Comparison of the vector systems for gene transduction into rat dendritic cells and peritoneal exudate cells

Specialized antigen-presenting cells (APC), known as dendritic cells (DC), play a pivotal role in initiating primary immune responses. It has been reported that several vector systems, including adenoviral vectors, retroviral vectors, Hemagglutinating Virus of Japan (HVJ)-related vectors, and electroporation, are able to transduce genes into mouse and human DC. This has not been achieved for rat DC. To our knowledge, there is no direct evidence to support the view that the currently used vector systems are able to transduce genes into mature DC. Because most, if not all, gene transfer studies investigating DC or DC-related cell populations are carried out using heterogeneous groups of cells, it is therefore very important to determine to what extent gene transduction occurs in rat DC, and also selected mature DC (CD161a+ fully mature DC). In this study, we provide evidence that none of 4 vector systems are able to transfer genes into fully mature rat DC, which are derived from bone marrow cells (BMC), driven by Flt3/Flk2 ligand and interleukin (IL)-6, and purified by CD161a. Nevertheless, the most efficient gene transduction was observed in the developing DC progenitor cells during the long-term culture of rat BMC, and its gene expression was successfully achieved after 2 weeks of culture only with a human immunodeficiency virus (HIV)-based lentiviral vector system. The most critical time point for lentiviral gene transduction was around the 7th day from the beginning of culture with lentiviral vectors. Rat peritoneal exudate cells (PEC) and another cell line (K562) were easily transducted by adenoviral vectors and lentiviral vectors.     

Studies on the most efficient vector systems for gene transduction into dendritic cells

Specialized antigen-presenting cells (APC), known as dendritic cells (DC), play a pivotal role in initiating primary immune responses. Several vector systems, including adenoviral vectors, retroviral vectors, hemagglutinating virus of Japan-related vectors, and the electroporation, have been shown to transduce genes into mouse and human but not rat DC. However, there is no direct evidence to support the view that the currently used vector systems are able to transduce genes into mature DC. Inasmuch as most, if not all, gene transfer studies investigating DC or DC-related cell populations are performed employing heterogeneous-groups of cells, it is therefore important to determine the extent to which gene transduction occurs in bona fide DC. In this study, we provide evidence that none of these vector systems are able to transfer genes into mature rat DC, which are derived from bone marrow cells (BMC), driven by Flt3/Flk2 ligand and IL-6, and purified with CD161a. Nevertheless, the most efficient gene transduction was observed with developing DC progenitor cells during long-term culture of rat BMC. Successful gene transfer was achieved after 2-week culture with an HIV-based lentiviral vector system.     

Transduction efficiency of adenoviral vectors into human glioma cells increased by association with cationic liposomes

Replication-deficient adenoviral vectors are promising agents for human gene therapy of the greater transduction efficiency than other vectors. However, there are distinct disadvantages, including high immunogenicity, which limits the administration to human organs, particularly the brain. Injection of adenoviral vectors into the human brain causes inflammatory responses and induces cerebral edema. The combined effect of adenoviral vectors and cationic liposomes in vitro was investigated in an effort to reduce the immune reaction against the antigens of adenoviral vectors. No toxicity of adenoviral vector-associated liposomes was observed within optimal lipid concentration. The transduction efficiency of the adenoviral vectors containing the beta-galactosidase gene increased almost 10-fold when associated with the cationic liposomes. Furthermore, greater cytotoxicity was induced when the adenoviral vector containing herpes simplex virus-thymidine kinase gene was combined with cationic liposomes than with only the adenoviral vector. These results suggest that the combination of adenoviral vectors and cationic liposomes allows the doses of adenoviral vectors to be reduced while maintaining transduction efficiency.     

Transduction of dendritic cell progenitors with a retroviral vector encoding viral interleukin-10 and enhanced green fluorescent protein allows purification of potentially tolerogenic antigen-presenting cells

BACKGROUND: Dendritic cells (DC) are important antigen-presenting cells that play critical roles in the initiation and modulation of immune responses. Genetic engineering of DC to express immunosuppressive molecules is a novel approach to the inhibition of allograft rejection. Retroviral delivery of viral interleukin (vIL)-10 to replicating myeloid DC progenitors (DCp) impairs their T-cell stimulatory capacity and promotes the induction of antigen-specific T-cell hyporesponsiveness. However, transduction efficiency with retroviral vectors is comparatively low. Enhanced green fluorescent protein (EGFP) is important both as a marker of gene transduction and for the selection of transduced cells. Our aims were to construct a retroviral vector encoding both vIL-10 and EGFP, to positively select transduced DC, and to assess the impact of these highly purified, vIL-10-secreting antigen-presenting cells on allogeneic T-cell responses. METHODS: DCp propagated from bone marrow of C57BL10 (H2b) mice in granulocyte-macrophage colony-stimulating factor (GM-CSF)+IL-4 were transduced with a retroviral vector encoding both vIL-10 and EGFP by centrifugal enhancement. Gene transfer efficiency was determined by flow cytometry. Transduced cells were flow sorted, and vIL-10 secretion was quantified by ELISA. DC function was assessed by the ability of the cells to induce naive allogeneic (C3H; H2k) T-cell proliferation and cytotoxic T lymphocyte generation. RESULTS: Retrovirally transduced DC expressed both vIL-10 and EGFP gene products. Approximately 20% of unsorted cells expressed EGFP, as determined by flow cytometry. vIL-10 was produced at a mean rate of 31 ng/40 hr/10(6) cells. After sorting, the incidence of EGFP+ DC was increased dramatically to at least 95%, and the production of vIL-10 was increased approximately three- to fourfold, to a mean of 107 ng/40 hr/10(6) cells. These highly purified, vIL-10-secreting DC exhibited markedly diminished capacity to induce allogeneic T-cell proliferative and cytotoxic responses. CONCLUSIONS: DCp retrovirally transduced to express both vIL-10 and EGFP can be rapidly identified and sorted to high levels of purity. The availability of highly enriched preparations of vIL-10-transduced DC facilitates studies of their immunoregulatory function and may enhance their therapeutic potential in transplantation or autoimmune disease.     

腺病毒与慢病毒载体对人脂肪间充质干细胞转导效率及基因表达的差异

目的：比较重组腺病毒、慢病毒载体介导增强型绿色荧光蛋白对体外培养的人脂肪来源干细胞（hADSCs）的转导效率和外源基因表达差异。方法：原代分离培养hADSCs并鉴定,应用Ad5F35-EGFP及LV—EGFP以MOI=0、25、50、100、200、400、800感染hADSCs,在1、3、7、14、21天应用荧光显微镜、流式细胞仪观察检测表达EGFP细胞阳性率。MTT法检测转导对hADSCs增殖的影响。YonKossa染色、油红O染色检测体外诱导转导EGFP的hADSCs向成骨及成脂方向分化能力。结果：hADSCs细胞表面标记CD31、CD34、CD45、CD106和HLA-DR阴性,CD29、CD44、CD49d、CD105、CD166阳性。荧光显微镜观察Ad5F35-EGFP感染hADSCs后（1～7天）可见EGFP高表达,此后逐渐减弱;LV—EGFP感染hADSCs后21天,仍可见EGFP高表达。流式细胞仪检测Ad5F35-EGFP、LV-EGFP对hADSCs转导效率与MOI值呈正相关。MTT显示Ad5F35-EGFP在高MOI值（800）检测A值与对照组相比差异显著（P〈0．01）。转导EGFP的hADSCs经体外诱导14天可向成骨、成脂方向分化。结论：与腺病毒相比,慢病毒能够更为安全、有效地感染体外培养hADSCs,并长期表达外源基因,是研究基因修饰hADSCs的理想载体。     

Minor histocompatibility antigens as risk factor for poor prognosis in kidney transplantation

Progress in transplantation has relied on similar human leukocyte antigen (HLA) matching between the donor and the patient, while the role of other immunologic factors like non-HLA markers including minor histocompatibility antigens (miHA) are currently in the forefront. miHA are polymorphic proteins that vary even in monozygotic twins. The best known is the H-Y antigen, but there are also other autosomal miHA and MICA (MHC class I chain-related gene A). miHA have been well studied in transplantation of hematopoietic precursors, but not in solid organ transplantation. The most important studies in this field relate to incompatibility of H-Y antigen as a risk factor in kidney transplantation, although the findings are still inconclusive. This review presents the role of minor histocompatibility antigens in solid organ transplantation, especially of the kidney.     

Hemopoietic histocompatibility (Hh-1) regulatory and structural genes of the f haplotype map to H-2

Natural resistance to bone marrow stem cell (BMC) grafts in lethally irradiated mice is a consequence of natural killer cell recognition and elimination of BMC that express hemopoietic histocompatibility (Hh) antigens inherited noncodominantly. The major Hh genetic region, Hh-1, maps to H-2. The phenotype of the BMC was determined by grafting BMC into panels of irradiated mice, and measuring splenic incorporation of the radioactive specific DNA precursor, 5-iodo-2'-deoxyuridine [125I] (IUdR) 5 days after cell transfer. Our previous analysis of Hh-1 antigen expression on BMC of intra-H-2 recombinant inbred strain mice indicated that Hh-1 regulatory genes (Hh-1r) map in the H-2S/H-2D interval. Nine recombinants described here shared the following: (1) the DL region was donated by H-2f that was associated with expression of determinant 2, shared with H-2d/Hh-1d, and (2) had crossovers in the S/D or Ea/S intervals or within Eb, and (3) had BMC that were Hh-1 null. F1 hybrids of crosses between mice of these recombinant strains with H-2d strain mice had BMC that expressed determinant 2, suggesting that a structural gene had been lost by the crossover event. Crosses with H-2b strain mice produced mice whose BMC were Hh-1 null, indicating that the H-2f/Hh-1f regulatory genes were still intact. We suggest that Hh-1r maps to the S/D interval of all haplotypes so far studied, even H-2f. The structural gene for the f haplotype is centromeric of Eb and may be Kf, based on recent data supporting the role of class I antigens in Hh antigen expression.     

Adenoviral-mediated gene transfer of ICP47 inhibits major histocompatibility complex class I expression on vascular cells in vitro

PURPOSE: Many viruses have evolved mechanisms to evade detection by the host immune system. The herpes simplex gene ICP47 encodes a protein that binds to the host antigen-processing transporter, inhibiting the formation of major histocompatibility complex class I (MHC-I) antigens in infected cells. MHC-I antigen expression is also important in acute allograft rejection. This study was designed to quantitate the effect of adenoviral-mediated gene transfer of ICP47 on MHC-I cell surface expression of human vascular cells. We hypothesized that the transduction of vascular cells with a replication-incompetent adenoviral vector that was expressing ICP47 (AdICP47) would inhibit constitutive and inducible MHC-I expression and thereby reduce the rate of cytolysis of ICP47-transduced vascular cells by sensitized cytotoxic T lymphocytes (CTL). METHODS: A replication-incompetent adenoviral vector, AdICP47, was created to express ICP47 driven by the cytomegalovirus immediate early promoter. Cultured human vascular endothelial and smooth muscle cells and human dermal fibroblasts were transduced with either AdICP47 or the control empty vector AddlE1. Cell surface constitutive and gamma-interferon-induced MHC-I expression were quantitated by flow cytometry. A standard 4-hour chromium release cytotoxicity assay was used to determine the percent cytolysis of transduced and nontransduced endothelial cells by sensitized CTL. Finally, to quantitate the specificity of the effect of ICP47 on MHC-I expression, adhesion molecule expression was quantitated in both transduced and nontransduced cells. RESULTS: Constitutive MHC-I expression in AdICP47-transduced endothelial cells was inhibited by a mean of 84% +/- 5% (SEM) in five experiments. After 48 hours of exposure to gamma-interferon, AdICP47-transduced cells exhibited a mean of 66% +/- 8% lower MHC-I expression than nontransduced cells. Similar inhibition in MHC-I expression was achieved in AdICP47-transduced vascular smooth muscle cells and dermal fibroblasts. Percent cytolysis of AdICP47-transduced endothelial cells by CTL was reduced by 72%. Finally, the specificity of the effect of transduction of ICP47 on vascular cell MHC-I expression was confirmed by a lack of significant change in either constitutive or tumor necrosis factor-induced vascular cell adhesion molecule/intercellular adhesion molecule expression. CONCLUSION: Transduction of vascular cells with AdICP47 strongly inhibits both constitutive and inducible MHC-I expression in human vascular cells. AdICP47-transduced cells exhibited a substantial reduction in cytolysis by CTL. Thus AdICP47 transduction holds promise as a technique to characterize the role of MHC-I expression in acute vascular allograft rejection in vivo and as a potential therapeutic intervention.     

Ex Vivo Adenoviral-Mediated Gene Transfer to Lung Isografts During Cold Preservation

Background. Although whole-organ gene transfer has been reported in heart and liver transplant models, it has not been well characterized in lung grafts. The aim of this study was to determine the feasibility of ex vivo gene transfer to rat lung isografts during cold preservation using an adenoviral vector.

Methods. F344 rats, divided into four groups, underwent orthotopic left lung transplantation. In group I, lung grafts were flushed with adenovirus carrying the β-galactosidase gene. After storage at 10°C, grafts were implanted in recipient animals. Group II underwent the same procedure but graft storage was at 4°C. Groups III (10°C) and IV (4°C) served as controls. On postoperative day 5, recipients were sacrificed, and native and transplanted lungs were examined.

Results. In group I, all animals showed successful, albeit patchy, gene expression. This occurred in 2 of 4 animals in group II, the other 2 showing no expression. Transduced cells were consistent morphologically with endothelial cells and pneumocytes. A minimal mononuclear inflammatory infiltrate was present. Control groups showed no transduction.

Conclusions. It is feasible to perform ex vivo adenoviral-mediated gene transfer to rat lung isografts during cold preservation.     

A single minor histocompatibility antigen encoded by UGT2B17 and presented by human leukocyte antigen-A*2902 and -B*4403

BACKGROUND: T-cell responses to minor histocompatibility antigens are mediators of graft-versus-host disease and organ graft rejection. We previously identified a human minor histocompatibility antigen that is recognized by CD8 cytotoxic T lymphocytes (CTLs) and encoded by the UDP glycosyltransferase 2 family, polypeptide B17 (UGT2B17) gene, which is highly expressed in the liver, colon, and small intestine. The UGT2B17 is presented by human leukocyte antigen (HLA)-A*2902, and the immunogenicity of this minor histocompatibility antigen results from differential protein expression in donor and recipient cells as a consequence of a UGT2B17 gene deletion. METHODS: An HLA-B*4403-restricted CD8 CTL clone was isolated from the same hematopoietic stem cell transplant recipient that exhibited an HLA-A*2902-restricted UGT2B17-specific response. The minor histocompatibility antigen recognized by the HLA-B*4403-restricted clone was identified, and the ability of the peptide to be presented by HLA-B*4402 was examined. RESULTS: The HLA-B*4403-restricted CTL clone recognized a peptide encoded by UGT2B17, which is identical to the peptide presented by HLA-A*2902. Peptide binding assays revealed this UGT2B17 peptide binds with comparable affinity to HLA-B*4402 as to HLA-B*4403. This patient had acute graft-versus-host disease involving liver and gastrointestinal tract, suggesting the T-cell response directed against UGT2B17 is involved in graft-versus-host disease. CONCLUSIONS: A single peptide encoded by UGT2B17 can be presented by HLA-A*2902, B*4402 and B*4403, and may serve as an immunodominant minor histocompatibility antigen in individuals with these HLA alleles that undergo transplantation of stem cells or organ grafts from UGT2B17 disparate donors.     

腺病毒载体介导NGF基因转染大鼠肌源性干细胞的实验研究

目的:探讨腺病毒载体介导神经生长因子(NGF)体外转染大鼠肌源干细胞(MDSCs)应用于基因治疗的可行性。方法:培养并纯化MDSCs细胞株,采用具有高效转染能力的腺病毒载系统将NGF基因导入MDSCs中,采用绿色荧光蛋白(EGFP)荧光技术检测转染率、实时荧光定量PCR (qPCR)检测目的基因NGF的表达情况。结果:...     

Altered distribution of MHC class II antigens on enterocytes during acute small bowel allograft rejection in rats

Class II major histocompatibility complex (MHC) antigen induction was investigated on enterocytes of heterotopic rat small bowel allografts in the Lewis-Brown Norway strain combination and on isografts in the Lewis-Lewis strain combination. I a antigens were detected with monoclonal antibodies using an immunoperoxidase technique. Generally, MHC class II antigens were not exhibited in the isografted group, with the exception of two long-term isografts that presented the same pattern as normal small bowel. In these cases, I a was expressed in a patchy distribution predominantly in the villi, and only very few enterocytes stained positive in Lieberkühn's crypts. Allografted rats showed a typical pattern of I a expression on the enterocytes during the rejection course. The initial expression was confined to the crypts, indicating a very early stage of rejection when compared to histological findings. More advanced stages of rejection were accompanied by increasing I a biosynthesis in the crypts and I a expression by the epithelium lining the villi. Cyclosporin (CyA) was not able to fully inhibit MHC class II antigen expression; however, the appearance of I a was delayed.     

Involvement of Male-Specific Minor Histocompatibility Antigen H-Y in Epidermal Equivalent Allograft Rejection

This study describes the involvement of male-specific minor histocompatibility antigen H-Y in vitro cultured epidermal equivalent (EE) rejection. Male and female Balb/c or C3H/HeN keratinocytes were isolated and cultured separately. Male EE were grafted onto adult male (isografts) and adult female (H-Y allografts) mice. As controls, Balb/c EE were grafted onto adult C3H/HeN (complete allografts) mice. Fourteen, 21, and 30 days postgrafting, histological studies showed well-organized cutaneous tissues with complete basement membranes (laminin and type IV collagen deposition) in H-Y allografts compared to the isografts. This cutaneous organization was altered 150 days postgrafting, which is a sign of the H-Y EE allograft rejection. Complete allografts were totally rejected 21 days postgrafting. Immunological studies revealed leucocyte infiltration of H-Y allografts. Significant infiltration was detected even 150 days postgrafting. Leucocyte phenotyping revealed the presence of Mac-1⁺, CD8⁺ and CD4⁺ cells in the H-Y allografts. Humoral immune analysis revealed the presence of circulating anti-H-Y allogeneic keratinocyte cytotoxic antibodies in female recipient sera. Our data suggest that male-specific minor histocompatibility antigen H-Y induces cellular and humoral activation of the recipient immune system even after grafting EE free of cutaneous active immune cells such as T lymphocytes and Langerhans cells.     

绿色荧光蛋白在人大肠癌HT-29c细胞的表达

目的 观察增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）在人大肠癌细胞的表达情况。方法 构建逆转录病毒载体质粒prkat EGFP/neo,用磷酸钙沉淀法转导293T细胞,48h获得高滴度病毒颗粒上清液并转染人大肠癌HT－29c细胞。荧光显微镜及流式细胞仪观察转染的EGFP基因在人大肠癌HT-29c细胞体内外的表达。结果 携带EGFP的逆转录病毒载体能有效地转染人大肠癌细胞,HT－29c细胞能稳定、高铲和持久地表达EGFP,与未来转染细胞比较,它们的生物学特性未改变（F＝0.62,P＞0.05）。在裸大鼠皮下肿瘤中,EGFP也能稳定、高效的表达。结论 外源性EGFP能在人大肠癌HT－29c细胞内进行有效的复制、转录和翻译,在体内外可持续、稳定、高效的表达。它能作为一种新的实验方法应用于肿瘤细胞生长、扩散和转移的研究。     

Altered distribution of MHC class II antigens on enterocytes during acute small bowel allograft rejection in rats

Abstract. Class II major histocompatibility complex (MHC) antigen induction was investigated on enterocytes of heterotopic rat small bowel allografts in the Lewis-Brown Norway strain combination and on isografts in the Lewis-Lewis strain combination. Ia antigens were detected with monoclonal antibodies using an immunoper-oxidase technique. Generally, MHC class II antigens were not exhibited in the isografted group, with the exception of two long-term isografts that presented the same pattern as normal small bowel. In these cases, I a was expressed in a patchy distribution predominantly in the villi, and only very few enterocytes stained positive in Lieberkühn's crypts. Allografted rats showed a typical pattern of I a expression on the enterocytes during the rejection course. The initial expression was confined to the crypts, indicating a very early stage of rejection when compared to histological findings. More advanced stages of rejection were accompanied by increasing I a biosynthesis in the crypts and I a expression by the epithelium lining the villi. Cyclosporin (CyA) was not able to fully inhibit MHC class II antigen expression; however, the appearance of I a was delayed.     

Prostate specific membrane antigen (PSMA) is a tissue-specific target for adenoviral transduction of prostate cancer in vitro

BACKGROUND: Adenovirus binds to the coxsackievirus and adenovirus receptor (CAR) as a first step in the process of cellular infection. This dependence on CAR potentially limits the use of adenovirus in gene therapy, since CAR is expressed in many tissues of the body, and expression of CAR may be low or lost upon progression of certain tumors. These limitations may be overcome by transductional targeting of adenovirus towards other cell surface molecules. We have evaluated the pantumoral epithelial cell adhesion molecule (EpCAM) and prostate specific membrane antigen (PSMA) as possible targets for adenoviral transduction of prostate cancer cells. METHODS: Bispecific antibodies, constructed as conjugates between an anti-adenovirus fiber knob Fab' fragment and anti-EpCAM or anti-PSMA monoclonal antibodies, were incubated with an eGFP-expressing adenovirus to retarget this vector. A cell panel, that includes two prostate cancer cell lines and four non-prostate control lines, were infected with serial dilutions of the retargeted vector and specificity of infection was determined. RESULTS: Receptor-specific transduction was obtained for both EpCAM and PSMA. PSMA-retargeting was shown to be selective for the prostate cancer cell lines. CONCLUSIONS: PSMA serves as a tissue-specific target for adenoviral vectors and may be applicable for gene therapeutical treatment of prostate cancer.     

Adenoviral-mediated overexpression of either membrane-bound human FasL or human decoy Fas can prolong pig islet xenograft survival in a rat transplant model

The success of pancreatic islet transplantation is limited because of the severe shortage of allogeneic pancreas donors. Accordingly, pig islets are considered to be an attractive, promising alternative. However, cell-mediated immunity, especially CD8+ cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, remains a formidable barrier to prevent long-term islet survival in xenograft recipients. Therefore, it is particularly important to explore methods to specifically prevent cell-mediated immunity against pig islets. Our group previously demonstrated that the overexpression of either membrane-bound human FasL or human decoy Fas antigen in pig endothelial cells prevented CTL xenocytotoxicity. In this study, we assessed the cytoprotective effects of adenoviral-mediated overexpression of either membrane-bound human FasL or human decoy Fas antigen in pig islets to inhibit CTL xenocytotoxicity. The CTL-mediated killing of pig islets infected with an adenoviral vector carrying either membrane-bound human FasL or human decoy Fas was significantly reduced compares with that of control pig islets transfected with adenoviral vector encoding enhanced green fluorescent protein (EGFP). Moreover, we transfected pig islets with these molecules to confirm their cytoprotective effects in in vivo studies. The significant long-term survival of pig islets expressing these molecules was elicited through days 3 to 5 posttransplantation. Thus, these results demonstrated that the remodeling of either death receptor or death ligand on pig islets by adenoviral gene transfer prevented innate cellular immunity against xeno-islet grafts facilitating long-term xenograft survival.     

The effects of cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in the rat

We have previously reported 5-30-fold increases in the expression of class I and class II major histocompatibility complex (MHC) antigens in rejecting heart and kidney allografts in the DA-to-PVG rat strain combination. We examine here the effects of immunosuppression with cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in this strain combination. Immunohistological studies and quantitative absorption analyses using monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens were used throughout. Heart allografts in cyclosporine-treated rats were examined on day 3,5,7,9,11, and 14 after transplantation, and kidney allografts in cyclosporine-treated rats were examined at day 7. In addition, untreated heart and kidney isografts were studied at days 1,3,5, and 7 after grafting. Immunohistological studies on frozen sections showed that cyclosporine-treated heart and kidney allografts showed no induction of class II MHC antigens, in contrast to untreated heart and kidney allografts. Class I MHC antigen induction did occur in spite of cyclosporine-therapy, but at levels lower than those seen in untreated allografts. Moreover, the pattern and degree of class I induction in the cyclosporine-treated allografts resembled very closely those seen in isografts, and so this induction was, in all probability, a consequence of the transplantation procedure rather than of specific immune responses. We also noted, in the cyclosporine-treated heart allografts, that all donor interstitial dendritic cells had disappeared and been replaced by recipient interstitial dendritic cells by the end of the second week after grafting. In addition, there was no reduction in the class II antigen content of kidney allografts treated for 7 days with cyclosporine. The absence of class II antigen induction in allografts where rejection is effectively suppressed with cyclosporine might be of clinical value in the differential diagnosis between rejection and cyclosporine toxicity in renal transplantation, and between active and inactive cellular infiltrates in heart transplantation.     

Adenovirus-mediated gene therapy to liver grafts: successful gene transfer by donor pretreatment

BACKGROUND: We have previously shown excellent adenoviral (Ad) gene transfection to transplanted liver grafts with the clamp technique (CT) where viral vector was delivered ex vivo and trapped in cold preserved liver grafts. In this study, we adopted a new gene therapy approach to achieve early transgene expression by donor pretreatment with viral vector and compared the efficacy of these two methods by using Ad vector encoding enhanced green fluorescent protein (AdEGFP) marker gene. METHODS: AdEGFP (1 x 10(9)plaque forming units) was delivered to the liver grafts by: (1) single intravenous injection to donor Lewis rats 48 hours before harvesting, (2) ex vivo cold infusion into the harvested liver with CT, or (3) a combination of both methods. Liver grafts were stored in University of Wisconsin solution at 4 degrees C for 18 hours and then orthotopically transplanted into syngeneic recipients, and the expression of EGFP was studied. RESULTS: With intravenous pretreatment of donor liver grafts, EGFP-expressing cells were detected as early as 3 hours after transplant, and moderate expression was seen by 12 hours. In contrast, EGFP was not detected until 12 to 24 hours after transplant with CT. High levels of EGFP-producing cells were seen with each technique at 7 days ( approximately 30% transfection efficiency). A combination of both methods did not enhance infectivity. Liver preservation injury was comparable between groups. CONCLUSIONS: Gene transfer by donor pretreatment with AdEGFP induces early and efficient gene transduction to liver grafts compared with back-table delivery with CT. This method is simple and provides early transgene expression in liver grafts that potentially could be used to deliver genes to decrease preservation injury or rejection.