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We examined the clinical features of bisphosphonate(BP)-related osteonecrosis of the jaws(BRONJ), a serious complication resulting from intravenous BP treatment for multiple myeloma and malignant tumors with bone metastasis. We retrospectively reviewed the medical records of 36 patients who received intravenous BP therapy for the above-mentioned conditions, at Sapporo Medical University Hospital between July 2006 and October 2008. BP therapy caused BRONJ in 7 of 24 patients, but did not affect the bones of the other 17 patients. The other 12 of the 36 patients involved in the study were prescribed BP only after they had undergone an oral examination and treatment for dental inflammation. Of these patients, 7 developed BRONJ with BP treatment, after tooth extraction or acute dental inflammation. Treating dental inflammation before prescribing BP prevented the development of BRONJ. BRONJ is highly intractable and does not resolve with the standard treatment for osteomyelitis. Therefore, preventive therapy, which can be achieved by cooperation between medical doctors and dentists, is currently the most effective strategy for BRONJ. Conservative treatment with antibiotics may also be useful for maintaining or improving the quality of life of BRONJ patients.

To explain the tissue-selective expression patterns of a distinct subclass of glutathione S-transferase (GST), transgenic mice expressing EGFP under control of a 2 kb promoter sequence in the 5'-flanking region of the mGstm5 gene were produced. The intent of the study was to establish whether the promoter itself or whether posttranscriptional mechanisms, particularly at the levels of mRNA translation and stability or protein targeting, based on unique properties of mGSTM5, determine the restricted expression pattern. Indeed, the transgene expression was limited to testis as the reporter was not detected in somatic tissues such as brain, kidney or liver, indicating that the mGstm5 proximal promoter is sufficient to target testis-specific expression of the gene. EGFP expression was also more restricted vis-a-vis the natural mGstm5 gene and exclusively found in germ but not in somatic cells. Real-time quantitative PCR (qPCR) data were consistent with alternate transcription start sites in which the promoter region of the natural mGstm5 gene in somatic cells is part of exon 1 of the germ cell transcript. Thus, the primary transcription start site for mGstm5 is upstream of a TATA box in testis and downstream of this motif in somatic cells. The 5' flanking sequence of the mGstm5 gene imparts germ cell-specific transcription.

Osteonecrosis of the jaw is a severe new complication in cancer patients with bone metastases receiving bisphosphonate. Currently, there is no effective treatment for bisphosphonate-related osteonecrosis of the jaw, and the pathogenesis of this complication has not been completely elucidated. It has been shown that a potential risk factor for the complication is dentoalveolar trauma including extraction of teeth during bisphosphonate therapy. Attention should be paid to dental care in patients prior to the initiation of bisphosphonate therapy, and extraction of teeth during bisphosphonate therapy should be avoided to prevent this complication. Therefore, the communication between general physicians prescribing bisphosphonate and dentists is important.

Summary The purpose of this clinical trial was to assess the toxicity and efficacy of docetaxel plus nedaplatin induction chemotherapy in patients with locally advanced oral squamous cell carcinoma (OSCC). Twenty-one patients were enrolled in this phase I/II clinical study. The toxicities, response rates, and the maximum tolerated dose of nedaplatin that could be safely given preoperatively were assessed. Patients received escalating doses of nedaplatin (60, 70, 80, 90 mg/m 2 ) combined with a fixed dose of docetaxel (60 mg/m 2 ) on day one. Dose-limiting toxicity (DLT), grade 4 leukopenia lasting for two days or more, was seen in one patient at dose level 3; no other DLT was observed at any dose level. The overall response rate was 66.7%. The response rate was 100% at nedaplatin dose level 4, while that at dose level 1 was low (33.3%). Given these results, the recommended dose of nedaplatin in this regimen combined with fixed dose docetaxel (60 mg/m 2 ) was determined to be 90 mg/m 2 . Docetaxel 60 mg/m 2 plus nedaplatin 90 mg/m 2 induction chemotherapy can be recommended for patients with locally advanced oral squamous cell carcinoma. Based on these results, an early phase II clinical study using this dose level was conducted; docetaxel plus nedaplatin induction chemotherapy appears to be a useful regimen for the treatment of OSCC. A late phase II clinical study is warranted.

Hematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However, the mechanisms involved in controlling the number and abilities of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in coculture with CD34+ cells and human stromal cells. Ihh mRNA was expressed in primary and telomerized human (hTERT) stromal cells, and its receptor molecules were detected in CD34+ cells. Ihh gene transfer into hTERT stromal cells enhanced their hematopoietic supporting potential, which was elevated compared with control stromal cells, as indicated by the colony-forming units in culture (CFU-Cs) (26-fold ± 2-fold versus 59-fold ± 3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's], 63-fold ± 37-fold versus 349-fold ± 116-fold). Engraftments of nonobese diabetic/severe combined immunodeficiency–ß2m–/– (NOD/SCID–ß2m–/–) repopulating cells (RCs) expanded on Ihh stromal cells were significantly higher compared with control coculture results, and engraftment was neutralized by addition of an antihedgehog antibody. Limiting dilution analysis indicated that NOD/SCID–ß2m–/– RCs proliferated efficiently on Ihh stromal cells, compared with control stromal cells. These results indicate that Ihh gene transfer could enhance the primitive hematopoietic support ability of human stromal cells.

Although naked plasmid injection is the safest and most convenient method for gene delivery, a major limitation of this approach is currently poor transgene expression. The CA promoter (chicken beta-actin promoter with cytomegalovirus, CMV, enhancer) is one of the strongest transcriptional control modules found; however, it is uncertain whether a CA promoter-based vector is efficient enough for naked gene therapy in a cardiovascular context.The beta-galactosidase (LacZ) expression provided by CA promoter plasmid (pCAZ2) injection into the skeletal muscle or the heart of Lewis rats was compared with CMV promoter plasmid or adenoviral vector (AxCAZ3). The effect of Simian virus 40 of the replication origin (SV40ori) deletion from pCAZ2 on transgene expression was also evaluated.pCAZ2 showed the highest LacZ expression in both skeletal muscle and heart in comparison with the CMV promoter-based vector 5 days after naked plasmid injection. LacZ expression in the heart obtained using 20 micro g of pCAZ2 was almost equivalent to that shown with AxCAZ3 at 6.0 x 10(9) optical particle units. The time course of transgene expression driven by CMV and CA promoters in the heart were similar, with the CA promoter providing significantly higher gene expression than the CMV promoter across all time points examined. SV40ori deletion from pCAZ2 did not affect transgene expression in either skeletal muscle or heart.Transgene expression mediated by naked CA promoter-based plasmid injection was shown to be quite efficient in the heart. We propose that the CA promoter vector is suitable for myocardial gene therapy.

In acute myocardial infarction (AMI), prognosis and mortality rate are closely related to the infarct size and the progression of postinfarction cardiac failure. Angiogenic gene therapy has presented a new approach for the treatment of AMI. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. We hypothesized that gene therapy using Ang1 for AMI might promote angiogenesis cooperatively with intrinsic VEGF, since high concentrations of circulating VEGF have been reported in AMI. To evaluate our hypothesis, we employed a rat AMI model and adenoviral Ang1 (HGMW-approved gene symbol ANGPT1) gene transfer to the heart. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with adenoviral Ang1 gene treatment compared with control infarcted hearts treated with saline or adenoviral vector containing the β-galactosidase gene. Furthermore, the Ang1 group showed significantly higher cardiac performance in echocardiography (55.0% of ejection fraction, P < 0.05 vs control) than the saline or adenoviral controls (36.0 or 40.5%, respectively) 4 weeks after myocardial infarction. The adenoviral delivery of Ang1 during the acute phase of myocardial infarction would be feasible to attenuate the progression of cardiac dysfunction in the rat model.

Objective. To compare the hematopoietic support provided by telomerized human mesenchymal stem cells (MSCs) and telomerized MSC-derived stromal cells. Methods. We transfected the human telomerase catalytic subunit (hTERT) gene into primary MSCs to establish hTERT-transduced MSCs (hTERT-MSCs). Stromal induction of hTERT-MSCs was performed by replacing the culture medium with Dexter-type culture medium. Hematopoietic support was examined by coculture with cord blood CD34 + cells. Results. The hTERT-MSCs were morphologically identical with the primary MSCs and expressed surface antigens including CD105, CD73, and CD166. hTERT-MSCs showed a similar doubling time as primary MSCs and continued to proliferate to over 80 population doublings (PD), although the primary MSCs underwent crisis in vitro at 16 PD. The osteogenic, chondrogenic, adipogenic, neurogenic, and stromal differentiation potential of hTERT-MSCs were maintained up to at least 40 PD. The degree of expansion of CD34 + cells and total number of colony-forming units in culture (CFU-C) upon 12-day coculture with the hTERT-MSC–derived stromal cells were nearly the same as those upon 12-day coculture with hTERT-MSCs (CD34, 33.0-fold±2.8-fold vs 36.1-fold±1.7-fold of the initial cell number; CFUs, 344.4-fold±62.5-fold vs 239.3-fold±87.0-fold; CFU-mix, 368.4-fold±113.7-fold vs 341.3-fold±234.3-fold). However, on day 18 of coculture, the number of cobblestone areas (CA) observed beneath the stromal cells was 15 times higher than that beneath hTERT-MSCs (CA, 146.9±54.6 vs 9.4±8.1, p Conclusion. Stromal induction of hTERT-MSCs exclusively enhanced the support of CA formation provided by hTERT-MSCs. Our human hTERT-MSCs will be useful for elucidating the mechanism of the formation of CAs.

Current clinical success rates of adenoviral vector (Adv)-based gene therapy of squamous cell carcinoma (SCC) of the head and neck remain unsatisfactory. A major problem with this approach is thought to be related to low Adv transduction efficiency due to weak expression of the adenovirus receptor, coxsackie-adenovirus receptor (CAR), in SCC. To improve the limited infectivity of Adv in oral SCC, we constructed mutated Adv incorporating the integrin-binding motif, RGD, in the HI loop of the fiber knob. The mutated Adv infected target cells through integrins commonly expressed in oral SCC. LacZ marker gene expression after infection with this mutated Adv (Adv-F/RGD) in oral SCC cell lines that showed reduced expression of CAR was approximately 5-10 times higher than that obtained with the parental Adv containing wild-type fiber knob (Adv-F/wt). In an in vitro study, transduction of oral cancer cell lines with Adv-F/RGD expressing human IL-2 (AxCAhIL2-F/RGD) resulted in greater production of cytokine than AxCAhIL2-F/wt infection. In an in vivo therapeutic xenograft model of oral SCC in nude mice, AxCAhIL2-F/RGD demonstrated antitumor effects superior to those of AxCAhIL2-F/wt. These data suggest that exploitation of genetically altered adenovirus vectors with integrin-binding motifs may offer significant improvements in oral SCC gene therapy.

BACKGROUND pRb2/p130 is one of the retinoblastoma (Rb) gene family and a suppressor oncogene. Immunohistochemically, the expression of pRb2/p130 was reported to be correlated inversely with the degree of malignancy in lung carcinoma and endometrial carcinoma. In the current study, the correlation between expression of pRb2/p130 and clinicopathologic factors in oral squamous cell carcinoma was investigated. METHODS One hundred twenty-two specimens from patients with oral squamous cell carcinoma were investigated by staining with a polyclonal antibody against pRb2/p130. The correlation between the expression of pRb2/p130 and various clinicopathologic factors was studied. RESULTS Positive staining for pRb2/p130 was observed in 61 of 122 cases (50.0%). pRb2/p130 expression was found to be correlated significantly with clinical stage (P = 0.050), cervical lymph node metastasis (P = 0.035), and tumor differentiation (P = 0.050). In the entire group a significantly reduced 5-year cumulative survival rate was observed in patients with pRb2/p130-negative tumors compared with patients whose tumors positively expressed pRb2/p130 (P = 0.0004). When tested with Cox proportional hazards regression analysis, the most significant independent prognostic factor for the entire group of 122 patients was found to be pRb2/p130 expression. CONCLUSIONS Expression of pRb2/p130 may be a good prognostic indicator in patients with oral squamous cell carcinoma and also may be utilized for the subclassification of tumors with the Grade 3 mode of carcinoma invasion. Cancer 2001;92:2117–25. © 2001 American Cancer Society.

We previously reported that pRb2/p130 gene, one of the Rb family members, was immunohistochemically abundantly expressed in well-differentiated oral squamous cell carcinomas, whereas in undifferentiated ones the expression was low. Oral malignant melanoma is extremely rare, however the prognosis is poor because it tends to locally invade tissue or metastasize and its biological behavior appears to be different from cutaneous malignant melanoma. The present study dealt with the expression of pRb2/p130, Rb, p53, and p16 in 13 cases of malignant melanoma of oral mucosa as revealed by immunohistochemical staining. The stage classification of the 13 patients was as follows; stage II: eight patients, stage III: three patients, and stage IV: two patients. pRb2/p130 was expressed in only two stage II-cases, neither of which have shown any evidence of recurrence or metastasis for over 14 years. Positive staining for Rb was found in three cases consisting of one stage II-case, one stage III-case, and one stage IV-case. p53 was expressed in two cases, one a stage II and the other a stage IV. Positive staining for p16 was found in seven cases consisting of four stage II-cases, two stage III-cases, and one stage IV-case. pRb2/p130 may be inversely correlated with the malignancy of oral malignant melanoma, but further study is needed.

Background: Ischemia and reperfusion (I/R) injury is one of the important causes of early graft dysfunction and graft loss. Among factors that may be involved in the etiology of I/R injury, apoptosis is an early event after I/R injury of heart. Thus, inhibition of myocardial apoptosis might be a rational therapeutic strategy to reduce the preservation injury in the heart transplantants. We previously reported that Bcl-xL gene transfer can confer resistance against warm I/R injury and result in the improvement of cardiac function and the reduction of infarct size in rat heart. These evidences indicate that Bcl-xL would be also useful as a cardiac protective alternative in heart cold preservation. In this study, we employed adenoviral Bcl-xL gene transfer to the heart, and evaluated the cardiac protection of Bcl-xL in the heart transplantation after long cold preservation.

Background: Sendai virus (SeV) is negative-stranded RNA virus, which is classified as a type I parainfluenza virus belonging to the family of Paramyxoviridae. SeV has been known as rodent respiratory virus, however it is considered to be non-pathogen for humans. Recently, efficient gene transduction into various types of cells and tissue has been reported. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. Previously we reported Ang1 gene therapy clearly promoted myocardial angiogenesis and prevented remodeling in a rat AMI model. In the present study, we created recombinant Sendai virus vector (SeVV) encoding human Ang1 and evaluated therapeutic effect in the cardiovascular diseases. We also evaluated the angiogenic effect of mesenchymal stem cells (MSCs) transplantation with SeVV-mediated Ang1 gene modification.

Background: Clinical trials for severe coronary ischemia and critical limb ischemia using vascular endothelial growth factor (VEGF) gene have been on going for several years. However, the indication of VEGF therapy for ischemic heart disease is only limited to chronic ischemia, and acute myocardial infarction (AMI) is exception for this therapy at present. High levels of circulating VEGF were reported to arise following AMI in a short period after the ischemic event and surmised that it protects the blood vessels from apoptosis, modulates vasomotor response, and supports vessel formation in the ischemic area, and to contribute to quick recovery from the ischemic state. In this regard, we attempted adenoviral VEGF gene therapy for rat myocardial infarction model. However, we found the increase mortality of animals receiving adenoviral VEGF. Therefore, we re-evaluated the adverse effect of myocardial administration of adenoviral vector encoding VEGF in AMI rat.

Background: Sendai virus (SeV), a rodent respiratory virus considered non-pathogenic for humans, is a negative-strand RNA virus, classified as a type I parainfluenza virus belonging to the family Paramyxoviridae. Recently, efficient gene transduction into various primary cultured cells and tissues by a recombinant Sendai virus vector (SeVV) has been reported. However, the feasibility of using SeVV in the context of myocardial gene therapy was not evaluated. In the present study, we evaluated SeVV-mediated gene-transfer into cardiomyocytes in vitro and in vivo