What do animal models have to tell us regarding Duchenne muscular dystrophy?

Animal models of DMD have played, and will continue to play, a key role in the understanding of the pathogenesis and treatment of Duchenne muscular dystrophy (DMD). The mdx mouse and GRMD dog are spontaneous dystrophin deficient mutants and have been the most widely used models to date. A number of other murine models have been created by exposure to mutagens or genetic manipulation. The animal models have allowed the development of a number of promising experimental therapeutic approaches to DMD that are now entering clinical trial, the majority of which would not have been developed without their use. However, there has been much debate about the merits of the different animal models, which will only be finally clear as we learn from the initial human clinical trials.

Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins.

One of the possible therapies for Duchenne muscular dystrophy (DMD) is the introduction of a functional copy of the dystrophin gene into the patient. For this approach to be effective, therapeutic levels and long-term expression of the protein need to be achieved. However, immune responses to the newly expressed dystrophin have been predicted, particularly in DMD patients who express no dystrophin or only very truncated versions. In a previous study, we demonstrated a strong humoral and cytotoxic immune response to human dystrophin in the mdx mouse. However, the mdx mouse was tolerant to murine dystrophin, possibly due to the endogenous expression of dystrophin in revertant fibres or the other nonmuscle dystrophin isoforms. In the present study, we delivered human and murine dystrophin plasmids by electrotransfer after hyaluronidase pretreatment to increase gene transfer efficiencies. Tolerance to murine dystrophin was still seen with this improved gene delivery. Tolerance to exogenous recombinant full-length human dystrophin was seen in mdx transgenic lines expressing internally deleted versions of human dystrophin. These results suggest that the presence of revertant fibres may prevent the development of serious immune responses in patients undergoing dystrophin gene therapy.

Enhanced in vivo delivery of antisense oligonucleotides to restore dystrophin expression in adult mdx mouse muscle.

The use of antisense oligonucleotides (AOs) to induce exon skipping leading to generation of an in-frame dystrophin protein product could be of benefit in around 70% of Duchenne muscular dystrophy patients. We describe the use of hyaluronidase enhanced electrotransfer to deliver uncomplexed 2'-O-methyl modified phosphorothioate AO to adult dystrophic mouse muscle, resulting in dystrophin expression in 20-30% of fibres in tibialis anterior muscle after a single injection. Although expression was transient, many of the corrected fibres initially showed levels of dystrophin expression well above the 20% of endogenous previously shown to be necessary for phenotypic correction of the dystrophic phenotype.

High-efficiency plasmid gene transfer into dystrophic muscle.

The efficiency of plasmid gene transfer in skeletal muscle is significantly enhanced by pretreatment with hyaluronidase and the application of an electrical field to the muscle following the injection of plasmid DNA, a process referred to as electrotransfer. However, the presence of increased levels of connective tissue in muscular dystrophies, such as Duchenne muscular dystrophy (DMD), may affect the efficiency of this process. Here we demonstrate that the efficiency of electrotransfer is not affected by increased levels of connective tissue in the mdx mouse model of DMD and that any damage induced by the electrotransfer process is not exacerbated in the dystrophic phenotype. However, increasing the concentration of hyaluronidase does not improve transfection efficiencies further. Unlike direct injection of plasmid DNA, the efficiency of electrotransfer is not dependent upon the sex and age of mice used. The combined treatment of hyaluronidase and electrotransfer results in highly efficient gene transfer in dystrophic muscle with limited muscle damage.

Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase -- increased expression with reduced muscle damage.

The efficiency of plasmid gene transfer to skeletal muscle can be significantly improved by the application of an electrical field to the muscle following injection of plasmid DNA. However, this electrotransfer is associated with significant muscle damage which may result in substantial loss of transfected muscle fibres. Reduction of the voltage used in the technique can result in a decrease in muscle damage, with a concomitant reduction in expression, but without a significant decrease in the number of transfected fibres. Pre-treatment of the muscle with a solution of bovine hyaluronidase greatly increases the efficiency of plasmid gene transfer when used in conjunction with electrotransfer, but not when used alone. This combination treatment results in greatly enhanced levels of transfected muscle fibres without the increases in muscle damage associated with the electrotransfer process.

Bexarotene metabolism in rat, dog, and human, synthesis of oxidative metabolites, and in vitro activity at retinoid receptors.

The metabolism of bexarotene, a rexinoid recently approved in the United States for treatment of cutaneous T-cell lymphoma, was studied using liver slices from untreated rats and dogs, liver microsomes from untreated and pretreated rats, and pooled human liver microsomes. Metabolite profiles were examined in bile and plasma from rats and dogs, and plasma from humans treated with bexarotene. Four metabolites, racemic 6-hydroxy-bexarotene, racemic 7-hydroxy-bexarotene, 6-oxo-bexarotene, and 7-oxo-bexarotene, were synthesized and their binding to, and transactivation of retinoid receptors were examined. Qualitatively similar metabolite profiles were observed in the microsomal and liver slice extracts; the predominant metabolites were 6-hydroxy-bexarotene and glucuronides of parent or hydroxylated metabolites. Pretreatment of rats with bexarotene induced hepatic microsomal bexarotene metabolism. The hydroxy and oxo metabolites were observed in plasma of rats, dogs, and humans treated with bexarotene and 6-hydroxy-bexarotene was a major circulating metabolite. The oxidative metabolites were more abundant relative to parent in plasma from humans than from rat or dog. The predominant biliary metabolites in rat and dog were bexarotene acyl glucuronide and a glucuronide of oxidized bexarotene, respectively. Since bexarotene elimination is primarily biliary in these species, these metabolites represent the main bexarotene metabolites in rats and dogs. The binding of synthetic metabolites to retinoid receptors was much reduced relative to parent compound. The metabolites exhibited minimal activity in transactivating retinoic acid receptors and had reduced activity at retinoid X receptors relative to bexarotene. Thus, while there is substantial systemic exposure to the oxidative metabolites of bexarotene, they are unlikely to elicit significant retinoid receptor activation following bexarotene administration.

Immune responses to dystropin: implications for gene therapy of Duchenne muscular dystrophy.

Introduction of dystrophin by gene transfer into the dystrophic muscles of Duchenne muscular dystrophy (DMD) patients has the possibility of triggering an immune response as many patients will not have been exposed to some (or all) of the epitopes of dystrophin. This could in turn lead to cytotoxic destruction of transfected muscle fibres. We assessed such concerns in the dystrophin-deficient mdx mouse using plasmid DNA as the gene transfer system. This avoids complications associated with the administration of viral proteins. Gene transfer of cDNAs encoding mouse full-length or a truncated minidystrophin did not evoke either a humoral or cytotoxic immune response. Mdx mice may be tolerant due to the presence of rare 'revertant' dystrophin-positive fibres in their skeletal muscles. In contrast, gene transfer of human full-length or minidystrophin provoked both humoral and cytotoxic responses leading to destruction of the transfected fibres. These experiments demonstrate the potential risk of deleterious effects following gene therapy in DMD patients and lead us to suggest that patients enrolled in gene therapy trials should ideally have small, preferably point, mutations and evidence of 'revertant' dystrophin-positive muscle fibres.

Periprosthetic breast capsules contain the fibrogenic cytokines TGF-beta1 and TGF-beta2, suggesting possible new treatment approaches.

Periprosthetic breast capsules composed of fibrotic collagenous material with increased collagen production are not dissimilar to other fibrotic conditions occurring in other organs. Fibrosis in the lung, liver, kidney, and skin has been associated with overproduction of the fibrogenic isoforms of transforming growth factor beta (TGF-beta1 and TGF-beta2). If periprosthetic breast capsules contained high levels of these cytokines, possibly new treatment approaches for capsular contraction could be proposed. Breast implant capsules of 35 patients harvested at the time of explantation were examined using indirect immunohistochemistry. Staining intensity for TGF-beta1 and TGF-beta2 was measured in all specimens. Immunohistochemical staining for TGF-beta1 and TGF-beta2 revealed that these two cytokines were present in all capsules analyzed. Minimal TGF-beta1 and TGF-beta2 were found in normal breast tissue. Levels of control vs. TGF-beta1 and control vs. TGF-beta2 were significant (p = 0.004 and p < 0.001 respectively). The presence of TGF-beta isoforms that are known to be fibrogenic may suggest new therapeutic approaches, which are being investigated for other fibrotic conditions.

Parotid selective lymphadenectomy in malignant melanoma.

Malignant melanoma of the head and neck can metastasize to lymph nodes within the parotid gland. Selective lymphadenectomy is the modern method of staging regional lymph node basins in clinically localized melanoma. This procedure involves intraoperative lymphatic mapping and directed, selective removal of the first draining nodes or sentinel lymph nodes (SLNs). Historically, the assessment of parotid lymph nodes would involve a superficial parotidectomy with facial nerve dissection. Since 1993, 28 patients with localized melanoma of the head and neck have demonstrated lymphatic drainage to parotid lymph nodes on preoperative lymphoscintigraphy. The overall success rate of parotid selective lymphadenectomy is 86% (24 of 28 patients). Of the 28 patients, there were 6 early patients in whom blue dye alone was utilized intraoperatively, and the success rate is 50% (3 of 6 patients). When blue dye and radiocolloid mapping techniques are combined, the parotid selective lymphadenectomy is successful in 95% of patients (21 of 22 patients). Four of the 24 patients (17%) had metastases to the SLNs and underwent therapeutic superficial parotidectomy and/or modified radical neck dissection. After completion of the therapeutic superficial parotidectomy, 1 of the 4 patients was found to have an additional parotid (nonsentinel) node with melanoma metastases. None of the patients incurred injury to the facial nerve by parotid selective lymphadenectomy. To date, 2 of 28 patients (7%) have had regional recurrence to the parotid gland. Failure of the SLN technique may occur when blue dye alone is used, when human serum albumin (not sulfur colloid) is the radiocolloid, when prior wide excision and skin graft is present before lymphatic mapping, and when all SLNs are not retrieved. We conclude that parotid selective lymphadenectomy is a safe and reliable alternative to superficial parotidectomy for staging clinically localized melanoma of the head and neck.

Lymphatic mapping and sentinel lymph node biopsy in patients with melanoma of the lower extremity.

Lymphatic mapping and sentinel lymph node biopsy is a new technique used in the surgical treatment of patients with malignant melanoma. The purpose of this study was to evaluate the results of this approach for patients with melanoma of the lower extremity. Between May of 1994 and June of 1997 at the H. Lee Moffitt Cancer Center and Research Institute, 85 consecutive patients with clinical stage I and II melanoma of the lower extremity underwent lymphatic mapping and sentinel lymph node biopsy. These nodes were identified in all 85 patients by intraoperative lymphatic mapping with both radiolymphoscintigraphy and a vital blue dye injection. Eleven patients (12.9 percent) had histologically positive sentinel lymph nodes, and 10 patients underwent inguinal complete lymph node dissections. All 10 patients had no further histologically positive lymph nodes confirmed by subsequent complete dissection. Among 74 patients with histologically negative sentinel lymph nodes, only 2 patients (2.7 percent) developed inguinal nodal metastases during a mean follow-up period of 21.8 months (range, 13.5 to 58.3 months). The sensitivity of lymphatic mapping and sentinel lymph node biopsy in this series was 100 percent and the specificity was 97.3 percent. Therefore, we conclude that the use of lymphatic mapping and sentinel lymph node biopsy can accurately stage patients with melanoma of the lower extremity and provide a rational surgical approach for these patients.

Evaluation of plasmid DNA for in vivo gene therapy: factors affecting the number of transfected fibers.

Gene transfer by intramuscular injection of plasmid DNA has potential application in gene therapy. We examined factors affecting the number of expressing fibers, in contrast to total expression, following injection of plasmid DNA. Barium chloride proved effective in inducing muscle necrosis and regeneration in mice, and this increased the number of fibers expressing a reporter gene. Coinjection of ion-channel modulators did not increase the number of positive fibers, but increasing dose and repeated administration of plasmid did. Importantly, the plasmid size (7-16 kb) did not affect the number of fibers expressing the transgene, in both normal and regenerating muscle.

Inflammatory responses following direct injection of plasmid DNA into skeletal muscle.

Transfer of genes by injection of plasmid DNA into skeletal muscle has a wide variety of applications ranging from treatment of neuromuscular disorders to genetic vaccination. We examined each component involved in the intramuscular injection of plasmid DNA in terms of the induction of inflammatory responses. The insertion of a needle and the injection of a relatively large volume of saline caused very little muscle damage except in rare cases. In contrast, barium chloride-induced regeneration of muscle, injection of lipopolysaccharide, plasmid backbone or plasmid expressing a neo-antigen (beta-galactosidase) all generated widespread inflammation of injected muscle, with mononuclear infiltrate, comprised largely of macrophages and with both CD4+ and CD8+ T lymphocytes, present. Such inflammation may hamper clinical application of this technology and may encourage undesirable immune responses in gene therapy trials. Inflammation was not greatly reduced by CD4- or CD8-depleting antibodies, suggesting this initial inflammation did not involve T cells, but methylation of plasmid DNA before injection substantially lessened the inflammatory response and resulted in longer term expression of the transgene.

Body image and psychological sequelae of silicone breast explantation: preliminary findings.

Twenty-two breast explantation (implant removal) and 20 cholecystectomy patients were assessed preoperatively and postoperatively and compared with 20 nonsurgical control subjects on several body-image measures, depression, self-esteem, and self-reported health status. Explantation patients had higher breast anxiety and upper torso dissatisfaction than either control group and levels were unaffected by implant removal. The discrepancy between self-rated ideal and current breast size increased substantially after implant removal for the explantation group, but did not change for controls. Overall appearance satisfaction level and positive appearance-related cognitions decreased as a function of surgery for explantation patients, but remained unchanged in cholecystectomy and nonsurgical controls. Depression levels were elevated in explantation patients and did not change as a function of surgery; self-reported health status level improved for the explantation group, but levels still remained below those of both control groups after explantation. Therapeutic indications for the elevated depression levels and unique body-image issues that patients undergoing explantation experience are discussed.

Sentinel lymph node biopsy in melanoma of the head and neck.

BACKGROUND: The sentinel lymph node is the first node or nodes to drain a cutaneous melanoma. Sentinel lymph node biopsy is performed to determine whether regional metastases are present. The authors' experience with the new technique of sentinel lymph node biopsy for melanoma of the head and neck is reported. PATIENTS AND METHODS: During the period of January of 1992 to December of 1995, 58 consecutive patients were identified from the melanoma database who had localization of the sentinel lymph node for primary cutaneous melanoma of the head and neck. Techniques for identification of the sentinel node(s) include preoperative lymphoscintigraphy and intraoperative Lymphazurin dye (vital blue dye) and technetium-99m-labeled sulfur colloid injection around the primary tumor site with Neoprobe mapping. RESULTS: Fifty-eight patients (13 female, 45 male), mean age 61 years, with melanoma of the head and neck with a mean Breslow thickness of 2.21 mm. (range, 0.82-6.87 mm.) and no regional lymphadenopathy underwent sentinel node mapping. The sentinel node was successfully identified in 55 patients (95 percent). Blue dye was visualized in 85 of 126 sentinel nodes excised (67 percent), whereas the remainder of the sentinel nodes were localized with the Neoprobe. Forty-nine patients who had successful mapping and sentinel node biopsy had no evidence of metastatic disease in the sentinel node or other nodes in the basin. Six of the fifty-five patients (11 percent) had evidence of micrometastatic disease, and all six had the sentinel node as the only site of metastasis. Five of six patients with micrometastases had a subsequent neck dissection and/or superficial parotidectomy. None of these patients had evidence of "skip metastases" with a negative sentinel node and higher level nodes positive for metastases. In total, 6 of the 18 sentinel nodes (33 percent) identified in these six patients contained micrometastatic disease, whereas none of the 139 other nodes sampled had any evidence of metastases. The exact probability that all six unpaired observations would consist of involvement of only the sentinel nodes is p = 0.0312. CONCLUSIONS: By combining the two mapping techniques in patients with melanoma of the head and neck, the sentinel node(s) can be mapped and identified individually, similar to melanoma in other locations. The sentinel nodes have been shown to contain the first evidence of regional metastatic melanoma. This staging information can be used to plan therapeutic node dissections and adjuvant therapy that may have a survival benefit in patients with stage III melanoma of the head and neck. Lymphatic mapping can be used to make the surgical care of the melanoma patient more conservative, so that only those patients with solid evidence of regional node metastases are subjected to the morbidity and expense of a complete node dissection and the toxicities of adjuvant therapy.

Outcome study of the psychological changes after silicone breast implant removal.

INTRODUCTION: The purpose of this longitudinal study is to determine if there are changes in psychological well-being after breast implant removal. METHODS: Thirty-seven women underwent breast implant removal and completed a preoperative baseline, early postoperative (4 to 9 months), and late postoperative (> 10 months) Brief Symptom Inventory, a measure of psychological distress. RESULTS: After breast implant removal the mean Global Severity Scores on the Brief Symptom Inventory of women undergoing implant removal increased over the three time periods, indicating increasing psychological distress. When the group was divided into those who had breast reconstruction after implant removal (implant replacement with saline-filled implants or TRAM flaps) and those who did not have reconstruction, both groups had increasing psychological distress, but the women who had reconstruction had slightly higher scores. Women with a history of psychiatric treatment showed the greatest increase in psychological symptoms. CONCLUSION: Breast implant removal did not have psychological benefits in this group of women. Breast reconstruction after removal did not appear to be psychologically beneficial to this cohort.

Immune responses, not promoter inactivation, are responsible for decreased long-term expression following plasmid gene transfer into skeletal muscle.

Long-term high-level in vivo gene expression appears to depend on the promoter chosen to drive the gene of choice. In many cases the promoter appears to 'switch off' some time after in vivo gene transfer. We demonstrate that, following intramuscular injection of beta-galactosidase reporter plasmids, promoter 'switch off' is due to elimination of fibres expressing the transferred reporter gene by activation of a Th1 (cytotoxic) immune response. This finding, in the absence of stimulation of the immune system by viral vector proteins, has implications not only for gene transfer experiments but for the future of muscle-directed gene therapy.

Comparison of psychological symptoms of women requesting removal of breast implants with those of breast cancer patients and healthy controls.

Concern about the safety of silicone breast implants has led many women with numerous physical and psychological symptoms to seek breast implant removal. This retrospective group comparison study describes the psychological profile of women requesting breast implant removal compared with two control groups. The Brief Symptom Inventory was used to compare psychological symptoms of three groups of women: a preoperative breast implant group requesting removal of implants (n = 78), a postoperative breast cancer group without breast implants (n = 64), and a control group with no known breast disease and unknown breast implant status (n = 68). Scores were compared on the Global Severity Index of the Brief Symptom Inventory as well as on nine subscales: somatization, obsessive-compulsiveness, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, and psychoticism. The breast implant group had significantly elevated Global Severity Index scores, as well as somatization, obsessive-compulsiveness, depression, hostility, and anxiety subscale scores, when compared with the other groups. Post hoc data analysis revealed that women who had implants after subcutaneous mastectomy as prophylaxis for breast cancer (n = 18) had a significantly different symptom profile and higher Global Severity Index scores than women who had cosmetic augmentation (n = 53). Additionally, women who had subcutaneous mastectomy and implants had significantly higher subscales of interpersonal sensitivity, phobic anxiety, paranoid ideation, and psychoticism than the cosmetic implant subjects. Women requesting removal of silicone breast implants had greater psychological distress than women who were recently diagnosed with breast cancer or controls with no known breast disease and unknown implant status. Within the implant group, however, women who had subcutaneous mastectomy showed greater psychological disturbance than those who had augmentation mammaplasty.

The clinical relevance of sentinel lymph nodes identified with radiolymphoscintigraphy.

BACKGROUND: The purpose of this case report is to illustrate the utility of radio-guided mapping of sentinel lymph nodes (SLN's) as demonstrated by the technique's successful identification of nodes containing metastatic disease that would have been left behind if only the visual-oriented vital blue dye mapping technique had been used. METHOD: The patient underwent preoperative lymphoscintigraphy and intra-operative lymphatic mapping using vital blue dye and radiolymphoscintigraphy using the Neoprobe (handheld gamma probe). Nodes which were blue and/or "hot" (i.e., radioactive counts were three times the background count) were considered SLN's. RESULTS: Four SLN's were harvested, all of which were "hot" but only one of which was both "hot" and blue. Pathology revealed that the two SLN's positive for metastatic disease were not blue. CONCLUSION: While the blue dye lymphatic mapping technique provides the surgeon with a visual road map in the identification of SLN's, the Neoprobe increases the success rate of localization when compared to vital blue dye mapping due to the reliable migration of radiocolloid to the SLN's in the regional basin. Radiolymphoscintigraphy also increases the accuracy and efficiency of the SLN harvest by providing a directed dissection to the level of the nodes in the basin. The Neoprobe increases the yield of SLN's, some of which are clinically relevant since they contain metastatic disease.

Transmembrane calcium flux regulates LDL oxidation by arterial smooth muscle cells.

Although clinical and experimental studies have supported a role of calcium in atherogenesis, the nature of this relationship has not been determined. The following investigation was performed to examine the effect of transmembrane calcium flux on the oxidative modification of low-density lipoprotein (LDL) by arterial smooth muscle cells (SMC). Confluent SMC were incubated in basal medium alone or in medium containing either nifedipine (0.25 microM), a calcium channel blocker, or dantrolene (10 microM), a blocker of calcium release from the endoplasmic reticulum. Cells were then suspended in medium with either physiologic (1.25 mM) or supraphysiologic (2.5 mM) calcium concentrations ([Ca2+]e) and were exposed to oxidized LDL (20 micrograms/protein/ml; 4.96 +/- 0.76 nmole malondialdehyde/mg LDL protein). Changes in cytosolic calcium ([Ca2+]i) were measured by spectrofluorometric analysis using a Fura 2-AM indicator. In similar studies, the cellular oxidation of native LDL was determined by fluorometric measurement of thiobarbituric acid-reactive substances in the media. Nifedipine and, to a lesser extent, dantrolene lowered steady-state [Ca2+]i at supraphysiologic [Ca2+]e (2.5 mM; P < 0.0002). Exposure of SMC to Ox-LDL increased [Ca2+]i (P < 10(7), which was further augmented by increasing [Ca2+]e (P < 10(-7). Nifedipine significantly reduced the calcium response to Ox-LDL proportionate to [Ca2+]e (P < 0.0002). A similar reduction in [Ca2+]i vs control was seen with dantrolene, but was independent of [Ca2+]e. TBARS assays revealed a significantly greater degree of cellular oxidation of native LDL following preincubation of SMC with Ox-LDL (P < 10(8). This effect was markedly inhibited by both nifedipine (P < 10(-8) and dantrolene (P < 10(-8), which showed some degree of synergism. These results indicate that the increase in cytosolic calcium in SMC exposed to Ox-LDL may occur through both membrane channels and reticular release. Inhibition of transmembrane calcium flux severely limits the cellular oxidation of low-density lipoprotein. These alternate means of calcium signal generation may allow a more varied response of the SMC to atherogenic stimuli and provide an opportunity for specific therapeutic intervention.