Role of oxidative stress,angiogenesis and chemo-attractant cytokines in the pathogenesis of ischaemic protection induced by remote ischaemic conditioning: Study of a human model of ischaemia-reperfusion induced vascular injury

Aims

We explored the effect of remote ischaemic conditioning (RIC) on endothelial function and on circulating mediators.

Diagnosis of congenital toxoplasmosis by two-dimensional immunoblot differentiation of mother and child immunoglobulin g profiles

Differentiation between the specific immunoglobulin G (IgG) response to Toxoplasma gondii by a mother and her newborn child is helpful in the diagnosis of congenital infection with T. gondii in newborns without T. gondii-specific IgM and/or IgA antibodies at birth. Previous methods include immunoblotting and complexing T. gondii antigen with the sera from the mother and child and comparing the bands after electrophoresis. We developed a two-dimensional immunoblotting (2DIB) method with T. gondii RH strain tachyzoite antigen and validated the method with sera from 11 children identified through the neonatal screening program for congenital toxoplasmosis in Denmark. The children were identified by using Toxoplasma-specific IgM antibodies at the screening test, but the presence of T. gondii-specific IgM and/or IgA antibodies could not be confirmed at the subsequent serum sample tested. The children were monitored for at least 12 months, and in seven of eight patients monitored for 12 months the results of the 2DIB-predicted congenital infection were confirmed by the presence of persistent Toxoplasma-specific IgG antibodies. 2DIB is a sensitive technique that allows early differentiation between passively transferred maternal T. gondii-specific IgG antibodies and antibodies synthesized by the newborn child.     

Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.     

Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo

Cellular protein trafficking has been studied to date only in vitro or with techniques that are invasive and have a low time resolution. To establish a gentle method for analysis of glucose transporter-4 (GLUT4) trafficking in vivo in fully differentiated rat skeletal muscle fibres we combined the enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week-old rats and peaked around 1 week after transfection. The gene gun was used subsequently with a plasmid coding for EGFP linked to the C-terminus of GLUT4 (GLUT4-EGFP). Rats were anaesthetised 5 days after transfection and insulin given i.v. with or without accompanying electrical hindleg muscle stimulation. After stimulation, the hindlegs were fixed by perfusion. GLUT4-EGFP-positive FDB fibres were isolated and analysed by confocal microscopy. The intracellular distribution of GLUT4-EGFP under basal conditions as well as after translocation to the plasma membrane in response to insulin, contractions, or both, was in accordance with previous studies of endogenous GLUT4. Finally, GLUT4-EGFP trafficking in quadriceps muscle in vivo was studied using time-lapse microscopy analysis in anaesthetised mice and the first detailed time-lapse recordings of GLUT4-EGFP translocation in fully differentiated skeletal muscle in vivo were obtained.     

DNA microarray technique for detection and identification of seven flaviviruses pathogenic for man

A flavivirus microarray was developed for detection and identification of yellow fever (YF), West Nile, Japanese encephalitis (JE), and the dengue 1-4 viruses, which are causing severe human disease all over the world. The microarray was based on 500-nucleotide probe fragments from five different parts of the seven viral genomes. A low-stringent amplification method targeting the corresponding regions of the viral genomic RNA was developed and combined with hybridization to the microarray for detection and identification. For distinction of the generated virus-specific fluorescence-patterns a fitting analysis procedure was adapted. The method was verified as functional for all seven flaviviruses and the strategy for the amplification, combined with the long probes, provided a high tolerance for smaller genetic variability, most suitable for these rapidly changing RNA viruses. A potentially high detection and identification capacity was proven on diverged strains of West Nile and dengue viruses. The lower limit for detection was equivalent, or better, when compared to routinely used RT-PCR methods. The performance of the method was verified on human patient samples containing dengue viruses, or normal human serum spiked with YF or JE viruses. The results demonstrated the ability of the flavivirus microarray to screen simultaneously a sample for several viruses in parallel, in combination with a good lower limit of detection.     

Frequent polymorphism of the mitochondrial DNA polymerase gamma gene (POLG) in patients with normal spermiograms and unexplained subfertility

BACKGROUND: Male fertility largely depends on the quality of sperm production, which may be affected by environmental and genetic factors. In this study, we explored a possible role of the polymerase gamma (POLG) gene polymorphism, recently reported to be associated with male infertility in some populations. METHODS: The polymorphic CAG repeat (usually 10 codons long) in the POLG gene was studied in 1298 male subjects: 429 patients with infertility/subfertility, and 869 controls (495 men from the general population with unknown fertility and 374 recent fathers). In all subjects, the POLG polymorphism was assessed in relation to their semen quality, and--in the fertile controls--with biological fecundity measured as waiting time-to-pregnancy (TTP) for the couples. In the patients lacking the common POLG allele, the outcome of the assisted reproductive techniques (ART) for the couples was evaluated. RESULTS: The absence of one (10/ not equal to 10) or both common POLG alleles (not equal to 10/not equal to 10) was more frequent among the subfertile patients than among fertile controls (P=0.021 and P=0.04 respectively). The estimated predictive value for infertility in a man homozygous for the POLG polymorphism was 15.5% (95% CI: 4.8-51%). There was a positive association with sperm concentration: 14.3% of the normospermic subfertile patients were homozygous for the absence of the common POLG allele (not equal to 10/not equal to 10), in comparison with 2.3% of unselected controls (P=0.001) and 0.9% of the fertile men (P=0.0001). No association with sperm motility, morphology and TTP was found. Spermatozoa of the three not equal to 10/not equal to 10 patients treated with IVF retained the ability to penetrate the egg, but the fertilization rate was low. Nine homozygous not equal to 10/ not equal to 10 patients were treated with ICSI, resulting in pregnancy in seven couples. CONCLUSIONS: The POLG gene polymorphism should be considered as a possible contributing factor in patients with unexplained subfertility and normal spermiograms. The oocyte penetration ability of sperm may be partially impaired in the not equal to 10/not equal to 10 patients but most of them can be successfully treated with ICSI.     

p107 is a suppressor of retinoblastoma development in pRb-deficient mice

Hemizygosity for the retinoblastoma gene RB in man strongly predisposes to retinoblastoma. In the mouse, however, Rb hemizygosity leaves the retina normal, whereas in Rb^−/− chimeras pRb-deficient retinoblasts undergo apoptosis. To test whether concomitant inactivation of the Rb-related gene p107 is required to unleash the oncogenic potential of pRb deficiency in the mouse retina, we inactivated both Rb and p107 by homologous recombination in embryonic stem cells and generated chimeric mice. Retinoblastomas were found in five out of seven adult pRb/p107-deficient chimeras. The retinal tumors showed amacrine cell differentiation, and therefore originated from cells committed to the inner but not the outer nuclear layer. Retinal lesions were already observed at embryonic day 17.5. At this stage, the primitive nuclear layer exhibited severe dysplasia, including rosette-like arrangements, and apoptosis. These findings provide formal proof for the role of loss of Rb in retinoblastoma development in the mouse and the first in vivo evidence that p107 can exert a tumor suppressor function.