Bb2Bb3 regulation of murine Lyme arthritis is distinct from Ncf1 and independent of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase

Several quantitative trait loci regulating murine Lyme arthritis severity have been mapped, including a highly significant linkage found on chromosome 5, termed Bb2Bb3. Within this region, the Ncf1 gene of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been identified as a major regulator of arthritis severity in rodent models of rheumatoid arthritis, an effect attributed to protective properties of reactive oxygen species. To assess the role of Ncf1 in Lyme arthritis, we introgressed Bb2Bb3 from severely arthritic C3H/He mice onto mildly arthritic C57BL/6 mice. This increased Lyme arthritis severity, whereas the reciprocal transfer conferred protection from disease. A single nucleotide polymorphism was identified in the Ncf1 gene that did not influence the protein sequence or expression of Ncf1. Although polymorphonuclear leukocytes from C57BL/6 mice generated a greater oxidative burst than polymorphonuclear leukocytes from C3H/He mice, studies with the Bb2Bb3 congenic mice demonstrated this difference was not linked to Ncf1 alleles. Furthermore, Lyme arthritis severity was not altered in mice lacking either the Ncf1 or Gp91phox subunits of the NADPH oxidase complex. Together, these results argue that Ncf1 is not a candidate gene for regulation of Lyme arthritis and reveal Lyme arthritis to be independent of NADPH oxidase activity, distinguishing it from other models of rheumatoid arthritis.     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Activation of lymphokine genes during stimulation of cloned T cells

To study the regulation of lymphokine production by T lymphocytes, we have characterized the activation of lymphokine genes in T cells by measuring the levels of lymphokine mRNA in cloned murine T lymphocytes after stimulation. Lymphokine mRNA was not detected in cells taken after seven days of maintenance culture. Following stimulation of T helper lymphocytes L2 and AD9.1 with concanavalin A, lymphokine mRNA appeared, reached peak levels and disappeared over a 43-h time period. A single stimulation event resulted in the induction of mRNA for interleukin 2 (IL 2), IL 3 and interferon gamma. Maximal mRNA levels were generally found at 6 h in the T helper lymphocytes, but could occur as late as 18 h. The lymphokine genes were expressed coordinately; however, in these cloned cells, IL 2 mRNA levels appeared to be lower than the other two mRNAs. Lymphokine titers in the supernatant fluids paralleled the appearance of mRNA but IL 2 titers began to fall after 12 h probably because of utilization of this lymphokine by the activated cells. In the cytolytic T lymphocyte, L3, qualitatively similar kinetics were found after stimulation by lectin or a clonotypic antibody with peak mRNA levels occurring later (18 h) with the antibody. These studies indicate a single stimulating event activates the lymphokine genes of T cells in a coordinate manner; the appearance of the lymphokines in supernatant fluids represents de novo synthesis of these proteins but the levels of lymphokines measured in supernatant fluids reflects both production and utilization rates, and exposure to IL 2 at the time of stimulation is not essential for the production of other lymphokines.     

Sensitivity of respiratory virus culture when screening with R-mix fresh cells

Use of R-Mix Fresh Cells has been shown to be a rapid and sensitive method for the detection and identification of respiratory viruses. We prospectively evaluated the impact of incorporation of R-Mix shell vials on the sensitivity and time to detection of seven respiratory viruses recovered in a comprehensive culture during the course of an entire respiratory season in a high-volume clinical laboratory. In this study, R-Mix shell vials were used as part of the culture of 3803 respiratory specimens. A total of 428 respiratory viruses were recovered. Staining of R-Mix vials after overnight incubation allowed initial detection of 274 of 279 influenza viruses, 33 of 38 parainfluenza viruses, 35 of 51 adenoviruses, and 52 of 60 respiratory syncytial viruses (RSVs). The time to reporting of all positive cultures after in-lab specimen receipt was 2.9 days on average and those initially detected in R-Mix cells were reported in 2.3 days on average. A combination of direct fluorescent-antibody (DFA) staining and virus culture was performed on a subset of 711 respiratory specimens. Of 152 viruses identified, 57 were observed only with DFA testing (55 RSV and 2 influenza A viruses) and 31 were recovered only in cell culture. After overnight incubation, R-Mix cells detected 87.1% of respiratory viruses not observed by DFA testing and 96.9% of viruses positive by both methods. The sensitivities of DFA testing and R-Mix cells for identification of influenza viruses were 70.5% and 96.7%, respectively. The R-Mix method detected influenza virus in 18 samples that were negative by DFA testing.     

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Ventral medulla pHi measured in vivo by 31P NMR is not regulated during hypercapnia in anesthetized rat

Chemoreceptors in the ventral medulla contribute to the respiratory response to hypercapnia. Do they ‘sense’ intracellular pH (pH_i)? We measured pH_i in the ventral medulla or cortex (control) using ³¹P-NMR obtained via a novel 3×5 mm² surface coil in anesthetized rats breathing air or 7% CO₂. During air breathing over 240 min, pH_i decreased slightly from 7.13±0.02 to 7.05±0.02 (SEM; n=5; 2 cortex, 3 ventral medulla). During 180 min of hypercapnia, cortical pH_i (n=4) decreased from 7.17±0.02 to 6.87±0.01 by 90 min and recovered by 150 min. Ventral medulla pH_i showed no such regulation. It decreased from 7.11±0.02 to 6.88±0.02 at 90 min and recovered only after cessation of hypercapnia (n=5), results consistent with pH_i being the chemoreceptor stimulus. However, non-chemoreceptor neurons that contribute to our medullary NMR signal also do not appear to regulate pH_i in vitro. Regional differences in pH_i regulation between cortex and ventral medulla may be due to both chemosensitive and non-chemosensitive neurons.     

Nucleus basalis lesions in neonate rats induce a selective cortical cholinergic hypofunction and cognitive deficits during adulthood

Summary Ibotenic acid was infused into the nucleus basalis magnocellularis (nBM) of 2-day old rats to eliminate immature cholinergic neurons before they develop functional synaptic connections in the neocortex. For bilaterally lesioned neonates, cognitive testing was initiated 2 months after lesioning and animals were sacrificed at 8 or 12 months of age. Lesioned animals exhibited a marked deficit in the retention of passive avoidance behavior, as well as in the acquisition of 2-way active avoidance behavior. Lesioned animals also made significantly more alternation errors than control animals in the Lashley III spatial maze and showed severe impairments in general learning, reference memory and working memory during 17-arm radial maze testing. For all 4 tasks, neonatally lesioned animals did not show any recovery to the performance level of control animals. Histological analysis of the subcortex from lesioned animals during adulthood revealed: (1) a substantial reduction in acetylcholinesterase-positive cells (presumably cholinergic) within the nucleus basalis, (2) decreased acetylcholinesterase staining in neocortex and (3) a gliosis essentially restricted to the globus pallidus. Surrounding brain regions were apparently not damaged as a direct result of excitotoxin infusion. Neurochemically, neonate nBM lesioning produced a long term cholinergic hypofunction as evidenced by significant reductions of 25% and 18% in frontal cortex chorine acetyltransferase (CAT) activity at 12 and 8 months of age, respectively. By contrast, prefrontal cortical concentrations of biogenic amines and their metabolites were unaffected, thus indicating a degree of neurochemical specificity for these neonatal nBM lesions. The persistant cortical cholinergic hypofunction in lesioned animals may be related to the long term deficits in learning/memory abilities that were also observed. It is suggested that neonatal nBM lesioning could provide a useful animal model for elucidating the plasticity of the developing brain after cortical anervation.