Nanoparticles based on N-trimethylchitosan: evaluation of absorption properties using in vitro (Caco-2 cells) and ex vivo (excised rat jejunum) models.

Among the chitosan derivatives, trimethylchitosan (TMC) has been shown to have penetration enhancement properties also in intestinal environment. In addition, the use of nanoparticulate systems has the advantage of protecting peptidic drugs from intestinal degradations, due to internalisation behaviour. Therefore, the aim of this paper was to evaluate nanoparticulate systems based on TMC. In particular the mucoadhesive and absorption enhancement properties of nanoparticles based on TMC with different quaternization degree (QD) intended for the intestinal administration of macromolecules (peptides) have been evaluated. Comparison with chitosan (CS.HCl) nanoparticles was made. The nanoparticles were loaded with fluorescein isothiocyanate dextran (FD4, MW 4400 Da), used as the model macromolecule. The intestinal penetration enhancement properties of nanoparticles were investigated in an in vitro Caco-2 cell model and an ex vivo rat jejunum model. The mucoadhesion of the nanosystems was evaluated using excised rat jejunum. All of the nanoparticulate systems interacted with the Caco-2 cells decreasing the transepithelial electric resistance (TEER) and increasing Lucifer Yellow (LY) Papp (paracellular pathway marker). All the nanosystems improved FD4 Papp, with the exception of the nanoparticles based on TMC with the highest QD. In this case an entrapment of nanoparticles into Caco-2 cells was supposed. Analogous results were obtained using the excised rat jejunum model. The increase in QD of TMC was seen to favour the mucoadhesion, resulting in a prolonged residence time on intestinal mucosa. The nanoparticle penetration into excised rat jejunum tissue, observed by means of CLSM, suggested that the mucoadhesive properties delayed the absorption of nanoparticles, however they produced an increase in the contact time with intestinal epithelium, offering a better chance for internalisation. The improvement of mucoadhesion and of nanoparticle internalisation with respect to chitosan nanosystems makes the TMCs nanosystems suitable carriers for the intestinal absorption of peptides.     

Ex vivo evaluation of prolidase loaded chitosan nanoparticles for the enzyme replacement therapy.

Prolidase loaded chitosan nanoparticles were set up in order to suggest an innovative therapeutic approach for Prolidase Deficiency (PD), a rare autosomal inherited disorder of the connective tissue. The satisfactory drug loading efficiency (42.6+/-2.1%) as well as the suitable physical characteristics (mean diameter of 365.5+/-35.1nm and a positive zeta-potential of 17.94+/-0.12mV) was achieved. In order to verify the compatibility of the chitosan nanoparticles with cells, the influence of the nanoparticles on the growth and the viability (MTT assay) of cultured skin fibroblasts were determined: the nanoparticles showed a good biocompatibility up to 5mug of chitosan/10,000 fibroblasts. Uptake of chitosan nanoparticles by fibroblasts was verified by confocal microscopy using FITC-labelled chitosan nanoparticles. The ex vivo experiments were performed by incubating different amounts of prolidase loaded chitosan nanoparticles with skin human fibroblasts from PD patients for scheduled times. The restored prolidase activity was quantitatively monitored by a capillary electrophoretic method and confirmed by cells morphological observations. Standing from the nanoparticles internalization, the enzymatic activity was progressively restored reaching the best value (about 66%) after 5 days of co-incubation. Moreover, prolidase loaded chitosan nanoparticles permitted to restore prolidase activity in PD fibroblasts for a prolonged period of time (8 days).     

Determination of platinum in plasma of patients affected by inoperable lung carcinoma treated with radiotherapy and concurrent low-dose continuous infusion ofcis-dichlorodiammine platinum(II)

Platinum microquantities were determined in plasma of patients affected by lung carcinoma during treatment with radiotherapy (RT) and concurrent low-dose continuous infusion ofcis-dichlorodiammineplatinum(II) (CDDP). RT was given at 50 Gy in continuous course; CDDP was continuously infused at 4 mg/m² daily for 100h/week for 5 weeks, and the infusions were separated by 68h of rest. The percentage of free drug versus total drug in plasma was about 3%. It did not vary with therapy duration and was not significantly different from that found in 5-day continuous infusions at much higher daily doses. Never-theless, maximal values of free Pt in plasma were very low and agreed with the low level of CDDP toxicity encountered on the present administration schedule.     

Relationships of serum leptin to clinical and anthropometric findings in obese patients

Background: The authors evaluated the relationship between leptin and the clinical, anthropometric and metabolic variables connected to the metabolic syndrome in obese individuals. Methods: A large group of patients with different degrees of obesity was investigated: body mass index (BMI) values, serum leptin, fasting glucose and insulin, triglycerides and HDL-cholesterol concentrations, insulin resistance index and blood pressure were measured. Results: On multiple regression analysis, serum leptin levels appeared to be positively correlated to the BMI and to the serum HDL-cholesterol concentration. Principal component factor analysis revealed three factors, explaining 61.3% of the total variance of the sample. General features of these factors were: factor 1 - BMI values and serum leptin and fasting glucose concentration; factor 2 - systolic and diastolic blood pressure and serum triglycerides and HDL-cholesterol concentration; factor 3 - fasting serum insulin concentration and insulin resistance index. Conclusions: In obese subjects multiple factors underlie the metabolic syndrome and therefore more than one mechanism may account for the clustering characteristics. In obese patients leptin loads only one factor, and therefore leptin does not appear to be a key feature in the metabolic syndrome. On the contrary, multiple correlation and factor analysis data give rise to the hypothesis that in obese patients, leptin may play a protective role against cardiovascular risk.     

Effects of S 21403 on hormone secretion from isolated rat pancreas at different glucose concentrations

The plasma cholesterol-lowering effect and mechanism thereof of a choleretic phloracetophenone or 2,4,6-trihydroxyacetophenone (THA) were investigated in hypercholesterolemic male hamsters. Intragastric administration of THA (300-600 micromol/kg) twice a day for 7 days to these animals caused a dose- and time-dependent decrease in both plasma cholesterol and triglyceride levels. THA at a dose of 400 micromol/kg reduced the cholesterol and triglyceride levels in plasma to 52% and 25% of the level in corresponding cholesterol-fed controls, respectively, with decreases in both plasma very low density lipoprotein and low density lipoprotein cholesterol but not in high density lipoprotein cholesterol. THA did not significantly alter total hepatic cholesterol content but significantly increased the excretion of both bile acids and cholesterol into the intestinal lumen for elimination. Corresponding to the increase in bile acid excretion, THA caused a seven-fold increase in hepatic cholesterol 7alpha-hydroxylase activity. These results suggest that THA exerts its cholesterol lowering effect by increasing hepatic cholesterol 7alpha-hydroxylase activity which increases hepatic conversion of cholesterol to bile acid for disposal via biliary secretion. This compound may have a potential for future development as a therapeutic agent for lowering lipids in hypercholesterolemic patients.     

Nephrogenic fibrosing dermopathy after liver transplantation successfully treated with plasmapheresis

Nephrogenic fibrosing dermopathy (NFD) is a recently described cutaneous fibrosing disorder associated with renal dysfunction. It appears similar to scleromyxedema but with some notable exceptions, including the lack of involvement of the face and absence of plasma cells on histology, systemic involvement, and paraproteinemia. Patients can present with thickened or edematous skin with indurated papules and plaques involving the extremities and the trunk. We report the first three cases of NFD after liver transplantation successfully treated with plasmapheresis. Two patients underwent liver transplantation for hepatitis C virus-induced cirrhosis and one for hepatitis B virus-induced cirrhosis. All the patients had encephalopathy, refractory ascites, and malnutrition prior to transplantation. Like those patients with NFD, all three of our patients had renal dysfunction and required hemodialysis before and after transplantation. Two were not dependent on dialysis at the time of diagnosis, however. These patients had excellent liver allograft function, but the other patient had allograft failure secondary to recurrent hepatitis C. Immunosuppression therapy consisted of basiliximab, mycophenolate mofetil, calcineurin inhibitor, and prednisone. The patients developed "woody" skin induration of the distal extremities, erythematous papules, and contractures at 1, 2, and 120 months after transplantation. Skin biopsies resembled NFD. No paraproteinemia was evident. One to three 5-day courses of plasmapheresis resulted in moderate to marked clinical improvement. The improvement of the kidney function in two of our patients did not appear to correlate with that of the skin disorder, because the kidney function was improving at the time the diagnosis of NFD was made. In conclusion, we report the first three cases of NFD after liver transplantation. Plasmapheresis was moderately successful in resolving the skin-indurated papules, severe skin induration, and associated joint contractures. Preliminary studies (unpublished data) show that decreasing plasma levels of transforming growth factor-beta1 after plasmapheresis appear to correlate with the amelioration of this clinical condition.     

Preventable infant mortality and quality of health care: maternal perception of the child's illness and treatment

This study used a qualitative methodology to analyze the discourse of mothers from Greater Metropolitan Belo Horizonte, Minas Gerais, Brazil, whose infant children had died from what were considered avoidable causes (diarrhea, malnutrition, and pneumonia), seeking to elucidate the factors associated with utilization of health care services. Identification of the illness by the mother was related to perception of specific alterations in the child's state of health. Analysis of the alterations helped identify the principal characteristics ascribed to each alteration and their relationship to the search for treatment. The authors also studied the mother's assessment of treatment received at health care facilities; 43.0% of the cases involved problems related to the structure of health care services or the attending health care professionals. In 46.0% of the cases, mothers associated the child's death with flaws in the health care service. The study group showed a variety of interpretations of illness, often distinct from the corresponding biomedical concepts. The fact that attending health care personnel overlooked or underrated the mother's perception of the illness and the lack of communications between health care personnel and the child's family had an influence on the child's evolution and subsequent death.     

Carpal tunnel syndrome modifies sensory hand cortical somatotopy: a MEG study

The adult somatosensory system has shown reorganizational abilities at cortical and subcortical levels after peripheral nerve lesions. In the present study the effects of carpal tunnel syndrome (CTS) are investigated as reflected on the somatotopy of the primary cortical hand representation. Position and intensity of cortical sources activated by the separate electrical stimulation of median nerve and Digits 1, 3, and 5 of both affected and non-affected hands are evaluated by magnetoencephalographic (MEG) technique. Correlation of MEG results with patient-, physician- and neurophysiological-oriented evaluations of CTS was carried out. Patients showed changes in cortical hand somatotopy in strict relationship to self-referred assessment of symptoms and hand disability in daily activities, including: 1) a more extended representation of the affected hand when paresthesias prevailed; and 2) a more restricted representation due to lateral shift of the little finger was observed when pain symptoms dominated the clinical picture. Contralateral to the side of CTS, the cortical sources activated by Digit 5-stimulation appeared significantly enhanced with respect to contralateral ones from non-affected hand. When comparing the amplitude of peripheral sensory nerve action potentials of median and ulnar nerves to that of cortical responses (i.e., ECD strengths of M20 and M30 components after stimulation of Digits 3 and 5), a significant selective amplification of M30 with respect to M20 and sensory nerve action potential (SNAP) appeared during Digit 3 stimulation compared to that observed for Digit 5. This has been interpreted as a central magnification mechanism in brain responsiveness, possibly revealing a safety factor enabling sensory perception despite the small peripheral signal due to nerve trunk dysfunction. Hum.     

Vascular endothelial growth factor and prognosis in patients with node-negative breast cancer

Vascular endothelial growth factor (VEGF) has emerged as one of the most important angiogenic growth factors from experimental in vitro and in vivo studies. In the present study, we investigated the relationship between VEGF expression and microvessel density (MVD) and defined their prognostic relevance on a series of 242 patients with node-negative breast cancer, using immunohistochemical methods. In parallel, estrogen and progesterone receptors were quantitatively assessed using the dextran-charcoal technique and cell proliferation was evaluated as S-phase cell fraction according to (3)H-thymidine-labeling index (TLI). The percentage of VEGF-expressing cells varied from 0-95% in the different tumors and was unrelated to menopausal status, tumor size or steroid receptor status. Conversely, a significant inverse relation was observed with patient age or tumor cell proliferation, albeit with very poor correlation coefficients. A significant relation was observed between VEGF expression and MVD (r(s) = 0.55, p < 0.001). Clinical outcome analyzed as a function of high and low VEGF expression showed slight differences in terms of both disease-free survival (DFS) and overall survival (OS) that never reached statistical significance. Moreover, the trend was paradoxically in favor of patients with highly VEGF-expressing tumors. Finally, DFS and OS curves, when analyzed as a function of VEGF expression or MVD, were superimposable. In conclusion, our study did not highlight a prognostic relevance of VEGF expression in patients with node-negative breast cancer, as already observed for MVD.     

Catecholamine production and tyrosine hydroxylase expression in peripheral blood mononuclear cells from multiple sclerosis patients: effect of cell stimulation and possible relevance for activation-induced apoptosis

Sympathoadrenergic mechanisms may play a role in multiple sclerosis (MS). We examined catecholamine (CA) levels and production and tyrosine hydroxylase (TH) expression in peripheral blood mononuclear cells (PBMCs) from MS patients, and the correlation between CA production and apoptosis in PBMCs. PBMCs from MS patients had increased norepinephrine (NE) levels. However, phytohaemagglutinin (PHA)-stimulated PBMCs from MS patients with active disease synthesized less dopamine (DA) than cells from both healthy controls and patients with inactive disease. PBMCs from patients with inactive disease showed lower expression of TH. Pharmacological inhibition of TH in cultured PBMCs stimulated with PHA reduced the percentage of apoptotic cells. Since a failure of activation-induced apoptosis in immune cells may be involved in MS, it is suggested that altered CA production by PBMCs may be implicated in such dysregulation.