Transcutaneous immunization using a dissolving microneedle array protects against tetanus, diphtheria, malaria, and influenza.

Transcutaneous immunization (TCI) is an attractive alternative vaccination route compared to the commonly used injection systems. We previously developed a dissolving microneedle array for use as a TCI device, and reported that TCI with the dissolving microneedle array induced an immune response against model antigens. In the present study, we investigated the vaccination efficacy against tetanus and diphtheria, malaria, and influenza using this vaccination system. Our TCI system induced substantial increases in toxoid-specific IgG levels and toxin-neutralizing antibody titer and induced the production of anti-SE36 IgG, which could bind to malaria parasite. On influenza HA vaccination, robust antibody production was elicited in mice that provided complete protection against a subsequent influenza virus challenge. These findings demonstrate that TCI using a dissolving microneedle array can elicit large immune responses against infectious diseases. Based on these results, we are now preparing translational research for human clinical trials.

Primary Ewing sarcoma of the spine mimicking a psoas abscess secondary to spinal infection.

STUDY DESIGN: A case of primary Ewing sarcoma of the lumbar spine is presented. OBJECTIVE: To present and review a rare case of primary Ewing sarcoma of the lumbar spine that required differentiation from spinal infection. SUMMARY OF BACKGROUND DATA: Primary Ewing sarcoma originating from the spinal column is very rare. Because Ewing sarcoma is one of the most aggressive bone tumors with high proliferative and invasive potential, its clinical symptoms and variety of imaging manifestations can mimic the pathologic findings of other diseases, including infectious diseases. METHODS: The clinical course, radiologic features, pathology and treatment outcome of a patient with primary Ewing sarcoma of the lumbar spine was documented. RESULTS: The magnetic resonance imaging findings showed an abnormal marrow signal at the L2 vertebra and significant enlargement of the unilateral iliopsoas muscle. Immunologic and molecular analysis of the surgical specimen provided a diagnosis of Ewing sarcoma. Laminotomy followed by multidisciplinary therapy including chemotherapy and radiation therapy was effective for treating this case. CONCLUSION: We report a case of Ewing sarcoma that mimicked a psoas abscess secondary to spinal infection. Abnormal magnetic resonance imaging images, as well as a confusing clinical course, made diagnosis difficult. When enlargement of the iliopsoas with a vertebral lesion is detected in a child with low back pain, Ewing sarcoma should be included in the differential diagnosis.

The membrane proteinase 3 expression on neutrophils was downregulated after treatment with infliximab in patients with rheumatoid arthritis.

Proteinase 3 (PR3) expression on neutrophils was examined in rheumatoid arthritis (RA) patients before and after antitumor necrosis factor (TNF)-alpha therapy. Membrane PR3 expression from patients with either an infection or RA significantly increased. Membrane PR3 expression on neutrophils from RA patients treated with infliximab (anti-TNF-alpha antibody) therapy was less than in those without such treatment in a resting state, but the expression later increased after stimulation in vitro. Membrane PR3 expression increased because of the stimulation of TNFalpha, whereas it was significantly suppressed by plasma or alpha(1)-proteinase inhibitor. The condition of patients with RA improved after treatment with infliximab. Membrane PR3 expression on neutrophils in RA patients was downregulated by infliximab. As a result, PR3 might play an important role in the neutrophil-mediated inflammatory reaction in patients with either RA or an infection.

Clinical trial of photodynamic therapy using acridine orange with/without low dose radiation as new limb salvage modality in musculoskeletal sarcomas.

Most patients with musculoskeletal sarcoma do not recover satisfactory limb function after limb salvage surgery. To achieve satisfactory improvement of limb function, we developed a unique surgical modality of photodynamic therapy using acridine orange (AO-PDT) and clinically applied it to patients with musculoskeletal sarcomas. Ten patients with primary musculoskeletal sarcomas were enrolled in the study. Of these, 6 had primary malignant soft tissue sarcoma and 4 had primary malignant bone tumor. In the AO-PDT procedure, intralesional or partially marginal tumor excision was initially conducted and microscopic curettage of the remnant tumor, which emitted green fluorescence under blue excitation after local administration of 1microg/ml AO solution, was performed using a fluorescence surgical microscope. Subsequently, blue light illuminated there for 10 minutes. The surgical wound was closed, followed by immediate X-ray irradiation of the resected area with 5 Gy in 5 out of 10 patients to enhance the effect of AO-PDT. The follow-up of the patients ranged from 24 to 48 months. All the patients (AO-PDT alone: 5, AO-PDT with 5-Gy radiation: 5) are alive; only one patient showed local recurrence of the tumor. The recurrence rate was 10%. None of the 5 patients treated by AO-PDT with radiation developed local tumor recurrence. The limb function in all the patients, except for one, recovered to the level before surgery. None of the patients clinically showed any local or systemic complications. AO-PDT may be a promising new limb salvage modality for preservation of excellent limb function in patients with musculoskeletal sarcoma.

Clinical outcome of a novel photodynamic therapy technique using acridine orange for synovial sarcomas.

Synovial sarcoma (SS) is one of common malignant soft-tissue tumors and is encountered most commonly in children and young adults. It frequently involves or invades major neurovascular structures and bones, and its local recurrence rate after simple resection has been reported to be as high as up to 80%. Because major nerves and vessels, as well as an adequate amount of bone, must be preserved to restore excellent limb function in cases of SS, a surgical technique entailing a low risk of local recurrence is needed. Based on the findings of recent experimental studies conducted by us using a mouse osteosarcoma model, we developed a novel therapeutic technique for SS, consisting of reduction surgery followed by photodynamic therapy using acridine orange (AO-PDT), with or without X-ray irradiation at 5 Gy. A preliminary study revealed that low-dose X-rays also excite AO like photons. After an initial study on cell cultures, this novel technique was applied to six cases of SS. A follow-up of the subjects to determine the clinical outcome revealed that none of the cases treated by AO-PDT, including the four cases treated by additional 5 Gy irradiation and the two cases not receiving any radiation, showed any evidence of recurrence or local/systemic complications during the follow-up period of 19-51 months after the surgery. Therefore, we believe that AO-PDT with 5 Gy irradiation may be an excellent novel therapeutic modality with reduction surgery to salvage excellent limb function in SS involving major nerves and vessels or bones.

Metastasis of malignant peripheral nerve sheath tumor to free vascularized myocutaneous flap.

The vascularized free myocutaneous flap graft, a recent advance in microsurgery, is a very useful reconstruction technique for covering massive soft tissue defects or muscular dysfunction caused by wide resection for malignant soft tissue tumors. We have used this technique to treat many patients after resection for malignant soft tissue tumors. Recently we encountered a case in which a malignant peripheral nerve sheath tumor (MPNST) metastasized to a vascularized free myocutaneous flap used for limb reconstruction surgery after wide resection of a primary lesion. To our knowledge, there have been no previous reports of metastasis of a malignant soft tissue tumor (or any other cancer) to a grafted cutaneous or myocutaneous flap.

Low-dose methotrexate inhibits lung metastasis and lengthens survival in rat osteosarcoma.

Lung metastasis is the most crucial event affecting the treatment of osteosarcoma and is dependent on tumor angiogenesis. To improve the prognosis for patients with osteosarcoma, prevention of lung metastasis is essential. Low-dose methotrexate is a useful drug for treating a variety of diseases. Low-dose methotrexate reportedly plays a role in antiangiogenesis for the synovial blood vessels in rheumatoid arthritis. However, whether low-dose methotrexate is correlated with tumor angiogenesis and metastasis is unclear. We investigated the inhibitory effect of methotrexate on lung metastasis in a rat osteosarcoma cell line with high metastatic potential, S-SLM. Two weeks after inoculation of S-SLM cells into male Fischer 344 rats, low-dose methotrexate (1.2 mg/kg once or twice a week) or saline was intraperitonealy injected for 4 weeks and the antimetastatic effect was evaluated. Low-dose methotrexate significantly reduced the number of lung metastatic nodules and the wet weight of the lungs. Immunohistochemical staining showed a decrease in microvessel density in the metastatic nodules. We also evaluated the effect of methotrexate on the proliferation of endothelial cells and S-SLM osteosarcoma cells in vitro. Methotrexate significantly inhibited the proliferation of endothelial cells at a lower concentration than that of S-SLM osteosarcoma cells. These data suggest that low-dose methotrexate inhibited lung metastasis of osteosarcoma through its antiangiogenic activity. Our results indicate that low-dose methotrexate is a promising drug for tumor dormancy therapy in patients with osteosarcoma and lung metastasis.

Thrombin inhibitor, argatroban, prevents tumor cell migration and bone metastasis.

It is well known that malignant cells show procoagulant activity, which is associated with their metastatic potential. Thrombin, the key enzyme of the blood coagulation system, is generated around tumor cells, promoting the migration and metastasis of tumor cells. In this study, we evaluated the effect of argatroban, a specific thrombin inhibitor, on the migration and metastasis of B16BL6 melanoma cells. In vitro argatroban dose-dependently inhibited cell migration, the maximum inhibition being observed in the presence of 10 microM argatroban (p < 0.0001). In order to investigate the antimetastatic effect of the thrombin inhibitor, we used an animal model that we have reported previously. C57BL6 mice which had received a bone (femur or tibia) transplanted into the dorsal subcutis were injected with B16 melanoma cells into the left heart ventricle. Intraperitoneal injection of argatroban (9 mg/kg/day for 4 weeks) significantly reduced the number of limbs with metastatic lesions as compared to a placebo (p < 0.05). These results suggest that argatroban was associated with reduced melanoma metastases by inhibiting cell migration. Our results showed that argatroban is effective for treatment of bone metastasis.

Surrounding muscle edema detected by MRI is valuable for diagnosis of intramuscular myxoma.

We recently experienced 4 cases of intramuscular myxoma and analyzed MRI findings, comparing them with histological ones. Results showed that all tumors were depicted with a homogeneous low signal intensity on T1-weighted images and a markedly high signal intensity on T2-weighted images, findings which are similar to those of cystic lesions like intramuscular ganglions. However, tumors were diffusely and finely enhanced on T1-weighted images with intravenous gadolinium administration. Three cases showed the characteristic fat rim and fat cap. A diffuse edematous lesion demonstrating intermediate signal intensity on T1-weighted images and high signal intensity on T2-weighted images was also found in the adjacent muscle surrounding the tumor in 3 of 4 cases. In this lesion, tumor invasion, diffuse and severe muscle degeneration, blood and lymphatic congestion with exudates, and reactive adipose tissue formation were observed. The present results suggested that for the clinical diagnosis of intramuscular myxoma with MRI examination, the combination of three MRI signs such as homogeneous low signal intensity on T1-weighted mages, markedly high signal intensity on T2-weighted images, and an enhancement effect with contrast medium are important, but the fat rim or fat cap, and the surrounding muscle edema as detected by MRI are also characteristic and allow for a more firm diagnosis. We presume that the diffuse and severe muscle fiber degeneration induced by tumors may cause such specific surrounding muscle edema.

Case report: case of clear cell sarcoma surviving with the primary lesion for 20 years after resection of a metastatic lymph node.

In this paper we report on an extremely rare case of clear cell sarcoma, which is a rare malignant soft tissue tumor, biologically similar to malignant melanoma. The patient has survived for 25 years after he noticed a small tumor mass at the wrist and for 20 years after a small sentinel lymph node metastasis in the axilla was removed by surgical resection. The patient visited us because the tumor in his wrist had increased rapidly in size during the previous year. We diagnosed the wrist tumor as a clear cell sarcoma, based on the typical histopathological findings and positivity of the tumor cells for HMB-45 and S-100 protein; the histopathological findings in the axillary soft tissue tumor resected 20 years previously were also compatible with clear cell sarcoma of the lymph node. Therefore, we concluded that the wrist tumor was the primary lesion of clear cell sarcoma and that the axillary tumor was a metastatic lesion in a sentinel lymph node. Generally, the prognosis of clear cell sarcoma is very poor, especially after the occurrence of lymph node metastasis. In this case, it is possible that certain immune mechanisms played a role in effectively suppressing the tumor growth for such a prolonged period, even after the development of lymph node metastasis.

Effect of retinoblastoma tumor suppressor gene expression on chemosensitivity of human osteosarcoma cell lines.

We examined the effects of inactivation of the RB gene on chemosensitivity of human osteosarcoma cell lines, using the MTT assay and calculating the inhibition index. Although the human osteosarcoma cell lines HOS and MG63 have a wild-type RB gene, SaOS-2 and OSrb (established from retinoblastoma patient) have no active RB gene. We used these 4 cell lines in growth inhibition assays for doxorubicin, cisplatin and methotrexate, and assessed the chemosensitivity. In the growth inhibition assay for methotrexate, cell lines lacking an active RB gene were more resistant than cell lines with an active RB gene.

Case report: recurrence of soft tissue MFH in bone due to minute intravenous tumor emboli detected by MRI.

We recently encountered a case with local recurrence of malignant fibrous histiocytoma (MFH) in the bone after wide resection, caused by minute intravenous tumor emboli which were retrospectively detected in MR imaging. The patient was a 69-year-old woman who initially noticed a mass in her left thigh. The tumor was diagnosed to be MFH, therefore a wide resection was performed; although the tumor was closely attached to the periosteum, it was not difficult to dissect the tumor subperiosteally from the cortex of the femur. The patient received postoperative brachytherapy, but no chemotherapy. Two years later, the tumor recurred with bony destruction of the femur. We reviewed the pre-operative films obtained by various imaging modalities, as well as the histology of the primary tumor, and found minute intravenous tumor emboli in the MR imaging obtained before surgery. Tumor emboli were also observed histologically in the small vessels of the surgically resected tumor. Such intravenous tumor emboli have recently been implicated in the development of regional bone metastasis near the site of the primary lesion in cases of malignant soft tissue tumors. Therefore, we concluded that the tumor recurrence in our case was caused by small tumor emboli invading the perforating veins of the femur. It is therefore emphasized that MR images should be carefully reviewed for the presence of such intravenous tumor emboli before surgery in cases of high-grade malignant sarcomas. As at the time of writing, our patient remains alive and disease-free, with no evidence of any local recurrence or distant metastasis after wide tumor resection for the recurrent tumor.

Case report: chondromyxoid fibroma arising at the clavicular diaphysis.

This report presents an extremely rare case of chondromyxoid fibroma arising at the clavicle. To the best of our knowledge, this may be the first case report demonstrating in detail the clinicopathological findings of chondromyxoid fibroma at the clavicle. The patient was a 34-year-old housewife. Radiography and CT demonstrated an osteolytic lesion with cortical thinning and expansion with partial destruction at the diaphysis of the left clavicle. MRI showed a homogeneous iso-signal intensity mass in T1-weighted imaging and a heterogeneous high-signal intensity in T2-weighted imaging. Histological findings of the widely resected tumor were consistent with chondromyxoid fibroma. The pre-operative diagnosis of chondromyxoid fibroma at an unusual location, as in this case, is difficult from the imaging examinations, or sometimes even from the histological examination of biopsy materials.

Telomerase activity and human telomerase reverse transcriptase mRNA expression are correlated with clinical aggressiveness in soft tissue tumors.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that extends telomere specific repeats on the ends of chromosomes. Telomerase activity has been detected frequently in various types of human tumors and has been associated with cell immortality and oncogenesis. Human telomerase reverse transcriptase (hTERT), a telomerase catalytic subunit, reportedly regulates telomerase activity. Little is known about telomerase activity and hTERT mRNA expression in soft tissue tumors. The objective of this study was to clarify the correlation between these two parameters and clinical aggressiveness in soft tissue tumors. METHODS: In 41 surgically resected soft tissue tumors, telomerase activity was measured by the fluorescence-based telomeric repeat-amplification protocol and hTERT mRNA expression was determined by real-time polymerase chain reaction. RESULTS: Telomerase activity was detected in 52% of sarcomas and in none of the benign soft tissue tumors (P < 0.05). Telomerase activity was found in 77% of 13 locally recurrent sarcomas and in 89% of 9 sarcomas with distant metastasis. The frequency of the presence of telomerase activity in those tumors was significantly greater compared with the frequency of telomerase activity in the other sarcomas (P < 0.05 and P < 0.01, respectively). All telomerase positive sarcomas expressed hTERT mRNA. The mean level of hTERT mRNA expression in sarcomas was significantly greater compared with the mean hTERT mRNA expression level in benign tumors (P < 0.05) and in locally recurrent sarcomas compared with primary sarcomas (P < 0.001). CONCLUSIONS: The results of the current study suggest that the detection of telomerase activity and the level of hTERT mRNA expression are useful markers for evaluating the clinical aggressiveness in soft tissue tumors.

Suppression of tumor growth and pulmonary metastasis in murine osteosarcoma using gene therapy.

We evaluated the effect of gene therapy in the murine osteosarcoma cell line, LM8, which preferentially metastasizes to the lungs. LM8 cells were transduced with the gene for a herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase (lacZ). We investigated the cytotoxicity of LM8 cells bearing an HSV-tk gene after treatment with ganciclovir (GCV). LM8 cells bearing an HSV-tk gene were more sensitive than non-transduced cells. The remarkable inhibition of tumor growth and pulmonary metastases was confirmed in vivo. Our findings indicated that GCV kills tumor cells transduced with HSV-tk in vitro and in vivo.