Effects of ropivacaine, bupivacaine and sufentanil in colon and pancreatic cancer cells in vitro.

The perioperative period is supposed to be a vulnerable period for cancer progression. Results of clinical studies indicate that the use of regional anesthesia can influence and improve oncological outcome of cancer patients. Uncontrolled cell proliferation and resistance to apoptotic cell death are important characteristics of solid tumors. The aim of this study was to investigate the effects of the clinically used local anesthetics ropivacaine or bupivacaine and the opioid analgesic sufentanil on cell proliferation, cell cycle distribution and apoptosis of colon (HT 29 and SW 480) and pancreatic (PaTu 8988t and PANC 1) cancer cell lines in vitro. Cell proliferation was measured by Cell Proliferation ELISA BrdU Assay. Apoptosis was analyzed by annexin V staining and cell cycle distribution was detected by flow cytometry. Ropivacaine, bupivacaine and sufentanil did not change apoptosis rate and cell cycle distribution in clinically concentration. Only high concentrations of ropivacaine or bupivacaine revealed antiproliferative potency. Protective effects of epidural anesthesia observed in clinical studies seem not to be based on direct effects of these drugs on cancer cells.

Effect of a novel nonviral gene delivery of BMP-2 on bone healing.

BACKGROUND: Gene therapeutic drug delivery approaches have been introduced to improve the efficiency of growth factors at the site of interest. This study investigated the efficacy and safety of a new nonviral copolymer-protected gene vector (COPROG) for the stimulation of bone healing. METHODS: In vitro, rat osteoblasts were transfected with COPROG + luciferase plasmid or COPROG + hBMP-2 plasmid. In vivo, rat tibial fractures were intramedullary stabilized with uncoated versus COPROG+hBMP-2-plasmid-coated titanium K-wires. The tibiae were prepared for biomechanical and histological analyses at days 28 and 42 and for transfection/safety study at days 2, 4, 7, 28, and 42. RESULTS: In vitro results showed luciferase expression until day 21, and hBMP-2-protein was measured from day 2 - day 10. In vivo, the local application of hBMP-2-plasmid showed a significantly higher maximum load after 42 days compared to that in the control. The histomorphometric analysis revealed a significantly less mineralized periosteal callus area in the BMP-2 group compared to the control at day 28. The rt-PCR showed no systemic biodistribution of luciferase RNA. CONCLUSION: A positive effect on fracture healing by nonviral BMP-2 plasmid application from COPROG-coated implants could be shown in this study; however, the effect of the vector may be improved with higher plasmid concentrations. Transfection showed no biodistribution to distant organs and was considered to be safe.

The use of ileocecal pouch with appendix as an urethral substitute in patients who are willing to have a orthotopic bladder replacement - point of technique.

INTRODUCTION: The authors describe the technique of orthotopic bladder replacement with an ileocecal pouch and unaltered appendix used as an orthotopic urethral substitute. Additional procedures with regard to the bothersome voiding symptoms will be described. MATERIAL AND METHODS: In a small cohort of 5 patients with muscle invasive bladder cancer with tumor involvement of the bladder neck or proximal urethra (2 males/3 females) we performed the following reconstruction. A low pressure reservoir was achieved by antimesenteric longitudinal transection of terminal ileum and cecum/colon ascendens and formation of a pouch. To develop the neourethra, the appendix together with it is accompanying mesentery was drawn through the pelvic floor and sutured to the bulbar urethra in males or formed as a complete neourethra in female patients respectively. RESULTS: There were no intraoperative nor early postoperative unwanted sequelae. Both male patients experienced recurrent anastomotic urethral stricture, consequently a Memokath stent and artificial urinary sphincter was implanted resulting in normal voluntary micturition. All female patients remained socially continent during the follow up period, one of them performing (clean intermittent catheterization) CIC. CONCLUSION: The technique described offers the possibility of orthotopic bladder replacement even in traditionally unsuitable, but highly motivated patients, who are requesting orthotopic bladder replacement for improved body image. It allows extension of urethral resection and provides additional continence support. However, additional measures such as urethral stenting, CIC or artificial urinary sphincter implantation may be necessary for long lasting success. Although, not being a routine method for urinary diversion this technique may be used in select patients.

Early weight gain and outcome in Henoch-Schönlein nephritis.

BACKGROUND: Intrauterine growth restriction seems to be a risk factor for an aggravated course of secondary renal diseases in children. Catch-up growth after birth may play a critical role. We tested if there is an association between an aggravated course of nephritis in Henoch-Schönlein Purpura (PSHN) and low birth weight or early weight gain during infancy. PATIENTS: We retrospectively analysed the clinical course of 34 children with PSHN. METHODS: Patients were sorted according their birth weight standard deviation score (SDS) in tertiles. Early weight gain was defined as gain of weight standard deviation score >0.67 between birth and 2 years of age. RESULTS: Patients with higher birth weight needed Cyclophosphamide in a higher rate than low birth weight children. In the high weight gain group (SDS gain >0.67) 9 of the 11 patients compared to 7 of 22 patients in the low weight gain group (SDS gain <0.67) presented with arterial hypertension during the initial manifestation of PSH nephritis (p=0.01). Median systolic blood pressure SDS in the high weight gain group was 1.54 (-1.39-4.71) versus 0.29 (0.52-4.05) in the low weight gain group (p=0.008). Nevertheless, other clinical parameters during first manifestation and follow-up were not relevantly different. CONCLUSION: In contrast to the data of children with idiopathic nephrotic syndrome or IgA nephropathy, this study does neither provide evidence for an association between low birth weight nor early weight gain and the later course of PSHN. Interestingly, early weight gain was associated with a higher systolic blood pressure during the initial manifestation of PSHN.

[Benefits and risks of tube feeding via gastrostoma in infants and children with peritoneal dialysis]. / Nutzen und Risiko eines Gastrostomas bei Säuglingen und Kleinkindern mit chronischer Peritonealdialyse.

BACKGROUND: Nutrition of children with end-stage renal disease and peritoneal dialysis (PD) is often difficult. Tube feeding via a gastrostoma is discussed controversially, and some authors consider this as a contraindication because of the risk of peritonitis. METHODS: In our centre 16 infants and children with end-stage renal disease were treated with PD and tube feeding over a gastrostoma in the last 12 years. The patients showed dystrophy (mean BMI -1.73 SDS) and were too small (mean body length -4.56 SDS). Seven of them (median age 11 months) received a gastrostoma before insertion of a Tenkhoff-catheter and start of PD. Nine children (median age 5 months) had PD primarily before insertion of the gastrostoma and start of tube feeding. RESULTS: Patients with start of PD while a gastrostoma was already inserted had 15 events with peritonitis in the observation time of 91 months (1.98 per patient year). Patients with primary start of PD had 12 events with peritonitis in a total time of 43 month (3.34 per patient year), after insertion while PD was already running the number of events fell significantly to 25 peritonitis events in a total of 271 months (1.11 per patient year, p < 0.01). The children had a benefit from tube feeding via a gastrostoma in regard of body weight (BMI + 1.61 SDS, p < 0.01) as well as growth (body height + 2.29 SDS, p < 0.05). CONCLUSION: Tube feeding via a gastrostoma is a good and safe option for alimentation, even under peritoneal dialysis. A decrease of PD-associated peritonitis under tube feeding was observed while physical development was positively influenced.

Virus-mediated release of endosomal content in vitro: different behavior of adenovirus and rhinovirus serotype 2.

Endosomal penetration by nonenveloped viruses might be accomplished by either local breakdown of the endosomal membrane (e.g., adenovirus) or formation of a membrane-spanning pore by capsid proteins. Uncoating of the nonenveloped virus human rhinovirus serotype 2 (HRV2) has been shown to occur from late endosomes and to be entirely dependent on the acidic pH in this compartment (Prchla, E., E. Kuechler, D. Blaas, and R. Fuchs. 1994. J. Virol. 68: 3713-3723). To investigate further the mechanism of uncoating of HRV2, an in vitro assay was established to test viruses or virus-derived peptides for their capacity to release cointernalized biotin-dextran of different molecular mass (10 and 70 kD) from isolated endosomes. The suitability of the assay was demonstrated by use of a fusogenic peptide derived from influenza virus hemagglutinin (GALA-INF3). Whereas adenovirus induced a low pH-dependent release of up to 46% of the internalized biotin-dextran and did not show any significant size selectivity (as expected for endosome disruption), HRV2 mediated release of 27% of the 10 kD dextran and only traces of the 70-kD dextran. Similarly, GALA-INF3-induced release of biotin-dextran was also size dependent. The potential role of the capsid protein VP1 in HRV2 uncoating in vivo was also substantiated in our in vitro system using an amphipathic, NH2-terminal peptide of VP1. Taken together, these data favor the model of a specific pore-forming mechanism for HRV2 uncoating which is in contrast to the membrane-disrupting mechanism of adenovirus.

[Pancreas. Congenital changes, acute and chronic pancreatitis]. / Pankreas. Angeborene Veränderungen, akute und chronische Pankreatitis.

The pancreas develops from ventral and dorsal buds, which undergo fusion. Failure to fuse results in pancreas divisum, which is defined by separate pancreatic ductal systems draining into the duodenum. Risk of developing pancreatitis is increased in pancreas divisum. MR cholangiopancreatography (MRCP) is the technique of choice for detecting it non-invasively. Annular pancreas is the result of incomplete rotation of the pancreatic bud around the duodenum with the persistence of parenchyma or a fibrous band encircling (stenosing) the duodenum. Acute pancreatitis is usually caused by bile duct stones or alcohol abuse. Contrast-enhanced multi-detector row CT is the method of choice to assess the extent of this disease. In acute pancreatitis, the role of MRCP is mainly limited to finding bile duct stones in patients with suspected biliary pancreatitis. Chronic pancreatitis results in relentless and irreversible loss of exocrine (and sometimes endocrine) function of the pancreas. MDCT even shows subtle calcifications. MRCP is the method of choice for non-invasive assessment of the duct. Inflammatory pseudotumor in chronic pancreatitis and groove pancreatitis are difficult to differentiate from pancreatic cancer. In these cases, multiple imaging methods such as MDCT, MRI and endosonography including biopsy may be used to make a diagnosis.

Matrix-based autologous chondrocyte implantation for cartilage repair with HyalograftC: two-year follow-up by magnetic resonance imaging.

OBJECTIVE: Monitoring of articular cartilage repair after matrix-associated autologous chondrocyte implantation with HyalograftC by a new grading system based on non-invasive high-resolution magnetic resonance imaging. PATIENTS AND METHODS: In 23 patients, postoperative magnetic resonance imaging (MRI) was performed between 76 and 120 weeks. In nine of these patients, five MRI examinations were performed at 4, 12, 24, 52 and 104 weeks after HyalograftC implant. The repair tissue was described with separate variables: degree of defect repair in width and length, signal intensity of the repair tissue and status of the subchondral bone. For these variables a grading system with point scale evaluation was applied. CONCLUSION: High-resolution MRI provides a non-invasive tool for monitoring the development of cartilage repair tissue following HyalograftC technology, shows a good correlation with clinical outcome and may help to differentiate abnormal repair tissue from a normal maturation process.

Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer.

Hypoxia-inducible factor 1alpha (HIF-1alpha) is a transcriptional factor that regulates genes involved in response to hypoxia and promotes neoangiogenesis, which are considered essential for tumor growth and progression. Using immunohistochemistry, we investigated the influence of HIF-1alpha expression on prognosis in 91 patients with cervical cancer stage pT1b. In univariate and multivariate analysis, patients with strong expression of HIF-1alpha had a significantly shorter overall survival time (P = 0.0307, log-rank test) and disease-free survival time (P < 0.0001, log-rank test) compared with those with moderate to absent HIF-1alpha expression. HIF-1alpha expression is a strong independent prognostic marker in early stage cervical cancer.

Nonviral gene delivery to the lung with copolymer-protected and transferrin-modified polyethylenimine.

Polyethylenimine (PEI) has been shown to efficiently mediate topical gene transfer to the lungs after either direct intratracheal instillation or nebulisation. Recently, the protection of polyplexes with novel copolymers of poly(ethylene glycol) (PEG) via electrostatic interaction has been reported. In this study, such coated PEI polyplexes were investigated for their stability and interaction with human plasma and bronchoalveolar lavage fluid (BALF). Further, their potential for gene delivery to the mouse lungs in vivo was examined. Plasma protein and mucin adsorption was effectively inhibited when polyplexes were coated with the novel copolymers. Gene transfer efficiency of the coated PEI polyplexes decreased as compared with uncoated PEI polyplexes when administered intratracheally to the lung. The higher the molecular weight of the copolymerized PEG was, the stronger the observed gene transfer reduction. Gene transfer decreased presumably due to reduced interaction of the coated gene vectors with the cell surface. To circumvent this problem, transferrin was combined with PEI/DNA polyplexes for specific binding to the cell surface. In this case, gene transfer efficiency decreased. Gene transfer of the copolymer-protected and transferrin-modified gene vectors increased as compared with the copolymer-protected gene vectors alone but did not reach the level of uncoated gene vectors. These data show that copolymers could be used to effectively shield polyplexes from interaction with components of the airway surface liquid (ASL). Increased gene delivery was found upon transferrin modification of the coated PEI polyplexes suggesting a targeting effect.

Activation of the complement system by synthetic DNA complexes: a potential barrier for intravenous gene delivery.

We have examined the complement-activating properties of synthetic cationic molecules and their complexes with DNA. Commonly used gene delivery vehicles include complexes of DNA with polylysine of various chain lengths, transferrin-polylysine, a fifth-generation poly(amidoamine) (PAMAM) dendrimer, poly(ethyleneimine), and several cationic lipids (DOTAP, DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE). These agents activate the complement system to varying extents. Strong complement activation is seen with long-chain polylysines, the dendrimer, poly(ethyleneimine), and DOGS (half-maximal at about 3 microM amine content in the assay used). Compared to these compounds, the other cationic lipids (in liposome formulations) are weak activators of the complement system (half-maximal approximately 50-100 microM positive charge in assay). Complement activation by polylysine is strongly dependent on the chain length. Short-chain oligolysines are comparable to cationic lipids in their activation of complement. Incubation of these compounds with DNA to form complexes reduces complement activation in virtually all cases. The degree of complement activation by DNA complexes is strongly dependent on the ratio of polycation and DNA (expressed as the charge ratio) for polylysine, dendrimer, poly(ethyleneimine), and DOGS. To a lesser degree, charge ratio also influences complement activation by monovalent cationic lipid-DNA complexes. For polylysine-DNA complexes, complement activation can be considerably reduced by modifying the surface of preformed DNA complexes with polyethyleneglycol (half-maximal approximately 20 microM amine content). The data suggests that, by appropriate formulation of DNA complexes, complement activation can be minimized or even avoided. These findings should facilitate the search for DNA complex formulations appropriate for reproducible intravenous gene delivery.

Branched cationic peptides for gene delivery: role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes.

To examine the suitability of synthetic peptides as DNA-binding and -compacting agents for receptor-mediated gene delivery, we have synthesized and characterized a series of branched oligocationic peptides that differ in the number and type (lysine, arginine, ornithine) of cationic amino acids in the DNA-binding moiety. The peptides were designed as branched molecules to provide a coupling site via a spacer for the attachment of effectors at a flexible distance from the DNA-binding moiety. This design provides torsional flexibility in the peptide backbone of the DNA-binding moiety to maximize cation-DNA phosphate interactions and also minimizes the potential for interference by the effector with DNA binding. The branched peptides bind DNA with affinities that increase with the number of cationic groups. The peptides compact DNA into microparticulate structures as judged by an ethidium bromide displacement assay, dynamic light scattering, and electron microscopy. In general, differences in DNA binding and compaction owing to variation in the cationic side chain were modest, with the rank order being arginyl > lysyl approximately ornithyl. Incorporation of tryptophans into the DNA-binding moiety had no major effect on apparent binding affinity but clearly reduced the DNA-compacting potency of the peptides. Compared with polylysine, the peptides and their DNA complexes are weak activators of the complement system. Complement activation by an octaarginyl peptide was stronger than that induced by an octalysyl peptide. The microparticulate peptide-DNA complexes are suitable for receptor-mediated gene delivery as evidenced by transferrinfection of K562 cells in the presence of chloroquine. The results obtained in gene delivery in vitro suggest that a minimum chain length of six to eight cationic amino acids is required to compact DNA into structures active in receptor-mediated gene delivery.

Language disorder in a right-hander after occlusion of the right anterior cerebral artery.

A right-handed woman developed left hemiparesis and a language disturbance. At autopsy, there was infarction in the territory of the right anterior cerebral artery, involving, among other structures, the supplementary motor area. This brain region has been considered to play a role in speech, but whether the language disorder that follows its destruction is truly aphasic is controversial. Our patient does not answer that question, but if her disturbed language is viewed as aphasic, she represents the fourth autopsy case of "crossed aphasia in a dextral" and the first, with or without autopsy, after right anterior cerebral artery occlusion.

The pathology of drop attacks: a case report.

A 65-year-old man had four drop attacks in several days and then a fixed stroke with quadriplegia. At autopsy infarction in the lower pons and upper medulla affected principally the corticospinal tracts. Tegmental destruction included reticular formation nuclei with rostral projections, but spared the lateral reticular formation nuclei, from which arise the descending reticulospinal tracts. This case is the first detailed autopsy report of a patient with drop attacks, and supports the view that at least some drop attacks are caused by transient ischemia of the corticospinal tracts.

Pharmacokinetics of orally administered zidovudine among patients with hemophilia and asymptomatic human immunodeficiency virus (HIV) infection.

Zidovudine (formerly azidothymidine, AZT) is used to treat certain patients infected with the human immunodeficiency virus (HIV). However, the clinical use of zidovudine (ZDV) in hemophilia patients may be complicated by the high incidence of chronic hepatitis in this patient population. To examine the pharmacokinetics of ZDV eight asymptomatic HIV-infected hemophilia patients received a single oral dose (300 mg). ZDV and its glucuronide metabolite (GZDV) were measured in serum by HPLC. ZDV was rapidly absorbed with a wide range of peak serum concentrations (2052 +/- 970 ng/ml) at 0.5 h. Peak GZDV serum concentrations were 4751 +/- 2269 ng/ml at 1 h. Both ZDV and GZDV declined in a biexponential manner over 4 h. After 4 h, the ZDV serum concentration decay in three patients continued a log-linear decline, while five patients demonstrated a tri-exponential curve which had a mean terminal elimination half-life of 4.8 +/- 2.8 h. No relationship between ZDV or GZDV kinetics and the degree of hepatic enzyme elevation was observed. Although a therapeutic window for ZDV has yet to be described, the wide range of serum concentrations that result from a standard dose suggests that clinical monitoring of ZDV levels may be of value in certain patients. In addition, the prolonged elimination half-life of ZDV in the present study may provide a rationale for less frequent dosing in certain patients.

Hyperreactivity to myelin basic protein in multiple sclerosis.

Multiple sclerosis patients, control patients with other neurological diseases, and normal volunteers were assayed in a short-term 51chromium release assay for cell-mediated cytotoxicity against lymphocyte targets coated with myelin basic protein. Multiple sclerosis patients, compared to the other two groups, were hyperreactive to myelin basic protein, both before and after in vitro boost with additional myelin basic protein. The boost served to augment the difference between multiple sclerosis patients and controls.

Lymphatic microvessel density in epithelial ovarian cancer: its impact on prognosis.

BACKGROUND: The influence of lymphatic microvessel density (MVD) on survival in epithelial ovarian cancer is still unknown, owing to the fact that until recently no reliable immunohistologic markers for lymphatic endothelium were available. MATERIALS AND METHODS: By using a polyclonal antibody staining podoplanin, a novel marker for lymphatic endothelium, lymphatic MVD in tissue samples of 90 patients with epithelial ovarian cancer treated by radical surgery and chemotherapy was investigated. Survival analysis was performed using univariate and multivariate analysis. Furthermore, lymphatic MVD was compared to MVD assessed by CD34 immunostaining. RESULTS: Lymphatic MVD was significantly lower than CD34 MVD (p < 0.0001). There was no significant association between lymphatic MVD and various histological and clinical parameters. Lymphatic MVD had no influence on overall survival and disease free survival (p = 0.4627 and p = 0.4337, respectively; log-rank test). CONCLUSION: The formation of lymphatic vessels has no influence on the progression of epithelial ovarian cancer.

Fragrance compounds and essential oils with sedative effects upon inhalation.

Fragrance compounds and essential oils with sedative effects influence the motility of mice in inhalation studies under standardized conditions. A significant drop in the motility of mice was registered following exposure to these fragrances. The same results were achieved when the mice were artificially induced into overagitation by intraperitoneal application of caffeine and subsequently subjected to inhalation of fragrance compounds and essential oils. These results proved the sedative effects of these fragrants via inhalative exposure in low concentrations. Blood samples were taken from the mice after a 1-h inhalation period. Chromatographic and spectroscopic methods were used to detect and characterize the actual effective compounds after solid-phase extraction. Serum concentrations of 42 different substances, including fragrance compounds, were found in low ranges (ng/mL serum). The results contribute to the correct interpretation of the term aromatherapy (i.e., a stimulating or sedative effect on the behaviour of individuals only upon inhalation of fragrance compounds).

European Society of Gene Therapy (ESGT) Seventh Meeting. 26-28 November 1999, Munich, Germany.

This meeting gave an excellent overview of the recent developments in gene therapy. Much research effort has focused on the improvement and de novo construction of gene vectors, on characterizing their mechanisms of action and on their interactions with, and in, living organisms. The continual improvements in understanding disease at a molecular level and the progress in cell biology, immunology and related fields have opened the way for novel gene therapy approaches. The gene therapeutic strategy has proven to be feasible and efficient in numerous preclinical (animal) models of a variety of diseases. On the other hand, applying this experience to humans has turned out to be difficult. Currently, the field is rapidly moving into clinical applications. No major limiting side effects have been observed in patients in the phase I and later stage trials presented. Nonetheless, years of preclinical and clinical research will be required before gene therapy can be considered a reliable and efficient therapeutic approach with broad applicability.

Safety evaluation of phytosterol esters. Part 4. Faecal concentrations of bile acids and neutral sterols in healthy normolipidaemic volunteers consuming a controlled diet either with or without a phytosterol ester-enriched margarine.

A study was conducted in 12 healthy males and 12 females (mean age 36 years) to assess the impact of a margarine enriched with phytosterol esters on faecal concentrations of bile acids and sterols. During the run-in period, volunteers consumed 40 g of a control margarine for 21 consecutive days if male, and for 28 days if female. Half of the volunteers were then randomly allocated to consume the control margarine for another 21 or 28 days, respectively. The remaining subjects consumed 40 g of a margarine containing 8.6 g vegetable oil phytosterol (46% (w/w) beta-sitosterol, 26% campesterol, 20% stigmasterol). Throughout the total study subjects consumed the same diet adjusted for individual energy requirements. The phytosterol ester-enriched spread significantly enhanced faecal neutral sterol concentrations from about 40 mg/g to 190 mg/g dry weight faeces. Faecal neutral sterol metabolites increased from about 30 mg/g to about 50 mg/g. The major parent sterols excreted were cholesterol, sitosterol, campesterol and stigmasterol. Sitosterol, campesterol and stigmasterol comprised 28%, 15% and 12% of the total faecal neutral sterols, reflecting the composition of the sterol enriched margarine. The major sterol metabolites excreted were metabolites formed by, predominantly, oxidation at the 3-position and metabolites saturated at the 5,6 position in a beta-configuration. Faecal secondary bile acid concentration was reduced by vegetable oil sterols from 7.6 mg/g dry faeces to 6.0 mg/g. Consumption of vegetable oil phytosterols slightly but significantly increased the faecal concentration of 4-cholesten-3-one. However, 4-cholesten-3-one concentration remained very low (< 2 mg/g) and in line with values reported in the literature for subjects fed high or low fat diets. No sterol oxides could be detected in the faeces. We conclude that in healthy adult males and females a high intake of vegetable oil phytosterol esters does increase the amount of neutral sterols in the faeces, as expected, but does not result in the increased formation of bile acids or sterol metabolites.