RESUMEN
In recent years, circulating tumor cells (CTCs) in metastatic cancer patients have been found to be a promising biomarker to predict overall survival and tumor progression in these patients. A relatively high number of CTCs has been correlated with disease progression and poorer prognosis. This study was designed to assess innate immune system function, known to be responsible for the immune defense against developing neoplasms, in metastatic cancer patients with CTCs. Our aim is to provide a link between indication of poorer prognosis, represented by the number of CTCs to the cytotoxic activity of natural killer cells, an important component of the innate immune system, and to represent a promising expanded approach to management of metastatic cancer patients with CTCs. Seventy-four patients, with metastatic breast, colorectal, or prostate cancer, were recruited for this study. Using a flow cytometric assay, we measured natural killer (NK) cell cytotoxicity against K562 target cells; and CTCs were enumerated using the CellSearch System. Toll-like receptors 2 and 4 expression was also determined by flow cytometry. We found that within each of our three metastatic cancer patient groups, NK cell cytotoxic activity was decreased in patients with a relatively high number of CTCs in peripheral blood compared to patients with a relatively low number of CTCs. In the breast and prostate cancer group, patients with CTCs greater than 5 had decreased NK cell cytotoxicity when compared to patients with less than 5 CTCs. In the colorectal cancer group, we found that 3 or more CTCs in the blood was the level at which NK cell cytotoxicity is diminished. Additionally, we found that the toll-like receptors 2 and 4 expression was decreased in intensity in all the metastatic cancer patients when compared to the healthy controls. Furthermore, within each cancer group, the expression of both toll-like receptors was decreased in the patients with relatively high number of CTCs, i.e. greater than 5 for the breast and prostate cancer group and greater than 3 for the colorectal cancer group, compared to the patients with relatively low number, i.e. less than 5 or 3, respectively. Treatment options to increase NK cell cytotoxic activity should be considered in patients with relatively high numbers of CTCs.
Asunto(s)
Neoplasias de la Mama/inmunología , Neoplasias Colorrectales/inmunología , Inmunidad Innata/inmunología , Células Neoplásicas Circulantes , Neoplasias de la Próstata/inmunología , Recuento de Células , Femenino , Citometría de Flujo , Humanos , Células K562 , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Masculino , Metástasis de la Neoplasia , Pronóstico , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The present investigation was designed to evaluate the renal microvascular endothelial cell responses following exposure to preformed antibodies against human leukocyte antigens (HLA) in the recipient. We hypothesize that activation of endothelial cell genes has a pivotal role in renal allograft survival. In this study, we used cultured human umbilical cord vein endothelial cells (HUVEC), human microvascular glomerular endothelial cells (HMGEC), activated with and without IFN-γ and TNF-α, and pre-transplant blood group O patient sera containing multispecific HLA class I and class II antibodies. Molecular HLA typing revealed the HMGEC haplotype to be HLA-A*01, HLA-A*68, HLA-B*14, HLA-B*35, HLA-C*04, HLA-C*08, HLA-DRß1*13, and HLA-DRß1*15. Flow cytometry was used for phenotypic characterization of both inactivated and activated HUVECs and HMGECs with IFN-γ and TNF-α. HUVECs were positive for HLA-ABC, HLA-DR/DQ, von Willebrand factor, endoglin, PECAM, ICAM, MCAM, integrin beta-3, thrombomodulin, E-selectin, VCAM-1, and tissue factor, and negative for alpha smooth muscle actin and P-selectin antibodies. HMGECs were positive for HLA-ABC, HLA-DR/DQ, von Willebrand factor, endoglin, ICAM, MCAM, integrin beta-3, thrombomodulin, VCAM-1, and tissue factor; and negative for PECAM, E-selectin, P-selectin, and for blood group antigens A and B. 42 samples were analyzed by real time PCR and categorized into the following groups: the control group (HMGEC only, n=12), group 1 (HMGECs incubated with patient sera, n=15), and group 2 (HMGECs activated by TNF-α and IFN-γ and incubated with patient sera, n=15). Expression levels of the vasoconstriction genes endothelin 1 (EDN1), endothelin 2 (EDN2), and endothelin receptor type A (EDNRA) were up-regulated in both groups 1 and 2 compared to the control group. The thrombomodulin (THBD) gene was also up-regulated in both groups 1 and 2 compared to the control. Chemokine genes CCL5 and CX3CL1 were up-regulated in both groups 1 and 2 compared to the controls; whereas, CCL2 was up-regulated only in group 2. Cytokine activity genes colony stimulating factor 2 (CSF2), tumor necrosis factor (TNF), tumor necrosis factor (ligand) superfamilymember 10 (TNFSF10), interleukin 1 beta (IL1B), and interleukin 6 (IL6) were up-regulated in both groups 1 and 2 compared to the control; whereas, IL11 was up-regulated only in group 1 and IFNB1 in group 2. Adhesion molecule genes intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and integrin beta 3 (ITGB3) were up-regulated in both groups 1 and 2 compared to the control; whereas, CDH5 and COL18A1 were up-regulated only in group 2. Anti-apoptosis genes BCL2A1, CFLAR, and SPHK1 were up-regulated only in group 2 compared to controls. Apoptosis and caspase-activation genes CASP, RIPK1, and FAS were up-regulated only in group 2 compared to the control. Angiopoietin 1 (ANGPT1) and prostaglandin I2 (prostacyclin) synthase (PTGIS) were down-regulated in both groups 1 and 2 compared to the control group. Our results indicate that expression of the endothelin gene in endothelial cells may contribute to vasoconstriction of blood vessels in post-renal allograft transplantation. In addition, thrombomodulin, by reducing thrombogenic activity, and interleukin 11, through its cytoprotective effects, may have a role in transplant accommodation in the presence of pre-formed HLA antibodies. This study showed that activation of the vasoconstriction genes, thrombomodulin gene, chemokine genes, cytokine activity genes, adhesion genes, anti-apoptosis genes, and apoptosis and caspase-activation genes could have consequential effects on renal allograft survival.
Asunto(s)
Endotelio Vascular/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Trasplante de Riñón , Riñón/irrigación sanguínea , Microvasos/fisiología , Antígenos de Superficie/metabolismo , Apoptosis/genética , Antígenos de Grupos Sanguíneos/metabolismo , Línea Celular , Citocinas/genética , Citocinas/metabolismo , Endotelina-1/metabolismo , Endotelina-2/metabolismo , Endotelio Vascular/citología , Supervivencia de Injerto/genética , Antígenos HLA/genética , Antígenos HLA/inmunología , Antígenos HLA/metabolismo , Haplotipos , Humanos , Microvasos/citología , Receptores de Endotelina/metabolismo , Trombomodulina/genética , Trombomodulina/metabolismo , Trasplante Homólogo , Regulación hacia Arriba , Vasoconstricción/genéticaRESUMEN
The present investigation was designed to show the effect of human leukocyte antigen (HLA) class II molecular allelic specificities in the recipient on the induction of humoral antibody rejection, identified by C4d peritubular capillary staining, as well as specific antibody identified by Luminex technology. Major histocompatibility complex (MHC) class II molecules are expressed on dendritic cells, macrophages, and B lymphocytes and they present antigenic peptides to CD4 positive T lymphocytes. Human renal peritubular and glomerular capillaries express class II MHC molecules upon activation. Expression of class II molecules on renal microvascular endothelial cells exposes them to possible interaction with specific circulating antibodies. We hypothesize that HLA-DQß1*0202 expression in recipients decreases the likelihood of antibody-mediated renal allograft rejection. We found that 80% (=25) of DQ2 positive haplotype recipients failed to induce humoral antibody renal allograft rejection and 20% (n=25) of DQ2 positive haplotype recipients induced humoral antibody renal allograft rejection (p=0.008). By contrast, 48% (n=46) of DQ2 negative haplotype recipients failed to induce a humoral antibody component of renal allograft rejection and 52% (n=46) of DQ2 negative haplotype recipients induced humoral antibody-mediated renal allograft rejection. Our results suggest that recipients who express the DQß1*0202 allele are less likely to induce a humoral antibody component of acute renal allograft rejection than are those expressing DQ1, DQ3, or DQ4 alleles. DQß1*0202 allele expression in recipients could possibly be protective against acute humoral allograft rejection and might serve as a future criterion in recipient selection and in appropriate therapy for acute renal rejection episodes.
Asunto(s)
Formación de Anticuerpos/inmunología , Rechazo de Injerto/inmunología , Cadenas beta de HLA-DQ/inmunología , Inmunidad Humoral/inmunología , Trasplante de Riñón , Alelos , Rechazo de Injerto/genética , Rechazo de Injerto/patología , Cadenas beta de HLA-DQ/genética , Prueba de Histocompatibilidad , Humanos , Inmunidad Humoral/genética , Trasplante HomólogoRESUMEN
The present investigation was designed to show the effect of molecular HLA class II DR and DQ allelic differences between donor and recipient on humoral antibody rejection identified by C4d peritubular capillary staining. The hypothesis is that expression of the DRß1*1501, DQß1*0602 allele in the donor kidney increases the likelihood of humoral antibody rejection. We found that 67% (n=18) of DR15 and/or DQ6 haplotype donor kidneys induced humoral antibody renal allograft rejection; 35% (n=40) of DR15 and/or DQ6 haplotype donor kidneys failed to induce humoral antibody renal allograft rejection (p=0.02). 42% (n=31) of C4d+ recipients had donors with DR15; 17% (n=42) of C4d recipients had donors with HLA-DR15 (p=0.01).We compared donor haplotype alleles of 4 C4d+ with 6 C4d- recipients by high resolution molecular typing; 3 of 4 C4d+ recipients had a donor with the DRß1*1501/DQß1*0602 allele. This allele was absent in all C4d- donors. 35% of C4d+ recipients had 2 DR mismatches when compared to 36% of C4d- recipients. Our results, suggest that the DRß1*1501, DQß1*0602 allele in the donor kidney increases the risk of humoral antibody episodes of acute rejection, and signals the need for C4d staining of renal biopsies. Future analysis of additional donor and recipient haplotypes will establish whether or not this is a useful predictor of humoral rejection episodes.