RESUMO
BACKGROUND: Domestic cats rarely develop hepatocellular carcinoma. The reason for the low prevalence is unknown. Reductions in hepatocellular ploidy have been associated with hepatic carcinogenesis. Recent work in mice has shown that livers with more polyploid hepatocytes are protected against the development of hepatocellular carcinoma. Hepatocyte ploidy in the domestic cat has not been evaluated. We hypothesized that ploidy would be reduced in peri-tumoral and neoplastic hepatocytes compared to normal feline hepatocytes. Using integrated fluorescence microscopy, we quantified the spectra of ploidy in hepatocellular carcinoma and healthy control tissue from paraffin embedded tissue sections. RESULTS: Feline hepatocytes are predominantly mononuclear and the number of nuclei per hepatocyte did not differ significantly between groups. Normal cats have a greater number of tetraploid hepatocytes than cats with hepatocellular carcinoma. CONCLUSIONS: Total hepatocellular polyploidy in normal cat liver is consistent with values reported in humans, yet cellular ploidy (nuclei per cell) is greater in humans than in cats. Tetraploid cat hepatocytes are predominantly mononuclear.
Assuntos
Carcinoma Hepatocelular/veterinária , Doenças do Gato/genética , Hepatócitos/citologia , Ploidias , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Doenças do Gato/patologia , Gatos , Feminino , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/veterinária , MasculinoRESUMO
UNLABELLED: The herpes simplex virus type 1 (HSV-1) UL20 gene encodes a 222-amino-acid nonglycosylated envelope protein which forms a complex with viral glycoprotein K (gK) that functions in virion envelopment, egress, and virus-induced cell fusion. To investigate the role of the carboxyl terminus of the UL20 protein (UL20p) in cytoplasmic virion envelopment, a cadre of mutant viruses was constructed and characterized. The deletion of six amino acids from the carboxyl terminus of UL20p caused an approximately 1-log reduction in infectious virus production compared to that of the wild-type virus. Surprisingly, a phenylalanine-to-alanine replacement at amino acid position 210 caused a gain-of-function phenotype, increasing infectious virus production up to 1 log more than in the wild-type virus. In contrast, the replacement of two membrane-proximal phenylalanines with alanines caused drastic inhibition of infectious virion production and cytoplasmic virion envelopment. Prediction of the membrane topology of UL20p revealed that these two amino acid changes cause retraction of the carboxyl terminus of UL20p from the intracellular space. Confocal microscopy revealed that none of the engineered UL20 mutations affected intracellular transport of UL20p to trans-Golgi network membranes. In addition, a proximity ligation assay showed that none of the UL20 mutations affected UL20p colocalization and potential interactions with the UL37 protein recently found to interact with the gK/UL20 protein complex. Collectively, these studies show that phenylalanine residues within the carboxyl terminus of UL20p are involved in the regulation of cytoplasmic virion envelopment and infectious virus production. IMPORTANCE: We have shown previously that the UL20/gK protein complex serves crucial roles in cytoplasmic virion envelopment and that it interacts with the UL37 tegument protein to facilitate cytoplasmic virion envelopment. In this study, we investigated the role of phenylalanine residues within the carboxyl terminus of UL20p, since aromatic and hydrophobic amino acids are known to be involved in protein-protein interactions through stacking of their aromatic structures. Characterization of mutant viruses carrying phenylalanine (Phe)-to-alanine (Ala) mutations revealed that the two membrane-proximal Phe residues were critical for the proper UL20p membrane topology and efficient virion envelopment and infectious virus production. Surprisingly, a Phe-to-Ala change located approximately in the middle of the UL20p carboxyl terminus substantially enhanced cytoplasmic envelopment and overall production of infectious virions. This work revealed that Phe residues within the UL20p carboxyl terminus are involved in the regulation of cytoplasmic virion envelopment and infectious virus production.
Assuntos
Citoplasma/virologia , Glicoproteínas/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Fenilalanina/metabolismo , Proteínas Virais/metabolismo , Vírion/metabolismo , Animais , Fusão Celular , Chlorocebus aethiops , Herpes Simples/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Microscopia Eletrônica , Mutação/genética , Fenótipo , Fenilalanina/genética , Células Vero , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Vírion/patogenicidade , Rede trans-GolgiRESUMO
Previous in vitro work demonstrated that Edwardsiella ictaluri produces an acid-activated urease that can modulate environmental pH through the production of ammonia from urea. Additional work revealed that expression of the E. ictaluri type III secretion system (T3SS) is upregulated by acidic pH. Both the urease and the T3SS were previously shown to be essential to intracellular replication. In this work, fluorescence microscopy with LysoTracker Red DND-99 (LTR) indicated that E. ictaluri-containing vacuoles (ECV) became acidified following ingestion by head kidney-derived macrophages (HKDM). In vivo ratiometric imaging demonstrated a lowered ECV pH, which fell to as low as pH 4 but subsequently increased to pH 6 or greater. Inhibition of vacuolar H(+)-ATPases by use of the specific inhibitor bafilomycin A1 abrogated both ECV acidification and intracellular replication in HKDM. Failure of an E. ictaluri urease knockout mutant to increase the ECV pH in the in vivo ratiometric assay suggests that ammonia produced by the urease reaction mediates the pH increase. Additionally, when the specific arginase inhibitor l-norvaline was used to treat E. ictaluri-infected HKDM, the ECV failed to neutralize and E. ictaluri was unable to replicate. This indicates that the HKDM-encoded arginase enzyme produces the urea used by the E. ictaluri urease enzyme. Failure of the ECV to acidify would prevent both upregulation of the T3SS and activation of the urease enzyme, either of which would prevent E. ictaluri from replicating in HKDM. Failure of the ECV to neutralize would result in a vacuolar pH too low to support E. ictaluri replication.
Assuntos
Edwardsiella ictaluri/patogenicidade , Infecções por Enterobacteriaceae/microbiologia , Doenças dos Peixes/microbiologia , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Vacúolos/fisiologia , Análise de Variância , Animais , Arginase/metabolismo , Modelos Animais de Doenças , Edwardsiella ictaluri/efeitos dos fármacos , Edwardsiella ictaluri/crescimento & desenvolvimento , Inibidores Enzimáticos/farmacologia , Ictaluridae , Testes de Sensibilidade Microbiana , Microscopia de FluorescênciaRESUMO
B7-H4 is a recently identified B7 family member. We previously showed that ovarian tumor and associated macrophages expressed B7-H4; tumor B7-H4+ macrophages and CD4+CD25+FOXP3+ regulatory T cells (Treg cells) suppressed tumor-associated antigen-specific T-cell immunity. To determine the pathologic relationship between B7-H4, macrophages, and Treg cells in the tumor environment, in addition to Treg cell numbers, we quantified B7-H4 expression in the tumor and tumor-associated macrophages in 103 patients with ovarian carcinoma. We observed that the intensity of B7-H4 expression in macrophages was significantly correlated with Treg cell numbers in the tumor. Further, both Treg cells and macrophage B7-H4, but not tumor B7-H4, were negatively associated with patient outcome. Tumor Treg cells enabled macrophages to spontaneously produce interleukin (IL)-10 and IL-6. Tumor macrophages stimulated B7-H4 expression in an autocrine manner through IL-10 and IL-6. Our previous work showed that tumor-associated macrophages spontaneously produced chemokine CCL22 to mediate Treg cell trafficking into tumor, and Treg cells induced B7-H4 on antigen-presenting cells (APC) including macrophages. Altogether, our data support the concept that there is a mechanistic interaction between Treg cells and macrophage, and that Treg cells may convey the suppressive activity to APCs through B7-H4 induction in human ovarian cancer.
Assuntos
Antígeno B7-1/imunologia , Neoplasias Ovarianas/imunologia , Linfócitos T Reguladores/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígeno B7-1/biossíntese , Feminino , Humanos , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Macrófagos/imunologia , Inibidor 1 da Ativação de Células T com Domínio V-SetRESUMO
Tumor-associated macrophages are a prominent component of ovarian cancer stroma and contribute to tumor progression. B7-H4 is a recently identified B7 family molecule. We show that primary ovarian tumor cells express intracellular B7-H4, whereas a fraction of tumor macrophages expresses surface B7-H4. B7-H4+ tumor macrophages, but not primary ovarian tumor cells, suppress tumor-associated antigen-specific T cell immunity. Blocking B7-H4-, but not arginase-, inducible nitric oxide synthase or B7-H1 restored the T cell stimulating capacity of the macrophages and contributes to tumor regression in vivo. Interleukin (IL)-6 and IL-10 are found in high concentrations in the tumor microenvironment. These cytokines stimulate macrophage B7-H4 expression. In contrast, granulocyte/macrophage colony-stimulating factor and IL-4, which are limited in the tumor microenvironment, inhibit B7-H4 expression. Ectopic expression of B7-H4 makes normal macrophages suppressive. Thus, B7-H4+ tumor macrophages constitute a novel suppressor cell population in ovarian cancer. B7-H4 expression represents a critical checkpoint in determining host responses to dysfunctional cytokines in ovarian cancer. Blocking B7-H4 or depleting B7-H4+ tumor macrophages may represent novel strategies to enhance T cell tumor immunity in cancer.
Assuntos
Antígeno B7-1/genética , Carcinoma/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias Ovarianas/metabolismo , Animais , Antígenos CD/fisiologia , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/imunologia , Antígeno B7-1/biossíntese , Antígeno B7-H1 , Biomarcadores , Carcinoma/imunologia , Feminino , Humanos , Imunofenotipagem , Interleucina-10/fisiologia , Interleucina-6/fisiologia , Macrófagos/classificação , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Células Tumorais Cultivadas , Inibidor 1 da Ativação de Células T com Domínio V-SetRESUMO
To directly dissect the role of each immune component in human tumor immunopathogenesis, we have studied the interaction between dendritic cells and T cells in the tumor environment of patients with ovarian carcinoma. We previously reported that functional plasmacytoid dendritic cells, but not functionally mature myeloid dendritic cells, accumulated in tumor microenvironments. We now show that tumor ascites macrophage-derived dendritic cells induced tumor-associated antigen-specific CD8+ T cells with effector functions. Strikingly, tumor ascites plasmacytoid dendritic cells induced interleukin-10+ CCR7+ CD45RO+ CD8+ regulatory T cells. Four characteristics have been identified in tumor plasmacytoid dendritic cell-induced CD8+ regulatory T cells: (a) induction of CD8+ regulatory T cells is independent of CD4+ CD25+ T cells; (b) CD8+ regulatory T cells significantly suppress myeloid dendritic cell-mediated tumor-associated antigen-specific T cell effector functions through interleukin-10; (c) repetitive myeloid dendritic cell stimulation can recover CD8+ regulatory T cell-mediated poor T cell proliferation, but not T cell effector function; (d) CD8+ regulatory T cells express functional CCR7, and efficiently migrate with lymphoid homing chemokine MIP-3beta. Primary suppressive CCR7+ CD45RO+ CD8+ T cells are found in the tumor environment of patients with ovarian cancers. Thus, tumor-associated plasmacytoid dendritic cells contribute to the tumor environmental immunosuppressive network. Collectively, tumors manipulate tumor microenvironmental dendritic cell subset distribution and function to subvert tumor immunity. The data are relevant to understanding tumor immunopathology as well as reevaluating tumor immunotherapeutic strategies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/imunologia , Células Dendríticas/imunologia , Neoplasias Ovarianas/imunologia , Sequência de Aminoácidos , Movimento Celular/imunologia , Células Dendríticas/citologia , Feminino , Humanos , Interleucina-10/imunologia , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária , Dados de Sequência Molecular , Células Mieloides/imunologia , Plasmócitos/imunologia , Receptores CCR7 , Receptores de Quimiocinas/imunologiaRESUMO
Ovarian carcinomas have a poor prognosis, often associated with multifocal i.p. dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites and tumors of patients with untreated ovarian carcinoma. We observed that malignant ascites fluid induced potent in vivo neovascularization in Matrigel assay. We detected a sizable amount of vascular endothelial cell growth factor (VEGF) in malignant ascites. However, pathologic concentration of VEGF is insufficient to induce in vivo angiogenesis. We show that ovarian tumors strongly express CXC chemokine stromal-derived factor (SDF-1/CXCL12). High concentration of CXCL12, but not the pathologic concentration of CXCL12 induces in vivo angiogenesis. Strikingly, pathologic concentrations of VEGF and CXCL12 efficiently and synergistically induce in vivo angiogenesis. Migration, expansion, and survival of vascular endothelial cells (VEC) form the essential functional network of angiogenesis. We further provide a mechanistic basis for explaining the interaction between CXCL12 and VEGF. We show that VEGF up-regulates the receptor for CXCL12, CXCR4 expression on VECs, and synergizes CXCL12-mediated VEC migration. CXCL12 synergizes VEGF-mediated VEC expansion and synergistically protects VECs from sera starvation-induced apoptosis with VEGF. Finally, we show that hypoxia synchronously induces tumor CXCL12 and VEGF production. Therefore, hypoxia triggered tumor CXCL12 and VEGF form a synergistic angiogenic axis in vivo. Hypoxia-induced signals would be the important factor for initiating and maintaining an active synergistic angiogeneic pathway mediated by CXCL12 and VEGF. Thus, interrupting this synergistic axis, rather than VEGF alone, will be a novel efficient antiangiogenesis strategy to treat cancer.
Assuntos
Quimiocinas CXC/farmacologia , Neoplasias Ovarianas/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Ascite/metabolismo , Ascite/patologia , Hipóxia Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quimiocina CXCL12 , Quimiocinas CXC/biossíntese , Quimiocinas CXC/fisiologia , Sinergismo Farmacológico , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Feminino , Humanos , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Ovarianas/metabolismo , Proteínas Recombinantes/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
CD4(+)CD25(+) regulatory T cells (Tregs) mediate peripheral T-cell homeostasis and contribute to self-tolerance. Their homeostatic and pathologic trafficking is poorly understood. Under homeostatic conditions, we show a relatively high prevalence of functional Tregs in human bone marrow. Bone marrow strongly expresses functional stromal-derived factor (CXCL12), the ligand for CXCR4. Human Tregs traffic to and are retained in bone marrow through CXCR4/CXCL12 signals as shown in chimeric nonobese diabetic/severe combined immunodeficient mice. Granulocyte colony-stimulating factor (G-CSF) reduces human bone marrow CXCL12 expression in vivo, associated with mobilization of marrow Tregs to peripheral blood in human volunteers. These findings show a mechanism for homeostatic Treg trafficking and indicate that bone marrow is a significant reservoir for Tregs. These data also suggest a novel mechanism explaining reduced acute graft-versus-host disease and improvement in autoimmune diseases following G-CSF treatment.
Assuntos
Células da Medula Óssea/metabolismo , Antígenos CD4/imunologia , Quimiocinas CXC/metabolismo , Receptores CXCR4/metabolismo , Receptores de Interleucina-2/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Sequência de Bases , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Quimiocina CXCL12 , Primers do DNA , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Regulatory T (T(reg)) cells mediate homeostatic peripheral tolerance by suppressing autoreactive T cells. Failure of host antitumor immunity may be caused by exaggerated suppression of tumor-associated antigen-reactive lymphocytes mediated by T(reg) cells; however, definitive evidence that T(reg) cells have an immunopathological role in human cancer is lacking. Here we show, in detailed studies of CD4(+)CD25(+)FOXP3(+) T(reg) cells in 104 individuals affected with ovarian carcinoma, that human tumor T(reg) cells suppress tumor-specific T cell immunity and contribute to growth of human tumors in vivo. We also show that tumor T(reg) cells are associated with a high death hazard and reduced survival. Human T(reg) cells preferentially move to and accumulate in tumors and ascites, but rarely enter draining lymph nodes in later cancer stages. Tumor cells and microenvironmental macrophages produce the chemokine CCL22, which mediates trafficking of T(reg) cells to the tumor. This specific recruitment of T(reg) cells represents a mechanism by which tumors may foster immune privilege. Thus, blocking T(reg) cell migration or function may help to defeat human cancer.
Assuntos
Movimento Celular/imunologia , Quimiocinas CC/metabolismo , Imunidade Celular/imunologia , Neoplasias Ovarianas/imunologia , Linfócitos T/imunologia , Animais , Ascite/imunologia , Linfócitos T CD4-Positivos , Quimiocina CCL22 , Quimiocinas CC/imunologia , Proteínas de Ligação a DNA , Células Dendríticas/imunologia , Feminino , Fatores de Transcrição Forkhead , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Camundongos , Microscopia Confocal/métodos , Receptores de Interleucina-2RESUMO
Angiogenesis is essential for both primary and metastatic tumor growth. Tumor blood vessel formation is complex and regulated by many factors. Ovarian carcinomas have a poor prognosis, often associated with multifocal intraperitoneal dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites of patients with untreated ovarian carcinoma. We observed high numbers of plasmacytoid dendritic cells (PDCs) and significant stromal-derived factor (CXCL-12/SDF)-1 in their malignant ascites, attracting PDCs into the tumor environment. We now show that tumor-associated PDCs induced angiogenesis in vivo through production of tumor necrosis factor alpha and interleukin 8. By contrast, myeloid dendritic cells (MDCs) were absent from malignant ascites. MDCs derived in vitro suppressed angiogenesis in vivo through production of interleukin 12. Thus, the tumor may attract PDCs to augment angiogenesis while excluding MDCs to prevent angiogenesis inhibition, demonstrating a novel mechanism for modulating tumor neovascularization. Because dendritic cells (DCs) have long been known to affect tumor immunity, our data also implicate DCs in regulation of tumor neoangiogenesis, suggesting a novel role of DCs in tumor pathology.
Assuntos
Células Dendríticas/fisiologia , Neovascularização Patológica/patologia , Neoplasias Ovarianas/irrigação sanguínea , Animais , Ascite/metabolismo , Ascite/patologia , Células Dendríticas/classificação , Células Dendríticas/metabolismo , Feminino , Humanos , Interleucina-12/antagonistas & inibidores , Interleucina-12/biossíntese , Interleucina-8/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Mieloides/metabolismo , Células Mieloides/patologia , Células Mieloides/fisiologia , Neovascularização Patológica/metabolismo , Neoplasias Ovarianas/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Suppression of dendritic cell function in cancer patients is thought to contribute to the inhibition of immune responses and disease progression. Molecular mechanisms of this suppression remain elusive, however. Here, we show that a fraction of blood monocyte-derived myeloid dendritic cells (MDCs) express B7-H1, a member of the B7 family, on the cell surface. B7-H1 could be further upregulated by tumor environmental factors. Consistent with this finding, virtually all MDCs isolated from the tissues or draining lymph nodes of ovarian carcinomas express B7-H1. Blockade of B7-H1 enhanced MDC-mediated T-cell activation and was accompanied by downregulation of T-cell interleukin (IL)-10 and upregulation of IL-2 and interferon (IFN)-gamma. T cells conditioned with the B7-H1-blocked MDCs had a more potent ability to inhibit autologous human ovarian carcinoma growth in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, upregulation of B7-H1 on MDCs in the tumor microenvironment downregulates T-cell immunity. Blockade of B7-H1 represents one approach for cancer immunotherapy.