Single-step, multiple retroviral transduction of human T cells

Background

Retroviral transduction of human peripheral blood T cells has considerable potential in the development of gene therapy strategies for immunological disorders. New vectors and experimental procedures have been developed for efficient transduction of several genes into human T cells.

Methods

Bicistronic retroviral vectors encoding distinct cell markers were used for the simultaneous multiple transduction of a human T‐cell line (MT‐2), as well as of human peripheral blood T cells from normal donors. Transduction efficiencies were evaluated by flow cytometry and double‐ and triple‐transduced cells were isolated by fluorescence cell sorting.

Results

Four new bicistronic retroviral vectors were developed that express different gene markers under the control of the internal ribosome entry site (IRES) of the encephalomyocarditis virus. These markers are, respectively, enhanced green fluorescent protein (EGFP), β‐galactosidase, and truncated versions of human nerve growth factor receptor (ΔNGFR) and human growth hormone receptor (ΔGHR). A single 1 h spinoculation infection, performed in the presence of polybrene and using transiently produced amphotropic retroviral particles, was sufficient to obtain transduction efficiencies consistently greater than 50% on human peripheral blood T lymphocytes which had been previously stimulated for 3 days with immobilized anti‐CD3. The transient production of viral particles encoding EGFP, ΔNGFR, and ΔGHR markers in the same viral supernatant has allowed up to three different genes to be introduced simultaneously into human T cells.

Conclusions

This study describes new experimental conditions for efficient single‐step multiple transduction of human primary T lymphocytes. The procedure could be of interest for the development of gene therapy approaches. Copyright © 2002 John Wiley & Sons, Ltd.

     

Lack of specificity of cell-surface protease targeting of a cytotoxic hyperfusogenic gibbon ape leukaemia virus envelope glycoprotein

Background

In a strategy termed “Protease Targeting”, retroviral vectors carrying an EGF infectivity‐blocking domain fused to the N‐terminus of the envelope SU via a MMP (matrix metalloproteinase)‐cleavable linker were successfully used to target gene delivery to EGF receptor‐(EGF‐R‐)positive tumour cells over‐expressing MMPs. In the current study, we aimed to investigate whether this strategy could be applied to (a) limit the cytotoxic activity of a hyperfusogenic GALV therapeutic gene, and (b) enhance the immune‐stimulatory properties of GALV via local, MMP‐mediated release human granulocyte‐macrophage colony stimulating factor (GM‐CSF).

Methods

We generated GALV envelope expression constructs displaying EGF or GM‐CSF blocking ligands at the N‐terminus of GALV envelope SU via a non‐cleavable, Factor Xa protease or MMP‐cleavable linker and investigated their cytotoxicity on MMP‐positive and negative cell lines.

Results

The unmodified hyperfusogenic GALV envelope was cytotoxic to all cell lines tested. The non‐cleavable linker GALV envelope constructs caused no cytotoxicity, demonstrating efficient inhibition by the displayed domains. Moderate activation of fusion of the protease‐cleavable linker constructs was observed in all cell lines, regardless of their level of MMP expression and of the specificity of the linker. High levels of the ‘blocking domain’ were detected in the cell supernatants due to dissociation of the surface unit (SU) from the transmembrane (TM) component of the GALV envelope glycoprotein TM.

Conclusions

Unlike protease targeting in the context of retroviral vectors, protease activation of the cytotoxicity of GALV envelope by cleavage of a fusion blocking ligand at the cell surface does not appear to be specifically mediated by cell‐surface MMPs. In addition, shedding of the SU‐fusion protein from the TM limits the general applicability of this strategy for cancer gene therapy. Specificity of cell‐cell fusion mediated by GALV envelope cannot be manipulated in the same fashion as virus‐cell fusion. Copyright © 2002 John Wiley & Sons, Ltd.

     

Transfer of maternally administered fusogenic liposome-DNA complexes into monkey fetuses in a pregnancy model

Background

Materno‐fetal transfer of intravenously administered liposome‐plasmid DNA complexes has been demonstrated only in mice. Studies on its materno‐fetal transfer in the pregnant monkey model is needed because of critical differences in placental structure between primates including humans and rodents.

Methods

The reporter plasmid pEGFP‐C1 was formulated in cationic lipid containing polybrene and vesicular stomatitis virus G protein. The fusogenic liposome‐plasmid DNA complexes were intradermally injected into pregnant common marmosets (N=2), a New World monkey, near term. DNA extracted from fetal tissues was subjected to PCR for detection of the egfp gene. Confocal microscopy and immunostaining were performed to determine the sites of transgene expression in the fetal organs.

Results

The egfp gene was detected in fetal blood and major organs (heart, liver, lung). The encoded protein was mainly produced in the endothelial cells of blood vessels in the fetal lungs.

Conclusions

This is the first report on materno‐fetal transfer of intradermally administered fusogenic liposome‐plasmid DNA complexes and fetal expression of a transgene in primates. Copyright © 2002 John Wiley & Sons, Ltd.

     

Syngeneic fibroblasts transfected with a plasmid encoding interleukin-4 as non-viral vectors for anti-inflammatory gene therapy in collagen-induced arthritis

Background

No effective long‐term treatment is available for rheumatoid arthritis. Recent advances in gene therapy and cell therapy have demonstrated efficiency in collagen‐induced arthritis (CIA). Interleukin‐4 (IL‐4) is already known to be efficient in CIA in systemic injection or administered by gene therapy. This study was designed to evaluate the effect of a non‐viral gene therapy of CIA, involving injection of syngeneic fibroblasts transfected with a plasmid encoding for IL‐4.

Methods

Immortalised fibroblasts from DBA/1 mice (DBA/1/0 cells) were transfected with a plasmid expressing IL‐4 cDNA (DBA/1/IL‐4 cells). Xenogeneic fibroblasts from Chinese hamster ovary (CHO) transfected with a plasmid expressing IL‐4 cDNA (CHO/IL‐4) were studied also. The cells were engrafted in mice developing CIA by subcutaneous injection of 3 × 10⁶ DBA/1/0 or DBA/1/IL‐4 or CHO/IL‐4 cells.

Results

Injection of DBA/1/IL‐4 cells, on days 10 and 25 after immunisation, was associated with a significant and lasting improvement in the clinical and histological evidence of joint inflammation and destruction as compared with DBA/1/0 and CHO/IL‐4 cells. DBA/1/IL‐4 cell treatment decreased also the production of IgG2a antibody to CII and the proliferation of CIIB‐specific nodal T cells. Later treatments (engraftments on days 23 and 35 after immunisation) exerted also an anti‐inflammatory effect, as evaluated on clinical and histological signs of CIA.

Conclusions

Taken together, these findings indicate that systemic administration of syngeneic cells transfected with an anti‐inflammatory cytokine gene, namely IL‐4, with a non‐viral method is effective in CIA and may attenuate the cytokine imbalance seen in this disease. Copyright © 2002 John Wiley & Sons, Ltd.

     

The species–area relationship does not have an asymptote!

Aim

To attack a widespread myth.

Location

World‐wide.

Methods

Simple mathematical logical and empirical examples.

Results

As both species and area are finite and non‐negative, the species–area relationship is limited at both ends. The log species–log area relationship is normally effectively linear on scales from about 1 ha to 10⁷ km². There are no asymptotes. At the intercontinental scale it may get steeper; at small scales it may in different cases get steeper or shallower or maintain its slope.

Main conclusion

The species–area relationship does not have an asymptote.

     

Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus expressing the 10A1 envelope protein

Background

Introduction of recombinant genes in the genome of primary lymphocytes by virtue of a replication‐deficient retrovirus can be used in immunological studies and for cell‐based gene therapy.

Methods

Packaging cells GP+E86 producing replication‐deficient retrovirus incorporating the genes of enhanced green fluorescent protein (eGFP), C2γ or C2ξ, were generated by calcium phosphate‐mediated transfection. Clones with the highest titres of retrovirus vector were isolated from them and their supernatants were used for transduction of PT67 cells. Primary mouse lymphocytes and T‐cell hybridoma MD.45 were transduced by centrifugation with retroviral stock. The retroviral content of packaging cell supernatants was determined by dot blotting and hybridization with a DNA probe.

Results

PT67 cells produced ～50 times more retrovirus vector than the original GP+E86 clones. When retroviral stocks of PT67 and GP+E86 cells were used at 1/50 dilution and undiluted, respectively (to normalize them forretroviral RNA content), the transduction efficiency of mouse T‐cell hybridoma was 40% and 5%, respectively. Centrifugation of target cells with retroviral stock at 2000 g for 60 min increased the percentage of transduced cells two‐ to three‐fold. Within a population of cells isolated from the draining lymph nodes of an immunized mouse and reactivated with an antigen, up to 60% of CD4⁺ T cells and up to 80% of B cells could be transduced with a transgene in replication‐deficient retrovirus packaged by PT67 cells using the optimized gene transfer protocol.

Conclusions

This protocol allows for the generation of packaging cells producing high titres of retrovirus vector. The 10A1 envelope protein is superior to the ecotropic one for the transduction of mouse lymphocytes. Copyright © 2002 John Wiley & Sons, Ltd.

     

Phenotypic rescue after adeno-associated virus-mediated delivery of 4-sulfatase to the retinal pigment epithelium of feline mucopolysaccharidosis VI

Background

Mucopolysaccharidosis VI (MPS VI), due to recessively inherited 4‐sulfatase (4S) deficiency, results in lysosomal storage of dermatan sulfate in numerous tissues. Retinal involvement is limited to the retinal pigment epithelium (RPE). This study aimed to determine whether recombinant adeno‐associated virus (AAV)‐mediated delivery of 4S would reverse the RPE pathology seen in MPS VI cats.

Methods

AAV.f4S, containing the feline 4S cDNA, was delivered unilaterally to eyes of affected cats by subretinal or intravitreal injection. Contralateral eyes received AAV with the green fluorescent protein (GFP) reporter gene as control. At 2–11 months post‐injection, the cats were sacrificed and the treatment effects were evaluated histologically.

Results

By ophthalmoscopy and histological analyses, GFP was evident as early as 4 weeks and persisted through the latest time point (11 months). Untreated and AAV.GFP‐treated diseased retinas contained massively hypertrophied RPE cells secondary to accumulation of dilated lysosomal inclusions containing dermatan sulfate. MPS VI eyes treated subretinally with AAV.f4S had minimal RPE cell inclusions and, consequently, were not hypertrophied.

Conclusions

AAV‐mediated subretinal delivery of f4S provided correction of the disease phenotype in RPE cells of feline MPS VI, supporting the utility of AAV as a vector for the treatment of RPE‐specific as well as lysosomal storage diseases. Copyright © 2002 John Wiley & Sons, Ltd.

     

Rescue of retroviral envelope fusion deficiencies by cationic liposomes

Background

Retroviral particles that are inappropriately enveloped can transduce target cells if pre‐associated with cationic liposomes. This study optimises and addresses the mechanism of liposome‐enhanced gene delivery, and explores the potential for such agents to compensate for fusion deficiency associated with chimaeric envelope proteins.

Methods

Particles bearing wild‐type, chimaeric or no envelope proteins were complexed with DOTAP or DC‐Chol/DOPE cationic liposomes and added to target cells for various times. Particle binding was determined by detection of cell‐associated capsid protein and infectivity was measured histochemically.

Results

Stable association of cationic liposomes with retrovirus particles significantly enhanced their binding rate to target cells in proportion to the increase of transduction kinetics for infectious virus. Binding of virus was equivalent with or without envelope protein and/or virus receptor, indicating that a non‐specific interaction precedes receptor recognition. Non‐infectious combinations were rescued by the intrinsic fusogenicity of the cationic liposomes, which enabled entry of the viral core, but left subsequent events unaltered. The optimised transduction rate with non‐enveloped particles and DOTAP approached that of amphotropic‐enveloped virus in some cases, although the effect was target‐cell‐dependent. DC‐Chol/DOPE was less potent at direct fusion but was able to enhance 600‐fold the receptor‐dependent action of chimaeric envelopes that were deficient in fusion by virtue of the addition of targeting domains.

Conclusions

These data have implications for the development of retroviral vector targeting strategies from the perspectives of the specificity of target cell interaction and compensating for chimaeric envelope fusion deficiency. Copyright © 2002 John Wiley & Sons, Ltd.

     

DNA transfection of macaque and murine respiratory tissue is greatly enhanced by use of a nuclease inhibitor

Background

Nuclease activity present within respiratory tissues contributes to the rapid clearance of injected DNA and therefore may reduce the transfection activity of directly injected transgenes. Most gene transfer technologies transduce or transfect murine tissues more efficiently than corresponding primate tissues. Therefore, it is prudent to assess the utility of novel gene transfer strategies in both rodent and primate models before proceeding with further development.

Methods

This study analyzed the effects of ATA (a nuclease inhibitor) on the direct transfection of macaque and murine lung tissue; compared the levels of DNase activity in murine, primate, and human lung fluids; and tested the inhibitory activity of ATA on the DNase activity present in these samples. Fluorescent microspheres were used to detect areas of transfection in lung.

Results

Intratracheal administration of a nuclease inhibitor (ATA) with naked DNA (0.5 µg ATA/g body weight) enhanced direct transfection efficacy in macaque lung by over 86‐fold and by over 54‐fold in mouse lung. Hematoxylin and eosin staining showed no apparent tissue toxicity. Moreover, macaque, human, and mouse lung fluids were found to possess similar levels of DNase activity and this activity was inhibited by similar concentrations of ATA. The authors also successfully pioneered the use of carboxylate‐modified microsphere tracers to identify areas of transfection and/or treatment.

Conclusion

This work provides evidence that using direct nuclease inhibitors will enhance lung transfection and that nuclease activity is present in all lung fluids tested, which can be inhibited by the use of direct DNase inhibitors. Copyright © 2002 John Wiley & Sons, Ltd.

     

Encapsulated cells producing retroviral vectors for in vivo gene transfer

Background

Because gene therapy of the future will primarily take an in vivo approach, a number of problems associated with its current implementation exist. Currently, repeated delivery of a vector in vivo is necessary to ensure adequate transfer of the therapeutic gene. This may lead to the development of an immune response against the vector, thus interfering with gene delivery. To circumvent this problem, retroviral vector packaging cells that permanently produce recombinant retroviral vector particles have been encapsulated.

Methods

Vector (pBAG)‐producing amphotropic cells were encapsulated in beads composed of polymerized cellulose sulphate. These capsules were analysed in vitro for expression of the vector construct using X‐gal staining, as well as for the release of particles by performing RT‐PCR from culture supernatant. Infectivity studies were performed in vitro and in vivo. The latter was assayed using histological sections of the microcapsule and the surrounding area stained for β‐galactosidase activity and by RT‐PCR.

Results

In culture, the virus‐producing cells inside the capsules remained viable and released virus into the culture medium for at least 6 weeks. To test whether these capsules, upon implantation into mice, also release vector virions that infect the surrounding cells, two different models were used. In the first, capsules were implanted in the fat pad of the mammary gland of Balb/c mice. The capsules were well tolerated for at least 6 weeks and a self‐limiting inflammatory reaction without any other gross immune response was observed during this period. Furthermore, the virus‐producing cells remained viable. In the second model, SCID mice were immunologically reconstituted by subcutaneous implantation of thymus lobes from MHC‐identical Balb/c newborn mice and gene transfer into lymphoid cells was achieved by retroviral vectors released by co‐implanted capsules.

Conclusion

The implantation of such capsules containing cells that continually produce retroviral vector particles may be of use for in vivo gene therapy strategies. The data presented demonstrate the feasibility of the concept. Copyright © 2002 John Wiley & Sons, Ltd.

     

Effect of the mediterranean diet with and without weight loss on markers of inflammation in men with metabolic syndrome

Objective:

Intervention studies on the Mediterranean Diet (MedDiet) have often led to weight loss, which may have contributed to the purported anti‐inflammatory effects of the MedDiet. To investigate the impact of the MedDiet consumed under controlled feeding conditions before (?WL) and after weight loss (+WL) on markers of inflammation in men with metabolic syndrome (MetS).

Design and Methods:

Subjects (N = 26, male, 24–65 years) with MetS first consumed a North American control diet for 5 weeks followed by a MedDiet for 5 weeks both in isocaloric feeding conditions. After a 20‐week weight loss period in free‐living conditions (10 ± 3% reduction in body weight, P < 0.01), participants consumed the MedDiet again under isocaloric‐controlled feeding condition for 5 weeks.

Results:

MedDiet ? WL significantly reduced plasma C‐reactive protein (CRP) concentrations (?26.1%, P = 0.02) and an arbitrary inflammatory score (?9.9%, P = 0.01) that included CRP, interleukin‐6 (IL‐6), IL‐18, and tumor necrosis factor‐α (TNF‐α) compared with the control diet. The MedDiet + WL significantly reduced plasma IL‐6 (?20.7%) and IL‐18 (?15.6%, both P ≤ 0.02) concentrations compared with the control diet but had no further significant impact on plasma CRP concentration. Participants with a reduction in waist circumference ≥8.5 cm after MedDiet + WL showed significantly greater reductions in inflammation markers than those with a change in waist circumference <8.5 cm.

Conclusions:

Thus, consuming MedDiet even in the absence of weight loss significantly reduces inflammation. However, the degree of waist circumference reduction with weight loss magnifies the impact of the MedDiet on other markers of inflammation associated with MetS in men.

     

Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells

Background

The recently developed heterologous macrolide‐ (E.REX system) and streptogramin‐ (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines.

Methods

In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic‐dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter.

Results

These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression.

Conclusions

Due to their modular set‐up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell‐specific key regulation components. Copyright © 2002 John Wiley & Sons, Ltd.

     

High-level gene transfer to the cornea using electroporation

Background

Methods for gene transfer to the cornea that yield high‐level expression without inflammation or trauma are currently lacking. Because electroporation has proven effective for gene transfer in other tissues in terms of expression levels and safety, this study quantitatively evaluated its use in the cornea.

Methods

To evaluate the use of electroporation in the mouse cornea, plasmids expressing either luciferase or green fluorescent protein were injected intracorneally or subconjunctivally and square‐wave electric pulses were immediately applied to the eyes. Gene expression was quantified at later times and trauma and inflammation were monitored visually and by measuring interleukin‐6 (IL‐6) production.

Results

The application of electric pulses to eyes injected with plasmid resulted in nanogram levels of gene product expression. At an optimal field strength of 200 V/cm, no trauma, corneal edema or inflammation was observed. However, at higher field strengths, corneal damage was detected. Compared with injection of DNA alone, up to 1000‐fold more gene product was produced using electroporation. Expression was detected as early as 6 h post‐electroporation, remained high for 3 days, and decreased by 7 days. Gene expression was detected over the entire surface of the cornea in both epithelial and stromal layers.

Conclusions

These results demonstrate that electroporation is an excellent method for delivering genes to multiple cell layers within the mouse cornea and that it results in extremely high levels of gene expression with little, if any, inflammatory response or tissue damage, making this a very useful technique for corneal gene transfer. Copyright © 2001 John Wiley & Sons, Ltd.

     

Critical role of inflammatory mast cell in fibrosis: Potential therapeutic effect of IL‐37

Background

Fibrosis involves the activation of inflammatory cells, leading to a decrease in physiological function of the affected organ or tissue.

Aims

To update and synthesize relevant information concerning fibrosis into a new hypothesis to explain the pathogenesis of fibrosis and propose potential novel therapeutic approaches.

Materials and Methods

Literature was reviewed and relevant information is discussed in the context of the pathogenesis of fibrosis.

Results

A number of cytokines and their mRNA are involved in the circulatory system and in organs of patients with fibrotic tissues. The profibrotic cytokines are generated by several activated immune cells, including fibroblasts and mast cells (MCs), which are important for tissue inflammatory responses to different types of injury. MC‐derived TNF, IL‐1, and IL‐33 contribute crucially to the initiation of a cascade of the host defence mechanism(s), leading to the fibrosis process. Inhibition of TNF and inflammatory cytokines may slow the progression of fibrosis and improve the pathological status of the affected subject. IL‐37 is generated by various types of immune cells and is an IL‐1 family member protein. IL‐37 is not a receptor antagonist; it binds IL‐18 receptor alpha (IL‐18Rα) and delivers the inhibitory signal by using TIR8. It has been shown that IL‐37 can be protective in inflammation and injury, and inhibits both innate and adaptive immunity.

Discussion

IL‐37 may be useful for suppression of inflammatory diseases induced by inhibiting MyD88‐dependent TLR signalling. In addition, IL‐37 downregulates NF‐κB induced by TLR2 or TLR4 through a mechanism dependent on IL‐18Rα.

Conclusion

This review summarizes current knowledge on the role of MC in inflammation and tissue/organ fibrosis, with a focus on the therapeutic potential of IL‐37‐targeting cytokines.

     

Preparation of dry powder dispersions for non-viral gene delivery by freeze-drying and spray-drying

Background

Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze‐drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non‐viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze‐drying and spray‐drying, a one‐step process for producing dry and respirable powders, as methods for preparing non‐viral respiratory gene delivery systems.

Methods

Lipid:polycation:pDNA (LPD) vectors comprising 1,2‐dioleoyl‐3‐trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP‐N1 in 3% lactose solution were either snap‐frozen and lyophilised or spray‐dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence.

Results

Lyophilisation produced large, irregularly shaped particles prior to (mean diameter ～21 µm) and following (mean diameter ～18 µm) coarse grinding. Spray‐drying produced uniformly shaped spherical particles (mean diameter ～4 µm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray‐dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage.

Conclusions

Spray‐drying has utility for producing stable, efficient and potentially respirable non‐viral dry powder systems for respiratory gene delivery. Copyright © 2002 John Wiley & Sons, Ltd.

     

A new red fluorescent protein that allows efficient marking of murine hematopoietic stem cells

Background

Genetic marking of hematopoietic stem cells (HSCs) with multiple fluorescent proteins (FPs) would allow analysis of their features, including interaction with adjacent cells. However, there are few red FPs that are comparable to green FPs in terms of low toxicity and high fluorescent intensity. This study has evaluated the usefulness of Kusabira Orange (KO) originated from the coral stone Fungia concinna as a red FP for marking of HSCs

Methods

A vector used was the MSCV‐type retroviral vector, DΔNsap that has the PCC4 cell‐passaged myeloproliferative sarcoma virus derived long terminal repeat devoid of a binding site for YY1 and the primer‐binding site derived from the dl587rev, respectively. The vector was cloned with the codon‐optimized KO cDNA for higher expression in mammalian cells (huKO) and converted to the corresponding retroviruses pseudotyped with the vesicular stomatitis virus G envelope protein, then transduced into c‐KIT⁺Sca‐1⁺Lineage^? cells obtained from C57BL/6 (Ly5.1) mice followed by transplantation into lethally irradiated Ly5.2 mice.

Results

Approximately 70% of donor‐derived cells highly expressed huKO at 16 weeks post‐transplantation. Furthermore, the high expression of huKO was also detected in serially transplanted mice, suggesting that expression of huKO per se had little deleterious effect on murine hematopoiesis. In double marking experiments, huKO‐expressing hematopoietic cells were easily distinguished from those expressing EGFP by flow cytometery and fluorescent microscope analysis.

Conclusions

Overall, the results obtained from the present study suggest that huKO can be used as a valuable and versatile red fluorescent marker for HSCs. Copyright © 2008 John Wiley & Sons, Ltd.

     

ERRγ enhances UCP1 expression and fatty acid oxidation in brown adipocytes

Objective:

Estrogen‐related receptors (ERRs) are important regulators of energy metabolism. Here we investigated the hypothesis that ERRγ impacts on differentiation and function of brown adipocytes.

Design and Methods:

We characterize the expression of ERRγ in adipose tissues and cell models and investigate the effects of modulating ERR? activity on UCP1 gene expression and metabolic features of brown and white adipocytes.

Results:

ERRγ was preferentially expressed in brown compared to white fat depots, and ERRγ was induced during cold‐induced browning of subcutaneous white adipose tissue and brown adipogenesis. Overexpression of ERRγ positively regulated uncoupling protein 1 (UCP1) expression levels during brown adipogenesis. This ERRγ‐induced augmentation of UCP1 expression was independent of the presence of peroxisome proliferator‐activated receptor coactivator‐1 (PGC‐1α) but was associated with increased rates of fatty acid oxidation in adrenergically stimulated cells. ERR? did not influence mitochondrial biogenesis, and its reduced expression in white adipocytes could not explain their low expression level of UCP1.

Conclusions:

Through its augmenting effect on expression of UCP1, ERRγ may physiologically be involved in increasing the potential for energy expenditure in brown adipocytes, a function that is becoming of therapeutic interest.

     

Intratumoral activation of cyclophosphamide by retroviral transfer of the cytochrome P450 2B1 in a pancreatic tumor model. Combination with the HSVtk/GCV system

Background

Pancreatic cancer is one of the most aggressive human tumors and the development of new therapeutic approaches is particularly urgent since current therapies are not effective. The use of pro‐drug‐activating genes is a possible approach for cancer gene therapy.

Methods

The present study evaluated the efficiency of the cytochrome P4502B1 (CYP2B1) suicide gene that encodes the enzyme responsible for activating the pro‐drug cyclophosphamide (CPA), in pancreatic tumor cells invitro and in vivo. The effects on tumor growth of the combination of two suicide systems, CYP2B1/CPA and herpes simplex virus thymidine kinase gene/ganciclovir (HSVtk/GCV), were also studied.

Results

Retroviral CYP2B1 transfer followed by CPA treatment highly sensitized pancreatic tumor cells NP‐9, NP‐18, and NP‐31, and led to stabilization of tumor growth in a pancreatic tumor model. Differences in tumor volume at the end of the treatment were statistically significant when compared with animals injected with CPA alone. The combination of both suicide systems CYP2B1/CPA and HSVtk/GCV in vitro resulted in a potentiation of the killing effect. However, no potentiation was achieved in vivo, although retardation in tumor growth was evident.

Conclusions

The results show that in situ transduction of pancreatic tumor cells with the CYP2B1 gene by retroviral vectors clearly increases the sensitivity to CPA. Moreover, they suggest that in order to achieve a potentiation on cell killing when the two suicide systems HSVtk/GCV and CYP2B1/CPA are combined, co‐expression of both genes in the same tumor cell would be necessary. Copyright © 2002 John Wiley & Sons, Ltd.