Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon

Animal studies with Streptococcus pneumoniae have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive lux transposon cassette, Tn4001 luxABCDE Km(r), that allows random integration of lux genes onto the bacterial chromosome was constructed. The cassette was designed so that the luxABCDE and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome. S. pneumoniae D39 was transformed with plasmid pAUL-A Tn4001 luxABCDE Km(r), and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical S. pneumoniae isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the lux construct was highly stable even after several days in vivo. We believe that this lux transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence.     

Comparison of the fertilizing capability of spermatozoa from ejaculates, epididymal aspirates and testicular biopsies using intracytoplasmic sperm injection

A prospective study was carried out to compare the fertilizing capability and pregnancy outcome following intracytoplasmic sperm injection (ICSI) using spermatozoa obtained from ejaculates, or surgically from epididymis or seminiferous tubules. A total of 77 ICSI cycles (one per patient) was included. In all, 28 patients had severe oligoasthenoteratozoospermia, 19 patients had obstructive azoospermia and 30 patients had non-obstructive azoospermia. The main outcome measures were fertilization rate per injected metaphase II oocyte and the clinical pregnancy rate per embryo transferred back to the female recipients. In patients with severe oligoasthenoteratozoospermia, the fertilization and pregnancy rates were 79 and 25 %. In patients with obstructive azoospermia, for whom epididymal spermatozoa were used, these were 75 and 28%, and in the non-obstructive group for which testicular spermatozoa were used for injection, they were 69 and 21% respectively. These rates were not significantly different in the three groups (P = 0.85 and P = 0.14 respectively), suggesting that spermatozoa from the ejaculates and epididymal or testicular biopsies are able to fertilize equally by using ICSI. Live birth per embryo transfer was significantly reduced in patients with non-obstructive azoospermia compared to the other two groups. The high abortion rate (50%) in the group in which testicular spermatozoa were used raises doubts about the developmental competence of such embryos.      

Characterization of the human jumonji gene

While constructing a cDNA library of human embryos, we have isolated a clone homologous to jumonji, a mouse gene required for neural tube formation. We have determined the complete coding sequence of the human homologue (JMJ) and deduced the amino acid sequence of the putative protein. We show here that human and mouse jumonji putative proteins are homologous and present 90% identity. During human embryogenesis, JMJ mRNAs are predominantly expressed in neurons and particularly in dorsal root ganglion cells. They are also expressed in neurons of human adult cerebral cortex. In view of these observations, we propose JMJ as a candidate gene for developmental defects of the central nervous system in the human. The human JMJ gene maps at position 6p24-6p23.      

Is the outcome of in-vitro fertilization and embryo transfer treatment improved by spontaneous or surgical drainage of a hydrosalpinx?

A pilot study was designed to examine whether the outcome of embryo transfer in women with a hydrosalpinx might be improved by surgical drainage of the hydrosalpinx at the time of oocyte collection for in- vitro fertilization treatment. A comparative, controlled but retrospective analysis of the results was performed of all women with infective tubal damage aged <40 years old, who had ovulatory cycles, a normal uterus and a partner with normal spermatozoa. A standardized treatment regimen was used. A maximum of three embryos were transferred. Hydrosalpinx was defined by prior hysterosalpingography and/or laparoscopy with transcervical dye injection. A total of 237 embryo transfer cycles in women with hydrosalpinges (tubal distension not visible in 151, visible but not drained in 30 and drained in 56) were compared with 705 embryo transfer cycles in women with tubal disease but no hydrosalpinx. Results were analysed in the first three cycles but also separately in the first cycle to check for bias. Success rates were higher in the first cycle, but did not significantly influence overall differences. Implantation rates were significantly reduced overall in the hydrosalpinx group (8.0 versus 13.2% for controls; P < 0.001), being 8.3% (P < 0.01) in the subgroup without evident tubal distension and 7.5% (not significant) in the drained hydrosalpinx group. This study shows that tubal damage with distal occlusion is associated with a marked reduction in embryo implantation, even in the absence of obvious fluid distension. Surgical drainage of distended hydrosalpinges appears to offer no benefit.      

Screening for complement deficiency in bacterial meningitis

Seventy-seven children with bacterial meningitis were screened for complement deficiency. Both the classical and the alternate pathways were normal in 75 patients. Transiently reduced total haemolytic activity of the classical pathway was documented in a boy with meningococcal meningitis. Total haemolytic activity of both the classical and the alternate pathways were reduced in another patient with pneumococcal meningitis: individual complement components determination indicated predominant activation of the alternate pathway.     

Identification of differentially expressed genes in aflatoxin B1- treated cultured primary rat hepatocytes and Fischer 344 rats

Aflatoxin B1 (AFB1), a mutagen and hepatocarcinogen in rats and humans, is a contaminant of the human food supply, particularly in parts of Africa and Asia. AFB1-induced changes in gene expression may play a part in the development of the toxic, immunosuppressive and carcinogenic properties of this fungal metabolite. An understanding of the-role of AFB1 in modulating gene regulation should provide insight regarding mechanisms of AFB1-induced carcinogenesis. We used three PCR- based subtractive techniques to identify AFB1-responsive genes in cultured primary rat hepatocyte RNA: differential display PCR (DD-PCR), representational difference analysis (RDA) and suppression subtractive hybridization (SSH). Each of the three techniques identified AFB1- responsive genes, although no individual cDNA was isolated by more than one technique. Nine cDNAs isolated using DD-PCR, RDA or SSH were found to represent eight genes that are differentially expressed as a result of AFB1 exposure. Genes whose mRNA levels were increased in cultured primary rat hepatocytes after AFB1 treatment were corticosteroid binding globulin (CBG), cytochrome P450 4F1 (CYP4F1), alpha-2 microglobulin, C4b-binding protein (C4BP), serum amyloid A-2 and glutathione S-transferase Yb2 (GST). Transferrin and a small CYP3A-like cDNA had reduced mRNA levels after AFB1 exposure. Full-length CYP3A mRNA levels were increased. When liver RNA from AFB1-treated male F344 rats was evaluated for transferrin, CBG, GST, CYP3A and CYP4F1 expression, a decrease in transferrin mRNA and an increase in CBG, GST, CYP3A and CYP4F1 mRNA levels was also seen. Analysis of the potential function of these genes in maintaining cellular homeostasis suggests that their differential expression could contribute to the toxicity associated with AFB1 exposure.