Monoclonal antibodies generated in carbonic anhydrase IX-deficient mice recognize different domains of tumour-associated hypoxia-induced carbonic anhydrase IX

Transmembrane carbonic anhydrase IX (CA IX) is frequently expressed in human tumours in response to hypoxia and may serve as a tumour marker and therapeutic target. So far, only a single monoclonal antibody (MAb) M75 with an epitope in the N-terminal proteoglycan (PG)-like region has been available for detection purposes. Attempts to produce MAbs against other parts of CA IX were unsuccessful due to the immunodominance of the PG region that significantly differs between human and mouse homologues. To overcome this problem, we used various forms of human CA IX antigen to immunize CA IX-deficient mice recently produced by targeted disruption of Car9 gene. Here, we describe new MAbs that react with human, but not mouse CA IX in different immunodetection settings, and show no cross-reactivity with CA I, II and XII. MAb IV/18 is directed to the PG region, while the other six antibodies bind to the CA domain, as determined by CA IX deletion variants. IV/18 recognizes a linear epitope, while anti-CA MAbs V/10, V/12, VII/20, VII/28, VII/32 and VII/38 react with conformational epitopes clustered into three antigenic sites. The new antibodies represent important tools for improving our knowledge of structure-function relationships in the CA IX molecule and a better understanding of the role of CA IX in cancer development. Moreover, the availability of the MAbs specific for distinct antigenic regions on two separate extracellular domains offers an opportunity to elaborate a sensitive assay that could be particularly important for CA IX detection in body fluids of cancer patients.     

Determination of K869, a Novel Oxime Reactivator of Acetylcholinesterase,in Rat Body Fluids and Tissues by Liquid-Chromatography Methods: Pharmacokinetic Study

Oxime reactivators of acetylcholinesterase (AChE) represent an integral part of standard antidote treatment of organophosphate poisoning. Oxime K869 is a novel bisquaternary non-symmetric pyridinium aldoxime with two pyridinium rings connected by a tetramethylene bridge where two chlorines modify the pyridinium ring bearing the oxime moiety. Based on in vitro assays, K869 is a potent AChE and butyrylcholinesterase (BChE) reactivator. For the investigation of the basic pharmacokinetic properties of K869 after its intramuscular application, new HPLC-UV and LC-MS/MS methods were developed and validated for its determination in rat body fluids and tissues. In this study, the SPE procedure for sample pretreatment was optimized as an alternative to routine protein precipitation widely used in oxime pharmacokinetics studies.K869 oxime is quickly absorbed into the central compartment reaching its maximum in plasma (39 ± 4 μg/mL) between 15 and 20 min. The majority of K869 was eliminated by kidneys via urine when compared with biliary excretion. However, only a limited amount of K869 (65 ± 4 ng/g of brain tissue) was found in the brain 30 min after oxime administration. Regarding the brain/plasma ratio calculated (less than 1%), the penetration of K869 into the brain did not exceed conventionally used oximes.     

Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency

The purinosome is a multienzyme complex composed by the enzymes active in de novo purine synthesis (DNPS) that cells transiently assemble in their cytosol upon depletion or increased demand of purines. The process of purinosome formation has thus far been demonstrated and studied only in human epithelial cervical cancer cells (HeLa) and human liver carcinoma cells (C3A) transiently expressing recombinant fluorescently labeled DNPS proteins. Using parallel immunolabeling of various DNPS enzymes and confocal fluorescent microscopy, we proved purinosome assembly in HeLa, human hepatocellular liver carcinoma cell line (HepG2), sarcoma osteogenic cells (Saos-2), human embryonic kidney cells (HEK293), human skin fibroblasts (SF) and primary human keratinocytes (KC) cultured in purine-depleted media. Using the identical approach, we proved in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency that various mutations of ATIC and ADSL destabilize to various degrees of purinosome assembly and found that the ability to form purinosomes correlates with clinical phenotypes of individual ADSL patients. Our results thus shown that the assembly of functional purinosomes is fully dependent on the presence of structurally unaffected ATIC and ADSL complexes and presumably also on the presence of all the other DNPS proteins. The results also corroborate the hypothesis that the phenotypic severity of ADSL deficiency is mainly determined by structural stability and residual catalytic capacity of the corresponding mutant ADSL protein complexes, as this is prerequisite for the formation and stability of the purinosome and at least partial channeling of succinylaminoimidazolecarboxamide riboside-ADSL enzyme substrates-through the DNPS pathway.     

Distinct Immunoregulatory Mechanisms in Mesenchymal Stem Cells: Role of the Cytokine Environment

Mesenchymal stem cells (MSCs) represent a population of cells which have the ability to regulate reactivity of T and B lymphocytes by multiple mechanisms. The immunoregulatory activities of MSCs are strictly influenced by the cytokine environment. Here we show that two functionally distinct cytokines, interleukin-4 (IL-4) and interferon-γ (IFN-γ), significantly potentiate the ability of MSCs to inhibit IL-10 production by activated regulatory B cells (Bregs). However, MSCs in the presence of IL-4 or IFN-γ inhibit the IL-10 production by different mechanisms. Preincubation of MSCs with IFN-γ led to the suppression, but pretreatment with IL-4 of neither MSCs nor B cells resulted in the suppression of IL-10 production. The search for candidate regulatory molecules expressed in cytokine-treated MSCs revealed different patterns of the gene expression. Pretreatment of MSCs with IFN-γ, but not with IL-4, induced expression of indoleamine-2,3-dioxygenase, cyclooxygenase-2 and programmed cell death-ligand 1. To identify the molecule(s) responsible for the suppression of IL-10 production, we used specific inhibitors of the putative regulatory molecules. We found that indomethacine, an inhibitor of cyclooxygenase-2 (Cox-2) activity, completely abrogated the inhibition of IL-10 production in cultures containing MSCs and IFN-γ, but had no effect on the suppression in cell cultures containing MSCs and IL-4. The results show that MSCs can inhibit the response of B cells to one stimulus by different mechanisms in dependence on the cytokine environment and thus support the idea of the complexity of immunoregulatory action of MSCs.     

Analysis of a cell line derived from a chicken hepatoma induced by virus MC29

A cell line derived from a transplantable chicken hepatoma induced by virus MC29 was established. This cell line grew permanently over 2 years and could be easily recovered from frozen state. Morphology of cells did not change during the period of cultivation. Karyological analysis revealed the hypodiploid stemline with reduced numbers of microchromosomes. The cell line was not transplantable in 1-day-old chicks, but induced multiple tumour nodules in the mesenterium and liver about 8 weeks postinoculation. The cell line is virus productive. The analysis of viral proteins with the gag specific determinants showed the presence of pr76 and p110.     

In vitro antiplatelet activity of flavonoids from Leuzea carthamoides

Plants and their secondary metabolites, including flavonoids, exhibit a wide range of biological effects. Consequently, natural substances are receiving an increased attention in medicinal research. Owing to these facts, in vitro antiplatelet activity of ethanol summary extract and four flavonoids from Leuzea carthamoides was determined in human platelet-rich plasma. Arachidonic acid (AA), adenosine diphosphate (ADP), collagen (COL), and thrombin were used as agonists of platelet aggregation. The summary extract showed a significant inhibition of the aggregation induced by COL and ADP. Of the tested flavonoids, eriodictyol (1) and patuletin (2) influenced COL- and AA-induced aggregation. Their IC(50) values are presented. Flavonoid glycosides eriodictyol-7-beta-glucopyranoside (3) and 6-hydroxykaempferol-7-O-(6'-O-acetyl-beta-D[small cap]-glucopyranoside) (4) were found to be weak antiplatelet agents. These results confirmed the fact that glucosylation decreases the antiplatelet activity. Quantitative composition of tested flavonoids in L. carthamoides extract was also determined. Though two of the tested flavonoids inhibited platelet aggregation, further evaluation of L. carthamoides, in order to discover other antiplatelet active compounds and possible adverse health effects, is needed.     

Intervention for cardiovascular risk factors decreases adipocyte fatty acid-binding protein levels in males — a pilot study

Cardiovascular disease (CVD) remains the leading cause of mortality in developed countries. According to the 2012 European Guidelines on Cardiovascular Disease Prevention in Clinical Practice, family history is a cornerstone for risk stratification of CVD. First-degree relatives are persons in whom CVD should be assessed and targeted intervention should be performed. The aims of this pilot study were (i) to determine risk factors (RFs) for cardiovascular disease (CVD) in a group of first-degree relatives of patients with CVD at baseline and after 1 year, (ii) to measure adipocyte fatty acid-binding protein (A-FABP) levels as a potential connecting link between metabolic disease and atherosclerosis, and (iii) to determine the impact of targeted intervention on these parameters. The study comprised 62 asymptomatic subjects (41 males; mean age of 53.8±8.3 years). Preventive examinations and interventions were carried out at baseline and at 1-year follow-up to assess RFs and evaluate A-FABP levels. At 1 year, males had significantly lower levels of cholesterol (median 5.18 vs 4.67, p=0.005), HDL (median 1.24 vs 1.14, p=0.021), LDL (median 3.08 vs 2.46, p=0.021), ApoB (median 0.99 vs 0.82, p=0.012) and A-FABP (median 19.84 vs 16.73, p = 0.015). In females after 1 year, only significantly lower levels of fibrinogen (median 3.10 vs 2.79, p=0.043) were found. All subjects were clinically examined or contacted by phone after a mean of 36.7 months (range, 11–55). Over that time, no serious complications were noted. In males, intervention for RFs leads to lower levels of A-FABP as a potential RF linking metabolic syndrome to atherosclerosis.