Differential activation of subtype purinergic receptors modulates Ca(2+) mobilization and COX-2 in human microglia

We have studied modulation of purinergic receptors (P(2Y) and P(2X) subtypes) on changes in intracellular Ca(2+) [Ca(2+)](i) and expression and production of COX-2 in human microglia. Measurements using Ca(2+)-sensitive spectrofluorometry showed adenosine triphosphate (ATP) to cause rapid transient increases in [Ca(2+)](i). Application of ATP plus the P(2X) antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), or treatment with adenosine diphosphate-beta-S (ADP-beta-S), a selective P(2Y) agonist, led to a considerable prolongation in [Ca(2+)](i) responses compared with ATP. The prolonged time courses were consistent with sustained activation of store-operated channels (SOC) since SKF96365, an inhibitor of SOC, blocked this component of the response. RT-PCR data showed that microglia expressed no COX-2 either constitutively or following treatment of cells with ATP (100 microM for 8 h). However, treatment using ATP plus PPADS or with ADP-beta-S led to marked expression of COX-2. The enhanced COX-2 with ATP plus PPADS treatment was absent in the presence of SKF96365 or using Ca(2+)-free solution. Immunocytochemistry, using a specific anti-COX-2 antibody, also revealed a pattern of purinergic modulation whereby lack of P(2X) activation enhanced the production of COX-2 protein. These results suggest that modulation of subtypes of purinergic receptors regulates COX-2 in human microglia with a link involving SOC-mediated influx of Ca(2+).     

Age-related changes in the distribution of Na(v)1.1 and Na(v)1.2 in rat cerebellum

Modification of sodium channel availability and behavior is obviously a good candidate for alteration of action potential observed during aging. In the present study, we revealed age-related alterations in the expression of voltage-gated Na+ (Na(v)) channel in rat cerebellum by immunohistochemistry. In the cerebellar cortex of aged rats, Na(v)1.1 immunoreactivity in Purkinje cell bodies was highly increased, whereas granule cells showed lower staining intensity. In the cerebellar nuclei of aged rats, Na(v)1.1 and Na(v)1.2 expression was specifically increased in the cerebellar output neurons, which was confirmed by image analysis. The first demonstration of age-related changes in Na(v) channel expression contributes to our understanding of the mechanisms responsible for alteration in synaptic transmission during aging.     

Effects of morphine on mechanical allodynia in a rat model of central neuropathic pain

We examined whether morphine reduced the behavioral signs of neuropathic pain below level induced by T13 spinal hemisection in rats. In order to examine the effect of morphine on the mechanical allodynia, morphine alone, morphine with naloxone and saline were administered intraperitoneally and intrathecally and behavioral tests were conducted. In systemic injection, mechanical allodynia was reduced only when a higher concentration of morphine (5 mg/kg) was used. Intrathecally injected morphine (0.5, 1, 2, 5 microg) reduced mechanical allodynia dose-dependently. It is suggested that systemic morphine has limited effect on mechanical allodynia but direct spinal administration of morphine is more effective in controlling central pain following spinal cord injury.     

Serial measurement of surface expressions of CD63, P-selectin and CD40 ligand on platelets in atherosclerotic ischemic stroke. A possible role of CD40 ligand on platelets in atherosclerotic ischemic stroke

BACKGROUND: Platelet activation is a key step in the progression of atherosclerosis. The CD40 ligand (CD40L) on platelets may be a critical factor to develop the acute vascular events from atheroma. METHODS: To determine the role of CD40L on platelets in atherosclerotic ischemic stroke, we serially measured the expressions of CD63, P-selectin and CD40L on platelets in patients with atherosclerotic ischemic stroke (n = 25) and compared them with those in patients with asymptomatic carotid stenosis (n = 20) and in normal subjects (n = 24). RESULTS: The expressions of CD63 and P-selectin on platelets were significantly higher in patients with atherosclerotic ischemic stroke (n = 25) than in normal subjects (n = 24). The extents of surface expressions of CD63 and P-selectin on platelets showed no significant differences between atherosclerotic ischemic stroke and asymptomatic carotid stenosis. However, the CD40L expression on platelets was significantly higher in atherosclerotic ischemic stroke when compared to that in asymptomatic carotid stenosis. CONCLUSIONS: In our data, among the population with large artery atherosclerosis, the patients with symptomatic ischemic events showed a significantly elevated expression of CD40L on platelets compared to those without ischemic events. Therefore, the upregulation of CD40L on platelets may be a specific marker of platelet activation to provoke ischemic stroke from large artery atherosclerosis.     

Hematopoietic chimerism and central tolerance created by peripheral-tolerance induction without myeloablative conditioning

Allogeneic hematopoietic chimerism leading to central tolerance has significant therapeutic potential. Realization of that potential has been impeded by the need for myeloablative conditioning of the host and development of graft-versus-host disease (GVHD). To surmount these impediments, we have adapted a costimulation blockade-based protocol developed for solid organ transplantation for use in stem cell transplantation. The protocol combines donor-specific transfusion (DST) with anti-CD154 mAb. When applied to stem cell transplantation, administration of DST, anti-CD154 mAb, and allogeneic bone marrow leads to hematopoietic chimerism and central tolerance with no myeloablation and no GVHD. Tolerance in this system results from deletion of both peripheral host alloreactive CD8+ T cells and nascent intrathymic alloreactive CD8+ T cells. In the absence of large numbers of host alloreactive CD8+ T cells, the transfusion that precedes transplantation need not be of donor origin, suggesting that both allospecific and non-allospecific mechanisms regulate engraftment. Agents that interfere with peripheral transplantation tolerance impair establishment of chimerism. We conclude that robust allogeneic hematopoietic chimerism and central tolerance can be established in the absence of host myeloablative conditioning using a peripheral transplantation tolerance protocol.     

1,25-Dihydroxyvitamin D3 stimulates osteopontin expression in rat kidney

Osteopontin (OPN) is a secreted phosphoprotein expressed constitutively in the descending thin limb (DTL) and papillary surface epithelium (PSE) of the kidney. Although its function is not fully established, a role for OPN in the regulation of calcium-mediated or calcium-dependent processes has been proposed. The aim of this study was to examine the effects of 1,25-dihydroxyvitamin D(3) (vitD), a hormone involved in the regulation of calcium homeostasis, on renal OPN expression. Four groups of rats were studied: acute vehicle (single intraperitoneal [i.p.] injection of 0.1 ml 10% ethanol-90% propylene glycol, 12 h before being killed); acute vitD (single injection of vitD, 2 ng/g i.p., 12 h before being killed); chronic vehicle (daily subcutaneous [s.c.] injection of 0.1 ml 10% ethanol-90% propylene glycol for 7 days); and chronic vitD (daily s.c. injection of vitD, 0.5 ng/g, for 7 days). Kidneys were processed for light and electron microscope immunocytochemistry, in situ hybridization, and Western blot analysis. In vehicle-treated animals, OPN mRNA and protein were expressed primarily in the DTL and PSE. In the acute vitD group, OPN mRNA and immunoreactivity appeared in the thick ascending limb (TAL) of the inner stripe of the outer medulla, and increased slightly in the DTL and PSE. The proximal tubules exhibited strong OPN immunoreactivity, but no hybridization signal. In the chronic vitD group, there was a marked increase in OPN mRNA and immunoreactivity in the distal tubule, including the TAL, as well as in the DTL and PSE. A weak hybridization signal and immunostaining were also observed in some proximal tubules. Administration of vitD causes a marked increase in OPN mRNA and protein in the rat kidney, mainly in the distal nephron, but also in the DTL, PSE, and proximal tubules. These results indicate that vitD is involved in the regulation of OPN expression in the kidney.     

Nasopharyngeal carcinoma: posttreatment changes of imaging findings

OBJECTIVE: The purpose of this study was to determine the radiologic results of nasopharyngeal carcinoma (NPC) that showed complete responses on follow-up imaging studies after radiation therapy. MATERIALS AND METHODS: This study is a retrospective review of 23 patients (18 male, 5 female, aged 15-71 years; mean age, 48.5 years) affected with NPC, from August 1995 to July 2000, who were examined with magnetic resonance imaging or computed tomography scan before and after either radical radiotherapy or chemoradiotherapy. The median follow-up was 24.7 months and ranged from 12 to 48 months. We analyzed the primary tumors by ascertaining/measuring tumor size, depth, middle ear effusion, skull base invasion, and lymphadenopathy. The treatment responses for primary tumors were classified as either atrophy, scar (asymmetric elevation without enhancement), or normalized. The tumor response and the appearance of bone regeneration in the previous destructive part of the skull base were also recorded. RESULTS: The 23 patients consisted of 12 superficial tumors, for whom treatment results were normalized in 10, atrophy in 1, and scar in 1 and 11 deep tumors for whom treatment results were scar in 6, normalized in 3, and atrophy in 2. Skull base invasion was detected in 6 patients, 5 of whom showed complete healing of skull base destruction after radiotherapy. However, the other patient exhibited an unusual hyperostotic change in the skull base mimicking fibrous dysplasia of the skull base. CONCLUSIONS: The superficial tumors tended to be radiologically normalized even when they were large. However, the deep tumors mainly changed to scar after radiation therapy. On the other hand, skull base invasion could be normalized after radiotherapy.     

Laser Punch-Out for Acne Scars

Patients with acne scars want smooth facial skin. However, achieving this is difficult with dermabrasion or chemical peeling. Nor can acne scars be covered with cosmetics, due to their ice-picked or cobblestone appearance. Laser resurfacing is more effective and safer than other conventional methods due to its precision with depth control and variable methods of surface cutting. Even depth resurfacing with a laser shows unsatisfactory results, therefore, for the deep-sited acne scar the cutting methods have to be changed according to the depth and pattern of the scar. For 2 years, starting in January 1996, we treated 71 patients with a high-powered CO₂ laser (Ultrapulse). Different resurfacing methods were applied according to the depth and pattern of the scars. For mild depressed scars, even depth resurfacing was done. For moderate-depth acne scars, the shoulder technique was also used. For the deepest and ice-picked scars, the laser punch-out was combined. Laser resurfacing was carried out at 300–500 mJ, with two to five passes. Laser punch-out was done at 500 mJ, with three to seven continuous passes on the ice-picked scar. From the pathologic findings of acne scars showing that there was thick intradermal scar, we knew that laser punch-out was necessary for improvement of acne scars. Depth-wide, the ice-picked scars improved by over 80% and the sharp demarcated margin of the acne scar faded out. Most of the patients with acne scars were satisfied with laser resurfacing. Only six patients had a second laser treatment, with an interval of 12 months. There were no hypertrophic scars after laser resurfacing, but erythema lasted for 3–12 months. Patients taking oral retinoic acid were not contraindicated for laser resurfacing but required special caution because they had atrophic skin and delayed wound healing. Laser resurfacing is the most versatile method for acne scars, with a high-powered CO₂ laser. The laser punch-out method is better than even depth resurfacing for improving deep acne scars and can be combined with the shoulder technique or even depth resurfacing according to the type of acne scar.     

Anticoagulant activity of sulfoalkyl derivatives of curdlan

Curdlan is a natural beta-1,3-glucan produced by Agrobacterium biovar 1. In this study, the anticoagulant activity of sulfoalkyl derivatives of curdlan was investigated by carrying out activated partial thromboplastin time (APTT) assay and compared with that of o-sulfonated curdlan. Approximately 100-fold higher concentration of o-sulfonated curdlan than heparin was required to obtain the same level of the clotting time. Anticoagulant activity of curdlan derivatives was dependent on the degree of sulfation in prolonging the clotting time. However, the chain length of the substituent did not play a role in prolonging the clotting time. The curdlan derivatives enhanced thrombin inhibition by mediating through antithrombin III. The inhibition of thrombin by o-sulfonated curdlan was found to be approximately 10-fold weaker than that by heparin.     

beta-Cell dysfunction rather than insulin resistance is the main contributing factor for the development of postrenal transplantation diabetes mellitus

BACKGROUND: Our study was undertaken to investigate the pathogenesis and possible risk factors for postrenal transplantation diabetes mellitus (PTDM). METHODS: We recruited 114 patients with normal glucose tolerance (NGT) and performed both 75-g oral glucose tolerance tests (OGTT) and short insulin tolerance tests 1 week before and 9-12 months after transplantation. RESULTS: The subjects were classified into three groups by World Health Organization criteria on the basis of OGTT after transplantation: (a) 36 (31.6%) subjects with normal glucose tolerance; (b) 51 (45.7%) subjects with impaired glucose tolerance (IGT); and (c) 27 (23.7%) subjects with postrenal transplantation diabetes mellitus. Dosages of steroid and cyclosporine were equivalent among the three groups. Before transplantation, the fasting and 2-hr plasma glucose and proinsulin/insulin (PI/I) ratios were significantly higher in the IGT and PTDM groups than in the NGT group, but the insulin sensitivity index (ISI) was not significantly different among the three groups. In addition, the area under the curve-insulin on OGTT was significantly lower in the PTDM group than in the NGT group. After transplantation, however, the ISI was increased in all groups. Furthermore, the ISI and PI/I ratios revealed significantly higher values in the PTDM group than in the NGT group after transplantation. CONCLUSIONS: These results revealed that fasting and 2-hr plasma glucose levels, as well as the proinsulin/insulin ratio before transplantation, are both possible indicators of beta-cell dysfunction and may be predictors for the development of PTDM. Furthermore, beta-cell dysfunction, rather than insulin resistance, was proven to be the main factor for the pathogenesis of PTDM.