Long-term survival of a patient with amyotrophic lateral sclerosis (ALS) who received autologous adipose-derived mesenchymal stem cells.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a progressive debilitating neurodegenerative disease with a life expectancy of 3-5 years from initial symptoms. We report a case of ALS who received autologous adipose-derived mesenchymal stem cells (ADSC) and was followed up for 7 years. CASE REPORT: A 46-year-old man noticed weakness of his legs, difficulties on going down the stairs and coughing during eating in 2009. After complete workout, a diagnosis of ALS was confirmed. His ALS Functional Rating Scale-R (ALSFS-R) was 43. Symptoms rapidly progressed and he coughed and choked during eating. Starting in 2013, the patient received a total of six intravenous infusions of autologous ADSC. Changes in electromyogram, nerve conduction, and ALSFS-R were assessed. RESULTS: Soon after the administration, he noticed that he did not cough during conversation or eating food. Although he had difficulty in walking down the stairs, he remained well without coughing, dysarthria, or dysphagia. His ALSFS-R increased up to 45. Fascicular potentials were not detected in any muscles examined including trapezius muscle and rectus femoris muscles. The patient was well for 7 years after ADSC therapy by the time of this report and more than 10 years from the time of onset. CONCLUSIONS: The present case suggests that autologous ADSC can be administered safely and may be potentially useful in patients with ALS. Further investigations are warranted in order for the results to be generalized to other ALS patients.

Improving the electron spin properties of nitrogen-vacancy centres in nanodiamonds by near-field etching.

The nitrogen-vacancy (NV) centre in diamond is a promising candidate for quantum computing applications and magnetic sensing applications, because it is an atomic-scale defect with stable coherence time (T2) and reliable accessibility at room temperature. We demonstrated a method for improving the NV spin properties (the full width half maximum (FWHM) value of the magnetic resonance spectrum and T2) through a near-field (NF) etching method under ambient conditions. The NF etching method, based on a He-Cd ultraviolet laser (325 nm), which is longer than the absorption edge of the oxygen molecule, enabled selective removal of defects on the nanodiamond surface. We observed a decrease in the FWHM value close to 15% and an increase in T2 close to 25%. Since our technique can be easily reproduced, a wide range of NV centre applications could be improved, especially magnetic sensing applications. Our results are especially attractive, because they have been obtained under ambient conditions and only require a light source with wavelength slightly above the O2 absorption edge.

MR Imaging Findings of a Leiomyosarcoma of the Thoracic Spine: A Case Report.

We report a case of leiomyosarcoma of the thoracic spine. Primary leiomyosarcoma is a malignant connective tissue tumor originating from smooth muscle cells. Leiomyosarcoma frequently occurs in the uterus, retroperitoneal space, gastrointestinal tract, and deep soft tissues; primary leiomyosarcoma of the bone is rare. The MR imaging including intravoxel incoherent motion (IVIM) imaging findings of the current case indicated a low diffusion coefficient and high blood flow, which were in concurrence with high cell density on histology and increased vascularity by angiography. Although some benign tumors such as osteoblastoma and giant cell tumor would show similar findings on IVIM imaging, these additional imaging features may narrow the differential diagnosis of spinal tumors.

Antimicrobial photodynamic therapy using a diode laser with a potential new photosensitizer, indocyanine green-loaded nanospheres, may be effective for the clearance of Porphyromonas gingivalis.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) is a new treatment method for the removal of infectious pathogens using a photosensitizer and light of a specific wavelength, e.g., toluidine blue with a wavelength of about 600 nm. We explored a new photosensitizer and focused on indocyanine green (ICG), which has high absorption at a wavelength of 800-805 nm. We investigated the bactericidal effect of PDT on Porphyromonas gingivalis using a new photosensitizer, ICG-loaded nanospheres with an 805 nm wavelength low-level diode laser irradiation. METHODS: We designed ICG-loaded nanospheres coated with chitosan (ICG-Nano/c) as a photosensitizer. A solution containing Porphyromonas gingivalis (10(8)  CFU/mL) with or without ICG-Nano/c (or ICG) was prepared and irradiated with a diode laser or without laser irradiation as a negative control. The irradiation settings were 0.5 W with a duty ratio of 10%, for 3-100 ms in repeated pulse (RPT) or continuous wave mode. CFU were counted after 7 d of anaerobic culture. RESULTS: We observed that ICG-Nano/c could adhere to the surface of P. gingivalis. When ICG-Nano/c was used for aPDT, irradiation with RPT 100 ms mode gave the lowest increase in temperature. Laser irradiation with ICG-Nano/c significantly reduced the number of P. gingivalis (i.e., approximately 2-log10 bacterial killing). The greatest bactericidal effect was found in the RPT 100 ms group. However, laser irradiation (RPT 100 ms) with ICG, as well as without photosensitizer, had no effect on the number of bacteria. CONCLUSIONS: Within the limits of this study, ICG-Nano/c with low-level diode laser (0.5 W; 805 nm) irradiation showed an aPDT-like effect, which might be useful for a potential photodynamic periodontal therapy.

Resident macrophages are involved in intestinal transplantation-associated inflammation and motoric dysfunction of the graft muscularis.

Gut manipulation and ischemia/reperfusion evoke an inflammatory response within the intestinal muscularis that contributes to dysmotility. We hypothesize that resident macrophages play a key role in initiating the inflammatory cascade. Isogenic small bowel transplantation was performed in Lewis rats. The impact of recovery of organs on muscularis inflammation was investigated by comparing cold whole-body perfusion after versus prior to recovery. The role of macrophages was investigated by transplantation of macrophage-depleted gut. Leukocytes were counted using muscularis whole mounts. Mediator expression was determined by real-time RT-PCR. Contractility was assessed in a standard organ bath. Both organ recovery and ischemia/reperfusion induced leukocyte recruitment and a significant upregulation in IL-6, MCP-1, ICAM-1 and iNOS mRNAs. Although organ recovery in cold ischemia prevented early gene expression, peak expression was not changed by modification of the recovery technique. Compared to controls, transplanted animals showed a 65% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted isografts exhibited significant less leukocyte infiltration and only a 19% decrease in contractile activity. In summary, intestinal manipulation during recovery of organs initiates a functionally relevant inflammatory response within the intestinal muscularis that is massively intensified by the ischemia reperfusion injury. Resident muscularis macrophages participate in initiating this inflammatory response.

Acute rejection in allogeneic rodent small bowel transplantation causes smooth muscle dysfunction via an inflammatory response within the intestinal muscularis.

INTRODUCTION: Isogeneic intestinal transplantation elicits an inflammatory response within the intestinal muscularis that is associated with dysmotility. Usually the inflammation and the postoperative motor dysfunction resolve within a few days after small bowel transplantation (SBTx). However, the onset of acute rejection in allogeneic SBTx is again associated with dysmotility. We hypothesized that dysmotility during acute rejection is based on coexpression of kinetically active mediators by the alloreactive leucocyte infiltrate. MATERIALS AND METHODS: Rat SBTx (BN to Lew and BN to BN) was performed without immunosuppression. Animals were sacrificed at 1, 4, and 7 days after SBTx. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by reverse transcriptase polymerase chain reaction. Muscle contractility was assessed in a standard organ bath. RESULTS: Transplanted animals showed a significant inflammatory response within the muscularis at day 1 after SBTx. However, allogeneic transplanted animals exhibited a significant second inflammatory peak at day 7 (mRNA induction: iNOS 150-fold; tumor necrosis factor-alpha 18-fold; interferon-gamma 397-fold), parallel to the onset of rejection. This change was associated with a significant leukocyte infiltration. Compared to controls, allogeneic transplanted animals showed a 29% decrease in smooth muscle contractility on days 1 and 4 and a 71% decrease of contractility on postoperative day 7. CONCLUSIONS: The motor dysfunction of transplanted small bowel during acute rejection is associated with an inflammatory reaction in the intestinal muscularis. The initial unspecific inflammation process immediately after transplantation is reactivated and intensified during acute rejection.

Mechanism and impact of organ harvesting and ischemia-reperfusion injury within the graft muscularis in rat small bowel transplantation.

INTRODUCTION: Inflammatory events within the gut muscularis contribute to dysmotility. We hypothesized that manipulation during organ harvesting initiated an inflammatory response via muscularis macrophages and that this cascade was amplified during reperfusion. METHODS: Small bowel transplantation was performed in Lewis rats. To investigate the impact of organ harvesting on muscularis inflammation, cold whole-body perfusion was performed after versus prior to organ harvesting. The role of macrophages was investigated by transplantation of the macrophage-depleted gut. Leukocyte infiltration was investigated in muscularis whole mounts. Mediator mRNA expression was determined by real-time reverse transcriptase polymerase chain reaction. Contractility was assessed in a standard organ bath. RESULTS: Organ harvesting and ischemia-reperfusion induced leukocyte recruitment and mRNA upregulation in the muscularis: interleukin-6 12217-fold, monocyte chemoattractant protein-1 62-fold, ICAM-1 12-fold, cyclooxygenase-2: 8-fold, iNOS: 150-fold. Although organ harvesting with cold ischemia prevented early gene expression, peak expression at 3-hour reperfusion was not changed by modification of the harvesting technique. Compared to controls, transplanted animals showed a 63% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted gut exhibited significantly fewer leukocytes and only a 16% decrease in contractility. CONCLUSIONS: Gut manipulation during organ harvesting initiates an inflammatory response within the muscularis that is massively intensified during reperfusion. This change contributes to muscular dysfunction. Furthermore, the results suggested that resident macrophages play a key role in initiating this process.

Indoleamine 2,3-dioxygenase is necessary for cytolytic activity of natural killer cells.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan catabolic enzyme that is widely distributed in various tissues. In peripheral blood mononuclear cells (PBMCs), production of IDO by macrophages or dendritic cells has been reported to inhibit T-cell activation and proliferation. In the present study, we have determined that other phenotypes of PBMCs also express IDO. In cultures of PBMCs, IDO was found predominantly in monocyte by immunohistochemistry. Reverse transcriptase polymerase chain reaction analysis showed that IDO mRNA was expressed in T lymphocytes, B lymphocytes and natural killer (NK) cells and that expression was increased upon activation with interferon-gamma. The cytotoxicity of NK cells against K562 and HepG2 cells was reduced by IDO inhibitor. These results suggest that IDO in NK cells is essential for NK cells to generate killing activity against cancer cells.

DeltaFosB, but not FosB, induces delayed apoptosis independent of cell proliferation in the Rat1a embryo cell line.

The fates of Rat1a cells expressing FosB and DeltaFosB as fusion proteins (ER-FosB, ER-DeltaFosB) with the ligand binding domain of human estrogen receptor were examined. The binding of estrogen to the fusion proteins resulted in their nuclear translocation and triggered cell proliferation, and thereafter delayed cell death was observed only in cells expressing ER-DeltaFosB. The proliferation of Rat1a cells, but not cell death triggered by ER-DeltaFosB, was completely abolished by butyrolactone I, an inhibitor of cycline-dependent kinases, and was partly suppressed by antisense oligonucleotides against galectin-1, whose expression is induced after estrogen administration. The cell death was accompanied by the activation of caspase-3 and -9, the fragmentation of the nuclear genome and cytochrome c release from the mitochondria, and was suppressed by zDEVD-fmk and zLEHD-fmk but not zIETD-fmk. The cell death was not suppressed by exogenous His-PTD-Bcl-x(L) at all, suggesting involvement of a Bcl-x(L)-resistant pathway for cytochrome c release.

Experimental small bowel transplantation using a newborn intestine in rats: IV. Effect of cold preservation on graft neovascularization.

BACKGROUND/PURPOSE: University of Wisconsin (UW) solution is one of the most superior organ preservation solutions for liver, kidney, and pancreas; however, it still is controversial for intestinal preservation. Here, the authors studied the efficacy of preservation with 2 kinds of solutions, UW and modified TOM (m-TOM) solutions in our experimental newborn intestinal transplantation model. UW solution was used as a standard intracellular and m-TOM solution as an extracellular preservation solution. Lactated ringer (LR) solution was used as a control. METHODS: Newborn intestine, which were preserved in these solutions for 24 or 48 hours, were transplanted in the subcutaneous spaces of the syngeneic recipients without surgical vascular anastomosis and histologically examined 14 days after grafting. The preserved grafts were evaluated histologically by use of light and electron microscopy just after preservation. The biochemical parameters such as LDH and serotonin also were measured in the supernatants of preservation solutions. RESULTS: Fresh newborn grafts were revascularized successfully at a rate of 80% (16 of 20). After 24 hours of preservation, 65% (13 of 20), 75% (15 of 20), and 85% (17 of 20) of the grafts were observed to be revascularized in LR, m-TOM, and UW solutions, respectively. After 48 hours of preservation, 60% (12 of 20), 80% (16 of 20), and 80% (16 of 20) of the grafts also were revascularized in the respective solutions (no statistic difference among the groups). The cold-preservation did not affect the neovascularization of newborn intestine until 48 hours. Histologic findings of the preserved intestine and biochemical analyses showed that UW and m-TOM solutions kept villous architectures of the preserved grafts, however, might be harmful to enterochromaffin cells. CONCLUSION: Long-time preservation of newborn intestine did not interfere with neovascularization and maturation. J Pediatr Surg 36:1805-1810.

Electroretinogram contact lens electrode with tri-color light-emitting diode.

PURPOSE: This study was performed to evaluate a new electroretinogram (ERG) contact lens electrode containing four light-emitting diodes (LEDs) that are used for both stimulus and background light. METHODS: The luminance of each LED could be changed independently and used as stimulus light. Red, blue, bright white, and flickering ERGs were recorded in 12 normal subjects and two patients with progressive cone dystrophy. The long-duration light stimuli separated the on- and off-responses of the ERG. This equipment is not according to the ISCEV standard. RESULTS: The tri-color LED electrode contact lens can efficiently produce and record ERG responses. Off-responses were recordable separately from on-responses by lengthening the stimulus duration. CONCLUSION: This combined stimulus-electrode system is compact and portable. Combined with the portable amplifier and the recorder, the ERGs can be recorded easily in an operating room, at patients' bedside, and in remote locations away from clinics and hospitals.

Dendritic cell vaccination with MAGE peptide is a novel therapeutic approach for gastrointestinal carcinomas.

The MAGE gene is selectively expressed in cancer tissues such as melanoma or gastrointestinal carcinomas, whereas no expression is observed in normal tissues except testis. There are several reports of successful induction of HLA class I-restricted antitumor CTLs using MAGE peptides, and some clinical trials with these immunogenic peptides were reported as effective for some patients with malignant melanoma. However, there are no similar studies in gastrointestinal carcinomas, which are important neoplasms. Autologous dendritic cells (DCs) were generated ex vivo and were pulsed with MAGE-3 peptide, depending on the patient's HLA haplotype (HLA-A2 or A24). Patients were immunized with DC pulsed with MAGE-3 peptide every 3 weeks at four times. Twelve patients with advanced gastrointestinal carcinoma (six stomach, three esophagus, and three colon) were treated, and no toxic side effects were observed. Peptide-specific CTL responses after vaccination were observed in four of eight patients. Improvement in performance status was recognized in four patients. Tumor markers decreased in seven patients. In addition, minor tumor regressions evidenced by imaging studies were seen in three patients. These results suggested that DC vaccination with MAGE-3 peptide is a safe and promising approach in the treatment of gastrointestinal carcinomas.

Green fluorescent protein-transgenic rat: a tool for organ transplantation research.

The purpose of this study is to evaluate green fluorescent protein (GFP) transgenic rats for use as a tool for organ transplantation research. The GFP gene construct was designed to express ubiquitously. By flow cytometry, the cells obtained from the bone marrow, spleen, and peripheral blood of the GFP transgenic rats consisted of 77, 91, and 75% GFP-positive cells, respectively. To examine cell migration of GFP-positive cells after organ transplantation, pancreas graft with or without spleen transplantation, heart graft with or without lung transplantation, auxiliary liver and small bowel transplantation were also performed from GFP transgenic rat to LEW (RT1(1)) rats under a 2-week course of 0.64 mg/kg tacrolimus administration. GFP-positive donor cells were detected in the fully allogenic LEW rats after organ transplantation. These results showed that GFP transgenic rat is a useful tool for organ transplantation research such as cell migration study after organ transplantation without donor cell staining.

Increased expression of P-cadherin mRNA in the mouse peritoneum after carbon dioxide insufflation.

BACKGROUND: Although systemic responses to carbon dioxide (CO(2)) pneumoperitoneum have been studied, there have been few reports of local responses within the peritoneum. We investigated the expression of mRNA for adhesion molecules involved in cell-cell interactions, including ICAM-1, VCAM-1, CD44, E-cadherin, P-cadherin, and N-cadherin, after the induction of a CO(2) pneumoperitoneum in mice. METHODS: Mice were treated with CO(2) pneumoperitoneum (4-6 mmHg for 30 min) and then killed after 24 h, 48 h, and 72 h. The peritoneum of the abdominal wall was resected, and total RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform extraction procedure, cDNA were synthesized by reverse transcription. Expression of the mRNA for each gene was normalized to that of b-actin for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of P-cadherin mRNA was significantly increased at 48 h (p = 0.007) and returned to the control level by 72 h after CO(2) pneumoperitoneum. The expression of CD44 increased gradually, reaching a peak at 48 h and returning to the control value by 72 h after CO(2) pneumoperitoneum. Expression of ICAM-1 mRNA was not changed significantly after the application of CO(2). CONCLUSION: The expression of P-cadherin mRNA in the peritoneum can be induced to repair injuries to mesothelial cells caused by CO(2) pneumoperitoneum.

Orthotopic intestinal transplantation using the cuff method in rats: a histopathological evaluation of the anastomosis.

Segmental small intestine transplantation (SIT) in rats, using a cuff technique, has achieved a high success rate. However, there have been few reports on the influence of the foreign body reaction to polyethylene cuff on vessel anastomoses and graft after SIT. This study involves the histopathological examination of the site of cuff anastomosis and grafts in the short- and long-term survival of segmental SIT. The data obtained from the suture anastomosis model also served as a control. One week after heterotopic segmental SIT using the cuff technique, orthotopic continuations were carried out in syngeneic combination. Twenty-five of 30 rats surviving >200 days (83.3%) were examined for vessel anastomosis. All arterial anastomoses were patent, but the portovenous anastomoses in 10 grafts (33%) were totally occluded and were associated with the formation of collateral vessels. Histopathological examination demonstrated good patency of the artery and vein anastomotic site in the short term, but granulation, fibrosis, and neovascularization at the anastomosis site surrounding the cuffs in the long-surviving group. However, the grafts appeared to be intact, with normal features of the villi. On the contrary, the site of the sutured anastomosis in the long-survival rats showed no inflammatory reaction. Although a polyethylene cuff caused foreign body reaction, the graft blood supplies were maintained by collateral vessels. Considering the low mortality and high success rate, polyethylene cuff is good for short-term study and an alternative method for long-term SIT experiments.

Anti-apolipoprotein A-I autoantibody: characterization of monoclonal autoantibodies from patients with systemic lupus erythematosus.

OBJECTIVE: The autoantibody to apolipoprotein A-I (apoA-I), a major constituent of high density lipoproteins (HDL), has been detected in sera of patients with systemic lupus erythematosus (SLE). We established a series of monoclonal anti-apoA-I antibodies (MAAI) from 2 patients with SLE and report the reactivities of MAAI with oxidized HDL, anionic substances, and blood coagulation factors. METHODS: Peripheral blood B cells from patients with SLE were immortalized by Epstein-Barr virus, and B cells secreting anti-apoA-I antibodies (AAI) were fused with mouse myeloma cells. Six MAAI reactive with human apoA-I in both ELISA and immunoblotting analysis were established. The reactivities of MAAI with HDL, ssDNA and dsDNA, phospholipids such as cardiolipin (CL), and coagulation factors were examined by ELISA. RESULTS: Although all MAAI bound effectively to apoA-I after the protein had been denatured and transferred to the filter membrane (in immunoblotting analyses), they bound less effectively to apoA-I present in HDL. Both oxidation of HDL in the presence of Mn2+ and an association of apoA-I with autoxidized trilinolein strongly enhanced the binding of MAAI to apoA-I, suggesting that MAAI recognize a defined region of apoA-I, which is exposed upon interacting with oxidatively modified lipids. MAAI showed a functional heterogeneity in their cross-reactivity with self-components: some MAAI were shown to cross-react with anionic substances such as CL and ssDNA, and one MAAI was shown to bind effectively to thrombin. CONCLUSION: We identified a novel family of AAI that shows preferential binding to apoA-I in oxidatively modified HDL. These AAI are composed of antibodies with heterogeneous cross-reactivities to various self-components such as anionic phospholipids, ssDNA, and thrombin.