The past and presence of gene targeting: from chemicals and DNA via proteins to RNA.

The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Subcutaneous immunotherapy with purified Der p1 and 2 suppresses type 2 immunity in a murine asthma model.

BACKGROUND: Allergen-specific immunotherapy can induce long-term suppression of allergic symptoms, reduce medication use, and prevent exacerbations of allergic rhinitis and asthma. Current treatment is based on crude allergen extracts, which contain immunostimulatory components such as ß-glucans, chitins, and endotoxin. Use of purified or recombinant allergens might therefore increase efficacy of treatment. AIMS: Here, we test application of purified natural group 1 and 2 allergens from Dermatophagoides pteronyssinus (Der p) for subcutaneous immunotherapy (SCIT) treatment in a house dust mite (HDM)-driven mouse model of allergic asthma. MATERIALS AND METHODS: HDM-sensitized mice received SCIT with crude HDM extract, a mixture of purified Der p1 and 2 (DerP1/2), or placebo. Upon challenges, we measured specific immunoglobulin responses, allergen-induced ear swelling response (ESR), airway hyperresponsiveness (AHR), and inflammation in bronchoalveolar lavage fluid (BAL) and lung tissue. RESULTS: ESR measurement shows suppression of early allergic response in HDM-SCIT- and DerP1/2-SCIT-treated mice. Both HDM-SCIT and DerP1/2-SCIT are able to suppress AHR and eosinophilic inflammation. In contrast, only DerP1/2-SCIT is able to significantly suppress type 2 cytokines in lung tissue and BAL fluid. Moreover, DerP1/2-SCIT treatment is uniquely able suppress CCL20 and showed a trend toward suppression of IL-33, CCL17 and eotaxin levels in lung tissue. DISCUSSION: Taken together, these data show that purified DerP1/2-SCIT is able to not only suppress AHR and inflammation, but also has superior activity toward suppression of Th2 cells and HDM-induced activation of lung structural cells including airway epithelium. CONCLUSIONS: We postulate that treatment with purified natural major allergens derived from HDM will likely increase clinical efficacy of SCIT.

Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is overexpressed on most carcinomas. Dependent on the tumour type, its overexpression is either associated with improved or worse patient survival. For ovarian cancer, however, the role of EpCAM remains unclear. METHODS: Cell survival of ovarian cancer cell lines was studied after induction or repression of endogenous EpCAM expression using siRNA/cDNA or artificial transcription factors (ATF) consisting of engineered zinc-fingers fused to either a transcriptional activator or repressor domain. RESULTS: Two ATFs were selected as the most potent down- and upregulator, showing at least a two-fold alteration of EpCAM protein expression compared with control. Downregulation of EpCAM expression resulted in growth inhibition in breast cancer, but showed no effect on cell growth in ovarian cancer. Induction or further upregulation of EpCAM expression decreased ovarian cancer cell survival. CONCLUSION: The bidirectional ATF-based approach is uniquely suited to study cell-type-specific biological effects of EpCAM expression. Using this approach, the oncogenic function of EpCAM in breast cancer was confirmed. Despite its value as a diagnostic marker and for immunotherapy, EpCAM does not seem to represent a therapeutic target for gene expression silencing in ovarian cancer.

Transcription factors and molecular epigenetic marks underlying EpCAM overexpression in ovarian cancer.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is overexpressed on carcinomas, and its downregulation inhibits the oncogenic potential of multiple tumour types. Here, we investigated underlying mechanisms of epcam overexpression in ovarian carcinoma. METHODS: Expression of EpCAM and DNA methylation (bisulphite sequencing) was determined for ovarian cancer cell lines. The association of histone modifications and 16 transcription factors with the epcam promoter was analysed by chromatin immunoprecipitation. Treatment with 5-Aza-2'-deoxycytidine (5-AZAC) was used to induce EpCAM expression. RESULTS: Expression of EpCAM was correlated with DNA methylation and histone modifications. Treatment with 5-AZAC induced EpCAM expression in negative cells. Ten transcription factors were associated with the epcam gene in EpCAM expressing cells, but not in EpCAM-negative cells. Methylation of an Sp1 probe inhibited the binding of nuclear extract proteins in electromobility shift assays; such DNA methylation sensitivity was not observed for an NF-κB probe. CONCLUSION: This study provides insights in transcriptional regulation of epcam in ovarian cancer. Epigenetic parameters associated with EpCAM overexpression are potentially reversible, allowing novel strategies for sustained silencing of EpCAM expression.

Pompe disease: current state of treatment modalities and animal models.

Pompe disease is a rare autosomal recessive lysosomal storage disease caused by deficiency of acid-alpha-glucosidase (GAA). This deficiency results in glycogen accumulation in the lysosomes, leading to lysosomal swelling, cellular damage and organ dysfunction. In early-onset patients (the classical infantile form and juvenile form) this glycogen accumulation leads to death. The only therapy clinically available is enzyme replacement therapy, which compensates for the missing enzyme by i.v. administration of recombinant produced enzyme. The development of clinically relevant animal models gained more insight in the disease and allowed evaluation of recombinant enzyme therapy. Several therapies are currently under investigation for Pompe disease, including gene therapy. This review gives an overview of the available knockout mouse models, of the in vitro and in vivo studies performed using recombinant produced enzyme. Furthermore, it describes current therapeutic approaches for Pompe disease as well as experimental therapies like gene correction therapy.

An SV40 large T-antigen immortalized human umbilical vein endothelial cell line for anti-endothelial cell antibody detection.

OBJECTIVE: Anti-endothelial cell antibodies in serum of patients with different inflammatory diseases can be detected by a whole cell enzyme-linked immunosorbant assay, using primary cultures of human umbilical vein endothelial cells. To avoid repeated isolation, it would be of great value if an immortal endothelial cell line could be used to perform anti-endothelial cell antibody assays. METHODS: In this study endothelial cells from human umbilical and iliac veins and arteries were transfected with a plasmid containing the Simian Virus 40 large T-antigen. Endothelial cell line(s) derived from this procedure were compared with human umbilical vein endothelial cells in the anti-endothelial cell antibody assay. RESULTS: After transfection, clones of homologous cell populations showed an extended lifespan, before entering a period of crisis. In one human umbilical vein endothelial cell clone a subpopulation of cells escaped crisis and became immortal (EVLC2). Telomerase was activated in this endothelial cell line, resulting in maintenance of the telomere length. There was a significant correlation between anti-endothelial cell antibody testing on human umbilical vein endothelial cells and on the cell line EVLC2. CONCLUSION: The Simian Virus 40 large T-antigen immortalized human umbilical vein endothelial cell line EVLC2 may be useful for the detection of anti-endothelial cell antibodies.

Telomere length in breast cancer patients before and after chemotherapy with or without stem cell transplantation.

High-dose chemotherapy and peripheral blood stem cell transplantation (PBSCT) may accelerate telomere length loss in haematopoietic stem cells. As data including pre-and post-treatment samples are lacking, we studied leukocyte telomere length and telomerase activity before and after treatment in breast cancer patients randomized to receive 5 adjuvant courses FEC (5-FU, epirubicin and cyclophosphamide) (n = 17), or 4 x FEC followed by high-dose cyclophosphamide, thiotepa, carboplatin and autologous PBSCT (n = 16). Haemoglobin, MCV, leukocyte-and platelet numbers were assessed prior to (t(0)), 5 months after (t(1)) and 9 months after chemotherapy (t(2)); these parameters were decreased at t(1)and t(2)compared to t(0)(high-dose: all parameters; standard-dose: leukocytes and platelets), and all parameters were lower after high-dose than standard-dose treatment at t(1). Paired individual leukocyte samples of t(0)and t(1)showed telomere length change (determined by telomere restricted fragment (TRF) assay) ranging from +0.8 to -2.2 kb, with a decreased TRF length in 9 patients of both groups. Telomerase activity (determined by TRAP assay) was below detection limit in leukocyte samples of t(0)and t(1). Thus, standard-and high-dose chemotherapy negatively affect haematological reconstitution in this setting. In individual patients, telomere length can be remarkably changed following haematological proliferative stress after treatment.

The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice: a new model to study carcinoma-directed immunotherapy.

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a M(r) 38,000 transmembrane antigen also known as 17-1A or Ep-CAM, is commonly used for targeted immunotherapy of carcinomas because it is strongly expressed by most carcinomas. EGP-2 is, however, also expressed in most normal epithelia. To evaluate anti-EGP-2-directed treatment-associated effects on tumors and on EGP-2-positive normal tissue, we generated EGP-2-expressing transgenic mice. A 55-kb DNA fragment consisting of the 14-kb genomic coding sequence of the human EGP-2 gene with approximately 10-kb-upstream and approximately 31-kb-downstream sequences was isolated and used to direct EGP-2 expression in an epithelium-specific manner. In the EGP-2 transgenic mice, EGP-2 appeared to be specifically expressed in all of those epithelial tissues that also express EGP-2 in humans, whereas all of the other tissues were negative. The specific in vivo localization of the i.v. administered anti-EGP-2 monoclonal antibody MOC31 was studied in EGP-2-positive and -negative tumors induced s.c. in this EGP-2 transgenic mouse model. Immunohistochemical analysis showed specific localization of MOC31 in the EGP-2-positive tumors but not in the EGP-2-negative tumors. No anti-EGP-2 monoclonal antibody localization was observed in any of the EGP-2-positive normal mouse tissues, which indicated a limited in vivo accessibility. In conclusion, an EGP-2 transgenic mouse model has been generated that expresses the EGP-2 antigen as in humans and, therefore, can serve as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities in both tumors and normal tissue.

Scanning electron microscopic analysis of endothelial cell coverage and quality in large vessels from multi-organ donors: effects of preservation on endothelial cell integrity.

Endothelial cell integrity (coverage and quality) of large donor vessels is important because these vessels are used for vascular reconstructions in solid-organ transplantation. Disruption of the endothelial cell monolayer will initiate blood coagulation and may lead to thrombosis of large vessels, often resulting in the loss of the transplanted organ. Iliac arteries and veins, removed from 10 heart-beating multi-organ donors at the end of the donor procedure, were analyzed using scanning electron microscopy at three different time points of preservation. Endothelial cell coverage and quality were determined immediately after removal from the donor, after 10 h (time of transplantation) and 7 d storage in 'University of Wisconsin' cold preservation solution (UW). Endothelial cell coverage decreased during the preservation of arteries, but was maintained in veins. Storage of the veins for 7 d in plastic bags showed a decreased endothelial cell coverage compared to storage in glass vials. Early removal of the blood vessels and proper storage, free floating and in clean UW, may improve maintenance of the endothelial cell integrity. These findings may be important in order to reduce the risk of thrombosis and, consequently, organ failure after transplantation. Furthermore, vessels with maintained endothelial cell integrity after 7 d may be used for in vitro research.

Telomerase activity in needle biopsies from prostate cancer and benign prostates.

BACKGROUND: Telomerase activation is thought to be essential for the immortality of cancer cells. It may be a prognostic factor in small volume well differentiated prostate cancers and hence a guide for the aggressiveness of the approach. The length of the chromosome tips (telomeres) are maintained by a specific enzyme (telomerase) independently of the normal cell division cycle. Although telomerase is not expressed in most normal human tissues, it is expressed in most human tumours. For the detection of telomerase in small prostate needle biopsy samples a recently developed telomeric repeat amplification protocol (TRAP) assay was used. The aim of the present study was: to measure telomerase activity in human prostate samples, and to evaluate the applicability of this assay on specimens from a prostate biopsy. MATERIALS AND METHODS: From 36 patients referred for lower urinary tract symptoms (LUTS) or suspicion of having prostate cancer a total of 288 prostate biopsy samples were obtained (8 in each patient). When the digital rectal examination was abnormal and/or when the PSA level was elevated in L.U.T.S., or asymptomatic patients' tissue samples were obtained by transrectal ultrasound (TRUS) guided biopsies. Samples were tested for telomerase activity by a modified TRAP and forwarded for histology. RESULTS: In 19 out of 36 patients prostate cancer was diagnosed on histology. In 11 of these 19 tumours substantial telomerase activity was detected, whereas only very low telomerase activity existed in 2 of 17 samples from benign prostatic hypertrophy (BPH) patients. In this small series the relative telomerase activity in prostate cancer correlated with histopathological grade. CONCLUSIONS: Our results show the applicability of a TRAP assay to measure telomerase activity in small needle biopsied prostate samples. In poorly differentiated and metastatic cancer we observed that levels of telomerase activity were high. To establish accuracy and to distinguish the 'relative good from the ugly' further study is needed.

The ubiquitin-activating enzyme E1-like protein in lung cancer cell lines.

The UBE1L gene isolated from the chromosome 3p21 region has an extremely reduced level of mRNA in lung cancer. Sequence analysis showed a 45% homology to the human ubiquitin-activating enzyme E1 at the amino acid level. To further characterize the protein product, we generated UBE1L protein-specific antibodies. Immunoblot analysis revealed a full-length gene product of approximately 112 kDa. Assessment of the level and distribution pattern of the UBE1L protein in normal and tumor tissue using the generated antibodies showed that the UBE1L protein was present in normal lung cells and non-lung cancer cell lines, but was undetectable in all 14 human lung cancer cell lines analyzed. This difference in expression of the UBE1L protein between normal lung tissue and lung tumor-derived cell lines suggests a possible involvement of an E1-like protein in the origin and/or progression of lung tumors.

Characterization of immortalized human umbilical and iliac vein endothelial cell lines after transfection with SV40 large T-antigen.

Most in vitro studies of human endothelial cells have relied on cells derived from human umbilical veins (HUVEC); however, heterogeneity of primary cultured endothelial cells can make critical interpretation of results difficult. Several endothelial cell lines have been produced to serve as a more constant source of endothelial cells. In this study, we characterized the endothelial cell lines EVLB3 and EVLC2 derived from HUVEC, and EVLK1 and EVLK2 derived from human iliac vein endothelial cells (HIVEC). These cell lines maintained the typical endothelial cell cobblestone morphology and appeared to be growth factor independent. They lost PECAM-1 and von Willebrand factor, GP96 was reduced to the level of vascular smooth muscle cells (SMC), but aSMC-actin was far less than in vascular SMC. Antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were comparable with young endothelial cells, and mRNA was present for tPA, PAI-1, tissue factor (TF), tissue factor pathway inhibitor and thrombomodulin. This study revealed that mRNA and protein expression of coagulation and fibrinolytic factors was influenced by the stage of cell confluence. No differences could be detected between the endothelial cell lines derived from HUVEC and HIVEC. These cell lines may be a useful tool for studies on cellular interactions of fibrinolytic components or exploring the regulation of TF expression.

An EGP-2/Ep-CAM-expressing transgenic rat model to evaluate antibody-mediated immunotherapy.

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), also known as 17-1A or Ep-CAM, is a 38-kDa transmembrane antigen, commonly used for targeted immunotherapy of carcinomas. Although strongly expressed by most carcinomas, EGP-2 is also expressed in most simple epithelia. To evaluate treatment-associated effects and side-effects on tumor and normal tissue respectively, we generated an EGP-2-expressing transgenic Wistar rat. To express the cDNA of the EGP-2 in an epithelium-specific manner, the 5' and 3' distal flanking regions of the human keratin 18 (K18) gene were used. EGP-2 protein expression was observed in the liver and pancreas, whereas EGP-2 mRNA could also be detected in lung, intestine, stomach and kidney tissues. In this rat, EGP-2-positive tumors can be induced by injecting a rat-derived carcinoma cell line transfected with the GA733-2 cDNA encoding EGP-2. Transgenic rats were used to study specific in vivo localization of an i.v. anti-EGP-2 monoclonal antibody, MOC31, applied i.v. Immunohistochemical analyses showed the specific localization of MOC31 in s.c. induced EGP-2-positive tumors, as well as in the liver. In contrast, in EGP-2-transgenic rats, MOC31 did not bind to EGP-2-negative tumors, the pancreas, or other normal tissues in vivo. In conclusion, an EGP-2-transgenic rat model has been generated that serves as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities.

Regulation of spontaneous and TNF/IFN-induced IL-6 expression in two human ovarian-carcinoma cell lines.

Autocrine and paracrine production of interleukin-6 (IL-6) is considered to be involved in the ongoing proliferation of ovarian-cancer cells. In view of the variability in IL-6 expression between various ovarian-cancer cells, we questioned whether differences in IL-6-gene regulation might be observed in ovarian tumor cells with and without IL-6 expression. The CAOV-3 cell line spontaneously secreted IL-6, which was enhanced by tumor necrosis factor-alpha (877 +/- 89 vs. 8,452 +/- 1,762 pg/ml, x +/- sd, p < 0.01). The electrophoretic mobility shift assay (EMSA) demonstrated that basic IL-6 expression was associated with DNA binding of activator protein-1 (AP-1) and nuclear factor IL-6 (NF-IL6). Nuclear factor kappa-B (NF-KB), which consisted mainly of p65-NF-KB was induced in response to TNF-alpha stimulation. A2780 cells did not express IL-6, either spontaneously or after stimulation with TNF-alpha. EMSAs, showed spontaneous AP-1 but no NF-IL6 or NF-KB DNA binding. TNF-alpha stimulation enhanced AP-1 and induced NF-KB but no NF-IL6 DNA binding in these cells. NF-IL6 protein, however, was detected in nuclear extracts of these cells by Western blotting. In contrast, IL-6-promoter transfection studies showed no difference in promoter activation between CAOV-3 and A2780. This study reveals that differential IL-6-gene expression observed in ovarian-cancer cell lines is independent of NF-IL6 activation.

Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles.

OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a 10:1 ratio for concentrations of SAINT-2pp/DOPE: plasmid. Using these conditions, cell concentrations were lowered step-wise and we were able to transfect as few as one thousand cells with both methods. For detection of transfection of a small number of cells a sensitive assay was needed (Luciferase). A plasmid containing the neomycin resistance gene was used to determine the transfection rate expressed in colony forming units by counting colonies after selection. At low plasmid concentrations this transfection rate was within the same range for both electroporation and SAINT-2pp/DOPE transfection. Fluorescent in situ hybridisation of metaphase chromosomes of transfected endothelial cells using the plasmid as a probe showed that stable integration was possible with both methods. CONCLUSIONS: Electroporation and a synthetic amphiphile, SAINT-2pp, provide the possibility of transfecting small numbers of cells resulting in stable integration of low plasmid concentrations. The availability of this technology is important in order to obtain functional endothelial cell lines from various human blood vessels for research purposes.

Telomerase activity as a biomarker for (pre)neoplastic cervical disease in scrapings and frozen sections from patients with abnormal cervical smear.

PURPOSE: To evaluate the diagnostic value of semi-quantitative telomerase activity assessment in cervical scrapings together with human papillomavirus (HPV) typing for detection of (pre)neoplastic cervical lesions and to compare telomerase activity in cervical scrapings and frozen specimens from the same patients. PATIENTS AND METHODS: A cross-sectional study was performed in 161 patients referred for an abnormal cervical cytology report. In cervical scrapings, telomerase activity was determined by modified telomere repeat amplification protocol (TRAP) assay and HPV typing by polymerase chain reaction (PCR) with general and type-specific primers. Final diagnosis was made by pathologic examination of biopsy and/or loop excision specimens. RESULTS: Telomerase activity was detectable in assessable scrapings from one of nine (11%) patients without cervical intraepitheleal neoplasia (CIN), in three of 26 (12%) with CIN I, eight of 35 (22%) with CIN II, 18 of 62 (29%) with CIN III, and four of 13 (31%) with cancer. Sensitivity and negative predictive value of the TRAP assay for CIN II/III and cancer lesions were 25% and 28%, respectively, while specificity for no CIN or CIN I was 89%. In representative frozen sections, frequency of detectable telomerase activity was related to grade of CIN/cancer; none of 21 normal cervices, none of two CIN I, two of 12 (17%) CIN II, 10 of 31 (32%) CIN III, and 18 of 21 (86%) cervical cancer lesions were telomerase-positive (P < .0005). Telomerase activity levels in paired scrapings and frozen sections appeared to be only weakly related; telomerase-positive sections with negative scrapings and vice versa (only in CIN III) were observed. In oncogenic HPV-negative scrapings (n = 14), no telomerase activity was detected, but in frozen sections, telomerase activity levels appeared to be unrelated to presence of specific HPV types. CONCLUSION: Telomerase activity is more frequent in higher grade CIN/cervical cancer lesions. Telomerase activity assessment in cervical scrapings has a low sensitivity for CIN II/III and/or cervical cancer and does not appear to be useful in primary screening for cervical cancer. However, increased telomerase activity in frozen CIN sections may be a possible marker of progressive disease.

Spinal Cord Stimulation and the Induction of c-fos and Heat Shock Protein 72 in the Central Nervous System of Rats.

For more than a decade, spinal cord stimulation (SCS) has been used as an adjuvant treatment for patients who are unresponsive to conventional therapies for angina pectoris. Many studies showed that SCS has both electro-analgesic and anti-ischemic effects. Nonetheless, the biological substrates by which SCS acts have not yet been unraveled, although recently areas in the brain have been described that show changes in blood flow, following SCS, and during provocation of angina. In search of a putative mechanism of action of SCS, we hypothesized that SCS affects processing of nociceptive information within the central nervous system (CNS). Moreover, it may alter the limbic system activity that maintains the balance between sympathetic and parasympathetic activity in the heart. Hence, we have developed a rat model to investigate its suitability for studying the induction of neural activity during SCS. To characterize neural activity, we used the expression of both the immediate early gene c-fos and the heat shock protein 72 (HSP72). c-Fos was used to identify structures in the CNS affected by SCS, and HSP72 was applied in order to ascertain whether SCS might operate as a stressor. In 20 halothane-anesthetized male Wistar rats, two electrodes were placed epidurally, one at the C7 level and the other at the T2 level. Two days after surgery, the rats were either stimulated "treated" animals, n = 10) or used as controls ("unstimulated" = "sham," n = 10) in random order. Furthermore, we studied the effect of SCS on behavior in five treated and five control rats. Three hours after stimulation, the rats were euthanized and the brain and spinal cord were removed. The treated group showed regional increased c-fos expression in regions of the limbic system (periaqueductal gray, paraventricular hypothalamic nucleus, paraventricular thalamic nucleus, central amygdala, agranular and dysgranular insular cortex, (peri)ambiguus, nucleus tractus solitarius, and spinal cord) that are involved in the processing of pain and cardiovascular regulation, among other things. Moreover, in both treated rats and controls, HSP72-expression was found in the endothelium of the enthorhinal cortex, the amygdala, and the ventral hypothalamus, but not in the neurons. Finally, treated animals were significantly more alert and active than controls. In conclusion, the rat model we developed appears to be suitable for studying potential mechanisms through which SCS may act. In addition, SCS affects c-fos expression in specific parts of the brain known to be involved in regulation of pain and emotions. HSP72-expression is limited to the endothelium of certain parts of the CNS and thereby excludes physical stress effects as a potential mechanism of SCS.

Novel pyridinium surfactants for efficient, nontoxic in vitro gene delivery.

Novel, double-chained pyridinium compounds have been developed that display highly efficient DNA transfection properties. The transfection efficiency of several of these compounds is enhanced by an order of magnitude, when compared with the transfection efficiency accomplished with the widely used cationic lipid system, lipofectin. Most importantly, the pyridinium compounds were found to be essentially nontoxic toward cells. Using various reporter genes, such as beta-galactosidase and pNEO (a gene construct that renders cells resistent to antibiotic derivatives of neomycin like G418), we demonstrate that the enhanced efficiency relates to the fact that a relative higher number of cells in the population is transfected (approximately 50% in the case of COS cells) by the pyridinium derivatives, whereas the delivery of DNA per cell is also enhanced. Furthermore, application of the pyridinium derivatives shows little cellular preference in their ability to transfect cells. By systematically modifying the structure of the pyridinium amphiphile, i.e., by changing either the headgroup structure or the alkyl chains, some insight was obtained that may lead to unraveling the mechanism of amphiphile-mediated transfection, and thus to protocols that further optimize the carrier properties of the amphiphile. Our results reveal that unsaturated alkyl chains enhance the transfection properties of the pyridinium-based amphiphiles. Preliminary experiments suggest that the structure-dependent improvement of transfection efficiency, when comparing pyridinium derivatives with lipofectin, likely relates to the mechanism of delivery rather than the packaging of the amphiphile/DNA complex.

Methodological aspects of glucose monitoring with a slow continuous subcutaneous and intravenous ultrafiltration system in rats.

A method for the continuous ultrafiltration of venous blood or subcutaneous fluid is demonstrated with glucose monitoring in the living rat. Ultrafiltrate was withdrawn at a constant flow rate of approximately 100 nl/min. Glucose content of the ultrafiltrates was electrochemically determined with a flow injection analysis method and a bi-enzyme reactor. After glucose loading, the time course of glucose in the ultrafiltrate from the jugular vein was virtually identical, whereas that from the subcutaneous compartment was attenuated and the peaks blunted as compared to glucose levels in concomitantly assayed arterial blood. Our study demonstrates the potential of low rate ultrafiltration for monitoring metabolism with biosensor technology in vivo.