RESUMEN
We have investigated the influence of ex vivo expansion of human CD34(+) cord blood cells on the expression and function of adhesion molecules involved in the homing and engraftment of haematopoietic progenitors. Ex vivo expansion of umbilical cord blood CD34(+) cells for 6 d in the presence of interleukin 3 (IL-3), IL-6 and stem cell factor (SCF) or IL-11, SCF and Flt-3L resulted in increased expression of alpha 4, alpha 5, beta 1, alpha M and beta 2 integrins. However, a significant decrease in the adhesion of progenitor cells to fibronectin was observed after the ex vivo culture (adhesion of granulocyte-macrophage colony-forming units (CFU-GM) was 22 +/- 4% in fresh cells versus 5 +/- 2% and 2 +/- 2% in each combination of cytokines). Incubation with the beta 1 integrin-activating antibody TS2/16 restored adhesion to fibronectin. Transplantation of ex vivo expanded umbilical cord blood CD34(+) cells was associated with an early delayed engraftment in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Incubation of cells with the monoclonal antibody TS2/16 before transplantation almost completely abrogated NOD/SCID repopulating ability of both fresh and expanded CD34(+) cells. The seeding efficiency of fresh and expanded CD34(+) cells was similar, but markedly reduced after incubation with the TS2/16 monoclonal antibody. Our results show that functional activation of beta 1 integrins could overcome the decreased very late antigen (VLA)-4- and VLA-5-mediated adhesion observed after ex vivo expansion of haematopoietic progenitors. However, in vivo, these effects induced an almost complete abrogation of the homing and repopulating ability of CD34(+) UCB cells.
Asunto(s)
Antígenos CD34 , Integrinas/metabolismo , Leucocitos Mononucleares/fisiología , Animales , División Celular , Células Cultivadas , Sangre Fetal/inmunología , Sangre Fetal/metabolismo , Fibronectinas/metabolismo , Citometría de Flujo/métodos , Humanos , Integrina alfa4beta1 , Leucocitos Mononucleares/trasplante , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de Fibronectina/metabolismo , Receptores Mensajeros de Linfocitos/metabolismoRESUMEN
Stromal cell-derived factor-1alpha (SDF-1alpha) is a potent chemoattractant for hematopoietic progenitor cells (HPC), suggesting that it could play an important role during their migration within or to the bone marrow (BM). The integrin VLA-4 mediates HPC adhesion to BM stroma by interacting with CS-1/fibronectin and VCAM-1. It is required during hematopoiesis and homing of HPC to the BM. As HPC migration in response to SDF-1alpha might require dynamic regulation of integrin function, we investigated if SDF-1alpha could modulate VLA-4 function on BM CD34(hi) cells.CD34(hi) BM cells and hematopoietic cell lines were tested for the effect of SDF-1alpha on VLA-4-dependent adhesion to CS-1/fibronectin and VCAM-1, as well as to BM stroma. CD34(hi) BM cells that adhered to VLA-4 ligands after SDF-1alpha treatment were characterized in colony-forming and long-term culture-initiating cell (LTC-IC) assays.SDF-1alpha rapidly (1 minute) and transiently upregulated the adhesion of CD34(hi) BM cells and hematopoietic cell lines to both CS-1/fibronectin and VCAM-1, and to BM stromal cells. The upregulation of VLA-4-dependent cell adhesion by SDF-1alpha targeted primitive LTC-IC as well as committed CD34(hi) cells. SDF-1alpha-triggered enhancement in VLA-4 function was inhibited by pertussis toxin (PTx) and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Instead, activation of p44/42 MAP kinases by SDF-1alpha did not functionally correlate with enhancement of VLA-4-dependent cell adhesion. Modulation of VLA-4-mediated CD34(hi) BM cell adhesion by SDF-1alpha could play a key role in their migration within and to the BM and therefore influence their proliferation and differentiation.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Quimiocinas CXC/fisiología , Fibronectinas/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Integrinas/fisiología , Fragmentos de Péptidos/metabolismo , Receptores Mensajeros de Linfocitos/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Línea Celular , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Quimiotaxis/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/metabolismo , Humanos , Integrina alfa4beta1 , Leucemia Megacarioblástica Aguda/patología , Hígado/citología , Hígado/embriología , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Receptores CXCR4/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Células del Estroma/fisiología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
The clinical outcome of Helicobacter pylori infection may be associated with the cagA bacterial genotype. To investigate the cagA status of H. pylori-infected patients and the relationship between cagA and peptic ulcer disease, gastric biopsy specimens from 103 Caucasian patients in Brazil were analyzed by PCR. Since allelic variation in cagA exists and distinct H. pylori subgenotypes may circulate in different regions, PCR using primers for a variable 3' region of the cagA gene according to a Japanese methodology and for a consensus cagA 3' region used in Western methods was used for cagA detection. cagA was present in 53 (71%) of 75 H. pylori-positive cases when analyzed by the consensus region method and was associated with duodenal ulcer disease (P = 0.02), but not with gastric ulcer (P = 0.26), when compared to patients with duodenitis or gastritis. The variable region PCR method was able to detect 43 (57%) cagA-positive cases within the same group of H. pylori-positive patients and showed three subtypes of cagA (A, B/D, and C) that were not associated with clinical outcome. However, in 8 (18%) of the cases, more than one subtype was present, and an association between patients with multiple subtypes and disease outcome was observed when compared to patients with isolated subtypes (P = 0.048). cagA was a marker of H. pylori strains for duodenal ulcer disease in our population, and in spite of the differences in the 3' region of the cagA gene, the Japanese methodology was able to detect the cagA status in most cases. The presence of multiple subgenotypes of cagA was associated with gastric ulcer.
Asunto(s)
Antígenos Bacterianos , Proteínas Bacterianas/genética , Variación Genética , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/genética , Úlcera Péptica/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil , Femenino , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , Fumar , Población BlancaRESUMEN
OBJECTIVE: The term Barrett's esophagus refers to a premalignant condition that is characterized by the replacement of the esophageal squamous mucosa by a columnar-lined one. Preliminary studies have demonstrated reversal of Barrett's mucosa after endoscopic coagulation with different techniques associated with acid inhibition. However, most of these studies have shown that residual Barrett's glands are found underneath the new squamous epithelium in up to 40% of patients. The goal of our study is to verify whether complete restoration of Barrett's mucosa can be achieved by the combination of high power setting argon plasma coagulation and omeprazole. METHODS: A total of 33 patients (mean age: 55.2 yr, range: 21-84 yr; 21 men and 12 women) with histologically demonstrated Barrett's esophagus (mean length: 4.05 cm, range: 0.5-7 cm) were treated. Fourteen cases presented with low-grade dysplasia and one with high-grade dysplasia. All of the extent, or until a maximum of 4 cm, of the Barrett's mucosa was cauterized in each session using argon beam coagulation at a power setting of 65-70 W. All patients received 60 mg omeprazole during the treatment period. RESULTS: Complete restoration of squamous mucosa was obtained in all 33 cases after a mean of 1.96 sessions (range, 1-4). Endoscopic results were histologically confirmed. Nineteen (57.5%) patients experienced moderate to severe chest pain and odyno-dysphagia lasting for 3-10 days after the procedure. Five of these cases experienced high fever and a small volume of pleural effusion, and three patients developed esophageal strictures that needed to be dilated. Another patient developed pneumomediastinum and subcutaneous emphysema without evidences of perforation. After a mean follow-up of 10.6 months there was one endoscopic, as well as histological, recurrence of Barrett's mucosa in a patient with an ineffective laparoscopic fundoplication. CONCLUSIONS: High power setting argon plasma coagulation combined with intensive acid suppression is an effective treatment for the total endoscopic ablation of Barrett's esophagus, at least in the short term. Long-term follow-up of treated patients in whom gastroesophageal reflux is surgically or medically alleviated seems mandatory before drawing definitive conclusions about this therapy.
Asunto(s)
Esófago de Barrett/cirugía , Coagulación con Láser/métodos , Adulto , Anciano , Anciano de 80 o más Años , Antiulcerosos/uso terapéutico , Argón , Esófago de Barrett/tratamiento farmacológico , Esófago de Barrett/patología , Terapia Combinada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Omeprazol/uso terapéutico , Cuidados Posoperatorios , Complicaciones Posoperatorias/epidemiologíaRESUMEN
BACKGROUND/AIMS: The introduction of laparoscopic cholecystectomy has increased the need for preoperative diagnosis of common bile duct stones. The purpose of this study is to verify the sensitivity of the liver function tests in the detection of duct stones. METHODOLOGY: We evaluated 438 patients (223 retrospectively and 215 prospectively) who underwent endoscopic papillotomy for bile duct stones in two different services. In every case, blood samples for liver function tests levels were collected prior to endoscopic retrograde cholangiopancreatography. RESULTS: The most sensitive test was gamma-glutamyl transpeptidase, that was abnormal in 92.2% of the cases. Alkaline phosphatase was elevated in 74.7% of the patients with choledocholithiasis. The least sensitive parameter was AST, altered in only 50.8% of times. The sensitivity of all liver tests for the diagnosis of choledochal stones taken together was 94.3%. CONCLUSIONS: Liver function tests are very sensitive in the detection of common bile duct stones, however these blood tests are in the normal range of about 5% of endoscopically treated patients.
Asunto(s)
Pruebas Enzimáticas Clínicas , Cálculos Biliares/diagnóstico , Cálculos Biliares/enzimología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/sangre , Biomarcadores/sangre , Femenino , Cálculos Biliares/sangre , Cálculos Biliares/cirugía , Humanos , Hígado/enzimología , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Esfinterotomía Endoscópica , gamma-Glutamiltransferasa/sangreRESUMEN
1. The effect of the method employed to prepare liposomes and their lipid composition were evaluated in terms of the encapsulation efficiency and pharmacokinetic features of two oligodeoxynucleotides of a 21 mer: the normal (N-Odn) and the phosphorothioate (S-Odn) oligodeoxynucleotide. 2. Liposomes were prepared by the classical method of multilamellar vesicles (MV) and by the dehydration-rehydration method (DR). Two lipid mixtures were used to prepare liposomes--the predominant lipid being phosphatidylcholine (PC) and sphingomyelin (SM) respectively. 3. The DR method for liposome preparation provided the highest encapsulation efficiency, regardless of liposome lipid composition and the type of oligodeoxynucleotide involved (N-Odn or S-Odn). 4. The pharmacokinetics of free and liposome encapsulated oligodeoxynucleotides was studied in mouse following i.v. administration. Liposome encapsulated oligodeoxynucleotides exhibited a significantly lower plasma clearance and longer half-life and residence time than free oligodeoxynucleotides. The method used to obtain the liposomes affected plasma clearance, which was lower for liposomes elaborated by the DR method than for liposomes prepared with the MV method. The use of S-Odn in place of N-Odn decreased the plasma clearance of oligodeoxynucleotide when administered encapsulated in liposomes, regardless of the lipid composition and method used to obtain the liposomes.
Asunto(s)
Composición de Medicamentos/métodos , Lípidos/química , Liposomas/química , Oligodesoxirribonucleótidos/farmacocinética , Animales , Inyecciones Intravenosas , Ratones , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/sangre , Tionucleótidos/sangre , Tionucleótidos/química , Tionucleótidos/farmacocinéticaAsunto(s)
Trombosis Coronaria/tratamiento farmacológico , Fibrinolíticos/uso terapéutico , Trombina/antagonistas & inhibidores , Angina de Pecho/tratamiento farmacológico , Fibrinolíticos/farmacología , Heparina/farmacología , Hirudinas/farmacología , Humanos , Infarto del Miocardio/tratamiento farmacológico , Pautas de la Práctica en MedicinaRESUMEN
The effects of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine H-7 (a cAMP-dependent protein kinase and protein kinase C inhibitor), n-(2-[methylamino]ethyl)-5-isoquinoline-sulfonamide H-8 (a cAMP- and cGMP-dependent protein kinase inhibitor) and indomethacin (IND, a cyclooxygenase inhibitor) on both the spontaneous metastatic ability of 3LL (Lewis lung carcinoma) tumor cells and anti-tumor host response were studied. The study of tumor progression showed that H-7 and H-8 (2 mg kg(-1) day(-1) , i.p., for 8 days) significantly reduced the mean number of metastases (0.8 +/- 0.2 and 1.0 +/- 0.7, respectively, P < 0.05) with respect to the number of lung metastases (4.2 +/- 2.1) observed in the control group. In turn, the highest tumor-specific cytotoxicity response (50% increase vs. non-treated target cells) was observed when both animal and tumor cells were treated with H-8. This suggests that the protein kinase inhibitors could inhibit tumor progression toward lung metastases formation by blocking the immunosuppressor mechanism triggered by agents that increase intracellular cAMP.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores Enzimáticos/farmacología , Isoquinolinas/farmacología , Animales , Carcinoma Pulmonar de Lewis/patología , División Celular/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Progresión de la Enfermedad , Indometacina/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Trasplante de Neoplasias , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
B16 melanoma-bearing mice were treated with anti-interleukin 4 antibody, indomethacin or its combination to evaluate the ability of the primary tumor to induce lung metastasis and the antitumor host response. Flow cytometry of tumor cells incubated with sera from tumor-bearing mice showed B16 melanoma to induce a significant antitumor humoral response (39.0 +/- 1.1% positive cells versus 1.8 +/- 0.9% in the control). The treatment of tumor-bearing mice with antimouse anti-interleukin 4 monoclonal antibody plus indomethacin significantly increased (P < .01) the mean value of lung metastasis (from 6.1 +/- 3.0 in the controls to 50.8 +/- 21.8). Also, a significant increase in natural cytotoxicity against tumor cells was observed when both peripheral blood mononuclear cells and splenocytes were used as effector cells. In contrast, an antibody-dependent cellular cytotoxicity decrease was found with effector cells from both normal and tumor-bearing mice. In the former, the antibody-dependent cellular cytotoxicity decrease was 49.4% and 58.4% (P < .05) for peripheral blood mononuclear cells and splenocytes, whereas in the second case the decrease was 40.7% (P < .05) and 29.1% (P < .01), respectively. These results suggest that an efficient antibody-dependent cellular cytotoxicity response might be necessary to secure an effective host antitumor immune response.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Indometacina/farmacología , Interleucina-4/fisiología , Melanoma Experimental/inmunología , Antagonistas de Prostaglandina/farmacología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica , Neoplasias Pulmonares/secundario , Masculino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
The efficiency of both anionic and cationic liposomes as vectors for in vivo human alpha1-antitrypsin (AAT) gene transfer was studied in mice with and without an associated partial hepatectomy. The pTG7101 plasmid, containing the full-length human AAT gene, was encapsulated in small liposomes bearing 10% of negatively (phosphatidylserine, PS) or positively (DOTAP) charged lipids. The results indicated that the DNA/lipid ratio was increased in cationic liposomes by inclusion of monosialoganglioside-GM1. The expression of human protein after in vivo gene transfer was quantified in mouse plasma by an ELISA procedure, and revealed that both anionic and cationic liposomes mediated the presence of human protein in mouse plasma for 2-3 weeks. This effect was prolonged (>5 months) when a partial hepatectomy was performed after treatment. In addition, it was observed that the efficacy of liposome-mediated gene transfer was more limited when the plasmid was externally associated to cationic liposomes.
Asunto(s)
Liposomas/metabolismo , alfa 1-Antitripsina/genética , Animales , Aniones/metabolismo , Cationes/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica , Técnicas de Transferencia de Gen , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Immunization of C57BL/6 mice with tumor-derived membrane-proteins encapsulated in sized liposomes (0.2 microgram/mouse) and composed by phosphatidylcholine or sphingomyelin, significantly reduced the mean values of spontaneous lung metastasis from both B16 (0.7 +/- 0.5 and 1.2 +/- 0.6, respectively) and 3LL (4.8 +/- 2.5 and 7.2 +/- 4.1, respectively) tumors, with respect to control (HEPES) groups (4.8 +/- 1.1 and 19.0 +/- 4.4, respectively). However, no significant antimetastatic effect was observed using free tumor-derived proteins (2 micrograms/mouse) or liposome vehicle alone. Specific humoral immune response after the vaccination was studied by flow cytometry of tumor cells incubated with a pooled sample from each group of immunized mice and FITC-conjugate antimouse immunoglobulins. The results showed that the highest number of positive tumor cells was identified using sera from immunized mice with sized liposomes encapsulating tumor-derived proteins whereas the immunization with the protein fraction in free form failed to induce this effect. In addition, an increased cytotoxicity towards 3LL and B16 tumor cells can also be observed when tumor cells were incubated with spleen effector cells plus specific immunosera. In conclusion, our results show that antitumor active vaccination, using sized liposomes as adjuvants, induces an antitumor host response and a significant inhibition of tumor progression.
Asunto(s)
Antígenos de Neoplasias/administración & dosificación , Inmunización , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos de Superficie/administración & dosificación , Membrana Celular/inmunología , Técnicas In Vitro , Liposomas , Neoplasias Pulmonares/inmunología , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/secundario , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Bazo/inmunologíaRESUMEN
A plasmid (pTG7101) containing the full-length human alpha 1-antitrypsin gene was encapsulated in small liposomes and used for "in vivo" gene transfer to mouse hepatocytes, by i.v. injection (100 ng DNA/mouse and dose). The expression of human protein was evaluated by microspectrophotometry after human alpha 1-antitrypsin immunoperoxidase reaction on liver cryosections and the presence in mouse plasma of de novo synthesized protein was detected by ELISA analysis. Our results indicate that a single dose of encapsulated plasmid induces the expression of human alpha 1-antitrypsin in mouse hepatocytes and a large effect (70%) remains two weeks after treatment. However, no effect was observed when mice were treated with buffer or free plasmid (100 ng/mouse) plus an equivalent lipid dose of empty liposomes. In addition, whereas no additive effect was observed after repetitive treatment-doses, the partial hepatectomy three hours after a single treatment-dose, significantly increased the presence of human alpha 1-antitrypsin in mice plasma.