Sequence-based typing of ade B as a potential tool to identify intraspecific groups among clinical strains of multidrug-resistant Acinetobacter baumannii

Sequence analysis of an 850-bp fragment internal to the aspecific drug efflux gene ade B revealed 11 sequence types (STs) among a collection of 50 multidrug-resistant Acinetobacter baumannii (MDRAB) strains, including members of pan-European clones I, II, and III. The delineation of STs conformed with the intraspecific grouping of these strains previously determined by different DNA fingerprinting methods. Larger strain collections need to be screened to further explore the potential of sequence-based ade B typing as a universal tool for the monitoring of MDRAB clones.     

Development of the NADPH-diaphorase-positive neurons in the sympathetic ganglia

Changes in the distribution of NADPH-diaphorase (NADPH-d) were studied in neurons of the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglia (CG) in newborn, 10-, 20-day-old, 1-month-old, 2-month-old and 6-month-old rats, mice and kittens. NADPH-d-positive neurons were revealed in all sympathetic ganglia in kittens but not in rodents from birth onwards. In kittens, the largest population of NADPH-d-positive cells was found in the SG, the smallest in the SCG (<1%) and we observed only a few cells in the CG. The proportion of NADPH-d-positive cells in the SG increased from 3.1±0.15% in newborn kittens to 9.3±0.63% in 20-day-old animals and decreased further from 8.1±0.75% in 30-day-old kittens to 3.4±0.54% in 2-month-old animals. The content of NADPH-d-positive cells in the CG and SCG did not change during development. There were no differences in cross-sectional area between neurons located in different ganglia of animals from the same age group under study. We conclude that the development of NADPH-d-positive neurons in different sympathetic ganglia has its own time dynamics and is completed by the end of the second month of life.     

SDF‐1/CXCR4/CXCR7 is pivotal for vascular smooth muscle cell proliferation and chronic allograft vasculopathy

Chronic rejection remains a major obstacle in transplant medicine. Recent studies suggest a crucial role of the chemokine SDF‐1 on neointima formation after injury. Here, we investigate the potential therapeutic effect of inhibiting the SDF‐1/CXCR4/CXCR7 axis with an anti‐SDF‐1 Spiegelmer (NOX‐A12) on the development of chronic allograft vasculopathy. Heterotopic heart transplants from H‐2bm12 to B6 mice and aortic transplants from Balb/c to B6 were performed. Mice were treated with NOX‐A12. Control animals received a nonfunctional Spiegelmer (revNOX‐A12). Samples were retrieved at different time points and analysed by histology, RT‐PCR and proliferation assay. Blockade of SDF‐1 caused a significant decrease in neointima formation as measured by intima/media ratio (1.0 ± 0.1 vs. 1.8 ± 0.1, P < 0.001 AoTx; 0.35 ± 0.05 vs. 1.13 ± 0.27, P < 0.05 HTx). In vitro treatment of primary vascular smooth muscle cells with NOX‐A12 showed a significant reduction in proliferation (0.42 ± 0.04 vs. 0.24 ± 0.03, P < 0.05). TGF‐β, TNF‐α and IL‐6 levels were significantly reduced under SDF‐1 inhibition (3.42 ± 0.37 vs. 1.67 ± 0.33, P < 0.05; 2.18 ± 0.37 vs. 1.0 ± 0.39, P < 0.05; 2.18 ± 0.26 vs. 1.6 ± 0.1, P < 0.05). SDF‐1/CXCR4/CXCR7 plays a critical role in the development of chronic allograft vasculopathy (CAV). Therefore, pharmacological inhibition of SDF‐1 with NOX‐A12 may represent a therapeutic option to ameliorate chronic rejection changes.     

Bicuculline attenuates the MLR-induced suppression of dorsal horn neurons receiving group III afferent input

In decerebrate cats, we found that iontophoretic application of bicuculline, a GABA(A) receptor antagonist, attenuated the mesencephalic locomotor region-induced suppression of dorsal horn neurons responding to group III afferent input from the hindlimb. Our findings raise the possibility that GABA release onto dorsal horn neurons in the spinal cord may play an important role in the attenuation of the exercise pressor reflex by central command during exercise.     

First-pass metabolism of ethanol in human beings: effect of intravenous infusion of fructose.

Intravenous infusion of fructose has been shown to enhance reduced form of nicotinamide adenine dinucleotide reoxidation and, thereby, to enhance the metabolism of ethanol. In the current study, the effect of fructose infusion on first-pass metabolism of ethanol was studied in human volunteers. A significantly higher first-pass metabolism of ethanol was obtained after administration of fructose in comparison with findings for control experiments with an equimolar dose of glucose. Because fructose is metabolized predominantly in the liver and can be presumed to have virtually no effects in the stomach, results of the current study support the assumption that only a negligible part of first-pass metabolism of ethanol occurs in the stomach.     

Quantitative analysis of human immunoregulatory cytokines by electrochemiluminescence method

Quantitative analysis of human immunoregulatory cytokines in physiological media and cell cultures plays an important role in fundamental and clinical research. Here we describe the quantification of interleukin (IL)-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) in human serum and peripheral blood mononuclear cell (PBMC)-conditioned medium by electrochemiluminescence method (ECL). We demonstrate that this approach allows to detect cytokine concentration from 1 pg/ml. The high sensitivity in combination with accuracy and wide range of determined concentration indicates that ECL meets the standards of quantitative analysis of cytokines. Simplicity and short time of procedure, small assay volume and high reproducibility make ECL method competitive in practical use with conventional quantitative methods of cytokine detection.     

Mycalosides B-I,eight new spermostatic steroid oligoglycosides from the sponge Mycale laxissima

New steroidal oligoglycosides mycalosides B-I (2-9) have been isolated from the polar extract of the Caribbean sponge Mycale laxissima, and their structures have been elucidated by 1D and 2D NMR ((1)H, (13)C, DEPT, COSY-45, COSY-RCT, HSQC, HMBC, and NOESY spectra) and MALDI-TOF mass spectrometry. Mycalosides B (2) and C (3) were shown to be 27- and 28-nor derivatives of the previously known tetraoside mycaloside A (1). Mycaloside D (4) differs from 1 only in the presence of an additional acetyl group in the carbohydrate moiety. Mycaloside E (5) was structurally identified as a 28-nor-4-dehydroxy derivative of 1. Mycalosides F-H (6-8), differing from each other by the structures of their side chains and nonacetylated (7, 8) or acetylated (6) tetrasaccharide carbohydrate moieties, have new 5(6)-unsaturated 3 beta,4 beta,21-trihydroxy-15-keto-steroidal aglycons. Mycaloside I (9) is a tetraoside of a new 7,24(28)-diunsaturated 3beta,15 beta,29-trihydroxystigmastane aglycon. It was established that the total fraction of the mycalosides as well as mycalosides A (1) and G (7) inhibit the fertilization of eggs by sperm of the sea urchin Strongylocentrotus nudus preincubated with these compounds.     

Effect of elevated K(+), hypotonic stress, and cortical spreading depression on astrocyte swelling in GFAP-deficient mice

Glial fibrillary acidic protein (GFAP) is the main component of intermediate filaments in astrocytes. To assess its function in astrocyte swelling, we compared astrocyte membrane properties and swelling in spinal cord slices of 8- to 10-day-old wild-type control (GFAP(+/+)) and GFAP-knockout (GFAP(-/-)) mice. Membrane currents and K(+) accumulation around astrocytes after a depolarizing pulse were studied using the whole-cell patch-clamp technique. In vivo cell swelling was studied in the cortex during spreading depression (SD) in 3 to 6-month-old animals. Swelling-induced changes of the extracellular space (ECS) diffusion parameters, i.e., volume fraction alpha and tortuosity lambda, were studied by the real-time iontophoretic tetramethylammonium (TMA(+)) method using TMA(+)-selective microelectrodes. Morphological analysis using confocal microscopy and quantification of xy intensity profiles in a confocal plane revealed a lower density of processes in GFAP(-/-) astrocytes than in GFAP(+/+) astrocytes. K(+) accumulation evoked by membrane depolarization was lower in the vicinity of GFAP(-/-) astrocytes than GFAP(+/+) astrocytes, suggesting the presence of a larger ECS around GFAP(-/-) astrocytes. Astrocyte swelling evoked by application of 50 mM K(+) or by hypotonic solution (HS) produced a larger increase in [K(+)](e) around GFAP(+/+) astrocytes than around GFAP(-/-) astrocytes. No differences in alpha and lambda in the spinal cord or cortex of GFAP(+/+) and GFAP(-/-) mice were found; however, the application of either 50 mM K(+) or HS in spinal cord, or SD in cortex, evoked a large decrease in alpha and an increase in lambda in GFAP(+/+) mice only. Slower swelling in GFAP(-/-) astrocytes indicates that GFAP and intermediate filaments play an important role in cell swelling during pathological states.     

TREC/KREC Levels and T and B Lymphocyte Subpopulations in COVID-19 Patients at Different Stages of the Disease

Background: T and B cell-mediated immunity can be assessed using T cell receptor excision circle (TREC) and Kappa-deleting recombination excision circle (KREC) analysis, respectively, and successful implementation of this method requires evaluation of the correlation between the TREC frequencies and T cell subsets as well as KREC levels and B lymphocyte subsets. The aim of the present study was to evaluate the correlation between the TREC/KREC concentrations and T/B lymphocyte subsets at different stages of COVID-19. Methods: We examined 33 patients in the acute stage of COVID-19 (including 8 patients with poor outcomes) and 33 COVID-19 survivors. TREC/KREC concentrations were measured using quantitative real-time PCR. T/B lymphocyte subsets were determined using flow cytometry. Results: Blood TREC and KREC levels were found to be significantly lower in the acute stage of COVID-19 compared to control values. Moreover, a zero blood TREC level was a predictor of a poor disease outcome. Reductions in CD3⁺CD4⁺CD45RO⁻CD62L⁻ and CD3⁺CD8⁺CD45RO⁻CD62L⁻ T cell counts (as well as in the main fractions of B1 and B2 B cells) indicated a favorable outcome in COVID-19 patients in the acute stage of the disease. Decreased CD3⁺CD4⁺CD45RO⁻CD62L⁺ and CD3⁺CD8⁺CD45RO⁻CD62L⁺ T cell frequencies and increased CD3⁺CD8⁺CD45RO⁻CD62L⁻ cell counts were found to indicate a poor outcome in patients with acute COVID-19. These patients were also found to have increased B1 cell counts while demonstrating no changes in B2 cell counts. The levels of effector T cell subsets an naïve B cells were normal in COVID-19 survivors. The most pronounced correlations between TREC/KREC levels and T/B cell subsets counts were observed in COVID-19 survivors: there were positive correlations with naïve T and B lymphocytes and negative correlations with central and effector memory T cell subsets. Conclusions: The assessment of correlations between TREC and T cell subsets as well as KREC levels and B cell subset counts in patients with acute COVID-19 and COVID-19 survivors has shown that blood concentrations of TREC and KREC are sensitive indicators of the stage of antigen-independent differentiation of adaptive immunity cells. The results of the TREC and KREC analysis correlated with the stages of COVID-19 and differed depending on the outcome of COVID-19.