A laboratory study of the effects of processing blood through a cell salvage device and leucocyte depletion filter on levels of pro-inflammatory cytokines and bradykinin.

We investigated changes in concentrations of interleukin-1ß, interleukin-6, tumour necrosis factor-α and bradykinin in blood during passage through a cell salvage device and a leucocyte depletion filter, with or without application of subatmospheric pressure across the filter. Blood samples from 19 healthy women undergoing scheduled caesarean section showed concentrations of cytokines and bradykinin in blood filtered under gravity flow that were equal to or significantly lower than those of pre-operative venous blood samples. They were also significantly lower than that in postoperative orthopaedic shed blood, which is commonly reinfused after orthopaedic surgery. A minority of samples taken from blood that had been filtered using subatmospheric pressure showed raised interleukin-6 concentrations. We suggest that use of a leucocyte depletion filter for cell-salvaged blood with gravity flow is likely to be safe with regard to concentrations of cytokines and bradykinin. However, this may not hold true for the filter used with subatmospheric pressure. If transfusion of salvaged blood using a leucocyte depletion filter seems to induce hypotension, elevation of interleukin-6 should be suspected.

Identification of differentially expressed genes using annealing control primer-based GeneFishing in human squamous cell cervical carcinoma.

AIMS: To compare different gene expression patterns between squamous cell cervical carcinoma (SCC) and normal cervical tissue in Korean women and to identify those genes that are specifically or predominantly expressed in SCC by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR). MATERIALS AND METHODS: Cervical cancer specimens were obtained from patients enrolled at the Department of Obstetrics and Gynecology, Kang Nam St. Mary's Hospital, Catholic University of Korea. We used a common reference that was mixed with an equal amount of RNA extracted from patients without cervical cancer. The profiles of expressed genes were compared between the SCC and normal cervix identified using GeneFishing differentially expressed gene kits, screened by a BLAST search, and confirmed by semi-quantitative reverse transcription-PCR (RT-PCR). RESULTS: Almost 100 differentially expressed genes were identified in the control and SCC samples. Using 60 arbitrary ACPs, 50 differentially expressed genes were identified, and 30 up-regulated and 20 down-regulated expressed genes were sequenced. Among 50 clones selected by ACP-based GeneFishing PCR, six genes with different expression patterns were determined and confirmed by semi-quantitative RT-PCR. The functional roles of two up-regulated genes, fibrillarin and calgranulin A, and one down-regulated gene, clusterin, were previously identified. However, the functional roles of two up-regulated genes and one down-regulated gene were not identified. CONCLUSION: We identified distinctive gene expression profiles in Korean women with SCC using ACP-based GeneFishing PCR.

Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions.

We investigated clinical efficacy of green tea extracts (polyphenon E; poly E and (-)-epigallocatechin-3-gallate [EGCG]) delivered in a form of ointment or capsule in patients with human papilloma virus (HPV) infected cervical lesions. Fifty-one patients with cervical lesions (chronic cervicitis, mild dysplasia, moderate dysplasia and severe dysplasia) were divided into four groups, as compared with 39 untreated patients as a control. Poly E ointment was applied locally to 27 patients twice a week. For oral delivery, a 200 mg of poly E or EGCG capsule was taken orally every day for eight to 12 weeks. In the study, 20 out of 27 patients (74%) under poly E ointment therapy showed a response. Six out of eight patients under poly E ointment plus poly E capsule therapy (75%) showed a response, and three out of six patients (50%) under poly E capsule therapy showed a response. Six out of 10 patients (60%) under EGCG capsule therapy showed a response. Overall, a 69% response rate (35/51) was noted for treatment with green tea extracts, as compared with a 10% response rate (4/39) in untreated controls (P<0.05). Thus, the data collected here demonstrated that green tea extracts in a form of ointment and capsule are effective for treating cervical lesions, suggesting that green tea extracts can be a potential therapy regimen for patients with HPV infected cervical lesions.

Sterically stabilized anti-G(M3), anti-Le(x) immunoliposomes: targeting to B16BL6, HRT-18 cancer cells.

Various tumor-associated antigens have been identified as carbohydrates bound to lipids or to proteins expressed on tumor cell membranes. We prepared tumor-specific immunoliposomes by coupling anticarbohydrate antibodies, such as antiganglioside G(M3) antibody (DH2) or anti-Le(x) antibody (SH1), to polyethylene glycol (PEG)-coated liposomes. In vitro and in vivo targetability of anti-G(M3) and anti-Le(x) immunoliposomes to B16BL6 mouse melanoma cells and HRT-18 human colorectal adenocarcinoma cells were monitored with a fluorescence microscopy, and analyzed by biodistribution assay of the immunoliposome in mice bearing the tumor tissues. The antibody coupling to the PEG liposomes did not greatly diminish the circulation time of the liposome in the C57BL/6 mouse model. In vitro cytotoxicity of doxorubicin encapsulated in liposomes was enhanced by antibody coupling, but still behind free doxorubicin. However, in vivo antitumor therapeutic efficacy of doxorubicin encapsulated in the immunoliposomes was far greater than the free drug or in conventional liposomes. Doxorubicin encapsulated in anti-G(M3) immunoliposomes was able to reduce in vivo tumor growth and metastasis of B16BL6 mouse melanoma cells more greatly than any other formulations of the drug. This study suggests that tumor-associated antigens can be good target molecules for tumor-specific delivery of liposomal drugs or other synthetic drug delivery systems.

Pharmacodynamics of insulin in polyethylene glycol-coated liposomes.

To reduce the injection frequency and toxicity of intravenously administered protein drugs, it is necessary to develop safe and sustained injectable delivery systems. In this study, to evaluate liposomes as safe and sustained injectable delivery systems of proteins, we chose insulin as a model protein drug and tested its incorporation efficiency and pharmacodynamics in various liposomes with and without polyethylene glycol (PEG)-derivatized phospholipid. The liposomes coated with PEG showed 3-fold higher efficiency of insulin incorporation than did the liposomes without PEG. Moreover, among the liposomes coated with PEG, dipalmitoylphosphocholine (DPPC) liposomes showed higher incorporation efficiency than did dimyristoylphosphocholine (DMPC) liposomes. For pharmacodynamic study, insulin (2 IU/kg) was administered in various formulations, such as insulin alone in phosphate-buffered saline and insulin in the DPPC liposomes with and without PEG, to streptozotocin-treated diabetic rats. The pharmacodynamics of insulin alone, however, could not be measured due to the immediate death of rats caused by hypoglycemic shock. In contrast, all the rats treated with liposomal insulin survived, probably by the sustained release of insulin from liposomes. Pharmacodynamics of liposomal insulin showed that PEG-coated liposomes induced the lowest level of blood glucose-the nadir-1 h later than did the liposomes without PEG. These results indicate that PEG-coated liposomes could be developed as a relatively safe and sustained injectable delivery system for insulin with improved incorporation efficiency. Moreover, it is suggested that the liposomes coated with PEG might have a potential as safe injectable delivery systems for other protein and peptide drugs.

An easy technique for long-term central venous catheterization and subcutaneous tunneling of the silastic catheter in neonates and infants.

Venous access is a common problem in the care of seriously ill infants and neonates on pediatric and pediatric surgical services, and recently there has been a high increase in indications for central venous lines. With the development of many types of silastic catheters, a new safe and fast method of catheter insertion, subcutaneous tunneling and fixation has been developed. Our method is based on Yoffa's (1965)1 description. In the past we used infraclavicular subclavian venipuncture,2 but now we prefer to use the supraclavicular subclavian venipuncture as the first choice in children. Thirty-one catheters were placed in 27 consecutive patients ranging in age from two days to ten years. Perioperative complications were nil, and those catheterized for a long period of time had relatively few complications.

Staple line covering procedure after thoracoscopic bullectomy for the management of primary spontaneous pneumothorax.

BACKGROUND: Thoracoscopic bullectomy together with a pleural adhesive procedure is generally accepted as the standard for the definitive treatment of primary spontaneous pneumothorax (PSP). The purpose of this study was to evaluate whether the results of a thoracoscopic bullectomy followed by coverage of the staple line with cellulose mesh and fibrin glue could be comparable with those of adhesive procedures described in the literature. METHODS: Between May 2000 and February 2003, we performed 227 thoracoscopic surgeries on 219 patients with PSP using a single technique. After the bullectomy, the staple line was covered with cellulose mesh and fibrin glue. The postoperative status was evaluated with a mean follow-up of 46 months. RESULTS: The mean patient age was 24.3 years and 90.9 % of the 219 patients were male. Recurrent pneumothorax (37.4 %) was the most common operative indication, followed by persistent air leakage of more than 5 days (28.2 %). The mean duration of postoperative chest tube drainage was 1.6 days and the mean postoperative hospital stay was 3.8 days. Six patients experienced surgical complications (2.2 %); there was air leakage of more than 3 days in two cases, a small apical dead space in one case, a fever-associated wound problem in one case, and a reoperation due to air leakage of more than 7 days in two cases. Eleven patients (4.8 %) suffered a recurrence of pneumothorax during the follow-up period. Of these, nine cases required readmission and three (1.3 %) of these cases required a reoperation. CONCLUSIONS: Given the nature of a meticulous thoracoscopic bullectomy followed by coverage with cellulose mesh and fibrin glue, good surgical results can be expected without the need for a pleural adhesive procedure.

Gene expression profiles in squamous cell cervical carcinoma using array-based comparative genomic hybridization analysis.

Our aim was to identify novel genomic regions of interest and provide highly dynamic range information on correlation between squamous cell cervical carcinoma and its related gene expression patterns by a genome-wide array-based comparative genomic hybridization (array-CGH). We analyzed 15 cases of cervical cancer from KangNam St Mary's Hospital of the Catholic University of Korea. Microdissection assay was performed to obtain DNA samples from paraffin-embedded cervical tissues of cancer as well as of the adjacent normal tissues. The bacterial artificial chromosome (BAC) array used in this study consisted of 1440 human BACs and the space among the clones was 2.08 Mb. All the 15 cases of cervical cancer showed the differential changes of the cervical cancer-associated genetic alterations. The analysis limit of average gains and losses was 53%. A significant positive correlation was found in 8q24.3, 1p36.32, 3q27.1, 7p21.1, 11q13.1, and 3p14.2 changes through the cervical carcinogenesis. The regions of high level of gain were 1p36.33-1p36.32, 8q24.3, 16p13.3, 1p36.33, 3q27.1, and 7p21.1. And the regions of homozygous loss were 2q12.1, 22q11.21, 3p14.2, 6q24.3, 7p15.2, and 11q25. In the high level of gain regions, GSDMDC1, RECQL4, TP73, ABCF3, ALG3, HDAC9, ESRRA, and RPS6KA4 were significantly correlated with cervical cancer. The genes encoded by frequently lost clones were PTPRG, GRM7, ZDHHC3, EXOSC7, LRP1B, and NR3C2. Therefore, array-CGH analyses showed that specific genomic alterations were maintained in cervical cancer that were critical to the malignant phenotype and may give a chance to find out possible target genes present in the gained or lost clones.

Development of anticancer gene vaccine interact with human papillomavirus oncoprotein inhibition.

Adeno-associated virus (AAV) Rep 78 protein is known to inhibit the promoter site of several oncogenes and viral genes, including the human papillomavirus (HPV) type 16 E6 transforming genes. The biochemical studies of Rep 78 have been reported, but the effects of Rep 78 gene-mediated inhibition of HPV 16 E6 promoter activity on the various human cervical carcinoma cells have not been characterized. pEGFP-N1 vector, cloned by AAV-mediated Rep 78, is transfected into cervical carcinoma cells. Transfection efficiency of Rep 78 was approximately 30-60% different. Messenger RNA (mRNA) and protein expression of Rep 78 gene was significantly higher on day 1 of the transfection of Rep 78 DNA in CaSki cells, and DNA level of HPV 16 E6 was decreased on day 1 of the transfection. The growth of CaSki cervical cancer cells was only 10-15% inhibited by Rep 78, and the other cervical cells, HeLa, HeLaS3, HT3, and QGU, were unaffected by Rep 78 transfection. In spite of the high efficiency of Rep 78 gene transformation and expression rate, we could not show the significant growth inhibition in various cervical cancer cell lines. Taken together, long-term expression of Rep 78 strategy might be needed for cervical carcinoma gene therapy using AAV vector.

Distinctive cell cycle regulatory protein profiles by adenovirus delivery of p53 in human papillomavirus-associated cancer cells.

In this study, microarray analyses were performed to determine the time course of gene expression profiles in SiHa cells after infection with an adenovirus-expressing p53 (Adp53). We then investigated the consequences of Adp53 gene transfer on the expression level of six genes associated with cell cycle control and on apoptosis and cell cycle arrest in SiHa cells and compared these results with those from CaSki and HeLa cells. Gene expression profiling of the p53-targeted genes in SiHa cells revealed that p21, p53, and mdm2 protein expression was significantly upregulated at 24 and 48 h. Western blot results revealed that p21 and p53 expression levels had significantly increased after Adp53 infection. Cyclin-dependent kinase 4 levels were decreased 48 h after treatment in SiHa and CaSki cells. Proliferating cell nuclear antigen levels were unchanged after Adp53 infection. Only SiHa cells exhibited significant cell death. Cell cycle arrest at the G1 phase was induced in the SiHa and HeLa cells but was not induced at the G2/M and S phases in the CaSki cells. These data support the notion that the understanding of p53-dependent apoptosis and cell growth arrest could be applicable to advanced strategies in the development of preferential tumor cell-specific delivery.

Diagnostic approach using the expression profiling of the P53 tumor suppressor gene and its related proteins in ovarian epithelial tumors.

The initial aim of this study was to examine the expression profiles of P53 and its upstream genes, downstream genes, and cell cycle regulators to determine whether these markers are useful for making a differential diagnosis among the benign, borderline, and malignant ovarian epithelial tumors. Between borderline and malignant tumors, the increased expression levels of P53, Bax, Cyclin E, and cyclin-dependent kinase-2 as well as the decreased expression levels of growth arrest and DNA damage (GADD45) and murine double minute-2 (MDM2) were significantly associated with malignancy (P < 0.01, each). Using the receiver operating curve (ROC), the most reliable cutoff value of the added-up staining scores of those markers was 4.5 with 79% sensitivity and 89% specificity for malignancy. Between benign and borderline tumors, the P21 and Bax expression levels were significantly higher in borderline tumors, whereas the Bcl-2 expression level was much higher in benign tumors (P < 0.01, each). Using the ROC, the cutoff value of the added-up staining scores used to discriminate between the two groups was 2.5 with 70% sensitivity and 74% specificity for borderline tumors. Thus, for the differential diagnosis between borderline and malignant tumors, the cutoff value 4.5 of the cumulative staining scores can be used. However, the cutoff value 2.5 for discrimination between benign and borderline tumors may not be useful because of its relatively low sensitivity and specificity. In addition, the P53, GADD45, Cyclin E, and MDM2 expression levels in malignant ovarian tumors might be useful for determining the histologic grade and type.

Cellular process classification of human papillomavirus-16-positive SiHa cervical carcinoma cell using Gene Ontology.

This study utilized mRNA differential display and the Gene Ontology (GO) analysis to characterize the multiple interactions of a number of genes involved in human papillomavirus (HPV)-16-induced cervical carcinogenesis. We used HPV-16-positive cervical cancer cell line (SiHa) and normal human keratinocyte cell line (HaCaT) as a control. Each gene has several biological functions in the GO, and hence, we chosen the several functions for each gene. and then, the specific functions were correlated with gene expression patterns. The results showed that 157 genes were up- or down-regulated above two-fold and organized into mutually dependent subfunction sets depending on the cervical cancer pathway, suggesting the potentially significant genes of unknown function. The GO analysis suggested that cervical cancer cells underwent repression of cancer-specific cell-adhesive properties. Also, genes belonging to DNA metabolism such as DNA repair and replication were strongly down-regulated, whereas significant increases were shown in protein degradation and in protein synthesis. The GO analysis can overcome the complexity of the gene expression profile of the HPV-16-associated pathway and identify several cancer-specific cellular processes as well as genes of unknown function. Also, it can become a major competing platform for the genome-wide characterization of carcinogenesis.

Production of Poly(3-hydroxybutyrate) by fed-batch culture of recombinant Escherichia coli with a highly concentrated whey solution.

Fermentation strategies for the production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes were developed. The pH-stat fed-batch cultures of E. coli CGSC 4401 harboring pJC4, a stable plasmid containing the A. latus PHA biosynthesis genes, were carried out with a concentrated whey solution containing 280 g of lactose equivalent per liter. Final cell and PHB concentrations of 119.5 and 96.2 g/liter, respectively, were obtained in 37.5 h, which resulted in PHB productivity of 2.57 g/liter/h.

Coiling of lumbar epidural catheters.

BACKGROUND: The difficulties in threading an epidural catheter to vertebral levels remote to the puncture level have been well documented. This study was undertaken to determine the length that a single orifice epidural catheter can be threaded into the lumbar space without coiling (coiling length), and whether this is affected by the direction of the epidural needle bevel. METHODS: Forty-five young male patients scheduled for surgery under epidural analgesia were enrolled. The epidural space was identified using a midline approach at the L(2-3) or L(3-4) interspace with the loss of resistance to air technique. A 19-G single-orifice epidural catheter (Flextip Plus, Arrow International, Inc, Reading, PA, USA) was inserted through a Tuohy needle oriented either cephalad (n=20) or caudad (n=25). During insertion, the path and the position of the catheter tip was determined by fluoroscopy using iohexol dye. RESULTS: The median coiling length was 2.8 cm, ranging from 1.0 to 8.0 cm. Only 13% of epidural catheters could be threaded 4 cm beyond the tip of the needle without coiling. No significant difference was found in coiling length between the cephalad group (2.9 cm) and the caudad group (2.5 cm). CONCLUSION: This study demonstrates that coiling length is independent of whether the bevel of the Tuohy needle is directed cephalad or caudad. We recommend that an optimal insertion depth of an end-hole single orifice catheter is 3 cm.

The laryngeal mask airway in infants and children.

PURPOSE: To compare the effectiveness of various laryngeal mask airway (LMA) sizes and their performance during positive pressure ventilation (PPV) in paralyzed pediatric patients. METHODS: Pediatric patients (n = 158), < 30 kg, ASA 1 or 2 were studied. After paralysis, an LMA of the recommended size was inserted and connected to a volume ventilator. Fibreoptic bronchoscopy (FOB) was performed and graded: 1, larynx only seen; 2, larynx and epiglottis posterior surface seen; 3, larynx, and epiglottis tip or anterior surface seen--visual obstruction of epiglottis to larynx: < 50%; 4, epiglottis down-folded, and its anterior surface seen--visual obstruction of epiglottis to larynx: > 50%; 5, epiglottis down-folded and larynx not seen directly. Inspiratory and expiratory tidal volumes (V(T)), and airway pressure were measured by a pneumo-tachometer, and the fraction of leakage (F(L)) was calculated. In 79 cases, LMA was used for airway maintenance throughout surgery. RESULTS: Successful LMA placement was achieved in 98% of cases: three failures were due to gastric insufflation. For LMA # 1, 1.5, 2, and 2.5, FOB grades [median (range)] were 3(1-5), 3(1-5), 1(1-5) and 1(1-3) respectively. In smaller LMAs, the cuff more frequently enclosed the epiglottis (P < .001). F(L) of LMA # 1 was higher than those of LMA # 1.5 and LMA # 2.5 (P < .05), and F(L) of LMA # 2 was higher than that of LMA # 2.5 (P < .05). In the 79 patients, the number of patients experiencing complications decreased as LMA size increased (P < .05). CONCLUSION: Use of the LMA in smaller children results in more airway obstruction, higher ventilatory pressures, larger inspiratory leak, and more complications than in older children.

Cephalic spreading levels after volumetric caudal epidural injections in chronic low back pain.

The volumetric caudal epidural steroid injection has been advocated to facilitate the delivery of medications to the lesion site. This study was aimed to examine the actual spreading patterns of this technique, using epidurogram. A total of 32 patients with chronic low back pain accompanied by radiculopathy of various causes (degenerative spondylosis, herniated nucleus pulposus, spondylolisthesis, and spinal stenosis) were included. The volumetric caudal epidural injection of the 10 mL mixture of contrast medium 5 mL, 0.5% bupivacaine 1 mL, triamcinolone 1.5 mL (60 mg) and normal saline 25 mL was performed. Immediately after the cessation of the first spread, the subsequent solution of another 10 mL of contrast medium 5 mL, 0.5% bupivacaine 1 mL and normal saline 4 mL was injected. This procedure was repeated serially until the total volume to be 50 mL. Continuous fluoroscopic imaging was obtained after each injection. Average time taken to complete the study was 37 sec per every 10 mL. The spreading levels of the mixture were distributed mainly at mid to lower lumbar area in the majority of the patients. During the subsequent injections, the levels were not changed significantly. This was thought to be due to the minimal resistance in cephalad direction, anatomic variations and Starling effect of epidural space.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by metabolically engineered Escherichia coli strains.

The recombinant Escherichia coli strain, equipped with the newly cloned Aeromonas PHA biosynthesis genes, could produce a terpolymer of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB-co-3HV-co-3HHx)] from dodecanoic acid plus odd carbon number fatty acid. In addition, the orf1 gene of Aeromonas hydrophila was found to play a critical role in assimilating the 3HV monomer and in regulating the monomer fraction in the terpolymer.

The effect of introducer gauge, design and bevel direction on the deflection of spinal needles.

This study was performed to determine how the use of an introducer affects the extent to which a needle deflects during a spinal or combined spinal-epidural injection. A polystyrene block was used to simulate the paraspinal area of the back. A line was drawn perpendicular to the edge of the block to use as a guide and to measure the deflection. The use of an introducer needle decreased the deflection in all the bevelled needles (p < 0.001). Depending on the direction of both the bevels, the deflection decreased as the introducer bevel was changed from the same direction, to right-angles to bevel direction and then to a direction opposite to that of the spinal needle (p < 0.05). Deflection was decreased when a thick introducer was used (p < 0.001). The use of an introducer increased the deflection of the pencil-point needle only in the deflection direction of the introducer (p < 0.001). The 18-gauge Tuohy needle with a "backhole" deflected more than the corresponding needle without a backhole (p < 0.001), and the spinal needle inserted through the Tuohy needle with a backhole deflected more (p = 0.002). Besides the tip type and gauge, the deflection of a spinal needle depends upon the use of introducer, its gauge and bevel direction. The deflection of a Tuohy needle depends upon its design, gauge and the presence of a backhole.

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by metabolically engineered Escherichia coli strains.

Recombinant Escherichia coli strains harboring a plasmid containing a novel artificial polyhydroxyalkanoate (PHA) operon consisting of the Aeromonas PHA biosynthesis related genes and Ralstonia eutropha reductase gene were developed for the production of poly(3-hydroxybutyrate-co-hydroxyhexanoate) [P(3HB-co-3HHx)] from dodecanoic acid. By applying stepwise reduction of dissolved oxygen concentration (DOC) during the fermentation, the final dry cell weight, PHA concentration, and PHA content of 79 g/L, 21.5 g/L, and 27.2 wt %, respectively, were obtained in 40.8 h, which resulted in the PHA productivity of 0.53 (g/L)/h. The 3HHx fraction slowly increased during the fed-batch culture to reach a final value of 10.8 mol %. The 3HHx fraction in the copolymer could be increased by 3-fold when the Aeromonas hydrophila orf1 gene was coexpressed with the PHA biosynthesis genes.