Generation and characterization of single-chain antibody fragments specific against transmembrane envelope glycoprotein gp46 of maedi-visna virus

A single-chain antibody fragments (scFv) was developed directed against transmembrane envelope glycoprotein gp46 of the virus maedi-visna, by the application of the antibody phage display library. To get specific scFv binders, the library was panned against the biotinylated peptide of 20 amino acids corresponding to the principal immunodominant domain of gp46 protein. The number of positively binding scFvs was evaluated by scFv-phage ELISA, BstN1 fingerprinting and DNA sequencing. The scFvs were expressed in soluble form and purified by immobilized metal affinity chromatography (IMAC) with a yield of 2-2.5 mg/l. Two scFvs have shown to recognize gp46 and gp150 proteins in Western blot analysis. The scFvs also recognized the virus in infected cells as shown by immunofluorescence assay. The affinity of the obtained antibody fragments to gp46 peptide was measured by surface plasmon resonance, and the resulting K(A) was in the 10(6)-10(7)lmol(-1) range. The application of characterized scFvs for expression as intrabodies in intracellular immunization against virus maedi-visna infection and for the diagnosis of this virus is discussed.     

Plasma testosterone and catecholamine responses to physical exercise of different intensities in men

Summary Plasma testosterone, noradrenaline, and adrenaline concentrations during three bicycle ergometer tests of the same total work output (2160 J·kg^–1) but different intensity and duration were measured in healthy male subjects. Tests A and B consisted of three consecutive exercise bouts, lasting 6 min each, of either increasing (1.5, 2.0, 2.5 W·kg^–1) or constant (2.0, 2.0, 2.0 W·kg^–1) work loads, respectively. In test C the subjects performed two exercise bouts each lasting 4.5 min, with work loads of 4.0 W·kg^–1. All the exercise bouts were separated by 1-min periods of rest.Exercise B of constant low intensity resulted only in a small increase in plasma noradrenaline concentration. Exercise A of graded intensity caused an increase in both catecholamine levels, whereas, during the most intensive exercise C, significant elevations in plasma noradrenaline, adrenaline and testosterone concentrations occurred. A significant positive correlation was obtained between the mean value of plasma testosterone and that of adrenaline as well as noradrenaline during exercise.It is concluded that both plasma testosterone and catecholamine responses to physical effort depend more on work intensity than on work duration or total work output.This work was performed within the Scientific Exchange Programme between the Institute of Experimental Endocrinology, Slovak Academy of Sciences in Bratislava and Medical Research Centre, Polish Academy of Sciences, Warsaw/Project 10.4/     

Hemoperfusion study with carbamazepine in vitro

Sorption efficacy of active charcoal, Amberlite XAD-2 and Amberlite XAD-4 in hemoperfusion with carbamazepine was investigated in vitro. Authors carried out 15 hemoperfusions with carbamazepine in a closed system using the above mentioned sorbents; The perfusion solution used was 3,000 ml of 0.9% NaCl. The carbamazepine concentration in the solution was 52.5+/-3.0 mg/L. In addition, two in vitro hemoperfusions with carbamazepine using Amberlite XAD-2 sorbent and 1.500 ml of human plasma as a perfusion solution, were also performed. The concentration of carbamazepine in plasma was 50.4+/-1.2 mg/L. Individual hemoperfusions lasted 5 hours. Zero values of carbamazepine were achieved after 120 min of Amberlite XAD-2 or Amberlite XAD-4 hemoperfusion in 0.9% NaCl and after 240 min of hemoperfusion in human plasma. The results gathered suggest that the most effective in vitro hemoperfusion with carbamazepine was obtained with Amberlite XAD-2 or Amberlite XAD-4 and the least effective was with active charcoal. The authors recommend the use of hemoperfusion with Amberlite XAD-2 or Amberlite XAD-4 for acute intoxications with carbamazepine in humans. In addition, authors successfully performed 5-hour hemoperfusion using Amberlite XAD-4 in vivo, in an 18-year-old girl, who suffered from acute carbamazepine intoxication after a suicidal attempt. This treatment led to the rapid survival.     

Downregulation of plasma membrane expression/cytoplasmic accumulation of beta-catenin predicts shortened survival in non-small cell lung cancer. A clinicopathologic study of 100 cases

The E-cadherin-catenin complex proteins function in cell-cell adhesion and have been reported to be dysregulated in various human malignancies. Beta catenin is a cytoplasmic protein that associates with tyrosine kinase receptors and modulates cytoskeletal dynamics. It also plays a role in the Wnt signaling pathway. During neoplastic transformation, the phosphorylation of beta-catenin causes a loss of intercellular adhesions resulting in increased tumor cell motility and invasiveness. Tissue sections from 100 cases of non-small cell lung cancer (NSCLC) were immunostained with a monoclonal beta-catenin antibody. There were 47 squamous cell carcinomas (SCC) and 53 adenocarcinomas (AC) in the study group. Plasma membrane/cytoplasmic beta-catenin immunoreactivity was scored for intensity and distribution and correlated with tumor stage, grade and survival. Plasma membrane/cytoplasmic immunoreactivity for beta-catenin protein was observed in 71 (71%) of 100 NSCLC. 44 (94%) of 47 SCC and 27 (51%) of 53 AC expressed beta catenin. On univariate analysis, loss of beta catenin expression correlated with high tumor stage (p = 0.025), large tumor size (p = 0.02) and decreased patient survival (p = 0.04). The loss of beta catenin expression associated with high grade NSCLC reached near significance (p = 0.07). On multivariate analysis, the loss of beta catenin expression independently predicted shortened overall patient survival in NSCLC (p = 0.05). Beta catenin expression loss is associated with advanced tumor stage and is an independent predictor of shortened patient survival in NSCLC.     

Radioimmunoassay of measles virus antibodies in SSPE

A sensitive radioimmunological assay (RIA) was introduced for detection of measles virus IgG and IgM antibodies. The hyperimmune response to measles virus could be demonstrated more precisely by RIA than by haemagglutination inhibition (HI). The ratio between RIA and HI antibody titres was decidedly higher in sera and cerebrospinal fluids (CSF) of patients with subacute sclerosing panencephalitis (SSPE) than in those of other groups tested.     

Age-related changes in the response to vasoconstrictor and dilator agents in isolated beagle coronary arteries

Summary Responses to vasoactive agents were compared in helical strips of coronary arteries isolated from beagles of 30 days, 3 months, 2 years and 12 years old. Serotonin contracted the arterial strips dose-dependently, the contraction being greater in the arteries of proximal portion than in the distal arteries. The contraction increases with age from 3 months to 12 years, although EC50 values did not differ. Angiotensin II contracted distal coronary arteries to a greater extent than the proximal ones. Age did not alter the peptide-induced contraction. In prostaglandin F₂-contracted coronary arteries, acetylcholine-induced relaxations, dependent on endothelium, were less in the arteries from senescent beagles than in those from adult beagles (2 years old). Histamine relaxed the infant beagle arteries to a lesser extent than the adult and senescent beagle arteries. Histamine-induced relaxations were attenuated selectively by cimetidine. Relaxations caused by adenosine and prostaglandin I₂ did not differ in coronary arteries from beagles of different ages. It may be concluded that greater responsiveness to serotonin of senescent beagle coronary arteries is due preferentially to increased function of serotonergic receptors rather than impaired function of the arterial endothelium responsible for the release of relaxing factor(s), although some impairment of the function is supposed, on the basis of interferences with acetylcholine-induced relaxation in the aged beagle arteries. Histaminergic H₂ receptor function appears to develop in beagle coronary arteries until 3 months of age. Send offprint requests to N. Toda     

Krill Oil Supplementation Reduces Exacerbated Hepatic Steatosis Induced by Thermoneutral Housing in Mice with Diet-Induced Obesity

Preclinical evidence suggests that n-3 fatty acids EPA and DHA (Omega-3) supplemented as phospholipids (PLs) may be more effective than triacylglycerols (TAGs) in reducing hepatic steatosis. To further test the ability of Omega-3 PLs to alleviate liver steatosis, we used a model of exacerbated non-alcoholic fatty liver disease based on high-fat feeding at thermoneutral temperature. Male C57BL/6N mice were fed for 24 weeks a lard-based diet given either alone (LHF) or supplemented with Omega-3 (30 mg/g diet) as PLs (krill oil; ω3PL) or TAGs (Epax 3000TG concentrate; ω3TG), which had a similar total content of EPA and DHA and their ratio. Substantial levels of TAG accumulation (~250 mg/g) but relatively low inflammation/fibrosis levels were achieved in the livers of control LHF mice. Liver steatosis was reduced by >40% in the ω3PL but not ω3TG group, and plasma ALT levels were markedly reduced (by 68%) in ω3PL mice as well. Krill oil administration also improved hepatic insulin sensitivity, and its effects were associated with high plasma adiponectin levels (150% of LHF mice) along with superior bioavailability of EPA, increased content of alkaloids stachydrine and trigonelline, suppression of lipogenic gene expression, and decreased diacylglycerol levels in the liver. This study reveals that in addition to Omega-3 PLs, other constituents of krill oil, such as alkaloids, may contribute to its strong antisteatotic effects in the liver.     

Structural volumetry in NPH diagnostics and treatment—future or dead end?

Neurosurgical Review - To assess automated volumetric analysis as a potential presurgical diagnostic tool or as a method to potentially shed light on normal pressure hydrocephalus (NPH)...     

Intercellular Interactions of an Adipogenic CXCL12-Expressing Stromal Cell Subset in Murine Bone Marrow

Bone marrow houses a multifunctional stromal cell population expressing C-X-C motif chemokine ligand 12 (CXCL12), termed CXCL12-abundant reticular (CAR) cells, that regulates osteogenesis and adipogenesis. The quiescent pre-adipocyte-like subset of CXCL12⁺ stromal cells (“Adipo-CAR” cells) is localized to sinusoidal surfaces and particularly enriched for hematopoiesis-supporting cytokines. However, detailed characteristics of these CXCL12⁺ pre-adipocyte-like stromal cells and how they contribute to marrow adipogenesis remain largely unknown. Here we highlight CXCL12-dependent physical coupling with hematopoietic cells as a potential mechanism regulating the adipogenic potential of CXCL12⁺ stromal cells. Single-cell computational analyses of RNA velocity and cell signaling reveal that Adipo-CAR cells exuberantly communicate with hematopoietic cells through CXCL12-CXCR4 ligand-receptor interactions but do not interconvert with Osteo-CAR cells. Consistent with this computational prediction, a substantial fraction of Cxcl12-creER⁺ pre-adipocyte-like cells intertwines with hematopoietic cells in vivo and in single-cell preparation in a protease-sensitive manner. Deletion of CXCL12 in these cells using Col2a1-cre leads to a reduction of stromal-hematopoietic coupling and extensive marrow adipogenesis in adult bone marrow, which appears to involve direct conversion of CXCL12⁺ cells to lipid-laden marrow adipocytes without altering mesenchymal progenitor cell fates. Therefore, these findings suggest that CXCL12⁺ pre-adipocyte-like marrow stromal cells prevent their premature differentiation by maintaining physical coupling with hematopoietic cells in a CXCL12-dependent manner, highlighting a possible cell-non-autonomous mechanism that regulates marrow adipogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).