Diagnosis and Treatment of Leprosy in Taiwan during the COVID-19 Pandemic: A Retrospective Study in a Tertiaty Center.

Currently, over 200,000 new cases of leprosy are reported annually worldwide. Although leprosy was thought to have been eradicated in Taiwan, a few new cases still occur annually. Protean clinical manifestations of leprosy and immunological reactions result in delayed diagnoses. In addition, drug-resistant leprosy is emerging and poses treatment challenges. In this retrospective study, we collected and analyzed the clinicopathological features, leprosy type, treatment response, and relapse rate of patients with leprosy in our hospital between January 2009 and November 2022. We found that 54% of patients were Indonesian, and borderline lepromatous leprosy was predominant (39%); moreover, histoid leprosy and the Lucio phenomenon were also reported. Polymerase chain reaction analysis identified four positive cases, including a dapsone-resistant (4%) case. Our findings indicated good control of leprosy and a lower rate of dapsone resistance than that reported by the World Health Organization (4% vs. 13%) from 2009 to 2015. We found that the patient profile in terms of the treatment duration, recurrence rate, systemic symptoms, and neurological symptoms did not differ between before and during the pandemic. We report the recent advances in leprosy diagnosis, drug-resistant gene mutations, post-exposure prophylaxis, vaccination, and the effect of coronavirus disease 2019 on leprosy to facilitate updated leprosy diagnosis and management.

Pannexin 1 Modulates Angiogenic Activities of Human Endothelial Colony-Forming Cells Through IGF-1 Mechanism and Is a Marker of Senescence.

BACKGROUND: We examined the role of Panxs (pannexins) in human endothelial progenitor cell (EPC) senescence. METHODS: Young and replication-induced senescent endothelial colony-forming cells (ECFCs) derived from human circulating EPCs were used to examine cellular activities and senescence-associated indicators after transfection of short interference RNA specific to Panx1 or lentivirus-mediated Panx1 overexpression. Hind limb ischemia mice were used as in vivo angiogenesis model. Protein and phospho-kinase arrays were used to determine underlying mechanisms. RESULTS: Panx1 was the predominant Panx isoform in human ECFCs and upregulated in both replication-induced senescent ECFCs and circulating EPCs from aged mice and humans. Cellular activities of the young ECFCs were enhanced by Panx1 downregulation but attenuated by its upregulation. In addition, reduction of Panx1 in the senescent ECFCs could rejuvenate cellular activities with reduced senescence-associated indicators, including senescence-associated ß-galactosidase activity, p16INK4a (cyclin-dependent kinase inhibitor 2A), p21 (cyclin-dependent kinase inhibitor 1), acetyl-p53 (tumor protein P53), and phospho-histone H2A.X (histone family member X). In mouse ischemic hind limbs injected senescent ECFCs, blood perfusion ratio, salvaged limb outcome, and capillary density were all improved by Panx1 knockdown. IGF-1 (insulin-like growth factor 1) was significantly increased in the supernatant from senescent ECFCs after Panx1 knockdown. The enhanced activities and paracrine effects of Panx1 knockdown senescent ECFCs were completely inhibited by anti-IGF-1 antibodies. FAK (focal adhesion kinase), ERK (extracellular signal-regulated kinase), and STAT3 (signal transducer and activator of transcription 3) were activated in senescent ECFCs with Panx1 knockdown, in which the intracellular calcium level was reduced, and the activation was inhibited by supplemented calcium. The increased IGF-1 in Panx1-knockdown ECFCs was abrogated, respectively, by inhibitors of FAK (PF562271), ERK (U0126), and STAT3 (NSC74859) and supplemented calcium. CONCLUSIONS: Panx1 expression is upregulated in human ECFCs/EPCs with replication-induced senescence and during aging. Angiogenic potential of senescent ECFCs is improved by Panx1 reduction through increased IGF-1 production via activation of the FAK-ERK axis following calcium influx reduction. Our findings provide new strategies to evaluate EPC activities and rejuvenate senescent EPCs for therapeutic angiogenesis.

A Common East Asian aldehyde dehydrogenase 2*2 variant promotes ventricular arrhythmia with chronic light-to-moderate alcohol use in mice.

Chronic heavy alcohol use is associated with lethal arrhythmias. Whether common East Asian-specific aldehyde dehydrogenase deficiency (ALDH2*2) contributes to arrhythmogenesis caused by low level alcohol use remains unclear. Here we show 59 habitual alcohol users carrying ALDH2 rs671 have longer QT interval (corrected) and higher ventricular tachyarrhythmia events compared with 137 ALDH2 wild-type (Wt) habitual alcohol users and 57 alcohol non-users. Notably, we observe QT prolongation and a higher risk of premature ventricular contractions among human ALDH2 variants showing habitual light-to-moderate alcohol consumption. We recapitulate a human electrophysiological QT prolongation phenotype using a mouse ALDH2*2 knock-in (KI) model treated with 4% ethanol, which shows markedly reduced total amount of connexin43 albeit increased lateralization accompanied by markedly downregulated sarcolemmal Nav1.5, Kv1.4 and Kv4.2 expressions compared to EtOH-treated Wt mice. Whole-cell patch-clamps reveal a more pronounced action potential prolongation in EtOH-treated ALDH2*2 KI mice. By programmed electrical stimulation, rotors are only provokable in EtOH-treated ALDH2*2 KI mice along with higher number and duration of ventricular arrhythmia episodes. The present research helps formulate safe alcohol drinking guideline for ALDH2 deficient population and develop novel protective agents for these subjects.

Deferoxamine accelerates endothelial progenitor cell senescence and compromises angiogenesis.

Senescence reduces the circulating number and angiogenic activity of endothelial progenitor cells (EPCs), and is associated with aging-related vascular diseases. However, it is very time-consuming to obtain aged cells (~1 month of repeated replication) or animals (~2 years) for senescence studies. Here, we established an accelerated senescence model by treating EPCs with deferoxamine (DFO), an FDA-approved iron chelator. Four days of low-dose (3 µM) DFO induced senescent phenotypes in EPCs, including a senescent pattern of protein expression, impaired mitochondrial bioenergetics, altered mitochondrial protein levels and compromised angiogenic activity. DFO-treated early EPCs from young and old donors (< 35 vs. > 70 years old) displayed similar senescent phenotypes, including elevated senescence-associated ß-galactosidase activity and reduced relative telomere lengths, colony-forming units and adenosine triphosphate levels. To validate this accelerated senescence model in vivo, we intraperitoneally injected Sprague-Dawley rats with DFO for 4 weeks. Early EPCs from DFO-treated rats displayed profoundly senescent phenotypes compared to those from control rats. Additionally, in hind-limb ischemic mice, DFO pretreatment compromised EPC angiogenesis by reducing both blood perfusion and capillary density. DFO thus accelerates EPC senescence and appears to hasten model development for cellular senescence studies.

Timing of Adjuvant Radiotherapy After Keloid Excision: A Systematic Review and Meta-Analysis.

BACKGROUND: No consensus exists regarding the appropriate timing of adjuvant radiotherapy administration after surgical excision of keloids. OBJECTIVE: This study investigated the appropriate timing of adjuvant radiotherapy. MATERIALS AND METHODS: A systematic review and meta-analysis of randomized controlled trials and observational cohort studies was performed. A pooled estimate of the incidence rate was performed using a random-effects model. Subgroup analyses based on different anatomic region, biologically effective dose, keloid length, and radiotherapy regimen were also conducted. RESULTS: Sixteen observational cohort studies (1,908 keloid lesions) met the inclusion criteria. The incidence rate was significantly lower in the group treated with electron beam therapy more than 24 hours after surgery (3.80%; 95% confidence interval [CI], 1.78%-8.13%) than that in the group treated with the same therapy within 24 hours of surgery (37.16%; 95% CI, 20.80%-66.37%; p < .0001), but no significant difference was observed between the groups regarding brachytherapy and x-ray treatments. CONCLUSION: Immediate adjuvant radiotherapy did not significantly reduce the incidence rate of recurrent keloids.

Development of a selective three-dimensional HPLC system for enantiomer discriminated analysis of lactate and 3-hydroxybutyrate in human plasma and urine.

For the enantiomer discriminated determination of lactate (LA) and 3-hydroxybutyrate (3HB) in various complicated samples, a three-dimensional HPLC (3D-HPLC) system has been designed and developed by investigating the separation of the target analytes from unknown substances observed in the real target matrices. LA and 3HB were pre-column derivatized with 4-nitro-7-piperazino-2,1,3-benzoxadiazole for the sensitive fluorescence detection and introduced into the 3D-HPLC system composed of reversed-phase, mixed-mode and enantioselective separations. The present method was validated by calibration curves, precision and accuracy using standard solutions and human samples, and sufficient values were obtained. Using the method, the levels of d-LA, l-LA, d-3Hb and l-3HB were determined, and their concentrations were 9.9, 1004.2, 79.7 and 2.1 µM in the human plasma and 16.0, 86.6, 8.7 and 4.8 µM in the human urine, respectively. The present 3D-HPLC system could selectively determine trace amounts of the target hydroxy acid enantiomers without disturbance of the intrinsic interfering substances in complicated matrices and the applications to various disease samples are expected.

Determination of phenylalanine enantiomers in the plasma and urine of mammals and á´ -amino acid oxidase deficient rodents using two-dimensional high-performance liquid chromatography.

A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice.

Determination of temporal changes in serum and urinary lactate and 3-hydroxybutyrate enantiomers in mice with nephrotoxic serum nephritis by multi-dimensional HPLC.

Nephrotoxic serum (NTS) nephritis occurs in three stages: inflammation, early kidney damage, and severe kidney damage. We quantified the temporal changes in the enantiomers of lactate (LA) and 3-hydroxybutyrate (3HB) in serum and urine during the progression of autoimmune kidney damage in mice with NTS nephritis. Two-dimensional and three-dimensional HPLC were used to quantify the enantiomers. The serum and urinary levels of LA and 3HB enantiomers significantly changed during the progression of NTS nephritis. Specifically, d-LA was significantly higher in the serum (131.8 ±â€¯30.6, 123.7 ±â€¯27.2, 109.3 ±â€¯15.6 vs. 51.2 ±â€¯7.5 µM; p < 0.05) and urine (222.2 ± 34.8, 197.4 ± 53.9, 214.8 ± 68.9 vs. 100.8 ± 37.7 µmol/g creatinine; p < 0.05) of the week 0 (W0), week 1 (W1), and week 2 (W2) groups than the normal group. The l-3HB/d-3HB ratio was significantly lower in the W0, W1, and W2 groups than the normal group in serum (0.0362 ±â€¯0.0082, 0.0346 ±â€¯0.0065, 0.0323 ±â€¯0.0033 vs. 0.0602 ±â€¯0.0214; p < 0.05) and urine (0.0591 ± 0.0304, 0.1524 ± 0.0365, 0.1232 ± 0.1066 vs. 0.3273 ± 0.1394; p < 0.05). The changes in serum and urinary d-LA and l-3HB/d-3HB ratios were stable before severe kidney damage. In conclusion, we successfully determined the levels of LA and 3HB enantiomers in NTS nephritis by HPLC. Serum and urinary d-LA contents and l-3HB/d-3HB ratios may have potential as biomarkers of early autoimmune kidney injury.

S-Phase Kinase-associated Protein-2 Rejuvenates Senescent Endothelial Progenitor Cells and Induces Angiogenesis in Vivo.

Cell cycle slowdown or arrest is a prominent feature of cellular senescence. S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, is a key regulator of G1/S transition. We investigated whether Skp2 plays a role in the regulation of endothelial progenitor cell (EPC) senescence, which is closely associated with aging-related vasculopathy. Replication-induced senescent EPCs demonstrated more pronounced senescence markers and lower Skp2 levels in comparison with those of their younger counterparts. Depletion of Skp2 induced increases in senescence-associated ß-galactosidase (SA-ßGal) activity and a reduction of telomere length and generated a senescent bioenergetics profile, whereas adenoviral-mediated Skp2 expression reversed the relevant senescence. EPCs isolated from older rats displayed a reduced proliferation rate and increased SA-ßGal activity, both of which were significantly reversed by Skp2 ectopic expression. In addition to reversing senescence, Skp2 also rescued the angiogenic activity of senescent EPCs in the ischemic hind limbs of nude mice. The results revealed that ectopic expression of Skp2 has the potential to rejuvenate senescent EPCs and rescue their angiogenic activity and thus may be pivotal in the development of novel strategies to manage aging-related vascular disease.

Mitochondrial fission protein 1 up-regulation ameliorates senescence-related endothelial dysfunction of human endothelial progenitor cells.

BACKGROUND: We investigated the contribution of mitochondrial dysfunction to the senescence of human endothelial progenitor cells (EPCs) expanded in vitro and the underlying molecular mechanism. METHODS AND RESULTS: Serial passage increased cell doubling time and those cells reaching the doubling time for more than 100% were defined as senescent EPCs, of which the activity of therapeutic angiogenesis was attenuated in mouse ischemic hindlimbs. The senescent cells, in medium free of glucose and bicarbonate, showed impaired activity in migration and tube formation. Flow cytometry indicated increased content of reactive oxygen species, mitochondria, and calcium, while bioenergetic analysis showed increased oxygen consumption and reduced ATP content. Examination of mitochondrial network showed that senescence increased the length of the network and ultrastructure analysis exhibited elongated mitochondria. Immunoblotting of the senescent EPCs demonstrated decreased expression level of fission protein1 (Fis1). In rat EPCs, the Fis1 level was decreased in the animals aged 24 months or older, compared to those of 3 months. Silencing of Fis1 in the young EPCs using Fis1-specific siRNA leads to appearance of phenotype resembling those of senescent cells, including elevated oxidative stress, disturbed mitochondrial network, reduced mitochondria membrane potential, decreasing ATP content, lower proliferation activity, and loss of therapeutic potential in ischemic hindlimbs. Fis1 over-expression in senescent EPCs reduced the oxidative stress, increased the proliferation, and restored the cobble stone-like morphology, senescence, bioenergetics, angiogenic potential, and therapeutic activity. CONCLUSION: In human EPCs, down-regulation of Fis1 is involved in mitochondrial dysfunction and contributes to the impaired activity of EPCs during the senescence process. Enhanced expression of Fis1 in senescent EPCs restores the youthful phenotype.

KC21 Peptide Inhibits Angiogenesis and Attenuates Hypoxia-Induced Retinopathy.

Desmogleins (Dsg2) are the major components of desmosomes. Dsg2 has five extracellular tandem cadherin domains (EC1-EC5) for cell-cell interaction. We had previously confirmed the Dsg2 antibody and its epitope (named KC21) derived from EC2 domain suppressing epithelial-mesenchymal transition and invasion in human cancer cell lines. Here, we screened six peptide fragments derived from EC2 domain and found that KR20, the parental peptide of KC21, was the most potent one on suppressing endothelial colony-forming cell (ECFC) tube-like structure formation. KC21 peptide also attenuated migration but did not disrupt viability and proliferation of ECFCs, consistent with the function to inhibit VEGF-mediated activation of p38 MAPK but not AKT and ERK. Animal studies showed that KC21 peptides suppressed capillary growth in Matrigel implant assay and inhibited oxygen-induced retinal neovascularization. The effects were comparable to bevacizumab (Bev). In conclusion, KC21 peptide is an angiogenic inhibitor potentially useful for treating angiogenesis-related diseases.

Enantioselective and simultaneous determination of lactate and 3-hydroxybutyrate in human plasma and urine using a narrow-bore online two-dimensional high-performance liquid chromatography system.

For the enantioselective and simultaneous analysis of lactate and 3-hydroxybutyrate, a validated online two-dimensional high-performance liquid chromatography system using 4-nitro-7-piperazino-2,1,3-benzoxadiazole as a fluorescent derivatization reagent has been developed. For the reversed-phase separation in the first dimension, a Capcell Pak C18 ACR column (1.5 × 250 mm, particle size 3 µm) was used, and the target fractions were isolated by their hydrophobicity. In the second dimension, a polysaccharide-coated enantioselective column, Chiralpak AD-H (2.0 × 250 mm, 5 µm), was used. The system was validated by the calibration curve, intraday precision, interday precision, and accuracy using standards and real human samples, and satisfactory results were obtained. The present method was applied to human plasma and urine, and in the plasma, trace amounts of d-lactate (8.4 µM) and l-3-hydroxybutyrate (1.0 µM), besides high levels of l-lactate (860.9 µM) and d-3-hydroxybutyrate (59.4 µM), were successfully determined. In urine, trace levels of d-lactate (3.7 µM), d-3-hydroxybutyrate (2.3 µM), and l-3-hydroxybutyrate (3.3 µM) in addition to a relatively large amount of l-lactate (15.4 µM) were observed. The present online two-dimensional high-performance liquid chromatography system is useful for the simultaneous determination of all the lactate and 3-hydroxybutyrate enantiomers in human physiological fluids, and further clinical applications are ongoing.

Reduction of connexin43 in human endothelial progenitor cells impairs the angiogenic potential.

Our previous work showed that arsenic trioxide down-regulated Cx43 and attenuated the angiogenic potential of human late endothelial progenitor cells (EPC). However, the relation between Cx43 and angiogenic activity of the EPC remained unclear. In the study, human late EPC were treated with siRNA specific to Cx43 (Cx43siRNA). The expression profiles as well as activity of the treated cells were examined. In parallel, the angiogenic potential of human EPC treated with Cx43siRNA was evaluated using murine hind limb ischemic model. The results showed that, in the EPC treated with Cx43siRNA, the activity of migration, proliferation, and angiogenic potential were attenuated, accompanied by reduction in vascular endothelial growth factor (VEGF) expression. In hind limb ischemia mice, EPC treated with Cx43siRNA lost the therapeutic angiogenic potential. VEGF supplementation partially recovered the activity impaired by Cx43 down-regulation. In conclusion, reduced Cx43 expression per se in the EPC causes decreased expression of VEGF and impaired angiogenic potential of the cells. Prevention of Cx43 reduction is a potential target to maintain the angiogenic potential of the EPC.

Therapeutic angiogenesis of human early endothelial progenitor cells is enhanced by thrombomodulin.

OBJECTIVE: We examined the effect of thrombomodulin (TM) domains 2 and 3 (TMD23) on human early endothelial progenitor cells (EPCs). METHODS AND RESULTS: TM was expressed and released by human EPCs cultured from peripheral blood mononuclear cells (PBMCs). Addition of TMD23 (100 ng/mL) to the cultured PBMCs increased the colony-forming units, chemotactic motility, matrix metalloproteinase activity, and interleukin-8 secretion but decreased tumor necrosis factor-α (TNF-α) release. Analysis of the signal pathway showed that TMD23 activated Akt. Inhibition of phosphatidylinositol-3 kinase-Akt blocked the effects of TMD23 on chemotactic motility, matrix metalloproteinase-9, interleukin-8, and TNF-α. In hindlimb ischemia mice, laser Doppler perfusion imaging of the ischemic limb during the 21 days after arterial ligation showed that the perfusion recovered best with intraperitoneal infusion of TMD23 plus local injection of early EPCs, followed by either infusion of TMD23 or injection of the cells. Animals without either treatment had the worst results. Animals treated with TMD23 also had lower circulating and tissue levels of TNF-α. CONCLUSION: TM is expressed and released by human circulating EPCs. Exogenous TMD23 enhances the angiogenic potential of early EPCs in vitro through activation of phosphatidylinositol-3 kinase-Akt pathway. Coadministration of TMD23 plus early EPCs augments therapeutic angiogenesis of the EPCs in ischemic tissues.

Fluorescent gold nanoclusters as a biocompatible marker for in vitro and in vivo tracking of endothelial cells.

We have been investigating the fluorescent property and biocompatibility of novel fluorescent gold nanoclusters (FANC) in human aortic endothelial cells (HAEC) and endothelial progenitor cells (EPC). FANC (50-1000 nmol/L) was delivered into cells via the liposome complex. The fluorescence lasted for at least 28 days with a half-life of 9 days in vitro. Examination of 12 transcripts regulating the essential function of endothelial cells after a 72 h delivery showed that only the vascular cell adhesion molecule 1 and the vascular endothelial cadherin were down-regulated at high concentration (500 nmol/L). In addition, no activation of caspase 3 or proliferating cell nuclear antigens was detected. 3-[4,5-Dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) assay demonstrated that, unlike the markedly suppressed viability in cells treated with quantum dots, FANC had minimal effect on the viability, unless above 500 nmol/L, at which level a minor reduction of viability mainly caused by liposome was found. Tube formation assay showed no impaired angiogenesis in the EPC treated with FANC. In vivo study using hindlimb ischemic mice with an intramuscular injection of FANC-labeled human EPC showed that the cells preserved an angiogenic potential and exhibited traceable signals after 21 days. These findings demonstrated that FANC is a promising biocompatible fluorescent probe.

Localization of cholecystokinin and vasoactive intestinal peptide in lower biliary tract in cats following electroacupuncture on right Qimen (LR14) and Riyue (GB 24): an immunohistochemistry study.

Accumulating evidence has shown that control of the motility of the sphincter of Oddi (SO) involves a complex interaction between nerves, neurotransmitters and gastrointestinal hormones such as vasoactive intestinal peptide (VIP) and cholecystokinin (CCK). Our previous studies demonstrated that electroacupuncture (EA) modulated the SO motility in cats and rabbits through activation of nonadrenergic non-cholinergic (NANC) pathway. This study was designed to investigate the changes of neurotransmitters such as CCK and VIP in lower biliary tract in cats receiving EA stimulation. After cats were anesthetized with intramuscular injection of ketamine hydrochloride, they were prepared to conduct EA stimulation on right Qimen (LR14) and Riyue (GB 24). The parameters of EA were 6 pulses/ 3 sec and 45 pulses/ 3 sec alternatively in frequency, 1-2 mA in intensity and 20 min in stimulation duration. After the completeness of EA stimulation, visceral organs such as gallbladder, duodenum and the sphincter of Oddi were removed and frozen for immunohistochemistry localization of CCK and VIP. The results showed that the distribution of CCK-labeled cells in duodenum, gallbladder and SO were more and distinct after EA than before EA stimulation. Whereas, the VIP-labeled cells were significantly more and distinct in duodenum and SO, but not in gall bladder. We conclude that EA regulates the biliary motility though increasing the distribution of CCK- and VIP-containing cells in duodenum and the sphincter of Oddi.

Chinese medicinal herbs Muh-Shiang Bin-Lang-Wan increases the motility of sphincter of Oddi in anesthetized rabbits through activation of M1 muscarinic receptors.

The sphincter of Oddi (SO) plays an important role in regulating the bile flow into the duodenum. This study was designed to investigate the effect of Chinese Medicinal Herbs Muh-Shiang-Bin-Lang-Wan (MSBLW) and their mechanism of action on regulating the motility of SO in rabbits. The activity of SO in anesthetized rabbits was measured by using a continuously perfused open-tip manometric method. The rabbits were administered with different doses of MSBLW through naso-gastric tubes. The SO motility before and after the administration of MSBLW were recorded, and analyzed with a computer equipped with an off line analysis software. The results showed that the SO activity, in terms of tonic pressure and phasic contraction pressure, were significantly changed. A significant lower tonic pressure and a higher phasic contraction pressure were noticed 40-60 min after administration of MSBLW with a peak response at 0.5-1.0 gm range. The responses were blocked by pretreatment of muscarinic receptors (M1) antagonist, pirenzepine (10 mg/kg, orally). We conclude that MSBLW is effective in increasing the SO motility in rabbits through activation of M1 muscarinic receptors. However, potential application of MSBLW in the treatment of human biliary disorders needs further evaluation.

Effect of calcium channel antagonist diltiazem and calcium ionophore A23187 on cyclosporine A-induced apoptosis of renal tubular cells.

Calcium channel antagonists have been reported to have a favorable impact on cyclosporin A (CsA)-treated kidney transplant recipients. However, it is not clear whether this is because of their direct effect on antagonizing the toxicity of CsA to renal tubular cells. In this study, we have used Madin-Darby canine kidney tubular cells as a model to examine the effect of diltiazem, a calcium channel antagonist, on CsA-induced apoptosis. Moreover, to investigate the possible regulation of CsA cytotoxicity by intracellular calcium level, the effect of the calcium ionophore A23187 on CsA-induced apoptosis was also examined. We found that treatment of CsA (20 microM) alone caused 20-30% cell death, which was apparently (30-40%) enhanced by diltiazem at 100 microg/ml, accompanied by more severe DNA fragmentation, activation of caspases, and a decreased level of Bcl-2. The caspase inhibitor ZVAD-fmk or Bcl-2 overexpression was capable of suppressing apoptosis induced by the synergistic effect of diltiazem and CsA. Moreover, the survival rate of cells treated with CsA (30 microM) alone remained only 30%, however, it was markedly (approximately 40%) elevated by co-treatment with A23187 (75 ng/ml). The rescue of cells from CsA-induced apoptosis by A23187 was correlated with AKT activation, BAD phosphorylation, and caspase-3 inactivation. Taken together, our results suggest that the reported favorable impact of diltiazem on kidney grafts is likely not because of its direct protection on renal tubular cells. Instead, it enhances the toxicity of CsA to renal tubular cells. In addition, our findings raise a possibility that the intracellular calcium level and the AKT pathway may participate in the regulation of CsA cytotoxicity.