Characterization of two blood-brain barrier mimicking cell lines: distribution of lectin-binding sites and perspectives for drug delivery.

In the present study plant lectins with distinct sugar specificities were applied to two blood-brain barrier (BBB) mimicking cell lines, namely human ECV304 and porcine brain microvascular endothelial cells PBMEC/C1-2 in order to elucidate their glycosylation pattern and to evaluate the lectin-cell interaction for lectin-mediated targeting. The bioadhesive properties of fluorescein-labeled lectins were investigated with monolayers as well as single cells using fluorimetry and flow cytometry, followed by confirmation of the specificity of binding. For PBMEC/C1-2 layers highest binding capacity was found for wheat germ agglutinin (WGA), followed by Dolichus biflorus agglutinin (DBA) whereas single cell experiments revealed a predominance of DBA only. Analyzing ECV304 monolayers and single cells, WGA yielded the strongest interaction without any changes during cultivation. The binding capacities of the other lectins increased significantly during differentiation. As similar results to primary cells and brain sections were observed, both cell lines seem to be suitable as models for lectin-interaction studies. Thus, an additional focus was set on the mechanisms involved in uptake and intracellular fate of selected lectins. Cytoinvasion studies were performed with WGA for human ECV304 cells and WGA as well as DBA for PBMEC/C1-2 cells. For both lectins, the association rate to the cells was dependent on temperature which indicated cellular uptake.

Alteration of the glycosylation pattern of monocytic THP-1 cells upon differentiation and its impact on lectin-mediated drug delivery.

In the present study, human monocytic THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA) in order to obtain macrophage-like cells. Before and after treatment, plant lectins with distinct sugar specificities were applied in order to elucidate the glycosylation patterns of both monocytic and macrophage-like cell types and to follow changes during differentiation. As a result of flow-cytometric analyses, for untreated as well as for PMA-differentiated cells WGA yielded the highest binding rate without significant changes in the binding capacity. For the other lectins, divergent results were obtained which point to reorganization of sugar residues on the cell surface during differentiation. Additionally, cytoinvasion being beneficial for enhanced drug absorption was studied with WGA which had displayed a high binding capacity together with a high specificity. For both untreated and PMA-differentiated cells decreased fluorescence intensity at 37 degrees C as compared to 4 degrees C was observable pointing to internalization and accumulation within acidic compartments. Moreover, WGA-functionalized PLGA nanoparticles were prepared, and their uptake evaluated. Uptake rates of 55% in case of PMA-differentiated cells suggested that WGA-grafted drug delivery systems might be an interesting approach for treatment of infectious diseases provoked by parasites, facultative intracellular bacteria, or viruses such as HIV.

Strategies to improve drug delivery in bladder cancer therapy.

Bladder cancer is the ninth most common malignancy in the world featuring very high gender variability in occurrence. Current options for bladder cancer therapy include surgery, immunotherapy, chemotherapy and radiotherapy with a trend towards multimodal treatments. However, successful management remains a challenge for urologists and oncologists because of the high risk for recurrence and progression. Particularly in the field of bladder cancer chemotherapy, efficacy of treatment might be improved by advanced drug delivery strategies aimed at prolonged residence time within the bladder cavity and increased permeability of the bladder wall during intravesical instillation. Moreover, a deeper understanding of the biology of bladder carcinogenesis and malignant progression stimulated the development of a new generation of anticancer drugs for targeted therapies that might result in increased treatment specificity together with lower toxic potential and higher therapeutic indices. This review discusses the available strategies for 'targeted therapy', focusing on molecular targets, and for 'controlled delivery', comprising all other approaches towards improved drug delivery.

Alteration of the glycocalyx of two blood-brain barrier mimicking cell lines is inducible by glioma conditioned media.

Recent studies showed that glioma conditioned medium is able to induce blood-brain barrier properties in in vitro models. In this regard, it was investigated whether glioma conditioned medium can also influence the lectin-binding capacity of blood-brain barrier in vitro models. For the presented study cell lines PBMEC/C1-2 and ECV304 were chosen because it was previously shown that glioma conditioned medium was able to induce specific blood-brain barrier properties in these cell lines. Six different plant lectins (WGA, STL, LCA, UEA-I, DBA, PNA) with distinct sugar specificities were applied in order to elucidate the glycosylation patterns of cell line PBMEC/C1-2 and ECV304. Lectin-binding studies were carried out with monolayers as well as with single cells. In the case of PBMEC/C1-2 monolayers, results showed a significant increase of the binding of lectins WGA, STL, UEA-I, DBA and PNA after application of 25 pmol lectin when cultured in media containing soluble factors derived from glioma cell line C6, whereas the binding capacity for LCA remained similar. For ECV304 monolayers, a significant decrease of WGA, STL and LCA was observable, whereas UEA-I binding increased in comparison to cells grown in the corresponding basal growth medium without soluble C6 factors. Single cell studies showed less significant, but similar changes in the lectin-interactions with the cell surfaces. In conclusion, it was shown that soluble factors derived from glioma cell line C6 can modulate the "glycocalyx" of blood-brain barrier mimicking cell lines.

Lectin binding patterns reflect the phenotypic status of in vitro chondrocyte models.

In vitro studies using chondrocyte cell cultures have increased our understanding of cartilage physiology and the altered chondrocytic cell phenotype in joint diseases. Beside the use of primary cells isolated from cartilage specimens of donors, immortalized chondrocyte cell lines such as C-28/I2 and T/C-28a2 have facilitated reproducible and standardized experiments. Although carbohydrate structures appear of significance for cartilage function, the contribution of the chondrocyte glycocalyx to matrix assembly and alterations of the chondrocyte phenotype is poorly understood. Therefore, the present study aimed to evaluate the glycoprofile of primary human chondrocytes as well as of C-28/I2 and T/C-28a2 cells in culture. First, the chondrocytic phenotype of primary and immortalized cells was assessed using real-time reverse transcriptase polymerase chain reaction, immunofluorescence, and glycosaminoglycans staining. Then, a panel of lectins was selected to probe for a range of oligosaccharide sequences determining specific products of the O-glycosylation and N-glycosylation pathways. We found that differences in the molecular phenotype between primary chondrocytes and the immortalized chondrocyte cell models C-28/I2 and T/C-28a2 are reflected in the glycoprofile of the cells. In this regard, the glycocalyx of immortalized chondrocytes was characterized by reduced levels of high-mannose type and sialic acid-capped N-glycans as well as increased fucosylated O-glycosylation products. In summary, the present report emphasizes the glycophenotype as an integral part of the chondrocyte phenotype and points at a significant role of the glycophenotype in chondrocyte differentiation.

Long-term outcome of laparoscopic Nissen-Rossetti fundoplication for neurologically impaired and normal children.

BACKGROUND: This study aimed to assess the long-term effects of laparoscopic Nissen-Rossetti fundoplication (LNF) on clinical and pH evaluations of children with gastroesophageal reflux disease (GERD) according to neurologic status. METHODS: The study examined 127 children (73 neurologically impaired and 54 neurologically normal with primary GERD) who consecutively underwent LNF from 1992 to 2003. The follow-up protocol included evaluations at 3, 15, and more than 36 months (long-term evaluation) postoperatively, which consisted of physical examination and 24-h pH monitoring. Recurrences were defined as abnormal pH-metry exhibited by symptomatic children. RESULTS: The long-term follow-up period averaged 5.5 years. Of the 73 neurologically impaired children, 9 (12%) had GERD recurrences, which occurred during the evaluation period and required redo surgery in four cases, including two Bianchi procedures. In the neurologically normal group, one recurrence (2%) occurred 3 months after surgery. The long-term complications in this group included dyspepsia (n = 3), occasional dysphagia (n = 2), gas bloat syndrome (n = 1), and alimentary disorders (n = 2). CONCLUSIONS: For children with primary GERD, LNF is a long-term efficient procedure. For the neurologically impaired children, the results were good, with more than 85% of the children symptom free after 5 years, although repeated evaluations are required to diagnose late recurrences related to evolving dysmotility disorders.

Lectin binding studies on C-28/I2 and T/C-28a2 chondrocytes provide a basis for new tissue engineering and drug delivery perspectives in cartilage research.

The present study was performed to evaluate the applicability of plant lectins as mediators of bioadhesion in cartilage research using human chondrocyte cell lines C-28/I2 and T/C-28a2. The bioadhesive properties of fluorescein-labelled lectins with different carbohydrate specificities were investigated by flow cytometry. Specificity of the lectin-cell interactions was ascertained by competitive inhibition using complementary carbohydrates. As compared to that of other lectins, the interaction between wheat germ agglutinin (WGA) and chondrocytic cells was characterised by remarkable cytoadhesion, adequate binding strength and a high degree of specificity for N-acetyl-glucosamine as contained in hyaluronan chains. We therefore suggest WGA to be a promising candidate for mediating bioadhesion to low-adhesive scaffolds in cartilage tissue engineering. Moreover, the WGA-association rate of C-28/I2 and T/C-28a2 cells was dependent on temperature indicating cellular uptake of membrane-bound WGA. Intracellular enrichment was confirmed by confocal microscopy. Equilibration of intracellular pH gradients with monensin resulted in the reversal of quenching effects indicating accumulation of WGA within acid compartments of chondrocytic cells. Thus, WGA might be internalised into chondrocytes together with hyaluronan via the CD44 receptor-mediated endocytosis pathway and accumulated within lysosomes. This physiological process could represent a feasible pathway to target WGA-functionalised drug delivery devices into chondrocytes.

Postpneumonectomy syndrome in children: advantages and long-term follow-up of expandable prosthesis.

BACKGROUND/PURPOSE: Pneumonectomy in children can be complicated by a severe mediastinal shift, which leads to bronchial stretching resulting in severe respiratory failure. This postpneumonectomy syndrome can be corrected by inserting a prosthesis in the empty side of the chest. METHODS: Forty-two children, from 6 months to 15 years old, underwent a pneumonectomy. Seven of these patients were treated surgically for severe manifestations of postpneumonectomy syndrome. First insertion of an expandable prosthesis was followed up in 5 cases by its replacement with a breast prosthesis in adolescence. The expandable prosthesis was injected periodically with saline solution to maintain the mediastinum in a midline position as the children grew. RESULTS: The mean delay between pneumonectomy and first prosthesis implantation was 5 years (range, 11 months to 8 years). Pulmonary function tests showed a substantial improvement in the obstructive syndrome in all patients except one, in whom the functional improvement was moderate. The mean follow-up after the expandable prosthesis implantation was 6 years (range, 6 months to 10 years) and all patients are doing well. CONCLUSIONS: The insertion of an intrathoracic prosthesis can dramatically improve the clinical symptoms and reduce the functional obstructive syndrome. The expandable prosthesis allowed for progressive, well-tolerated recentering of the mediastinum and adjustment for growth.

Interleukin-8 increases acetylcholine response of rat intestinal segments.

BACKGROUND: Interleukin-8 (IL-8) is a pro-inflammatory cytokine highly expressed in inflammatory bowel diseases, but whose effects on intestinal motility are unknown. AIM: To characterize the role of IL-8 in the contraction of rat intestinal segments. METHODS: Contractile response to acetylcholine (ACh 10-6 M) in terminal ileal segments (including mucosa) from Wistar rats was measured before and after incubation (15, 30, 60 or 90 min) with IL-8 (1 ng/mL), and after 60 min of incubation with different doses of IL-8 (0, 0.1, 0.5, 1, 10 and 100 ng/mL). The effects of blocking neural transmission with tetrodotoxin (TTX) and inhibiting protein synthesis (cycloheximide) were tested. The contractile response of longitudinal muscle-myenteric plexus preparations (i.e. without mucosa) was measured after 60 min of incubation with 0.1 and 1 ng/mL of IL-8. RESULTS: IL-8 increased ileal contraction induced by ACh 10(-6) M. This augmentation was significant after 60 min of incubation (58%, P=0.01) and persisted after 90 min (18%, P=0.04). A 60-min incubation period showed a dose-related effect, beginning at 0.5 ng/mL (30%, P=0.003) and reaching a peak at 1 ng/mL (58%, P=0.01). The same effect was also observed on colonic segments. TTX did not affect the IL-8 increase of ACh-induced contractions, which was completely abolished by cycloheximide. IL-8 had no significant effect on longitudinal muscle-myenteric plexus preparations. CONCLUSION: In vitro, IL-8 increases contractile response of the ileum to ACh in a dose-dependent manner. This effect is not neurally mediated, but seems to involve protein synthesis by intestinal mucosa.

Laparoscopic adrenalectomy in children.

BACKGROUND: The aim of this study was to describe the indications and results of laparoscopic adrenalectomy in children. METHODS: This retrospective multicenter study included six children (four boys, two girls) aged 2-16 years (mean, 9.5). Three children had hypertension. In three cases, the adrenal mass was discovered incidentally. The tumors were bilateral in two children, right-sided in two cases, and left-sided in two cases. The mean tumor size was 4 cm (range, 1-7). Each child underwent MIBG scintigraphy and MRI before the operation. RESULTS: Four right and two left adrenal glands were resected by laparoscopy (transperitoneal), and two left glands were resected by retroperitoneoscopy. Two conversions were necessary for two left adrenal glands (one retroperitoneoscopy, one laparoscopy). Two partial resections and six adrenalectomies were performed. Histological examination of the tumors revealed two ganglioneuromas, one neuroblastoma, and five pheochromocytomas (two bilateral). One child had an involved node (pheochromocytoma). Both bilateral pheochromocytomas had von Hippel-Lindau disease. There was no morbidity. Mean hospitalstay was 6 days. Postoperative evaluation at 1 month was normal in all children. CONCLUSION: The indications for laparoscopic adrenalectomy in children are benign tumors and pheochromocytomas. In these cases, laparoscopic adrenalectomy is feasible and safe, even in cases of pheochromocytoma.

[Severe pancreaticoduodenal trauma: review of a series of 30 patients]. / Traumatismes fermés duodéno-pancréatiques sévères. A propos d'une série de 30 patients.

Retrospective study of a series of 30 patients (mean age: 25.5 years), including 8 children with severe duodenopancreatic trauma, treated over a period of 15 years. This series consisted of 14 cases of duodenal perforation, 3 cases of duodenal haematoma, 11 cases of isolated pancreatic lesions (including 5 isthmic ruptures) and 2 cases of associated duodenal and pancreatic lesions. Injuries were due to road accidents in 60% of cases. Eight patients were considered to have multiple injuries. Twelve patients required emergency surgery. Eighteen were observed in a surgical unit. Two duodenal haematomas were operated. Duodenal perforations were operated urgently in 8 cases and electively in 6 cases. The surgical procedure consisted of simple suture (n = 3), suture combined with diversion (n = 7), or resection-anastomosis (n = 4). Five patients with pancreatic contusion were operated, in a context of acute pancreatitis in four cases and for associated lesions in one case. Isthmic ruptures were treated by left pancreatic resection. This was a rare lesion (1.8 to 9% of organ lesions). Duodenal haematomas justify first-line medical treatment, while duodenal perforations must be operated. The presence of a lesion of the pancreatic duct frequently justifies pancreatic resection.

[The place of laparoscopy in pediatric surgery]. / La place de la coelioscopie en chirurgie pédiatrique.

The development of laparoscopy in children is designed to decrease the risks of this technique by respecting certain rules (particularly open laparoscopy). The indications are beginning to be well defined for certain frequent diseases (appendicitis, impalpable testes, gastro-oesophageal reflux) suitable for prospective studies, but remain much more subjective in other indications (splenectomy, lumboscopy) due to the limited paediatric series.

[Endoscopic treatment of residual vesico-ureteral reflux after reimplantation in children: twelve cases]. / Traitement endoscopique du reflux vésico-urétéral résiduel après réimplantation chez l'enfant: a propos de 12 observations.

OBJECTIVE: To evaluate the safety and efficacy of endoscopic treatment of vesicoureteric reflux in children persisting after surgical reimplantation. MATERIAL AND METHODS: 12 children between the ages of 17 and 103 months were treated by endoscopic injection after failure of Cohen ureterovesical reimplantation. An associated uropathy was present in 4 children. Reflux was unilateral in every case. The operation was performed as a day-only procedure. All children were reviewed at 3 months. RESULTS: No early complications were observed. The follow-up cystography was normal in 10 children. The same degree of reflux persisted in 2 children, requiring a second ureterovesical reimplantation. A meatal stricture occurred 2 years after injection. The mean follow-up was 24.7 months. The success rate of the technique was 75% in this series. CONCLUSION: This technique is reliable, simple and can be performed in the day hospital. However, these good results must not mask the risk of long-term recurrence, requiring prolonged surveillance.

[Complicated appendicitis in children: laparoscopy or Mac Burney incision?]. / Appendicites compliquées de l'enfant: laparoscopie ou Mac Burney?

PURPOSE: The aim of this retrospective study was to compare open (Mc Burney incision group MB n = 92) and laparoscopic (group LAP n = 58) appendectomy for complicated appendicitis (abscess or peritonitis). MATERIAL AND METHOD: 150 children, with a mean age of 8.5 years, were operated in our department from January 1990 to April 1996. Only children with complicated appendicitis and positive bacteriology of peritoneal fluid were included in this study. All children received parenteral antibiotics for an average of 5.4 days. RESULTS: The mean operating time was significantly longer in the LAP group (63 vs 43 min p < 0.0001). The conversion rate in the LAP group was 12%. The mean hospital stay was 8.4 days (3-29) without any difference between the 2 groups. There was no significant difference for the complication rate in the 2 groups, except for wound infections which were more frequent in the MB group (p = 0.008). Late postoperative complications occurred in 3 cases in the MB group (none in the LAP group) (NS). There were 2 small bowel obstructions and a wound dehiscence. CONCLUSION: Laparoscopic appendectomy is a safe procedure for complicated appendicitis in children, but the greatest short-term benefit is cosmetic. Long-term results have to be evaluated, particularly with regards to the long-term complication rate.

[Bilateral pneumothorax and hemorrhagic rectocolitis]. / Pneumothorax bilatéral et rectocolite hémorragique.

BACKGROUND: Respiratory diseases associated with ulcerative colitis are rare. CASE REPORT: A 14-year-old girl with ulcerative colitis was admitted for a left pneumothorax. She was given corticoidsteroids and enemas of 5 aminosalicylic acid. The pneumothorax was not controlled by pleural drainage and a pleural irritation was performed under thoracoscopy. Recurrence of pneumothorax led to surgical pleurectomy. The following day, a right pneumothorax occurred, also requiring pleurectomy. The pulmonary biopsies showed constrictive bronchiolitis. A restrictive syndrome was confirmed by functional pulmonary examinations. Total colectomy was performed nine months later for the ulcerative colitis. COMMENTS: Ulcerative colitis can be associated with bronchial pathology (bronchitis, bronchiectasis). Occurrence of pneumothorax has never been described; it can be a fortuitous association, but the histological features are not very different from those described in association with ulcerative colitis. The treatment of pneumothorax is difficult if the ulcerative colitis requires corticosteroids.

Laparoscopic nissen fundoplication with simultaneous percutaneous endoscopic gastrostomy in children.

BACKGROUND: The aim of the study was to evaluate the results of laparoscopic Nissen fundoplication (LNF) with simultaneous percutaneous endoscopic gastrostomy (PEG) in children with gastroesophageal reflux (GER) disease documented by upper gastrointestinal contrast and/or pH monitoring and/or esophageal endoscopy. METHODS: Fifteen LNF + PEGs were performed in children with pathologic antecedents: ten neurologically impaired children, two ORL (otorhinolaryngeal) pathologies. Two cases of AIDS, and one neuroblastoma. In one case, disruption of the fundoplication occurred during insufflation of the stomach. The child was reoperated on the 3rd day using an open procedure, so she was excluded from the results of the LNF. RESULTS: Two children had postoperative complications: one with cardiac insufficiency, one case of dehydration. Fourteen LNFs were controlled at 3 months by gastroesophageal X-ray and pH-metry. The 14 gastroesophageal X-rays were normal in 12 cases; gastroesophageal reflux was present in two cases. Twelve pH monitorings were analyzed (two technical failures), the median time pH <4 was 0.2% (0-20). Only one pH monitoring was pathologic (pH <4: 20%). This recurrent reflux to led to a second LNF with a good clinical result. CONCLUSIONS: In conclusion, it is possible to perform LNF and PEG during the same operative procedure. Short-term results are satisfactory with 14% recurrent GER. Long-term results need to be evaluated.

Randomized, single-blinded trial of laparoscopic versus open appendectomy in children: effects on postoperative analgesia.

BACKGROUND: The benefit of laparoscopy to patients has been clearly established in adults undergoing cholecystectomy. Results are less clear for appendectomy. The current study was undertaken to compare the respective 3-day postoperative periods after laparoscopic and open appendectomy in children. METHODS: Sixty-three children (aged 8-15 yr) scheduled for appendectomy were randomly assigned to two groups: open and laparoscopic. Postoperative evaluation included delay of postoperative recovery (walking and feeding), pain assessment by visual analog scale during the 3 subsequent days, amount of nalbuphine administered via a patient-controlled analgesia system during the first 48 h and responses by children, parents, and nurses on the overall quality of analgesia. RESULTS: There was no difference between groups for demographic data (particularly macroscopic aspect of appendix) analgesia, sedation, delay before eating and walking, incidence of urinary retention, nausea, vomiting. Operative time was long (P < or = to 0.05) in the laparoscopic group (54 +/- 17 min) than in the open group (39 +/- 18 min). Thirty five percent of the children had pain at the shoulder in the LAP group versus ten percent in the open group (P < or = 0.05). CONCLUSIONS: Laparoscopy did not improve analgesia and postoperative recovery after appendectomy in children.