Zinc Finger Nucleases: Tailor-made for Gene Therapy.

Genome editing with the use of zinc finger nucleases has been successfully applied to variety of a eukaryotic cells. Furthermore, the proof of concept for this approach has been extended to diverse animal models from Drosophila to mice. Engineered zinc finger nucleases are able to target specifically and manipulate disease-causing genes through site-specific double strand DNA breaks followed by non-homologous end joining or homologous recombination mechanisms. Consequently, this technology has considerable flexibility that can result in either a gain or loss of function of the targeted gene. In addition to this flexibility, gene therapy by zinc finger nucleases may enable persistent long term gene modification without continuous transfection- a potential advantage over RNA interference or direct gene inhibitors. With systemic viral delivery systems, this gene-editing approach corrected the mutant factor IX in models of mouse hemophilia. Moreover, phase I clinical trials have been initiated with zinc finger nucleases in patients with glioblastoma and HIV. Thus, this emerging field has significant promise as a therapeutic strategy for human genetic diseases, infectious diseases and oncology. In this article, we will review recent advances and potential risks in zinc finger nuclease gene therapy.

Peptide-based Antifungal Therapies against Emerging Infections.

Acquired drug resistance to mycotic infections is rapidly emerging as a major medical problem. Opportunistic fungal infections create therapeutic challenges, particularly in high risk immunocompromised patients with AIDS, cancer, and those undergoing transplantation. Higher mortality and/or morbidity rates due to invasive mycosis have been increasing over the last 20 years, and in light of growing resistance to commonly used antibiotics, novel antifungal drugs and approaches are required. Currently there is considerable interest in antifungal peptides that are ubiquitous in plant and animal kingdoms. These small cationic peptides may have specific targets or may be multifunctional in their mechanism of action. On the basis of recent advances in protein engineering and solid phase syntheses, the utility and potential of selected peptides as efficient antifungal drugs with acceptable toxicity profiles are being realized. This review will discuss recent advances in peptide therapy for opportunistic fungal infections.

Improvement in lip appearance (lateral and frontal aspects) following mandibular setback surgery.

INTRODUCTION: The post-operative facial profile is critical for patients who undergo orthognathic surgery. The present study investigated the improvement in lip appearance (lateral and frontal aspects) following mandibular setback surgery. MATERIAL AND METHODS: Thirty-one patients with mandibular prognathism underwent mandibular setback surgery. Lateral and posteroanterior cephalograms were obtained before surgery (T0) and more than 1 year after surgery (T1). The landmarks (soft and hard tissues) and linear distances were compared by statistical analysis. RESULTS: The lateral cheilion (Ch), point B (B), and pogonion (Pog) were significantly setbackin the horizontal plane: 5.59, 11.49, and 12.35 mm, respectively. In the vertical plane, B and Pog did not move significantly. The Ch moved significantly downward by 3.23 mm on average. The setback ratios of soft tissue/hard tissue, soft tissue of B/B, and soft tissue of Pog/Pog were 0.96. The Ch/Pog ratio was 0.45. The width of the frontal Ch was significantly reduced by 3.17 mm. CONCLUSIONS: The relationship between the corresponding soft and hard tissues of the chin was approximately 1. The relationship between the lip corner and chin bone was nearly 50%. The width of the lip corner was also significantly reduced.

Appearance on face reading (cheek line) after orthognathic surgery.

The cheek line (face reading) is an aesthetic element of the facial profile. The purpose of our study was to investigate the changes in the cheek line after mandibular setback surgery. Forty patients (20 female and 20 male, mean (SD) age 22 (5) years) were diagnosed with mandibular prognathism and treated by intraoral vertical ramus osteotomy alone. Cephalograms were obtained before operation (T1), at least a year postoperatively (T2), and final surgical changes over a year (T2-T1). The cheek line and landmarks (soft and hard tissues) were compared using the paired t test. The hypothesis was that the cheek line did not change significantly after mandibular setback. At the time of the final follow-up (T2-T1), the mean (SD) horizontal setback of pogonion (Pog) was 12.3 (3.5) mm for women and 11.7 (4.3) mm for men. The ratios of soft:hard tissue, labrale inferius:incisor inferius, labiomental sulcus:point B, soft tissue Pog:Pog, and cheek point:Pog in women were 0.96, 0.98, 0.98, and 0.08, and in men 0.91, 1.01, 0.94, and 0.13, respectively. The nasolabial and cervicomental angles in women were significantly increased by 11.1° and 11.4°, respectively, and in men the nasolabial angle was significantly increased by 11.1° and the mentolabial angle reduced by 9.9°. The cheek line (T2-T1) was moved significantly forwards. The hypothesis was therefore rejected. In conclusion, the cheek line was advanced significantly after isolated mandibular setback.

Diabetes-associated mesenteric vascular hypertrophy is attenuated by angiotensin-converting enzyme inhibition.

In experimental diabetes, the mesenteric vascular tree undergoes hypertrophy, and this is associated with an increase in mesenteric angiotensin-converting enzyme (ACE) levels. The aim of this study was to determine if inhibition of mesenteric ACE by ACE inhibition would influence diabetes-associated mesenteric vascular hypertrophy. Control or streptozocin-induced diabetic rats were randomized to receive no drug or the ACE inhibitor perindopril. In addition, other diabetic rats were randomized to receive either low-dose insulin that does not alter glycemic control or high-dose insulin, administered as a silastic pellet to achieve euglycemia. After 3 weeks, animals were killed for measurement of mesenteric ACE, vessel weight, and wall:lumen ratio. Diabetes was associated with increased mesenteric ACE levels, increased vessel weight, and an increase in the wall:lumen ratio. ACE inhibition, despite no effect on glycemic control, food intake, urinary urea excretion, or gut weight, prevented the increase in mesenteric ACE levels and attenuated mesenteric vascular hypertrophy as assessed by weight or wall:lumen ratio. The increase in staining by an antibody to the endothelial product, von Willebrand factor, in diabetic rats was totally prevented by perindopril treatment. Euglycemia but not low-dose insulin therapy in the diabetic rats normalized mesenteric vessel ACE, weight, and wall:lumen ratio. In conclusion, ACE inhibition may have a specific role in preventing diabetes-associated vascular hypertrophy, an important process in the genesis of micro- and macrovascular diabetic complications.

In situ hybridization to show sequential expression of osteoblast gene markers during bone formation in vivo.

We investigated the sequence of expression of osteoblast gene markers during bone formation in vivo by in situ hybridization. Cylindrical lesions were induced in the femora of sheep with titanium analytic bone implants that allow removal of serial core samples to study bone formation. At 2 weeks (2W), granulation tissue made up of spindle-shaped cells had partially replaced the blood clot. Islands of osseous tissue, first noted in the periphery of the ingrowing tissue at 3W, became the predominant tissue by 6W. The surfaces of newly forming bone at 3W were apposed by cuboidal cells, which in some areas were several layers thick. By 6W, most of the cells lining bone trabeculae had assumed a flattened morphology. The temporal and spatial distribution of osteoblast gene markers was examined by in situ hybridization with nonradioactive digoxigenin probes for alpha 1(I) procollagen, alkaline phosphatase (ALP), osteopontin (OP), and bone Gla protein (BGP). The spindle-shaped cells in the granulation tissue expressed mRNA for alpha 1(I) procollagen, ALP, and OP but not BGP, suggesting that they may be osteoblast precursor cells. alpha 1(I) procollagen mRNA was strongly expressed by all cells on the surface of bone, with a peak intensity at 3W and then reducing sharply by 6W. Initially, only pockets of cuboidal cells on bone surfaces expressed ALP mRNA, with a peak intensity at 5W. Similarly, only a proportion of cuboidal cells expressed OP mRNA early in bone formation, but the number of cells expressing OP mRNA increased with time. Clumps of cuboidal cells expressed BGP mRNA only when bone was present, and the degree of expression increased with the amount of bone formed. This model allows the study of temporal and spatial sequence of gene expression in cells participating in osteogenesis. The temporal sequence is similar to that shown in vitro in other models of mineralization. The geographic localization of cells expressing mRNA for alpha 1(I) procollagen, ALP, OP, and BGP implies subspecialization of osteoblasts in bone formation.

Parathyroid hormone-related protein mRNA and protein expression in multiple myeloma: a case report.

Multiple myeloma frequently leads to complications, such as osteolytic lesions, hypercalcemia, and pathological fractures. Increased bone resorption in myeloma is due to osteoclast activation. The nature of the osteoclast activator(s) remains unclear. We describe a case of multiple myeloma with marked hypercalcemia and skeletal complications that progressed rapidly despite chemotherapy. The patient had marked hypercalcemia at diagnosis (4.5 mmol/l), and elevated parathyroid hormone-related protein (PTHrP) levels were found in plasma. Analysis of the bone marrow trephine biopsy showed PTHrP gene transcription and protein in myeloma cells. These results provide strong evidence for the production of significant amounts of PTHrP by human myeloma cells. PTHrP has been measured as elevated in the plasma of patients with myeloma and might be an important contributor to the skeletal complications in this disease.

Parathyroid hormone-related protein: immunohistochemical localization in cancers and in normal skin.

An immunoperoxidase method has been developed to detect parathyroid hormone-related protein (PTHrP) in histological specimens of tumors and of normal skin. A rabbit polyclonal antiserum against PTHrP-(1-16) was used that did not cross-react with PTH-(1-34) either under radioimmunoassay conditions or at the high antiserum concentrations used in neutralizing biologic activity. PTHrP antigen was detected in the keratinocyte layer of normal skin and in 100% of 34 samples of squamous cell cancers but in only one of six breast cancers, and none of 15 other adenocarcinomata. It was also detected in four of four samples of renal cortical carcinoma and two of two of melanoma, both of which can be associated with hypercalcemia, and three of three small cell carcinomata of the lung. Immunologic detection of PTHrP could be useful in the diagnosis of tumors of squamous cell origin, particularly in the cytological differentiation of lung cancers, where it may be of value in distinguishing between squamous cell and small cell carcinoma on the one hand and poorly differentiated adenocarcinoma on the other.

The roles of reactive oxygen species and endogenous opioid peptides in ischemia-induced arrhythmia of isolated rat hearts.

Although the formation of oxygen-derived free radicals (or reactive oxygen species; ROS) and the release of endogenous opioid peptides (EOP) have been independently reported to be the major arrhythmogenic factors in ischemic hearts, possible relations between these two factors have seldom been investigated. Thus, we studied whether the ROS and EOP were related in the progression of ischemia-induced arrhythmias. Isolated rat hearts perfused in the Langendorff mode were treated with dynorphin A1-13 (kappa EOP receptor agonist), and/or allopurinol (xanthine oxidase inhibitor), before the onset of ischemia induced by ligating the left coronary arteries. Ischemic period lasted for 30 min, during which cardiac rhythms were recorded. At the end of ischemia, hearts were analyzed for the glutathione and ascorbate levels. Allopurinol (100 nmoles/heart) was effective in reducing the severity of arrhythmia (arrhythmia score: Mean +/- SEM 3.00 +/- 0.80 for allopurinol, 5.75 +/- 0.41 for placebo, p < 0.01), while dynorphin (10 micrograms/heart) potentiated the arrhythmia (6.71 +/- 0.52, p < 0.05 vs. placebo). Coadministration of allopurinol and dynorphin was capable of reducing arrhythmia (5.57 +/- 0.65) when compared with the administration of dynorphin alone (6.71 +/- 0.52, p < 0.05). Tissue oxidative stress was evaluated by the concentrations of glutathione (GSH) and ascorbate. Allopurinol did not significantly elevate tissue GSH concentrations (1.46 +/- 0.05 mumoles/g wet wt) in ischemic hearts, while dynorphin alone significantly decreased the GSH concentrations (0.96 +/- 0.08, p < 0.05) when compared with the placebo (1.32 +/- 0.03). The dynorphin-induced GSH decrease cannot be reversed by coadministration with allopurinol (0.90 +/- 0.104). Allopurinol significantly elevated tissue ascorbate levels (0.16 +/- 0.01) when compared with placebo (0.10 +/- 0.01, p < 0.05). Interestingly, dynorphin alone also elevated the tissue ascorbate concentrations (0.16 +/- 0.02). Coadministration of allopurinol and dynorphin further spiked the ascorbate levels (0.28 +/- 0.05, p < 0.01). In conclusion, the results suggested that ischemia-induced arrhythmia mechanisms might involve the formation of superoxide and other ROS, which were probably generated from the release of EOP (or EOP/EOP receptor interactions). Superoxide, the formation of which can be inhibited by allopurinol that exerted antiarrhythmic effect, was probably scavenged by ascorbate in myocardial ischemia. The ROS resulting from EOP/EOP receptor interactions were probably scavenged by glutathione system. Elevated ascorbate levels in dynorphin-treated hearts might result from the compensatory synthesis induced by decreased glutathione levels.

Transforming growth factor beta 1 stimulates bone formation and resorption in an in-vivo model in rabbits.

The influence of TGF beta 1 on bone was studied in a titanium device implanted into the tibia of rabbits. TGF beta 1 was infused via an Alzet osmotic pump calibrated to deliver at a rated of 200ng daily for 2 weeks before replacement. A hollow channel is incorporated into the device into which tissue can grow, and the histological sequence of events was observed over 6 weeks. In control samples, the rod-shaped piece of tissue at 2W consisted of spindle-shaped cells in the center, flanked at both ends by islands of trabecular bone lined by cuboidal osteoblasts and osteoclasts. By 4W, ingrowth of bone reached the center if the specimen, by which time, the bone surfaces were apposed by a single layer of flattened osteoblasts. However, osteoclastic resorption continued unabated so that by 6W, only a thin layer of cortical bone remained, enclosing a marrow cavity with hemopoietic elements. Significant differences were observed in samples continuously infused with TGF beta 1. At 2W, trabecular bone had reached further towards the center of the specimen and the granulation tissue was made up of cells that were more plump and cuboidal compared to the spindle cells of control sample. At 3W, there was increased bone volume and osteoid seams were thicker, covering a greater extent of the trabeculae surfaces. At 4W, the bony trabeculae were up to 3 times thicker than control trabeculae. There was very active bone resorption with many multinucleate osteoclasts and multilayered aggregates of cuboidal osteoblasts lining bony surfaces. Yet at 6W, the morphological appearance was similar to control samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Marrow development and its relationship to bone formation in vivo: a histological study using an implantable titanium device in rabbits.

During embryogenesis, the creation of marrow sinusoids is intimately related to the coupled processes of osteogenesis and osteoclastic resorption. We set out to further define the relationship between bone formation and marrow development by implanting an intraosseous titanium device into the tibiae of rabbits which permits the examination of bone formation under standardized and reproducible conditions as well as allowing repeated sampling of new bone. A hollow channel is incorporated into the device into which tissue can grow. The device was left in place for 6 weeks to allow osseous integration to occur, after which the initial rod of new tissue growth was removed and subsequent histological and immunohistological sequence of events observed over the next 7 weeks. Interpretation of its morphological changes was further aided by concurrent histomorphometric studies. Because the channel was in direct continuity with the marrow cavity and isolated from the endosteum, immediate marrow regeneration was expected, following dissolution of the blood clot. Instead, our studies indicated that hemopoietic marrow cells, including the erythroid and myeloid series as well as megakaryocytes, did not appear until 3 weeks after implantation of the chamber when the newly formed bone had been remodeled to form an expanded marrow cavity. This intraosseous device is a useful in vivo model for studying the development of bone marrow hemopoietic and nonhemopoietic stromal cells and our results confirm the previous observation that influx of marrow cellular elements follow the formation of bone during endochondral as well as intramembranous ossification.

Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model.

Expression of parathyroid hormone-related protein (PTHrP) messenger RNA (mRNA) and protein was investigated throughout the developmental progression of endochondral bone formation in mouse and intramembranous bone formation in an in vivo model in rabbit, using in situ hybridization and immunohistochemistry. Endochondral bone formation was investigated in a developing embryo, newborn, and adult mouse. In fetal long bones through to newborn (day 7), PTHrP mRNA and protein were consistently expressed in chondrocytes within the proliferative, transitional, and hypertrophic zones. In addition, high levels of PTHrP were also detected in osteoblasts on the surface of trabecular bone surfaces. Similarly, at the adult stage (week 7), PTHrP mRNA and protein were consistently expressed in chondrocytes at epiphyseal ends of the subarticular cartilage, within cortical periosteum, as well as in osteoblasts located at the metaphyseal trabecular bone surfaces. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in preosteoblasts prior to trabecular bone formation (1-week bone harvest). As bone formed (2-, 3-, and 4-week bone tissue harvests), PTHrP mRNA and protein were highly expressed in actively synthesizing osteoblasts and in those osteocytes embedded within the superficial layers of the bone matrix. Lining osteoblasts and osteocytes buried deeply in the bone matrix displayed weak or no signal for PTHrP. The pattern of spatial and temporal expression of PTHrP demonstrated in cartilage cells and osteoblasts in the two systems suggests an important role of PTHrP in both endochondral and intramembranous bone formation.

In situ hybridization of parathyroid hormone-related protein in normal skin, skin tumors, and gynecological cancers using digoxigenin-labeled probes and antibody enhancement.

We describe a novel procedure for in situ hybridization that combines the use of digoxigenin-labeled oligonucleotide probes with an antibody enhancement step that can be performed on formalin-fixed, paraffin-embedded tissues. Addition of a second antibody enhances the visibility of parathyroid hormone-related protein (PTHrP) mRNA expression from barely to highly discernible and interpretable, with virtually no nonspecific background expression. This technique has allowed visualization of PTHrP mRNA in normal human skin and epithelium-derived tumors. PTHrP mRNA expression was confined to the basal and spinous keratinocyte layers of skin. There was strong hybridization in the spinous keratinocyte layer and a low level of hybridization in the basal layer. An extensive panel of positive and negative controls included poly d(T) probe to indicate total mRNA present in the sections. Squamous cell carcinomas and basal cell carcinomas of the skin, from pathology archives, were examined for the presence of PTHrP mRNA. The results reflected previous immunohistochemical studies, with every squamous cell carcinoma hybridizing strongly with the PTHrP probes. The basal cell carcinomas showed no expression of PTHrP mRNA, although the total mRNA signal was very strong. The localization of PTHrP mRNA in the tumors of the gynecological tract also reflected the immunohistochemical findings, with expression found in the squamous cell carcinomas but not in the adenocarcinomas. In situ hybridization with digoxigenin-labeled oligonucleotide probes and antibody enhancement has provided a sensitive, highly specific procedure for detection of PTHrP mRNA in tumors and normal tissue.

Glutathione concentration in oral cancer tissues.

Glutathione (GSH) concentrations in human epidermoid carcinoma tissues were measured by high performance liquid chromatography. The mean glutathione content of 26 epidermoid carcinoma intratumor tissue specimens was 24.36 nmol/mg protein, which was significantly higher than that in adjacent non-tumor tissue parts (3.04 nmol/mg protein). The mean concentration found in normal oral mucosa was 4.80 nmol/mg protein. Tissue GSH levels were not correlated with the age of the patients or tumor size. Additionally, cellular GSH levels in nine different cell lines were found to spread over a wide range from 0.97 to 50.97 nmol/mg protein. Elevated GSH levels in cancer tissues were probably due to their abnormal proliferative activities. These results indicate that the glutathione level of oral tissues may be a useful marker for oral cancer, which is in agreement with findings from lung squamous cell carcinoma, cervical squamous cell carcinoma and other squamous cell carcinomas.

Lectin-binding affinities of human epidermal tumors and related conditions.

Paraffin-embedded sections from a variety of epidermal lesions were stained with fluorescein isothiocyanate-labeled concanavalin A and examined by a fluorescence microscope. Seventy-six normal, hyperplastic, and neoplastic tissues were examined. Lectin binding was demonstrated in all malignant tumors, the fluorescence being confined to the plasma membrane of the tumor cells. Normal and hyperplastic tissues either failed to stain or showed a grossly diminished level of fluorescence. The distinction between malignant and normal of hyperplastic cells was clear-cut and definite.

Impact of complementary parathyroid scintigraphy and ultrasonography on the surgical management of hyperparathyroidism.

BACKGROUND: An ongoing debate exists regarding the relative merits of full versus limited neck exploration in the surgical management of parathyroid adenomata. The aim of this study was to assess the impact of localization studies on the subsequent surgical management of hyperparathyroidism. METHODS: The accuracy of complementary ultrasonography and 201TI/99mTc parathyroid subtraction scintigraphy in hyperparathyroidism was evaluated retrospectively during a 10-year period in patients referred for localization studies. Surgical and pathologic confirmation of the diagnosis was possible in 121 patients, and these data formed the basis of this study. Operative procedure, times, outcome, and complications were recorded. RESULTS: The sensitivity, specificity, and accuracy for combined scintigraphy and ultrasonography were 86%, 98%, and 96%, respectively. Limited neck exploration was performed in 61 of 121 patients, and 60 patients underwent full neck exploration. In primary hyperparathyroidism 59 of 105 patients underwent limited and 46 underwent full neck exploration with average operative times of 70 and 109 minutes, respectively. (p < 0.0001). Complications developed in five patients who underwent full neck exploration. CONCLUSIONS: Confident localization of parathyroid adenomata facilitated successful limited neck exploration in most of the patients, questioning the need for full neck exploration in all patients with primary hyperparathyroidism.

p53 protein immunoreactivity in fibrohistiocytic tumors of the skin.

Abnormal expression of the 53 kDa nuclear phosphoprotein produced by the p53 gene is observed in many human cancers. p53 nuclear immunoreactivity is found commonly in tumor cells. Immunohistochemistry was performed using a monoclonal antibody, DO-7 (DAKO, Denmark; cat. no. M7001; 1:100 dilution), to investigate p53 protein immunoreactivity in a group of cutaneous fibrohistiocytic tumors that are known to be locally aggressive. The study group consisted of dermatofibrosarcoma protuberans (DFSP) (n = 14) and atypical fibroxanthoma (AFX) (n = 7). Cases of dermatofibroma (DF) (n = 16) formed the benign control group. Intense nuclear immunostaining for p53 protein was observed in 71% of DFSP and 86% of AFX. None of the dermatofibromas showed strong p53 nuclear immunostaining. Statistical analyses revealed significant differences in p53 immunoreactivity between DFSP and DF (P = 0.0001, chi 2 test) and between AFX and DF (P = 0.0001, chi 2 test). In conclusion, increased p53 protein immunoreactivity is found in DFSP and AFX but not in DF. These differences in p53 immunoreactivity suggest that increased expression of the protein may be important in the pathogenesis of the more aggressive group of fibrohistiocytic tumors.

Mucin-producing cholangiocarcinoma: an autopsy study in Hong Kong.

Fifty autopsy cases (35 male and 15 female) of mucin-secreting cholangiocarcinoma in Chinese were reviewed. The peak incidence was in the 7th decade for males and in the 6th for females. Massive (37), multinodular (8), diffuse (1) and hilar (4) types were recognized grossly. The hilar tumours arose from the main intrahepatic ducts and the other types originated from smaller ducts. The overall association with stones was 20% and clonorchiasis 92%. Cirrhosis occurred in only 4% of cases. There was an association between the degree of mucin secretion and the presence and severity of clonorchiasis.

The longevity of Clonorchis sinensis.

The persistence of Clonorchis sinensis infestation of the bile duct for a period of at least 26 years without neoplasia supervening is reported in a Chinese immigrant to Australia.

Recurrent pyogenic cholangitis: a necropsy study.

Forty-six necropsy cases of recurrent pyogenic cholangitis (RPC) among Chinese in Hong Kong were reviewed. Intrahepatic biliary pigment stones or bile sludge were present in all cases and liver abscesses were common (80%). Histologically the early lesion was marked by acute inflammation in portal tracts with frequent pylethrombophlebitis and the advanced chronic lesion by periductal fibrosis. Most patients died from chronic persistent hepatic suppuration or disseminated infection. The pathogenesis is not fully understood; the present study favours the view that a protein-deficient diet initiates intrahepatic stone or sludge formation with biliary obstruction; subsequent bacterial infection probably originates from the portal system.