PMC:29007 / 4463-12209 JSONTXT

{"project":"2_test","denotations":[{"id":"11056717-856499-23491501","span":{"begin":7037,"end":7039},"obj":"856499"}],"text":"Materials and methods\n\nAnimals\nMale Golden-Syrian hamsters weighing 80–140g (Harlan Animals, \t\t\t Indianapolis, Indiana, USA) were housed in a controlled environment and were \t\t\t exposed to 12 h light–dark cycles. The animals had unrestricted access to water \t\t\t and standard rodent chow throughout the experiments. This study was approved by \t\t\t the Institutional Animal Care and Use Committee at the Veterans Affairs Medical \t\t\t Center, Washington, DC.\n\nBacteria\nEscherichia coli (O18:K1:H7) were obtained from Dr Alan S \t\t\t Cross, Division of Communicable Diseases and Immunology, Walter Reed Army \t\t\t Institute of Research, Washington, DC, USA. The bacteria were grown in 100 ml \t\t\t of LB Broth (Fisher Scientific, Pittsburgh, Pennsylvania, USA) at 37°C in \t\t\t a shaker water bath to log phase and stored in 250 μ l aliquots at \t\t\t -70°C until use.\nOn the day of an experiment, a 250 μ l aliquot of bacteria was \t\t\t thawed and grown in 100 ml LB broth at 37°C in a shaker water bath to log \t\t\t phase. The optical density of the specimen was measured at 600 nm on a Stasar \t\t\t III spectrophotometer (Gilford Instruments, Oberlin, Ohio, USA) and quantified \t\t\t by interpolation on a previously constructed curve of optical density plotted \t\t\t against colony forming units (cfu). Additional specimens were taken from the \t\t\t stock solution, and diluted and plated to confirm the counts estimated by \t\t\t spectrophotometry.\n\nIntra-abdominal pellets\nBacterial suspensions of 2.0 × 108, 1.0 × \t\t\t 109, 2.0 × 109, or 4.0 × 109 cfu/ml \t\t\t E coli were pipetted in 0.5 ml aliquots into 8 mm plastic embedding \t\t\t molds (Shandon-Upshaw, War-rington, Pennsylvania, USA). Each pellet for \t\t\t implantation was made by adding 0.5 ml sterile molten agar at 50°C to the \t\t\t bacterial suspension, after which the mixture was allowed to solidify at room \t\t\t temperature. The final number of viable colony forming units of bacteria in \t\t\t each pellet was 1.0 × 108, 5.0 × 108, 1.0 \t\t\t × 109, or 2.0 × 109 cfu/pellet.\n\nExperimental protocol\n\nMortality studies\nIndividual hamsters were assigned to four groups (n \t\t\t\t = 16/group) to receive progressively increasing inocula of bacteria. After \t\t\t\tadequate anesthesia with 50 mg/kg pentobarbital via intraperitoneal injection, \t\t\t\tthe abdomen of each animal was prepared with 70% alcohol and incised in the \t\t\t\tmidline. Bacterial sepsis was induced by implanting one pellet in the right \t\t\t\tlower quadrant of the peritoneal cavity of each animal. The abdominal incisions \t\t\t\twere then closed with non-absorbable suture. Animals were caged individually, \t\t\t\tgiven unrestricted access to water and rodent chow and monitored for mortality \t\t\t\tover a 72 h period.\n\nTotal iCT studies\nAfter intraperitoneal implantation of agar pellets with \t\t\t\tprogressively increasing doses of E coli, separate groups (n \t\t\t\t = 10/group) were killed for serum total immunoreactive (i)CT determinations. \t\t\t\tSince mortality was evident but not prohibitively high at 12 h, we chose this \t\t\t\ttimepoint to determine serum total iCT levels. Therefore, 12 h after animals \t\t\t\twere challenged with E coli, they were anesthetized with \t\t\t\tintraperitoneal pentobarbital (50 mg/kg) and exsanguinated by open cardiac \t\t\t\tpuncture. The blood was collected in individual tubes and centrifuged at \t\t\t\t3000 rpm for 15 min. The serum specimens were transferred to individual glass \t\t\t\ttubes, sealed with parafilm and stored at -70°C until \t\t\t\tradioimmunoassay.\nSerum was also obtained from a patient with documented \t\t\t\tGram-negative sepsis and was stored at -70°C to be assayed with the \t\t\t\thamster serum samples following G-75 Sephadex gel filtration for the purpose of \t\t\t\tcomparison of molecular forms as described below.\n\nMetabolic studies\nMale hamsters (n = 16/group) underwent intraperitoneal \t\t\t\timplantation of agar pellets impregnated with 2 × 109 cfuE \t\t\t\tcoli (O18: K1: H7), according to the above implantation protocol. This \t\t\t\thigh dose was chosen for its ability to induce a significant increase of ProCT \t\t\t\tat 12 h in the proceeding experiments. Animals were killed in the previously \t\t\t\tdescribed fashion at 3, 6, 12 or 24 h after septic challenge. Their sera were \t\t\t\tanalyzed for serum total iCT per the radioimmunoassay described below, as well \t\t\t\tas for total serum total calcium and serum albumin using a standard serum \t\t\t\tmultichannel analyzer.\n\nRadioimmunoassay\nThe samples were allowed to warm to room temperature and were \t\t\t\tpipetted into labeled glass test tubes in 1.0 ml aliquots, to which 100 μ l \t\t\t\tdextran blue (B-2000, 2 000 000 Da; Sigma Chemical Co, St Louis, Missouri, USA) \t\t\t\twas added. Five milliliter glass columns were rinsed with 1M ammonium \t\t\t\thydroxide:acetonitrile (1:1) and deionized water, after which fine-grade \t\t\t\tpolyacrylamide gel columns (5ml) were prepared (BioGel P-2; 100–200 mesh; \t\t\t\tBio-Rad Laboratories, St Louis, Missouri, USA) using a glass bead to support \t\t\t\tthe gel. The samples were applied to the columns and eluted with 0.1 M ammonium \t\t\t\tbicarbonate containing 0.1% Triton X-100 (Pierce, Rockford, Illinois, USA). The \t\t\t\tspecimens containing dextran blue were then recovered in their original test \t\t\t\ttubes, to which ethyl alcohol was added in a 2:1 volume ratio. These mixtures \t\t\t\twere then centrifuged at 3000 rpm for 30 min, after which the supernatant for \t\t\t\teach was decanted into new tubes and the pellet discarded. The solvent was \t\t\t\tremoved using a Savant SpeedVac Plus (SC110A) over 2–4 h. The residue for each \t\t\t\tsample was then reconstituted to the original specimen volume using gelatin \t\t\t\tbuffer (0.2% gelatin in borate buffer with 0.01% merthiolate and 0.1% Triton \t\t\t\tX-100). Using these techniques, peptide recovery is approximately 80%.\nThe radioimmunoassay design was similar to that previously \t\t\t\treported [19]. Initially, hamster serum total iCT from \t\t\t\tgel filtration studies was determined by using an antiserum to the \t\t\t\tcarboxyl-terminal portion of mature human CT, Ab4. This antiserum reacts with \t\t\t\tthe CT molecule, whether it is in the free, amidated, 32-amino acid mature \t\t\t\tform, or within its precursor propeptides [ie procalcitonin, the conjoined \t\t\t\tcalcitonin:calcitonin carboxypeptide-I (CT:CCP-1), or the free immature, \t\t\t\tunamidated CT]. Subsequent studies were performed with a new antibody, R1B4, \t\t\t\twhich has ten times the crossreactivity of Ab-4 with the prohormone. The buffer \t\t\t\twas 0.2% gelatin (0.13 M H3BO3 containing 9 g NaCl, 2 g \t\t\t\tgelatin, 1 ml Triton-X 100 and 0.1 g merthiolate/l at pH = 7.5). The antiserum was \t\t\t\tpreincubated with standards or unknowns (20–100 μ l) in 0.2 ml at 4°C \t\t\t\tfor 4 days. After addition of 50 μ l 125I-hCT, and 200 μ l gelatin \t\t\t\tbuffer, incubation was continued for 2 days. After adding 50 μ l goat \t\t\t\tanti-rabbit IgG bound to iron particles, incubation was continued in 0.5 ml for \t\t\t\t1 day. Bound and free hormone were separated with magnetic tube racks. Maximum \t\t\t\tbound =37%; sensitivity =1g; 50% B/Bo =50pg.\n\nGel filtration\nSimilarly to work previously reported [20], \t\t\t\tconstituted extracts, in 1–10 ml 0.2% gelatin or 0.2% HSA, were applied to \t\t\t\tcalibrated 2.5 × 100 cm columns containing G-75 superfine Sephadex \t\t\t\t(Pharmacia Biotech, Piscataway, New Jersey, USA) suspended in 0.1% human serum \t\t\t\talbumin (1 g HSA, 0.1 mol NH4HCO3 and 0.1 g merthiolate/l at \t\t\t\tpH = 7.5) at 4°C. One hundred fractions (120 drops or 5.5 ml/tube) were \t\t\t\tcollected during 48 h in 16× 100 mm borosilicate glass culture tubes. The \t\t\t\tvoid volume (VV) was based on the peak elution volume (EV) of blue dextran, and \t\t\t\tthe salt volume (SV) was based on the peak EV of Na125I. The Kav for \t\t\t\tindividual components was determined according to the formula: Kav = \t\t\t\t(EV–VV)/(SV–VV).\n\n"}

{"project":"Colil","denotations":[{"id":"T1","span":{"begin":7037,"end":7039},"obj":"856499"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Materials and methods\n\nAnimals\nMale Golden-Syrian hamsters weighing 80–140g (Harlan Animals, \t\t\t Indianapolis, Indiana, USA) were housed in a controlled environment and were \t\t\t exposed to 12 h light–dark cycles. The animals had unrestricted access to water \t\t\t and standard rodent chow throughout the experiments. This study was approved by \t\t\t the Institutional Animal Care and Use Committee at the Veterans Affairs Medical \t\t\t Center, Washington, DC.\n\nBacteria\nEscherichia coli (O18:K1:H7) were obtained from Dr Alan S \t\t\t Cross, Division of Communicable Diseases and Immunology, Walter Reed Army \t\t\t Institute of Research, Washington, DC, USA. The bacteria were grown in 100 ml \t\t\t of LB Broth (Fisher Scientific, Pittsburgh, Pennsylvania, USA) at 37°C in \t\t\t a shaker water bath to log phase and stored in 250 μ l aliquots at \t\t\t -70°C until use.\nOn the day of an experiment, a 250 μ l aliquot of bacteria was \t\t\t thawed and grown in 100 ml LB broth at 37°C in a shaker water bath to log \t\t\t phase. The optical density of the specimen was measured at 600 nm on a Stasar \t\t\t III spectrophotometer (Gilford Instruments, Oberlin, Ohio, USA) and quantified \t\t\t by interpolation on a previously constructed curve of optical density plotted \t\t\t against colony forming units (cfu). Additional specimens were taken from the \t\t\t stock solution, and diluted and plated to confirm the counts estimated by \t\t\t spectrophotometry.\n\nIntra-abdominal pellets\nBacterial suspensions of 2.0 × 108, 1.0 × \t\t\t 109, 2.0 × 109, or 4.0 × 109 cfu/ml \t\t\t E coli were pipetted in 0.5 ml aliquots into 8 mm plastic embedding \t\t\t molds (Shandon-Upshaw, War-rington, Pennsylvania, USA). Each pellet for \t\t\t implantation was made by adding 0.5 ml sterile molten agar at 50°C to the \t\t\t bacterial suspension, after which the mixture was allowed to solidify at room \t\t\t temperature. The final number of viable colony forming units of bacteria in \t\t\t each pellet was 1.0 × 108, 5.0 × 108, 1.0 \t\t\t × 109, or 2.0 × 109 cfu/pellet.\n\nExperimental protocol\n\nMortality studies\nIndividual hamsters were assigned to four groups (n \t\t\t\t = 16/group) to receive progressively increasing inocula of bacteria. After \t\t\t\tadequate anesthesia with 50 mg/kg pentobarbital via intraperitoneal injection, \t\t\t\tthe abdomen of each animal was prepared with 70% alcohol and incised in the \t\t\t\tmidline. Bacterial sepsis was induced by implanting one pellet in the right \t\t\t\tlower quadrant of the peritoneal cavity of each animal. The abdominal incisions \t\t\t\twere then closed with non-absorbable suture. Animals were caged individually, \t\t\t\tgiven unrestricted access to water and rodent chow and monitored for mortality \t\t\t\tover a 72 h period.\n\nTotal iCT studies\nAfter intraperitoneal implantation of agar pellets with \t\t\t\tprogressively increasing doses of E coli, separate groups (n \t\t\t\t = 10/group) were killed for serum total immunoreactive (i)CT determinations. \t\t\t\tSince mortality was evident but not prohibitively high at 12 h, we chose this \t\t\t\ttimepoint to determine serum total iCT levels. Therefore, 12 h after animals \t\t\t\twere challenged with E coli, they were anesthetized with \t\t\t\tintraperitoneal pentobarbital (50 mg/kg) and exsanguinated by open cardiac \t\t\t\tpuncture. The blood was collected in individual tubes and centrifuged at \t\t\t\t3000 rpm for 15 min. The serum specimens were transferred to individual glass \t\t\t\ttubes, sealed with parafilm and stored at -70°C until \t\t\t\tradioimmunoassay.\nSerum was also obtained from a patient with documented \t\t\t\tGram-negative sepsis and was stored at -70°C to be assayed with the \t\t\t\thamster serum samples following G-75 Sephadex gel filtration for the purpose of \t\t\t\tcomparison of molecular forms as described below.\n\nMetabolic studies\nMale hamsters (n = 16/group) underwent intraperitoneal \t\t\t\timplantation of agar pellets impregnated with 2 × 109 cfuE \t\t\t\tcoli (O18: K1: H7), according to the above implantation protocol. This \t\t\t\thigh dose was chosen for its ability to induce a significant increase of ProCT \t\t\t\tat 12 h in the proceeding experiments. Animals were killed in the previously \t\t\t\tdescribed fashion at 3, 6, 12 or 24 h after septic challenge. Their sera were \t\t\t\tanalyzed for serum total iCT per the radioimmunoassay described below, as well \t\t\t\tas for total serum total calcium and serum albumin using a standard serum \t\t\t\tmultichannel analyzer.\n\nRadioimmunoassay\nThe samples were allowed to warm to room temperature and were \t\t\t\tpipetted into labeled glass test tubes in 1.0 ml aliquots, to which 100 μ l \t\t\t\tdextran blue (B-2000, 2 000 000 Da; Sigma Chemical Co, St Louis, Missouri, USA) \t\t\t\twas added. Five milliliter glass columns were rinsed with 1M ammonium \t\t\t\thydroxide:acetonitrile (1:1) and deionized water, after which fine-grade \t\t\t\tpolyacrylamide gel columns (5ml) were prepared (BioGel P-2; 100–200 mesh; \t\t\t\tBio-Rad Laboratories, St Louis, Missouri, USA) using a glass bead to support \t\t\t\tthe gel. The samples were applied to the columns and eluted with 0.1 M ammonium \t\t\t\tbicarbonate containing 0.1% Triton X-100 (Pierce, Rockford, Illinois, USA). The \t\t\t\tspecimens containing dextran blue were then recovered in their original test \t\t\t\ttubes, to which ethyl alcohol was added in a 2:1 volume ratio. These mixtures \t\t\t\twere then centrifuged at 3000 rpm for 30 min, after which the supernatant for \t\t\t\teach was decanted into new tubes and the pellet discarded. The solvent was \t\t\t\tremoved using a Savant SpeedVac Plus (SC110A) over 2–4 h. The residue for each \t\t\t\tsample was then reconstituted to the original specimen volume using gelatin \t\t\t\tbuffer (0.2% gelatin in borate buffer with 0.01% merthiolate and 0.1% Triton \t\t\t\tX-100). Using these techniques, peptide recovery is approximately 80%.\nThe radioimmunoassay design was similar to that previously \t\t\t\treported [19]. Initially, hamster serum total iCT from \t\t\t\tgel filtration studies was determined by using an antiserum to the \t\t\t\tcarboxyl-terminal portion of mature human CT, Ab4. This antiserum reacts with \t\t\t\tthe CT molecule, whether it is in the free, amidated, 32-amino acid mature \t\t\t\tform, or within its precursor propeptides [ie procalcitonin, the conjoined \t\t\t\tcalcitonin:calcitonin carboxypeptide-I (CT:CCP-1), or the free immature, \t\t\t\tunamidated CT]. Subsequent studies were performed with a new antibody, R1B4, \t\t\t\twhich has ten times the crossreactivity of Ab-4 with the prohormone. The buffer \t\t\t\twas 0.2% gelatin (0.13 M H3BO3 containing 9 g NaCl, 2 g \t\t\t\tgelatin, 1 ml Triton-X 100 and 0.1 g merthiolate/l at pH = 7.5). The antiserum was \t\t\t\tpreincubated with standards or unknowns (20–100 μ l) in 0.2 ml at 4°C \t\t\t\tfor 4 days. After addition of 50 μ l 125I-hCT, and 200 μ l gelatin \t\t\t\tbuffer, incubation was continued for 2 days. After adding 50 μ l goat \t\t\t\tanti-rabbit IgG bound to iron particles, incubation was continued in 0.5 ml for \t\t\t\t1 day. Bound and free hormone were separated with magnetic tube racks. Maximum \t\t\t\tbound =37%; sensitivity =1g; 50% B/Bo =50pg.\n\nGel filtration\nSimilarly to work previously reported [20], \t\t\t\tconstituted extracts, in 1–10 ml 0.2% gelatin or 0.2% HSA, were applied to \t\t\t\tcalibrated 2.5 × 100 cm columns containing G-75 superfine Sephadex \t\t\t\t(Pharmacia Biotech, Piscataway, New Jersey, USA) suspended in 0.1% human serum \t\t\t\talbumin (1 g HSA, 0.1 mol NH4HCO3 and 0.1 g merthiolate/l at \t\t\t\tpH = 7.5) at 4°C. One hundred fractions (120 drops or 5.5 ml/tube) were \t\t\t\tcollected during 48 h in 16× 100 mm borosilicate glass culture tubes. The \t\t\t\tvoid volume (VV) was based on the peak elution volume (EV) of blue dextran, and \t\t\t\tthe salt volume (SV) was based on the peak EV of Na125I. The Kav for \t\t\t\tindividual components was determined according to the formula: Kav = \t\t\t\t(EV–VV)/(SV–VV).\n\n"}