Qualitative Histologic Assessment vs. Geomorphometric Analysis of Nerve Fiber Shape after the Intraneural Application of Liposomal Bupivacaine / Evaluación Histológica Cualitativa Versus Análisis Geomorfométrico de la Forma de la Fibra Nerviosa Después de la Aplicación Intraneural de Bupivacaína Liposomal

SUMMARY: The preserved form of all components of the nerve fiber is a prerequisite for the proper conduction of the nerve impulse. various factors can change the shape of nerve fibers. In everyday practice, qualitative histological analysis is the gold standard for detecting changes in shape. Geometric morphometry is an innovative method that objectively enables the assessment of changes in nerve fibers' shape after local anesthetics action. A total of sixty sciatic nerves were used as material, which was intraneural injected with saline solution in the control group (n=30), and a solution of 1.33 % liposomal bupivacaine (n=30) in the test group. After the animals were sacrificed, nerve samples were taken and histological preparations were made. The preparations were first described and examined using a qualitative histological method, after which digital images were made. The images were entered into the MorphoJ program and processed using the method of geometric morphometry. Qualitative histological examination revealed no differences in nerve fibers after intraneurally applied physiological solution and liposomal bupivacaine. Using the method of geometric morphometry, a statistically significant change in the shape of axons was found after intraneurally applied saline solution and liposomal bupivacaine (p=0.0059). No significant differences in histological changes were found after the qualitative histological analysis of nerve fiber cross-section preparations. A statistically significant change in the shape of nerve fiber axons was observed after geometric morphometric analysis of digital images after intraneural application of saline and liposomal bupivacaine.

La forma conservada de todos los componentes de la fibra nerviosa es un requisito previo para la conducción correcta del impulso nervioso. Varios factores pueden cambiar la forma de las fibras nerviosas. En la práctica diaria, el análisis histológico cualitativo es el estándar de oro para detectar cambios de forma. La morfometría geométrica es un método innovador que permite evaluar objetivamente los cambios en la forma de las fibras nerviosas después de la acción de los anestésicos locales. Se utilizó como material un total de sesenta nervios ciáticos, que se inyectaron intraneuralmente con solución salina en el grupo control (n=30), y una solución de bupivacaína liposomal al 1,33 % (n=30) en el grupo de prueba. Después de sacrificados los animales, se tomaron muestras de nervios y se realizaron preparaciones histológicas. Primero se describieron y examinaron las preparaciones utilizando un método histológico cualitativo, después de lo cual se tomaron imágenes digitales. Las imágenes fueron ingresadas al programa MorphoJ y procesadas mediante el método de morfometría geométrica. El examen histológico cualitativo no reveló diferencias en las fibras nerviosas después de la aplicación intraneural de solución fisiológica y bupivacaína liposomal. Usando el método de morfometría geométrica, se encontró un cambio estadísticamente significativo en la forma de los axones después de la aplicación intraneural de solución salina y bupivacaína liposomal (p = 0,0059). No se encontraron diferencias significativas en los cambios histológicos después del análisis histológico cualitativo de las preparaciones de secciones transversales de fibras nerviosas. Se observó un cambio estadísticamente significativo en la forma de los axones de las fibras nerviosas después del análisis de morfometría geométrica de imágenes digitales después de la aplicación intraneural de solución salina y bupivacaína liposomal.

Comparison of amphotericin B lipid complex (Abelcet) and liposomal amphotericin B (Ambisome) on rat kidney: a morphological evaluation / Comparasión de la anfotericina B complejo lipídico (Abelcet) y la anfotericina B liposomal (AmBisome) en el riñón de rata: una evaluación morfológica

The aim of our study was to compare the nephrotoxic effects of liposomal amphotericin B (Ambisome) and amphotericin B lipid complex (Abelcet) on rat kidneys at short (14 days) and long term (28 days) treatment applications. Thirty-six male Wistar rats were included and divided into six groups (n=6). Groups 1 and 4 are composed as control groups by administrating intraperitoneal (ip) 0, 9 molar Serum physiologic for a period of 14 and 28 days respectively. Group 2 and 3 are treated with 5 mg/kg Ambisome and 5 mg/kg Abelcet for 14 days respectively, group 5 and 6 are treated with same agents for 28 days respectively. Then, the rats were transcardially perfused, samples were taken from cortex and medulla regions of kidneys. The micrographs of group 1 and 4 were seen as normal. For short term treatment, some morphological changes were seen in proximal tubule cells in group 3 whereas in group 2 the graphs were observed as normal. However, after long term drug using in group 5 and 6 there were vacuolization, increased lysosomal structures and deep basal folding's into tubular cells lumen. These experiments establish that renal damage were seen in short and long term use of Abelcet and long term use of Ambisome.

El objetivo de nuestro estudio fue comparar los efectos nefrotóxicos de la anfotericina B liposomal (AmBisome) y anfotericina B en complejo lipídico (Abelcet) sobre riñones de ratas, en el tratamiento de aplicación a corto (14 días) y largo plazo (28 días). Fueron incluidas en el estudio 36 ratas Wistar machos, divididas en seis grupos (n = 6). Los Grupos 1 y 4 fueron grupos de control mediante la administración intraperitoneal (ip) de 0, 9 molar de suero fisiológico durante un periodo de 14 y 28 días respectivamente. Los Grupos 2 y 3 fueron tratados con 5 mg/kg de ambisome y 5 mg/kg abelcet durante 14 días respectivamente, y finalmente los grupos Grupos 5 y 6 tratados con los mismos agentes durante 28 días, respectivamente. Luego, las ratas fueron perfundidas vía transcardíaca, y se tomaron muestras de la corteza y la médula renal. Las micrografías de los grupos 1 y 4 se observaron normal. En el tratamiento a corto plazo, algunos cambios morfológicos se observaron en las células del túbulo proximal en el grupo 3, mientras que en el grupo 2 los gráficos se observaron normales. Sin embargo, después de utilizar la droga a largo plazo en los grupos 5 y 6 hubo vacuolización, aumento de las estructuras lisosomales y un profundo plegamiento basal de las células del lumen tubular. Estos experimentos establecen que el daño renal se produce en el uso a corto y largo plazo de Abelcet, y largo plazo de Ambisome.

Gene transfer using liposome-complexed adenovirus seems to overcome limitations due to coxsackievirus and adenovirus receptor-deficiency of cancer cells, both in vitro and in vivo

Use of adenoviruses as vehicle for gene therapy requires that target cells express appropriate receptors such as coxsakievirus and adenovirus receptor (CAR). We show here that CAR-deficiency in cancer cells, that limits adenoviral gene delivery, can be overcome by using adenovirus complexed with the liposome, Ad-PEGPE [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000]. We first confirmed that CT-26 mouse colon cancer cells are deficient in CAR by RT-PCR, and then showed that CT-26 cells infected with Ad-GFP/PEGPE exhibited highly enhanced expression of green fluorescent protein (GFP), compared with those infected with Ad-GFP. GFP expression depends on the dose of liposome and adenovirus. Luciferase expression in livers treated with Ad-luc/PEGPE was about 1,000-fold less than those infected with Ad-luc. In a liver metastasis mouse tumor model developed by intrasplenic injection of CT-26 cells, luciferase expression following i.v. injection of Ad-luc/PEGPE was significantly higher in tumors than in adjacent non-neoplastic liver. Following systemic administration of Ad-GFP/PEGPE, GFP expression increased in tumors more than in adjacent liver while the reverse was true following administration of Ad-GFP. In the latter case, GFP expression was higher in liver than in tumors. This study demonstrates that systemic delivery of PEGPE-adenovirus complex is an effective tool of adenoviral delivery as it overcomes limitation due to CAR deficiency of target cells while reducing hepatic uptake and enhancing adenoviral gene expression in tumors.

Liposomal daunorubicin and dexamethasone as a treatment for multiple myeloma: the DD Protocol

CONTEXTO E OBJETIVO: A daunorrubicina lipossomal tem sido usada no tratamento em várias doenças hematológicas malignas, incluindo mieloma múltiplo (MM). O objetivo deste estudo foi avaliar a eficácia, efeitos colaterais e toxicidade da daunorrubicina lipossomal and dexametasona no Protocolo DD. TIPO DE ESTUDO E LOCAL: Estudo prospectivo, realizado nos hospitais Sírio Libanês, São Camilo, Brasil e no Hospital Alemão Oswaldo Cruz. MÉTODOS: 20 pacientes com MM ativo receberam daunoxome (25-30 mg/m²/dia) por três dias consecutivos, mensal, por quatro meses (total de quatro ciclos), e dexametasona, 10 mg a cada seis horas por quatro dias consecutivos (dia 1 - 4, 9 - 12 e 17 - 20), também mensal. RESULTADOS: A mediana entre o diagnóstico e o início do protocolo DD foi de 13 meses. Quinze pacientes receberam alguma quimioterapia anterior ao protocolo DD. Uma redução maior que 50% do pico monoclonal sérico foi observada em seis paciente após o primeiro ciclo do DD (30%), em seis pacientes após o segundo ciclo (30%), em quatro pacientes após o terceiro ciclo (20%) e em quatro pacientes não houve redução (20%). No início do protocolo, 17 pacientes (85%) apresentavam anemia e em 12 destes pacientes (70%) a anemia foi corrigida. Doença progressiva foi observada em três pacientes (15%), um apresentava resposta mínima, quatro pacientes (20%) apresentaram resposta parcial e 12 (60%) apresentaram resposta completa. A toxicidade hematológica foi aceitável.Toxicidade em trato gastrointestinal foi leve, consistindo em náusea (10%) e anorexia (15%), sem episódios de vômito. CONCLUSÃO: Este tratamento apresentou uma baixa toxicidade, uma boa taxa de resposta e pode ser usado previamente ao transplante de medula óssea autogênico.

Effective glycemic control achieved by transplanting non-viral cationic liposome-mediated VEGF-transfected islets in streptozotocin-induced diabetic mice

Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microgram/microliter cDNA and 25 microliter effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.

CpG DNA, liposome and refined antigen oral cholera vaccine.

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.

Liposome entrapped allergen reduces plasma histamine in sensitized mice.

Immunotherapy of allergic diseases is associated with problems of adverse systemic reactions. We have shown earlier that liposome entrapped allergen (LEA) is effective in inducing IgG response and restricting IgE response in immunized mice. This mode of treatment may be more effective and safer if it can prevent anaphylaxis. To determine this feature, mice were administered allergen preparations repeatedly and later challenged with the same allergen. Mice given liposomal preparation showed lower specific IgE response as compared to the mice given free allergen or alum adsorbed allergen of Artemisia scoparia. Specific IgG response was higher in mice immunized with LEA. The mice immunized with liposomal preparation survived whereas others injected with free allergen or alum adsorbed allergen died probably due to anaphylaxis. High levels of histamine were observed in mice injected with free allergen as compared to the mice injected LEA. The increase in plasma histamine level may be the cause of anaphylaxis during allergen challenge. In conclusion, LEA could be used as a safe and effective mode of immunotherapy for allergy diseases, since it reduces plasma histamine levels considerably thereby reducing the chances of anaphylaxis.

Utilidade terapeutica dos lipossomas / Therapeutic utility of lipossomes

Neste artigo e feita uma revisao da utilidade dos lipossomas como carreadores de medicamentos e seus demais usos na terapeutica. Sao descritos alguns aspectos relativos a preparacao dos lipossomas, suas caracteristicas e propriedades que tornam os lipossomas uma das mais importantes ferramentas da terapeutica moderna

The differential effect of liposomal amphotericin B on human erythrocytes and promastigotes of Leishmania sp / Efecto diferencial de la anfotericina B ligada a liposomas sobre eritrocitos humanos y promastigotes de Leishmania sp

In this work, it was studied the effect that amphotericin B bound to liposomes (L-AmB) exerted on the potassium permeability of human erythrocytes and also on the rate of growth on culture of promastigotes of Leishmania sp. Liposomal amphotericin B was prepared by adding AmB to sonicated liposomes composed of egg-phosphatidylcholine (PC) or dipalmitoyl-phosphatidylcholine (DPPC). Incubation of human erythrocytes with free AmB (dissolved in organic solvent) led to a rapid enhancement of potassium efflux from erythrocytes. This permeabilizing effect of amphotericin B in erythrocytes decreased when the polyene antibiotic was added dissolved in a salt solution but it was negligible when AmB was added bound to liposomes (L-AmB). Liposomal AmB preparations made of PC or DPPC were equally effective in reducing AmB -induced potassium efflux from red cells to a minimum. By contrast, it was found that exposure of Leishmania promastigotes to liposomal AmB (PC or DPPC L-AmB) led to an inhibition of cell growth rate in a magnitude comparable to that exerted by AmB added in salt solution. Short term measurements of the magnitude of cell lysis after treating Leishmania promastigotes with amphotericin B revealed that the lytic effect of AmB added in aqueous solution, organic solvent or as PC-Liposomal AmB can be detected much earlier than that exerted by DPPC-Liposomal AmB. These results are discussed in terms of the differential capacity of AmB binding to liposomes of different composition and also on the characteristics of the mechanism of incorporation of AmB molecules to the membrane of target cells

Increased efficacy of liposomes-encapsulated praziquantel in treatment of opisthorchiasis in hamsters.

An increased efficacy of liposomes-encapsulated praziquantel was observed in the treatment of hamster opisthorchiasis. A single intracardial dose of 1.5 mg/kg of encapsulated praziquantel is as effective as an intracardial dose of 30 mg/kg or an oral dose of 100 mg/kg of free praziquantel. The suppressing activity of both free and liposomes-encapsulated praziquantel significantly decrease as the infection times increase from 1 to 5 weeks, suggesting that the young liver flukes are more susceptible to praziquantel than the adult flukes.