Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes.

BACKGROUND: Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. Many factors can affect the quality of PBMCs, and careful consideration and validation of an appropriate PBMC isolation and cryopreservation method is important for well-designed clinical studies. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. To address potential differences in sample outcome, we isolated, cryopreserved, and compared PBMCs using parallel protocols differing only in the use of one of two common tube types for isolation. METHODS: Whole blood was processed in parallel using both Cell Preparation Tubes™ (CPT, BD Biosciences) and Lymphoprep™ Tubes (Axis-Shield) and assessed for yield and viability prior to cryopreservation. After thawing, samples were further examined by flow cytometry for cell yield, cell viability, frequency of 10 cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production. RESULTS: No significant differences in cell recovery, viability, frequency of immune cell subsets, or T cell functionality between PBMC samples isolated using CPT or Lymphoprep tubes were identified. CONCLUSION: CPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.

BDNF mRNA expression of peripheral blood mononuclear cells was decreased in depressive patients who had or had not recently attempted suicide.

BACKGROUND: Recent reports have suggested a pathophysiological role for brain-derived neurotrophic factor (BDNF) in major depression and suicide. We evaluate BDNF mRNA in peripheral blood mononuclear cells (PBMCs) of patients with major depression who had or had not recently attempted suicide. METHODS: BDNF mRNA expression was examined in PBMCs of 30 major depressed patients without recent suicide attempts, 30 major depressed patients with recent suicide attempts, and 30 healthy controls. All patients were diagnosed with major depressive disorder (MDD) and they were either medication-naïve or medication-free. BDNF mRNA expression was measured using real-time quantitative PCR. RESULTS: BDNF mRNA expression was significantly decreased in PBMCs of MDD patients, with or without a history of suicide attempts, when compared with healthy controls (Z=-5.233, p<0.001). Suicidal MDD patients had more decrease in BDNF mRNA expression (chi(2)=45.881, df=2, p<0.001). LIMITATIONS: The sample size of the present study was relatively small. Although suicidal MDD patients had higher HDRS scores than non-suicidal patients, the comparison of BDNF mRNA between them could not be adjusted for HDRS score. CONCLUSIONS: Our study suggests that the BDNF mRNA expression is reduced in PBMCs of patients with major depression. This alteration of BDNF mRNA expression was noteworthy in recent suicide attempters. Such a decrease in BDNF mRNA might be associated with BDNF decrease in serum or plasma, and might also correspond to a BDNF decline in the brains of MDD patients.

Improved islet survival and funtion with rat endothelial cells in vitro co-culture.

OBJECTIVE: Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To preserve its function, transplanted islets must be revascularized because arterial and venous connections are disrupted during islet isolation. The current paradigm is that islet revascularization originates from the transplant recipient. This study was designed to test whether the function of isolated islets can be retained by co-culture with thoracic aorta endothelial cells in vitro. METHODS: Sprague-Dawley rats were used in this study. The endothelial cells (ECs) were isolated from the thoracic aorta. The viability of the isolated islets was assessed by acridine orange/propidium iodide (AO/PI) double staining. The islets were either placed in standard cultures (group A) or in co-cultures with ECs (group B). Islet viablity was assessed by an insulin release assay. RESULTS: The islets in group B exhibited normal morphology with >90% staining positive as detected by AO/PI with 7 days. Insulin release assays showed a significantly higher simulation index (SI) in group B compared with group A (P < .05) except on the first day. CONCLUSION: This study suggested that co-cultrue of freshly isolated rat islets with ECs improves postculture survival and islet function in vitro.

An optimized technique for separation of human decidual leukocytes for cellular and molecular analyses.

PROBLEM: Human decidua contains a significant number of leukocytes, phenotypically and functionally different from peripheral blood. In vitro studies of decidual leukocytes require constant improvement of cell isolation technique in order to obtain highly purified viable and functionally active cells. METHOD OF STUDY: Optimized isolation procedure of decidual leukocytes, based on non-enzymatic cell dispersion, Percoll gradient centrifugation and purification step involving immunomagnetic beads is compared with a similar procedure with Lymphoprep gradient. RESULTS: The method based on Percoll gradient gave a viable leukocyte fraction with higher amount of alphabetaT- and gammadeltaT cells compared with Lymphoprep gradient. The leukocytes exhibited a well-preserved ultrastructure and surface marker expression and were suitable for molecular analysis. The magnitude and the kinetics of their proliferative response were comparable with peripheral blood lymphocytes. CONCLUSIONS: The results provide an optimized method for isolation and enrichment of decidual leukocytes, suitable for phenotypic, ultrastructural, molecular and functional analyses.

IL-1 and TNF-alpha release in human polymorphonuclear leukocytes after exposure to calcium hydroxide treated Porphyromonas endodontalis lipopolysaccharide / 대한치과보존학회지

Bacterial lipopolysaccharide (LPS) plays a major role in stimulating the synthesis and release of the principal osteoclast-activating cytokines, namely, interleukin 1 and tumor necrosis factor-alpha from immune cells. Although monocytes/macrophages are the main producers of these cytokines, recent evidence has indicated that polymorphonuclear leukocytes (PMN) have the ability to release IL-1 and TNF-alpha. Calcium hydroxide has been shown to be an effective medicament in root canal infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)2 may also be the result of direct inactivation of LPS. The purpose of this study was to investigate whether treatment of Porphyromonas endodontalis LPS with calcium hydroxide alters its biological action as measured by human PMN secretion of IL-1 and TNF-alpha, and it was compared with Escherichia coli LPS. P. endodontalis ATCC 35406 was cultured in anaerobic condition, and LPS was extracted using the hot-phenol water extraction method and purified. Purchased E. coli LPS was also purified. 100 microg/ml of each LPS in pyrogen free water were incubated with 25mg/ml Ca(OH)2 at 37degrees C for 7 days. The supernatants were subjected to ultrafiltration, and the isolates were lyophilized and weighed. PMNs were obtained from peripheral blood by centrifugation layered over Lymphoprep. The cells were resuspended (4x106 cells/ml) in RPMI 1640 followed by treatment with various concentrations of LPS (0, 0.1, 1, 10microg/ml) for 24 hours at 37degrees C in 5% CO2 incubator. The supernatants of cells were collected and the levels of IL-1alpha, IL-1beta and TNF-alpha were measured by enzyme-linked immunosorbent assay. The results were as follows; 1. The levels of IL-1alpha, IL-1beta, TNF-alpha from PMN treated with each LPS were significantly higher than those released from unstimulated PMN of the control group (p0.05). 3. The levels of secretion for all three cytokines were affected in a dose-dependent manner in PMN stimulated with each LPS (p0.05). 4. The levels of all three cytokines released from PMN stimulated with P. endodontalis LPS were significantly lower than those released from PMN stimulated with E. coli LPS (p<0.05).

The influence of interfacial forces and duration of filing on root canal shaping.

The aim of this study was to investigate the possible relationship between interfacial forces, duration of instrumentation and effectiveness of root canal shaping by filing. All factors were standardised except the manner of manipulation of the files and the dependent variables (interfacial force and duration of filing). The outcome measured was the final prepared canal shape. The study tested the hypothesis that if interfacial forces and duration of filing had predictive value for effective canal shaping, a relationship between them should be apparent. If it was not, it could be inferred that the remaining uncontrollable variable, "manner of file manipulation" had a dominating influence. 18 operators used a standard filing technique to instrument a root canal each in single rooted teeth matched for anatomy. The teeth were mounted in a custom-made transducer designed to measure laterally applied interfacial forces between file and dentine. The length of time taken with each instrument in the series was measured giving a total duration of instrumentation. The final canal shape measured by standardised "before" and "after" radiographic images of the roots was judged against an "ideal" canal shape calculated from the size of instruments used and based on the preliminary canal shape. Although some trends were revealed, no obvious relationship was demonstrated between interfacial force, duration of filing and final canal shape. Operators clearly used a range of interfacial forces characteristic for each individual and for the different file sizes. Both light and heavy forces could produce a satisfactory canal shape. Use of light or heavy forces did not obviously affect speed of preparation. Tactile discrimination was not dependent on use of light or heavy forces. It was inferred that the "manner of file manipulation" remained undefined and was probably the dominant factor influencing outcome of canal shaping.

Role of autocrine stimulation for the effects of cyclic AMP on protein and lipid phosphorylation in platelets activated by particles.

We have compared responses in platelets stimulated with the particulate materials, Intralipid (liposome-suspension) and a potential contrast medium IEEC (1'-(ethyloxycarbonyloxy)-ethyl-5-acetyl-amino-3-(N-methyl-acetyla mino)-2,4,6-triiodo-benzenecarboxylate coated with human serum albumin), with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP-removing system of creatine phosphate/creatine phosphokinase; RGDS to prevent fibrinogen/fibronectin binding to GPIIb/IIIa; SQ 29.548 as a TXA2 receptor antagonist; cyproheptadine as a serotonin receptor antagonist; BN 52021 as a platelet-activating factor receptor antagonist). The pattern of tyrosine-phosphorylated proteins, phosphorylation of initial lipids and phosphorylation of pleckstrin (P47) were used as markers for early signal transducing responses, while secretion of ADP+ATP and beta-N-acetyl-glycosaminidase were used as final responses. Intralipid showed no platelet activation except for some weak tyrosine protein phosphorylation that was inhibited by elevated cAMP. IEEC induced strong platelet activation that was partly inhibited by increased levels of cAMP and IAS. The inhibition of elevated cAMP seemed to be due to removal of the G protein-mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure effect from IEEC which is not affected by elevated cAMP.

A single step density gradient separation for large scale enrichment of mobilized peripheral blood progenitor cells collected for autotransplantation.

Peripheral blood leukocytes are becoming the preferred source of hematopoietic progenitor/stem cells for autologous transplantation. However, in vitro purging procedures are complex and expensive when applied to peripheral blood progenitor cells harvests. This is mainly due to the large quantities of nucleated cells present in leukapheresis collections. Aiming to reduce total cellularity without significant loss of CD34+ cells, we developed an in vitro cell separation procedure based on ficoll/metrizoate gradient used at a final density of 1.067 g/ml. To obtain this density, standard Lympho-prep (1.077 g/ml) was diluted with normal saline solution (NaCl 9 g/l). Twenty-six leukapheresis collections (median cellularity 21.1 x 10(9), range 2.8-60) from 14 patients with non-Hodgkin's lymphoma, multiple myeloma or plasma cell leukemia were processed (median two leukaphereses per patient). Mean (+/- s.d.) recovery of total nucleated cells, CD34+ cells and CFU-GM was 20.9 +/- 10%, 74.7 +/- 22% and 70.5 +/- 19%, respectively. Cumulative per patient progenitor cell recovery was always above 50%, and as high as 80% in 10/14 patients, while total cellularity was reduced to a median 21.5% (10-33%) of pre-separation values. Contaminating neoplastic cells, identified by immunofluorescence in five collections, were reduced by 1-2 logs. The results indicate that our density gradient separation is an effective method to reduce total cellularity prior to immunological purging, without significant loss of progenitor cells.

Cardiovascular and electrocardiographic effects of iopentol in left ventricular angiography. Comparison of the low-osmolar, non-ionic iopentol (Imagopaque 350) and the hyper-osmolar, ionic metrizoate meglumine-Na-Ca (Isopaque Coronar 370) in patients with coronary heart disease.

UNLABELLED: The aim of the study was to evaluate and compare the hemodynamic and electrocardiographic effects following injection of the non-ionic, low-osmolar contrast medium iopentol (Imagopaque 350, Nycomed Imaging AS, Oslo, Norway) and the ionic, hyper-osmolar contrast medium metrizoate meglumine-Na-Ca (Isopaque Coronar 370, Nycomed Imaging AS, Oslo, Norway) when used for left ventricular angiography. The study was performed in a double-blind, randomized manner in 82 patients with severe coronary heart disease. The patients who received iopentol experienced less adverse events and subjective discomfort of lesser intensity than those who received metrizoate (p = 0.0001). Both contrast media induced a biphasic change in left ventricular (LV) systolic pressure, with an initial fall followed by a prolonged rise, but the alterations were statistically significantly more pronounced with metrizoate than with iopentol. The changes in LV end-diastolic pressure (p = 0.023), and LV negative dP/dt (p = 0.002) were significantly more pronounced with metrizoate than with iopentol. Cardiac output and heart rate increased more with metrizoate, while stroke volume was equally increased by both agents. A prolonged increase in the QT-interval, throughout the 10-min observation period, was seen only after injection of metrizoate (p = 0.0006 for comparison between contrast media). CONCLUSION: Iopentol was well tolerated and induced markedly less severe hemodynamic and electrocardiographic alterations than did metrizoate in patients with severe coronary heart disease.

Contrast media leakage to the cerebrospinal fluid after intravenous injection. Comparison of stroke patients and controls.

We have tested a method to evaluate leakage of i.v. injected contrast media (CM) through the 3 partitions between blood and cerebrospinal fluid (CSF) in controls and in patients with acute cerebrovascular disease (ACBVD) to detect differences between normal brains and brains with ischemic lesions. High-osmolar (HOCM) and low-osmolar (LOCM) CM were used. In 55 patients and in 41 controls who underwent CT after i.v. contrast administration, lumbar CSF was collected 1 hour after injection and the iodine content in the CSF was measured. The concentration of iodine in CSF was very low, between 0.57 and 11.20 ng/l, and no significant difference could be found between patients and controls or between HOCM and LOCM. We conclude that under the conditions mentioned above, iodine detected in the human lumbar CSF does not reflect the true leakage of contrast agent through the blood-brain barrier.

Effects of iodinated x-ray contrast media on renal epithelial cells in culture.

RATIONALE AND OBJECTIVES: To study cellular mechanisms that cause contrast media nephropathy, an in vitro system for proximal and distal tubular cells has been established to evaluate the influence of x-ray contrast media on tubular function. METHODS: Confluent cell cultures of the two renal cell lines, proximal tubule (LLC-PK1) and distal tubule (MDCK), were exposed for 20 hours to 0 to 100 mg iodine/mL of the ionic monomer metrizoate, the ionic dimer ioxaglate, and the non-ionic monomer iohexol. Toxicity was assessed by electron microscopy, cell viability, and biochemical assays of brush-border and lysosomal marker enzymes. RESULTS: The results demonstrated a concentration-dependent toxic effect from the contrast media on cellular appearance consisting of an increased vacuolization and on the activity of brush-border and lysosomal marker enzymes in cells and in culture media. CONCLUSION: The results, in which the nonionic x-ray contrast media iohexol appeared to be less toxic than the ionic x-ray contrast media investigated, demonstrated that defined renal cells in culture are valuable tools in studies regarding renal toxicity of x-ray contrast media.

The effect of various contrast media on the activation of plasminogen by streptokinase or recombinant tissue plasminogen activator in vitro.

RATIONALE AND OBJECTIVES: Radiologic contrast media (CM) may influence processes of coagulation and fibrinolysis. In the current study, the effects of various CM on the formation of plasmin were examined in an in vitro buffer system. METHODS: The effects of three clinically relevant concentrations of seven different iodine-containing CM and gadolinium-DTPA on streptokinase (SK) or recombinant tissue plasminogen activator (rt-PA)-induced plasmin formation was monitored using a plasmin-sensitive chromogenic substrate. RESULTS: Contrast media generally had an inhibitory effect at the plasminogen activation step; this effect was particularly noticeable with the ionic CM. CONCLUSIONS: Contrast media influence plasminogen activation by SK and rt-PA in vitro. Ionic CM have a more pronounced inhibitory effect than the nonionic media. The ionic Gd-DTPA shows a less inhibitory effect than the ionic iodine-containing CM. However, they must be regarded separately because of the different chemical composition of the magnetic resonance imaging and x-ray CM.

Image quality and safety in pediatric urography using an ionic and a non-ionic iodinated contrast agent.

The safety and diagnostic efficacy of the new non-ionic, monomeric contrast medium iopentol (Imagopaque) were evaluated and compared with those of the ionic medium metrizoate (Isopaque), in urography in children in a randomized, double-blind, parallel study. The trial comprised 59 children aged from 3 months to 8 years; children with predefined risk factors were not included. The difference in attenuation between the renal and perirenal tissue, assessed from film density measurements, was chosen as the main variable. Diagnostic efficacy was also evaluated subjectively from demarcation of the kidney and the pelvic structures. There was no statistically significant difference between the groups in the diagnostic efficacy. Serious adverse events did not occur in either group. Mild to moderate, transient adverse events were significantly less frequent in the iopentol group than in the metrizoate group, but in no case was medical treatment required. There were no changes or trends toward changes of clinical importance in either group, in serum chemistry variables measured in blood samples taken 2 min after injection of contrast medium compared to baseline values.

A comparison between IEEC, a new biodegradable particulate contrast medium, and iohexol in a tumor model of computed tomography imaging of the liver.

RATIONALE AND OBJECTIVES: Higher contrast between normal and pathologic tissues in the liver may enable detection of smaller lesions in computed tomography (CT). This can be obtained using a liver-specific contrast medium. The authors evaluate a new agent, IEEC (1'-Ethyloxycarbonyloxy)-ethyl-5-acetylamino-3-(N-methyl-acetylami no)-2,4,6- triiodo-benzenecarboxylate), in an animal model, as a potential contrast agent for CT scanning of the liver. The IEEC particulate contrast medium used is based on a prodrug ester design of metrizoic acid and accumulates rapidly in the liver. The particles are quickly degraded into well-known metabolites and excreted from the body. METHODS: Two groups of rabbits were inoculated with VX2-carcinoma directly into the liver by laparotomy. Computed tomography imaging studies were carried out 9 and 11 days after the inoculation. The investigation was designed as a crossover study. The first group was imaged both as controls (without contrast medium) and with the particulate contrast medium on the 9th day and with iohexol on the 11th day. The second group was imaged with iohexol on the 9th day and as controls, and with the particulate contrast medium on the 11th day. The contrast medium was administered in a dose of 100 mgI/kg. Iohexol was administered in a dose of 570 mgI/kg according to a standard clinical scheme in use at a radiology department for dynamic CT. Changes in normal liver/lesion contrast and the conspicuity of tumors were assessed. On completion of imaging studies on day 11, all animals were killed. The liver was removed and evaluated for the presence of tumors. RESULTS: At macroscopic inspection, all rabbits were found to have tumors ranging from 2 to 14 mm in diameter. The size and location of the tumors corresponded well with the CT images. In the images where the particulate contrast medium was used, the attenuation in the normal liver parenchyma and the contrast between normal liver and lesion was significantly higher compared with the images where iohexol was used or the controls. For all tumor sizes, the lesion detection capability with the particulate contrast medium was significantly higher compared with iohexol (P < .005) and controls (P < .05). CONCLUSIONS: VX2-carcinoma in rabbit liver is a useful model for studying the efficacy of contrast media in CT imaging. The particulate contrast medium IEEC improved visualization of liver tumors.

Early effect of gadopentetate and iodinated contrast media on rabbit kidneys.

RATIONALE AND OBJECTIVES: The authors compared the physiologic and nephrotoxic effects of the magnetic resonance imaging contrast medium gadopentetate with two conventional radiographic contrast media. METHODS: Rabbits were injected intravenously with one of the following solutions: 1) gadopentetate (0.1 M); 2) iohexol (300 mg I/mL); 3) metrizoate (300 mg I/mL); and 4) NaCl (0.9%). Blood samples were taken before and 5, 15, 45, 90, and 180 minutes after injection of the solutions and were analyzed for creatinine, aldosterone, and contrast media levels. Urine was sampled before and 1, 2.5, and 5 hours after injection of the solutions, and creatinine, leucine amino peptidase (LAP), alkaline phosphatase (ALP), gamma glutaryl transferase (GGT), and N-acetyl beta-D-glucosaminidase (NAG) activities were quantified. RESULTS: Contrast media clearance was similar for gadopentetate, iohexol, and metrizoate. Plasma aldosterone was significantly higher in the two groups injected with iodinated contrast agents compared with the gadopentetate and saline groups in the 3-hour samples. During the 5 hours after injection, the excretion of brushborder enzymes LAP, ALP, and gamma GT was significantly higher for all contrast media compared with pre-contrast values and 0.9% NaCl controls. NAG, a lysosomal enzyme from tubular cells, showed a significant increase compared with pre-contrast values for all contrast media. CONCLUSIONS: Intravenous injection of gadopentetate in rabbits showed nephrotoxicity of the same order as that of conventional iodinated contrast media.