RESUMEN
Despite its rapid enzymatic inactivation and therefore limited activity in vivo, Gemcitabine is the standard drug for pancreatic cancer treatment. To protect the drug, and achieve passive tumor targeting, we developed a liposomal formulation of Gemcitabine, GemLip (Ø: 36 nm: 47% entrapment). Its anti-tumoral activity was tested on MIA PaCa-2 cells growing orthotopically in nude mice. Bioluminescence measurement mediated by the stable integration of the luciferase gene was employed to randomize the mice, and monitor tumor growth. GemLip (4 and 8 mg/kg), Gemcitabine (240 mg/kg), and empty liposomes (equivalent to 8 mg/kg GemLip) were injected intravenously once weekly for 5 weeks. GemLip (8 mg/kg) stopped tumor growth, as measured via in vivo bioluminescence, reducing the primary tumor size by 68% (SD +/- 8%; p < 0.02), whereas Gemcitabine hardly affected tumor size (-7%; +/- 1.5%). In 80% of animals, luciferase activity in the liver indicated the presence of metastases. All treatments, including the empty liposomes, reduced the metastatic burden. Thus, GemLip shows promising antitumoral activity in this model. Surprisingly, empty liposomes attenuate the spread of metastases similar to Gemcitabine and GemLip. Further, luciferase marked tumor cells are a powerful tool to observe tumor growth in vivo, and to detect and quantify metastases.
Asunto(s)
Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Animales , Antimetabolitos Antineoplásicos/química , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Tumoral , Química Farmacéutica , Desoxicitidina/administración & dosificación , Desoxicitidina/química , Desoxicitidina/uso terapéutico , Portadores de Fármacos , Composición de Medicamentos , Azul de Evans , Liposomas , Luciferasas/genética , Luminiscencia , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Permeabilidad , GemcitabinaRESUMEN
Changes in the expression of various activation-dependent surface markers have been reported for polymorphonuclear neutrophils (PMN) isolated from synovial fluid of patients with inflammatory joint diseases. We extend these findings to the expression of CD66 molecules and several other surface markers. Three members of the CD66 family, namely CD66a, CD66b, and CD66c, showed an up to fourfold up-regulation on synovial fluid PMN compared with peripheral blood PMN (PBG) of the same patients; CD59 was increased twofold, the expression of CD16 did not change, whereas CD62L was reduced by more than 50% on synovial fluid PMN. It is interesting that CD66a, CD66b, and CD66c showed a coordinated expression on PBG of patients and controls and a coordinated up-regulation on synovial neutrophils. In contrast, after in vitro stimulation of peripheral blood PMN with phorbol myristate acetate, CD66c was much less up-regulated compared with CD66a and CD66b. All samples of synovial fluid PMN exhibited an additional increase in the expression of CD66a, CD66b, and CD66c when stimulated with phorbol myristate acetate in vitro. Prostaglandins are known to inhibit various responses of neutrophils to inflammatory stimuli. We could show that prostaglandins inhibit N-formyl-methionyl-leucyl-phenylalanine-induced up-regulation of CD66 on peripheral blood PMN in a concentration-dependent manner.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos de Diferenciación/biosíntesis , Regulación de la Expresión Génica , Neutrófilos/metabolismo , Isoformas de Proteínas/biosíntesis , Líquido Sinovial/inmunología , Adulto , Anciano , Alprostadil/farmacología , Antígenos CD/genética , Antígenos de Diferenciación/genética , Artritis/inmunología , Artritis/patología , Artritis Psoriásica/inmunología , Artritis Psoriásica/patología , Artritis Reactiva/inmunología , Artritis Reactiva/patología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Antígenos CD59/biosíntesis , Antígenos CD59/genética , Moléculas de Adhesión Celular , Dinoprostona/farmacología , Femenino , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/genética , Humanos , Selectina L/biosíntesis , Selectina L/genética , Masculino , Persona de Mediana Edad , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Isoformas de Proteínas/genética , Receptores de IgG/análisis , Líquido Sinovial/citología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Antibodies to CD66 recognize at least five members (CD66a-e) of the carcinoembryonic antigen (CEA) family. Recombinant human single-chain Fv fragments (scFvs) that bind specifically to CD66a (biliary glycoprotein) were obtained from a naive human scFv library. The scFvs bound to the N-domain of CD66a on Chinese hamster ovary (CHO) transfectants but did not bind to freshly isolated peripheral granulocytes or to dimethylsulfoxide-treated HL-60 cells. In contrast, scFvs bound well to granulocytes that were short-term activated with N-formyl-Met-Leu-Phe or phorbol 12-myristate 13-acetate and to human HL-60 cells that were treated with all-trans-retinoic acid to induce granulocytic differentiation. Quantification of antigenic sites showed that the activation-dependent CD66a epitopes were expressed on nearly all of the CD66a molecules on CHO-biliary glycoprotein transfectants, but they were detected only on a portion of the molecules on activated polymorphonuclear neutrophils and differentiated HL-60 cells. Binding of CD66a scFvs to their neoepitopes on prestimulated PMNs induced respiratory burst, suggesting that CD66a is capable of delivering transmembrane signals in these cells.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación/inmunología , Epítopos , Fragmentos de Inmunoglobulinas/inmunología , Neutrófilos/inmunología , Animales , Antígenos CD/análisis , Antígenos de Diferenciación/análisis , Células CHO , Moléculas de Adhesión Celular , Cricetinae , Células HL-60 , Células HeLa , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Proteínas Recombinantes/inmunología , Estallido Respiratorio , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
The carcinoembryonic antigen (CEA) family comprises a group of glycoproteins including the classical CEA, nonspecific cross-reacting antigens (NCA), and biliary glycoprotein (BGP). CEA glycoproteins have been identified in many glandular and mucosal tissues. In view of their putative role in cell adhesion, protein sorting, and signal transduction, CEA glycoproteins are thought to be involved in embryogenesis, architectual integrity, and secretory mechanisms of glandular epithelia. Since there are few data available on the expression of CEA-like proteins in human skin, the aim of this study was to immunohistochemically specify and localize the CEA glycoproteins in cutaneous adult and fetal glands using a panel of well-characterized antibodies. The secretory parts of eccrine sweat glands expressed CEA, NCA-90, and BGP, whereas apocrine glands remained unreactive for CEA glycoproteins. The ductal epithelia of both eccrine and apocrine glands contained CEA and NCA-90. Sebaceous glands were stained for BGP only. Electron microscopy of sweat glands showed CEA glycoprotein expression in cytoplasmic organelles and on microvilli lining the ductal surface. In sebaceous glands, BGP were demonstrated in small vesicles and along the cell membranes of differentiating sebocytes. Fetal development of cutaneous glands was associated with early expression of CEA glycoproteins. Additionally, mice transgenic for human CEA were shown to express CEA in sweat glands. The overall distribution of CEA glycoproteins in cutaneous glands was consistent with that in epithelia of other glandular tissues.
Asunto(s)
Envejecimiento/metabolismo , Antígeno Carcinoembrionario/química , Glicoproteínas/fisiología , Glándulas Sebáceas/química , Piel/química , Glándulas Sudoríparas/química , Adulto , Animales , Feto/metabolismo , Humanos , Ratones , Ratones Transgénicos , Glándulas Sebáceas/ultraestructura , Piel/embriología , Piel/ultraestructura , Glándulas Sudoríparas/ultraestructuraRESUMEN
On the basis of compelling preclinical data in cats and dogs, we initiated a clinical gene therapy study in nine patients with advanced solid tumors using xenogeneic fibroblasts secreting human interleukin (IL)-2 (Vero-IL-2 cells). Cohorts of three successive patients with tumors accessible to computed tomography- or ultrasound-guided injection were treated repeatedly with 5 x 10(5), 5 x 10(6), or 5 x 10(7) Vero-IL-2 cells. The endpoints of the study were feasibility, toxicity, and the clinical and biological effects of this novel approach to immunotherapy of cancer. Histopathological, immunological, and molecular analyses were performed on biopsy specimens of tumors and blood samples before, during, and after treatment. Treatment was well tolerated, and toxicity consisted of transient fever in one patient and short-lived, mild itching and erythema in two others. One patient with soft-tissue sarcoma showed a reduction of >90% and >50% of the volume of two distant, noninjected metastases, lasting for 29+ and 26 months, respectively. Four other patients showed stabilization of their disease for 3-9 months; of these patients, one with melanoma developed marked vitiligo. We conclude that repeated injections of < or =5 x 10(7) Vero-IL-2 cells are feasible and safe in heavily pretreated patients with advanced solid tumors. An additional evaluation of an intratumoral application of Vero-IL-2 seems warranted.
Asunto(s)
Citocinas/genética , Terapia Genética , Interleucina-2/genética , Adulto , Anciano , Animales , Biopsia , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/terapia , Complejo CD3/genética , Chlorocebus aethiops , Femenino , Terapia Genética/efectos adversos , Humanos , Inmunohistoquímica , Interleucina-2/administración & dosificación , Interleucina-2/metabolismo , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Radiografía , Células Tumorales Cultivadas/metabolismo , Células Vero , Vitíligo/inducido químicamenteRESUMEN
Monoclonal antibodies (MoAbs) were produced against the fluorescence marker fluorescein isothiocyanate (FITC). FITC was used as a hapten to label different proteins and the anti-FITC MoAbs were used to identify these labelled proteins in a solid-phase radioimmunoassay and in cellular radioimmuno-binding assays for the demonstration of antigens and antibodies.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Fluoresceínas/inmunología , Radioinmunoensayo/métodos , Tiocianatos/inmunología , Animales , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Fusión Celular , Línea Celular , Gonadotropina Coriónica/análisis , Gonadotropina Coriónica/inmunología , Neoplasias del Colon/análisis , Fluoresceína-5-Isotiocianato , Haptenos/inmunología , Humanos , Indicadores y Reactivos , Ratones , Ratones Endogámicos BALB CRESUMEN
A combination of fluorescence-activated cell sorting and HAT medium selection has been used to establish bispecific antibody (biAbs)-producing hybrid hybridomas. For this purpose hypoxanthine-guanine phosphoribosyl transferase (HGPRT)-deficient mutants were isolated from a hybridoma line (D11-DG2) producing anti-CEA antibodies by 8-azaguanine treatment. The resulting HAT-sensitive hybrid cells were stained with the fluorescence marker tetramethyl rhodamine isothiocyanate (TRITC) and fused by polyethylene glycol (PEG) with HAT-non-sensitive unstained hybrid cells producing antibodies to horseradish peroxidase (POD). Fluorescent fused hybrid hybridomas as well as non-fused stained anti-CEA cells were separated from the unstained anti-POD cells using a fluorescent activated cell sorter (FACS). Finally, non-fused enzyme-deficient anti-CEA cells were eliminated by cultivation in HAT selection medium which permits only an outgrowth of HAT-resistant hybrid hybridoma cells containing the genes for producing both antibodies.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Antígeno Carcinoembrionario/inmunología , Peroxidasa de Rábano Silvestre/inmunología , Hibridomas/inmunología , Animales , Azaserina , Antígeno Carcinoembrionario/aislamiento & purificación , Separación Celular , Células Cultivadas , Medios de Cultivo , Citometría de Flujo , Colorantes Fluorescentes , Peroxidasa de Rábano Silvestre/aislamiento & purificación , Hipoxantina , Hipoxantinas , Ratones , Rodaminas , TimidinaRESUMEN
Gene and immunotherapeutic approaches to treat human malignant tumors are reviewed. Special attention is given to the different strategies of cancer gene therapy and to recent aspects of cytokine-supported tumor immunotherapy or tumor-specific vaccination. The limitations of these therapy approaches are critically discussed especially with respect to immune escape mechanisms.
Asunto(s)
Terapia Genética , Inmunoterapia , Neoplasias/terapia , Animales , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Citocinas/uso terapéutico , Humanos , Melanoma/inmunología , Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Escape del TumorRESUMEN
Monoclonal antibody D11-DG2 (DG2) against carcinoembryonic antigen (CEA) was examined for suitability for radioimmunodetection of human tumors grown in nude mice. Antibodies DG2 and a control antibody of the same IgG1 subclass were labeled with 131I and injected into mice bearing one of three types of CEA-containing tumors (cell lines LS 174T, HT-29 and Rec S) and/or a CEA-negative tumor (Rec R). Gamma-camera imaging and distribution studies revealed that CEA-containing tumors selectively accumulate DG2 but Rec R does not. As the tumors differ in CEA-content, the highest accumulation of 131I-DG2 (corresponding to the best scintigraphic imaging) was found in LS 174T tumors, intermediate in Rec S and lowest in HT-29 tumors. The mean tumor-to-blood ratios on the sixth day after antibody administration were 4.6, 3.2, and 2.1, respectively, in the control experiments the value of this parameter was always lower than 1. The results showed the applicability of DG2 for immunoscintigraphic studies in patients. Furthermore, a positive correlation was found between the uptake of anti-CEA antibody and CEA-content in the tumors.
Asunto(s)
Anticuerpos Monoclonales , Antígeno Carcinoembrionario/inmunología , Neoplasias Experimentales/diagnóstico por imagen , Animales , Antígeno Carcinoembrionario/análisis , Humanos , Radioisótopos de Yodo , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neoplasias Experimentales/inmunología , Cintigrafía , Trasplante HeterólogoRESUMEN
On the basis of compelling preclinical data in cats and dogs we initiated a clinical gene therapy study in nine patients with advanced solid tumors using xenogeneic fibroblasts secreting human IL-2 (Vero-IL-2 cells). Cohorts of three successive patients with tumors accessible to CT- or ultrasound-guided injection were treated repeatedly with 5 x 10(5), 5 x 10(6), or 5 x 10(7) Vero-IL-2 cells. Endpoints of the study were feasibility, toxicity, and clinical and biological effects of this novel approach to immunotherapy of cancer. Histopathological, immunological and molecular analyses were performed on biopsy specimens of tumors and blood samples from before, during and after treatment. Low levels of serum antibodies to Vero cells developed in 2/9 patients. Analysis of tumor biopsies showed increased expression of CD3 mRNA and enhanced tumor infiltration with varying lymphocyte subpopulations after treatment. In addition, monoclonal alterations of the TCR repertoire of blood and tumor lymphocytes were observed. Treatment was well tolerated and toxicity consisted of transient fever in one patient and short-lived, mild itching and erythema in two others. One patient with soft tissue sarcoma showed a more than 90% and more than 50% reduction of the volume of two distant, non-injected metastases, respectively, lasting for 22+ months. Four other patients showed stabilization of their disease for three to nine months, among whom was a patient with melanoma who developed marked vitiligo. We conclude that repeated injection of up to 5 x 10(7) Vero-IL-2 cells was safe and showed biological and clinical activity in heavily pretreated patients with advanced solid tumors. Further evaluation of intratumoral application of Vero-IL-2 seems warranted.
Asunto(s)
Terapia Genética/métodos , Interleucina-2/genética , Neoplasias/terapia , Adulto , Anciano , Animales , Antígenos CD/análisis , Gatos , Chlorocebus aethiops , Protocolos Clínicos , Citocinas/análisis , Perros , Femenino , Terapia Genética/efectos adversos , Humanos , Inmunoterapia/efectos adversos , Inmunoterapia/métodos , Interleucina-2/biosíntesis , Masculino , Persona de Mediana Edad , Neoplasias/inmunología , Neoplasias/patología , Transfección/métodos , Trasplante Heterólogo , Células VeroRESUMEN
The authors present their experience gained in preparing, isolating and labeling antibodies with radionuclides for the purpose of using them in immunoscintigraphy. The experimental part includes results obtained with different labeled antibodies and their F/ab/2 fragments in distribution studies, involving also immunoscintigraphic imaging of tumors. The clinical part presents results of immunoscintigraphy obtained with the commercial antibody kits Iodomab and Imacis in patients with tumors of the digestive tract.
Asunto(s)
Anticuerpos Monoclonales , Neoplasias Gastrointestinales/diagnóstico por imagen , Radioisótopos de Yodo , Animales , Humanos , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , CintigrafíaAsunto(s)
Antígenos de Neoplasias , Moléculas de Adhesión Celular , Glicoproteínas de Membrana/fisiología , Antígenos CD , Antígenos de Diferenciación Mielomonocítica/fisiología , Antígenos CD11/metabolismo , Antígeno Carcinoembrionario/genética , Adhesión Celular/inmunología , Proteínas Ligadas a GPI , Glicosilación , Granulocitos/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Transducción de Señal/inmunologíaAsunto(s)
Antígenos CD/fisiología , Glicoproteínas/fisiología , Animales , Antígeno Carcinoembrionario/genética , Moléculas de Adhesión Celular , Cromosomas Humanos Par 19/genética , Células Epiteliales/metabolismo , Granulocitos/metabolismo , Humanos , Células Asesinas Activadas por Linfocinas/metabolismo , Células Asesinas Naturales/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Activación Neutrófila/inmunología , Linfocitos T/metabolismoAsunto(s)
Antígeno Carcinoembrionario/fisiología , Empalme Alternativo , Antígenos CD11/metabolismo , Moléculas de Adhesión Celular/metabolismo , Cromosomas Humanos Par 19/genética , Granulocitos/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Neisseria gonorrhoeae/inmunología , Neisseria meningitidis/inmunología , Activación Neutrófila/inmunologíaAsunto(s)
Antígenos CD/fisiología , Antígeno Carcinoembrionario/fisiología , Bacterias/metabolismo , Cromosomas Humanos Par 19/genética , Células Epiteliales/metabolismo , Glicosilación , Humanos , Macrófagos del Hígado/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/diagnóstico , Neoplasias/metabolismoAsunto(s)
Antígenos de Neoplasias , Moléculas de Adhesión Celular , Glicoproteínas de Membrana/fisiología , Antígenos CD , Antígenos de Diferenciación Mielomonocítica/fisiología , Antígeno Carcinoembrionario/genética , Cromosomas Humanos Par 19/genética , Células Epiteliales/metabolismo , Proteínas Ligadas a GPI , Glicosilación , Granulocitos/metabolismo , Humanos , Inflamación/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína/genéticaAsunto(s)
Antígenos CD/genética , Glicoproteínas beta 1 Específicas del Embarazo/genética , Empalme Alternativo , Antígenos CD/biosíntesis , Antígenos CD/sangre , Cromosomas Humanos Par 19/genética , Femenino , Glicosilación , Humanos , Hígado/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Placenta/metabolismo , Embarazo , Glicoproteínas beta 1 Específicas del Embarazo/biosíntesis , Estructura Terciaria de Proteína/genéticaRESUMEN
We present a novel concept of an implantable active microport based on micro technology that incorporates a high-resolution volumetric dosing unit and a drug reservoir into the space of a conventional subcutaneous port. The controlled release of small drug volumes from such an "active microport" is crucial e.g. for innovative methods in cancer treatment or pain therapy. Our microport system delivers a flow rate in the range of 10-1,000 mul/h and enables a patient-specific release profile. The core of our device is a two-stage piezoelectric micropump. It features a backpressure-independent volumetric dosing capability i.e. a stable flow rate is ensured up to a backpressure of 30 kPa. The stroke volume and hence the resolution of the mircopump is voltage controlled and can be preset between 10 and 200 nl. A miniaturized high-performance electronic control unit enables freely programmable dosing profiles. This electronic circuit is optimized for both energy consumption and weight which are both essential for a portable device. The data of an implemented pressure sensor are used to permanently monitor the dosing process and to detect a potential catheter occlusion. A polyurethane soft lithography process is introduced for the fabrication of the prototype. Therewith, a compact multilayer system has been developed which measures only 50 x 35 x 25 mm(3).