Lanreotide vs octreotide LAR for patients with advanced gastroenteropancreatic neuroendocrine tumors: An observational time and motion analysis.

BACKGROUND: Lanreotide and octreotide acetate suspension for injectable (LAR) are both recommended for clinical use in patients with locally advanced or metastatic gastroenteropancreatic neuroendocrine tumors. However, each agent possesses unique attributes in terms of their drug-delivery characteristics. The study objective was to compare overall drug-delivery efficiency between lanreotide and octreotide LAR in gastroenteropancreatic neuroendocrine tumor patients. METHODS: This study employed an observational time and motion design among patients treated with lanreotide or octreotide LAR across five US cancer centers. Baseline patient data collection included age, disease grade and duration, prior therapies and performance status. Drug-delivery time (drug preparation and administration), total patient time and resource use data were collected for gastroenteropancreatic neuroendocrine tumors receiving lanreotide (n = 22) or octreotide LAR (n = 22). Following each administration, qualitative data on the drug-delivery experience was collected from patients and nurses. RESULTS: Lanreotide was associated with a significant reduction in mean delivery time (2.5 min; 95% CI:2.0 to 3.1) compared to octreotide LAR (6.2 min; 95%CI: 4.4 to 7.9; p = 0.004). The mean total patient time for lanreotide and octreotide LAR was comparable between groups (32.1 vs. 36.6 minutes; p = 0.97). Nurses reported increased concerns with octreotide LAR related to needle clogging (p = 0.034) and device failures (p = 0.057). Overall, lanreotide had a median satisfaction score of 5.0 compared to a score of 4.0 with octreotide LAR (p = 0.03). CONCLUSIONS: Lanreotide was associated with significant reductions in drug-delivery time compared to octreotide LAR, which contributed to an improvement in overall healthcare efficiency. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT03017690.

Effect of rev on the cytoplasmic localization of intron-containing human immunodeficiency virus type 1 RNA.

Human immunodeficiency virus type 1 (HIV-1) proteins are expressed from both intron-containing and completely spliced RNAs. Rev, an HIV-1 regulatory protein, is necessary for the expression of intron-containing RNAs. The effect of Rev on the subcellular localization of intron-containing HIV-1 RNA was examined by in situ RNA hybridization. In the presence of Rev, intron-containing HIV-1 RNA accumulated at the nuclear membrane and within the cytoplasm of transfected cells. In the absence of Rev, intron-containing HIV-1 RNA accumulated within the nucleus. In approximately 20% of the cells transfected in the absence of Rev, intron-containing HIV-1 RNA was also found in the cytoplasm. Differences in the subcytoplasmic localization of intron-containing HIV-1 RNA in the presence and absence of Rev were not observed using in situ RNA hybridization. To determine the effect of Rev on RNA localization within the cytoplasm, an extensive fractionation protocol involving both hypotonic and detergent lysis was used. In the presence of Rev, 40.9 +/- 4.6% of the cytoplasmic intron-containing HIV-1 RNA was released by hypotonic lysis. A similar fractionation profile was seen for several other translated viral and cellular RNAs. However, in the absence of Rev, only 16.5 +/- 5.1% of the cytoplasmic intron-containing HIV-1 RNA was released on hypotonic lysis (P < 0. 005). Thus the cytoplasmic fractionation pattern of this RNA was altered in the absence of Rev.

Activation of a cryptic splice donor in human immunodeficiency virus type-1.

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function as cis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5' splice donor of HIV-1 was mutated in the context of other gag sequences. Following transient transfection, RNA expression by RT-PCR was analyzed. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild-type 5' splice donor and the tat splice acceptor was used. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of the RNA product containing the cryptic splice donor revealed that the Rev was required for the cytoplasmic accumulation of unspliced RNA, while spliced RNA was Rev independent. Transfection of a wild-type construct also demonstrated usage of the cryptic splice donor. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor may retain Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in the determining the Rev dependence of viral RNAs.

Effect of Rev on the intranuclear localization of HIV-1 unspliced RNA.

Human immunodeficiency virus type 1 (HIV-1) Rev is a 19-kDa regulatory protein which binds to unspliced and partially spliced HIV-1 RNAs. Export, splicing, stability, and translation of HIV-1 RNAs are influenced by Rev. To further understand the effect of Rev on HIV-1 RNA splicing, the intranuclear localization of unspliced HIV-1 RNA and a cellular splicing factor was examined in the presence and absence of Rev. Splicing component-35 (SC-35) is an essential SR protein splicing factor which localizes into 20-40 nuclear granules (Fu, X. D., and Maniatis, T. Nature 343 (6257), 437-441, 1990). Laser scanning confocal microscopy was utilized to examine the colocalization of unspliced HIV-1 RNA and SC-35-containing granules. In the presence of Rev, many of the SC-35-containing granules were colocalized on their edges or completely colocalized with HIV-1 unspliced RNA speckles. In the absence of Rev, however, little colocalization of the unspliced HIV-1 RNA speckles and the SC-35-containing granules was observed. Quantitative RT-PCR was utilized to examine the effect of Rev on the level of fully spliced HIV-1 RNA. In the presence of Rev, a decrease in the level of fully spliced HIV-1 RNA was observed. Thus both the intranuclear localization and posttranscriptional processing of HIV-1 unspliced RNA are affected by Rev.

Involvement of human immunodeficiency virus type-1 splice sites in the cytoplasmic accumulation of viral RNA.

To define the role of human immunodeficiency virus type 1 splice sites in the cytoplasmic accumulation of viral RNAs, sequential deletion mutagenesis on an infectious proviral clone of HIV-1 was performed. Deletion of the majority of intron sequences, containing previously identified CRS, did not attenuate CRS activity. Retention of either the first or second tat intron preserved CRS activity. RNAs containing splice donor sequences, in the absence of known downstream splice acceptor sequences, retained CRS activity. Unexpectedly, these splice donors were still utilized for splicing. These results indicate that the major HIV-1 splice donors can function as CRS and function to negatively regulate the cytoplasmic accumulation of HIV-1 RNAs in COS cells.

Characterization of Rev function using subgenomic and genomic constructs in T and COS cells.

The effect of the human immunodeficiency type 1 (HIV-1) Rev protein on the splicing and cytoplasmic accumulation of HIV-1 RNAs was investigated in COS and T cells. Subgenomic and genomic constructs were used which expressed varying levels of complexity in their potential RNA constituents. Using all constructs, in both cell types, an inhibitory effect of Rev on the level of fully spliced HIV-1 RNAs could be demonstrated. An increase in the nuclear level of unspliced pre-mRNA was seen in the presence of Rev with genomic constructs. Thus, the inhibitory effect on splicing was not merely due to enhancement of nuclear export of the pre-mRNA with these constructs. In both cell types, a positive effect of Rev on the cytoplasmic accumulation of HIV-1 RNAs could also be seen. However, in T cells, the Rev-dependent RNAs were still capable of accumulating at a reduced level in the cytoplasmic fraction in the absence of Rev. The identity of the cell type, construct, and RNA species impacted on the phenotypic manifestation of Rev function.

Retrovirus-mediated transfer of viral IL-10 gene prolongs murine cardiac allograft survival.

A murine heterotopic, nonvascularized cardiac allograft model was used to examine the effects of the immunosuppressive cytokine, viral IL-10 (vIL-10), delivered by gene transfer on graft rejection. Retroviral-mediated gene transfer and expression of vIL-10 significantly prolonged allograft survival, without conventional systemic immunosuppression, from 12.1 +/- 0.8 days to 39.4 +/- 2.5 days (p < 0.0001). The effect was specific, dose dependent, and restricted to the site of transplantation. PCR analysis demonstrated specific expression of the transferred gene within the allograft. Analysis of the cellular infiltrate in the allografts showed a reduction in T cells and alloantigen-specific cytotoxic T cells and IL-2 producing helper T cells. Thus, the transient local expression of a gene encoding an immunosuppressive protein within a graft can generate local immunosuppression, making gene therapy a viable approach for facilitating transplantation.

Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immunosuppression with TGF-beta 1 or vIL-10.

The application of gene transfer techniques to organ transplantation offers the potential for modulation of immunity directly within an allograft without systemic side effects. Expression vectors and promoter elements are important determinants of gene transfer and expression. In this study, various vectors (naked plasmid DNA, retroviral vector, herpes simplex viral vector, and adenoviral vector) with various promoters (RSV-LTR, SV40, MuLV-LTR, HCMVie1) were directly compared to demonstrate the successful gene transfer and expression of beta-galactosidase in murine myoblasts in vitro and within murine heterotopic, nonvascularized cardiac isografts or allografts in vivo. Expression of transferred genes was not toxic to cells and strength of expression varied according to the type of vector. Plasmid DNA was expressed in myocytes, retroviral vector was expressed in the graft infiltrating cells, and herpes simplex and adenoviral vectors were expressed in both myocytes and graft-infiltrating cells. Preliminary studies evaluated the ability of these vectors to deliver immunologically important signals. Allografts injected with pSVTGF-beta 1, a plasmid-encoding transforming growth factor beta 1 (TGF-beta 1) under the control of the SV40 promoter, showed significant prolongation of graft survival of 26.3 +/- 2.5 days compared with 12.6 +/- 1.1 days for untreated allografts, and 12.5 +/- 1.5 days for the allografts injected with control plasmid (P < 0.05). Allografts injected with MFG-vIL-10, a retroviral vector encoding viral interleukin-10 under the control of the MuLV-LTR, showed prolongation of graft survival of 36.7 +/- 1.3 days versus 12.6 +/- 1.1 days for the untreated allograft, and 13.5 +/- 2.0 days for the allografts injected with control retroviral vector (P < 0.001). Both vectors were transcriptionally active in vivo and did not appear to have toxic effects. Gene therapy for transplantation can induce transient expression of immunologically relevant molecules within allografts that impede immune activation while avoiding the systemic toxicity of conventional immunosuppression.

Gene transfer for transplantation. Prolongation of allograft survival with transforming growth factor-beta 1.

OBJECTIVE: The authors tested the ability of plasmid gene transfer to express transforming growth factor-beta 1 (TGF-beta 1), prolong allograft survival, and evaluate promoter effects on gene expression. SUMMARY BACKGROUND DATA: Delivery of immunosuppressants directly to allografts using gene transfer and gene therapy approaches may inhibit immune activation while avoiding the systemic toxicity of conventional immunosuppression. Candidate genes include soluble cytokines, which could be expressed at low levels throughout the graft while inducing a local immunosuppressive effect. Transforming growth factor-beta 1 is a soluble cytokine that has pleiotropic immunosuppressive effects. METHODS: Cardiac grafts from syngeneic (CBA/J, H-2k) or allogenic (C57BL/6, H-2b) donors were placed into CBA/J recipients. Purified plasmid DNA-encoding murine TGF-beta 1 or beta-galactosidase (Lac Z) under the control of RSV, SV40, MMTV, or pancreatic elastase promoters was injected into grafts at surgery. The Lac Z expression was determined by histologic examination and TGF-beta 1 expression by graft survival. Cytotoxic T lymphocyte and flow cytometric analyses were performed to evaluate the immunosuppressive effects of TGF-beta 1 in vitro. RESULTS: Plasmid DNA-encoding TGF-beta 1 prolonged survival from 12.6 +/- 1.1 days to 26.3 +/- 2.5 days (p < 0.02, Student's t test). The SV40 promoter was superior to the MMTV promoter in its ability to prolong survival. The effects of the plasmids were specific because Lac Z, antisense TGF-beta 1 inserts, or pancreatic elastase promoter did not prolong allograft survival. Histologic examination demonstrated Lac Z expression at least 14 days post-transplant in myocardial cells. Both RSV and SV40 promoters were effective in this respect, while a control null promoter was not. Toxicity testing showed that gene transfer of TGF-beta 1 did not alter survival or histology of syngeneic grafts. In addition, plasmids and purified TGF-beta 1 protein were not toxic to myoblasts in vitro. Recombinant TGF-beta 1 inhibited cytotoxic T lymphocyte generation and altered T cell surface receptor expression and subset expansion in vitro. CONCLUSION: Gene transfer/therapy with plasmid DNA encoding TGF-beta 1 in vivo achieves immunologic effects that prolong allograft survival. Multiple promoters effectively induce plasmid expression, which is achieved in cardiac myocytes for at least 2 weeks without toxicity or adverse systemic effects. Transforming growth factor-beta 1 inhibits immune responses by different mechanisms, revealed by in vitro analysis of T cell cytolytic function, subset distribution, and receptor display.