Alexander Stoff

Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma

PurposeAlternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo.Experimental designHMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival.ResultsIntravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone.ConclusionInjected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

Promotion of incisional wound repair by human mesenchymal stem cell transplantation.

Abstract:  The purpose of this study was to determine the effect of transplanted human mesenchymal stem cells (hMSCs) on wound healing. In this model, full‐thickness cutaneous wounds were created by incision in the skin of adult New Zealand white rabbits and treated by transplanted hMSCs into the wounds. Wound healing was evaluated by histological analysis and tensiometry over time. A total of 15 New Zealand white rabbits with 10 wounds per animal were examined in this study. Animals were treated with hMSCs and euthanised at 3, 7, 14, 21 and 80 days after manipulation. The hMSCs were labelled with a fluorescent dye (CM‐DiI), suspended in phosphate‐buffered saline and used to treat full‐thickness incisional wounds in rabbit skin. Tensiometry and histology were used to characterise the wound‐healing rate of the incisional wounds. These results showed that transplanted hMSCs significantly inhibited scar formation and increased the tensile strength of the wounds. Importantly, MSCs from genetically unrelated donors did not appear to induce an immunologic response. In conclusion, human mesenchymal stem cell therapy is a viable approach to significantly affect the course of normal cutaneous wound healing and significantly increase the tensile strength.

Transpalpebral decompression of endocrine ophthalmopathy by intraorbital fat removal (Olivari technique): experience and progression after more than 3000 operations over 20 years.

Background: Graves’ ophthalmopathy is a chronic, multisystem disorder characterized by increased intraorbital fat tissue and hypertrophic extraocular muscles caused by an autoimmune process. Graves’ ophthalmopathy represents the most frequent extrathyroidal manifestation of Graves’ disease. Clinical findings are impaired ocular motility, diplopia, lid retraction, and impaired visual acuity up to optic neuropathy, with menacing blindness. Methods: Transpalpebral decompression by intraorbital fat removal was first described by Olivari in 1988. From 1984 to 2004, a consecutive series of 1635 patients (3210 eyes) with Graves’ ophthalmopathy underwent this operation at the authors’ institution. The medical records of 1374 patients (84 percent) could be evaluated retrospectively. Results: Postoperatively, the majority of patients showed significant improvements of major symptoms such as ocular protrusion, diplopia, decreased visual acuity, swelling of the eyelids, retrobulbar pressure, and headache. In addition, complications—most of them temporary and reversible—were rare. Because the osseous orbita is not touched, no complications, such as penetration of the dura, infection of the sinus maxillaris, meningitis, irritation of the infraorbital nerve, or obstruction of the lacrimal system, were observed. However, the high number of additional eyelid corrections (average, 2.5 individual corrections) following the decompression indicated the complexity of surgical treatment in endocrine orbitopathy. Conclusion: Transpalpebral decompression has proved to be reliable, effective, and safe, with good, lasting results leading to an improvement not only in visual function but also in the patients personal well-being and social life, with a high-benefit–to–low-risk ratio.

Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-β1 and TGF-β2 precursor proteins, but an increase in expression of TGF-β3 precursor protein and TGF-β type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy

The development of novel therapeutic strategies is imperative for the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on ‘xenotype’ knob switching with non-human adenovirus have been exploited. Herein, we present the characterization of a novel vector derived from a recombinant Ad5 vector containing the canine adenovirus serotype 1 (CAV-1) knob (Ad5Luc1-CK1), the tropism of which has not been previously described. We compared the function of this vector with our other chimeric viruses displaying the CAV-2 knob (Ad5Luc1-CK2) and Ad3 knob (Ad5/3Luc1). Our data demonstrate that the CAV-1 knob can alter Ad5 tropism through the use of a CAR-independent entry pathway distinct from that of both Ad5Luc1-CK2 and Ad5/3-Luc1. In fact, the gene transfer efficiency of this novel vector in ovarian cancer cell lines, and more importantly in patient ovarian cancer primary tissue slice samples, was superior relative to all other vectors applied in this study. Thus, CAV-1 knob xenotype gene transfer represents a viable means to achieve enhanced transduction of low-CAR tumors.

Cancer-specific targeting of a conditionally replicative adenovirus using mRNA translational control.

BackgroundIn view of the limited success of available treatment modalities for a wide array of cancer, alternative and complementary therapeutic strategies need to be developed. Virotherapy employing conditionally replicative adenoviruses (CRAds) represents a promising targeted intervention relevant to a wide array of neoplastic diseases. Critical to the realization of an acceptable therapeutic index using virotherapy in clinical trials is the achievement of oncolytic replication in tumor cells, while avoiding non-specific replication in normal tissues. In this report, we exploited cancer-specific control of mRNA translation initiation in order to achieve enhanced replicative specificity of CRAd virotherapy agents. Heretofore, the achievement of replicative specificity of CRAd agents has been accomplished either by viral genome deletions or incorporation of tumor selective promoters. In contrast, control of mRNA translation has not been exploited for the design of tumor specific replicating viruses to date. We show herein, the utility of a novel approach that combines both transcriptional and translational regulation strategies for the key goal of replicative specificity.MethodsWe describe the construction of a CRAd with cancer specific gene transcriptional control using the CXCR4 gene promoter (TSP) and cancer specific mRNA translational control using a 5′-untranslated region (5′-UTR) element from the FGF-2 (Fibroblast Growth Factor-2) mRNA.ResultsBoth in vitro and in vivo studies demonstrated that our CRAd agent retains anti-tumor potency. Importantly, assessment of replicative specificity using stringent tumor and non-tumor tissue slice systems demonstrated significant improvement in tumor selectivity.ConclusionsOur study addresses a conceptually new paradigm: dual targeting of transgene expression to cancer cells using both transcriptional and mRNA translational control. Our novel approach addresses the key issue of replicative specificity and can potentially be generalized to a wide array of tumor types, whereby tumor selective patterns of gene expression and mRNA translational control can be exploited.

Gene transfer to carcinoma of the breast with fiber-modified adenoviral vectors in a tissue slice model system

Successful adenoviral (Ad) vector-mediated strategies for breast cancer gene therapy and virotherapy have heretofore been hindered by low transduction efficiency. This has recently been understood to result from a relative paucity of expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on primary tumor cells. To further investigate this issue, we evaluated the expression of CAR on breast cancer cell lines as well as primary breast cancer cells. With the exception of one patient sample, CAR expression was notably higher in the tumor cells from patients compared to CAR expression in the tumor cell lines. Furthermore, we explored CAR-independent targeting strategies to breast cancer tissue by exploring a panel of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs for their utility in breast cancer gene therapy and virotherapy. These targeting motifs included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7), and were tested using the breast cancer tissue slice model, which is the most stringent substrate system available. Of all the tested tropism modified Ad vectors, Ad5/3 exhibited the highest transductional efficiency in breast cancer. These preclinical results suggest that Ad5/3 is the most useful modification to achieve higher clinical efficacy of breast cancer gene therapy and virotherapy.

Abdominoplasty with Total Abdominal Liposuction for Patients with Massive Weight Loss

BackgroundMassive weight loss after bariatric surgery is associated with significant skin excess, laxity, and ptosis over the abdomen. Good results have been achieved with abdominoplasty and circumferential lipectomy. However, blood transfusions are sometimes needed, and patients may require long hospital stays. Furthermore, morbidity rates are high. Total abdominal liposuction performed with abdominoplasty allows for the preservation of lymphatic vessels below Scarpa’s fascia and eliminates the need for upper flap undermining. This study aimed to evaluate this technique in patients with anterior abdominal redundancy attributable to massive weight loss after bariatric surgery.MethodsThe charts of 60 patients treated between December 2001 and October 2004 were retrospectively reviewed. All the patients had undergone previous bariatric surgery as well as subsequent total abdominal liposuction and abdominoplasty.ResultsThe average amount of wetting solution used was 3.1 l, and the average total aspirate was 2.5 l. The mean pannus weight was 3,649 g, and the average dimension was 48 × 25 × 6 cm. No patient required a blood transfusion. The median in-hospital stay was 1 day, with 42% of the patients treated as outpatients. The median follow-up period was 3 months. Morbidity was 22%. Factors associated with the development of complications were weight of the pannus, transverse dimension of the pannus, and body mass index. All the patients were satisfied with the results.ConclusionsTotal abdominal liposuction followed by abdominoplasty is adequate treatment for anterior abdominal redundancy for patients with massive weight loss.

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 (AdΔ24) 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 AdΔ24 compared to Adnull. AdΔ24-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.

A comparison of a new skin closure device and intradermal sutures in the closure of full-thickness surgical incisions.

Background: A novel topical skin adhesive system was developed to close the outermost layer of skin in an expeditious manner. To determine its clinical utility, a clinical investigation was undertaken to demonstrate equivalence of a new adhesive skin closure system (Prineo Skin Closure System) to intradermal sutures in wound closure. Methods: The investigation included 83 patients who underwent elective abdominoplasty, circumferential body lift procedures, and breast reconstruction with deep inferior epigastric perforator flaps. Incisions were divided in half, and each half was randomized to wound closure with the new skin closure system, including a pressure-sensitive adhesive mesh tape for wound edge approximation and next-generation cyanoacrylate or intradermal sutures. Postoperative evaluations took place at 24 hours, 7 days, 12 to 25 days, 90 days, 6 months, and 12 months. Results: The new skin closure system was found to be equivalent to intradermal sutures for the continuous approximation of wounds. The upper limit of the two-sided 90 percent confidence interval for difference in proportions was 10.9 percent. The mean time to closure for the new skin closure system was 1.46 minutes, approximately 5 minutes faster than that for intradermal sutures (p < 0.0001). Both treatments had similar incision healing and cosmetic outcomes. No quantitative or qualitative differences of clinical significance were evident between the treatment groups. Conclusions: The Prineo Skin Closure System can be considered equivalent to intradermal sutures for full-thickness surgical incisions with regard to safety and effectiveness. The ease and speed of application contribute to shortened operative times (4.5 times faster than intradermal sutures). CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.