Evaluation of efficient chemoembolization mixtures by magnetic resonance imaging therapy monitoring: an experimental study on the VX2 tumor in the rabbit liver.

To find effective chemoembolization mixtures, we tested combinations of carboplatin with the embolizates Spherex and Gelfoam in comparison to a therapy with NaCl-solution, a treatment with the cytostatic drug only, and a therapy with each of the embolizates alone. The experiments were carried out using as a model the VX2 tumor in the liver of male chinchilla rabbits (five for each group). Carboplatin was revealed by the 3-(4,5-dimethylthiazole-2)-yl-2,5-diphenyltetrazolium bromide test to be a potent cytostatic drug for VX2 rabbit tumor cells. We used magnetic resonance imaging to examine the tumor volume and signal intensity enhancement up to 15 min after Gd-DTPA administration within the tumor and liver before and after the different therapies. These parameters allowed us to evaluate tumor growth and vitality as well as liver injury for the different therapy types. The results found by magnetic resonance imaging corresponded very well to those obtained by histological analysis of the tumors. The chemoembolization therapies were significantly more efficient than the other therapies, as indicated by the reduction of signal intensity enhancement after contrast agent administration within the tumor and by the histologically determined necrotic fraction after therapy. In addition, we found a significant decrease of the tumor volume and no significant live injury for a therapy with Carboplat and Gelfoam.

Cellular uptake of liposomes targeted to intercellular adhesion molecule-1 (ICAM-1) on bronchial epithelial cells.

Previously, it was demonstrated that immunoliposomes, bearing anti-intercellular adhesion molecule-1 (ICAM-1) antibodies (mAb F10.2), can specifically bind to different cell types expressing ICAM-1. In this study, we have quantified the amount of immunoliposomes binding to IFN-gamma activated human bronchial epithelial cells (BEAS-2B) in vitro and studied the subsequent fate of cell-bound anti-ICAM-1 immunoliposomes. We demonstrate that binding of the immunoliposomes to the epithelial cells depends on the liposome concentration used. After binding to the cell surface, the anti-ICAM-1 immunoliposomes are rapidly internalised by the epithelial cells. Sixty percent of cell-bound immunoliposomes were internalised by the epithelial cells within 1 h of incubation at 37 degrees C. The results indicate that ICAM-1 targeted immunoliposomes may be used as carriers for the intracellular delivery of anti-inflammatory drugs to sites of inflammation characterised by an increased expression of ICAM-1.

Efficacy of cationic liposome-mediated gene transfer to mesangial cells in vitro and in vivo.

Mesangial cells represent a major target for gene transfer approaches to the kidney. To establish a liposome-based system for transfection of mesangial cells we analyzed the efficacy and toxicity of different cationic liposomes and other nonviral transfection methods in primary cultures of rat and human mesangial cells using the Escherichia coli beta-galactosidase (lacZ) gene as a marker. In addition, an expression vector containing a human renin cDNA under the control of the cytomegalovirus immediate-early promoter/enhancer was generated, introduced into mesangial cells, and assayed in a system of transient gene expression. In vivo, gene transfer was studied after infusion of liposome/DNA complexes in the kidney of rats via the renal artery. Transfection efficiency ranged from 5.5% with DMRIE Liposomes in rat mesangial cells to 1.1% with LipofectAmine liposomes in human mesangial cells. Cytotoxicity following transfection was dependent on the transfection method. Transfection with the human renin expression vector led to the secretion of 11 pg/10(4) cells/48 h human renin in rat mesangial cells, 3,600 pg/10(4) cells/48 h in 293 cells, and 113 pg/10(4) cells/48 h human renin in opossum kidney cells. In vivo, infusion of liposomes was accompanied by nephrotoxicity and did not result in marker gene expression. Together the data demonstrate that cationic liposomes are useful tools for transferring genes into mesangial cells, including human mesangial cells. Cationic liposomes provide a functional system for the synthesis and secretion of human renin in mesangial cells and other mammalian kidney cells. The current limitation of the evaluated liposomes for an efficient in vivo gene transfer to mesangial cells is the toxicity upon intrarenal arterial administration.

Improvement in 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd) concentration by 5-fluorouracil-polyethylene-glycol-liposomes in abdominal stop-flow: treatment of VX2 liver-tumor-bearing rabbits.

The application of liposome-encapsulated cytostatics results in higher concentrations in tumor tissue. This effect can be further increased by blood flow retardation with longer retention time in the tumor and by arterial administration. In abdominal stop-flow therapy, a separate partial circulation with a defined flow is realized via a roller pump under hypoxic conditions. Forty chinchilla rabbits with VX-2 liver tumors were treated either intra-aortally (stop-flow therapy) or systemically with 50 mg 5-FU or 5-FU-PEG liposomes. During therapy, pH and pO2 were measured at regular intervals. After 20 minutes, concentrations of 5-FU and its metabolite FdUrd were determined by HPLC in different organs and the liver tumor. Compared to the i.v. application of monosubstances, the combination of i.a. 5-FU-PEG liposomes and flow retardation increased the concentration in tumor tissue by a factor of 44 and even 100-fold in the para-aortal lymph nodes (LN). The concentration of 5-FU and FdUrd was increased by flow reduction, intraaortal application and liposomal encapsulation of 5-FU.

Mitochondria as subcellular targets for clinically useful anthracyclines.

Due to the widespread use of anthracyclines as antitumor agents, a large number of investigations have been reported analyzing clinical and molecular aspects of these quinone antibiotics. While the high affinity of anthracyclines towards chromosomal DNA has been held responsible for their antitumor activity, an increasing amount of data is being accumulated showing that these drugs also target mitochondria thus interfering with major mitochondrial functions. Since this toxicity of anthracyclines towards mitochondria is associated with side effects significantly limiting their chemotherapeutic dose, the corresponding underlying mechanisms need to be understood. Bioenergetic failure, enzyme inhibitions, lipid peroxidations, induction of membrane disorders as well as the initiation of oxidative stress are being attributed to the accumulation of anthracyclines at or inside mitochondria. In this review the wide spectrum of possible mode of actions of these antibiotics leading to mitochondrial dysfunctions will be presented and discussed.

Global cerebral ischemia in the rat: online monitoring of oxygen free radical production using chemiluminescence in vivo.

Using online in vivo chemiluminescence (CL), we studied for the first time continuously the production of reactive oxygen species (ROS) after global cerebral ischemia and the relationship of ROS production to CBF. In anesthetized rats equipped with a closed cranial window, the CL enhancer, lucigenin (1 mM), was superfused onto the brain topically. CL was measured through the cranial window with a cooled photomultiplier, and CBF was measured simultaneously with laser-Doppler flowmetry. Reperfusion after 10 min (n = 8) of global cerebral ischemia led to a CL peak to 188 +/- 77% (baseline = 100%) within 10 +/- 4 min. After 2 h of reperfusion, CL had returned to 102 +/- 28%. Reperfusion after 20 min (n = 8) of ischemia increased CL to 225 +/- 48% within 12 +/- 3 min. After 2 h, CL was still increased (150 +/- 44%, p < 0.05 compared with 10 min of ischemia). CL after 10 min of ischemia was neither affected by brain topical free CuZn-superoxide dismutase (SOD) (100 U/ml, n = 3) nor by i.v. administration of free CuZn-SOD (104 U/kg, followed by 104 U/kg/h, n = 3). The CBF hyperfusion peak on reperfusion preceded the CL peak in all experiments by several minutes. In additional in vitro experiments we investigated the source of CL: Intracellular loading of lucigenin was demonstrated in cultured CNS cells, and a very similar pattern of CL as in the in vivo preparation after ischemia developed in rat brain slices after 15 min of hypoxia, which was unaffected by free CuZn-SOD (100 U/ml) but strongly attenuated by liposome-entrapped CuZn-SOD. We conclude that lucigenin-enhanced CL is a promising tool to study ROS production continuously from the in vivo brain of experimental animals and brain slices, and that the CL signal most likely derives from the intracellular production of superoxide. The production of ROS is preceded by reperfusion, is burst-like, and is dependent on the duration of the ischemic interval.

Short-term neuropathological aspects of in vivo suicide gene transfer to the F98 rat glioblastoma using liposomal and viral vectors.

To date, only few preclinical protocols on liposomal suicide gene transfer in tumors have been published, none of which directly compared viral to liposomal vectors in terms of immunoreactivity and efficacy. We thus studied the neuropathological alterations in 80 rats being treated for glioblastoma using liposomal and, for comparison, adenoviral and retroviral suicide gene transfer approaches to identify vector-associated efficacy and toxicity for further clinical studies. 62 rats served as controls. F98 tumors were established in Fisher rats and transfected in vivo with the thymidine kinase gene of herpes simplex virus (HSVtk) by a single intratumoral application and an implanted intratumoral continuous delivery system. Three days later ganciclovir was given intraperitoneally for 14 days. The animals were sacrificed 17 days post completed gene transfer. Brains were examined histologically and immunohistochemically using markers for immunocompetent cells. Ten animals showed complete tumor regression; they all belonged to the liposomal and adenoviral groups. In 6 of 10 experimental groups considerable numbers of lymphocytes along the margins of the regression cavities could be observed. Control animals of the liposomal and adenoviral groups showed only little lymphocytic infiltration, underlining the minimal immunogenicity of these carriers. In contrast, the retroviral control group featured a high lymphocyte infiltration. In summary, this study indicates that, in terms of both efficacy and immunoreaction, liposomes are as appropriate as adenoviruses in the treatment of rat glial tumors using suicide gene transfer strategies.

Superoxide-mediated reduction of the nitroxide group can prevent detection of nitric oxide by nitronyl nitroxides.

Nitronyl nitroxides (NN), a class of compounds which react with nitric oxide forming imino nitroxides, were applied in different systems for the detection of nitric oxide. Addition of a NN to planar monolayers of bovine aortic endothelial cells (BAEC) activated by Ca2+ ionophore A23187 immediately resulted in a strong decrease of the ozone-mediated .NO chemiluminescence. Simultaneously, a rapid diminution of the electron spin resonance (ESR) signal intensity of the NN (without detectable formation of the corresponding imino nitroxide) was observed; superoxide dismutase partially inhibited this decrease in the NN concentration. Model experiments using hypoxanthine/xanthine oxidase in aqueous solution and KO2 in dimethylsulfoxide as sources of O2.- revealed that there is a rapid reduction of nitronyl nitroxides by superoxide. The second order rate constant for the reaction of the water soluble NN with O2.- was determined to be 8.8 x 10(5) M-1s-1, which is more than two orders of magnitude higher than the value reported previously for reaction with .NO (Woldman et al., BBRC 202, 195-203, 1994). Reduction of the nitronyl nitroxide was also observed in the presence of glutathione, ascorbic acid or rabbit liver microsomes. Incorporation of both nitronyl and imino nitroxides into liposomes strongly decreased reduction by superoxide and other reductants, however, in the presence of microsomes, there was no protective effect by liposomal encapsulation of NN. The results indicate that in biological systems (in addition to other reducing agents) the presence of superoxide can prevent the detection of nitric oxide using nitronyl nitroxides.

Carboplatin-liposomes as activators of hematopoiesis.

Recombinant colony stimulating factors are studied in clinical trials with the purpose being the relief of the side effects of high dose chemotherapy and to make an optimized treatment regimen possible. The broad use of such factors is hindered by their relatively high costs, their short elimination half-lives, the occurrence of mild side effects, and the limitation of their action to the stimulation of mainly neutrophils. Therefore, exogenous preparations inducing an endogenous activation of hematopoiesis are being sought. In experiments in mice we have shown that carboplatin-liposomes injected intraperitoneally in a single dose of 100 mg/kg led to a strong two-peak increase in white blood cell counts. A maximum 10-fold elevation compared to controls of free carboplatin or empty liposomes was observed on day 2 and was probably due to the release and mobilization of cells from storage compartments. The second peak of about a 6-fold increase occurred on day 7-8 and can be seen as an indicator of bone marrow stimulation. Differentiation of blood cells revealed that neutrophils, lymphocytes and platelets multiplied. We presume that this effect of carboplatin-liposomes is due to a relatively fast uptake of these vesicles by macrophages as their natural target. Within these cells carboplatin is metabolized, leading to an almost total loss of antineoplastic activity against the murine P388 leukemia. Concomitantly, cytokines are apparently induced in and released from macrophages producing secondarily hematopoietic growth factors either directly or in combination with other cytokines. An involvement of macrophages is indicated by the fact that an intraperitoneal pretreatment of mice with zymosan caused a partial but significant suppression of hematopoietic stimulation. In an in vitro colony forming assay of serum of mice treated 1, 3, or 7 days with carboplatin-liposomes, the number of colonies increased 20-fold compared to serum from saline treated animals. Additionally, a combined intraperitoneal treatment of mice with 100 mg/kg of cyclophosphamide followed by carboplatin-liposomes one hour later demonstrated that prevention of cytostatic-induced leukopenia is possible by this method. Although the mechanism of stimulation of hematopoiesis by carboplatin-liposomes is still partially unknown our results suggest that there should be further development of such a preparation for possible use in the treatment of cancer or other inherited or acquired hematopoietic disorders.

Stealth liposomal 5-fluorouracil with or without degradable starch microspheres for hepatic arterial infusion in the treatment of liver metastases. An animal study in VX-2 liver tumor-bearing rabbits.

PURPOSE: Regional application of cytostatics in liver metastases leads to increased concentrations in tumor tissue. Flow retardation by temporary occlusion and drug targeting via liposome encapsulation (PEG liposomes) will further increase tumor concentrations. MATERIALS AND METHODS: Liver tumor-bearing rabbits were submitted to i.v. or i.a. therapy with or without liposome-encapsulated 5-fluorouracil (5-FU). I.a. groups were additionally treated with or without degradable starch microspheres. Tumor concentrations were calculated by HPLC as area under the curve (AUC). RESULTS: A comparison with i.v.-applied 5-FU yielded the following increasing concentrations: 5-FU-PEG liposomes i.v. 6-fold, 5-FU i.a. 20-fold, 5-FU i.a. + DSM 226-fold, 5-FU-PEG liposomes i.a. 319-fold, 5-FU-PEG liposomes i.a. + DSM 2203-fold. CONCLUSION: The intratumoral concentration of 5-FU was increased up to 2203 times the intravenous dose by combination of regional application via the hepatic artery with temporary embolization by degradable starch microspheres and drug targeting by liposome encapsulated 5-FU.

Paclitaxel partitioning into lipid bilayers.

Paclitaxel (taxol) is diterpenoid anticancer drug with a new mechanism of cytostatic action. It is under investigation in clinical trials for treatment of various types of human cancer. A major difficulty in developing paclitaxel as a chemotherapeutic agent in its poor water solubility. In order to improve the bioavailability of paclitaxel, novel vehicle systems such as mixed micelles or liposome-based formulations are being developed. In this study we determined the partition coefficient of paclitaxel partitioning into small unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine using two different methods, namely high-sensitivity titration calorimetry and fluorescence spectrometry. We measured a partition coefficient of Kp approximately equal to 9,500 M-1, a partition enthalpy of Delta H = -25 +/- 3 kcal mol-1 and a free energy of binding of Delta G = -7.9 kcal mol-1. The binding reaction is enthalpy-driven, which can be explained by van der Waals interactions between the hydrophobic drug and the strong temperature dependence of the partition equilibrium. A temperature increase of 10 degrees C reduces the paclitaxel solubility in the lipid phase by a factor of 4.

[Magnetosomes as biological model for iron binding: relaxivity determination with MRI]. / Magnetosomen als biologisches Modell der Eisenbindung: Messung der Relaxivität in der MRT.

PURPOSE: In vitro characterization of iron-containing bacterial particles (magnetosomes) as superparamagnetic contrast agents for MRI. MATERIAL AND METHODS: Different concentrations of magnetosomes were examined with a 1.5 T clinical whole-body MR system at 21 degrees C using the transit/receive extremity coil. Both longitudinal and transversal relaxivities (R1 and R2) of the magnetosomes were determined by an inversion recovery snapshot gradient recall echo (IR FLASH) with various inversion times and a multi echo spin echo sequence. Atomic absorption spectrometry (AAS) and electron microscopy were used as reference standard. RESULTS: Longitudinal and transverse relaxivities of the magnetosomes were calculated to be R1 = 7.688 mmol -1 s -1 and R2 = 147.67 mmol -1 s -1, respectively. The corresponding iron concentrations were determined in all dilutions using AAS, while the magnetosomes were morphologically delineated by electron microscopy. CONCLUSION: Magnetosomes represent a new and interesting class of iron-containing contrast agents warranting further evaluation in cellular cultures and animal models. Magnetosomes may be suited for displaying the vector distribution and gene expression of new molecular therapies.

Carboplatin-liposomes (CPL) in immunodeficient mice: improved antitumor activity for breast carcinomas and stimulation of hematopoiesis.

Carboplatin-liposomes (CPL) have been shown to possess a strong stimulatory activity on the hematopoiesis in immunocompetent mice. As we were interested in studying this pharmacological characteristic in parallel with any antitumour effects which might be expected for the encapsulated cytostatic, we used a panel of six human breast carcinomas xenotransplanted to nude mice. The antitumor activity as well as the hematopoietic effects of the vesicles were studied in comparison to, and in combination with, the free drug. Carboplatin was encapsulated into reverse phase evaporation vesicles (REV) and injected i.p. as a single dose of 75 mg kg-1 into tumor-free and breast-carcinoma-bearing animals, respectively. Following a single application of CPL in nude mice, a significant increase of the WBC numbers to about three times for that of the normal level could be observed over a period of at least 28 days. The elevation was due to an increase in both circulating granulocytes and lymphocytes. The peripheral effect was accompanied by a relative decrease of spleen cellularity, while the number of bone marrow cells was hardly affected. There was no influence detectable on circulating blood cells in SCID mice. However, a rather high toxicity of CPL for this immunodeficient mouse strain was noticed. In the panel of breast carcinomas used, free carboplatin and CPL displayed a different pattern of therapeutic efficiencies. In four of the five tumor models tested, a combination of the free with the liposomal drug showed a significant inhibition of tumor growth while effectively preventing a drug-induced leukopenia.(ABSTRACT TRUNCATED AT 250 WORDS)

Therapeutic evaluation of liposome-encapsulated Daunoblastin in murine tumor models.

Daunoblastin in free and liposome-encapsulated form was tested in the L1210 murine leukemia and the intramuscularly transplanted 276A sarcoma. Both therapeutic (% ILS, tumor volume inhibition) and toxicologic (leukocytes, body weight difference) parameters were evaluated. The liposome preparations showed similar therapeutic effects as the free substance but caused a lower toxicity with a lower mortality rate, higher leukocyte values and smaller body weight reduction. Longer sonication time with the output of more smaller unilamellar vesicles had no influence on the parameters in the solid model, but resulted in shorter ILS values in the L1210 model. Administration of empty liposomes immediately before liposomal Daunoblastin did not result in better antineoplastic activity but yielded higher leukocyte values.

Influence of liposomes rich in unsaturated or saturated fatty acids on the growth of human xenotransplanted mammary carcinomas and on the levels of heart type fatty acid binding protein.

A panel of 4 human mammary carcinomas passaged in nude mice were subjected to intraperitoneal application of cholesterol-free liposomes enriched with linoleic (unsaturated fatty acid) or stearic acid (saturated fatty acid). The liposomes were examined with regard to their influence on the tumor growth and level of heart type fatty acid binding protein (FABP). Liposomes with different fatty acid composition influenced the growth of mammary carcinomas 3366, BO, 4000 and 4151 in distinct ways. Liposomes with a high content of stearic acid significantly inhibited the growth of mammary carcinomas 3366 and BO, whereas mammary carcinomas 4000 and 4151 were not affected. The growth of mammary carcinoma 3366 was moderately increased after supplementation of liposomes rich in linoleic acid, the tumor BO was significantly inhibited and the growth of MaCa 4000 and 4151 was unchanged. Liposome treatment led to a significant increase in heart type FABP in mammary carcinomas 3366 and BO regardless of whether the animals were treated with liposomes rich in stearic or linoleic acid. Such significant changes of FABP level could not be observed in mammary carcinomas 4000 or 4151. We suggest that the lipid-mediated growth modulation seems to be dependent on an increase of heart type FABPs in these tumor models.

[Macrophage depletion inhibits leukocyte recruitment in experimental melanin-induced uveitis (EMIU)]. / Makrophagendepletion hemmt die Leukozytenrekrutierung bei experimenteller Melanininduzierter Uveitis (EMIU).

BACKGROUND: To study the role of macrophages in experimental melanin-induced uveitis (EMIU), we used the method of intravital microscopy to analyse changes in leukocyte adhesion to iris venules of live rats with EMIU after pretreatment with liposomal clodronate. MATERIALS AND METHODS: EMIU was induced in Lewis rats (n = 48) by intraperitoneal immunisation with bovine crude melanosomes emulsified in complete Freund's adjuvant (CFA) and pertussis toxin (PTX). Control animals received CFA and PTX only (n = 12) or no injection (n = 6). Animals were treated with liposomal clodronate (DMDP-lip) or empty liposomes on days -2, +1, 4, 6 and 8. Using IVM, postcapillary iris venules of rats were examined to quantify leukocyte rolling and firm adhesion to the vascular endothelium. RESULTS: Depletion of macrophages caused a decreased percentage of rolling leukocytes on day 8 (2 +/- 1.1% vs 15.2 +/- 1.6%, DMDP-lip vs EMIU, mean +/- SEM, ANOVA, p < 0.05) and day 10 (2.6 +/- 0.3% vs 14.2 +/- 1.6%). A significant decline in the number of firmly adherent leukocytes was detected on days 8 and 10 (88 +/- 13/mm2 vs 175 +/- 18/mm2 and 129 +/- 13/mm2 vs 372 +/- 31/mm2, DMDP-lip vs EMIU). Treatment with empty liposomes showed no changes in leukocyte firm adhesion. CONCLUSIONS: Elimination of macrophages prevents the induction of EMIU. In autoimmune-mediated uveitis, macrophages play a crucial role in the initiation of leukocyte-endothelium interaction.

[Preparation, properties and therapeutic effect of Daunorubicin-containing liposomes]. / Darstellung, Eigenschaften und therapeutische Wirksamkeit von daunorubicinhaltigen Liposomen.

Preparation and analytic characterization of uni- and multilamellar lipid vesicles with different lipid composition, charge and size, containing Daunorubicin are described. A critical review is given for the methods of preparation of lipids and other amphiphilic compounds. Variations of the lipid composition, charge and size of the liposomes do not alter the therapeutic effectiveness.

Hepatic arterial infusion in the treatment of liver metastases with PEG liposomes in combination with degradable starch microspheres (DSM) increases tumor 5-FU concentration. an animal study in CC-531 liver tumor-bearing rats.

UNLABELLED: The regional application of cytostatics in liver metastases leads to increased concentrations in the tumor tissue. The effect of flow retardation by temporary occlusion and drug targeting with liposome encapsulation (PEG liposomes) on tumor 5-fluorouracil (5-FU) concentrations was investigated. MATERIALS AND METHODS: Tumor-bearing rats were submitted to i.v. or intraarterial (i.a.) therapy with liposome-encapsulated or non-encapsulated 5-FU. The i.a. groups were additionally treated with or without Spherex® degradable starch microspheres (DSM). The tumor 5-FU concentrations were determined by high-performance liquid chromatography (HPLC) as area under the curve (AUC). RESULTS: A comparison with i.v. in administered 5-FU yielded the following increases tumor concentrations: 5-FU-PEG liposomes i.v. 27-fold, 5-FU i.a. 19-fold, 5-FU i.a. + DSM 1760-fold, 5-FU-PEG liposomes i.a. 110-fold, 5-FU-PEG liposomes i.a. + DSM 7665-fold. CONCLUSION: Liver intratumoral 5-FU concentration increases to >7,500 times that following i.v. administration by a combination of regional administration via the hepatic artery with temporary embolization by DSM and drug targeting by liposome-encapsulated 5-FU.

Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems.

The ability of nonviral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications. In recent years, several efforts were focused on the elucidation of the gene transfer mechanisms and on the development of multicomponent systems in order to improve both targeted gene delivery and transfection efficiency. The transport of the therapeutic DNA from the cytoplasm into the nucleus is an inefficient process and is considered as the major limiting step in nondividing cells. One of the strategies to improve nuclear uptake of DNA is taking advantage of the cellular nuclear import machinery. Synthetic peptides containing a nuclear localization signal (NLS) are bound to the DNA so that the resulting DNA-NLS complex can be recognized as a nuclear import substrate by specific intracellular receptor proteins. In this review, we critically summarize recent studies applying this approach with a particular focus on NLS-sequence specificity. Implications of the observed results are also discussed in regards to future developments of this technology.

In vivo gene transfer: focus on the kidney.

Renal gene transfer techniques are being developed as a novel experimental approach to understand the pathogenesis of renal disease and to potentially develop new therapeutic tools. We review the currently available technology to introduce foreign genetic material into renal tissue, i.e., retroviral, adenoviral, and liposomal transfer systems with their respective advantages and caveats. Today, the transfer efficiency of these methods appears to be sufficiently high to study the effects of transduced genes on renal function and morphology in rat kidney. This will allow (i) the elucidation of the function of genes on the course of renal disease in experimental animal models and (ii) the modulation of local expression of endogenous genes which presumptively contribute to renal pathology in these models. One strategy to accomplish this aim is the use of recombinant DNA technology to design antisense DNA constructs or oligonucleotides, which interfere with the renal expression of target genes. We will also discuss some of the shortcomings of the currently used techniques with respect to potential therapeutic use of gene transfer systems and gene modulation.