Rapamycin as an inhibitor of osteogenic differentiation in bone marrow-derived mesenchymal stem cells

Background An autograft of cultured bone marrow-derived mesenchymal stem cells has already been used in clinical practice. In those patients whose bone marrow cannot be used, a cell allograft with the use of immunosuppressant drugs will be an option in the future. However, little is known about the effects of immunosuppressant drugs on mesenchymal stem cells. This study assessed the effects of immunosuppressant drugs on osteogenic differentiation of mesenchymal stem cells and analyzed the manner in which immunosuppressant drugs modulate the osteogenic effect of dexamethasone. Methods Rat bone marrow cells were cultured with or without dexamethasone as an osteogenic supplement. In each experimental group, one of three immunosuppressants (rapamycin, cyclosporine A, or FK506) was added. As a control, cells were cultured without immunosuppressants. Histologically, mineralization was assessed by alizarin red S staining and phase-contrast microscopy. Biochemically, alkaline phosphatase activity, calcium content, and osteocalcin content were assessed. Results On histological analysis, no mineralized nodules were seen on alizarin red S staining or phase-contrast microscopy in the groups not treated with dexamethasone, except in the group that was treated with FK506. Mineralized nodules were seen in the groups treated with dexamethasone, except in the group that was treated with rapamycin. On biochemical analysis, it was found that, compared to the control group, rapamycin reduced alkaline phosphatase activity and the calcium content of mesenchymal stem cells; FK506 increased alkaline phosphatase activity, calcium content, and osteocalcin content; and cyclosporine A had negligible effects. Dexamethasone increased alkaline phosphatase activity, calcium content, and osteocalcin content, but these effects were decreased by rapamycin. Conclusions Rapamycin did not have an osteogenic effect on mesenchymal stem cells, but inhibited the effect of osteogenic differentiation induced by dexamethasone. In contrast, FK506 had an osteogenic effect on mesenchymal stem cells. Therefore, FK506 might be more useful than rapamycin in allogeneic transplantation of mesenchymal stem cells.     

Axonal regeneration of retinal ganglion cells in the cat geniculocortical pathway

The optic nerve of anesthetized cats was completely cut and the autologous sciatic nerve was transplanted. Sixty days later some populations of retinal ganglion cells were shown to regenerate the axon with retrograde HRP labeling. We verified that ganglion cells that had projected to the lateral geniculate nucleus (LGN) were able to regenerate through the transplant with a double-labeling method: diI was injected into the LGN prior to the transplantation, and dextran-fluorescein was injected into the graft after axonal regeneration. Intracellular injection of HRP into regenerating ganglion cells in an in vitro preparation revealed that the two major cell types projecting to the LGN, and β, regenerated axons and showed normal dendritic morphology.     

K1 Antigen, Serotype and Antibiotic Susceptibility of Escherichia coli Isolated from Cerebrospinal Fluid, Blood and Other Specimens from Japanese Infants

K1 antigens, serotypes and antibiotic susceptibilities of Escherichia coli isolates from neonates and infants were investigated. The presence of K1 antigen was tested by the K1-specific phage method. The number of K1 positive strains was 27 (84%) of 32 isolates from cerebrospinal fluid, 11 (25%) of 44 from blood and 4 (22%) of 18 from other specimens. Fourteen (33%) of the K1 positive strains were serotyped as O16:H6, and 8, 7 and 5 were serotyped as O18ac:H7, O1:H7 and O7:H-, respectively. One of 5 of the K1 negative strains were distributed into 30 different combinations of O and H antigens. The ampicillin resistance rates were 19% in K1 positive strains and 45% in K1 negative ones. The incidence of chloramphenicol resistance was the same in K1 positive and negative strains (21%). Ampicillin resistance was not noted in O16: H6 strains, but the incidence of antibiotic resistance was high (65% to ampicillin and 53% to chloramphenicol) in the rough-type strains.     

Creatine kinase release from regenerated muscles after eccentric contractions in rats

The purpose of this study was to test the hypothesis that an increase in plasma creatine kinase (CK) activity after eccentric contractions (ECC) would be attenuated in regenerated muscle fibres. Adult male Wistar rats (aged 12–14 weeks) were randomly assigned to a treatment group (n =14) or a control group (n = 10). In the treatment group, 1.2% barium chloride solution (BaCl₂) was injected into the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles to induce degeneration and subsequent regeneration. The same amount of isotonic saline solution was injected into TA and EDL for the control group. Histological observation showed that approximately 50% of the fibres in the transverse sections of both muscles underwent necrosis 2 days after BaCl₂ injection. The CK activity increased about tenfold at 2–4 h after BaCl₂ injection. At 4 weeks after BaCl₂ injection, when the regeneration process was almost complete, the TA and EDL of anaesthetized rats from both groups were subjected to ECC in which maximal dorsiflexion was caused by nerve electrical stimulation and the flexed foot was forcibly extended by a lever arm connected to a motor. This action was performed in 2 sets of 30 repetitions. Maximal isometric torque of the dorsiflexors decreased to about 15% (P<0.01) of the pre-ECC value immediately after the exercise. Blood samples were collected before and 2, 4, 12, 24, 48 h after ECC. The CK activity increased significantly (P < 0.01) and peaked at 2–4 h after ECC, and there was no significant difference in the amount of CK increase between the treatment [1007 (SEM 120) IU · I^–1] and the control [1064 (SEM 120) IU · 1^–1 group. Contrary to the hypothesis, CK release after ECC was not attenuated in muscle regenerated from BaCl₂-induced myonecrosis.     

Public, Experts, and Acceptance of Advanced Medical Technologies: The Case of Organ Transplant and Gene Therapy in Japan

In 1997, after long social debates, the Japanese government enacted a law on organ transplantation from brain-dead bodies. Since 1993, on gene therapy, administrative agencies have issued a series of guidelines. This study seeks to elucidate when people became aware of the issues and when they formed their opinions on organ transplant and gene therapy. At the same time, it aims to examine at which point in time experts, those in university ethical committees and in academic societies, consider these technologies became accepted among the public. A self-administered questionnaire was sent by mail to a stratified random sampling of 3000 people nationwide in Japan. Another questionnaire was sent both to the member societies of the Japanese Association of Medical Sciences and to the ethical committees of all the medical schools in Japan. Results of the surveys indicated that many of the public remained undecided on the desirability of organ transplant or gene therapy at the time of enactment of official guidelines. A substantial part of them formed their opinions in subsequent periods, especially around the time of first implementation and thereafter. Experts of the academic societies and of the university ethical committees regarded the time of implementation as an important factor in the acceptance of the technologies in society. Since many people formed their opinion during the period of technological implementation, communications efforts to facilitate public understanding of science and technology, as well as to advance practical discussion on policy alternatives in this period can play a key role in determining the fate of technological innovation and ethical debates in medicine.     

Anterior chamber angle vascularization in Sturge-Weber syndrome Report of a case

The case of a 20-year-old woman with a left-sided facial hemangioma and a homolateral glaucoma is reported, complete with the histology of a trabeculectomy specimen. Her left eye had an episcleral hemangioma and goniodysgenetic features in the anterior chamber angle, while the intraocular pressure was measured to be 45 mmHg. The left optic disc showed a large cupping and the left visual field was constricted. The right eye had no glaucomatous changes. Histological examination of the trabeculectomy specimen by both light and electron microscopy showed multiple congenital anomalies. There was a cluster of blood vessels in the trabecular meshwork. Abnormal accumulations of fine granular extracellular matrixes were observed in both the juxtacanalicular connective tissue and around the vascular structures. The lumen of Schlemm's canal was subdivided into three or four parts with few giant vacuole structures. The endothelial cells lining the inner wall of Schlemm's canal contained a well-formed basal lamina with many villi projecting into the lumen. These findings suggest that the multiple anomalies observed in the trabecular tissue may contribute to the manifestation of glaucoma in Sturge-Weber syndrome.     

The role of activation of the sympathetic nervous system in the central pressor action of calcitonin gene-related peptide in conscious rats

The effects of intracerebroventricular (i.cv.) administration of calcitonin gene-related (CGRP) on blood pressure and heart rate (HR), and the underlying mechanisms were studied in conscious rats. CGRP (0.1–3.0 nmol i.cv.) increased mean arterial blood pressure (MABP) and HR. CGRP (3.0 nmol i.cv.) also significantly increased both plasma norepinephrine and epinephrine concentrations. Pretreatment with 16.5 nmol i.cv. CGRP(8–37), a specific CGRP receptor antagonist, significantly inhibited the i.cv. CGRP (1.0 nmol)-induced increases in MABP and HR. Phenoxybenzamine inhibited the i.c.v. CGRP-induced increase in MABP, while propranolol suppressed the tachycardiac response to i.cv. CGRP. Chemical sympathectomy by 6-hydroxydopamine inhibited the increases in MABP and HR produced by i.cv CGRP. These results suggest that the central pressor and tachycardiac effects of i.c.v. CGRP are mediated by catecholamine release due to stimulation of sympathetic nervous system activity, possibly via specific CGRP receptors in the central nervous system.This study was presented, in part, at the 14th Scientific Meeting of the International Society of Hypertension, held in Madrid, Spain in June, 1992 Correspondence to: Y. Ouchi at the above address     

Preoperative respiratory physiotherapy for a patient with severe respiratory dysfunction and annuloaortic ectasia.

A 23-year-old man with Marfan syndrome, who had undergone surgery for pectus excavatum and scoliosis and who had severe respiratory dysfunction, was referred for surgical repair of annuloaortic ectasia. The preoperative pulmonary function test revealed severe obstructive and restrictive respiratory dysfunction, with forced expiratory volume in one second of 650 ml and vital capacity of 1,220 ml. These parameters improved after 4 months respiratory physiotherapy. A modified Bentall's procedure was performed after respiratory physiotherapy. A tracheostomy made on the 7th postoperative day (POD) appeared to improve respiratory condition and he was weaned off mechanical ventilation on the 14th POD. The lower limits of pulmonary function for open heart surgery have not been established clearly; however, our case will help elucidate these limits of respiratory function for open heart surgery. Preoperative respiratory physiotherapy improved parameters of pulmonary function test and may decrease the morbidity of postoperative pulmonary complications in a patient with severe respiratory dysfunction.