RESUMEN
Serositis is a rare manifestation of chronic GvHD (cGvHD). No risk factors or laboratory changes associated with this syndrome have been recognized to date, and outcomes have not been described in a large series. We searched our institutional database for patients undergoing allogeneic hematopoietic cell transplant identified as having serositis or pericarditis. Laboratory studies from prior to diagnosis, at diagnosis and post diagnosis of serositis, as well as outcomes from invasive procedures were included. Twenty patients met criteria for cGvHD-associated serositis, and all but three patients had a prior diagnosis of cGvHD. Fifteen were male, and the complication occurred in the setting of immunosuppressant taper in 12 cases. Ten patients required invasive interventions, including pericardial window or stripping. A significant increase in blood monocytes and decrease in serum albumin were identified at diagnosis compared with pre-diagnosis. Out of 20 patients, 17 were treated with steroids, with 12 demonstrating a complete response. These data suggest that cGvHD-associated serositis occurs mainly in the setting of treated as opposed to de novo cGvHD and biomarkers associated with the syndrome include a decrease in albumin and an increase in absolute monocyte count. Outcome data from larger series are required to better understand the optimal management of this rare complication.
Asunto(s)
Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/terapia , Pericarditis/diagnóstico , Pericarditis/terapia , Serositis/diagnóstico , Serositis/terapia , Adulto , Anciano , Aloinjertos , Enfermedad Crónica , Femenino , Enfermedad Injerto contra Huésped/sangre , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/terapia , Humanos , Masculino , Persona de Mediana Edad , Pericarditis/sangre , Serositis/sangreRESUMEN
Clinical efficacy of adenovirus-mediated cancer gene therapy has been limited thus far. To improve its oncolytic effect, a replication-competent adenoviral vector was previously constructed to express high levels of p53 at a late time point in the viral life cycle. p53 expression from this vector improved tumor cell killing and viral spread in vitro. However, p53 function is antagonized by cellular mdm2 and adenoviral E1b-55kD, both of which are known to bind to and inactivate p53. Therefore, a new vector (Adp53W23S) that expresses a modified p53 transgene, which does not bind to E1b-55kd and mdm2, was constructed. The modified p53 protein was demonstrated to have a substantially prolonged half-life, and its localization was predominantly nuclear. Viral replication was unaffected by expression of the modified p53 and cancer cell killing was improved in vitro. However, in a xenograft model, efficacy was not significantly different from control virus. In conclusion, expression of a degradation-resistant p53 transgene late in the life cycle of a replication-competent adenovirus improves p53 stability and cancer cell killing in vitro. However, other factors, such as the adenoviral E1b-19kD and E1a proteins, which oppose p53 function, and limitations to viral spread need to be addressed to further improve in vivo efficacy.
Asunto(s)
Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Replicación Viral , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1B de Adenovirus/genética , Muerte Celular , Línea Celular Tumoral , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Neoplasias/virología , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Successful cancer therapy using replicating viral vectors relies on the spread of virus from infected to uninfected cells. To date, there has been limited clinical success in the use of replicating adenoviruses. In animal models, established xenograft tumors are rarely eliminated despite the persistence of high viral titers within the tumor. Hypoxia is a prevalent characteristic of solid tumors, whereas adenovirus naturally infects tissues exposed to ambient oxygen concentrations. Here, we report that hypoxia (1% oxygen) reduces adenoviral replication in H1299 and A549 lung cancer cells, BxPC-3 pancreatic cancer cells, LNCaP prostate cancer cells and HCT116 colon cancer cells. However, hypoxia does not reduce cell viability or restrict S-phase entry. Importantly, the production of E1a and fiber proteins under hypoxic conditions was substantially decreased at 24 and 48 h compared to room air controls. In contrast, Northern analysis showed similar levels of E1a mRNA in room air and hypoxic conditions. In conclusion, a level of hypoxia similar to that found within solid tumors reduces the replication of adenoviral vectors by reduction of viral protein expression without a reduction in mRNA levels. To further improve oncolytic therapy using a replicating adenovirus, it is important to understand the mechanism through which hypoxia and the virus interact to control expression of viral and cellular proteins, and consequently to develop means to overcome decreased viral production in hypoxic conditions.
Asunto(s)
Adenoviridae/fisiología , Vectores Genéticos , Neoplasias/virología , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Adenoviridae/genética , Northern Blotting , Hipoxia de la Célula/fisiología , Regulación hacia Abajo , Terapia Genética/métodos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neoplasias/metabolismo , Neoplasias/patología , ARN Mensajero/genética , ARN Viral/genética , Fase S/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Strategies to target viral replication to tumor cells hold great promise for the treatment of cancer, but even with replicating adenoviruses complete tumor responses are rarely achieved. To evaluate replicating adenoviral vectors, we have used A549 human lung cancer nude mouse xenografts as a model system. Intratumoral injection of wild-type adenovirus (Ad309) significantly reduced tumor growth from day 14 (p = 0.04) onward; however, tumor volumes reached a plateau at day 50. At 100 days, high levels of titratable virus were present within persistent viable tumors. In contrast to viral injection into established tumors, when tumor cells were infected in vitro with wild-type virus and then mixed with uninfected tumor cells, 1% of infected cells was sufficient to prevent tumor establishment. An E1b-19kD-deleted viral mutant (Ad337) was more efficient than Ad309 in this cell-mixing model. Just 1 cell in 1000 infected with Ad337 prevented tumor growth. However, although better than wild-type virus, Ad337 was unable to eradicate established flank tumors. These data suggest that although replicating adenoviruses exhibit significant oncolytic activity, barriers within the established tumor, such as connective tissue and tumor matrix, may limit the spread of virus. Strategies to enhance viral spread through established tumors are therefore likely to greatly improve the therapeutic efficacy of replicating adenoviruses.
Asunto(s)
Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Animales , División Celular , Eliminación de Gen , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Replicating adenoviral vectors are a promising new modality for cancer treatment and clinical trials with such vectors are ongoing. Targeting these vectors to cancer cells has been the focus of research. However, even if perfect targeting were to be achieved, a vector still must effectively kill cancer cells and spread throughout the bulk of the tumor. The adenoviral E1b-19kD protein is a potent inhibitor of apoptosis and may therefore compromise the therapeutic efficacy of an adenoviral vector. In this study we have investigated if an E1b-19kD gene deletion could improve the ability of a replicating adenoviral vector to spread through and kill cancer cells. In several lung cancer cell lines an E1b-19kD-deleted virus (Ad337) induced substantially more apoptosis than did a wild-type virus (Ad309), and tumor cell survival was significantly reduced in three of four cell lines. In addition, the apoptotic effects of cisplatin or paclitaxel were augmented by Ad337, but inhibited by wild-type virus. The number of infectious virus particles in the supernatant of infected cells was increased with Ad337 compared with wild-type virus, indicating enhanced early viral release. Ad337, in contrast to Ad309, induced significantly larger plaques after infection of A549 cells. This well-described large plaque phenotype of an E1b-19kD mutant virus is likely the result of early viral release and enhanced cell-to-cell viral spread. Loss of E1b-19kD function caused only minor cell line-specific increase or decrease in viral yield. We conclude that deletion of the E1b-19kD gene may enhance the tumoricidal effects of a replicating adenoviral vector.
Asunto(s)
Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Apoptosis/genética , Eliminación de Gen , Proteínas E1B de Adenovirus/metabolismo , Antineoplásicos/farmacología , Cisplatino/farmacología , Vectores Genéticos/genética , Humanos , Paclitaxel/farmacología , Células Tumorales Cultivadas , Ensayo de Placa Viral , Replicación ViralRESUMEN
It has been proposed that an adenovirus with the E1b-55kD gene deleted has a selective advantage in replicating in cancer cells that have mutations in the p53 gene (Bischoff et al., 1996). We have explored this hypothesis in several lung cancer cell lines, and evaluated potential mechanisms that might regulate the replication of Ad338, an E1b-55kD-deleted virus, with the objective of developing a rational approach for targeting gene therapy to lung tumors. Our data show that Ad338 replicates poorly in three lung cancer cell lines with various p53 mutations (H441, H446, and Calu1), yet this virus replicates to a high level in a lung cancer cell line with wild-type p53 (A549) and in a normal lung fibroblast line (IMR90). Viral DNA replication, expression of viral proteins, and shutoff of host cell proteins were not important variables in limiting the replication of the E1b-55kD-deleted virus. However, the cell lines resistant to host cell protein shutoff were also the most resistant to the cytopathic effect induced by mutant and wild-type virus and the only cells to survive for 8 days following infection. The E1b-55kD protein clearly has an important role in viral replication beyond its interaction with p53. Thus, an E1b-55kD-deleted virus cannot be used to specifically target viral replication to p53-mutated lung cancer cells.
Asunto(s)
Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Terapia Genética , Vectores Genéticos , Neoplasias Pulmonares/terapia , Adenoviridae/fisiología , Secuencia de Bases , Supervivencia Celular , Efecto Citopatogénico Viral , Cartilla de ADN , Replicación del ADN/genética , Genes Virales , Humanos , Neoplasias Pulmonares/virología , Proteína p53 Supresora de Tumor/genética , Replicación Viral/genéticaRESUMEN
The hair follicle provides an excellent system in which to study growth and differentiation. Hair keratins are useful tissue-specific molecular markers for these events. By comparing a second mouse Type I hair keratin cDNA clone, MHKA-2, with our previously described MHKA-1, we have been able to contrast the nucleotide sequences and corresponding deduced amino acid sequences of the smallest (mHa4) and the largest (mHa1) major Type I hair keratins. Both nucleotide sequences and both deduced amino acid sequences share high identity but have distinct segments suitable for generation of specific molecular probes. Comparison of amino acid sequences adjacent to the central helical domains has demonstrated homologous subdomains, designated H1 and H2, in the Type I hair keratin nonhelical termini. Although there is only 56% amino acid identity in the carboxy-terminal nonhelical domains, a common sequence, T-------CGPC----R, has been identified in this domain, suggesting a possible common functional role for this portion of the molecule. In addition, it appears that mHa4 may differ in part from mHa1 by deletion of a segment between the H2 subdomain and the conserved sequence. Staining of mouse and human hair follicles with AmHa1, a monospecific polyclonal antibody for mHa1, and AE13, an antibody specific for all Type I hair keratins, suggests differential expression of individual Type I hair keratins in both species. This supports our hypothesis that distinct functional requirements are satisfied by the multiplicity of hair keratins.