Histological changes in the olfactory bulb and rostral migratory stream due to interruption of olfactory input.

OBJECTIVE: Periglomerular and granule cells in the adult mammalian olfactory bulb modulate olfactory signal transmission. These cells originate from the subventricular zone, migrate to the olfactory bulb via the Rostral Migratory Stream (RMS), and differentiate into mature cells within the olfactory bulb throughout postnatal life. While the regulation of neuroblast development is known to be affected by external stimuli, there is a lack of information concerning changes that occur during the recovery process after injury caused by external stimuli. To address this gap in research, the present study conducted histological observations to investigate changes in the olfactory bulb and RMS occurring after the degeneration and regeneration of olfactory neurons. METHODS: To create a model of olfactory neurodegeneration, adult mice were administered methimazole intraperitoneally. Nasal tissue and whole brains were removed 3, 7, 14 and 28 days after methimazole administration, and EdU was administered 2 and 4 h before removal of these tissues to monitor dividing cells in the RMS. Methimazole-untreated mice were used as controls. Olfactory nerve fibers entering the olfactory glomerulus were observed immunohistochemically using anti-olfactory marker protein. In the brain tissue, the entire RMS was observed and the volume and total number of cells in the RMS were measured. In addition, the number of neuroblasts and dividing neuroblasts passing through the RMS were measured using anti-doublecortin and anti-EdU antibodies, respectively. Statistical analysis was performed using the Tukey test. RESULTS: Olfactory epithelium degenerated was observed after methimazole administration, and recovered after 28 days. In the olfactory glomeruli, degeneration of OMP fibers began after methimazole administration, and after day 14, OMP fibers were reduced or absent by day 28, and overall OMP positive fibers were less than 20%. Glomerular volume tended to decrease after methimazole administration and did not appear to recover, even 28 days after recovery of the olfactory epithelium. In the RMS, EdU-positive cells decreased on day 3 and began to increase on day 7. However, they did not recover to the same levels as the control methimazole-untreated mice even after 28 days. CONCLUSION: These results suggest that the division and maturation of neuroblasts migrating from the RMS was suppressed by olfactory nerve degeneration or the disruption of olfactory input.

Long-term effects of oral L-carnitine supplementation on anemia in chronic hemodialysis.

BACKGROUND: The therapeutic role of L-carnitine (LC) on the anemia of chronic hemodialized patients is still controversial. In order to clarify the long-term effects of LC administration on renal anemia, an open, observational 12-month study was performed. METHODS: Twenty stable outpatients undergoing hemodialysis were administered LC 900 mg p.o. daily for 12 months. The recombinant human erythropoietin (rHuEPO) dose was adjusted monthly when necessary to maintain the target hemoglobin (Hb) levels. RESULTS: The free LC level increased, while the acyl/free LC ratio decreased significantly 3 months after administration and was then maintained until the end of the study. There was no difference in Hb levels and the erythropoietin resistance index (ERI) during the study period. However, it was observed that ERI decreased significantly in 7 out of 18 patients (responders) 5 months after LC administration and was maintained thereafter (almost 40% reduction of the rHuEPO dose). The acyl/free carnitine ratio at baseline was the most contributing factor distinguishing responders from nonresponders. CONCLUSION: Although the beneficial effect of LC supplementation on renal anemia was not observed in all patients, at least 40% of the patients (responders) showed a significant improvement in ERI after long-term LC administration.

The effects of oral L-carnitine supplementation on physical capacity and lipid metabolism in chronic hemodialysis patients.

BACKGROUND: It is well known that the physical activity in chronic hemodialysis patients decreases compared to that in normal subjects. In order to investigate the effects of L-carnitine on physical capacity and lipid metabolism, a cardiopulmonary exercise test using a bicycle ergometer was performed before and after 3 months of oral L-carnitine supplementation under double-blind conditions. METHODS AND RESULTS: A total of 20 stable outpatients undergoing hemodialysis treatment were randomly divided into 2 groups: controls receiving placebo and patients receiving 900 mg L-carnitine p.o. daily. The levels of free and acyl carnitine increased significantly from 22.9 ± 7.3 to 149.9 ± 51.8 µmol/l and from 16.0 ± 2.8 to 100.3 ± 50.2 µmol/l, respectively, in the L-carnitine group; however, there was no significant change in other plasma lipid profiles. The exercise time was decreased and the heart rate at the anaerobic threshold was increased in the control group 3 months after the study period, but there were no such changes observed in the L-carnitine group. The minute ventilation/CO2 output slope increased significantly from 38.9 ± 7.8 to 43.8 ± 11.8 in the L-carnitine group. It has been speculated that a shift in the energy source occurs from carbohydrate to lipid, in terms of an increase of oxygen demand. CONCLUSION: L-Carnitine supplementation might have some beneficial effects on the physical capacity of chronic hemodialysis patients due to the improvement of the lipid metabolism in the muscle.

Clinical study of therapeutic angiogenesis by autologous peripheral blood stem cell (PBSC) transplantation in 92 patients with critically ischemic limbs.

Patients with critically ischemic limbs due to maintenance hemodialysis and diabetes are increasing in number markedly in Japan. The difficulty of treating critically ischemic limbs is well recognized. Despite active medication and surgical therapy, many critically ischemic limbs are amputated. Ninety-two patients with critically ischemic limbs were treated by transplantation of autologous peripheral blood stem cells (PBSCs). The stem cells were mobilized into the peripheral blood by administration of granulocyte colony stimulating factor (G-CSF). The mobilized mononuclear cells were separated by an apheresis technique using a centrifuge. The separated mononuclear cells contained approximately 4.0 x 10(7) CD34-positive cells. The collected cell suspension was divided into aliquots of 0.5-1.0 ml and transplanted into the muscle of ischemic limbs at 50-70 transplantation points. At 1.5 months after PBSC transplantation, a strong immunostaining of CD34-positive cells and factor VIII, as well as capillary formation, was observed in the muscles into which stems cells had been transplanted. In each patient tested, the serum vascular endothelial growth factor (VEGF) level increased after stem cell transplantation; the mean VEGF level increased by 176%. Of 11 diabetic patients (DM) who were not receiving hemodialysis (HD), there were no amputees regardless of their Fontaine classification. Of 19 patients in the HD(+)DM(-) category, there were no amputations in Fontaine stage I, II, and III patients, whereas three limbs and one toe were amputated in Fontaine stage IV patients. Of 13 patients in the HD(-)DM(+) category, none of the Fontaine stage I, II, or III patients underwent amputation, but six Fontaine stage IV patients underwent amputation. Of 49 patients in the HD(+)DM(+) category, 38 (78%) were classified as Fontaine stage IV, 71% (27/38) of whom had a toe or a limb amputated. In nine patients over 80 years of age, one toe and one limb were amputated. Nondiabetic, nondialyzed patients with ischemic limbs are strongly indicated for stem cell transplantation regardless of Fontaine classification. Therapeutic angiogenesis is effective for critically ischemic limbs resulting from hemodialysis and diabetes until Fontaine stage III, but is of limited effectiveness for stage IV cases.

Blood purification therapy for sepsis.

Accumulating evidences of underlining pathogenesis of sepsis have contributed to the therapeutic strategy for sepsis. Not only endotoxin and cytokine, but also signal transduction through Toll-like receptors could be a strategic target for the management of sepsis. Blood purification therapy including polymyxin B-immobilized hemoperfusion cartridge and continuous hemodiafiltration has shown the beneficial effect on patients with sepsis in Japan. Although they were initially designed to remove endotoxin and cytokines respectively, they might eliminate unexpected mediators responsible for sepsis. Further elucidation of mechanism and randomized controlled studies are needed to establish the role of blood purification therapy in sepsis.

Artificial liver support at present and in the future.

Liver failure is a fatal disease. Liver transplantation is the only established treatment for liver failure; however, donor shortages remain problematic. In the United States and Europe, artificial livers as a bridge to liver transplantation are being considered. In Japan, we have taken a different approach to the treatment of end-stage liver diseases because of the characteristics of the health-care insurance system, regulated by the government. Furthermore, cadaveric liver transplantations are unsuited to the social mores of Japanese culture. Practically speaking, we believe that plasma exchange (PE) and continuous hemodiafiltration (CHDF) are the most effective therapies for the treatment of liver failure, although randomized controlled studies are needed to determine their effects. Overall, we believe that the first line of treatment for liver failure should be PE and CHDF, and the second line should be bioartificial liver support. In the near future, we hope that both gene therapy and regenerative medicine will contribute to the development of a functional artificial liver.

Prevention of limb amputation in patients with limbs ulcers by autologous peripheral blood mononuclear cell implantation.

There are many cases of amputation of ischemic limbs of dialysis patients due to diabetes, despite the availability of medicine therapy and vascular by-pass operations. As there is extensive ruin of the vascular bed due to diabetes, vascular regeneration therapy by stem cell implantation is effective. Thirty patients with ischemic limbs due to diabetes (not including type-I) and on dialysis for chronic renal failure (19 cases), diabetes (5 cases), dialysis patients without diabetes (4 cases), and arteriosclerosis obliterans (ASO, 2 cases) were treated by autologous peripheral blood stem cell (PBSC) implantation where imminent amputation was under consideration. Granulocyte Colony Stimulate Factor (G-CSF: 5 microg/kg/day) was administered subcutaneously for 4 days before PBSC collection, that was carried out using a centrifuge (Spectra and/or CS3000) via the vein. The collected PBSC, containing 4.2 x 10(7) of CD 34 positive cells, was divided into units of 0.5-1.0 mL and implanted, without any purification, to the ischemic area of the limbs in about 65 points. In 21 cases, normalization of limb temperature was observed by thermograph, and symptoms also improved. The result of this first attempt of PBSC implantation is that we were able to save 22 ischemic limbs. This is the first large report of the application of regenerative medicine to peripheral ischemic limbs.

Potential roles of the nucleotide exchange factor ECT2 and Cdc42 GTPase in spindle assembly in Xenopus egg cell-free extracts.

The ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho family of small GTPases. ECT2 contains motifs of cell cycle regulators at its N-terminal domain. We previously showed that ECT2 plays a critical role in cytokinesis. Here, we report a potential role of XECT2, the Xenopus homologue of the human ECT2, in spindle assembly in cell-free Xenopus egg extracts. Cloned XECT2 cDNA encodes a 100 kDa protein closely related to human ECT2. XECT2 is specifically phosphorylated in M phase extracts. Affinity-purified anti-XECT2 antibody strongly inhibited mitosis in Xenopus cell-free extracts. Instead of bipolar spindles, where chromosomes are aligned at the metaphase plane in control extracts, the addition of anti-XECT2 resulted in the appearance of abnormal spindles including monopolar and multipolar spindles as well as bipolar spindles with misaligned chromosomes. In these in vitro synthesized spindle structures, XECT2 was found to tightly associate with mitotic spindles. The N-terminal half of XECT2 lacking the catalytic domain also strongly inhibited spindle assembly in vitro, resulting in the formation of mitotic spindles with a low density. Among the representative Rho GTPases, a dominant-negative form of Cdc42 strongly inhibited spindle assembly in vitro. These results suggest that the Rho family GTPase Cdc42 and its exchange factor XECT2 are critical regulators of spindle assembly in Xenopus egg extracts.

Rho exchange factor ECT2 is induced by growth factors and regulates cytokinesis through the N-terminal cell cycle regulator-related domains.

The ECT2 protooncogene plays a critical role in cytokinesis, and its C-terminal half encodes a Dbl homology-pleckstrin homology module, which catalyzes guanine nucleotide exchange on the Rho family of small GTPases. The N-terminal half of ECT2 (ECT2-N) contains domains related to the cell cycle regulator/checkpoint control proteins including human XRCC1, budding yeast CLB6, and fission yeast Cut5. The Cut5-related domain consists of two BRCT repeats, which are widespread to repair/checkpoint control proteins. ECT2 is ubiquitously expressed in various tissues and cell lines, but elevated levels of ECT2 expression were found in various tumor cell lines and rapidly developing tissues in mouse embryos. Consistent with these findings, induction of ECT2 expression was observed upon stimulation by serum or various growth factors. In contrast to other oncogenes whose expression is induced early in G1, ECT2 expression was induced later, coinciding with the initiation of DNA synthesis. To test the role of the cell cycle regulator/checkpoint control protein-related domains of ECT2 in cytokinesis, we expressed various ECT2 derivatives in U2OS cells, and analyzed their DNA content by flow cytometry. Expression of the N-terminal half of ECT2, which lacks the catalytic domain, generated cells with more than 4N DNA content, suggesting that cytokinesis was inhibited in these cells. Interestingly, ECT2-N lacking the nuclear localization signals inhibited cytokinesis more strongly than the derivatives containing these signals. Mutational analyses revealed that the XRCC1, CLB6, and BRCT domains in ECT2-N are all essential for the cytokinesis inhibition by ECT2-N. These results suggest that the XRCC1, CLB6, and BRCT domains of ECT2 play a critical role in regulating cytokinesis.

Successful treatment of ulcerative colitis with leukocytapheresis using non-woven polyester filter.

Ulcerative colitis is a chronic inflammatory disease of the rectum and colon. Although the pathogenesis of ulcerative colitis is not fully elucidated, cell-mediated immunity plays an important role in disease pathogenesis. Leukocytapheresis is a newly emerging therapy to eliminate activated leukocyte from systemic circulation. We have studied the effects of leukocytapheresis on patients with ulcerative colitis who had failed to respond to conventional therapy. A total of 51 patients with ulcerative colitis were treated with apheresis using a non-woven polyester fiber filter (Finecell, Asahi Medical Co.,Tokyo, Japan) originally developed as a microcoagulation elimination filter for massive transfusion. Of the 51 patients, 33 (64.7%) achieved clinical remission manifested by clinical activity and colonoscopic findings without any adverse effects. This result suggested that leukocytapheresis using Finecell might serve as an alternative therapy for ulcerative colitis as other leukocytapheresis using centrifugation or column.