Microbiological examinations and in-vitro testing of different antibiotics in therapeutic endoscopy of the biliary system

BACKGROUND AND STUDY AIMS: Prior to endoscopic therapeutic procedures, no antibiotic prophylaxis is administered routinely. Because of the reported incidence of infectious complications, which may reach up to 10%, a prospective study was undertaken to investigate the effects of a prophylactic dose of cefuroxime on the incidence of bacteremia and clinical signs of infection, but no significant effects could be demonstrated. In addition to this published work, blood and bile cultures obtained in this trial were also investigated, and the in-vitro susceptibility to several antibiotics was tested in order to recommend the appropriate substances. PATIENTS AND METHODS: Ninety-nine consecutive patients (51 men, 48 women; mean age 61.4 +/- 17 years) with biliary obstruction who underwent an endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous transhepatic cholangiography with drainage (PTCD) were included. Sequential blood cultures were taken before and up to 60 minutes after the endoscopic intervention. Bile cultures were obtained in 56 patients with biliary drainage. Aerobic and anaerobic cultures were prepared from all obtained specimens and the isolated organisms were identified. In the case of positive cultures, an in-vitro resistance test for 15 different antibiotics was performed. RESULTS: The incidence of bacteremia was 11.1% (n = 11), and 16 bacteria were isolated. Twelve different microorganisms were detected, with Escherichia coli found in four cases. From 41 positive out of 56 prepared bile cultures (73.2%), 91 isolates were found with 25 different species. A single agent was detected in eight cases (19.5%), while a mixed growth, with pathogens ranging from two to six species, was found in 33 cases (80.5%). The seven most frequently isolated germs were E. coli and Enterococcus (each n = 19), Klebsiella (n = 10), Streptococcus viridans (n = 9), Staphylococcus epidermidis (n = 5), Morganella morganii (n = 4), and Bacteroides fragilis (n = 3), representing 76% of all agents. Examination for fungal infection revealed positive cultures of Candida albicans in 16.1% of bile cultures (nine of 56). Interestingly, the use of proton-pump inhibitors (PPIs), with a consequent rise in the gastric pH value, led to an increase in the rate of bacteremia to 26.2% (five of 19) compared to the other patients not on PPIs (n = 80), who developed bacteremia in only six cases (7.5%; p = 0.02). In-vitro testing of different antibiotics was carried out in 73 isolates. Imipenem showed the best antimicrobial activity (98.4%), followed by trimethoprim and sulfamethoxazole (90%), amoxicillin plus clavulanic acid (87.3%), vancomycin (82.4%), and ofloxacin (76.9%). CONCLUSIONS: Escherichia coli was found to be the pathogen most frequently detected in blood and bile following endoscopic interventions in the biliary tract. Enterococci, Klebsiella and Streptococcus viridans were found in bile cultures with an incidence exceeding 10%. In view of the in-vitro test results, possible side effects, and contraindications, amoxicillin plus beta-lactamase inhibitors or quinolones are considered to be suitable antibiotics for the prophylaxis of biliary infections.     

Interleukin (IL)-10, IL-12, and interferon-gamma production in primary and memory immune responses to varicella-zoster virus

Varicella immunization provided the opportunity to examine the kinetics of interleukin (IL)-10, IL-12 and interferon (IFN)-gamma production elicited during primary in vivo sensitization with proteins of varicella-zoster virus (VZV), a common human herpesvirus. VZV-specific IFN-gamma release and T cell proliferation were elicited by immunization and persisted through 15 months of follow-up. The induction of VZV-specific T cells and IgG antibodies was accompanied by transient increases in IL-10 and IL-12 production. T cell proliferation to VZV was significantly lower in adults at 15 months than in vaccinated children or naturally immune subjects and correlated with lower IFN-gamma responses in individual vaccinees. After primary immunity was induced, continued IL-12 production was not necessary to maintain the predominant Th1-type response elicited by VZV. Cytokine profiles observed during primary in vivo sensitization to VZV suggest that parallel increases in IFN-gamma and IL-10 may be important in the induction of immunity to some viral pathogens.     

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

Experience in the study of the effects of lead on the health status of children in Belovo]

Lead releases in Belovo town containing metallurgy enterprise had reached 120 tons/year earlier, but in recent years have decreased to 9 tons/year. Reduction of the production induced decrease of lead levels in the ambient air from 0.7-2.3 mg/m3 in 1994 to 0.001-0.24 mg/m3. Lead concentration in the soil ranges from 30 to 3000 mg/kg. Lead levels were measured in serum of 91 children, in hair of 67 ones and in teeth of 15 children. Serum lead levels in children aged 7-8 years varied from 0.5 to 39 mg/dl, with an average of 9.9 mg/dl (SD is 5.2 mg/dl), geometric mean is 8.5 mg/dl and error of geometric mean is 3.3. 46% of the children had serum lead levels exceeding the normal one (10 mg/dl). Average lead level in the hair equaled 4.5 mg/g (SD is 4.9 mg/g). The children living in towns with higher environmental lead levels demonstrated more frequent anxiety and changes in higher psychic functions. The major points influencing the serum lead level are proximity to highway, dietary load of goods grown near the residence, mother's smoking. Biokinetic model describing lead transfer into the blood helped to evaluate various modes of the enterprise functioning and efficiency of some environmental protection measures. The most efficient are measures aimed to lower dietary intake of lead, less efficiency is associated with measures reducing lead levels in air, dust and soil.     

Preservation with 8-bromo-cyclic GMP improves pulmonary function after prolonged ischemia

BACKGROUND: Cyclic guanosine monophosphate (cGMP) is a potent second messenger for the nitric oxide pathway in the pulmonary vasculature. Increased cytosolic cGMP levels elicit pulmonary vasodilatation resulting in decreased pulmonary vascular resistance and maximized pulmonary function after ischemia-reperfusion injury. We hypothesized that the addition of a membrane-permeable cGMP analogue (8-bromo-cGMP) to a Euro-Collins (EC) preservation solution would ameliorate pulmonary reperfusion injury better than prostaglandin E1 injection alone after prolonged hypothermic ischemia. METHODS: All lungs from New Zealand White rabbits (weight, 3 to 3.5 kg) were harvested en bloc, flushed with EC solution, and reperfused with whole blood for 30 minutes. Group 1 lungs (immediate control) were immediately reperfused. Group 2 lungs (control) were stored inflated at 4 degrees C for 18 hours before reperfusion. Groups 3 and 4 lungs were flushed with EC solution containing 200 micromol/L 8-bromo-cGMP and stored at 4 degrees C for 18 and 30 hours, respectively. Fresh, nonrecirculated venous blood was used to determine single-pass pulmonary venous-arterial oxygen gradients at 10-minute intervals. Assays for cGMP, cyclic adenosine monophosphate, nitric oxide synthase activity, and myeloperoxidase were performed on all lung tissue samples. Wet to dry weight ratios were determined after 2 weeks of passive desiccation. RESULTS: Oxygenation (venous-arterial oxygen gradient), pulmonary artery pressure, pulmonary vascular resistance, and edema formation were significantly improved in groups 3 and 4 (addition of 8-bromo-cGMP to EC plus 18 or 30 hours of hypothermic ischemia). Hypothermic storage (groups 2, 3, and 4) decreased both nitric oxide synthase activity and myeloperoxidase levels compared with immediate reperfusion (group 1). CONCLUSIONS: These results suggest that the addition of a membrane-permeable cGMP analogue to an EC pulmonary flush solution improves pulmonary function after prolonged storage compared with EC and prostaglandin (E1) preservation alone. The finding of myeloperoxidase reduced levels after hypothermic storage and subsequent reperfusion may suggest a more important role for pulmonary hemodynamic control in mitigating pulmonary reperfusion injury.     

Radionuclides in the Kara sea bottom sediments

The contents and distribution of natural (214Pb, 214Bi, 228Ac, and 208Tl) and technogenic (90Sr, 137Cs, and 60Co) radionuclides in the Kara Sea bottom sediments was analyzed by using the materials collected by R/V "Pomor" (Murmansk Marine Biological Institute). In 1994, no high (critical) concentrations of technogenic radioisotopes were found in the sea sediments, while the spots (regions) of elevated 137Cs content found in 1984 were not confirmed in 1994. It was proposed that the main sources of entry of technogenic radionuclides in the sea sediments in the last years are the flow of the Ob'-Yenisei waters and container burials of radioactive waste in the sea, which appeared to have been markedly corroded. The latter is confirmed by detection in some places of 60Co, which was not previously found in the sediments.     

CD34+, kit+, rhodamine123(low) phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells

We hypothesized that human hematopoietic cells displaying a CD34+, kit-, rhodamine123(low) phenotype would be highly enriched for cells with stem-like properties. To test this hypothesis, we employed fluorescence activated cell sorting (FACS) to isolate cells with this phenotype from normal light density marrow mononuclear cells (MNC). CD34+, kit+, rhodamine123(low) cells comprised from 0.05-0.01% of the total MNC population. They were small, had scant cytoplasm, and contained nuclei with dense, hyperchromatic chromatin and inconspicuous nucleoli. Additional immunophenotyping revealed that these cells were CD33-, CD38-, CD20-, and glycophorin A-. When plated in semisolid cultures containing optimal concentrations of IL-3, GM-CSF, KL, EPO, IL-6, and IL-1 these cells did not form colonies. However, when cultured over irradiated stromal cells, cobblestone areas were observed to form after 3 weeks, and harvested cells were able to initiate long-term cultures. To further demonstrate that these cells were indeed stem like, we also tested their ability to engraft and mature in immunocompromised (SCID) mice. Irradiated (400 cGy) SCID mice were transplanted with 2 x 10(3) candidate stem cells which were then injected with recombinant human growth factors every other day. Two months post-transplant the animals were sacrificed. PCR and FACS analysis of marrow and spleen cell samples revealed the presence of cells expressing human CD45 consistent with engraftment of human stem cells and the establishment of murine-human chimerism. Moreover, MNC isolated from transplanted mice formed unambiguously human BFU-E, CFU-GM and B cell colonies when stimulated with the appropriate growth factors. Accordingly, we have identified a relatively rapid and simple mechanism for isolating primitive human hematopoietic cells with stem cell-like properties. We anticipate that this strategy will be useful for experimental and therapeutic applications that require human stem cells in quantity.     

The anatomy of a hypoxic operator in Saccharomyces cerevisiae

Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.     

Rapid inhalation induction in children: 8% sevoflurane compared with 5% halothane

BACKGROUND: Ultrasonography (US) by acknowledged experts enhances the diagnostic performance and reduces the rate of negative laparotomies in patients with suspected acute appendicitis (AA). METHODS: The diagnostic accuracy and clinical impact of routine US performed by surgical residents was prospectively studied in 504 unselected patients admitted for AA. Clinical and US findings were correlated with laparotomy findings and pathological outcome in 135 patients (113 cases with proven AA, prevalence 22.4%) and clinical as well as follow-up data were compared in the remainder. RESULTS: The overall accuracy, sensitivity, and specificity of the clinical diagnosis of AA were 84.9%, 51.3%, and 94.6% and those of US were 93. 6%, 83.1%, and 96.6%. Joint evaluation of the results from clinical evaluation and US further improved diagnostic performance (accuracy 93.4%, sensitivity 84.1%, specificity 96.2) and significantly reduced the rate of diagnostic errors to 3.4% (p < 0.001) and unnecessary laparotomies to 9.6% (p < 0.01) in patients with suspected AA. CONCLUSIONS: Ultrasonographic evaluation of the patient with suspected AA is considered to be of value in surgical practice.     

Transgenic A1 adenosine receptor overexpression increases myocardial resistance to ischemia

Activation of myocardial A1 adenosine receptors (A1AR) protects the heart from ischemic injury. In this study transgenic mice were created using the cardiac-specific alpha-myosin heavy chain promoter and rat A1AR cDNA. Heart membranes from two transgene positive lines displayed approximately 1,000-fold overexpression of A1AR (6,574 +/- 965 and 10,691 +/- 1,002 fmol per mg of protein vs. 8 +/- 5 fmol per mg of protein in control hearts). Compared with control hearts, transgenic Langendorff-perfused hearts had a significantly lower intrinsic heart rate (248 beats per min vs. 318 beats per min, P < 0. 05), lower developed tension (1.2 g vs. 1.6 g, P < 0.05), and similar coronary resistance. The difference in developed tension was eliminated by pacing. Injury of control hearts during global ischemia, indexed by time-to-ischemic contracture, was accelerated by blocking adenosine receptors with 50 microM 8-(p-sulfophenyl) theophylline but was unaffected by addition of 20 nM N6-cyclopentyladenosine, an A1AR agonist. Thus A1ARs in ischemic myocardium are presumably saturated by endogenous adenosine. Overexpressing myocardial A1ARs increased time-to-ischemic contracture and improved functional recovery during reperfusion. The data indicate that A1AR activation by endogenous adenosine affords protection during ischemia, but that the response is limited by A1AR number in murine myocardium. Overexpression of A1AR affords additional protection. These data support the concept that genetic manipulation of A1AR expression may improve myocardial tolerance to ischemia.