A rapidly growing fibroadenoma in a pregnant woman: A case report.

BACKGROUND: Fibroadenomas are the most common benign breast lesions in women. They present as a unilateral mass and can rapidly enlarge in size through hormonal changes. Fibroadenomas could be classified as small or giant, and as simple or complex. They are classified as 'giant' when the size exceeds 5 cm and/or weight 500 gram; and as 'complex' if one of the following characteristics is present: cysts with a size >3 mm, epithelial calcifications, sclerosing adenosis and papillary apocrine metaplasia. Giant fibroadenomas can cause compression of surrounding breast tissue or breast asymmetry, requiring surgical excision in order to preserve a normal breast shape. CASE: A 26-year-old pregnant woman was referred with a palpable mass of her right breast. The mass rapidly increased in size to a diameter of 13 cm during the second trimester of her pregnancy. A tru-cut biopsy confirmed a fibroadenoma. The rapid growth and compression of normal breast tissues indicated a lumpectomy during her pregnancy. The mass was easily excised without any consequences for the pregnancy. Pathological examination showed a complex giant fibroadenoma. CONCLUSION: A unique case of a pregnant woman with rapid progression of a fibroadenoma that met the criteria of a complex and giant fibroadenoma, was presented. This case emphasizes the importance of timely surgical intervention, even during pregnancy, to prevent permanent breast tissue damage.

Gadolinium Enhancement of the Aneurysm Wall in Extracranial Carotid Artery Aneurysms.

BACKGROUND AND PURPOSE: The natural history and optimal treatment of extracranial carotid artery aneurysms are unknown. Gadolinium enhancement of the aneurysm wall may reflect aneurysm wall inflammation and instability. In this study, we investigated the feasibility of extracranial carotid artery aneurysm wall imaging and explored a potential relationship of aneurysm wall enhancement with aneurysm growth and the presence of (silent) brain infarcts and white matter lesions. MATERIALS AND METHODS: Fourteen conservatively treated patients with 15 asymptomatic extracranial carotid artery aneurysms underwent gadolinium-enhanced 3T MR imaging at 2 time points with a 12-month interval. Primary outcome was growth of the aneurysm sac (≥2.0 mm); secondary outcomes were the presence of (silent) brain infarcts and white matter lesions at baseline and follow-up. MR images were reviewed by 2 independent observers, and inter- and intraobserver reproducibility was assessed. RESULTS: Seven (50%) patients were men; the median age was 55 years (range, 40-69 years). Eleven extracranial carotid artery aneurysms (73%) were saccular (median size, 11 mm; range, 5.0-38.5 mm), and 4 were fusiform (median size, 21.5 mm; range, 10.0-40.0 mm). Eleven of 15 aneurysms (73%) exhibited gadolinium enhancement at baseline. Four aneurysms (27%) showed growth at follow-up imaging, 2 gadolinium-positive (+) and 2 gadolinium-negative (-) (P = .245). Three patients (21%) had ipsilateral brain infarcts at baseline; 1 of them showed a new silent brain infarct at follow-up imaging (gadolinium+). Nine patients (64%) showed bilateral white matter lesions at baseline. In 3 patients, increased white matter lesion severity was observed at follow-up (2 gadolinium+). All observations showed excellent inter- and intraobserver reproducibility. CONCLUSIONS: In this explorative study, we demonstrated that extracranial carotid artery aneurysm wall imaging was feasible. Future well-powered studies are needed to investigate whether extracranial carotid artery aneurysm gadolinium enhancement predicts aneurysm growth and thromboembolic complications.

Effect of bilateral carotid occlusion on cerebral hemodynamics and perivascular innervation: An experimental rat model.

We aimed to investigate the effect of chronic cerebral hypoperfusion on cerebral hemodynamics and perivascular nerve density in a rat model. Bilateral common carotid artery (CCA) ligation (n = 24) or sham-operation (n = 24) was performed with a 1-week interval. A subgroup (ligated n = 6; sham-operated n = 3) underwent magnetic resonance imaging (MRI) before the procedures and 2 and 4 weeks after the second procedure. After termination, carotids were harvested for assessment of complete ligation and nerve density in cerebral arteries that were stained for the general neural marker PGP 9.5 and sympathetic marker TH by computerized image analysis. Five rats were excluded because of incomplete ligation. MRI-based tortuosity of the posterior communicating artery (Pcom), first part of the posterior cerebral artery (P1) and basilar artery was observed in the ligated group, as well as an increased volume (p = 0.05) and relative signal intensity in the basilar artery (p = 0.04; sham-group unchanged). Immunohistochemical analysis revealed that compared to sham-operated rats, ligated rats had increased diameters of all intracircular segments and the extracircular part of the internal carotid artery (p < 0.05). Ligated rats showed a higher general nerve density compared to controls in P1 (10%, IQR:8.7-10.5 vs. 6.6%, IQR:5.5-7.4, p = 0.003) and Pcom segments (6.4%, IQR:5.8-6.5 vs. 3.2%, IQR:2.4-4.3, p = 0.003) and higher sympathetic nerve density in Pcom segments (3.7%, IQR:2.8-4.8 vs. 1.7%, IQR:1.3-2.2, p = 0.02). Bilateral CCA occlusion resulted in redistribution of blood flow to posteriorly located cerebral arteries with remarkable changes in morphology and perivascular nerve density, suggesting a functional role for perivascular nerves in cerebral autoregulation.

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'.

Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD.

BACKGROUND: Goblet cell metaplasia, a common feature of chronic obstructive pulmonary disease (COPD), is associated with mucus hypersecretion which contributes to the morbidity and mortality among patients. Transcription factors SAM-pointed domain-containing Ets-like factor (SPDEF) and forkhead box protein A2 (FOXA2) regulate goblet cell differentiation. This study aimed to (1) investigate DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation and (2) compare this in airway epithelial cells from patients with COPD and controls during mucociliary differentiation. METHODS: To assess DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation, primary airway epithelial cells, isolated from trachea (non-COPD controls) and bronchial tissue (patients with COPD), were differentiated by culture at the air-liquid interface (ALI) in the presence of cytokine interleukin (IL)-13 to promote goblet cell differentiation. RESULTS: We found that SPDEF expression was induced during goblet cell differentiation, while FOXA2 expression was decreased. Importantly, CpG number 8 in the SPDEF promoter was hypermethylated upon differentiation, whereas DNA methylation of FOXA2 promoter was not changed. In the absence of IL-13, COPD-derived ALI-cultured cells displayed higher SPDEF expression than control-derived ALI cultures, whereas no difference was found for FOXA2 expression. This was accompanied with hypomethylation of CpG number 6 in the SPDEF promoter and also hypomethylation of CpG numbers 10 and 11 in the FOXA2 promoter. CONCLUSIONS: These findings suggest that aberrant DNA methylation of SPDEF and FOXA2 is one of the factors underlying mucus hypersecretion in COPD, opening new avenues for epigenetic-based inhibition of mucus hypersecretion.

Continuous EEG Monitoring for Early Detection of Delayed Cerebral Ischemia in Subarachnoid Hemorrhage: A Pilot Study.

INTRODUCTION: Early identification of delayed cerebral ischemia (DCI) in patients with aneurysmal subarachnoid hemorrhage (aSAH) is a major challenge. The aim of this study was to investigate whether quantitative EEG (qEEG) features can detect DCI prior to clinical or radiographic findings. METHODS: A prospective cohort study was performed in aSAH patients in whom continuous EEG (cEEG) was recorded. We studied 12 qEEG features. We compared the time point at which qEEG changed with the time point that clinical deterioration occurred or new ischemia was noted on CT scan. RESULTS: Twenty aSAH patients were included of whom 11 developed DCI. The alpha/delta ratio (ADR) was the most promising feature that showed a significant difference in change over time in the DCI group (median -62% with IQR -87 to -39%) compared to the control group (median +27% with IQR -32 to +104%, p = 0.013). Based on the ROC curve, a threshold was chosen for a combined measure of ADR and alpha variability (AUC: 91.7, 95% CI 74.2-100). The median time that elapsed between change of qEEG and clinical DCI diagnosis was seven hours (IQR -11-25). Delay between qEEG and CT scan changes was 44 h (median, IQR 14-117). CONCLUSION: In this study, ADR and alpha variability could detect DCI development before ischemic changes on CT scan was apparent and before clinical deterioration was noted. Implementation of cEEG in aSAH patients can probably improve early detection of DCI.

The role of neutrophil gelatinase associated lipocalin (NGAL) as biological constituent linking depression and cardiovascular disease.

Depression is more common in patients with cardiovascular disease than in the general population. Conversely, depression is a risk factor for developing cardiovascular disease. Comorbidity of these two pathologies worsens prognosis. Several mechanisms have been indicated in the link between cardiovascular disease and depression, including inflammation. Systemic inflammation can have long-lasting effects on the central nervous system, which could be associated with depression. NGAL is an inflammatory marker and elevated plasma levels are associated with both cardiovascular disease and depression. While patients with depression show elevated NGAL levels, in patients with comorbid heart failure, NGAL levels are significantly higher and associated with depression scores. Systemic inflammation evokes NGAL expression in the brain. This is considered a proinflammatory effect as it is involved in microglia activation and reactive astrocytosis. Animal studies support a direct link between NGAL and depression/anxiety associated behavior. In this review we focus on the role of NGAL in linking depression and cardiovascular disease.

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.

Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression.

Suppressors of interictal discharges in idiopathic childhood occipital epilepsy of Gastaut.

Idiopathic childhood occipital epilepsy of Gastaut (ICOE-G) is a rare form of epilepsy, characterized by visual hallucinations, periods of blindness, motor seizures, and migraine-like symptoms. A characteristic EEG feature is fixation-off sensitivity: epileptiform discharges are suppressed by visual input. Here, we present an 11-year-old girl suffering from ICOE-G, who was studied to identify potential additional suppressors of the epileptiform discharges.

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.

Dynamic lattice distortions in Sr2RuO4: microscopic studies by perturbed angular correlation spectroscopy and ab initio calculations.

Applying time differential perturbed angular correlation (TDPAC) spectroscopy and ab initio calculations, we have investigated possible lattice instabilities in Sr(2)RuO(4) by studying the electric quadrupole interaction of a (111)Cd probe at the Ru site. We find evidence for a dynamic lattice distortion, revealed from the observations of: (i) a rapidly fluctuating electric-field gradient (EFG) tensor showing non-Arrhenius relaxation, (ii) an anomalous temperature dependence of the quadrupole interaction frequency, and (iii) a monotonic increase of the EFG asymmetry (η) below 300 K. We argue that the observed dynamic lattice distortion is caused by strong spin fluctuations associated with the inherent magnetic instability in Sr(2)RuO(4).

Secretion of thymidine kinase to increase the effectivity of suicide gene therapy results in the loss of enzymatic activity.

Low efficiency of gene transfer is one of the major limitations of gene therapy. A solution to this problem may be transmission; by modification of the transgene, the gene product can be secreted and internalized by the surrounding cells. Cancer gene therapy using the herpes simplex thymidine kinase (HSV-TK) suicide gene is a promising treatment, and TK has been used in clinical trials with some success. However, this kind of therapy has limited efficacy due to the low level of gene transfer reached. A modified TK protein, capable of migrating from the producing cell to neighboring cells, would result in a greater proportion of cells affected by the treatment. As a first step towards transmission, we constructed a secretory form of HSV-TK by including the Igkappa leader peptide in the gene. An endoplasmatic reticulum export signal was added to the construct to further improve its secretion. Secretion and protein production in cancer cells, the enzymatic activity of the modified proteins and the ability of the modified TK to sensitize cancer cells to ganciclovir were tested. Addition of the Igkappa leader resulted in high levels of secretion of HSV-TK, with up to 70% of the total amount of protein secreted. Inclusion of an ER export signal did not further improve secretion. The enzyme activity of the secreted TK however, was decreased when compared to native TK. This study is the first to report on secretion of TK, and provides a first step in a novel strategy to improve the efficiency of cancer gene therapy. The loss of function in secreted TK however, may present a major hurdle in the development of a transmitted form of TK.

A comparative study on the immunotherapeutic efficacy of recombinant Semliki Forest virus and adenovirus vector systems in a murine model for cervical cancer.

Currently, various therapeutic strategies are being explored as a potential means to immunize against metastatic malignant cells or even primary tumours. Using recombinant viral vectors systems or protein-based immunization approaches, we are developing immunotherapeutic strategies against cervical cancer or premalignant cervical disease, as induced by high-risk type human papillomaviruses (HPVs). We previously demonstrated that immunization of mice with recombinant replication-defective Semliki Forest virus (rSFV) encoding a fusion protein of HPV16 E6 and -E7 (SFV-eE6,7) induces strong cytotoxic T-lymphocyte (CTL) activity and eradication of established HPV-transformed tumours. In this study, we compared the antitumour efficacy of SFV-eE6,7 with that of a recombinant adenovirus (rAd) type 5 vector, expressing the same antigen construct (Ad-eE6,7). Prime-boosting with SFV-eE6,7 resulted in higher precursor CTL frequencies and CTL activity compared to prime-boosting with Ad-eE6,7 and also in murine tumour treatment experiments SFV-eE6,7 was more effective than Ad-eE6,7. To elicit a therapeutic effect with Ad-eE6,7, 100/1000-fold higher doses were needed compared to SFV-eE6,7. In vivo T-cell depletion experiments demonstrated that these differences could not be explained by the induction of a different type of effector cells, since CD8+ T cells were the main effector cells involved in the protection against tumour growth in both rSFV- and rAd-immunized mice. Also comparable amounts of in vivo transgene expression were found upon immunization with rSFV and rAd encoding the reportor gene luciferase. However, anti-vector responses induced by a single injection with rAd resulted in a more than 3-log decrease in luciferase expression after a second injection of rAd. With rSFV, transgene expression was inhibited by only one to two orders of magnitude in preinjected mice. As an antigen-specific booster immunization strongly increases the level of the CTL response and is essential for efficient induction of immunological memory, it is likely that (part of) the difference in efficacy between rSFV and rAd type 5 can be ascribed to a diminished efficacy of the booster immunization in the case of rAd due to anti-vector antibody responses.

Nuclear imaging of hormonal receptor status in breast cancer: a tool for guiding endocrine treatment and drug development.

Breast cancer is a commonly occurring disease in women and a major cause of morbidity and mortality. In the past decades, the development of medical endocrine therapies has led to a significant improvement in treatment outcome for this type of cancer. This therapy is targeting specific hormone receptors that are overexpressed by the tumor cells. In breast cancer, estrogen and progesterone receptors are important targets and therefore the receptor status of the tumor strongly determines treatment outcome. However, the receptor status can change during the course of the disease and consequently therapy resistance can occur. Therefore, insight in the current receptor status of the tumor is essential for optimal treatment. Nuclear imaging techniques like positron emission tomography (PET) and single photon emission computed tomography (SPECT), could provide the means to monitor the receptor status of tumors and the receptor occupancy by medical endocrine drugs in a non-invasive manner. Thus, these imaging techniques could offer a tool to guide therapy management in the individual patient. Nuclear imaging techniques for some of the relevant receptors for treatment of breast cancer are currently available. These imaging techniques could also aid the development of novel treatment strategies like modulation of hormone receptor expression. This review will address the role of hormone receptors in breast cancer treatment, the available nuclear imaging methods for monitoring the receptor status, the potential role of nuclear imaging in therapy management and drug development.

Fusion of herpes simplex virus thymidine kinase to VP22 does not result in intercellular trafficking of the protein.

Suicide gene therapy is a promising approach for the treatment of cancer. Current protocols, however, suffer from low efficiency. We tried to alleviate this problem by developing a transgene that will spread from the initially transduced cell to the surrounding cells (transmission). We used herpes simplex virus (HSV) VP22 as a signal for cellular uptake of HSV-1 thymidine kinase (TK). By co-culturing naive cells with cells producing a TK-VP22 fusion protein, we detected intercellular trafficking of this protein. We used a variety of techniques, including two-color flow cytometry and cytotoxicity assays to detect the presence of TK in the non-producing cells. We confirmed intercellular migration of VP22. We did not detect any intercellular trafficking of the TK-VP22 fusion protein, by various fixation methods or flow cytometry. In ganciclovir sensitivity assays, we found no difference between the efficiency of TK (IC50=3.15+/-0.76 microg/ml) and TK-VP22 (IC50=2.27+/-0.59 microg/ml). Using a cell-free enzyme activity assay we showed that fusion of TK to VP22 did not change the enzyme activity. In conclusion, we described novel and robust methods to detect intercellular trafficking. From our data we concluded that protein transmission of TK by VP22 for gene therapy is not likely to be successful. In addition, we described a useful and quantifiable assay to measure the enzymatic activity of TK and TK fusion proteins, and described some common properties of VP22 fusion proteins that may explain the different results that have been obtained by others.

Highly efficient and carcinoma-specific adenoviral replication restricted by the EGP-2 promoter.

Although some successes have been reported using adenoviral vectors for the treatment of cancer, adenoviral cancer gene therapy is still hampered by the lack of sufficient tumor cell killing. To increase the efficiency, adenoviruses have been modified to replicate specifically in tumor tissues by using tumor specific promoters controlling genes essential for adenoviral replication. However, many conditionally replicating adenoviral vectors replicate in one tumor type only, which limits their application. The epithelial glycoprotein-2 (EGP-2) promoter is active in a broad variety of carcinomas, the most common type of cancer. We utilized this promoter to restrict adenoviral replication. In this report we demonstrate that the potency of the replication-competent adenovirus AdEGP-2-E1 to specifically lyse EGP-2 positive cells is comparable to wild-type adenovirus (AdWT). In addition, we show that in vivo AdEGP-2-E1 replicates as efficient as AdWT in EGP-2 positive tumor cells. On the contrary, in EGP-2 negative cell lines as well as in primary human liver samples, the replication was attenuated up to 4-log in comparison to wild-type virus. This report clearly shows the potency of the EGP-2 promoter to mediate highly efficient and specific adenoviral replication for carcinoma gene therapy.

Employment of liver tissue slice analysis to assay hepatotoxicity linked to replicative and nonreplicative adenoviral agents.

Whereas virotherapy has emerged as a novel and promising approach for neoplastic diseases, appropriate model systems have hampered preclinical evaluation of candidate conditionally replicative adenovirus agents (CRAds) with respect to liver toxicity. This is due to the inability of human viral agents to cross species. We have recently shown the human liver tissue slice model to be a facile means to validate adenoviral replication. On this basis, we sought to determine whether our ex vivo liver tissue slice model could be used to assess CRAd-mediated liver toxicity. We analyzed and compared the toxicity of a conditionally replicative adenovirus (AdDelta24) to that of a replication incompetent adenovirus (Adnull [E1-]) in mouse and human liver tissue slices. To accomplish this, we examined the hepatic apoptosis expression profile by DNA microarray analyses, and compared these results to extracellular release of aminotransferase enzymes, along with direct evidence of apoptosis by caspase-3 immunhistochemical staining and TUNEL assays. Human and mouse liver tissue slices demonstrated a marked increase in extracellular release of aminotransferase enzymes on infection with AdDelta24 compared to Adnull. AdDelta24-mediated liver toxicity was further demonstrated by apoptosis induction, as detected by caspase-3 immunohistochemical staining, TUNEL assay and microarray analysis. In conclusion, concordance of CRAd-mediated apoptosis in both the human and the mouse liver tissue slice models was demonstrated, despite the limited replication ability of CRAds in mouse liver slices. The results of this study, defining the CRAd-mediated apoptosis gene expression profiles in human and mouse liver, may lay a foundation for preclinical liver toxicity analysis of CRAd agents.

The carcinoma-specific epithelial glycoprotein-2 promoter controls efficient and selective gene expression in an adenoviral context.

Adenoviral vectors are widely used in cancer gene therapy. After systemic administration however, the majority of the virus homes to the liver and the expressed transgene may cause hepatotoxicity. To restrict transgene expression to tumor cells, tumor- or tissue-specific promoters are utilized. The tumor antigen epithelial glycoprotein-2 (EGP-2), also known as Ep-CAM, is expressed in many cancers from different epithelial origins. In this study, the EGP-2 promoter was shown to restrict the expression of luciferase and thymidine kinase in an adenoviral context in different cell lines. In vivo, the EGP-2 promoter mediated efficient expression of luciferase in tumors but showed a 3-log lower activity in liver tissue when compared with the cytomegalovirus (CMV) promoter. Similarly, the EGP-2 promoter mediated specific cell killing after ganciclovir treatment in EGP-2-positive cells. Moreover, in vivo, this treatment regiment did not cause any rise in the liver enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), demonstrating absence of liver toxicity. In contrast, CMV-mediated expression of thymidine kinase in combination with ganciclovir treatment resulted in high ASAT and ALAT values. This study demonstrates the value of the EGP-2 promoter to restrict transgene expression to a broad range of tumor types, thereby preventing liver toxicity.

Enhanced transduction of fibroblasts in transplanted kidney with an adenovirus having an RGD motif in the HI loop.

Application of gene therapy to the renal graft has a powerful potential to improve the outcome of kidney transplantation and eliminate detrimental side effects associated with systemic therapy, through local expression of immunoregulatory or other protective molecules. However, the search for the optimal vector is still ongoing. In this study, we used a modified adenovirus that has an Arg-Gly-Asp (RGD) motif inserted in the HI loop of the fiber knob, as a successful strategy to transduce the renal graft. Donor Lewis rat kidneys were infused via the renal artery with a solution containing either a fiber-modified adenovirus (AdTL-RGD) or an unmodified adenovirus (AdTL), or with saline. Syngeneic recipients were killed after 3, 7 or 14 days. Efficiency, selectivity, localization, time course of gene expression and side effects were studied using biochemical and immunohistological techniques. Enhanced gene expression was achieved selectively in the transplanted kidney by AdTL-RGD, when compared to AdTL. Transgene expression lasted for at least 2 weeks. With the AdTL-RGD vector, the transgene was abundantly expressed in the renal interstitial fibroblasts. An increase in the number of cytotoxic T lymphocytes accompanied the use of either vector, when compared to saline. These data convincingly show enhanced and selective gene transfer to the fibroblasts of transplanted kidneys using an RGD-modified adenovirus, providing a highly efficient vector system for future therapeutic interventions.