JLP belongs to the JIP family whose members serve as scaffolding proteins that link motor proteins and their cargo for intracellular transport. Although JLP is mainly cytoplasmic, it accumulates as a ...focus in the perinuclear region when stimulated by extracellular stimuli. Focus formation, which changes the nucleus shape and concentrates the nuclear pores, depends on p38MAPK activation and the dynein retrograde motor protein complex. Extracellular stimuli trigger the tethering of PLK1 to the centrosome by JLP, leading to centrosome maturation and microtubule array formation. The centrosome localization domain of JLP is important for the binding of the centrosome and the formation of the JLP focus and the microtubule array. Furthermore, the formation of the JLP focus and the microtubule array is interdependent and important for the transport of NF-κB p65 to the nucleus and its unloading therein. In conclusion, JLP exhibits multiple functions in the nuclear translocation of NF-κB p65.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common neoplasm worldwide. Despite advances in multimodality treatments involving surgery, radiation and chemotherapy, the five-year ...survival rate has remained at ~50% for the past 35 years. Therefore, the early detection of recurrent or persistent disease is extremely important. Replication-incompetent HIV-1-based lentiviral vectors have emerged as powerful and safe tools for gene delivery. Commonly, HNSCC is a locoregional disease that presents at or close to the body surface. Thus, HNSCC is amendable to intratumoral injections of gene therapy vectors aimed at correcting defects associated with tumor suppressor genes to induce the direct cytotoxicity of cancer cells or immune modulation to promote antitumor immunity. Current investigations analyzing HNSCC gene mutations and stem cell markers and the cancer immunoediting concept are creating exciting therapeutic opportunities for lentiviral and other gene transfer vectors. The present review reports specific examples of the current applications of lentiviral vectors in HNSCC.
RNA interference is an evolutionarily conserved process of gene silencing that in plants serves as a natural defense mechanism against exogenous viral agents. RNA interference is becoming an ...important tool for the study of biological processes through reverse genetics and has potential for therapeutic applications in humans; however, effective delivery is still a major issue. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) have been introduced into cells by transfection of chemically synthesized and RNA expression via plasmid cassettes utilizing RNA polymerase III transcription. The employment of siRNA/shRNA for gene knockout requires an efficient stable transfection or transduction process. Here, we report the successful construction of lentiviral vectors to express shRNA stably in human cells. We demonstrate that lentiviral vectors expressing siRNA directed to the reporter gene luciferase, when stably transduced into human cells without drug selection, are capable of protecting the cells from infection by a lentiviral vector encoding humanized firefly luciferase as a reporter gene. We observed 16- to 43-fold reduction of gene expression in infected cells transduced with shRNA vectors relative to cells transduced with control vectors. This model system demonstrates the utility of lentiviral vectors to stably express shRNA as both a cellular gene knockout tool and as a means to inhibit exogenous infectious agents such as viruses in human cells.
Dendritic cells are professional antigen-presenting cells that initiate, regulate and shape the induction of specific immune responses. The ability to use dendritic cells in the induction of ...antigen-specific tolerance, antigen-specific immunity or specific differentiation of T-helper subsets holds great promise in dendritic cell-based immunotherapy of various diseases such as cancer, viral infections, allergy, as well as autoimmunity. Replication-incompetent HIV-1-based lentiviral vector is now emerging as a promising delivery system to genetically modify dendritic cells through antigen recognition, costimulatory molecules and/or polarization signals for the manipulation of antigen-specific immunity in vivo. This article discusses some of the recent advances in the uses of lentiviral vectors in dendritic cell-based immunotherapy.
We examined the potential of ex vivo gene therapy to enhance bone repair using lentiviral vectors encoding either enhanced green fluorescent protein (EGFP) as a reporter gene or bone morphogenetic ...protein-2 (BMP-2) downstream of either the cytomegalovirus immediate early (CMV) promoter or the murine leukemia virus long terminal repeat (RhMLV) promoter derived from a murine retrovirus adapted to replicate in a rhesus macaque. In vitro, rat bone marrow stromal cells (BMSCs) transduced with Lenti-CMV-EGFP or Lenti-RhMLV-EGFP demonstrated over 90% transduction efficiency at 1 week and continued to demonstrate stable expression for 8 weeks. ELISA results demonstrated that lentivirus-mediated gene transfer into BMSCs induced stable BMP-2 production in vitro for 8 weeks. Increased EGFP and BMP-2 production was noted with the RhMLV promoter. In addition, we implanted BMSCs transduced with Lenti-RhMLV-BMP-2 into a muscle pouch in the hind limbs of severe combined immune deficient mice. Robust bone formation was noted in animals that received Lenti-RhMLV-BMP-2 cells at 3 weeks. These results demonstrate that lentiviral vectors expressing BMP-2 can induce long-term gene expression in vitro and new bone formation in vivo under the control of the RhMLV promoter. Prolonged gene expression may be advantageous when developing tissue engineering strategies to repair large bone defects.
CD3ζ has emerged as a clinically important immunological marker in head and neck squamous cell carcinoma (HNSCC) with reduced level of expression reported in both tumor infiltrating lymphocytes and ...peripheral blood lymphocytes. In this prospective study (power = 0.99,
α
= 0.05), CD3ζ expression was compared in 47 HNSCC patients and 53 controls using standardized flow cytometric method. There was no statistical difference in the percentages of the CD3 ε+ T‐cell subset present in the peripheral blood mononuclear cells of the HNSCC patients and the healthy controls; however, T cells from the HNSCC patients produced a significantly weaker IFN‐γ response in comparison to the healthy controls, when they were stimulated by the recall viral CEF peptide antigen. All patients were followed up for at least 3 years with a median follow‐up of 45 months. Levels of CD3ζ‐chain expression were measured at 117 follow‐up visits at six‐month intervals. Receiver operating characteristic curve identified the optimal cut off as a 12% increase in post treatment CD3ζ‐chain expression from the baseline levels to confirm absence of HNSCC with the area under curve of 0.81 (95% CI = 0.68–0.94) for predicting absence of HNSCC. The specificity, sensitivity and positive predictive value were 81.25% 79.21% and 97.56%, respectively. Three‐year disease specific survival (DSS) was significantly lower (
p
= 0.007) at 63.2% for patients who showed <12% increase in CD3ζ‐chain level as compared to 96.2% for patients who had ≥12% increase. Our results indicate that the change in CD3ζ‐chain expression from the baseline is an independent predictor of residual and recurrent HNSCC.
What's new?
There is an urgent need for biomarkers that allow early detection of relapse in head and neck squamous cell carcinoma (HNSCC). CD3ζ may be one such marker, as lymphocytes from HNSCC patients express reduced levels of this protein. In this study, the authors found that patients whose CD3ζ levels increased by less than 12% after treatment had significantly lower survival. These results indicate that measurement of CD3ζ over time may allow oncologists to detect and treat recurrence early enough to improve outcomes.
The mouse NKR-P1 family includes at least three genes: NKR-P1A, -B, -C. Neither surface expression nor function of the NKR-P1B gene product has previously been shown. Here, we demonstrate that the ...SJL/J allele of the NKR-P1B gene product is expressed on SJL/J NK cells, and is recognized by PK136 mAb. Interestingly, the same mAb does not recognize the NKR-P1B gene product of C57BL/6. We have also generated a novel mAb, 1C10, that recognizes an activation receptor on SJL/J NK cells. Activation of the NKR-P1B receptor-inhibited 1C10 mAb induced redirected lysis and recruited SHP-1, indicating that NKR-P1B is an inhibitory receptor. Therefore, the mouse NKR-P1 gene family, like the Ly49 family, includes both activation and inhibitory receptors.
Dendritic cells (DCs) are effective in stimulating and controlling the outcome of T cell responses. Human immunodeficiency virus type 1-based lentiviral vectors can achieve sustained transduction of ...genes/antigens in dividing and nondividing cells, thus representing a candidate vector for stable expression of antigens in DCs. We previously established conditions for transduction of purified cytokine mobilized rhesus CD34(+) cells in vitro, and transplantation of the autologous transduced cells in a nonhuman primate model in vivo. In the present study, we transplanted DCs derived from EGFP-transduced CD34(+) cells into nonmyeloablated rhesus macaques. Transplantation of DCs stably expressing EGFP into autologous animals induces persistent, long-lived (up to 100 weeks) EGFP-specific T cell responses. Of note, no humoral responses against EGFP are detected in the transplanted animals. These studies provide, to our knowledge, the first demonstration that lentiviral transduction of CD34(+) progenitor cells subsequently differentiated to DCs is capable of priming a specific T cell response in a nonhuman primate in vivo. Taken together, our data provide formal in vivo evidence that lentivirus-transduced dendritic cells represent a potential approach in eliciting cellular immune responses in primates.
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