Pre- and postoperative evaluation of plasma creatine kinase in 142 children subjected to "uncomplicated" anesthesia in muscle biopsy

Pre- and postoperative serum CK activity is evaluated in 142 children submitted, "uneventfully", to diagnostic muscle biopsy under halothane (77 patients), ketamine (50 patients) or "local" (15 patients) anaesthesia. The purpose was to ascertain whether or not anaesthesia-induced-rhabdomyolysis (AR) was an asymptomatic (and unrecognized) complication of "uneventful" anaesthesia. The majority of patients with low preoperative CK values showed a slight increase of serum CK activity on the first postoperative day. On the contrary, a postoperative decrease was observed in the majority of patients with high preoperative values (namely in almost all ketamine patients and in 2/3 of halothane-patients). In no case postoperative increase reached a value suggesting the occurrence of AR even though a postoperative value of 16480 U/I was observed in a patient with Duchenne muscular dystrophy after halothane anaesthesia. Sudden interruption of motor activity induced by general anaesthesia seems to be the most important factor in reducing the release of CK from muscle. When preoperative release is low, any further postoperative reduction is not sufficient to balance the moderate increase of CK produced by the surgical procedure; the opposite should happen in patients presenting with high preoperative release. So far as anaesthetics are concerned, our data seem to suggest that ketamine has a higher "protective" role compared to halothane.     

Cystic fibrosis: prenatal diagnosis and carrier detection by DNA analysis

The analysis of restriction fragment length polymorphism (RFLP)s was used to detect 11 polymorphisms that are linked to cystic fibrosis in 42 Australian families with at least one child with cystic fibrosis. The data from all the families were fully informative in regard to the gene for cystic fibrosis (CF). Prenatal assessment was performed for 24 of these families: seven fetuses were assessed to be homozygous for cystic fibrosis, 13 fetuses were heterozygous and three fetuses were free of the CF gene. Of the seven pregnancies in which it was predicted that the infant would be affected by cystic fibrosis, two were continued electively; both have come to term and the infants each were shown to have cystic fibrosis at birth. Of the 17 pregnancies in which it was predicted that the infant would not be affected by cystic fibrosis, 13 have come to term and all the infants but one (who has not yet been followed-up) have been shown to be unaffected by cystic fibrosis at birth. The polymerase chain reaction has been used to amplify the CS.7 and KM.19 loci close to the CF gene. This procedure allows a polymorphic site in each locus to be analysed in a much shorter time (one or two days rather than 10 days) and allows the use of very small test-samples, such as dried blood on filter paper ("Guthrie blood spots"). Our observations confirm the results of overseas studies and indicate that these techniques are eminently useful for prenatal diagnosis and the detection of carriers in the vast majority of Australian families with cystic fibrosis.     

Development of a molecular-beacon assay to detect the G1896A precore mutation in hepatitis B virus-infected individuals

The 1896 precore (PC) mutation is the most frequent cause of hepatitis B virus e-antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection. Detection of the 1896 PC mutation has application in studies monitoring antiviral therapy and the natural history of the disease. Identification of this mutation is usually performed by direct sequencing, which is both costly and laborious. The aim of this study was to develop a rapid, high-throughput assay to detect the 1896 PC mutation using real-time PCR and molecular-beacon technology. The assay was initially standardized on oligonucleotide targets and plasmids containing the wild-type (WT) and PC mutation and then tested on plasma samples from children with HBV DNA of >10(6) copies/ml. Nine individuals were HBeAg negative and suspected to harbor HBeAg mutations, while 12 children were HBeAg positive and selected as controls. Ninety percent (19 of 21) of plasma samples tested with molecular beacons were in complete agreement with sequencing results. The remaining 10% (2 of 21) of samples were identified as heterogeneous mixtures of WT and mutant virus by molecular beacons, though sequencing found only a homogeneous mutant in both cases. Overall, the 1896 PC mutation was detected by this assay in 55.5% of the children with HBeAg-negative infection. In summary, this assay is a rapid, sensitive, and specific technique that effectively discriminates WT from 1896 PC mutant HBV and may be useful in clinical and epidemiological studies.     

Survival and Implantation of Escherichia coli in the Intestinal Tract

Preliminary experiments established that a 0.5-ml inoculum that is introduced directly into the stomach of mice was cleared rapidly into the small intestine. Bicarbonate buffer, but not skim milk, protected such an inoculum from stomach acid until at least 90% of it had entered the small intestine. Passage and survival of various Escherichia coli strains through the mouse gut were tested by introducing a buffered bacterial inoculum directly into the stomach, together with the following two intestinal tracers: Cr(51)Cl(3) and spores of a thermophilic Bacillus sp. Quantitative recovery of excreted bacteria was accomplished by collecting the feces overnight in a refrigerated cage pan. The data show that wild-type E. coli strains and E. coli K-12 are excreted rapidly (98 to 100% within 18 h) in the feces without overall multiplication or death. E. coli varkappa1776 and DP50supF, i.e., strains certified for recombinant DNA experiments underwent rapid death in vivo, such that their excretion in the feces was reduced to approximately 1.1 and 4.7% of the inoculum, respectively. The acidity of the stomach had little bactericidal effect on the E. coli K-12 strain tested, but significantly reduced the survival of more acidsensitive bacteria (Vibrio cholerae) under these conditions. Long-term implantation of E. coli strains into continuous-flow cultures of mouse cecal flora or into conventional mice was difficult to accomplish. In contrast, when the E. coli strain was first inoculated into sterile continuous-flow cultures or into germfree mice, which were subsequently associated with conventional mouse cecal flora, the E. coli strains persisted in a large proportion of the animals at levels resembling E. coli populations in conventional mice. Metabolic adaptation contributed only partially to the success of an E. coli inoculum that was introduced first. A mathematical model is described which explains this phenomenon on the basis of competition for adhesion sites in which an advantage accrues to the bacterium which occupies those sites first. The mathematical model predicts that two or more bacterial strains that compete in the gut for the same limiting nutrient can coexist, if the metabolically less efficient strains have specific adhesion sites available. The specific rate constant of E. coli growth in monoassociated gnotobiotic mice was 2.0 h(-1), whereas the excretion rate in conventional animals was -0.23 h(-1). Consequently, limitation of growth must be regarded as the primary mechanism controlling bacterial populations in the large intestine. The beginnings of a general hypothesis of the ecology of the large intestine are proposed, in which the effects of the competitive metabolic interactions described earlier are modified by the effects of bacterial association with the intestinal wall.     

Immunogenicity and protective efficacy of the alpha C protein of group B streptococci are inversely related to the number of repeats.

Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates. Alpha C protein is a protective cell surface-associated protein of GBS. This protein contains a repeat region flanked by N and C termini. Variable expression of tandem repeating units of alpha C proteins had been found among clinical isolates of GBS. We examined the effect of the number of repeats on the immunogenicity of the alpha C protein and its ability to elicit protection from GBS infection in a neonatal mouse model. Mice were immunized with purified alpha C proteins of constructs containing various numbers of repeats (n = 1, 2, 9, and 16) and the N- and C-terminal regions. Both the N-terminal and the repeat regions contain protective and opsonic epitopes. Antibody responses to the alpha C protein constructs with various numbers of repeats were tested with enzyme-linked immunosorbent assay plates coated with either native, nine-repeat alpha C protein or "repeatless" N-terminal antigen. An inverse relationship was found between the number of repeats and the immunogenicity of the alpha C protein; this effect was most pronounced on titers of antibody to the N-terminal region. An inverse relationship was also observed between the number of repeats and protective efficacy, i.e., mouse dams immunized with 5 microg of one- or nine-repeat alpha C protein transferred protective immunity to 65 or 11% of their pups, respectively (P < 0.0001). Thus, the presence of multiple repeats appears to lessen the antibody response to the complete alpha C protein, and especially the antibody response to its N-terminal region, and suggests a mechanism whereby repeat elements contribute to the evasion of host immunity.     

Effects of anonymity,gender, and erotophilia on the quality of data obtained from self-reports of socially sensitive behaviors

This study examined the effects of anonymity, gender, and erotophilia on the quality of self-reports of socially sensitive health-related behaviors. A sample of 155 male and 203 female undergraduate students was randomly assigned to an anonymous and a confidential (i.e., nonanonymous) assessment condition. Gender, erotophilia, self-reports (of substance use, sexual behaviors, illegal activity), and perceived item threat were assessed by questionnaire. Data quality was strongly affected by experimental condition and gender. Thus, terminations were more frequent in the confidential condition and among women. In the confidential condition, women were significantly more likely to prefer not to respond to sensitive items compared to men. Both female gender and confidential condition were associated with lower frequency reports of sensitive health behaviors, and greater perceived threat of the assessment questions. Self-reported engagement in sensitive behaviors was positively related to both perceived question threat and erotophilia. Path analyses suggest that question threat mediates the effects of anonymity manipulations and gender on data quality (item refusal, termination), and that erotophilia mediates the effects of gender on incidence and frequency self-reports. The results indicate that anonymous assessments as well as male gender are associated with better data quality.     

The haemotoxicity of mitomycin in a repeat dose study in the female CD-1 mouse

Mitomycin (MMC), like many antineoplastic drugs, induces a predictable, dose-related, bone marrow depression in man and laboratory animals; this change is generally reversible. However, there is evidence that MMC may also cause a late-stage or residual bone marrow injury. The present study in female CD-1 mice investigated the haematological and bone marrow changes induced by MMC in a repeat dose study lasting 50 days. Control and MMC-treated mice were dosed intraperitoneally on eight occasions over 18 days with vehicle, or MMC at 2.5 mg/kg, autopsied (n = 6-12) at 1, 7, 14, 28, 42 and 50 days after the final dose and haematological changes investigated. Femoral nucleated bone marrow cell counts and levels of apoptosis were also evaluated and clonogenic assays carried out; serum levels of FLT3 ligand (FL) were assessed. At day 1 post-dosing, MMC induced significant reductions in RBC, Hb and haematocrit (HCT) values, and there were decreases in reticulocyte, platelet, and femoral nucleated cell counts (FNCC); neutrophil, lymphocyte and monocyte values were also significantly reduced. On days 7 and 14 post-dosing, all haematological parameters showed evidence of a return towards normal values, but at these times, and at day 28, values for RBC and FNCC remained significantly reduced in comparison with controls. At days 42 and 50 post-dosing, many haematological parameters in MMC-treated mice had returned to control levels; however, there remained evidence of late-stage effects on RBC, Hb and HCT values, and FNCC also continued to be significantly decreased. Results for granulocyte-macrophage colony-forming units and erythroid colonies showed a profound decrease immediately post-dosing, but a return to normal values was evident at day 50. Serum FL concentrations demonstrated very significant increases in the immediate post-dosing period, but a return to normal was seen at day 50 post-dosing; a relatively similar pattern was seen in the number of apoptotic femoral marrow nucleated cells. The histopathological examination of kidney tissues from MMC animals at day 42 and 50 post-dosing showed evidence of hydronephrosis with cortical glomerular/tubular atrophy and degeneration. It is therefore concluded that MMC administered on eight occasions over 18 days to female CD-1 mice at 2.5 mg/kg induced profound changes in haematological and bone marrow parameters in the immediate post-dosing period with a return to normal levels at day 50 post-dosing; however, there was evidence of mild but significant late-stage/residual effects on RBC and FNCC, and on cells of the erythroid lineage in the bone marrow.