Maternal death due to extended spectrum beta-lactamase-producing E. coli: a warning for the future?

We describe a maternal death due to necrotising fasciitis caused by an extended spectrum beta-lactamase-producing Escherichia coli resistant to routinely used antimicrobial agents. Necrotising fasciitis is a rare complication of septicaemia with a high mortality. Signs of infection were insidious and masked by the use of routine analgesic agents and concurrent preeclampsia. The incidence of infection with extended spectrum beta-lactamase-producing organisms is increasing both in the United Kingdom and globally and will need to be considered in the obstetric setting. The use of the current Modified Early Warning Scores was of limited help in this case. Where there is no response to routine antibiotics within 12h, microbiological review is indicated.

Gene expression analysis of calcineurin isoforms in T-lymphocytes--a method applied on kidney-transplant recipients.

BACKGROUND: Tacrolimus exerts its immunosuppressive effect through inhibition of the intracellular enzyme calcineurin phosphatase (CaN). In this study, we set-up a validated real-time PCR method to measure the gene expression of the two major isoforms of the catalytic subunit of CaN in T-lymphocytes. METHODS: 20 stable kidney-transplant recipients, 10 early kidney-transplant recipients and 10 healthy non-medicated subjects had blood drawn and T-lymphocytes were isolated using E-rosette gradient centrifugation method. The cell counts were analyzed by DNA quantification using Hoeschst 33285. Gene expressions were analyzed using real-time PCR for CaN Aalpha, CaN Abeta and the reference genes CD3E and PPIB. RESULTS: The real-time PCR method was found to be with high efficiencies and low intra- and inter-assay variabilities. No statistically significant differences were found in the gene expression levels of the two reference genes among the three groups. The two major isoforms of CaN A were expressed in equal amounts in the T-lymphocytes. CONCLUSION: We found no significant difference in the reference genes between the three groups, but looking at the data there was a trend towards an up-regulation of CD3E. PPIB appears to be the more stable of the two reference genes tested in our study.

Temporal profile of calcineurin phosphatase activity during acute allograft rejection in the heterotopic rat heart transplantation model.

BACKGROUND: Regardless of the extensive worldwide use of calcineurin inhibitors, little is known about the behavior of calcineurin phosphatase (CaN) during acute allograft rejection. The aim of this study was to investigate the temporal profile of CaN during acute allograft rejection and reveal if it can be utilized as a pharmacodynamic marker to identify and monitor the rejection process. METHODS: The heterotopic cervical rat heart transplantation model was used (dark Agouti to Lewis). We performed 25 control isogeneic and 46 allogeneic transplantations. Rats were sacrificed at various postoperative time points. CaN activity was measured in isolated peripheral blood and spleen mononuclear cells and in graft heart homogenates. CaN activity was measured as the release of radiolabeled phosphate from a previously phosphorylated 19 amino acid peptide. RESULTS: We have shown that CaN's activity levels are not significantly altered during acute allograft rejection in peripheral blood mononuclear cells and in spleen-isolated mononuclear cells. CaN's intragraft activity decreased with time in both rejectors and controls, and was significantly lower in the allogeneic group. CONCLUSIONS: CaN failed as a pharmacodynamic biomarker of acute allograft rejection in the heterotopic rat heart transplantation model. Further research is required in order to reveal the precise role of CaN during acute allograft rejection.

Calcineurin activity in tacrolimus-treated renal transplant patients early after and 5 years after transplantation.

The pharmacodynamic (PD) action of tacrolimus (FK) within the T-cell is inhibition of calcineurin phosphatase (CaN). Determination of CaN activity provides us with an important PD marker. Eleven renal transplant patients treated with FK were investigated on day 14 following transplantation and 5 years later. Blood samples drawn before as well as 1, 2, 3, and 4 hours after oral intake of FK were analyzed for CaN activity and blood FK concentrations. Twenty healthy subjects had one blood sample drawn for CaN activity, which was measured as the release of (32)P from a phosphorylated peptide. Radioactivity of (32)P was quantitated by liquid scintillation counting with the results converted to units of CaN utilizing a calibration curve. On day 14, we observed significant inhibition of CaN activity at T:1, 2, and 3 compared with the predose level (P = .002; P = .015; P = .015). Furthermore, all measured CaN activities were significantly different from those observed in healthy nonmedicated subjects. In contrast, at 5 years posttransplant only the CaN activity at T:2 was significantly inhibited compared with the predose level (P = .02). Additionally, all CaN activities at this time were not significantly different from CaN activities in the healthy subjects. We were not able to demonstrate individual CaN activity profiles in the patients. The lack of CaN inhibition at 5 years after transplantation despite relevant drug concentrations, probably reflected the lower drug dose used long after transplantation. This result raises the question of whether CaN inhibition is necessary to hold graft function and whether FK possess CaN-independent mechanisms of action.

24-h monitoring of calcineurin phosphatase activity in healthy subjects.

The calcineurin inhibitors cyclosporine and tacrolimus are the cornerstone immunosuppressants used in solid organ transplantation. Studies investigating calcineurin (CaN) activity in renal transplanted patients have been published, but basic properties of the enzyme activity in healthy subjects remain to be described. The aim of this study was to investigate whether CaN displays circadian variation or sex difference is present in healthy subjects. Twenty subjects had blood samples drawn every 4 h for a 24-h period. CaN activity was determined in whole blood as the release of 32P from a phosphorylated peptide. Activity of the 32P was quantitated by liquid scintillation and results converted to units CaN utilizing a calibration curve. We found no circadian variation in CaN activity and no difference between the two sexes. The clinical importance of these findings is that blood samples for calcineurin activity can be drawn without taking the exact time of day into consideration, but only considering the time of drug intake.

Comparison of the temporal profile of calcineurin inhibition by cyclosporine and tacrolimus in renal transplant patients.

Calcineurin phosphatase (CaN) activity has been the focus of several recent studies on renal transplant patients as the calcineurin inhibitors tacrolimus (FK) and cyclosporine (CsA) are still the cornerstone in the immunosuppressive treatment. The aim of this study was to compare the temporal inhibition profiles of CaN using CsA or FK in two groups of renal transplant patients. Nineteen tacrolimus-treated and 10 cyclosporine-treated renal transplant patients had blood samples drawn before and at 1, 2, 3, 4, and 6 hours after ingestion of drug. CaN activity was measured as the release of 32P from a previously phosphorylated peptide and radioactivity quantitated by liquid scintillation counting. Results were expressed as units CaN. Blood concentrations of tacrolimus were determined with an IMx method and of CsA with an EMIT assay. FK-treated patients showed maximal inhibition of CaN activity at 1 to 3 hours, returning to the predose level 4 hours after drug intake. CsA-treated patients showed a gradual decrease in CaN activity with a nadir after 3 hours, failing to return to predose levels during the observation period. Both groups showed a significant rise in drug blood concentrations. To conclude, we have demonstrated that two widely used immunosuppressants, CsA and FK, show different CaN inhibitory profiles in renal transplant patients.

Calcineurin phosphatase activity and immunosuppression. A review on the role of calcineurin phosphatase activity and the immunosuppressive effect of cyclosporin A and tacrolimus.

The mode of immunosuppressive action of tacrolimus (FK506) and cyclosporin A has been elucidated. Both drugs bind to proteins in the cytoplasm to form complexes, which in turn inhibit the phosphatase activity of calcineurin, an important limiting step in the activation of T cells. The association between drug uptake (pharmacokinetics) and enzyme inhibition (pharmacodynamics) is under current investigation. Great variations in the correlation between blood drug levels and enzyme inhibition could indicate that monitoring calcineurin phosphatase activity for treatment might be superior to monitoring blood drug levels.

Congenital syphilis following negative antenatal screening.

We report a case of severe early congenital syphilis in the infant of a mother who acquired syphilis in mid pregnancy. The mother had received full antenatal care including serological screening for syphilis. Congenital syphilis is re-emerging in a number of industrialised countries and this report demonstrates that some of the most serious cases cannot be prevented by routine antenatal screening.

Comparison of the lymphatic transport of a lipophilic drug from vehicles containing alpha-tocopherol and/or triglycerides in rats.

The applicability of alpha-tocopherol as a lymphotropic carrier for a highly lipophilic drug has been evaluated. Transport to the intestinal lymph of the highly lipophilic model drug, Lu28-179, in rats after administration to the stomach in an alpha-tocopherol emulsion was compared with lymphatic transport after administration of a sesame oil emulsion and an alpha-tocopherol/sesame oil emulsion. Lymphatic transport of the triglycerides and of alpha-tocopherol was determined. A conscious rat model was used, and the mesenteric lymph was collected. There was no significant difference between the cumulative masses of triglyceride from the two emulsions containing triglyceride 24 h after administration. Administration of an alpha-tocopherol emulsion seemed to induce mobilization of endogenous triglyceride. The lymphatic transport of alpha-tocopherol was less than 1 mg 24 h after administration of both emulsions containing alpha-tocopherol. The absorption of Lu28-179 from the alpha-tocopherol emulsion was very low, with a lymphatic recovery of 0.05%. When administered in an alpha-tocopherol/sesame oil emulsion, the recovery of Lu28-179 increased sevenfold to 0.35%. However, after administration of Lu28-179 in a sesame oil emulsion, the lymphatic recovery increased a further 13-fold to 4.5%. In conclusion, the study showed that alpha-tocopherol did not promote lymphatic absorption of Lu28-179 and thus was not a good lymphotropic carrier, as compared with sesame oil. Alpha-tocopherol in combination with sesame oil was not a good lymphotropic carrier either. The non-absorbed alpha-tocopherol fraction in the intestine might be able to prevent the absorption of Lu28-179.

The effect of alpha-tocopherol on the in vitro solubilisation of lipophilic drugs.

alpha-Tocopherol is an excellent solvent for many poorly soluble drugs. The aim of this work was to study whether or not the presence of alpha-tocopherol has an influence on the solubilisation of poorly soluble drugs in simulated intestinal fluids (SIF). The solubilizing capacity of mixed micelles containing alpha-tocopherol towards three lipophilic drugs was investigated. The solubilisation of alpha-tocopherol in an aqueous micellar phase was increased by the addition of monoglycerides (MG) and free fatty acids (FFA), preferably of medium chain length, as compared to a simple bile salt solution. The addition of alpha-tocopherol to mixed micellar solutions seems to have an effect on the solubilizing capacity, which can be correlated to the partition coefficient of the drug to be solubilised. A positive effect on the solubilisation of griseofulvin and felodipine was found. For a highly lipophilic drug (Lu28-179), a positive effect on solubilisation was observed only in media containing MG and FFA of medium chain length. Generally, alpha-tocopherol cannot be considered an important factor for the solubilisation of highly lipophilic drugs in SIF. The presence of lipolytic digestion products (LDP) of the proper chain length in relation to the drug to be solubilised is much more important.

Genetic variation at the growth hormone locus in a wild pig intercross; test of association to phenotypic traits and linkage to the blood group D locus.

A polymorphism in the TATA-box of the porcine growth hormone (GH) gene was analysed in a wild pig/Large White intercross, in which 129 markers had been scored previously. Linkage analyses demonstrated that the GH locus belonged to a linkage group on chromosome 12 together with a previously unassigned marker, the erythrocyte antigen D (EAD) locus. The linear order of this linkage group is EAD-GH-S0096-S0090-S0106-arachidonate 12-lipoxygenase (ALOX12)-inhibin beta A (INHBA). The length of the linkage group was estimated at 93 cM (sex average). The effects of the GH genotype on growth and fat deposition traits were investigated using phenotypic data from the 191 F2 animals. No significant effect of GH was detected, and we therefore conclude that this locus does not play a major role in defining the genetic differences between the wild and Large White pigs for these traits.

A physically anchored linkage map of pig chromosome 1 uncovers sex- and position-specific recombination rates.

A linkage map of pig chromosome 1 (SSC1) has been constructed using 15 polymorphic markers. Eleven markers constitute PCR-based microsatellites (three associated with coding sequences), six markers have also been mapped by in situ hybridization, and homologues to four of the markers have been mapped in human. The physical assignments show that almost the entire SSC1 is covered by the linkage map, which measures 164 cM (sex-averaged). In a large region on the q arm, representing about 40% of the chromosome, there is a significant excess of male recombination. In contrast, there is a significantly higher recombination rate in females in both terminal regions. In the penultimate marker intervals on the q arm as well as the p arm, females show a fivefold excess of recombination compared to males. The rate of genetic recombination in relation to the physical DNA content is 1 cM per 2-4 Mb over at least 80% of the chromosome. In the terminal part of the q arm, however, there is a tremendous increase in the recombination rate, with 1 cM equaling about 0.4 Mb. Two homologous chromosome segments in human could be detected, ESR-CGA on human chromosome 6 (HSA6) and IFNA-RLN-GRP78 on human chromosome 9 (HSA9). Since the porcine blood group locus EAA is located close to (or possibly within) the latter conserved segment and the suggested human counterpart, the ABO system, is similarly close to the segment on HSA9, our data provide indirect evidence that these blood group systems are homologous.

A survey of birds in Denmark for the presence of Pneumocystis carinii.

One hundred eighty-three toluidine blue O-stained necropsy lung imprint smears from different avian species were examined microscopically for Pneumocystis carinii. No cyst forms of the organism could be identified. Seventy-eight serum samples from a total of 155 chickens were examined by a competition enzyme-linked immunosorbent assay (ELISA) for antibodies to P. carinii; 53 serum samples were from individual chickens, and 25 samples were pools of sera from two to five chickens. Diluted 1:50, the 78 serum samples showed a specific ELISA-inhibition of 4% to 56% (the 95% confidence limit being 25% to 30% inhibition). Diluted 1:50, nine serum pools representing 34 chickens and 17 of the 53 individual serum samples (32.1%) showed an inhibition greater than 30%. No specific pneumocyst DNA could be detected in serum from 13 of the 53 chickens using polymerase chain reaction and dihydrofolate reductase gene as a specific probe. Specific antibodies to a 116,000-molecular-weight antigen of rat pneumocysts were shown in two (13.3%) of 15 individual chicken serum samples. The results indicate that P. carinii organisms do not commonly reside in the lungs of birds, although some birds may be exposed to external sources of organisms.

Characterization of 24 porcine (dA-dC)n-(dT-dG)n microsatellites: genotyping of unrelated animals from four breeds and linkage studies.

Twenty-four PCR primer pairs were designed for the detection of porcine microsatellites. Polymorphism was investigated in 76 unrelated animals from four different breeds: Duroc, Landrace, Hampshire, and Yorkshire. Compared with human microsatellites, a general lower heterozygosity was detected; however, for each microsatellite a significant variation between breeds in number of alleles and heterozygosity was seen. Mean heterozygosity was found to be significantly higher (P < 0.01%) in the Yorkshire breed than in the other three breeds. Linkage analyses with the CEPH linkage packet were performed in a backcross family comprising 45 animals, of which 43 had informative meioses. Ten of the microsatellites could be assigned to six different linkage groups, demonstrating that linkage mapping with microsatellites can be carried out with great efficiency in a relatively small number of animals. Four of the linkage groups represent Chromosomes (Chrs) 4, 6, 7, and 8 respectively, while two linkage groups are unassigned.

Antibiotic treatment of sinusitis in general practice. A double-blind study comparing ofloxacin and erythromycin.

The present study compared ofloxacin and erythromycin in a double-blind study with parallel groups for clinical efficacy and the number and severity of adverse reactions in patients treated in general practice for acute or chronic sinusitis. All patients lived in medium-sized to large towns and rural districts in the northern and western parts of Sealand, Funen and eastern Jutland, Denmark. Three hundred and nineteen patients were enrolled in the study, of whom 280 were clinically evaluable. From this total number, 136 patients were treated with ofloxacin (31 males and 105 females) and 144 patients were treated with erythromycin (40 males and 104 females). Following administration of either ofloxacin 400 mg once daily or erythromycin 500 mg twice daily for 7-14 days, 94.9% of the ofloxacin-treated group and 94.4% of the erythromycin-treated group were cured of their infections. There was no difference in clinical efficacy. Complications occurred in 18 of 155 patients in the ofloxacin-treated group and 32 of 164 in the erythromycin-treated group (P < 0.05), corresponding to 27 and 45 symptoms respectively (P < 0.01). This difference in adverse reactions principally involved gastrointestinal symptoms, i.e. 19 in the ofloxacin-treated group and 41 in the erythromycin-treated group (P < 0.01). Present findings show that the two antibiotics are comparable as to clinical efficacy, but the frequency of adverse reactions is significantly higher in the erythromycin-treated group due to the greater incidence of gastrointestinal adverse reactions.

On-the-job training.

The quality of training given by the security department has a direct impact on the job performances of each security officer. The author discusses how on-the-job training can be a vital part of the training effort when done properly and effectively.

Incidence of spontaneous bacterial peritonitis in patients with ascites. Diagnostic value of white blood cell count and pH measurement in ascitic fluid.

During a 21-month period, 65 consecutive patients admitted with ascites were included in a prospective study of the incidence of spontaneous bacterial peritonitis, and paracentesis was performed on admission. The ascitic fluid was cultured, ascitic leucocytes were counted and pH was measured. Bacterial growth was found in five patients with chronic liver disease, who were diagnosed as having spontaneous bacterial peritonitis (SBP), since no intra-abdominal focus could be demonstrated. Thus, the incidence of SBP in this material was 7.7% (95% confidence limits: 2.5-17%). SBP was caused by Escherichia coli (n = 3), coagulase negative staphylococcus (n = 1), and Bacteroides species (n = 1). Abdominal tenderness, abnormal intestinal sounds, fever and hepatic encephalopathy were equally frequent in the group with SBP and in patients with sterile ascites. Infection was not anticipated in any of the patients with SBP. In contrast to several previous studies, neither ascites pH nor ascites leucocyte counts were any help in obtaining a rapid diagnosis. Survival time of patients with SBP was significantly shorter than of patients without SBP.