RESUMEN

BACKGROUND/OBJECTIVES: The current world-wide obesity epidemic partially results from a vicious circle whereby maternal obesity during pregnancy predisposes the offspring for accelerated weight gain and development of metabolic syndrome. Here we investigate whether low-grade inflammation, characteristic of the obese state, provides a causal role for this disastrous fetal programming in mice. METHODS: We exposed pregnant and lactating C57BL/6JBom female mice to either high-fat diet (HFD), or continuous infusion of lipopolysaccharide (LPS), a potent trigger of innate immunity, and studied offspring phenotypes. RESULTS: Both maternal LPS or HFD treatments rendered the offspring hyperphagic and inept of coping with a HFD challenge during adulthood, increasing their adiposity and weight gain. The metabolic effects were more pronounced in female offspring, while exposed male offspring mounted a larger inflammatory response to HFD at adulthood. CONCLUSIONS: This supports our hypothesis and highlights the programming potential of inflammation in obese pregnancies.

Asunto(s)

Dieta Alta en Grasa/efectos adversos , Desarrollo Fetal/fisiología , Inflamación/fisiopatología , Resistencia a la Insulina/fisiología , Obesidad/fisiopatología , Aumento de Peso/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal , Fenómenos Fisiologicos de la Nutrición Prenatal/fisiología

RESUMEN

BACKGROUND AND PURPOSE: High-dose intravenous immunoglobulin (IVIg) is an established treatment for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Although Fc receptors on natural killer cells have been suggested as a target for IVIg, the pharmacological effects are not yet clarified. We hypothesize that IVIg therapy, dependent on the plasma IgG level, suppresses the cytotoxic capacity by a reduction in numbers of NK cells and their Fc receptor CD16. PATIENTS AND METHODS: Ten consecutive patients with CIDP in maintenance therapy with IVIg were studied before and immediately after the infusion of 0.7-2.0 g/kg IVIg. Peripheral blood mononuclear cell samples from these patients were analyzed immediately after isolation using flow cytometry and cytotoxicity assays. RESULTS: We found that following IVIg treatment, the cytotoxic activity of NK cells in CIDP patients was suppressed, partly caused by a dose-dependent decline in the number of circulating NK cells. In addition, a dose-dependent blockage of CD16 occurred. CONCLUSIONS: The study implies that IVIg infusion induces a substantial decline in the number of peripheral NK cells and a suppression of NK-cell-mediated cytotoxicity. We propose that these impairments of the NK cells contribute to the therapeutic effect of IVIg in CIDP.

Asunto(s)

Inmunoglobulinas Intravenosas/administración & dosificación , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/tratamiento farmacológico , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/inmunología , Receptores Fc/metabolismo , Adulto , Anciano , Pruebas Inmunológicas de Citotoxicidad , Relación Dosis-Respuesta Inmunológica , Femenino , Proteínas Ligadas a GPI/efectos de los fármacos , Proteínas Ligadas a GPI/metabolismo , Humanos , Inmunoglobulina G/sangre , Inmunoglobulinas Intravenosas/sangre , Inmunosupresores/farmacología , Células Asesinas Naturales/patología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/fisiopatología , Receptores Fc/fisiología , Receptores de IgG/efectos de los fármacos , Receptores de IgG/metabolismo , Adulto Joven

RESUMEN

Human herpesvirus 6B (HHV-6B) is the causative agent of the common childhood febrile illness, exanthema subitum. The virus is predominantly regarded as a T-cell tropic virus, although in reality it has the ability to infect a wide variety of cell types including monocytes, macrophages and dendritic cells (DC). Although DC are important immune regulators, the modulating effects of HHV-6B on DC are controversial. Here, we examine the phenotypic and functional consequences of HHV-6B infection of DC. The addition of HHV-6B to immature DC led to expression of the nuclear viral p41 protein and cell surface expression of the viral glycoprotein gp60/110 consistent with HHV-6B infection. Nevertheless, HHV-6B did not induce noticeable cytopathogenic effects or cell death in infected DC. Importantly, HHV-6B infection induced a partial phenotypic maturation of immature DC as demonstrated by a substantial increase in the expression of HLA-DR, CD86 and CD40, whereas only a minor increase in CD80 and CD83 was observed. This phenotypic maturation was, however, not followed by functional maturation, because HHV-6B infection did not induce IL-10 and IL-12p70 production in immature DC. However, infected DC were still able to react to bacteria-derived stimuli such as lipopolysaccaharide by an even more pronounced production of IL-10 and IL-12p70 when compared to that of uninfected DC.

Asunto(s)

Células Dendríticas/inmunología , Células Dendríticas/virología , Herpesvirus Humano 6/inmunología , Interleucina-10/biosíntesis , Interleucina-12/biosíntesis , Infecciones por Roseolovirus/inmunología , Antígenos CD/inmunología , Antígeno B7-1/inmunología , Antígeno B7-2/inmunología , Antígenos CD40/inmunología , Células Dendríticas/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Antígenos HLA-DR/inmunología , Humanos , Inmunoglobulinas/inmunología , Inmunofenotipificación , Interleucina-10/inmunología , Interleucina-12/inmunología , Glicoproteínas de Membrana/inmunología , Microscopía Confocal , Infecciones por Roseolovirus/sangre , Infecciones por Roseolovirus/virología , Antígeno CD83

RESUMEN

Endotoxaemia elicits a massive inflammatory insult affecting the beta2 integrin CD18. Being an adhesion molecule, CD18 is pivotal in inflammation and, moreover, exiting data suggest that CD18 is a lipopolysaccharide (LPS) receptor. Early LPS-induced inflammation is regulated by the signal regulatory protein (SIRPalpha), which is identical to the porcine panmyelocytic marker swine CD workshop 3 (SWC3), and LPS-induced downregulation of SIRPalpha has been described in vitro. The dynamic SIRPalpha/SWC3 and CD18 expression on peripheral blood mononuclear cells (PBMC) in vivo during LPS-induced inflammation is the focus of this study. Pigs were randomized into LPS (n = 12) or control (n = 6) groups. At start 0 min, LPS infusion was stepwise (2.5-15 mug/kg/h, 30 min) followed by maintenance infusion (2.5 mug/kg/h, 330 min). PBMC were isolated at 0, 60, 240 and 360 min, and two-colour flow cytometry was performed using monoclonal antibodies identifying SWC3 and CD18. Viability was tested using 7-amino-actinomycin D. LPS dramatically changed the relative distribution of circulating myeloid cells. At 60 min monocytes disappeared. This was followed by reappearance of a distinct population with low CD18 and SIRPalpha/SWC3 expression. Cell sorting showed that the appearing population comprised band neutrophils and apoptotic/dead cells. The remaining monocytes expressed less CD18 at 360 min than the controls (P = 0.03). The appearance of a distinct cell population comprising apoptotic cells and band neutrophils consistent with LPS-induced apoptosis, and decreased CD18 expression on monocytes suggests that early CD18 downregulation is profitable for the host in a situation with an intense LPS stimulus.

Asunto(s)

Antígenos CD18/biosíntesis , Endotoxemia/inmunología , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/inmunología , Receptores Inmunológicos/biosíntesis , Animales , Apoptosis/fisiología , Regulación hacia Abajo , Endotoxemia/inducido químicamente , Endotoxemia/metabolismo , Femenino , Citometría de Flujo , Lipopolisacáridos/toxicidad , Porcinos

RESUMEN

In a mouse model, we demonstrate how to obtain a direct, unbiased estimate of the total number of adoptively transferred cells in a variety of organs at different time points. The estimate is obtained by a straightforward method based on the optical fractionator principle. Specifically, non-stimulated C57BL/6J mouse splenocytes were labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and adoptively transferred to normal C57BL/6J mice by intravenous injection. The total number of CFSE-positive cells was subsequently determined in lung, spleen, liver, kidney, and inguinal lymph node at six different time points following adoptive transfer (from 60 s to 1 week), providing a quantitative estimate of the organ distribution of the transferred cells over time. These estimates were obtained by microscopy of uniform samples of thick sections from the respective organs. Importantly, the samples were chosen and prepared in accordance with the optical fractionator principle. We demonstrate that the method is simple, precise, and well suited for quantitative immunological studies.

Asunto(s)

Traslado Adoptivo , Técnicas Inmunológicas , Animales , Biometría , Recuento de Células , Separación Celular , Femenino , Fluoresceínas , Colorantes Fluorescentes , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Especificidad de Órganos , Bazo/citología , Succinimidas , Factores de Tiempo

RESUMEN

Histamine (HDC) inhibits formation and release of phagocyte-derived reactive oxygen species, and thereby protects natural killer (NK) and T cells against oxidative damage. Thus, the addition of histamine may potentially improve the efficacy of interleukin-2 (IL-2). We have explored this potential mechanism clinically in two randomised phase II trials in metastatic renal cell carcinoma (mRCC). In parallel with the clinical trial in Denmark (n=63), we obtained serial blood samples and tumour biopsies searching for a potential histamine effect in situ. At baseline and on-treatment weeks 3 and 8, we monitored the 'good guys' (i.e. NK and T cells) and 'bad guys' (i.e. monocytes/macrophages and neutrophils) simultaneously in blood (n=59) and tumour tissue (n=44). Patients with high number of monocytes and neutrophils in peripheral blood had very poor survival, with apparently no benefit from either IL-2 alone or IL-2/HDC treatment. Blood monocytes (r=-0.36, P=0.01) and neutrophils (r=-0.46, P=0.001) were negatively correlated with cytotoxicity, whereas blood NK cells were positively correlated with cytotoxicity (r=0.39, P=0.002). Treatment with IL-2 alone resulted in a significantly higher number of circulating monocytes (P=0.037) and intratumoral macrophages (P=0.005) compared with baseline. In contrast, IL-2/HDC resulted in an unchanged number of circulating monocytes and intratumoral macrophages, and in addition, a significantly increased number of intratumoral CD56(+) NK cells (P=0.008) and CD8(+) T cells (P=0.019) compared with baseline. The study provides evidence that circulating monocytes and neutrophils are powerful negative prognostic factors for IL-2-based immunotherapy and establishes a biological rationale for the potential use of histamine in conjunction with IL-2 in mRCC.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Monocitos/efectos de los fármacos , Metástasis de la Neoplasia/tratamiento farmacológico , Neutrófilos/efectos de los fármacos , Ensayos Clínicos Fase II como Asunto , Femenino , Histamina/administración & dosificación , Humanos , Inmunohistoquímica , Interleucina-2/administración & dosificación , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Masculino , Monocitos/inmunología , Neutrófilos/inmunología , Pronóstico , Ensayos Clínicos Controlados Aleatorios como Asunto , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Resultado del Tratamiento

RESUMEN

We have recently shown that adoptively transferred, IL-2-activated natural killer (A-NK) cells are able to eliminate well-established B16-F10.P1 melanoma lung metastases. However, some B16-F10.P1 lung metastases were resistant to infiltration by the A-NK cells and also resistant to the A-NK cell treatment. The infiltration-resistant (I-R) B16-F10.P1 metastases had a unique "compact" morphology compared to the "loose" morphology of the infiltration-permissive (I-P) metastases. Here, we show that I-P loose tumors and I-R compact tumors are also found in lung metastases of mouse Lewis lung carcinoma (3LL), MCA-102 sarcoma, and MC38 colon carcinoma as well as rat MADB106 mammary carcinoma origin. Furthermore, the infiltration resistance of the compact tumors is not restricted to A-NK cells, since PHA and IL-2 stimulated CD8+ T-cells (T-LAK cells) also infiltrated the compact tumors poorly. Analyses of tumors for extracellular matrix (ECM) components and PECAM-1(+) vasculature, revealed that the I-R lesions are hypovascularized and contain very little laminin, collagen and fibronectin. In contrast, the I-P loose tumors are well-vascularized and they contain high amounts of ECM components. Interestingly, the distribution pattern of ECM components in the I-P loose tumors is almost identical to that of the normal lung tissue, indicating that these tumors develop around the alveolar walls which provide the loose tumors with both a supporting tissue and a rich blood supply. In conclusion, tumor infiltration by activated NK and T cells correlates with the presence of ECM components and PECAM-1(+) vasculature in the malignant tissue. Thus, analysis of the distribution of ECM and vasculature in tumor biopsies may help select patients most likely to benefit from cellular adoptive immunotherapy.

Asunto(s)

Matriz Extracelular , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/inmunología , Metástasis de la Neoplasia/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Carcinoma Pulmonar de Lewis , Matriz Extracelular/química , Matriz Extracelular/inmunología , Femenino , Neoplasias Pulmonares/patología , Subgrupos Linfocitarios/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Metástasis de la Neoplasia/patología , Ratas

RESUMEN

NK cells have been shown to play an important role in the lungs with regards to tumor cell clearance and resistance of this organ to metastases. Here, we have investigated whether NK cells play a similar role in organs other than the lungs. We conclude that while organ-resistance to metastases correlates well with the NK activity of the host, a clear correlation between NK activity and clearance of tumor cells is found only in the lungs. We also demonstrate that activation of NK cells with the TLR 3 ligand poly I:C results in a substantial increase in the number of organ-associated NK cells. This increase may explain the increased resistance to metastasis seen in many organs after poly I:C treatment. Finally, we present data showing that NK cells activated ex vivo with IL-2 are able to localize to lung tumors following iv adoptive transfer and to significantly reduce the tumors they infiltrate. We conclude that NK cells, which currently are under intense investigation owing to their newly discovered immunoregulatory functions, remain very potent antitumor killer cells capable of killing not only circulating tumor cells, but also well-established micro metastases.

Asunto(s)

Células Asesinas Naturales/inmunología , Metástasis de la Neoplasia/inmunología , Neoplasias/inmunología , Animales , Humanos

RESUMEN

A majority of colorectal adenocarcinomas displays diminished MHC class I expression, making them particularly vulnerable for NK cell-mediated killing. Generally, these tumors also show a substantial inflammatory infiltrate. Most inflammatory cells, however, reside in the tumor stroma, where they do not have direct contact with tumor cells in the tumor epithelium. In this study, we investigated the correlation between colorectal tumor MHC class I aberrations and infiltration of NK cells. We studied 88 tumor specimens obtained from 88 colorectal cancer patients for locus-specific HLA aberrations and correlated these data to infiltration of CD4, CD8+ and CD56+ lymphocytes. The lymphocyte markers were individually combined with laminin as a second marker to facilitate quantification in the different tumor compartments, i.e. tumor epithelium and tumor stroma. Locus-specific partial or total HLA class I loss was detected in 72% of the tumors studied. Twenty-eight percent had no HLA loss at all. Mean overall intra-epithelial infiltration of CD56+ lymphocytes was 7 cells/mm(2) compared to 76 cells/mm(2) for CD8 and 19 cells/mm(2) for CD4+ lymphocytes. Locus-specific partial or total loss of tumor cell MHC class I expression was positively correlated with the intra-epithelial infiltration of CD8+ cells (P = 0.01), but not with CD4+ or CD56+ lymphocytes. Triple immunofluorescence staining showed that these cells were CD8 and granzyme-B positive T-lymphocytes. Our data showed that colorectal tumors are sparsely infiltrated by CD56+ cells compared to CD8+ T-cells and that loss of MHC is associated with T-cell infiltration instead of NK cell infiltration. Considering the fact that MHC loss is quite common in colorectal cancer and that, due to local absence of NK cells, it is unlikely that there has been selection for NK-escape variants, improvement of the intra-epithelial infiltration/migration of NK cells may be an important basis for the development of an effective adjuvant NK-based immunotherapy of colorectal cancer.

Asunto(s)

Adenocarcinoma/inmunología , Neoplasias Colorrectales/inmunología , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Movimiento Celular/inmunología , Regulación hacia Abajo , Femenino , Antígenos HLA/análisis , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias

RESUMEN

The aim of the present study was to investigate the in vivo antiproliferative effect of interferon alpha (IFN-alpha) in patients with metastatic renal cell carcinoma (mRCC). Core needle biopsies of metastatic and/or the primary kidney cancer were obtained before interleukin-2 (IL-2)- and IFN-alpha-based immunotherapy in 34 patients and repeated after 5 weeks in 25 patients. Tumour proliferation was assessed by use of the anti-Ki-67 antibody MIB-1 and evaluated in multiple, random systematic sampled fields of vision. Ki-67 labelling index (LI) at baseline was median 13.6% (range 1.2-85.0) and median 10.6% (range 1.3-48.6%) at week 5 with a median overall decline of 15.2% (range -95 to +258%) from baseline to week 5. There was no difference between responding and nonresponding patients. Ki-67 LI at week 5 was significantly correlated to survival. Thus, median survival of patients with Ki-67 LI 10.6% (P=0.016). Baseline or change in Ki-67 LI did not correlate to survival. These data suggest that IFN-alpha in vivo has only modest effect on tumour proliferation in patients with mRCC. Tumour Ki-67 (MIB-1) reactivity after 1 month of immunotherapy appears to be a significant predictor of patient survival.

Asunto(s)

Antineoplásicos/inmunología , Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Interferón-alfa/inmunología , Interferón-alfa/uso terapéutico , Antígeno Ki-67/análisis , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/inmunología , Adulto , Anciano , Biopsia con Aguja , Carcinoma de Células Renales/patología , División Celular , Femenino , Humanos , Interleucina-2/inmunología , Interleucina-2/uso terapéutico , Antígeno Ki-67/inmunología , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Análisis de Supervivencia

RESUMEN

Flow cytometric measurement of intracellular cytokines in T cells exposed to antigen is a widely used method for quantification of an antigen-specific T-cell response. As the frequency of antigen-specific T cells is often very low, any improvement in signal to noise ratio is of great importance. Thus, in this study, the ability of antigen-pulsed dendritic cells (DCs) to increase the number of antigen-specific, interferon-gamma (IFN-gamma)-producing CD4+ T cells measurable both in fresh peripheral blood and in reconstituted frozen blood mononuclear cell (MNC) samples was evaluated. Cytomegalovirus (CMV) was used as antigen in a 10 h assay, using cells from both CMV-seropositive and -seronegative donors. When reconstituted frozen samples were analysed, the general response towards CMV lysate in CMV-seropositive donors was 23-86% lower compared to the corresponding fresh blood samples. Antigen-pulsed DCs could not improve the sensitivity of the intracellular cytokine-detection assay when fresh peripheral blood samples were used. Interestingly, however, the addition of CMV lysate-pulsed DCs to cryopreserved MNC samples substantially increased the frequency of specifically induced IFN-gamma-producing cells to a level comparable to the frequency found in the corresponding fresh blood samples.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Citometría de Flujo , Interferón gamma/análisis , Adulto , Animales , Análisis Químico de la Sangre , Linfocitos T CD4-Positivos/virología , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/inmunología , Células Dendríticas/virología , Femenino , Humanos , Inmunoglobulina G/sangre , Interferón gamma/inmunología , Líquido Intracelular/química , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Manejo de Especímenes

RESUMEN

Typically autologous dendritic cells (DCs) intended for vaccination are generated from bone marrow derived stem cells or blood monocytes, loaded with antigen and introduced into the organism. However, addition of serum to DC culture medium is often necessary. Thus, serum proteins will be taken up and presented by the DCs together with other antigens. If heterologous serum is used, some of the serum proteins might be antigenic and thus induce a strong immune response when introduced in the recipient. We used the murine model of malignant melanoma, B16, to investigate the consequences of addition of fetal calf serum (FCS) to the medium for culturing murine DCs. The results showed that vaccination of mice with DCs cultured in vitro in the presence of FCS but in the absence of extraneous tumour antigens, protected the mice from challenge with B16 tumour cells similarly cultured in FCS. This protection could not be elicited by vaccination with FCS alone. Interestingly, the protective effect of DC vaccination was abolished when the challenging B16 tumour cells were free of serum proteins. Thus, these results show that DCs grown in the presence of FCS are able to induce immunity, which may be mistaken to be tumour immunity.

Asunto(s)

Proteínas Sanguíneas/inmunología , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Sangre Fetal/inmunología , Vacunación , Animales , Anticuerpos/sangre , Bovinos , Línea Celular , Femenino , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/prevención & control

RESUMEN

Circulating malignant cells in peripheral blood are thought to be precursors and surrogate markers of distant metastases and hence markers of a poor clinical outcome. In this study, we used the detection of MART-1 and tyrosinase (TYR) mRNA with a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to identify circulating melanoma cells. Blood samples were obtained from 35 patients with metastatic melanoma before, during and after treatment with interleukin-2, interferon-alpha and cisplatin. In addition, MART-1 and TYR protein was identified by immunohistochemistry in consecutive biopsies from 15 of the patients. Analysis of three daily blood samples for 3 days demonstrated that four out of 11 patients examined were negative for both markers on all occasions, and two patients were positive for both markers on all occasions but one. The remaining five patients showed sporadic low positive results for one or the other of the two markers. By comparing the immunohistochemistry results from consecutive biopsies with the RT-PCR results, we demonstrated that patients with MART-1 and TYR protein in their tumour cells had circulating MART-1 and TYR mRNA in 77% and 54% of the cases, respectively. During treatment, the majority of patients who were positive for MART-1 and TYR mRNA converted to being negative. However, these conversions did not significantly correlate with objective response. The presence of TYR mRNA in one of the first two samples showed a trend towards being an independent prognostic factor for poor survival.

Asunto(s)

Biomarcadores de Tumor/sangre , Interleucina-2/análogos & derivados , Melanoma/sangre , Monofenol Monooxigenasa/sangre , Proteínas de Neoplasias/sangre , Células Neoplásicas Circulantes/efectos de los fármacos , Neoplasias Cutáneas/sangre , Adulto , Anciano , Antígenos de Neoplasias , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cisplatino/administración & dosificación , Femenino , Humanos , Inmunoterapia , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interleucina-2/administración & dosificación , Antígeno MART-1 , Masculino , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/secundario , Persona de Mediana Edad , Monofenol Monooxigenasa/genética , Análisis Multivariante , Proteínas de Neoplasias/genética , Valor Predictivo de las Pruebas , Pronóstico , ARN Mensajero/sangre , Proteínas Recombinantes/administración & dosificación , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética

RESUMEN

BACKGROUND: Natural killer (NK) cells constitute an essential component of the innate immune system in the defence against infected and malignant cells. In this study the in vitro effect on NK cell activity of three different local anesthetics with different lipid solubility was investigated. METHODS: Venous blood from seven healthy volunteers was incubated with three amide local anesthetics with three different concentrations of lipid solubility: lidocaine 0.50, 1.00 and 2.00 mg/ml, ropivacaine 0.375, 0.75 and 1.50 mg/ml, and bupivacaine 0.25, 0.50 and 1.00 mg/ml. After 1 h of incubation, mononuclear cells were isolated and cryopreserved until tested for NK cell cytotoxicity in a 4-h 51Cr-release assay against K-562 target cells. Natural killer cell cytotoxicity of mononuclear cells incubated with isotonic saline was used as the control. RESULTS: A significant suppression in NK cell cytotoxicity was demonstrated for all three local anesthetic agents when the NK cell cytotoxicity was compared with the cytotoxicity estimated after incubation with the isotonic saline (P<0.004). Moreover a significant lipid solubility-dependent effect (P=0.0001) as well as an overall concentration-dependent effect (P<0.0001) on the NK cell cytotoxicity was found. CONCLUSION: The results of the present in vitro study suggest a negative association between the estimated NK cell cytotoxicity and the lipid solubility as well as the concentrations of the three local anesthetic agents tested.

Asunto(s)

Anestésicos Locales/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Adulto , Amidas/farmacología , Anestésicos Locales/química , Bupivacaína/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Células Asesinas Naturales/inmunología , Lidocaína/farmacología , Lípidos , Análisis Multivariante , Ropivacaína , Solubilidad

RESUMEN

The aim of the present study was to analyse lymphocyte subsets in consecutive peripheral blood samples and consecutive tumour tissue core needle biopsies performed before and during interleukin-2 based immunotherapy, and to correlate the findings with objective response and survival. Twenty-six patients with metastatic renal cell carcinoma were treated with low dose s.c. interleukin-2, interferon-alpha and histamine. A total of 250 blood samples and 62 core needle biopsies from 23 and 19 of these patients, respectively, were analysed. After 2 weeks of treatment, a significant positive correlation between absolute number of peripheral blood lymphocytes (P=0.028), CD3 (P=0.017), CD57 (P=0.041) and objective response was demonstrated. There was no correlation between any peripheral blood leukocyte subsets and survival. Cytotoxicity of peripheral blood mononuclear cells was not correlated to objective response or survival. Within the tumour tissue at baseline, a significant positive correlation between CD4 (P=0.027), CD8 (P=0.028), CD57 (P=0.007) and objective response was demonstrated. After one month of immunotherapy, a significant positive correlation between intratumoral CD3 (P=0.026), CD8 (P=0.015), CD57 (P=0.009) and objective response was demonstrated. A significant positive correlation between intratumoral baseline CD4 (P=0.047), baseline CD57 (P=0.035), CD3 at one month (P=0.049) and survival was demonstrated. These data provide novel in vivo evidence of the possible contribution of lymphocyte subsets in the tumour reduction in responding patients during interleukin-2 based immunotherapy. Confirmation of the results requires further studies including a larger number of patients.

Asunto(s)

Carcinoma de Células Renales/inmunología , Inmunoterapia , Interleucina-2/uso terapéutico , Neoplasias Renales/inmunología , Recuento de Linfocitos , Subgrupos Linfocitarios , Linfocitos Infiltrantes de Tumor/clasificación , Adulto , Anciano , Biopsia , Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/terapia , Femenino , Humanos , Inmunofenotipificación , Neoplasias Renales/sangre , Neoplasias Renales/mortalidad , Neoplasias Renales/patología , Neoplasias Renales/terapia , Tablas de Vida , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Análisis de Supervivencia , Resultado del Tratamiento

RESUMEN

In this article we report about the role that tumor structure and extracellular matrix (ECM) may play in immunotherapy and in gene therapy using adenoviruses. We performed studies in a rat model for colorectal cancer, CC531, and in specimens of human colorectal cancer. The tumors were composed of two compartments, tumor cell nests surrounded by stromal cells. ECM proteins were expressed in the stromal part, where the blood vessels were also located. Furthermore, in several tumors, the tumor cell nests were surrounded by basal membrane-like structures. Therefore, in vascular approaches to treat cancer, therapeutic agents on their route to tumor cells may be hampered by ECM to reach tumor cells. We found that immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor. Adenoviruses, when intravascularly injected, did not reach tumor cells in the CC531 rat model. Tumor cells were only infected, and even then in limited numbers, in cases of intratumoral injection. We hypothesize that ECM in a tumor is a barrier both for immune cells and for adenoviruses to make direct contact with these tumor cells, and thus limits colorectal tumor therapy.

Asunto(s)

Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , Matriz Extracelular/metabolismo , Terapia Genética/métodos , Adenoviridae/genética , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Antineoplásicos/inmunología , Formación de Anticuerpos/genética , Neoplasias Colorrectales/terapia , Femenino , Genes Reporteros , Vectores Genéticos/administración & dosificación , Vectores Genéticos/metabolismo , Humanos , Inmunohistoquímica , Células Asesinas Naturales , Operón Lac , Laminina/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Masculino , Persona de Mediana Edad , Ratas , Ratas Wistar , Linfocitos T , Distribución Tisular

RESUMEN

The circulatory pattern of IL-2 activated natural killer (A-NK) cells was studied in C57BL/6 mice bearing 10 day-old pulmonary and subcutaneous (s.c.) metastases of the B16 melanoma in order to evaluate the roles of the concentration of A-NK cells in the blood and of tumor blood flow on accumulation of A-NK cells in tumors. Kinetic studies of the presence of A-NK cells in peripheral blood after adoptive transfer revealed that these cells rapidly disappear from the blood. Via intravital microscopy of animals with exposed lung tissue, we have shown that the vast majority of transferred A-NK cells become efficiently arrested within the lung microcirculation at their first encounter with this organ, thereby explaining the fast disappearance of the cells from the bloodstream. Despite the low number of A-NK cells circulating in the blood, systemically injected A-NK cells (20 million per mouse) localized significantly (70-80 million cells/g) into most pulmonary metastases within 8-16 hours. In contrast, very few A-NK cells (< 0.2 million cells/g) were found in the s.c. metastases. Based on measurements of tumor blood flow (showing a classic inverse relationship between tumor size and tumor blood flow) and the blood concentration of A-NK cells, we estimated the highest intratumoral density of A-NK cells that theoretically can be generated by A-NK cells transported to the tumor by way of the blood. In s.c. tumors, the observed density of A-NK cells was at all times lower (10-50 fold) than the estimated density, indicating that only a few percent of the A-NK cells arriving at these tumors become retained in them. In contrast, the observed density of A-NK cells in pulmonary metastases was at all times higher (2-3 fold) than the estimated density. This finding indicates that A-NK cells might not reach the pulmonary metastases solely by way of the blood stream. In conclusion, i.v. injected A-NK cells become immediately entrapped in the lungs and, consequently, circulate poorly. While lung metastases become significantly infiltrated by i.v. injected A-NK cells, metastases in organs down-stream from the lungs become poorly infiltrated. We hypothesize that only a part of the A-NK cells found in lung metastases 8-16 hours following injection reach these metastases by way of the blood-vascular system. They might also migrate into the metastases from the surrounding normal lung tissue.

Asunto(s)

Interleucina-2/farmacología , Células Asesinas Activadas por Linfocinas/inmunología , Neoplasias Pulmonares/irrigación sanguínea , Melanoma Experimental/irrigación sanguínea , Neoplasias Cutáneas/irrigación sanguínea , Animales , Recuento de Células , Inmunoterapia Adoptiva , Células Asesinas Activadas por Linfocinas/efectos de los fármacos , Células Asesinas Activadas por Linfocinas/patología , Células Asesinas Activadas por Linfocinas/trasplante , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Microcirculación , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/secundario , Bazo/citología , Bazo/efectos de los fármacos

RESUMEN

Dendritic cells (DCs) represent a group of antigen-presenting leukocytes which are very effective in activating resting T-cells. DCs are present in almost all tissues of the body, but they are generally difficult to isolate. The study of human DCs has recently become greatly facilitated due to the advent of methods for isolation and in vivo generation of DCs from blood. Experiments in animal models have shown that DCs loaded with tumour antigens may induce effective immune responses against cancer. Now the potential of vaccination with tumour antigens presented on DCs is being evaluated in cancer patients. Preliminary clinical studies have shown encouraging results.

Asunto(s)

Células Dendríticas , Neoplasias/terapia , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/ultraestructura , Antígenos de Neoplasias/inmunología , Separación Celular , Células Dendríticas/inmunología , Células Dendríticas/ultraestructura , Humanos , Inmunoterapia , Activación de Linfocitos , Linfocitos T/inmunología

RESUMEN

The therapeutic potential of dendritic cells loaded with tumour antigens for the induction of effective immune responses against cancer is currently being tested in numerous clinical trials. In most cases, the dendritic cells are generated in vitro from peripheral blood monocytes. Many aspects of dendritic cell-based vaccination have not yet been examined in detail, and homologous mouse model systems may prove very valuable for optimizing clinical procedures. In the murine system, however, dendritic cells are usually isolated from either lymphoid tissues or bone marrow cultures. To date, murine monocyte-derived dendritic cells have been described only sporadically. Here, we describe a culture system for the generation of murine dendritic cells from adherent peripheral blood mononuclear cells by culturing in the presence of granulocyte-macrophage colony stimulating factor and interleukin-4. After 7 days of culture the nonadherent cells were harvested from the cultures. Most of these cells exhibited well-accepted characteristics of mature dendritic cells (e.g. veiled appearance, high expression of major histocompatibility complex class II and CD86) and stimulated vigorous proliferation of allogeneic T cells in a primary mixed leucocyte reaction following stimulation with bacterial lipopolysaccharide. Interestingly, staining the cells for expression of the putative antigen-uptake receptor DEC-205 revealed a distinct bimodal distribution.

Asunto(s)

Células Dendríticas/inmunología , Animales , Antígenos de Neoplasias/administración & dosificación , Vacunas contra el Cáncer/administración & dosificación , Adhesión Celular , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Separación Celular , Células Dendríticas/citología , Femenino , Citometría de Flujo , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/terapia , Fenotipo , Linfocitos T/inmunología

RESUMEN

The dendritic cell (DC) is a professional antigen-presenting cell of central importance in immunity. In this paper, we examined DCs generated by 11-day culture of bone-marrow cells from the four mouse strains C57BL/6J, BALB/cA, C3H/HeN and B10.PL-H2u (73NS)/Sn with respect to cell yield as well as surface-marker phenotype and morphology. We also investigated the phenotypic changes and the T-cell stimulatory activity of the DCs induced by bacterial lipopolysaccharide (LPS). Morphologically, we observed low levels (5-10%) of granulocyte contamination of the cultures after a culture period of 11 days. Considerable strain-specific differences were found in the expression levels of the surface markers in addition to the differences in the ratio of the immature to mature DCs in the cultures that were not stimulated with LPS. Furthermore, we found that LPS strongly induces maturation of DCs in all strains investigated with the exception of the B10.PL strain.

Asunto(s)

Células de la Médula Ósea/citología , Células Dendríticas/citología , Variación Genética/inmunología , Animales , Recuento de Células , Diferenciación Celular/inmunología , Células Cultivadas , Inmunofenotipificación , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Especificidad de la Especie