reduced (P < 0.05) average gizzard pH from 2.89 to 1.69, whilst feeding xylanase increased (P < 0.001) gizzard pH from 2.04 to 2.40. In contrast, digital probe measurements showed no effect of xylanase on gizzard pH, while addition of 2500 FTU/kg phytase increased (P = 0.05) pH compared to 500 FTU/kg phytase with or without xylanase.5. These findings suggested that xylanase and high phytase doses have opposite effects on real-time gastric pH, while similarly improving performance of broilers.

CARRAGEENAN INDUCES CELL DEATH IN RATS BLOOD

Background. Because of its physical and chemical properties, carrageenan is fairly widely used. About 70 % of the carrageenan produced in the world is used in the food industry. Previous studies point to the development of oxidative stress in rats, by means of which carrageenan chronic enterocolitis was modeled.Objective. The aim of our study was to investigate the level of apoptosis and necrosis in the suspension of leukocytes in rats using 0.5 % and 1.0 % solutions of carrageenan.Methods. Annexin V (V) binding assays were performed using Annexin V Apoptosis Kit (Sigma Aldrich, USA), caspase rate in leukocyte-lymphocyte blood fractions was determined by spectrofotometry.Results. It was established that in the experimental application of carrageenan, the percentage of leukocytes with signs of apoptosis in both experimental groups statistically significantly increased. It was detected by the increased activity of effector caspase-3 in 1 month after the experiment in 1.5 times in the 2nd group and in 2.8 times in the 3rd group vs control data that point to caspase-dependent apoptotic pathway in case of carrageenan usage in rats.Conclusions. Oral use of carrageenan in rats was accompanied by the increase in the number of leukocytes with signs of apoptosis. The animals that consumed 1.0 % solution of carrageenan had more obvious increase in the activity of caspase-3 in serum relative to a group of rats consuming 0.5 % of carrageenan, proving the increase in the severity of apoptotic processes in intestine with the increase of the dose of carrageenan.

Влияние низкомолекулярной фракции (до 5 кда) кордовой крови человека на ростовые свойства культуры мезенхимальных стромальных клеток, подвергнутых криоконсервированию

The influence of cryopreservation on growth properties, immune phenotype and differentiation potential of mesenchymal stromal cells (MSCs) of human and rat was investigated, as well as the ability of human cord blood fractions below 5 kD (hCBF) to affect these indices in thawed cultures. It has been established that introduction of the hCBF with the concentration of 256-400 µg/ml to rat MSCs (rMSCs) culture caused the normalization of the cells growth properties reduced after cryopreservation that was not observed in human MSCs (hMSCs) culture. It has been shown that neither cryopreservation nor hCBF change the immune phenotype and ability for multi-lineage differentiation of hMSCs and rMSCs. The efficiency of low molecular weight fraction dependent on physiological characteristics of cells and its concentration in culture medium was concluded. Probl Cryobiol Cryomed 2018; 28(2): 131–138

Biobanking in a Challenging African Environment: Unique Experience from the SIREN Project.

Africa was previously insufficiently represented in the emerging discipline of biobanking despite commendable early efforts. However, with the Human, Heredity, and Health in Africa (H3Africa) initiative, biorepository science has been bolstered, regional biobanks are springing up, and awareness about biobanks is growing on the continent. The Stroke Investigative Research and Educational Network (SIREN) project is a transnational, multicenter, hospital and community-based study involving over 3000 cases and 3000 controls recruited from 16 sites in Ghana and Nigeria. SIREN aims to explore and unravel the genetic and environmental factors that interact to produce the peculiar phenotypic and clinical characteristics of stroke as seen in people of African ancestry and facilitate the development of new diagnostics, therapeutics, and preventative strategies. The aim of this article is to describe our experience with the development of the procedure for collection, processing, storage, and shipment of biological samples (blood, serum, plasma, buffy coat, red cell concentrates, and DNA) and brain imaging across coordinating and participating sites within the SIREN Project. The SIREN network was initiated in 2014 with support and funding from the H3Africa Initiative. The SIREN Biobank currently has 3015 brain images, 92,950 blood fractions (serum, plasma, red cell concentrates, and buffy coat) accrued from 8450 recruited subjects, and quantified and aliquoted good-quality DNA extracts from 6150 study subjects. This represents an invaluable resource for future research with expanding genomic and trans-omic technologies. This will facilitate the involvement of indigenous African samples in cutting-edge stroke genomics and trans-omics research. It is, however, critical to effectively engage African stroke patients and community members who have contributed precious biological materials to the SIREN Biobank to generate appropriate evidence base for dealing with ethical, legal, and social issues of privacy, autonomy, identifiability, biorights, governance issues, and public understanding of stroke biobanking in the context of unique African culture, language, and belief systems.

Aggressive Phenotype of Cells Disseminated via Hematogenous and Lymphatic Route in Breast Cancer Patients

Intratumoral heterogeneity of breast cancer remains a major challenge in successful treatment. Failure of cancer therapies can also be accredited to inability to systemically eradicate cancer stem cells (CSCs). Recent evidence points to the role of epithelial-mesenchymal transition (EMT) in expanding the pool of tumor cells with CSCs features. Thus, we assessed expression level as well as heterogeneity of CSCs markers in primary tumors (PT), lymph node metastasis (LNM), and circulating tumor cells (CTCs)–enriched blood fractions in order to correlate them with signs of EMT activation as well as clinicopathological data of breast cancer patients. Level of CSCs markers (ALDH1, CD44, CD133, OCT-4, NANOG) and EMT markers was quantified in PT (N=107), LNM (N=56), and CTCs-enriched blood fractions (N=85). Heterogeneity of CSCs markers expression within each PT and LNM was assessed by calculating Gini Index. Percentage of ALDH1-positive cells was elevated in PT in comparison to LNM (P = .005). However, heterogeneity of the four CSCs markers: ALDH1 (P = .019), CD133 (P = .009), OCT-4 (P = .027), and CD44 (P < .001) was decreased in LNM. Samples classified as mesenchymal (post-EMT) showed elevated expression of CSCs markers (OCT-4 and CD44 in PT; OCT-4 in LNM; ALDH1, OCT-4, NANOG, CD44 in CTCs). Patients with mesenchymal-like CTCs had worse prognosis than patients with epithelial-like or no CTCs (P = .0025). CSCs markers are enriched in PT, LNM, and CTCs with mesenchymal features, but their heterogeneity is decreased in metastatic lymph nodes. Mesenchymal CTCs phenotype correlates with poor prognosis of the patients.

Comprehensive analysis of blood cells and plasma identifies tissue-specific miRNAs as potential novel circulating biomarkers in cattle

BackgroundThe potential of circulating miRNAs as biomarkers of tissue function, both in health and disease, has been extensively demonstrated in humans. In addition, circulating miRNA biomarkers offer significant potential towards improving the productivity of livestock species, however, such potential has been hampered by the absence of information on the nature and source of circulating miRNA populations in these species. In addition, many miRNAs originally proposed as robust biomarkers of a particular tissue or disease in humans have been later shown not to be tissue specific and thus to actually have limited biomarker utility. In this study, we comprehensively analysed miRNA profiles in plasma and cell fractions of blood from cattle with the aim to identify tissue-derived miRNAs which may be useful as biomarkers of tissue function in this important food animal species.ResultsUsing small RNA sequencing, we identified 92 miRNAs with significantly higher expression in plasma compared to paired blood cell samples (n = 4 cows). Differences in miRNA levels between plasma and cell fractions were validated for eight out of 10 miRNAs using RT-qPCR (n = 10 cows). Among miRNAs found to be enriched in plasma, we confirmed miR-122 (liver), miR-133a (muscle) and miR-215 (intestine) to be tissue-enriched, as reported for other species. Profiling of additional miRNAs across different tissues identified the human homologue, miR-802, as highly enriched specifically in liver.ConclusionsThese results provide novel information on the source of bovine circulating miRNAs and could significantly facilitate the identification of production-relevant tissue biomarkers in livestock. In particular, miR-802, a circulating miRNA not previously identified in cattle, can reportedly regulate insulin sensitivity and lipid metabolism, and thus could potentially provide a specific biomarker of liver function, a key parameter in the context of post-partum negative energy balance in dairy cows.

Heat Shock Protein Response During a Prolonged Ultraendurance Cycling Event in Hot and Humid Conditions

The heat shock protein (HSP) response is critical to the cellular stress response. Circulating, extracellular stress‐inducible HSP72 is known to respond to exercise‐heat stress, but little is known about other, constitutively expressed and fundamental members of the HSP family. The aim of this study was to investigate the extracellular HSP27, HSP60, and HSP90α response during a prolonged cycling event in hot and humid conditions. Seventy‐six subjects (mean±SD: age, 51.64±10.47 years; height, 176.44±6.85 cm; mass, 86.78±13.93 kg) were recruited at the Hotter'n Hell 100 mile cycling event in Wichita Falls, Texas. Blood and urine samples, as well as data on body weight and gastrointestinal temperature (TGI) were recorded before (pre) and after (post) the event. Plasma was isolated from blood samples, stored at −80°C, and later analyzed with StressMarq Biosciences ELISAs for HSP27, HSP60, and HSP90α concentrations. Student's t‐test was used to test for significant differences of means. TGI significantly increased from pre to post (pre: 37.14±0.55 °C; post: 38.12±0.79 °C; p&lt;0.05). HSP27 and HSP90α were both significantly elevated post race (pre27: 2.73±0.51 ng × mL−1; post27: 3.46±0.73 ng × mL−1; pre90α: 18.03±3.27 ng × mL−1; post90α: 21.53±4.84 ng × mL−1; p &lt; 0.05). However, there was no significant difference in HSP60 plasma concentration from pre to post (pre: 44.05±7.38; post: 43.65±6.81; p=0.77). In conclusion, significant increases in circulating HSP27 and HSP90α suggest that chaperones required for thermotolerance and protein degradation or folding during heat stress increase in extracellular compartments post exercise‐heat stress. However, our data suggest that chaperones specifically involved in mitochondrial protein assembly or molecular danger signals like HSP60 are not elevated in blood fractions. Ongoing work will characterize cellular expression of these proteins in circulating peripheral blood mononuclear cells.Support or Funding InformationNew investigator start‐up, McNair Scholar ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chimerism Analysis in the Pediatric Setting: Direct PCR from Bone Marrow, Whole Blood, and Cell Fractions

Certain blood components and anticoagulants interfere with the PCR process and subsequent analysis. Here we demonstrate that reliable test results can be obtained for chimerism analysis despite omitting a DNA-extraction step and performing PCR and fragment analysis directly on bone marrow, whole blood, and individual cell fractions. For chimerism analysis, direct-tissue PCR is possible with the use of a robust, commercially available PCR mix containing a DNA polymerase capable of DNA amplification directly from the sample without the need for pretreatment. A total of 178 chimerism samples were processed directly, and results were compared to those obtained from the corresponding DNA sample. No differences were observed between the two sets of results. For the cell fraction-purity assessment, commercially available PCR kitswere used directly on T and B cells without the use of any additional lysing agent. A total of 53 purity samples and their corresponding DNA samples were analyzed and showed a correlation similar to thatobtained for the chimerism samples. The results show that chimerism testing and associated cell fraction-purity assessment can be performed reliably without the need for prior DNA extraction andthatthis method can easily be integrated into existing routine laboratory procedures.

Mercury and metallothioneins in blood fractions and tissues of captive Morelet's crocodiles in Quintana Roo, Mexico

Even though studies of heavy metals and their relation with metallothioneins (MTs) in reptile tissues have been conducted, research on heavy metals and MTs in organs and blood fractions of crocodylians is limited. To date there are no studies on the distribution of MTs in organs and their relation with mercury (Hg), or the concentration of MTs in plasma and erythrocytes of crocodylians. In order to understand the role of MTs in crocodiles, our aim was to assess the detoxification mechanisms for exposure to metals in Morelet's crocodile (Crocodylus moreletii) by using two biomarkers (Hg and MTs) in blood fractions and tissues, and comparing them with concentrations between two populations of crocodiles, one previously wild and currently captive (Theme Park) and another raised in a Wildlife Management Unit (WMU). The caudal scutes from the Theme Park showed higher concentrations of Hg than those from the skin in the WMU samples, and significant negative relationships were observed between the total length (TL) and Hg in the scutes. The significant negative relationship between Hg and hemoglobin (Hb) may be due to disorders in the oxidation process and even denature of this protein, while the positive trend observed between MTs and Hb is consistent with the detoxifying functions and the protection against oxidative damage. This study is the first to report Hg in the erythrocytes of crocodylians and the use of MTs for testing the potential of these biomarkers as a tool to assess Hg exposure in crocodile's habitats.

ЗМІНИ ПОКАЗНИКІВ КАРБОНІЛЬНО-ОКСИДАТИВНОГО СТРЕСУ В ЩУРІВ З ЕКСПЕРИМЕНТАЛЬНОЮ ІШЕМІЄЮ МІОКАРДА ПІД ВПЛИВОМ ДОКСИЦИКЛІНУ

Heart diseases, especially acute myocardial infarction (AMI), belong to the most severe illnesses that often lead to death. Despite a large number of studies, the biochemical mechanisms of AMI and post-infarction myocardial remodeling are poorly understood. Carbonyl-oxidative stress (COS) is one of the more important triggers of the post-infarction complications in these patients, so the neutralizing of the intermediates and final products of COS are a perspective direction in the treatment of AMI. Flavonoide antioxidants as well as inhibitors of carbonylation and glycation of proteins shown the cardioprotective effects but their use have some limitations. Recently, new studies have appeared concerning the cardioprotective action of the doxycycline (DC). This tetracycline antibiotic can inhibit matrix metalloproteinases and proteolysis in extracellular matrix. At the same time, the presence of a multiple-substituted phenol ring can provide the ability of DC to neutralize free radicals, so we hypothesized that it can inhibit the COS. This article compares the effects of small (4,2 mg/kg) and of large (16.8 mg/kg) doses of DC with the effects of classical antioxidants, corvitin and aminoguanidine. The COS-markers and activity of antioxidant enzymes were determined in the blood and subcellular heart fractions of the rats with pituitrin-isoproterenol-induced myocardial damage. It has been established that DC exhibits cardioprotective properties, reducing the formation of products of carbonyl and oxidative modification of biomolecules TBA-active substances, fluorescent end products of glycation (fAGE), aldehyde phenylhydrazones (AFH) and ketone phenylhydrazones (CPH), and a more pronounced effect was shown for the low doses of this drug. Simultaneously, the DC activates enzymes of antioxidant protection, first of all, glutathione peroxidase. Effects of small doses of DC are comparable or exceed the action of aminoguanidine and corvitin, so DC can be useful in the treatment of postinfarction heart failure.

Effect of different commercial fat sources on brain, liver and blood lipid profiles of rats in growth phase.

To investigate the fatty acid content of different fat sources and evaluate the effect of them on plasma and hepatic lipids and on the fatty acid profile of the brain tissue of Wistar rats. Thirty male albino Wistar rats received for 59 days, the following diets: diet added of margarine with low content of polyunsaturated fatty acids (PUFA); diet added of margarine with high content of PUFA; diet added of butter; diet added of hydrogenated vegetable fat; diet added of soybean oil. Fatty acid profile of the lipid sources, blood and hepatic lipids fractions and fatty acid profile of the brain tissue were determined. Margarine consumption of provided different responses as to concentrations of blood and hepatic lipid fractions. Intake of butter and hydrogenated increased LDL-c/HDL-c ratio, being the steepest increase promoted by hydrogenated vegetable fat, which also raised LDL-c levels expressively. All fats used in the treatments reduced the cerebral concentration of docosahexaenoic acid when compared to soybean oil (control). The different fat sources commonly consumed by population provided different responses in vivo. This is particularly relevant considering the role of these lipids in the incidence and prevention of cardiovascular diseases.

Plasma miR-155, miR-203, and miR-205 are Biomarkers for Monitoring of Primary Cutaneous T-Cell Lymphomas.

Primary cutaneous T-cell lymphomas (CTCL) affect the skin and tend to transform and spread. CTCL involves primarily the Mycosis fungoides (MF) and more aggressive Sezary syndrome (SS). Oncogenic microRNAs (miRs) are stable epigenetic inhibitors often deregulated in the tumour and detectable as biomarkers in non-cellular fractions of peripheral blood. The tumour-specific expression of miR-155, miR-203, and miR-205 was shown to correctly diagnose CTCL. We herein asked whether these microRNAs can be used as plasma biomarkers for clinical CTCL monitoring. Patients with CTCL (n = 10) and controls with non-malignant conditions (n = 11) repeatedly donated plasma samples every ca. five months. MicroRNAs were detected in the plasma samples by specifically-primed RT-PCR followed by multivariate analyses of the miR expression dynamics. We herein established the plasma miR-classifier for detecting CTCL based on the miR-155 upregulation and miR-203/miR-205 downregulation with 100% specificity and 94% sensitivity. The 3-miR-score in the consecutive samples coincided with the clinical outcome of MF and SS patients such as the therapy response or changes in the clinical stage or tumor size. Quantitation of the selected microRNAs in plasma is a specific and straightforward approach for evaluating CTCL outcome representing, thus, a valuable tool for CTCL diagnostics and therapy response monitoring.

Mouse Red Blood Cell–Mediated Rare Xenobiotic Phosphorylation of a Drug Molecule Not Intended to Be a Kinase Substrate

Phosphorylation of xenobiotics is rare, probably owing to a strong evolutionary pressure against it. This rarity may have attracted more attention recently as a result of intentionally designed kinase-substrate analogs that depend on kinase-catalyzed activation to form phosphorylated active drugs. We report a rare phosphorylated metabolite observed unexpectedly in mouse plasma samples after an oral dose of a Tankyrase inhibitor that was not intended to be a kinase substrate, i.e., (S)-2-(4-(6-(3,4-dimethylpiperazin-1-yl)-4-methylpyridin-3-yl)phenyl)-8-(hydroxymethyl)quinazolin-4(3H)-one (AZ2381). The phosphorylated metabolite was not generated in mouse hepatocytes. In vitro experiments showed that the phosphorylation of AZ2381 occurred in mouse whole blood with heparin as anticoagulant but not in mouse plasma. The phosphorylated metabolite was also produced in rat, dog, and human blood, albeit at lower yields than in mouse. Divalent metal ions are required for the phosphorylation since the reaction is inhibited by the metal chelator EDTA. Further investigations with different cellular fractions of mouse blood revealed that the phosphorylation of AZ2381 was mediated by erythrocytes but did not occur with leukocytes. The levels of 18O incorporation into the phosphorylated metabolite when inorganic 18O4-phosphate and γ-18O4-ATP were added to the mouse blood incubations separately suggested that the phosphoryl transfer was from inorganic phosphate rather than ATP. It remains unclear which enzyme present in red blood cells is responsible for this rare phosphorylation.

Compound-specific isotope analysis resolves the dietary origin of docosahexaenoic acid in the mouse brain

DHA (22:6n-3) may be derived from two dietary sources, preformed dietary DHA or through synthesis from α-linolenic acid (ALA; 18:3n-3). However, conventional methods cannot distinguish between DHA derived from either source without the use of costly labeled tracers. In the present study, we demonstrate the proof-of-concept that compound-specific isotope analysis (CSIA) by GC-isotope ratio mass spectrometry (IRMS) can differentiate between sources of brain DHA based on differences in natural 13C enrichment. Mice were fed diets containing either purified ALA or DHA as the sole n-3 PUFA. Extracted lipids were analyzed by CSIA for natural abundance 13C enrichment. Brain DHA from DHA-fed mice was significantly more enriched (-23.32‰ to -21.92‰) compared with mice on the ALA diet (-28.25‰ to -27.49‰). The measured 13C enrichment of brain DHA closely resembled the dietary n-3 PUFA source, -21.86‰ and -28.22‰ for DHA and ALA, respectively. The dietary effect on DHA 13C enrichment was similar in liver and blood fractions. Our results demonstrate the effectiveness of CSIA, at natural 13C enrichment, to differentiate between the incorporation of preformed or synthesized DHA into the brain and other tissues without the need for tracers.