Fuminori Kanaya

Sciatic Function Index, Nerve Conduction Tests, Muscle Contraction, and Axon Morphometry as Indicators of Regeneration

&NA; The purpose of this study was to determine which parameters were the best measure of nerve regencration, assuming that the sciatic functional index (SFI) represented the “gold standard.” Three different sciatic functional indexes and 11 commonly used electrophysiologic and morphologic indicators of regeneration were all determined in 24 rats 12 weeks after one of three lesions was created in the sciatic nerve. With linear regression analysis, only fiber/axon diameter ratio (D/d) and myelin thickness/axon diameter ratio showed statistically significant correlations with sciatic functional index (r = 0.55 and 0.53, respectively). The other 11 parameters had poorer correlation. Therefore, if sciatic functional index is the best measure of comprehensive nerve function, then other parameters are not. It is probable that each parameter measures some different component of the regeneration process. A stepwise multiple linear regression analysis produced a model that included D/d, nerve conduction velocity, and nerve action potential amplitude that gave a slightly better correlation (r = 0.67). The relatively poor correlation between sciatic functional index and the other parameters of nerve function indicates that all nerve regeneration studies must be interpreted carefully before comparisons are made. Furthermore, the best measure of nerve function remains unproved or undiscovered in the experimental animal.

Functional results of vascularized versus nonvascularized nerve grafting.

Functional results of vascularized nerve grafts (VNG), end-to-end (E-E) repair, and nonvascularized nerve grafts (NNG) were compared using 75 rats. For all three groups, the sciatic function index (SFI) was measured for 12 weeks. For the vascularized nerve graft and nonvascularized nerve graft groups, it was measured every 4 weeks to week 36. The vascularized nerve graft and end-to-end repair groups showed significantly better recovery than the nonvascularized nerve graft group 4 weeks after surgery, and this pattern continued throughout the experiment. Results from the vascularized nerve graft and end-to-end repair groups did not differ significantly. Twelve weeks after surgery, morphologic and electrophysiologic assessments were used to compare the experimental and contralateral legs. The vascularized nerve graft group showed significantly better recovery than the nonvascularized nerve graft group on all measures except muscle weight and axon count, even though the grafts were completed in a normal bed.

High efficacy of tumor-targeting Salmonella typhimurium A1-R on a doxorubicin- and dactolisib-resistant follicular dendritic-cell sarcoma in a patient-derived orthotopic xenograft PDOX nude mouse model

Follicular dendritic-cell sarcoma (FDCS) is a rare and recalcitrant disease. In the present study, a patient-derived orthotopic xenograft (PDOX) mouse model of FDCS was established in the biceps muscle of nude mice. The FDCS PDOX was resistant to both doxorubicin (DOX) and NVP-BEZ235, dactolisib (BEZ) an experimental agent which is a dual pan-phosphoinositide 3-kinase-mammalian target of rapamycin inhibitor. However, in contrast to DOX and BEZ, the FDCS PDOX was sensitive to the tumor-targeting bacterial strain, Salmonella typhimurium A1-R (S. typhimurium A1-R). The combination of S. typhimurium A1-R and either DOX or BEZ did not increase the antitumor efficacy of S. typhimurium A1-R, indicating that DOX and BEZ were not active in this PDOX model. The efficacy of S. typhimurium A1-R in this recalcitrant FDCS gives strong impetus to move bacterial therapy to clinical trials for this disease. The findings of the present study are of particular importance since it demonstrates that S. typhimurium A1-R is effective in a PDOX model of FDCS established from a patient who failed DOX therapy.

Mutations in CD96, a Member of the Immunoglobulin Superfamily, Cause a Form of the C (Opitz Trigonocephaly) Syndrome

The C syndrome is characterized by trigonocephaly and associated anomalies, such as unusual facies, psychomotor retardation, redundant skin, joint and limb abnormalities, and visceral anomalies. In an individual with the C syndrome who harbors a balanced chromosomal translocation, t(3;18)(q13.13;q12.1), we discovered that the TACTILE gene for CD96, a member of the immunoglobulin superfamily, was disrupted at the 3q13.3 breakpoint. In mutation analysis of nine karyotypically normal patients given diagnoses of the C or C-like syndrome, we identified a missense mutation (839C-->T, T280M) in exon 6 of the CD96 gene in one patient with the C-like syndrome. The missense mutation was not found among 420 unaffected Japanese individuals. Cells with mutated CD96 protein (T280M) lost adhesion and growth activities in vitro. These findings indicate that CD96 mutations may cause a form of the C syndrome by interfering with cell adhesion and growth.

Will Preoperative Atrophy and Fatty Degeneration of the Shoulder Muscles Improve after Rotator Cuff Repair in Patients with Massive Rotator Cuff Tears

Recently, retear rate after repair for massive cuff tear have been improved through devised suture techniques. However, reported retear rate is relevant to preoperative atrophy and fatty degeneration. The purpose of this study was to investigate whether preoperative atrophy and fatty degeneration of rotator cuff muscles improve by successful repair. Twenty-four patients with massive rotator cuff tear were evaluated on the recovery of atrophy and fatty degeneration of supraspinatus and infraspinatus muscle after surgery. Atrophy was classified by the occupation ratio and fatty degeneration by modified Goutalliers classification. Both were assessed on magnetic resonance imaging (MRI) before and after the operation. When the cuff was well repaired, improvement of the atrophy and fatty degeneration were observed in a half and a one-fourth of the cases, respectively. In retear cases, however, atrophy and fatty degeneration became worse. Improvement of atrophy and fatty degeneration of the rotator cuff muscles may be expected in the cases with successful achievement of rotator cuff repair for large and massive tear.

Improvement in motor function after brachial plexus surgery

Motor functional recovery of 52 patients with brachial plexus surgery followed up for more than 2 years was evaluated. Fifty-eight surgical procedures were done, including autologous nerve grafting (38 cases), neurolysis (14 cases), and neurotization (6 cases). Overall results, evaluated according to the 0 to 5 formula of the Medical Research Council, were as follows: good, 58%; fair, 15%; and poor, 27%. Good results were evident in 58% of patients with nerve grafts and in 64% of those with neurolysis. In patients with neurotization, no good recovery and only one fair recovery were seen. Patients with open injuries showed good recovery, whereas the group with closed injury showed good recovery in only 48%. Patients with closed injuries caused by traffic accidents showed a worse recovery than those caused by other means. Patients with closed injuries and nerve grafting done within 3 months of injury or neurolysis within 6 months showed better recovery.

Sensory and motor fiber differentiation with Karnovsky staining

We examined four acetylthiocholine methods based on Karnovskys procedure--two fast-acting requiring 1 hour and two slow-acting requiring 24 hours. We compared these with our modification, which requires less than an hour and is simple to use. Rabbit sciatic nerves and spinal cords were used to compare methods. Our modification showed clearer differentiation than other fast-acting methods and staining identical to slow-acting methods. In blind examination of radial nerve specimens stained with our method, motor and sensory fascicles were correctly identified, showing sensitivity and specificity of 100%. In 12 clinical cases, our method produced staining in the proximal stump as long as 16 months after injury and in the distal stump as long as 5 days after injury. In 10 of 12 patients, this staining helped in aligning motor fascicles to motor fascicles and sensory fascicles to sensory fascicles.

Search for active endogenous retroviruses: identification and characterization of a HERV-E gene that is expressed in the pancreas and thyroid

AbstractTo elucidate possible physiological functions of human endogenous retroviruses (HERVs) and their role in the pathogenesis of human diseases, we have developed a strategy to identify transcriptionally active HERV genes. By this approach, we have identified and isolated an active HERV-E gene that was mapped to 17q11. Although the gene was predicted to produce no intact viral particles due to the presence of stop codons, long open reading frames were retained in each gag and pol region. Northern blot analyses revealed in the pancreas (and thyroid) two major transcripts, 3.3 and 4.1kb in size, associated with 500- to 600-nucleotide-longer minor bands. Preferential expression in pancreas and thyroid gland tissues may suggest a role for this gene in physiological functions common to these tissues.

In Vivo Gene Transfer Between Interacting Human Osteosarcoma Cell Lines Is Associated With Acquisition of Enhanced Metastatic Potential

We report here in vivo gene transfer between cancer cells is associated with acquisition of high metastatic behavior. The 143B‐GFP cell line with high metastatic potential and the MNNG/HOS‐RFP cell line with low metastatic potential, both derived from the TE85 human osteosarcoma cell line, were either co‐transplanted or transplanted alone in the tibia in nude mice. Upon mixed transplantation of the two differently labeled sublines, resulting metastatic colonies are single colored either red or green, thereby demonstrating their clonality and enabling facile color‐coded quantification. When MNNG/HOS‐RFP and 143B‐GFP were co‐transplanted in the tibia, the number of lung metastases of MNNG/HOS‐RFP increased eight‐fold compared to MNNG/HOS‐RFP transplanted alone (P < 0.01). In contrast, no enhancement of MNNG/HOS‐RFP metastases occurred when MNNG/HOS‐RFP and 143B‐GFP were transplanted separately in the right and left tibiae, respectively. This result suggests that the presence of 143B‐GFP increased the metastatic potential of MNNG/HOS‐RFP within the mixed tumor. We observed transfer of the Ki‐ras gene from 143B‐GFP to MNNG/HOS‐RFP after they were co‐implanted suggesting the Ki‐ras played a role in increasing the metastatic potential of MNNG/HOS‐RFP in the presence of 143B‐GFP. These data suggest the possible role of in vivo gene transfer in enhancing the metastatic potential of cancer cells. The data also further demonstrated the power of color‐coded imaging to visualize cancer‐cell/cancer‐cell interactions in vivo. J. Cell. Biochem. 108: 362–367, 2009.

3‐Dimensional Tissue Is Formed From Cancer Cells In Vitro on Gelfoam®, But Not on MatrigelTM

Cell and tissue culture can be performed on different substrates such as on plastic, in Matrigel™, and on Gelfoam®, a sponge matrix. Each of these substrates consists of a very different surface, ranging from hard and inflexible, a gel, and a sponge‐matrix, respectively. Folkman and Moscona found that cell shape was tightly coupled to DNA synthesis and cell growth. Therefore, the flexibility of a substrate is important for cells to maintain their optimal shape. Human osteosarcoma cells, stably expressing a fusion protein of αv integrin and green fluorescent protein (GFP), grew as a simple monolayer without any structure formation on the surface of a plastic dish. When the osteosarcoma cells were cultured within Matrigel™, the cancer cells formed colonies but no other structures. When the cancer cells were seeded on Gelfoam®, the cells formed three‐dimensional tissue‐like structures. The behavior of 143B osteosarcoma cells on Gelfoam® in culture is remarkably different from those of these cells in monolayer culture or in Matrigel™. Tissue‐like structures were observed only in Gelfoam® culture. The data in this report suggest a flexible structural substrate such as Gelfoam® provides a more in vivo‐like culture condition than monolayer culture or MatrigelTM and that MatrigelTM does not result in actual three‐dimensional culture. J. Cell. Biochem. 115: 1362–1367, 2014.