A retrospective review of an epidural blood patch database: the incidence of epidural blood patch associated with obstetric neuraxial anesthetic techniques and the effect of blood volume on efficacy.

BACKGROUND: The optimal volume of blood required to treat post-dural puncture headache remains in question. In our institution a target volume of 30mL is used for an epidural blood patch unless the patient experiences pain during injection. METHODS: The institutional database was retrospectively reviewed for epidural blood patch and delivery statistics over a 15-year period to determine if the volume of blood administered during the procedure directly correlated with the number of epidural blood patches administered. The primary endpoint was defined as the need for a repeat epidural blood patch. RESULTS: There were 466 epidural blood patches performed on 394 patients, associated with 84 804 obstetric neuraxial procedures. Thirty-two percent (95% CI 28.3 to 34.9%) of patients who had an inadvertent dural puncture with an epidural needle received an epidural blood patch versus 0.19% (0.16% to 0.22%) of patients who received neuraxial anesthesia with no documented dural puncture with an epidural needle. All patients experienced relief of post-dural puncture headache, although 17% required two and 1.5% required three epidural blood patches. The mean±SD volume of blood administered was 20.5±5.4mL and only 35 patients (8.9%) received 30mL. CONCLUSION: Increasing blood volumes up to 30mL did not reduce the need for repeat epidural blood patch. Although the optimal volume of blood to administer during epidural blood patch placement remains unknown, our institution will continue to administer up to 30mL or until the patient experiences pain during epidural injection.

Adenovirus type 7 induces interleukin-8 in a lung slice model and requires activation of Erk.

Adenovirus (Ad), particularly Ad type 7 (Ad7), causes severe lung infection and pneumonia. Initially, Ad causes neutrophilic inflammation of the distal airways and alveoli. Interleukin-8 (IL-8) is the major lung neutrophil chemotaxin, and we have shown that Ad7 induces IL-8 release from the A549 alveolar epithelial cell line. We sought to determine whether ex vivo human and bovine lung tissue containing primary pneumocytes could be used as a more accurate and relevant model to study Ad acute inflammation. We found that cultured lung tissue preserved normal lung architecture for more than 10 days. IL-8 was generated upon exposure of the lung organ culture to Ad7. IL-8 production required activation of the Ras/Erk pathway, since a pharmacological inhibitor blocked the appearance of IL-8 in the medium. Both human and bovine lung explants supported replication of Ad7, and immunohistochemistry experiments demonstrated the presence of the Ad hexon antigen within alveolar epithelial cells. These findings show that our novel human lung organ culture accurately reproduces the in vivo infectious disease process. Thus, this organ culture model represents a valuable tool for studying the acute innate immune response to respiratory infections.

Adenovirus type 7 induces interleukin-8 production via activation of extracellular regulated kinase 1/2.

Infection with adenovirus serotype 7 (Ad7) frequently causes lower respiratory pneumonia and is associated with severe lung inflammation and neutrophil infiltration. Earlier studies indicated release of proinflammatory cytokines, specifically interleukin-8 (IL-8), by pulmonary epithelial cells following infection by Ad7. However, the mechanism of IL-8 induction by Ad7 is unclear. We have explored the role of the Ras/Raf/MEK/Erk pathway in the Ad7-associated induction of IL-8 using a model system of A549 epithelial cells. We found that Ad7 infection induced a rapid activation of epithelial cell-derived Erk. The MEK-specific inhibitors PD98059 and U0126 blocked Erk activation and release of IL-8 following infection with Ad7. Treatment with PD98059 is cytostatic and not cytotoxic, as treated cells regain the ability to phosphorylate Erk and secrete IL-8 after removal of the drug. The expression of a mutated form of Ras in A549 epithelial cells blocked the induction of IL-8 promoter activity, and MEK inhibitor blocked induction of IL-8 mRNA. These results suggest that the Ras/Raf/MEK/Erk pathway is necessary for the Ad7 induction of IL-8 and that induction occurs at the level of transcription. Further, the kinetics of Erk activation and IL-8 induction suggest that an early viral event, such as receptor binding, may be responsible for the observed inflammatory response.

A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein.

As the use of adenoviral vectors in gene therapy protocols increases, there is a corresponding need for rapid, accurate, and reproducible titer methods. Multiple methods currently exist for determining titers of recombinant adenoviral vector, including optical absorbance, electron microscopy, fluorescent focus assay, and the "gold standard" plaque assay. This paper introduces a novel flow cytometric method for direct titer determination that relies on the expression of the green fluorescent protein (GFP), a tracking marker incorporated into several adenoviral vectors. This approach was compared to the plaque assay using 10(-4)- to 10(-6)-fold dilutions of a cesium-chloride-purified, GFP expressing adenovirus (AdEasy + GFP + GAL). The two approaches yielded similar titers: 3.25 +/- 1.85 x 10(9) PFU/mL versus 3.46 +/- 0.76 x 10(9) green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7 x 10 days required by the plaque assay. These results indicate that the GFU/mL is an alternative functional titer method for fluorescent-tagged adenoviral vectors.

A national clinical indicator database: issues of reliability and validity.

The introduction of performance (clinical) indicators into the accreditation process by the Australian Council on Healthcare Standards is in keeping with global trends and has enabled the establishment of a National Aggregate Database reflecting standards of care in acute health care organisations. The database contains both quantitative and qualitative information on the processes and outcomes of patient care and changes in practice induced through indicator monitoring. Of fundamental importance to the integrity of the database are the issues of indicator validity, responsiveness and reliability. This paper considers these issues, drawing parallels, as appropriate, to other performance indicator programs and studies.

Bovine UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase. I. Purification and subunit structure.

UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) catalyzes the initial step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme was partially purified approximately 30,000-fold by chromatography of solubilized membrane proteins from lactating bovine mammary glands on DEAE-Sepharose, reactive green 19-agarose, and Superose 6. The partially purified enzyme was used to generate a panel of murine monoclonal antibodies. The anti-GlcNAc-phosphotransferase monoclonal antibody PT18 was coupled to a solid support and used to immunopurify the enzyme approximately 480,000-fold to apparent homogeneity with an overall yield of 29%. The purified enzyme has a specific activity of 10-12 micromol of GlcNAc phosphate transferred per h/mg using 100 mM alpha-methylmannoside as acceptor. The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine GlcNAc-phosphotransferase is a 540,000-Da complex composed of disulfide-linked homodimers of 166,000- and 51,000-Da subunits and two identical, noncovalently associated 56,000-Da subunits.

Bovine UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase. II. Enzymatic characterization and identification of the catalytic subunit.

The kinetic properties of UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) purified to homogeneity from lactating bovine mammary gland have been investigated. GlcNAc-phosphotransferase transferred GlcNAc 1-phosphate from UDP-GlcNAc to the synthetic acceptor alpha-methylmannoside, generating GlcNAc-1-phospho-6-mannose alpha-methyl, the structure of which was confirmed by mass spectroscopy. GlcNAc-phosphotransferase was active between pH 5.7 and 9.3, with optimal activity between pH 6.6 and 7.5. Activity was strictly dependent on Mg2+ or Mn2+. The Km for Mn2+ was 185 microM. The Km for UDP-GlcNAc was 30 microM, and that for alpha-methylmannoside was 63 mM. The enzyme was competitively inhibited by UDP-Glc, with a Ki of 733 microM. The 166-kDa subunit was identified as the catalytic subunit by photoaffinity labeling with azido-[beta-32P]UDP-Glc. Purified GlcNAc-phosphotransferase utilizes the lysosomal enzyme uteroferrin approximately 163-fold more effectively than the non-lysosomal glycoprotein ribonuclease B. Antibodies to GlcNAc-phosphotransferase blocked the transfer to cathepsin D, but not to alpha-methylmannoside, suggesting that protein-protein interactions are required for the efficient utilization of glycoprotein acceptors. These results indicate that the purified bovine GlcNAc-phosphotransferase retains the specificity for lysosomal enzymes as acceptors previously observed with crude preparations.

The Australian Council on Healthcare Standards care Evaluation Program.

With the assistance of the medical colleges, the Australian Council on Healthcare Standards (ACHS), through its Care Evaluation Program, has established clinical performance measures which will assist both internal and external review of care and enable hospitals to compare their quality of patient care with that of other hospitals.

Constrained and unconstrained postures of the mandible--a break with tradition?

Evaluation of detailed electrognathographic recordings of various movements of the mandible (and points on it) suggested interpretations at variance with some traditional concepts. Data previously accumulated from several sources were subjected to critical analysis. An objective was to assess the manner in which two rotations of the mandible influenced its postures during trials of mastication and deliberate lateral excursions. Rotations about a vertical and an antero-posterior axis appear to facilitate a greater functional posturing potential of the mandible than has previously been reported. In most instances the influence of the rotations upon mandibular posture is considerably greater than the size of the recorded angles would suggest. Some of the resulting postures seem to challenge concepts derived strictly from cadaverine anatomy. For example, during mastication the condyle does not simply glide forward and backward relative to the eminence but may be distracted downward away from it for a part of the time. Furthermore, the ability of a subject to effect a considerable variety of mandibular postures challenges traditional reliance upon constrained mandibular movements as a means of evaluating function.

Hospital characteristics associated with unplanned readmissions.

The rate of unplanned readmission of patients to hospitals has been introduced into the Australian Council on Healthcare Standards accreditation program as an internal flag of problems in patient management and outcome. An emphasis, in the indicator definition, is placed on the unexpected nature of the admissions to exclude those which are unplanned but simply due to progression of a disease, and are therefore not 'unexpected'. The association of hospital characteristics with unplanned readmissions was examined using logistic regression on the data collected from hospitals surveyed in 1993. The risk of unplanned readmission was significantly higher in public hospitals than in private hospitals. Hospital bed-size also reflected differences in the risk of unplanned readmission, being significantly higher for hospitals with over 200 beds than for those with 1-100 beds. In rural areas, the risk of unplanned readmission was significantly lower in hospitals with 101-200 beds and over 200 beds compared to hospitals with 1-100 beds (p for trend = .004). However, in metropolitan areas, the risk of unplanned readmission increased with the size of the hospitals (p for trend < .0001). Monitoring of unplanned readmissions prompted internal clinical review and action in 31 per cent of hospitals, demonstrating the indicator's usefulness as an internal quality tool. However, the use of unplanned readmissions as an external performance measure must take into account a hospital's characteristics and will remain of limited value in the absence of clinical information about the expected or unexpected nature of the readmissions.

Bioluminescent determination of 0.1 picomole amounts of guanine nucleotides.

A bioluminescence procedure for the determination of the guanylates has been optimized to allow measurement of 0.1 pmol amounts. Modifications of the Karl procedure include the use of purified firefly luciferase and nucleoside diphosphate kinase instead of a crude extract of firefly tails, the use of Tricine buffer instead of the inhibitory arsenate buffer, and optimization of the amounts of reagents and incubation times for each of the partial reactions. In the determination of GMP, background values varied widely with different lots of bovine guanylate kinase. Careful selection of a suitable lot of bovine brain guanylate kinase was essential for determination of lower amounts of guanylates. This establishes that selection of guanylate kinase must be based on experimental determination and not reported adenylate kinase activity. The wide variation in background was not eliminated by the inclusion of adenylate kinase inhibitors.

Reliability of histologic scoring for lupus nephritis: a community-based evaluation.

OBJECTIVE: To determine the reliability of the National Institutes of Health (NIH)-modified semiquantitative histologic scoring system for lupus nephritis. DESIGN: Cross-sectional study, repeated after 8 to 9 months. SETTING: Four community hospitals and one university medical center. PARTICIPANTS: Five pathologists, all experienced in reading renal biopsy specimens, assessed 25 specimens that had been obtained from patients with a clinical diagnosis of systemic lupus erythematosus and showed diffuse proliferative glomerulonephritis. MEASUREMENTS: Biopsy specimens were scored independently and blindly by pathologists for components of nephritis chronicity and activity. Reliability was measured by percentage agreement, intraclass correlation coefficient or kappa statistic, and individual reader effect on the group arithmetic mean. RESULTS: As scored by the readers, the mean chronicity index score varied from 2.3 to 4.8 on a 12-point scale (P = 0.001) and the mean activity index score varied from 5.8 to 11.4 on a 24-point scale (P = 0.0001). Pairs of readers gave scores within 1 point for the chronicity index and within 2 points for the activity index in 50% of cases, and risk group assignments based on chronicity index (three strata) and activity index (two strata) were concordant in 59% and 76% of cases, respectively. Intraclass correlation coefficients for inter-reader agreement were 0.58 for the chronicity index (P < 0.01) and 0.52 for the activity index (P < 0.01). Intrareader agreement was uniformly higher than inter-reader agreement, but mean intraclass correlation coefficients exceeded 0.70 for only 1 of the 10 index components. Repeated readings yielded chronicity index scores that were more than 1 point discordant in 45% of cases and activity index scores that were more than 2 points discordant in 43% of cases. Risk group assignment changed on the basis of chronicity index and activity index in 36% and 21% of cases, respectively. CONCLUSIONS: In a nonreferral setting, the NIH-modified scoring system for lupus nephritis is only moderately reproducible and, if used to prognosticate renal outcome, may result in erroneous predictions of risk for renal failure and response to therapy.

Transfer of pheromone-inducible plasmids between Enterococcus faecalis in the Syrian hamster gastrointestinal tract.

Pheromone-responsive plasmids are common to Enterococcus faecalis, transfer at high frequency in vitro, and carry cytolysin and other gene products implicated in the pathogenesis of enterococcal infection. A Syrian hamster model of enterococcal intestinal overgrowth was used to test for transfer of three isogenic plasmids differing in conjugative and cytolytic phenotypes. Transconjugants were found in 8 (44%) of 18 and 6 (35%) of 17 hamsters given donor strains containing cytolytic (pAM714) and noncytolytic (pAM771) pheromone-responsive plasmids. Of the 14 hamsters from which transconjugants were isolated from stool, 9 (64%) had transconjugants 1 day after donor strain inoculation. The frequency of transfer (mean +/- SD) for pAM714 and pAM771 was 1.4 +/- 2.2 x 10(-1) and 2.9 +/- 4.2 x 10(-2) transconjugants/donor, respectively (P > .20). Transconjugants were not recovered from hamsters receiving a cytolytic, nonconjugative plasmid (pAM930; transfer frequency < 2 x 10(-5) transconjugants/donor). Pheromone-responsive plasmid transfer between E. faecalis strains occurs at high frequency in the gastrointestinal tract of hamsters and may be one means by which enterococcal resistance and virulence factors disseminate.

Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans.

Cryptococcus neoformans var. neoformans (74 isolates) and C. neoformans var. gattii (44 isolates) were used to test urease activity after growth on both yeast extract-glucose-peptone agar (YEPG) and on YEPG supplemented with 100 microM EDTA. Every isolate grown on YEPG agar for 48 h at 30 degrees C produced a positive reaction within 1 h in a modified rapid urease assay at 37 degrees C. However, isolates grown on YEPG with 100 microM EDTA showed a distinct pattern which corresponded to their varietal status. All but 1 of 74 C. neoformans var. neoformans isolates (98.7%) produced a positive reaction within 1 to 4 h, while none of 44 C. neoformans var. gattii isolates produced a positive reaction within the same period. The urease inhibition results and the canavanine-glycine-bromthymol blue agar test results showed 100% correlation among isolates of C. neoformans var. gattii and 98.7% correlation among isolates of C. neoformans var. neoformans. Two representative isolates of C. neoformans var. gattii (serotypes B and C) were further tested for urease during a prolonged incubation period in urea broth. These isolates failed to show a positive reaction even after 11 h of incubation. The uptake of EDTA was negligible in the two varieties. Extracts of cells grown on YEPA agar showed a high level of urease activity in both varieties. Extracts of cells grown on the agar with 100 microM EDTA showed a marked reduction (86%) of urease activity in one isolate of C. neoformans var. gattii but showed only a 30% reduction in one isolate of C. neoformans var. neoformans. Based on these results, the differential effect of EDTA on the two varieties of C. neoformans appeared to be due to greater inhibition of urease synthesis in C. neoformans var. gattii.

Urease testing and yeast taxonomy.

When urease production was assayed by the hydrolysis of [14C]urea, all basidiomycetous yeasts tested, including the Cryptococcus vishniacii complex (previously reported urease negative), produced significant amounts of 14CO2. The Schizosaccharomycetaceae were the only urease-positive ascomycetous yeasts tested. Yarrowia lipolytica was urease negative. The stoichiometry of [14C]urea hydrolysis paralleled by Roberts' rapid urea hydrolysis (RUH) test indicated that causes of anomalous results in conventional urease testing include acidification and alkalinization of the test medium by products of endogenous metabolism and autolysis rather than urease activity. Anomalous results also occurred when cells were grown on media containing the chelating agent ethylenediaminetetraacetic acid (EDTA) prior to RUH. The addition of EDTA to a complex natural medium inhibited urease production in all yeasts reportedly growing at 35 degrees C (and all other yeasts tested), except Filobasidiella (Cr.) neoformans var. neoformans (NIH 12). The RUH test could differentiate at the varietal level: Fil. (Cr.) neoformans var. neoformans was about 10 times more resistant to EDTA in media used for the growth of cells prior to RUH testing than was Fil. neoformans var. bacillispora (Cr. neoformans var. gattii) (NIH 191). Urease production by Fil. neoformans var. bacillispora was specifically restored to half maximal activity by the addition of 22 microM Ni+2 (as NiCl2) to a growth medium containing 0.100 mM EDTA.

Noninvasive diagnosis of deep venous thrombosis.

Eighty-five patients suspected of having lower-extremity deep venous thrombosis (DVT) participated in a prospective study to test the diagnostic accuracy of four noninvasive techniques: Doppler ultrasonic flow study, electrical impedance plethysmography, the serial dilution protamine sulfate test, and an extensive physical examination. Ascending radiocontrast phlebography was the diagnostic standard of reference. We found that (1) when both Doppler and impedance examinations were positive, the diagnosis of DVT could be considered virtually certain; (2) impedance and Doppler examinations, when used in combination, were reliable screening tests capable of establishing or excluding the presence of thigh DVT; (3) physical examination and the serial dilution protamine sulfate test were unreliable screening techniques for DVT; (4) techniques other than the noninvasive methods investigated were needed to reliably detect or to exclude popliteal and call DVT.