Increase of serum C-reactive protein is an early indicator of subsequent symptomatic anastomotic leakage after anterior resection.

OBJECTIVE: This prospective study investigated the factors which might indicate anastomotic leakage after low anterior resection. METHOD: Thirty-three patients who underwent anterior resection for rectal carcinoma (n = 32) and severe dysplasia (n = 1), were monitored daily by serum C-reactive protein (CRP) and white blood cell count (WBC) estimations until discharge from hospital. Computed tomography (CT) scans were performed on postoperative days 2 and 7 and the amount of presacral fluid collection was assessed. All patients had a pelvic drain and the volume of drainage was measured daily. RESULTS: The level of the anastomosis was at a median 5 cm (3-12 cm) above the anal verge. There was no 30-day mortality. Nine (27.2%) of the 33 patients developed a symptomatic anastomotic leakage which was diagnosed at a median of 8 days (range 4-14) postoperatively. The serum CRP was increased in patients who leaked from postoperative day 2 onwards (P = 0.004 on day 2; P < 0.001 on day 3-8). The WBC was decreased in preoperatively irradiated patients on days 1-5 (P

Computed tomographic colonography: comparison of two workstations.

PURPOSE: To compare two commercially available computed tomography (CT) colonography systems with respect to interobserver variability, the influence of level of expertise, and the gradual reduction of reviewing time for each system. MATERIAL AND METHODS: Two residents and two radiologists using Siemens CTAPP Colography software and Viatronix V3D-Colon software reviewed supine and prone CT acquisitions from 24 patients in a primary 3D endoluminal view. The observers graded each case with respect to technical quality and diagnostic value, assessed the presence of pathology, and indicated the time spent on the viewing. RESULTS: Significant differences were found in technical quality (P < 0.001) and diagnostic value (P<0.001) depending on which system was used, with higher scores for the Viatronix software. The agreement between specialists tended to be higher than that between residents (kappa=0.63 (0.30-0.95) vs. kappa=0.51 (0.21-0.81)), and the residents gave significantly (P < 0.001) higher scores of technical quality. However, the level of expertise had no significant impact on the assessments. We noted extensive variability in pathological lesions found by the different observers. The number of findings did not differ between workstations, but the viewers tended to report larger polyp sizes with the Viatronix software. The time needed for viewing decreased significantly from the first to the last examination viewed by each observer. CONCLUSION: Both the evaluated systems present trustworthy images of the human colon, but in a primary 3D setting the Viatronix software is favored owing to the user-friendly interface, higher experienced technical quality, and better diagnostic value.

Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34(+) hematopoietic cells.

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However, the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However, 5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood, as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted, mediated by CD8(+) lymphocytes, and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959)

Mercury induces regional and cell-specific stress protein expression in rat kidney.

Cells respond to physiologic stress by enhancing the expression of specific stress proteins. Heat-shock proteins (hsps) and glucose-regulated proteins (grps) are members of a large superfamily of proteins collectively referred to as stress proteins. This particular stress-protein response has evolved as a cellular strategy to protect, repair, and chaperone other essential cellular proteins. The objective of this study was to evaluate the differential expression of four hsps in the renal cortex and medulla during experimental nephrotoxic injury using HgCl2. Male Sprague-Dawley rats received single injections of HgCl2 (0.25, 0.5, or 1 mg Hg/kg, i.v.). At 4, 8, 16, or 24 h after exposure, kidneys were removed and processed for histopathologic, immunoblot, and immunohistochemical analyses. Nephrosis was characterized as minimal or mild (cytoplasmic condensation, tubular epithelial degeneration, single cell necrosis) at the lower exposures, and progressed to moderate or severe (nuclear pyknosis, necrotic foci, sloughing of the epithelial casts into tubular lumens) at the highest exposures. Western blots of renal proteins were probed with monoclonal antibodies specific for 4 hsps. In whole kidney, Hg(II) induced a time- and dose-related accumulation of hsp72 and grp94. Accumulation of hsp72 was predominantly localized in the cortex and not medulla, while grp94 accumulated primarily in the medulla but not cortex. The high, constitutive expression of hsp73 did not change as a result of Hg(II) exposure, and it was equally localized in cortex and medulla. Hsp90 was not detected in kidneys of control or Hg-treated rats. Since hsp72 has been shown involved in cellular repair and recovery, and since Hg(II) damage occurs primarily in cortex, we investigated the cell-specific expression of this hsp. Hsp72 accumulated primarily in undamaged distal convoluted tubule epithelia, with less accumulation in undamaged proximal convoluted-tubule epithelia. These results demonstrate that expression of specific stress proteins in rat kidney exhibits regional heterogeneity in response to Hg(II) exposure, and a positive correlation exists between accumulation of some stress proteins and acute renal cell injury. While the role of accumulation of hsps and other stress proteins in vivo prior to or concurrent with nephrotoxicity remains to be completely understood, these stress proteins may be part of a cellular defense response to nephrotoxicants. Conversely, renal tubular epithelial cells that do not or are unable to express stress proteins, such as hsp72, may be more susceptible to nephrotoxicity.

Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilized for the separation of recombinant human growth hormone (hGH) variants on a C18 silica column at 55 degrees C using an isocratic mobile phase which contained 27% 1-propanol in a 25 mM potassium phosphate buffer, pH 6.5. Three of the obtained peaks were characterized by tryptic mapping and mass spectrometry; two of the peaks were found to contain oxidized hGH (dioxy Met14/Met125 and Met125 sulfoxide) while the third contained a deamidated form (Asn149-->Asp149 or Asn152-->Asp152). Compared to the European Pharmacopoeia RP-HPLC method of hGH analysis, this new method gives two additional peaks and a 50% reduction in the analysis time.

Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry.

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated 14C-labelled eltanolone (5beta-Pregnan-3alpha-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of 14C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC-ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography-mass spectrometry with an electron impact ion source (GC-MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).

Absorbed dose and image quality in examinations of the colon with digital and analogue techniques.

PURPOSE: Image quality and the absorbed dose to the patient are issues of primary interest in the change-over from the conventional analogue technique to the digital technique in the examination of the colon by means of fluoroscopy. The aim of this study was to compare the incident radiation and to evaluate the image quality in two different X-ray equipment types, one digital and one analogue. MATERIAL AND METHODS: A kerma-area product meter was used to measure the incident radiation to the patient. Both fluoroscopy and total-examination times were measured as was the number of images. An evaluation of image quality was made and statistically analysed. RESULTS AND CONCLUSION: No significant difference in the irradiation dose was observed between the two techniques. The fluoroscopy time was significantly lower with the conventional technique but the total-examination time decreased by 18% with the digital technique. The total number of images taken was higher with the digital technique (25 images compared to 19) owing to the limited field of the image intensifier. Significantly more noise and less sharpness were observed with the digital system but there was no significant difference in contrast or image quality in the various anatomical structures. Although the change-over to the digital system produced a reduction in sharpness and an increase in noise, and no significant dose saving was measured, the digital system was faster to work with and could well be used for diagnostic purposes.

Matrix-assisted laser desorption/ionization mass spectrometry of proteins extracted directly from sodium dodecyl sulphate-polyacrylamide gels.

A new strategy for the characterization of Coomassie Brilliant Blue stained SDS-PAGE separated proteins by UV-MALDI-MS is reported. The proteins are extracted directly from the polyacrylamide gel by treatment with an organic solvent mixture consisting of formic acid, acetonitrile, isopropanol and water in an ultrasonic bath. A fraction of the supernatant is then mixed directly with the matrix solution and measured by MALDI-MS. High quality spectra could be obtained from gels which were loaded with 6 pmol of myoglobin. Compared to other methods based on electroblotting or electroelution this method is much simpler and less time consuming. The sensitivity is higher than or comparable to the Coomassie Blue staining procedure for proteins up to about 25 kDa. Another advantage is that mass shifts due to charging effects of the membranes, which are common if membranes are mounted directly on the sample target, can be avoided. However, all proteins studied showed slightly higher masses than expected which reduces mass accuracy to 0.2-0.3%. This is presumably partly due to formylation of serine or threonine residues during incubation in formic acid. Gel electrophoresis induced modifications can contribute as well. The possibility of further characterizing the remaining part of the supernatant after extraction by means of proteolytic digestion is also demonstrated. The knowledge of both molecular weight of the whole protein and of the proteolytic fragments increases specificity for protein identification by searching in sequence databases.

Prediction of genital prolapse after Burch colposuspension.

OBJECTIVE: The aim of this prospective observational study was to investigate the gynecological and defecographic features in women with stress urinary incontinence operated with Burch colposuspension in order to analyze if the findings could predict subsequent development of genital prolapse. SUBJECT: Twenty-one women with urodynamically proven genuine stress urinary incontinence were consecutively operated with the Burch colposuspension during 1991-1992. No concomitant prolapse repair surgery was performed. METHODS: All were carefully examined in the lithotomy position at rest and with the Valsalva maneuver. The pelvic floor laxity was graded semiquantitatively. The defecography and the clinical examination were done preoperatively and repeated one year postoperatively. RESULTS: The clinical examination revealed a significant progression of rectoceles (p = 0.003) after the colposuspension. The colposuspension cured a significant number of cystoceles (p = 0.035). Six women (29%) had subsequent corrective prolapse surgery median 2 years after the colposuspension. The defecographic measurements showed a significant increase of the recto-vaginal distance (RVD) following the operation (p = 0.020). At the postoperative measurement the group with subsequent prolapse surgery had a significantly larger RVD as compared to the group without further surgery (p = 0.004). The kappa reliability test showed poor agreement between the defecographic and clinical assessment of the rectoceles. CONCLUSION: We failed to find any clinical or defecographic characteristic which could predict the development of surgery-demanding genital prolapse following colposuspension. The colposuspension seemed to accelerate the deterioration of the pelvic floor. However, only a minority of the patients developed symptomatic genital prolapse demanding corrective surgery. We suggest that only women with symptomatic prolapse should be considered for concomitant corrective surgery at the time of the colposuspension.

Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli.

A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz. Cys182-Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (Tv18-19) and the corresponding tryptic peptide from rhGH (T18+19) were identical. Electrospray mass spectrometry (ES MS) of Tv18+19 isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T18+19 from rhGH. A synthetic Tv18+19 peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragment in ES/MS compared to Tv18+19 isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cys182-SSS-Cys189) bridge not found in the corresponding T18+19 disulfide peptide. Furthermore, the synthetic Tv18+19 co-eluted in RP-HPLC with Tv18+19 isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic T18+19 and Tv18+19 peptides were performed. Using these data all protons were assigned. The major chemical shift changes (delta delta > 0.05 ppm) observed were for the beta-protons of Cys182 and Cys189 in Tv18+19 as compared to T18+19. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data rhGH-HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties.

Plain X-ray films and air enema films reflect severe mucosal inflammation in acute ulcerative colitis.

In a prospective study of 34 patients with active ulcerative colitis, the findings of inflammation on plain abdominal films and air enema films were compared to those at colonoscopy including biopsy within 10 days. The degree of inflammation on X-ray films was graded independently by two radiologists, at colonoscopy by one gastroenterologist and from histological slides from 6 different colon segments by one pathologist for each patient. Air enema films had a high sensitivity for endoscopically confirmed friable or ulcerated mucosa (0.91). There was a high specificity (0.86) when excluding inflammation in individual colon segments. Absence of fecal residue as an indication of active inflammation had the same positive predictive value, 0.95, as an abnormal air enema film, 0.98 for endoscopically confirmed inflamed mucosa. The presence of fecal residue or a normal air enema film excluded a friable or ulcerated mucosa at endoscopy with negative predictive values of 0.83 and 0.86, respectively. Patients who had had a complete colonoscopy (n = 16) were divided into groups with total, extensive or distal colitis. Air enema films underestimated the extent of inflammation in 8 of 16 patients compared to colonoscopy. Of 6 patients with distal disease only on air enema films, 5 had disease above the splenic flexure at endoscopy. In patients with ulcerative colitis (1) the presence of fecal residue and a normal air enema film exclude a friable or ulcerated mucosa with a high degree of certainty, and (2) the absence of fecal residue and an abnormal air enema film are predictors of the presence of endoscopically confirmed inflammation.

Bioavailability and disposition of terodiline in man.

Terodiline was concomitantly administered intravenously (12.5 mg) and orally ([2H]terodiline, 12.5 mg) to 10 healthy volunteers. In four of the subjects, a tracer dose of the intravenously given terodiline was 3H-labeled. In a separate study, six subjects were given [3H]terodiline orally. Estimated pharmacokinetic parameters were as follows: systemic clearance, 93 mL/min; renal clearance, 14 mL/min; volume of distribution at steady-state, 407 L; terminal half-life, 54 h; and mean residence time, 77 h. After intravenous infusion, a rapid distribution phase (half-life, 4.5 min) could be observed. The maximum serum concentration after the oral dose was 29 micrograms/L and the time to maximum concentration was 5 h (estimated by noncompartmental analysis). Absorption commenced within the first hour and by deconvolution the maximum rate of absorption was determined to occur between 1 and 3 h, and by 3.4 h 90% of the available dose had been absorbed. Calculation of bioavailability by noncompartmental AUC, two-compartmental analysis, urinary excretion, and 24-h oral/intravenous concentration ratio gave similar results (ANOVA test, not significant). About 75% and 25% of administered radioactivity could be recovered in urine and feces, respectively. Intact terodiline in feces accounted for about 1% of the dose. p-Hydroxyterodiline was quantitated in feces and accounted for about 5% of the dose. Another metabolite, 3,4-dihydroxyterodiline, which has not previously been detected in urine or serum, was also identified.

Air enema revisited in assessment of colitis.

Plain radiographs and air enema were performed in 37 patients with ulcerative colitis, 7 patients with proctitis, and 8 patients with Crohn's disease. The air enema was superior to plain radiographs for diagnosing colitis, and for delineating the extent of disease and the degree of mucosal involvement. The air enema is simple to perform and easy to evaluate as shown by an almost complete agreement between 2 observers.