Synthetic Glycopeptide-Based Delivery Systems for Systemic Gene Targeting to Hepatocytes

Purpose. To design, synthesize, and test synthetic glycopeptide-baseddelivery systems for gene targeting to hepatocytes by systemicadministration.Methods. All peptides were synthesized by the solid phase methoddeveloped using Fmoc chemistry on a peptide synthesizer. The bindingof galactosylated peptides to HepG2 cells and accessibility of thegalactose residues on particle surface was demonstrated by acompetition assay using ¹²⁵I-labeleld asialoorosomucoid and RCA lectinagglutination assay, respectively. DNA plasmid encoding chloramphenicolacetyl transferase (CAT) gene was complexed with a tri-galactosylatedpeptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in thepresence of an endosomolytic peptide (GM225.1) or endosomolyticlipopeptide (GM227.3) to obtain DNA particles of 100–150 nm insize. The plasmid/peptide complexes were added to HepG2 cell culturesor intravenously administered by tail vein injection into normal miceor rats. Plasmid uptake and expression was quantified by qPCR andELISA, respectively.Results. Multiple antennary glycopeptides that have the ability tocondense and deliver DNA plasmid to hepatocytes were synthesized andcomplexed with DNA plasmid to obtain colloidally stable DNA/peptidecomplexes. Addition of DNA/GM245.3/GM225.1 peptide complexes(1:3:1 (–/+/–)) to HepG2 cell cultures yielded CAT expression intransfected cells. The transfection efficiency was significantly reducedin the absence of galactose ligand or removal of endosomolytic peptide.Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5(–/+)) into the tail vein of normal rats yielded DNA uptake in theliver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3resulted in more stable DNA particles, and a 10-fold enhancement inliver plasmid uptake. CAT expression was detectable in liver followingintravenous administration of DNA/GM246.3 complexes. Addition ofendosomolytic lipopeptide GM227.3 into the complexes(DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–))) yielded a 5-fold increase inCAT expression. Liver expression was 8-fold and 40-fold higher thanlung and spleen, respectively, and localized in the hepatocytes only.The transfection efficiency in liver was enhanced by increasing DNAdose and injection volume. The plasmid uptake and expression in liverusing DNA/GM246.3/GM227.3 complexes was 100-200-fold higherthan DNA formulated in glucose. Tissue examination and serumbiochemistry did not show any adverse effect of the DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–)) complexes after intravenous delivery.Conclusions. Gene targeting to hepatocytes was achieved by systemicadministration of a well-tolerated synthetic glycopeptide-baseddelivery system. The transfection efficiency of this glycopeptide deliverysystem was dependent on peptide structure, endosomolytic activity,colloidal particle stability, and injection volume.     

Epidermoid carcinoma of the anorectum: Review of 31 cases

Summary Thirty-one cases of epidermoid carcinoma found in 106 cases of carcinoma of the rectum and anal canal are reviewed. Clinical presentation and management are presented. Probable factors in the greater than usual incidence of epidermoid carcinomas of the anorectum are discussed. Indications for radiation therapy, local excision, and abdominoperineal and inguinal block dissection are presented.     

Acute renal failure due to non-traumatic rhabdomyolysis

Seventeen patients with acute renal failure of diverse aetiology showed myoglobinuria and elevated levels of serum creatine phosphokinase (mean 119·2 Sigma u./ml) and adolase (mean 88·5 Sibley-Lehninger (SL)u./ml), indicating the presence of diffuse muscle cell injury. The primary conditions which led to rhabdomyolysis and acute renal failure were burns, eclampsia, prolonged labour, crush injury, epileptiform convulsions, status asthmaticus, viral myositis and intoxication with chemicals including copper sulphate, mercuric chloride and zinc phosphide. In 10 non-myoglobinuric patients with acute renal failure, serum creatine phosphokinase was normal (mean 8·9 Sigma u./ml) and serum aldolase was only slightly elevated (mean 11·2 SL u./ml). Although uric acid was elevated in both groups, the values were significantly higher in myoglobinuric (mean 0·728 ± 0·199 mmol/l) compared to non-myoglobinuric patients (mean 0·583 ± 0·093 mmol/l). During the oliguric phase, hypocalcaemia was observed in 82·2% of myoglobinuric patients and in 20% of non-myoglobinuric patients. Ten out of 15 patients with myoglobinuric renal failure developed hypercalcaemia during the diuretic phase whereas only 3 non-myoglobinuric patients showed a transient hypercalcaemia. Although the mean serum potassium was somewhat higher in the myoglobinuric patients, the difference between the 2 groups was not significant. It is concluded that acute renal failure associated with non-traumatic rhabdomyolysis is not infrequent and may occur in a variety of conditions where gross evidence of muscle injury is lacking.     

Gene expression signature predicts lymphatic metastasis in squamous cell carcinoma of the oral cavity

Metastasis via the lymphatics is a major risk factor in squamous cell carcinoma of the oral cavity (OSCC). We sought to determine whether the presence of metastasis in the regional lymph node could be predicted by a gene expression signature of the primary tumor. A total of 18 OSCCs were characterized for gene expression by hybridizing RNA to Affymetrix U133A gene chips. Genes with differential expression were identified using a permutation technique and verified by quantitative RT-PCR and immunohistochemistry. A predictive rule was built using a support vector machine, and the accuracy of the rule was evaluated using crossvalidation on the original data set and prediction of an independent set of four patients. Metastatic primary tumors could be differentiated from nonmetastatic primary tumors by a signature gene set of 116 genes. This signature gene set correctly predicted the four independent patients as well as associating five lymph node metastases from the original patient set with the metastatic primary tumor group. We concluded that lymph node metastasis could be predicted by gene expression profiles of primary oral cavity squamous cell carcinomas. The presence of a gene expression signature for lymph node metastasis indicates that clinical testing to assess risk for lymph node metastasis should be possible.     

Palliative management of hilar cholangiocarcinoma

Around 80% of the patients with hilar cholangiocarcinoma are candidates for palliative management due to extensive co-morbidity for major surgery, metastases or advanced loco-regional disease. The primary aim of treatment is to provide biliary drainage with long-term relief from pruritis, cholangitis, pain and jaundice. Endoscopically placed self-expanding metallic biliary stent has low procedure-related complications and is probably the modality of choice for patients with unresectable tumour on preoperative assessment. Percutaneous biliary drainage has comparable results and is an alternative when endoscopic expertise is not available or has failed or there are multiple isolated segments with cholangitis. Surgical cholangiojejunostomy provides lasting biliary drainage but has limitations of associated morbidity and mortality. In the absence of high-quality studies, comparing these modalities the choice of biliary drainage procedure should be guided by the available local expertise. Other modalities of treatment like radiotherapy, chemotherapy and photodynamic therapy currently remain investigational.     

Airway management of patients undergoing oral cancer surgery: a retrospective study

OBJECTIVE: This retrospective study aims to describe the airway management and benefits of nasotracheal intubation over tracheostomy in 260 patients with oral cancer undergoing surgery. METHODS AND RESULTS: The medical records of 260 patients undergoing surgery for oral cancer were reviewed for airway management during the perioperative period. Eighteen patients had previous surgery for oral cancer and were scheduled for flap reconstruction, recurrence or other complications. In 28 cases neck movement was restricted and decreased mouth opening was found in 50% of all patients because of a large growth or fixation of tissues of head and neck, oral cavity, pharynx or larynx by tumour, or radiation fibrosis. In 53 patients intubation was undertaken under spontaneous ventilation. In 20 cases the trachea was extubated in the immediate postoperative period. In 220 cases patients were extubated next morning in the intensive care unit. In none of the cases was elective tracheostomy under local anaesthesia performed before surgery for the maintenance of the airway for anaesthesia. Elective tracheostomies were done in 17 cases. Three patients remained intubated for 24-48 h because of a high suspicion of airway obstruction following extubation due to a large pectoralis major flap. These three patients received a tracheostomy because of increased oropharyngeal and laryngeal oedema. In three cases emergency tracheostomies were performed due to upper airway obstruction after extubation and in one case prolonged elective ventilation was required due to severe chest infection. CONCLUSION: Oral cancer patients have a potentially difficult airway but, if managed properly during perioperative period, morbidity and mortality can be reduced or avoided. Oral cancer patients can be managed safely without the routine use of a tracheostomy. Nasotracheal intubation is a safe alternative to tracheostomy in oral cancer patients except in some selected patients.     

Pulmonary interstitial emphysema and continuous positive airway pressure in a premature infant

Pulmonary interstitial emphysema PIE is a recognized sequel of high pressure mechanical ventilation. Nevertheless, with the improvement in perinatal medical care, PIE started to be seen in spontaneously breathing infants. We present a 29-week-old girl who developed PIE on the first day of life, while she was on nasal continuous positive airway pressure, and was successfully managed with high frequency oscillatory ventilation. There is a paucity of these cases in the literature. We report on the occurrence of this entity, early reorganization, and the possible associated or causation factors.