Involvement of Nax sodium channel in peripheral nerve regeneration via lactate signaling

Na_x, a sodium concentration‐sensitive sodium channel, is expressed in non‐myelinating Schwann cells of the adult peripheral nervous system, but the pathophysiological role remains unclear. We found that functional recovery of the hind paw responses from the sciatic nerve transection was delayed in Na_x knockout ( ) mice. Histological analyses showed a decrease in the number of regenerated myelinated axons in sciatic nerves. The delay in the recovery in mice was improved by lactate and inhibited by a monocarboxylate transporter inhibitor. In vitro experiments using cultured Schwann cells showed that lactate release was enhanced by endothelin (ET)‐1 and blocked by an ET receptor type B antagonist. Here, it is conceivable that Na_x was activated by ET‐1. The amount of lactate release by ET‐1 was lower in mice than in wild‐type mice. These results indicated that Na_x is functionally coupled to ET for lactate release via ET receptor type B and is involved in peripheral nerve regeneration.     

Evidence of different pharmacokinetics between cyclosporine and tacrolimus in renal transplant recipients: why cyclosporine is monitored by C2 level and tacrolimus by trough level

The clinical efficacy of calcineurin inhibitors administered to renal transplant recipients is considered to be a strong function of the area under the concentration time curve (AUC). Monitoring of blood concentrations for two similar calcineurin inhibitors, cyclosporine (CyA) and tacrolimus (TAC) are different. Namely, CyA blood concentration is usually monitored at two hours after administration (C₂), a surrogate for peak concentration (C_p), and TAC at trough concentration (C_t). We examined the behavior of blood concentration curves simultaneously for both CyA and TAC in renal transplant recipients with similar clinical backgrounds. Furthermore, we analyzed the correlation of C_p and C_t vs AUC implementing an area under the trough level, or area above the trough level as new pharmacokinetic parameters, so that C₂ for CyA and C_t for TAC has validated using controlled clinical data. We observed differences in the pharmacokinetics between,     

Falsely abnormally elevated blood trough concentration of tacrolimus measured by antibody-conjugated magnetic immunoassay in a renal transplant recipient: a case report

This report presents a falsely abnormally elevated blood trough concentration (C_t) of tacrolimus measured by antibody-conjugated magnetic immunoassay (ACMIA) methods in a renal transplant recipient. Because the C_t of tacrolimus was 78.5 ng/mL at day 2 after a 52-year-old man underwent renal transplantation, we stopped the tacrolimus extended-release formulation. However, because the abnormally elevated blood C_t continued in the range of 41.1-59.1 ng/mL, we then measured the tacrolimus concentration in a stored blood sample before renal transplantation, it was 43 ng/mL. Consequently, the day-7 blood sample was measured with both ACMIA and enzyme-linked immunoassay, showing C_t values of 42.8 ng/mL and 0.89 ng/mL, respectively. Because the abnormally elevated C_t was falsely measured by the ACMIA method, we restarted tacrolimus However, the calcineurin inhibitor was subsequently converted to cyclosporine at day 21 after renal transplantation. Although cyclosporine was also measured by ACMIA, there was not an abnormally elevated C_t. Subsequently, the tacrolimus concentration ratio in plasma and whole blood (P/B-tacrolimus concentration ratio) was measured by ACMIA in a posttacrolimus blood sample. The P/B-tacrolimus concentration ratio was 100%. In contrast, the P/B-tacrolimus concentration ratio was <30% in 2 control patients administered tacrolimus. It has been reported recently that there were cases showing falsely slightly elevated C_t of tacrolimus within the therapeutic range of concentrations. Therefore, we must be careful not to reduce the tacrolimus dose falsely. We consider confirmatory methods for a falsely abnormally elevated C_t of tacrolimus measured by ACMIA to (1) measure P/B-tacrolimus concentration ratio, (2) compare ACMIA with another measurement, and (3) evaluate a blood sample stored before tacrolimus administration.     

Clinical Significance of Peripheral Blood Lymphocyte Sensitivity to Glucocorticoids for the Differentiation of High-risk Patients With Decreased Allograft Function After Glucocorticoid Withdrawal in Renal Transplantation

Purpose

A reliable biomarker to differentiate high-risk recipients who will experience a decrease in allograft function after glucocorticoid withdrawal has not been established in renal transplantation. We examined the clinical significance of peripheral blood lymphocyte sensitivity to glucocorticoids in vitro for the differentiation of the high-risk patients after glucocorticoid reduction/withdrawal in renal transplant recipients.

Methods

The study included 44 renal transplant recipients with stable allograft function. Peripheral lymphocyte responses to suppressive effects of cortisol, methylprednisolone, cyclosporine, and tacrolimus in mitogen assay procedures in vitro were examined. Clinical outcome after glucocorticoid reduction/withdrawal was retrospectively compared between recipients with lymphocytes normally sensitive to the drugs and those with hyposensitivity. The receiver-operating characteristic (ROC) curve analysis was undertaken for setting the cutoff IC₅₀ values of the drugs against the T cell mitogen–induced lymphocyte proliferation to differentiate the high-risk recipients with decreased allograft function after glucocorticoid withdrawal.

Findings

The median (range) IC₅₀ value for cortisol in the recipients who showed decreased renal function due to glucocorticoid withdrawal was 10,000 (570.9–72,279.3) ng/mL (n = 9), which was significantly higher than the value of 351.6 (2.0–10,000) ng/mL in the recipients who had not experienced glucocorticoid withdrawal symptoms (n = 35) (P < 0.001). Similarly, the median (range) IC₅₀ value for methylprednisolone in the recipients who showed decreased renal function after glucocorticoid withdrawal was 69.1 (21.5–1442.7) ng/mL (n = 9), which was significantly higher than the value of 13.8 (0.7–1000) ng/mL in the recipients who had not experienced glucocorticoid withdrawal symptoms (n = 30) (P < 0.003). In contrast, there was no significant difference in the median IC₅₀ values of cyclosporine and tacrolimus between the 2 recipient subgroups. The ROC curve analyses for the IC₅₀ values of the immunosuppressive drugs estimated the cutoff value of cortisol and methylprednisolone to be 3580.0 and 21.5 ng/mL, respectively. The ROC AUCs for cortisol and methylprednisolone were 0.83 and 0.84, respectively. According to the cutoff IC₅₀ value, the incidence of decreased allograft function in the low cortisol sensitivity (IC₅₀ >3580.0 ng/mL) subgroup was 7 of 13 patients, which was significantly higher than that of the higher sensitivity subgroup of 2 of 31 (P = 0.0012). A similar case was observed using the cutoff IC₅₀ value of methylprednisolone (P = 0.0012), whereas recipient grouping according to the cutoff IC₅₀ values of cyclosporine and tacrolimus failed to differentiate the high-risk recipients with decreased allograft function after glucocorticoid withdrawal.

Implications

Glucocorticoid pharmacodynamics in lymphocytes of individual patient origin is a reliable biomarker for differentiation of renal transplant recipients who will experience a safe reduction/withdrawal of glucocorticoid.     

Evidence of different pharmacokinetics including relationship among AUC, peak, and trough levels between cyclosporine and tacrolimus in renal transplant recipients using new pharmacokinetic parameter--why cyclosporine is monitored by C(2) level and tacrolimus by trough level--

The clinical efficacy of calcineurin inhibitors administered to renal transplant patients is considered to be a strong function of the area under the concentration time curve (AUC). Interestingly, monitoring timings of blood concentrations for two similar calcineurin inhibitors, cyclosporine (CYA; Neoral) and tacrolimus (TAC; Prograf) are different. Namely, CYA blood concentration is usually monitored at 2 h after administration (C(2)) substituted for peak concentration (C(p)) and TAC at trough concentration (C(t)). In the literature, data describing such characteristics of CYA and TAC have been presented in the past. However, each of these patient groups had different backgrounds. We have attempted to examine the behavior of blood concentration curves simultaneously for both CYA and TAC by establishing controlled groups of renal transplant patients with similar clinical backgrounds. Furthermore, we have analyzed the correlation with C(p) and C(t) versus AUC implementing area under the trough level (AUTL), or area above the trough level (AATL) as new pharmacokinetic parameters, such that C(2) for CYA and C(t) for TAC have been verified using controlled clinical data. We have also found distinct differences in the pharmacokinetics between CYA and TAC with the relationships between AUC, C(p), and C(t).     

Enhanced Delivery and Antitumor Activity of Doxorubicin Using Long-Circulating Thermosensitive Liposomes Containing Amphipathic Polyethylene Glycol in Combination with Local Hyperthermia

Enhanced delivery of doxorubicin (DXR) to a solid tumor subjected to local hyperthermia was achieved by using long-circulating, thermosensitive liposomes (TSL) composed of dipalmitoyl phosphatidylcholine (DPPC)/distearoyl phosphatidylcholine (DSPC) (9:1, m/m) and 3 mol% amphipathic polyethylene glycol (PEG) in colon 26-bearing mice. Inclusion of 3 mol% of distearoyl phosphatidylethanolamine derivatives of PEG (DSPE-PEG, amphipathic PEG) with a mean molecular weight of 1000 or 5000 in DPPC/DSPC liposomes resulted in decreased reticuloendothelial system (RES) uptake and a concomitant prolongation of circulation time, affording sustained increased blood levels of the liposomes. Concomitantly, DXR levels in blood were also kept high over a long period. The presence of amphipathic PEG did not interfere with the encapsulation of DXR by the pH gradient method (>90% trapping efficiency) or with the temperature-dependent drug release from the liposomes. The optimal size of these liposomes was 180 – 200 nm in mean diameter for thermosensitive drug release and prolonged circulation time. The DXR levels in the tumor after injection of long-circulating TSL (DXR-PEG1000TSL or DXR-PEG5000TSL, at a dose of 5 mg DXR/ kg) with local hyperthermia were much higher than after treatment with DXR-TSL lacking PEG or with free DXR, reaching 7.0 – 8.5 DXR µg/g tumor (approximately 2 times or 6 times higher than that of DXR-TSL or free DXR, respectively). Furthermore, the combination of DXR-PEGTSL and hyperthermia effectively retarded tumor growth and increased survival time. Our results indicate that the combination of drug-loaded, long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

Fluoride uptake in human teeth from fluoride-releasing restorative material in vivo and in vitro: two-dimensional mapping by EPMA-WDX

Class V cavities were prepared in the upper and lower left second premolars from an individual under infiltration anesthesia. The cavities were filled with fluoride- releasing resin (TF). One month later, the teeth were extracted. As a control (in vitro), the upper and lower right second premolars were extracted at the time of the cavity preparation in vivo. Immediately after extraction, class V cavities were prepared and filled with TF, and immersed in normal saline solution for 1 month at 37 degrees C. All four premolars were bisected longitudinally and the fluoride uptake around the cavity wall on the cut surface was measured using an electron probe microanalyzer-wavelength dispersive X-ray method. The fluoride uptake was given in the form of a two-dimensional map. Comparison of the observed values of each corresponding part of the tooth in vivo and in vitro revealed no characteristic differences. The maps were quite analogous as a whole.     

Fluoride uptake around cavity walls; two-dimensional mapping by electron probe microanalysis

This study reports fluoride uptake around the cavity wall of teeth by two-dimensional mapping. Fluoride concentration was measured using the wavelength dispersive x-ray analysis (WDX) method. The buccal cavity wall of a human tooth was coated five times with 2% sodium fluoride solution at three-day intervals for 12 days, and then immersed in a normal saline solution at 37 degrees C. After one month, the tooth was bisected longitudinally through the center of the cavity surface perpendicular to the axial wall. On the polished surface of the cut tooth, the fluoride concentration was measured. Fluoride distribution maps around the cavity wall were drawn using a bundle of the observed analytical lines. Fluoride uptake from fluoride-releasing materials (conventional glass-ionomer cement, light-cured glass-ionomer cement, light-cured composite resin, light-cured bonding agent) around the cavity wall was investigated using the same method. The maps showed higher fluoride uptake in dentin than in enamel and a strong location dependence of fluoride uptake in a tooth, especially in the dentin. Fluoride uptake from the resin was greater than that from the cement. It was summarized from these results that a two-dimensional map of fluoride uptake can provide valuable information on the cariostatic properties of fluoride-releasing materials.     

Steroid withdrawal based on lymphocyte sensitivity to endogenous steroid in renal transplant recipients

Though steroid withdrawal is done in many renal transplant recipients, some patients must restart steroids. Little report has investigated steroid withdrawal under pharmacodynamic monitoring. We assessed lymphocyte sensitivity to endogenous cortisol as a biomarker for determining the safety of steroid withdrawal in renal transplant patients, as we hypothesized that patients hyposensitive to cortisol could not be sufficiently immunosuppressed by their intrinsic cortisol as a substitute for the reduced or withdrawn steroid. Lymphocyte sensitivity to cortisol was examined in 30 long stable renal transplant recipients. Lymphocyte sensitivity to cortisol and its relationship with the clinical outcome after steroid reduction and withdrawal was investigated. The lymphocyte sensitivities to cortisol were estimated as IC(50) of lymphocyte blastogenesis. The lymphocyte proliferation rate for concentration of serum cortisol compared between incident and non-incident groups. Serum creatinine levels (S-Cr) increased in a significantly higher number of patients hyposensitive to cortisol (IC(50)≧10000 ng/ml) than in normally sensitive patients (IC(50)<10000 ng/ml). The incidences of steroid withdrawal syndrome and necessity for increasing steroid dose or restarting steroid administration were also higher in the patients hyposensitive to cortisol. The patients in whom the lymphocyte proliferation rate was less than 60% did not show increase in S-Cr, experience steroid withdrawal symptoms, or require an increase in the steroid dose or restart of steroid administration. The patients who have the normal IC(50) values of cortisol, can withdraw steroid more safely. The lymphocyte sensitivity to cortisol may be a useful biomarker for selecting patients who can sustain steroid withdrawal.