Association of lysosomal activity with sensitivity and resistance to tumor necrosis factor in murine L929 cells.

The cytotoxic mechanism of action of tumor necrosis factor (TNF) was examined using murine L929 fibrosarcoma cells in vitro. Two cell lines were evaluated: parental TNF sensitive (L929S) (50% cytotoxic concentration, 2-6 ng/ml); and TNF resistant (L929R) (50% cytotoxic concentration, greater than 10,000 ng/ml). The latter resistant cell line was developed by serial passage in increasing concentrations of recombinant human TNF. Sensitive cells demonstrated cytolytic and cytostatic effects at TNF concentrations between 2 and 6 ng/ml, respectively. However, TNF failed to show any selective depression of RNA, DNA, or protein synthesis or ATP content in these cells until general cell death was apparent, as defined by the cell rounding and lifting off the plastic surface. The cytokine also failed to cause DNA single-strand breaks, as detected by alkaline elution techniques. TNF was also found to be no more active in glutathione-depleted cells than in target cells containing normal glutathione levels. In contrast, various nonspecific lysosomotropic agents such as ammonium chloride and D-saccharic acid lactone led to a marked inhibition of the cytotoxic action of TNF in vitro. Furthermore, significant differences in lysosomal enzyme activity were noted between L929S and L929R cells. The changes in L929R cells involved a 50% reduction in total lysosomal protein levels and a marked depression of beta-glucuronidase activity. In contrast, L929R lysosomal hexosaminidase activity was significantly elevated over the L929S cells. From these studies it is concluded that the antitumor activity of TNF does not involve specific inhibition of macromolecular synthesis, ATP production, or the level of reduced thiols. Instead, TNF cytotoxicity appears to require functional lysosomes, which are altered when TNF resistance develops in vitro.

Analysis of antigenic expression by primary and autologous metastatic human sarcomas using murine monoclonal antibodies.

The efficacy of monoclonal antibody therapy depends in part on the expression of the relevant tumor-associated antigens by both primary tumors and their metastases. Antigen expression by paired primary and autologous metastases from surgically excised osteogenic and soft-tissue sarcomas from 15 patients was studied using a panel of murine hybridoma monoclonal antibodies and indirect immunoperoxidase staining of formalin-fixed tissue sections. The panel included three antibodies (B3619, 17-9H3, OST6) recognizing sarcoma-associated antigens and an antibody recognizing an HLA-DR framework determinant (OKla1). In most cases, antibody binding to both primary and metastatic tumors was observed. However, marked heterogeneity of binding intensity between primary and metastatic tumors and of cells expressing antigens within tumors was noted. This occurred even though primary and metastatic tumors demonstrated homogeneous histology and cellular morphology. Differences were noted among patients as well as among metastases taken from an individual. A significant number of both primary and metastatic tumors contained cells that did not bind a particular antibody even in the presence of other cells that demonstrated significant antibody binding. Thus, strategies for single monoclonal antibody therapy may be limited by heterogeneity of intertumor and intratumor antigenic expression.

Antineoplastic effects of tumor necrosis factor alone and in combination with gamma-interferon on tumor biopsies in clonogenic assay.

Tumor colony-forming cells were grown from fresh biopsy specimens from 102 patients with a variety of nonhematologic malignant neoplasms and exposed in vitro to pharmacologically achievable doses of recombinant human tumor necrosis factor (rTNF). In 68 instances, the tumor specimens were also tested against recombinant human gamma-interferon (rIFN-gamma), as well as the combination of rTNF and rIFN-gamma. rTNF exhibited dose-dependent and tumor-type-dependent antitumor effects. Sensitivity to rTNF at doses of less than 100 U was observed in 28% of the tumors tested. A higher than average frequency of sensitivity was observed in colorectal and lung cancer. Resistance to rTNF was observed in 42% of the tumors, including 52% of the ovarian cancer specimens tested. In paired experiments, exposure of tumor specimens to rTNF and rIFN-gamma in combination often resulted in a greater antitumor effect than was observed with either agent alone, with at least subadditive effects seen in 62% of the specimens tested against the combination. Antagonism between rTNF and rIFN-gamma was observed in 18% of the studies. Overall, exposure to the combination of rTNF and rIFN-gamma reduced the dose of rTNF required for significant antitumor activity by about threefold. Normal bone marrow granulocyte-macrophage colony-forming cells were also tested against both rTNF and rIFN-gamma and the combination. The bone marrow progenitors were more sensitive to rTNF and the combination with rIFN-gamma than were the tumor cells; however, the significance of this comparison between two different in vitro assay systems is indeterminate. Based on our observations, rTNF warrants phase II clinical trials in selected solid tumors with definite emphasis on colorectal and lung cancer. Additionally, studies of the combination of rTNF and rIFN-gamma are indicated and will be of particular interest in endometrial and breast cancer.

Beta-carotene stimulates human leukocytes to secrete a novel cytokine.

The effect of beta-carotene on cytokine production by human peripheral blood leukocytes was tested. Beta-carotene stimulated the secretion of a novel cytotoxic cytokine when peripheral blood cells were exposed to carotenoid concentrations between 10(-6) and 10(-10) M. Beta-carotene-treated supernatants caused the cytolysis of four out of the six human tumor cell lines tested. Low level toxicity was also observed when normal diploid fibroblast lines were exposed to beta-carotene-treated leukocyte supernatants. The cytotoxic activity elicited by beta-carotene was found to be distinct from characterized cytokines based on both antisera neutralization and target cell specificity studies. This study demonstrates that beta-carotene can induce human leukocytes to secrete one or more cytokines that can manifest cytotoxic activity against human tumor cells in vitro.

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppress tumor necrosis factor secretion in vitro.

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppressed peripheral blood leukocyte secretion of tumor necrosis factor (TNF). The suppressive activities of all three fluids had similar characteristics: each was heat and acid stable, removed by adsorption on immobilized lectins, and abrogated the stimulatory effect of interferon-gamma. Inhibition of leukocyte TNF secretion was observed only when either serum or conditioned medium was added to leukocytes at the initiation of culture; delaying the addition by 2 h failed to suppress cytokine secretion. Suppression by all fluids was also found to be reversible by washing cells free of suppressive activity. Although serum, tumor, and fibroblast culture supernatants inhibited cytokine secretion, they failed to alter the cytotoxic activity of recombinant human TNF on murine L929 cells. This study suggests that factors which can inhibit TNF secretion are present in human blood and are secreted by both fibroblasts and tumor cells. These suppressive factors may play an important role in the regulation of TNF secretion and cytokine homeostasis.

Production of tumor necrosis factor alpha by resident and activated murine macrophages.

Alveolar macrophages (Am phi s), resident peritoneal macrophages (RPm phi s), and thioglycolate-elicited peritoneal macrophages (TGPm phi s) were isolated from C57BL/6 mice and incubated with lipopolysaccharide (LPS), stimulated cell supernatant, or recombinant interferon gamma (IFN-gamma) for 24 h. Tumor necrosis factor (TNF) in cell-free supernatants was measured by enzyme-linked immunosorbent assay. Amo phi s incubated with 10(3) ng/ml LPS produced 50 times more TNF than RPm phi s and 5 times more than TGPm phi s, and LPS alone induced maximum TNF production by Am phi s. Stimulated cell supernatant or recombinant IFN-gamma alone did not induce TNF production. A combination of LPS with stimulated cell supernatant or IFN-gamma had only a limited synergistic effect on TNF production by Am phi s. However, both LPS and stimulated cell supernatant or recombinant IFN-gamma induced maximum TNF production by RPm phi s and TGPm phi s. TGPm phi s showed greater sensitivity to LPS and stimulated cell supernatant or IFN-gamma with regard to TNF production than the other macrophage populations investigated.

Serum levels of tumor necrosis factor and IL-1 alpha and IL-1 beta in diabetic patients.

OBJECTIVE: To determine whether chronic hyperglycemia causes increased levels of serum tumor necrosis factor (TNF) and interleukin 1 alpha (IL-1 alpha) and IL-1 beta. RESEARCH DESIGN AND METHODS: Sera were obtained from 59 diabetic patients, 44 chronically ill nondiabetic patients, and 34 age-matched healthy control subjects. Mononuclear cells were isolated from a subgroup of diabetic patients and healthy control subjects. RESULTS: Except for a modest increase in the prevalence of detectable serum TNF levels in diabetic patients, the serum cytokines measured in this study did not appear to be altered in diabetes. In vitro TNF production by mononuclear cells was not altered in diabetic patients. However, in vitro IL-1 beta secretion, in response to lipopolysaccharides, was reduced. CONCLUSIONS: Diabetes mellitus is not associated with significant changes in serum levels of TNF, IL-1 alpha, or IL-1 beta. In vitro secretion of IL-1 beta in response to lipopolysaccharides may be reduced in diabetes.

Decreased serum triiodothyronine is associated with increased concentrations of tumor necrosis factor.

Previous studies in laboratory animals have shown that tumor necrosis factor-alpha (TNF) may alter thyroid function tests. To determine whether elevated serum TNF levels are associated with altered serum concentrations of T4, T3, free T4, rT3, and TSH, we measured these parameters in 29 nursing home residents with detectable serum TNF levels and compared the levels to those found in 36 patients with undetectable serum TNF levels. The 2 groups were matched for age, sex, clinical problems, use of medications, and nutritional status. Patients with detectable serum TNF levels had significantly lower serum T3 concentrations compared to those with undetectable levels [1.072 +/- 0.588 vs. 1.621 +/- 0.594 nmol/L (mean +/- SD); P less than 0.01]. Differences in other tests did not achieve statistical significance. Thyroid function tests were not significantly different when patients with detectable interleukin-1 alpha levels, another cytokine secreted during endotoxemia, were compared to those with undetectable levels. These observations taken together with the previous findings in laboratory animals suggest that some of the alterations in thyroid hormone levels seen in nonthyroidal illness are associated with elevated serum concentrations of TNF.

Modification of the clonogenic assay for the detection of lymphokine activated killer cell activity.

The soft agar clonogenic assay using [3H]thymidine uptake as an endpoint was adapted for the detection of human LAK activity against the Daudi lymphoma cell line and human tumor cells obtained by biopsy. Using Daudi cells as the target population the modified agar assay was more sensitive than the conventional 4 h 51Cr release assay. Use of a single layer agar assay allowed the assessment of LAK cytotoxic/cytostatic activity against Daudi lymphoma cells after cell to cell contact, while a two layer system permitted evaluation of the role of soluble mediators in LAK/target cell interactions. This study shows that LAK cells can either kill or inhibit the proliferation of Daudi cells by two mechanisms: one which requires cell-to-cell contact, and a second via soluble mediators. As determined by the use of neutralizing antisera, the soluble factor(s) are not tumor necrosis factor and interferon-gamma. Of the nine individual human tumor samples obtained by biopsy. 89% were sensitive to allogeneic LAK cells when the two populations were admixed. Of these nine tumors 44% were inhibited by LAK-derived soluble factors. The soft agar assay system should serve as a useful tool for determining the sensitivity of human tumors to LAK cells and for studying the mechanisms of LAK anti-tumor activity.

The incidence and effect on outcome of hypoxemia in hospitalized medical patients.

The effect on survival of episodic hypoxemia was prospectively studied in 100 patients hospitalized on general medicine services of a large, tertiary care, university hospital. Pulse oximetry monitoring (POM) was initiated within 24 hours of admission and was maintained for approximately 24 hours independent of patient management. Hypoxemia lasting for at least 5 consecutive minutes and resulting in an arterial oxygen saturation of less than 90% occurred in 26 of the 100 patients. No clinical characteristics were found that could reliably distinguish those patients who did develop hypoxemia from those who did not, though the small number of patients in many categories precludes drawing firm conclusions. However, severe desaturation was unlikely to occur in patients with normal chest roentgenograms. During the following 4 to 7 months, 8 patients (32%) suffering episodic hypoxemia died, while only 7 individuals (10%) without hypoxemia died, an increase in mortality that remained significant after adjustment for severity of illness. The relative risk of death associated with desaturation was 3.3 (95% CI 1.41 to 8.2). The severity of hemoglobin oxygen desaturation, expressed as the saturation-time index, correlated inversely with survival time.

Infected radial artery pseudoaneurysms occurring after percutaneous cannulation.

During a ten-month period in 1988 at our institution, we identified three infected radial artery pseudoaneurysms (RAPAs) associated with arterial lines. A retrospective chart review to 1983 identified three additional cases, all occurring in 1986. In the six-year period of 1983 through 1988, during which approximately 12,500 radial artery catheters were placed, the incidence of RAPA formation was 6/12,500 (0.048 percent). Five of the six cases were associated with Staphylococcus aureus infection. The duration of radial artery cannulation was significantly longer in patients who developed a pseudoaneurysm (12.5 days) than in those patients who did not suffer this complication (4.3 days). Patients in whom infected RAPAs occurred also tended to be older (mean, 71.6 years) than the average age (54 years) for all patients admitted to the intensive care unit (ICU). They also tended to have long stays in the ICU prior to development of RAPA, the shortest stay being 11 days and the average being 51 days. Risk factors for the development of this complication may include advanced age, longer duration of catheterization and hospitalization, and infection with S aureus.

A comparison of three pulmonary artery oximetry catheters in intensive care unit patients.

OBJECTIVE: To compare the clinical performance of three pulmonary artery oximetry catheters (Oximetrix 3, SAT-2, and HEMOPRO2) in intensive care unit (ICU) patients. DESIGN: Unblinded comparison of performance over 24 h using an IL-282 CO-oximeter as a criterion standard. SETTING: Multispecialty adult ICU at a university teaching hospital. PATIENTS: Thirty critically ill patients selected from those requiring hemodynamic monitoring for medical management. MEASUREMENTS AND MAIN RESULTS: By all measures, performance of the Oximetrix 3 and SAT-2 systems were comparable; bias +/- precision were -1.98 +/- 3.07 and +1.80 +/- 3.49, respectively, vs -2.28 +/- 5.24 for the HEMOPRO2. The Oximetrix 3 and SAT-2 systems demonstrated consistent performance over the range of saturations tested, though Oximetrix 3 tended to underestimate and SAT-2 tended to overestimate the CO-oximeter value. The HEMOPRO2 underestimated the CO-oximetry-derived saturation, although this was not constant across the range of values tested. The 95 percent confidence limits based on intrasubject variability were similar (+/- 4.59, +/- 5.66, and +/- 6.56 for the Oximetrix 3, SAT-2, and HEMOPRO2, respectively); however, the 95 percent confidence limits based on total variability, while similar for Oximetrix 3 (+/- 6.03) and SAT-2 (+/- 6.86), were larger for the HEMOPRO2 (+/- 10.30). The expected SD was similar for the three systems (2.03, 2.50, and 2.90 for the Oximetrix 3, SAT-2, and HEMOPRO2 systems, respectively). None of the systems equaled or exceeded (p greater than 0.05) the manufacturers' published specifications, which, in all cases, are listed as +/- 2 percent (saturation; 1 SD) when compared with bench oximetry. CONCLUSIONS: Although each system measures mixed venous oxygen saturation, the Oximetrix 3 and SAT-2 systems demonstrate closer agreement with CO-oximetry. However, none of these catheters provided statistically significant evidence that they would perform within +/- 2 percent of CO-oximetry. As a continuous monitor used to detect changes or trends, any of the three may be acceptable.

Detectable serum levels of tumor necrosis factor alpha may predict early mortality in elderly institutionalized patients.

OBJECTIVE: To determine if detectable serum tumor necrosis factor alpha (TNF) levels are associated with higher mortality in nursing home residents. SUBJECTS AND METHODS: The basal serum concentrations of TNF and interleukin-1 alpha (IL-1) were measured in 129 elderly nursing home patients (mean age of 89 years), and survival in the cohort was monitored over a 13-month period. RESULTS: At 4 months follow-up, seven out of 33 patients with detectable serum TNF levels had died (21.2%), and only three out of 96 patients with undetectable serum TNF levels had died (3.1%) (P less than 0.001). The difference in mortality remained significant up to 13 months of follow-up (P less than 0.05). Those with detectable serum TNF levels and those with undetectable levels were comparable in age, body mass index, hematocrit, lymphocyte counts, and serum level of albumin, prealbumin, and retinol-binding protein. When patients with detectable serum IL-1 levels were compared to those with undetectable levels, there were no significant differences in mortality over a 13-month period. CONCLUSION: Detectable serum TNF levels in elderly nursing home patients may be a predictor of early mortality.

Lack of an association between the presence of tumor necrosis factor or interleukin-1 alpha in the blood and weight loss among elderly patients.

To determine the association between blood cytokine levels and body weight loss in elderly patients, serum levels of cachectin/tumor necrosis factor alpha (TNF) and interleukin-1 alpha (IL-1) were measured with specific and sensitive ELISA systems. Of the 19 healthy young subjects, two (10.5%) had detectable levels of serum TNF and one (5.3%) was positive for IL-1. In the healthy elderly group, two of the 12 subjects (16.7%) had measurable TNF levels and four (33.3%) had elevated IL-1 levels. Of the 61 ambulatory elderly patients, 31.1% had serum that contained TNF and 22.9% had IL-1. Similar proportions were found in 127 nursing home patients. None of the common diseases examined in this study nor any commonly used medications were associated with increased serum cytokine levels. Patients with weight loss of more than 5 lbs were less likely to have elevated serum TNF and IL-1 levels compared to the rest of the group. It is concluded that although elevated levels of TNF or IL-1 may occur more frequently in older groups, there is no evidence for a causal relationship between these circulating cytokines and clinically significant weight loss in the elderly.

The risk of peripheral vein phlebitis associated with chlorhexidine-coated catheters: a randomized, double-blind trial.

OBJECTIVE: To evaluate the risk of phlebitis associated with chlorhexidine-coated polyurethane catheters in peripheral veins. DESIGN: A randomized, double-blinded trial comparing chlorhexidine-coated polyurethane catheters with uncoated polyurethane catheters. SETTING: A university hospital. PATIENTS: Adult medicine and surgery patients. INTERVENTIONS: Certified registered nurse anesthetists or an infusion team consisting of nurses and physicians inserted the catheters. Catheter insertion sites were scored twice daily for evidence of phlebitis. At the time catheters were removed, a quantitative blood culture was performed, and catheters were sonicated for quantitative culture. RESULTS: Of 221 evaluable catheters, phlebitis developed in 18 (17%) of 105 coated catheters, compared to 27 (23%) of 116 uncoated catheters (relative risk [RR], 0.74; 95% confidence interval [CI95], 0.43-1.26; P = .32). By survival analysis, chlorhexidine-coated catheters had a lower risk of phlebitis during the first 3 days (P = .06), but not when all catheters were considered in both patient groups (P = .31). In the absence of catheter colonization, the incidence of phlebitis was 21% (16/76) and 24% (20/86) for coated and uncoated catheters, respectively (P = .85), whereas in the presence of catheter colonization, the incidence of phlebitis was 14% (1/7) and 80% (4/5) for coated and uncoated catheters, respectively (RR, 0.18; CI95, 0.03-1.15; P = .07). CONCLUSION: The risk of phlebitis in the presence of catheter colonization was 82% lower for chlorhexidine-coated polyurethane catheters compared to otherwise identical uncoated catheters.

A novel approach to production of antitumor monoclonal antibodies: antibody to a cell surface glycoprotein associated with transformation by a human oncogene.

Transfection is a technique for inducing transformation of normal fibroblasts (NIH 3T3) with DNA (oncogenes) from human tumors. Our goal was to determine if these transformed cells expressed antigens associated with malignancy. NIH 3T3 cells were transfected with DNA fragments from a human acute lymphocytic leukemia (ALL 1-69), and transformed colonies were selected for growth in soft agar. Transfected cells containing human DNA sequences demonstrated by Southern blot analysis were used to immunize Balb/C mice. Monoclonal antibodies were produced and screened for binding to the parental leukemia (ALL 1-69), transfectant (17(2], and 3T3 cells in an enzyme-linked assay. A monoclonal antibody (IgM kappa) designated 17-9H3 bound to ALL 1-69 and secondary transfectant 17(2) but not to NIH 3T3 plasma membranes. Immunoperoxidase staining confirmed this binding pattern and demonstrated that the antigen was expressed on the cell surface. Expression of the antigen by transfectants directly correlated with the presence of a single 6.1 kilobase human DNA sequence. The antibody binding site of the antigen was inactivated by trypsin, glucosidase, and hyaluronidase. Binding of the 17-9H3 antibody was selective for acute lymphocytic leukemias (5/8) and osteogenic sarcomas (33/36), although other tumor types did demonstrate significant binding by immunoperoxidase staining. The majority of normal tissues did not bind 17-9H3 with the exception of some metabolically active cells (renal tubular epithelium, secretory epithelial cells, and cardiac smooth muscle), germ cells, Leydig cells of the testes, and some lymphoid cells. Monoclonal antibodies to oncogene-associated antigens may be potentially useful for cancer diagnosis and therapy and as probes for oncogene isolation.

Binding and biological effects of tumor necrosis factor and gamma interferon in human pancreatic carcinoma cells.

The cytotoxic/cytostatic effects of recombinant human tumor necrosis factor alpha (rhTNF) and gamma interferon (rhIFN-gamma) were studied in five human pancreatic tumor cell lines. During a 48-h incubation, MIA PaCa-2 cells were most sensitive to rhTNF (56% cytotoxicity, 500 U/ml), T3M4 cells were most sensitive to rhIFN-gamma (54% cytostasis, 250 U/ml), and ASPC-1 and COLO 357 cells were most sensitive to the combination of rhTNF and rhIFN-gamma (56 and 55% cytotoxicity, respectively, 250 U/ml of each cytokine). The PANC-1 cells were relatively insensitive to either the individual or the combined effects of these cytokines. All five cell lines exhibited specific, high-affinity receptors for 125I-labeled rhTNF (480-8,610 sites/cell) and rhIFN-gamma (2,050-6,280 sites/cell). The MIA PaCa-2 cells, which were the most sensitive to the inhibitory effects of rhTNF, also possessed the largest number of 125I rhTNF receptors; all other cell lines had a relatively low number of binding sites and low sensitivity. In contrast, no direct correlation could be made between the number of IFN-gamma binding sites and inhibitory sensitivity in any of the cell lines. Incubation of COLO 357 cells at 37 degrees C with either 125I rhTNF or 125I rhINF-gamma led to internalization of the respective 125I-labeled ligand. Our findings document the presence of cytokine receptors in human pancreatic carcinoma cells and suggest that postreceptor events rather than differences in receptor number or affinity more likely govern the responsiveness of pancreatic cancer cells to TNF and IFN-gamma.

Circulating levels of tumor necrosis factor and interleukin-1 in cystic fibrosis.

To assess the role of tumor necrosis factor (TNF) and interleukin-1 (IL-1) in the pathophysiology of cystic fibrosis (CF)-associated growth failure/cachexia and lung disease we measured height, weight, triceps skin fold, forced vital capacity, forced expiratory volume in 1 second, and plasma levels of TNF, interleukin-1-alpha (IL-1 alpha), interleukin-1-beta (IL-1 beta), and alpha-1-antitrypsin (A1AT) in 12 patients with CF, and in 12 age- and gender-matched healthy controls. The patients as a group had significantly lower values for the anthropomorphic measurements and lung function parameters as compared to controls. They also had higher circulating levels of A1AT than controls. TNF, however, was detected less frequently in patients than in controls. Neither group had detectable levels of circulating IL-1 alpha or IL-1 beta, which is consistent with the observation that CF patients infrequently present with fever. Potential explanations for these findings include compartmentalization of secreted TNF/IL-1, altered regulation of TNF/IL-1 secretion as a result of the chronic inflammatory state seen in CF, or increased degradation of TNF/IL-1, also a result of chronic inflammation. The role of these cytokines in the pathophysiology of CF remains unclear, but should be explored further; however it seems unlikely that circulating TNF plays a role in the growth failure/cachexia associated with CF.

Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A.

While the gene delivery vehicle is critical for the efficacy of human factor VIII gene therapy, optimization of the potency and duration of the factor VIII gene that is delivered is equally important in light of the poor transcription and translation characteristics of this gene. We discuss here a systematic approach to optimization of factor VIII complementary DNA expression by analysis of specific elements engineered into the transcription unit and other positions in the expression plasmid. Within the transcription unit we have engineered different 5' and 3' sequence modifications and tested them for factor VIII expression in human liver cells. These changes incorporate liver-specific promoter and enhancer sequences and regulatory elements affecting RNA export. Specifically, the thyroid hormone-binding globulin promoter and alpha 1 microglobulin/bikunin enhancer were tested and a synthetic 5' intron was compared to a 3' post-transcriptional regulatory element on factor VIII expression levels. For translation optimization, a leader sequence was designed to be of optimum length, have no RNA secondary structure and contain the optimal translation initiation sequence. Finally, we discuss areas for plasmid optimization, which include removal of near-consensus splicing sequences, the inclusion of strong transcription termination elements and the use of autonomous replicating plasmid sequences for episomal maintenance and enhanced plasmid retention for duration of gene expression.

Hemodynamic status following regional and general anesthesia for carotid endarterectomy.

We prospectively studied 23 patients undergoing carotid endarterectomy under regional (n = 13) or general (n = 10) anesthesia to determine the hemodynamic basis of increased frequency in the need for postoperative vasopressor support when regional anesthesia was used. Anesthesia and postoperative care were conducted without reference to hemodynamic data from pulmonary artery catheterization. Although mean arterial pressure was similar in the two groups postoperatively, 11 of the 13 patients undergoing regional anesthesia and 3 of the 10 patients undergoing general anesthesia required phenylephrine postoperatively. No patient required therapy postoperatively to reduce a systolic pressure exceeding 160 mm Hg. Mean arterial pressure remained below the preoperative baseline value in both groups (p < 0.05 with general anesthesia; p = 0.06 with regional anesthesia) during follow-up. In the general anesthesia group, systemic vascular resistance declined significantly below baseline (p < 0.05) following the operation, accompanied by a decline in mean arterial pressure (p < 0.05) and a higher cardiac output. Intraoperative fluid requirements were greater during general anesthesia than during regional anesthesia (p < 0.01). Pulmonary artery occlusion pressure was lower postoperatively than at baseline in both groups (p < 0.05). Pulmonary artery occlusion pressure was higher in the general anesthesia group despite the greater use of phenylephrine in the regional anesthesia group.