Development of the hybrid Sleeping Beauty: baculovirus vector for sustained gene expression and cancer therapy.

Antiangiogenesis is an appealing anticancer approach but requires continued presence of the antiangiogenic agents, which can be remedied by gene therapy. Baculovirus is an emerging gene delivery vector but only mediates transient expression (<7 days); thus, this study primarily aimed to develop a hybrid baculovirus for sustained antiangiogenic gene expression and cancer therapy. We first constructed plasmids featuring adeno-associated virus inverted terminal repeats (AAV ITRs), oriP/Epstein-Barr virus-expressed nuclear antigen 1 (EBNA1) or Sleeping Beauty (SB) transposon and compared their efficacies in terms of persistent expression. In human embryonic kidney (HEK293) cells, AAV ITR failed to prolong the expression while oriP/EBNA1 moderately extended the expression to 35 days. In contrast, the SB system led to stable expression beyond 77 days even without antibiotic selection. Given this finding, we constructed a hybrid SB baculovirus expressing the SB transposase and harboring the transgene cassette flanked by inverted repeat/direct-repeat (IR/DR) elements recognizable by SB. The hybrid SB baculovirus efficiently transduced mammalian cells and mediated an expression duration longer than that by conventional baculoviruses, thanks to the transgene persistence and integration. The SB baculovirus (Bac-SB-T2hEA/w) expressing the antiangiogenic fusion protein comprising endostatin and angiostatin (hEA) also enabled prolonged hEA expression. With sustained hEA expression, Bac-SB-T2hEA/w repressed the angiogenesis in vivo, hindered the growth of two different tumors (prostate tumor allografts and human ovarian tumor xenografts) in mice and extended the life span of animals. These data altogether implicated the potential of the hybrid SB-baculovirus vector for prolonged hEA expression and for the treatment of multiple types of angiogenesis-dependent tumors.

Baculovirus vectors for antiangiogenesis-based cancer gene therapy.

Baculovirus is an insect virus that is non-pathogenic to humans and has emerged as a promising gene therapy vector. Since solid tumor growth/metastasis critically relies on angiogenesis and hEA, a fusion protein comprising human endostatin and angiostatin, exhibits potent antiangiogenic and antitumor efficacy in mouse models; this study aimed to evaluate the feasibility of baculovirus for hEA expression and antiangiogenesis-based cancer gene therapy. Toward this end, we constructed Bac-hEA that mediated transient hEA expression and Bac-ITR-hEA that exploited the adeno-associated virus inverted terminal repeats (ITRs) for prolonged hEA expression. Western blot and ELISA analyses showed that both Bac-hEA and Bac-ITR-hEA expressed hEA in transduced mammalian cells, yet Bac-ITR-hEA only marginally prolonged the hEA expression. In comparison with Bac-hEA, nonetheless, Bac-ITR-hEA significantly enhanced the hEA expression level that concurred with augmented antiangiogenic properties, as demonstrated by cell proliferation, migration and tubule network formation assays. Importantly, intratumoral injection of Bac-ITR-hEA into prostate cancer mouse models, when compared with Bac-hEA, exerted stronger antiangiogenic effects in vivo, more potently inhibited tumor growth and significantly prolonged mouse survival. This study collectively supported the notion that hEA is an effective antiangiogenic protein and proved the potential of baculovirus as a vector for antiangiogenesis-based cancer therapy, which may be combined with chemotherapy, radiotherapy or gene therapies using other vectors.

Minimal mesangial lupus nephritis: a systematic review.

OBJECTIVES: To summarize the clinical presentation, histological features, treatment, and outcome of minimal change nephropathy (MCN) in patients with systemic lupus erythematosus (SLE). METHODS: We performed a systematic review of cases of MCN in SLE patients reported in the English literature from January 1985 to May 2009 by a Medline search. RESULTS: The estimated prevalence of MCN in biopsy-proven lupus nephritis is 2.3% in childhood and 1.1% in adults. There are 13 individual cases (12 women, one man) of SLE-related MCN reported in the literature. The mean age of nephritis onset was 32.7 years. In six (46%) patients, MCN was the initial manifestation of SLE. All patients presented with nephrotic syndrome and two (15%) had active urinary sediments. Renal function was impaired in eight (62%) patients and six (46%) patients had active lupus serology. All patients responded promptly to high-dose glucocorticoids but four (31%) had relapse of proteinuria during their course of SLE. None of the patients developed thromboembolic or infective complications. CONCLUSIONS: MCN is an uncommon histological class of lupus nephritis. Typically, patients present with heavy proteinuria, and transient renal dysfunction is common. The prognosis of MCN in SLE appears to be good because of its rapid response to glucocorticoids. Relapses of proteinuria may be reduced by the use of maintenance immunosuppression. Alkylating agents, calcineurin inhibitors, mycophenolate mofetil, and rituximab can be considered in glucocorticoid-dependent or refractory cases of SLE-related MCN.

Recombinant human arginase inhibits proliferation of human hepatocellular carcinoma by inducing cell cycle arrest.

Human hepatocellular carcinoma (HCC) has an elevated requirement for arginine in vitro, and pegylated recombinant human arginase I (rhArg-PEG), an arginine-depleting enzyme, can inhibit the growth of arginine-dependent tumors. While supplementation of the culture medium with ornithine failed to rescue Hep3B cells from growth inhibition induced by rhArg-PEG, citrulline successfully restored cell growth. The data support the roles previously proposed for ornithine transcarbamylase (OTC) in the arginine auxotrophy and rhArg-PEG sensitivity of HCC cells. Expression profiling of argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and OTC in 40 HCC tumor biopsy specimens predicted that 16 of the patients would be rhArg-sensitive, compared with 5 who would be sensitive to arginine deiminase (ADI), another arginine-depleting enzyme with anti-tumor activity. Furthermore, rhArg-PEG-mediated deprivation of arginine from the culture medium of different HCC cell lines produced cell cycle arrests at the G(2)/M or S phase, possibly mediated by transcriptional modulation of cyclins and/or cyclin dependent kinases (CDKs). Based on these results, together with further validation of the in vivo efficacy of rhArg-PEG against HCC, we propose that the application of rhArg-PEG alone or in combination with existing chemotherapeutic drugs may represent a specific and effective therapeutic strategy against HCC.

Baculovirus as a new gene delivery vector for stem cell engineering and bone tissue engineering.

Baculovirus has emerged as a novel vector for in vitro and in vivo gene delivery due to its low cytotoxicity and non-replication nature in mammalian cells, but the applications of baculovirus in the genetic modification of human mesenchymal stem cells (hMSCs) and tissue engineering are yet to be reported. In this study, we genetically engineered hMSCs with a baculovirus (Bac-CB) expressing bone morphogenetic protein-2 (BMP-2). Bac-CB transduction of hMSCs at a multiplicity of infection of 40 triggered effective differentiation of hMSCs into osteoblasts. Supertransduction at day 6 after initial transduction enhanced the BMP-2 expression and further accelerated the in vitro osteogenesis, as confirmed by alkaline phosphatase assay, Alizarin red staining and reverse transcription-polymerase chain reaction analysis of osteoblastic genes. Implantation of the supertransduced cells at ectopic sites in the nude mice resulted in efficient cell differentiation into osteoblasts at week 2 and induced progressive mineralization and partial bone formation at week 6, as confirmed by hematoxylin and eosin, immunohistochemical and Alizarin red staining. These data collectively demonstrated, for the first time, the potential of baculovirus in hMSCs engineering and implicated its use in bone tissue engineering.

Baculovirus transduction of human mesenchymal stem cell-derived progenitor cells: variation of transgene expression with cellular differentiation states.

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs). In this study, we further demonstrated, for the first time, that baculovirus can transduce adipogenic, chondrogenic and osteogenic progenitors originating from MSCs. The transduction efficiency (21-90%), transgene expression level and duration (7-41 days) varied widely with the differentiation lineages and stages of the progenitors, as determined by flow cytometry. The variation stemmed from differential transgene transcription (as revealed by real-time reverse transcription-polymerase chain reaction), rather than from variability in virus entry or cell cycle (as determined by quantitative real-time PCR and flow cytometry). Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic, osteogenic and chondrogenic pathways. The susceptibility to baculovirus transduction was higher for adipogenic and osteogenic progenitors, but was lower for chondrogenic progenitors. In particular, the duration of transgene expression was prolonged in the transduced adipogenic and osteogenic progenitors (as opposed to the MSCs), implicating the possibility of extending transgene expression via a proper transduction strategy design. Taken together, baculovirus may be an attractive alternative to genetically modify adipogenic and osteogenic progenitors in the ex vivo setting for cell therapy or tissue engineering.

Two novel mutations and evidence for haploinsufficiency of the ADAR gene in dyschromatosis symmetrica hereditaria.

BACKGROUND: Dyschromatosis symmetrica hereditaria (DSH, MIM 127400) is a dominantly inherited skin disease associated with mutations in ADAR, the gene that encodes a double-stranded RNA-specific adenosine deaminase. We previously reported two novel ADAR mutations (p.Q513X and p.R916W) and confirmed the role of ADAR in Chinese patients with DSH. Both haploinsufficiency and a dominant-negative effect have been suggested as the potential mechanism by which ADAR mutations cause DSH. OBJECTIVES: To identify ADAR mutations in two additional Chinese DSH families and to obtain insight into the pathogenic mechanism of heterozygous ADAR mutations. METHODS: For mutation detection, all ADAR exons and their flanking intronic sequences were amplified and sequenced. Mutations were further confirmed by restriction analysis. Direct sequencing of cDNA fragments produced by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR were used to examine the expression of ADAR in peripheral lymphocytes isolated from affected individuals. RESULTS: A small deletion, c.1555delT (p.C519fs), and a missense mutation, c.3116A>G (p.K1039R), were found in families A and B, respectively. In individuals carrying p.Q513X or p.C519fs, sequencing of cDNA fragments indicated almost total loss of mRNA expression from the mutant alleles, and real-time quantitative RT-PCR showed an approximately 50% reduction of ADAR expression. However, equal abundance of the wild-type and mutant cDNA sequences without reduction of ADAR expression was found in a patient with the missense p.R916W mutation. These results suggest that both the nonsense p.Q513X and frameshift p.C519fs mutations have generated null alleles probably by nonsense-mediated mRNA decay. CONCLUSIONS: Two novel ADAR mutations were found in Chinese patients with DSH. Evidence for ADAR haploinsufficiency as a mechanism underlying the molecular pathogenesis of DSH was obtained.

Remission of hepatocellular carcinoma with arginine depletion induced by systemic release of endogenous hepatic arginase due to transhepatic arterial embolisation, augmented by high-dose insulin: arginase as a potential drug candidate for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is auxotrophic for the semi-essential amino acid arginine, depletion of which leads to tumor death. In humans, arginine is not an essential amino acid since many adult somatic cells can re-synthesize it from other sources, such as citrulline. Enzymes capable of depleting arginine in vitro include the urea cycle enzyme arginase, which is found in abundance in human liver. For over three decades, arginase has not been considered as a potential drug candidate because of its low substrate affinity, short circulatory half-life and sub-optimal enzymatic activity at physiological pH, though its in vitro anti-tumor activities in certain tumors have been amply reported. Arginine deiminase, a bacterial enzyme from Mycoplasma hominus has been shown to induce HCC remission through the mechanism of arginine depletion. We report here an innovative treatment approach for the treatment of locally advanced and metastatic HCC with transhepatic arterial embolisation (TAE) of the liver tumor with lipiodol and gel foam as a means of inducing a leakage of hepatic arginase from the liver into the circulation. Hepatic arginase released into the systemic circulation rapidly depleted plasma arginine. High-dose insulin was included to induce a state of hypoaminoacidaemia to augment arginine depletion. With this protocol, we have treated seven patients with locally advanced and/or metastatic HCC. Five patients achieved arginine depletion, ranging from 0 to 20 microM (normal plasma level 100-120 microM); all had varying degrees of tumor remission in their primary tumors and extra-hepatic sites in the lymph nodes, lungs and bones, suggesting systemic anti-cancer effect of arginine depletion. The two non-responders did not show significant reduction in plasma arginine. Based on our findings, we propose that the urea cycle enzyme, arginase, is a good drug candidate for the treatment of HCC.

Treatment of malignant bone tumours by extracorporeally irradiated autograft-prosthetic composite arthroplasty.

Autogenous bone graft which has been either autoclaved or irradiated is commonly used in oriental countries as an alternative to allograft. We started to use the technique of extracorporeal irradiation of the resected specimen and reimplantation (ECIR) in 1991. There was, however, a high incidence of fracture of the irradiated bone and loss of articular cartilage. In an attempt to reduce these complications, we combined the irradiated autograft with a conventional arthroplasty. Between 1995 and 1998, 14 patients underwent limb salvage by this method. Seven had an osteosarcoma, two bony metastases, three a chondrosarcoma, one a malignant fibrous histiocytoma, and one a leiomyosarcoma. Ten tumours were located in the proximal femur, two in the proximal humerus, and two in the distal femur. One patient who had a solitary metastasis in the proximal part of the left femur died from lung metastases 13 months after operation. The remaining 13 patients were alive and without evidence of local recurrence or distant metastases at a mean follow-up of 43 months (28 to 72). Postoperative palsy of the sciatic nerve occurred in one patient, but no complications such as wound infection, fracture, or nonunion were seen. All host-irradiated bone junctions healed uneventfully within eight months. Using the Enneking functional evaluation system, the mean postoperative score for all 14 patients was 80% (57 to 93). The use of irradiated autograft prosthesis composites reduces the complications of ECIR and gives good functional results. It may be a good alternative in limb-salvage surgery, especially in countries where it is difficult to obtain allografts.

Avascular necrosis of femoral head after gamma-nailing for unstable intertrochanteric fractures.

We reported on 7 cases of avascular necrosis of the femoral head after treatment of an unstable intertrochanteric fracture with the Asian Pacific gamma-nail. The incidence was about 1.16% (7 of 604) in our series. Good reduction and good implant position were achieved in all 7 men. Avascular necrosis was found about 6 months to 3 years after the initial operation, and all the fractures were solidly united at the final diagnosis. The possible etiologies were initial high energy trauma and combining basal neck fracture and iatrogenic damage of the blood supply to the femoral head.

Cefuroxime-impregnated cement at primary total knee arthroplasty in diabetes mellitus. A prospective, randomised study.

We have performed a prospective single-blinded randomised study to evaluate the role of antibiotic-impregnated cement in the prevention of deep infection at primary total knee arthroplasty (TKA) in patients with diabetes mellitus. We studied prospectively 78 arthroplasties performed for osteoarthritis in such patients. They were randomly separated into two groups. In group 1 (41 knees), cefuroxime-impregnated cement was used while in group 2 (37 knees) cefuroxime was not added to the cement. The preoperative, intraoperative and postoperative management was the same for both groups. The mean follow-up was 50 months (26 to 88). There were no cases of deep infection in group 1, but five (13.5%) occurred in group 2 (p = 0.021). We conclude that cefuroxime-impregnated cement is effective in the prevention of deep infection at primary TKA in patients with diabetes mellitus.

Intertrochanteric fractures in adults younger than 40 years of age.

This study reviewed 66 intertrochanteric fractures in patients younger than 40 years old (average 33.0 years old; range 17-40 years old). In contrast to the usual population with intertrochanteric fractures, the factors male predominance (46/66), less pre-injury comorbidity (9/66), more outdoor high energy trauma (47/66), and more associated injuries (32/66) were evident. The distribution of associated injuries was wide. Some of them were life threatening. According to Boyd's classification, 20 were type I, 24 were type II, 13 were type III, and 9 were type IV. Twenty-nine were stable, and 37 were unstable. Stratified by the mechanism of injury, the difference in distribution between the subgroups was significant (p = 0.027, two-tail Fisher's exact test). Simple falls only caused Boyd type I and II fractures. Boyd type III or IV fractures were found more often after vehicular trauma or falls from a height. All the intertrochanteric fractures healed on average 70.5 days (range 31-213 days) after operation. The fractures resulting from vehicular trauma or fall from a height healed significantly more slowly (p = 0.02, univariant log-rank test). There were 6 intertrochanteric fracture-related complications. The mechanism of injury determines the character of intertrochanteric fractures in young adults. Given tougher bone stock, better healing ability, and less co-morbidity, proper management can lead to healing of all intertrochanteric fractures. The extent of functional recovery was also determined by the associated injuries.

Treatment of acute closed humeral shaft fractures with Ender nails.

The effect of semirigid Ender nails (EN) in the treatment of closed humeral shaft fractures was reviewed and analyzed. Clinical study was set retrospectively with detailed parameters. One hundred and eighteen closed humeral shaft fractures, treated with closed reduction and internal fixation with ENs, were collected. The follow-up period was 78 (24-175) months. The average operation blood loss was 105 cc, operation time was 57 min, hospital stay was 6.5 days, and union time was 10.5 weeks. The postoperative complications included three superficial infections, one iatrogenic radial nerve palsy, eight nail backouts, and eight nonunions. In our experience, for closed humeral shaft fractures fixed surgically, EN is a good choice for its simplicity and efficacy, but the fracture gap should be minimized after fixation and postoperative care should be closely observed.

Surgical treatment for peroneal nerve palsy.

BACKGROUND: Peroneal nerve palsy is the most frequently encountered mononeuropathy of the lower extremities. Although many studies advocated spontaneous resolution of peroneal nerve palsy, more recent studies confirmed obvious improvement with surgical treatment techniques. METHODS: This study reviewed the results obtained from surgically treated peroneal nerve palsy in 14 patients who were admitted to our hospital between 1990 and 1996. The patients consisted of 12 males and two females with an average age of 31 years (range, 12-68 years). Peroneal nerve palsy in these patients was caused by direct or indirect injury, as confirmed by clinical examination and electromyography. The status of the nerve was observed for at least 4 months and explored when the nerve failed to reveal evidence of recovery. The nerve was decompressed, repaired or reconstructed by nerve grafting, according to the status of the injury. Weber scales were used to assess the peripheral neuropathies preoperatively and postoperatively. RESULTS: At a mean of 23 months (range, 11-61 months), nerve palsy scores improved from an average of 5 points to 3.14 points. Despite the small number of patients, our results indicated that the time interval between onset of injury and date of surgical treatment (p < 0.05) was the most significant factor to influence the prognosis of surgery. Results obtained from surgery were not related to the method of treatment, length of nerve graft or mechanism of injury. CONCLUSIONS: Because peroneal nerve palsy does not always resolve spontaneously, this study favored early surgical exploration for peroneal nerve dysfunction, based on at least 4 months of follow-up.

Surgical treatment of full thickness rotator cuff tear in patients younger than 40 years.

BACKGROUND: Full thickness tear of the rotator cuff is a well-known entity in the middle-aged and elderly population and the results of surgical repair are well documented. Rotator cuff tear in patients under the age of 40 years is unusual and the cause and treatment are not well established. The present study reports 12 young patients with full thickness tear of the rotator cuff treated surgically. METHODS: Open anteroinferior acromioplasty and cuff repairs were performed on every patient after failure of nonsurgical treatment. The average follow-up was 59.5 months (range, 36-100 months). RESULTS: The average patient age at the time of surgery was 30.2 years (range, 17-39 years). Symptoms included pain and dysfunction in all patients and weakness in eight patients. The duration of symptoms before surgery ranged from 6 to 60 months (average, 22.8 months). Ten patients were able to return to their preinjury status within an average of 4.5 months (range, 3-9 months) after surgery. Three athletes were able to return to previous levels of competition. Nine patients had excellent functional results with regard to pain, motion, strength, function and satisfaction. Eleven patients were satisfied with the operation and had significant pain reduction compared with their preoperative status. CONCLUSIONS: An acute traumatic event was the etiology of the rotator cuff tears in this study. The results of surgical treatment in this younger group of patients were satisfactory and were comparable to the outcomes reported for surgical treatment of rotator cuff tears in older patients and athletes.

The effect of posterior capsulorrhaphy in primary total hip arthroplasty: a prospective randomized study.

Between 1994 and 1997, 180 cases of primary total hip arthroplasty (THA) were performed with the posterior (Moore) approach for a variety of indications and studies prospectively. The cases were separated randomly into 2 groups to evaluate the effect of posterior capsulorrhaphy in the prevention of postoperative dislocation. In group 1 (96 cases), closure of the arthroplasty was performed with a posterior capsulorrhaphy; in group 2 (84 cases), closure was performed without capsulorrhaphy. The follow-up period was 38 months (range, 12-60 months). No dislocations occurred in group 1, whereas 2 dislocations (2.3%) occurred in group 2. Although the factors affecting dislocation in primary THA are many, a posterior capsulorrhaphy may be helpful in the prevention of posterior dislocation of primary THA performed with a posterior approach.

The influence of contact alignment of the tibiofemoral joint of the prostheses in in vitro biomechanical testing.

OBJECTIVE: To investigate the influence of contact alignment of the tibiofemoral joint of the prostheses in in vitro biomechanical testing. DESIGN: An experimental set-up was used to measure the total contact areas of the tibiofemoral joint of the prostheses subjected to a compressive load, and the malalignment situations were simulated. BACKGROUND: The contact alignment of the femoral component related to the tibial component in the literature was not described clearly and the effect of malalignment on the testing method has not been reported well. METHODS: Three commercial knee prostheses (Omnifit, Genesis, and AMK) were used for testing under a compression load (3000 N) at flexion 0 degrees and 10 degrees. After aligning the normal contact alignment, the simulated malalignment was done to evaluate the influence on the total contact areas in these conditions relative to the normal contact alignment. The simulated malalignment includes the medial-lateral translation (0.5 and 1 mm), anterior-posterior translation (2 and 4 mm) and internal-external rotation (1 degrees, 3 degrees and 5 degrees ) of the femoral component relative to the tibial component. RESULTS: The ratios of total contact areas of malalignment relative to normal contact alignment ranged from 1.06 to 0.93 in medial-lateral malalignment in these three prostheses. In anterior-posterior malalignment, the ratios ranged from 0.69 to 0.79 in Omnifit, 0.93-0. 96 in Genesis and 0.96-1.04 in AMK. In internal-external malrotation, the ratios ranged from 0.90 to 1.03 in these prostheses. CONCLUSIONS: This study proposes that contact alignment would affect the contact characteristics, especially in anterior-posterior alignment when high conformity knee prosthesis is tested. The contact alignment must be standardized in the procedure in in vitro biomechanical testing, which will be more objective to evaluate the contact characteristics of different knee prostheses. RELEVANCE: This study revealed the importance of contact alignment of the tibiofemoral joint of the prosthesis in in vitro biomechanical testing. Many published reports of the biomechanical evaluations on different designs of knee prostheses would show different results due to contact alignment. Furthermore, this study indicates that the ideal contact characteristics of the tibiofemoral joint in original design will be changed when the prosthesis under the malalignment condition which was caused by surgery technique or soft tissues imbalance.

An in vitro comparison of the motion behavior of different bipolar endoprostheses.

The relative motion of 3 different bipolar endoprostheses was evaluated in vitro. A paired fresh acetabulum was frozen at 0 degrees C and defrosted 12 hours before the experiment. Three bipolar endoprostheses were evaluated: UNIQHIP system (United Orthopedics), UHR system (Osteonics), and AML system (Depuy). The surface roughness and spherical roundness of outer shells and inner heads of the bipolar prostheses were measured before the experiments. The acetabulum and outer shell of the bipolar prostheses were fixed on a Bionix 858 material testing system axially by separate fixation tools. The axial load of 1,400 N and 2,800 N was than applied on the specimen. The axis was rotated from 0 degrees to 90 degrees at the speed of 1 degree/s. All 3 outer shells were tested to this paired acetabulum randomly and separately. The frictional torque on the outer bearing surface of the different prostheses was recorded by the material testing system. The frictional torque on the inner bearing surface was also measured by the same procedure as was done for the outer bearing. The final results were statistically compared by the 1-way analysis of variance test method. Bipolar prostheses of the UHR system showed the largest frictional torque on outer bearing when it was loaded with 1,400 N and 2,800 N. The final results showed that all the bipolar prostheses had ideal motion behavior when functioning under the loading of 1,400 N. The frictional torque on the inner bearing was found to be larger than the frictional torque on the outer bearing in some prostheses when the loading was increased to 2,800 N. Thus, the bipolar endoprostheses functioned as unipolar prostheses. The only relative motion remained between the outer bearing surface and the acetabulum. This effect causes complications, such as implant protrusion in the acetabulum.