Characterization of AST-001 non-clinical pharmacokinetics: A novel selective AKR1C3-activated prodrug in mice, rats, and cynomolgus monkeys.

AST-001 is a chemically synthesized inactive nitrogen mustard prodrug that is selectively cleaved to a cytotoxic aziridine (AST-2660) via aldo-keto reductase family 1 member C3 (AKR1C3). The purpose of this study was to investigate the pharmacokinetics and tissue distribution of the prodrug, AST-001, and its active metabolite, AST-2660, in mice, rats, and monkeys. After single and once daily intravenous bolus doses of 1.5, 4.5, and 13.5 mg/kg AST-001 to Sprague-Dawley rats and once daily 1 h intravenous infusions of 0.5, 1.5, and 4.5 mg/kg AST-001 to cynomolgus monkeys, AST-001 exhibited dose-dependent pharmacokinetics and reached peak plasma levels at the end of the infusion. No significant accumulation and gender differences were observed after 7 days of repeated dosing. In rats, the half-life of AST-001 was dose independent and ranged from 4.89 to 5.75 h. In cynomolgus monkeys, the half-life of AST-001 was from 1.66 to 5.56 h and increased with dose. In tissue distribution studies conducted in Sprague-Dawley rats and in liver cancer PDX models in female athymic nude mice implanted with LI6643 or LI6280 HepG2-GFP tumor fragments, AST-001 was extensively distributed to selected tissues. Following a single intravenous dose, AST-001 was not excreted primarily as the prodrug, AST-001 or the metabolite AST-2660 in the urine, feces, and bile. A comprehensive analysis of the preclinical data and inter-species allometric scaling were used to estimate the pharmacokinetic parameters of AST-001 in humans and led to the recommendation of a starting dose of 5 mg/m2 in the first-in-human dose escalation study.

[Acute renal failure caused by rhabdomyolysis in children: a clinical analysis of 26 cases].

OBJECTIVE: To investigate the clinical features and prognosis of acute renal failure (ARF) caused by rhabdomyolysis (RM) in children. METHODS: A retrospective analysis was performed for the clinical data, laboratory examination, and prognosis of 26 RM children with ARF. RESULTS: The causes for all 26 RM children with ARF were non-traumatic diseases, and the three most common causes were infection (69%), diabetes (12%), and metabolic disease (8%). In the RM children with ARF, the five most frequent clinical manifestations were fever (69%), multiple organ dysfunction syndrome (69%), convulsion (46%), oliguria or anuria (35%), and tea-colored urine (27%). All 26 children had a serum creatine kinase (CK) level of >1 000 IU/L, among whom 26 had increased aspartate aminotransferase, 25 had increased alanine aminotransferase, 25 had increased creatine kinase isoenzyme, and 23 had increased lactate dehydrogenase. Serum myoglobin (Mb) was measured in 22 children and was found to increase in all these children. The mean time for CK to decrease to below 1 000 IU/L was 10±5 d. There was no significant difference in the time to CK recovery between the 10 children who were treated with conventional treatment as well as continuous venous-venous hemofiltration and those who were not treated with blood purification (P>0.05). Of all 26 RM children with ARF, 7 were withdrawn from the treatment, and 19 had normal renal function after treatment. CONCLUSIONS: ARF and multiple organ dysfunction syndrome are major complications in RM children. The major primary disease for RM children with ARF is infectious disease. CK is the major marker for the diagnosis of RM. Early diagnosis and appropriate treatment may reverse ARF and improve prognosis.

Short-term MyD88 inhibition ameliorates cardiac graft rejection and promotes donor-specific hyporesponsiveness of skin grafts in mice.

Recognition of evolutionarily conserved ligands by Toll-like receptors (TLRs) triggers signaling cascades in innate immune cells to amplify adaptive immune responses. Nearly all TLRs require MyD88 to transduce downstream signaling. MyD88 deficiency has been shown to promote the allograft acceptance in mice. However, direct evidence for therapeutic potential of MyD88 inhibitors remains lacking. Herein, we used a MyD88 inhibitor, namely ST2825, to explore its therapeutic potential and mechanisms in fully allogeneic skin and heart transplant models. Phenotypic maturation of dendritic cells stimulated by TLR ligands was alleviated by ST2825 in parallel with reduced T-cell proliferation in vitro. A short-course treatment with ST2825 significantly prolonged cardiac graft survival (mean survival time = 18.5 ± 0.92 days vs. 7.25 ± 0.46 days). ST2825-treated group had significantly reduced proinflammatory cytokines in allografts compared with control group. ST2825 combined with anti-CD154 induced long-term skin allograft acceptance in about one-third of recipients (>100 days). 'Skin-tolerant' recipients showed attenuated donor-specific IFN-γ responses, intact IL-4 responses, and compromised alloantibody responses. We conclude that MyD88 inhibitor ST2825 attenuates acute cardiac rejection and promotes donor-specific hyporesponsiveness in stringent skin transplant models. The direct evidence suggests that pharmacological inhibition of MyD88 hold promising potential for transplant rejection.

Possible benefit of splenectomy in liver transplantation for autoimmune hepatitis.

Liver transplantation for autoimmune hepatitis (AIH) is usually successful with excellent long-term outcomes, but primary disease may recur. The recurrence of AIH is a significant cause of graft loss. This study was to analyze the effect of splenectomy in preventing AIH relapse. The clinical courses of 12 patients who had transplantation for AIH were analyzed retrospectively. All patients were subjected to transplantation for end-stage liver disease caused by chronic AIH. Based on the duration of immunosuppressive treatment before liver transplantation, simultaneous splenectomy was performed in ten patients. Two patients underwent liver transplantation without splenectomy, one of them developed recurrent AIH and died from graft failure caused by AIH relapse. However, no episode of AIH recurrence was observed in patients who had undergone simultaneous splenectomy. Splenectomy might be an option to prevent AIH relapse in some patients with high risk factors.

PIII/PV-Catalyzed Beckmann Reaction and Sequential [2,3]-Sigmatropic Rearrangement to Construct 2-Amidopyridines.

An organophosphorus catalytic method for the synthesis of substituted 2-amidopyridines is reported. The method employs a small-ring organophosphorus-based catalyst and a hydrosilane reductant to drive the conversion of ketoximes and pyridine-N-oxides into 2-amidopyridines through sequential Beckmann rearrangement followed by [2,3]-sigmatropic rearrangement. The readily available ketoximes could be activated to nitrilium ions in PIII/PV redox catalysis and could efficiently participate in the domino reaction of pyridine-N-oxides, thus providing various substituted 2-amidopyridines in moderate to excellent yields. This presented strategy features excellent functional group tolerance and a broad substrate scope.

Differential proteomics analysis of the analgesic effect of electroacupuncture intervention in the hippocampus following neuropathic pain in rats.

BACKGROUND: Evidence is building steadily on the effectiveness of acupuncture therapy in pain relief and repeated acupuncture-induced pain relief is accompanied by improvement of hippocampal neural synaptic plasticity. To further test the cellular and molecular changes underlying analgesic effect of acupuncture, the global change of acupuncture associated protein profiles in the hippocampus under neuropathic pain condition was profiled. METHODS: The chronic constrictive injury (CCI) model was established by ligature of the unilateral sciatic nerve in adult Wistar rats. Rats were randomized into normal control (NC) group, CCI group, and CCI with electroacupuncture (EA) stimulation group. EA was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) in the EA group. Differentially expressed proteins in the hippocampus in the three groups were identified by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The functional clustering of the identified proteins was analyzed by Mascot software. RESULTS: After CCI, the thermal pain threshold of the affected hind footpad was decreased and was reversed gradually by 12 sessions of acupuncture treatment. Following EA, there were 19 hippocampal proteins identified with significant changes in expression (>2-fold), which are involved in metabolic, physiological, and cellular processes. The top three canonical pathways identified were "cysteine metabolism", "valine, leucine, and isoleucine degradation" and "mitogen-activated protein kinase (MAPK) signaling". CONCLUSIONS: These data suggest that the analgesic effect of EA is mediated by regulation of hippocampal proteins related to amino acid metabolism and activation of the MAPK signaling pathway.

Distinct metabonomic signatures of Polygoni Multiflori Radix Praeparata against glucolipid metabolic disorders.

OBJECTIVES: Glucolipid metabolic disorders (GLMD) promote a series of major chronic diseases. Polygoni Multilori Radix Preparata (PMRP) has been widely acknowledged in the prevention and treatment of GLMD. We previously reported that water extract (WE) of PMRP and its major bioactive constituents such as polysaccharides (POL) and 2,3,5,4´-tetrahydroxy-stilbene-2-O-ß-D-glucoside (TSG) could alleviate GLMD. The mitochondrial dysfunction is an important mechanism of GLMD, but the underlying mechanisms behind the regulation of mitochondria to alleviate GLMD by WE, POL from PMRP and TSG are still unknown. METHODS: In this study, we elucidated the effects of WE, POL, and TSG towards regulating the mitochondrial dysfunction and alleviating GLMD using mitochondrial metabonomics. A rat model of GLMD was established by high-sugar and high-fat (HS-HF) diet. Rats were intragastrically given WE, POL, and TSG for 12 weeks. The liver mitochondrial metabolites were analyzed by ultra-high-performance liquid chromatography/mass spectrometry followed by multivariate statistical analysis to identify the differential metabolites and metabolic pathways. KEY FINDINGS: The WE, POL, and TSG could significantly restore the level of endogenous metabolites in liver mitochondria toward normal status. In total, sixteen, seven, and fourteen differential metabolites were identified in the liver mitochondrial samples obtained from the WE, GOL, and TSG groups, respectively. These metabolites were found to be mainly involved in glycerol phospholipid, histidine, alanine, aspartic acid, glutamate metabolism, and arginine biosynthesis. CONCLUSIONS: PMRP could improve the liver mitochondrial function by regulating the mitochondrial metabolic pathways to alleviate GLMD. Therefore, the application of PMRP might be a promising mitochondrial regulator/nutrient for alleviating GLMD-associated diseases and the mitochondrial metabonomics might provide insights into the evaluation of the efficacies and mechanisms of action of drugs.

An improved purification approach with high cell viability and low cell loss for cryopreserved hepatocytes.

A modified purification procedure is described for effectively eliminating dead cells after hepatocyte cryopreservation. Isolated hepatocytes from six pig tissue samples were cryopreserved in liquid nitrogen for 2 weeks. After thawing, we developed a pre-incubation step prior to gradient centrifugation. The hepatocytes were subsequent cultured in suspension overnight (12-16 h), and then dead cells were eliminated by Ficoll 400 purification. The results showed that a high viability (mean of 96%) of cells was obtained, with a low viable cell loss in number (2-5%), by using this modified method.

Clinicopathological characteristics and surgical outcomes of sarcomatoid hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common type of cancer and the fourth leading cause of cancer-related death worldwide. Sarcomatoid HCC, which contains poorly differentiated carcinomatous and sarcomatous components, is a rare histological subtype of HCC that differs from conventional HCC. It is highly aggressive and has a poor prognosis. Its clinicopathological characteristics, surgical outcomes and underlying mechanisms of its highly aggressive nature have not been fully elucidated. AIM: To examine the clinicopathological characteristics and surgical outcomes of sarcomatoid HCC and explore the histogenesis of sarcomatoid HCC. METHODS: In total, 196 patients [41 sarcomatoid HCC and 155 high-grade (Edmondson-Steiner grade III or IV) HCC] who underwent surgical resection between 2007 and 2017 were retrospectively reviewed. The characteristics and surgical outcomes of sarcomatoid HCC were compared with those of patients with high-grade HCC. The histological composition of invasive and metastatic sarcomatoid HCCs was evaluated. RESULTS: Sarcomatoid HCC was more frequently diagnosed at an advanced stage with a larger tumor and higher rates of nonspecific symptom, adjacent organ invasion and lymph node metastasis than high-grade HCC (all P < 0.05). Compared with high-grade HCC patients, sarcomatoid HCC patients are less likely to have typical dynamic imaging features of HCC (44.4% vs 72.7%, P = 0.001) and elevated serum alpha-fetoprotein levels (> 20 ng/mL; 36.6% vs 78.7%, P < 0.001). The sarcomatoid group had a significantly shorter median recurrence-free survival (5.6 mo vs 16.4 mo, log-rank P < 0.0001) and overall survival (10.5 mo vs 48.1 mo, log-rank P < 0.0001) than the high-grade group. After controlling for confounding factors, the sarcomatoid subtype was identified as an independent predictor of poor prognosis. Pathological analyses indicated that invasive and metastatic lesions were mainly composed of carcinomatous components. CONCLUSION: Sarcomatoid HCC was associated with a more advanced stage, atypical dynamic imaging, lower serum alpha-fetoprotein levels and a worse prognosis. The highly aggressive nature of sarcomatoid HCC is perhaps mediated by carcinomatous components.

Hepatocyte isolation from resected benign tissues: Results of a 5-year experience.

AIM: To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease. METHODS: We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic duct calculi (n = 7) and liver hemangioma (n = 17). Only the samples weighing ≥ 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation. RESULTS: Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 10(6) hepatocytes/g liver, with 76.4% ± 10.7% viability. The 17 samples of liver hemangioma had significantly higher yield of cells (5.4 ± 1.71 × 10(6) cells/g vs 3.49 ± 2.31 × 10(6) cells/g, P < 0.05) than the samples of intrahepatic duct calculi. However, there seems to be no clear difference in cell viability (80.3% ± 9.67% vs 76.4% ± 10.7%, P > 0.05). We obtained a cell yield of 5.31 ± 1.87 × 10(6) hepatocytes/g liver when the samples weighed > 20 g. However, for the tissues weighing ≤ 20 g, a reduction in yield was found (3.08 ± 1.86 × 10(6) cells/g vs 5.31 ± 1.87 × 10(6) cells/g, P < 0.05). CONCLUSION: Benign diseased livers are valuable sources for large-number hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermore, the size of the tissues can affect the outcome of hepatocyte isolation.

Ibandronate to treat skeletal-related events and bone pain in metastatic bone disease or multiple myeloma: a meta-analysis of randomised clinical trials.

OBJECTIVE: Randomised controlled trials (RCTs) have given contradictory results about the efficacy and safety of ibandronate in treating metastatic bone disease (MBD) or multiple myeloma. This review meta-analysed the literature to gain a more comprehensive picture. DESIGN: Systematic review and meta-analysis of ibandronate compared with placebo or zoledronate. DATA SOURCES: PubMed, EMBASE and the Cochrane Library databases were systematically searched to identify RCTs published up to March 2015 evaluating ibandronate to treat MBD or multiple myeloma. REVIEW METHOD: 10 RCTs involving 3474 patients were included. Six RCTs were placebo-controlled and four compared ibandronate with zoledronate. The studies included in this review were mainly from European countries. RESULTS: Intravenous ibandronate (6 mg) or oral drug (50 mg) decreased the risk of skeletal-related events compared to placebo (risk ratio (RR) 0.80, 95% CI 0.71 to 0.90, p=0.002). It also reduced the bone pain score below baseline significantly more than did placebo at 96 weeks (weighted mean difference -0.41, 95% CI -0.56 to -0.27, p<0.001). The incidence of diarrhoea, nausea and adverse renal events was similar between the ibandronate and placebo groups, but ibandronate was associated with greater risk of abdominal pain. Ibandronate was associated with similar risk of skeletal-related events as another bisphosphonate drug, zoledronate (RR 1.02, 95% CI 0.82 to 1.26, p=0.87). The incidence of nausea, jaw osteonecrosis and fatigue was similar for the two drugs, but the incidence of adverse renal events was significantly lower in the ibandronate group. CONCLUSIONS: Ibandronate significantly reduces the incidence of skeletal-related events and bone pain in patients with MBD or multiple myeloma relative to placebo. It is associated with a similar incidence of skeletal-related events as zoledronate.

Comparison of Body, Auricular, and Abdominal Acupuncture Treatments for Insomnia Differentiated as Internal Harassment of Phlegm-Heat Syndrome: An Orthogonal Design.

Objective. To identify the optimum treatment protocol for insomnia among auricular, body, and abdominal needling methods. Methods. A three-factor (3 needling protocols) and three-level experimental scheme was designed based on orthogonal method. 54 patients of insomnia differentiated as internal harassment of phlegm-heat syndrome were given two courses of acupuncture treatment (each with 20 times of acupuncture). The therapeutic effects were evaluated by comparing the Pittsburgh sleep quality index (PSQI), Hamilton Depression Scale (HAMD) scores, and Hamilton Anxiety Scale (HAMA) scores of patients before treatment, after one course of treatment, and after two courses of treatment as well as one month after treatment. Results. Body, auricular, and abdominal acupuncture treatments all alleviated symptoms of insomnia, depression, and anxiety, but body and auricular acupuncture had stronger therapeutic effects. Conclusions. Body acupuncture at basic points shall be given priority in protocol selection for insomnia. The second-best choice is auricular acupuncture with basic points combined with points based on Traditional Chinese Medicine (TCM) theories. Abdominal needling with very quick effect can be an alternative protocol with basic points combined with syndrome differentiation points.

[Effect of warm acupuncture stimulation of Waiguan (TE 5) on post-stroke shoulder-hand syndrome].

OBJECTIVE: To evaluate the therapeutic effect of warm acupuncture (moxa-heated acupuncture) needle stimulation of Waiguan (TE 5) acupoint in the treatment of shoulder-hand syndrome (phase I) in patients with stroke. METHODS: Sixty stroke patients with shoulder-hand syndrome (phase I ) were equally randomized into control group and warm acupuncture group. Patients of the warm acupuncture group were treated by warm acupuncture stimulation of the affected TE 5 in combination with routine acupuncture stimulation of Jianyu (LI 15), Jianjing (GB 21), Quchi (LI 11), Wangu (SI 4), Yangchi (TE 4) and Hegu (LI 4), and rehabilitation training (passive and active upper-limb motion exercise for 30 min, once daily), and patients of the control group treated with routine acupuncture stimulation of the same acupoints mentioned above, and rehabilitation training. The treatment was conducted once daily, 5 times per week for two weeks. The patients' clinical conditions were evaluated by using Visual Analogue Scale (VAS, 0-10 points, shoulder pain degree), edema severity score (0 point:normal, 2 points: mild, 4 points: moderate, and 6 points: severe) and simplified Fugl-Meyer motor assessment scale (0, 1 and 2 points, upper-limb motor function) before and after the treatment. RESULTS: After the treatment, the scores of VAS and edema severity of the two groups were significantly decreased in comparison with pre-treatment in the same one group (P < 0.01), and the Fugl-Meyer motor scores were considerably increased (P < 0.01), suggesting an improvement of the shoulder-hand syndrome after two weeks' treatment. Of the two 30 patients in the control group and warm acupuncture group, 0 and 2 (6.7%) were cured, 7 (23.3%) and 25 (83.3%) experienced marked improvement, 17 (56.7%) and 2 (6.7%) were effective, 6 (20. 0%) and 1 (3.3%) invalid, with the effective rates being 80.0% and 96.7%, respectively. The effect of warm acupuncture group was superior to that of the control group (P < 0.05). CONCLUSION: Warm acupuncture combined with routine acupuncture and rehabilitation training is effective in improving shoulder pain, hand edema and limb motor function in stroke patients with shoulder-hand syndrome at phase I.

Reversible immortalization of human hepatocytes mediated by retroviral transfer and site-specific recombination.

AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications. METHODS: We successfully isolated primary human hepatocytes from surgically resected liver tissue taken from a patient with liver hemangiomas. The freshly isolated cells were then immortalized with retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40T) and hygromycin-resistance genes flanked by paired loxP recombination targets. RESULTS: The freshly isolated hepatocytes with high viability (85%) were successfully immortalized using retroviral gene transfer of SV40T. SV40T in the immortalized cells was then excised by Cre/loxP site-specific recombination. This cell population exhibited the characteristics of differentiated hepatocytes. CONCLUSION: We successfully established reversibly immortalized human hepatocytes, which will provide an unlimited supply of cells for practical applications.

Analysis on interrelation between electroacupuncture-induced cumulative analgesic effect and hypothalamic cholinergic activities in chronic neuropathic pain rats.

OBJECTIVE: To observe the effects of repeated electroacupuncture (EA) of Zusanli (ST36)- Yanglingquan (GB34) on hypothalamic acetylcholinesterase (AchE) and vesicular acetylcholine (ACh) transporter (VAChT) activities and choline acetyltransferase (ChAT) mRNA and muscarinic M1 receptor (M1R) mRNA expression in chronic constrictive injury (CCI) and/or ovariectomy (OVX) rats so as to reveal its underlying mechanism in cumulative analgesia. METHODS: A total of 103 female Wistar rats were randomly divided into normal control (n =15), CCI (n =15), CCI+EA2d (n =15), CCI+EA2W (n =15), OVX+CCI =13), OVX+CCI+EA2d (n =15), and OVX+CCI+EA2W groups (n =15). CCI model was established by ligature of the unilateral sciatic nerve with surgical suture. Memory impairment model was established by removal of the bilateral ovaries. Morris water test was conducted to evaluate the OVX rats' memory learning ability, and the thermal pain threshold (PT) of the bilateral paws was detected the next morning after EA. EA (2/15 Hz, 1 mA) was applied to bilateral ST36-GB34 for 30 min, once daily for 2 days or 2 weeks, respectively. Hypothalamic AChE activity was detected by histochemistry, VAChT immunoactivity was determined by immunohistochemistry, and ChAT mRNA and M1R mRNA expressions were assayed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In comparison with the normal control group, the AChE activity in hypothalamic arcuate nucleus (ARC) and supraoptic nucleus (SON) regions of CCI group, AChE activity in paraventricular nucleus (PVN), ARC, and SON regions of OVX+CCI group, and hypothalamic muscarinic M1R mRNA expression levels in both CCI and OVX+CCI groups were down-regulated significantly (P <0.05). Compared with the CCI group, the AChE activities in hypothalamic ARC and SON regions of CCI+EA2d and CCI+EA2W groups and PVN region of CCI+EA2W group and hypothalamic ChAT mRNA and M1R mRNA expression levels in CCI+EA2W group were up-regulated considerably (P <0.05). In comparison with the OVX+CCI group, the AChE activities in PVN, ARC, and SON regions and the expressions of hypothalamic ChAT mRNA and VAChT in ARC region of OVX+CCI+EA2W group were up-regulated remarkably (P <0.05). The effects in rats of CCI+EA2W group were evidently superior to those of OVX+CCI+EA2d group in up-regulating AChE activities in PVN, ARC, and SON regions, VAChT immunoactivity in ARC region, and expression levels of hypothalamic ChAT mRNA and M1R mRNA (P <0.05). Similar situations were found in OVX+CCI rats after EA2W. It suggested a cumulative effect after repeated EA of ST36-GB34. Comparison between CCI+EA2W and OVX+CCI+EA2W groups showed that the effects in rats of the former group were evidently better than those of the latter group in up-regulating AChE activity in ARC and SON regions and the expressions of hypothalamic ChAT mRNA and M1 mRNA (P <0.05), suggesting a reduction of EA2W effects after OVX. CONCLUSION: Repeated EA can significantly up-regulate AChE and VAChT activities and ChAT mRNA and M1R mRNA expressions in the hypothalamus of CCI and OVX+CCI rats, which may contribute to the cumulative analgesic effects of repeated EA and be closely related to the animals' neuromemory ability.

Effects of repeated electroacupuncture on beta-endorphin and adrencorticotropic hormone levels in the hypothalamus and pituitary in rats with chronic pain and ovariectomy.

OBJECTIVE: To explore the mechanism of electroacupuncture (EA)-induced cumulative analgesic effects on chronic pain in rats with or without ovariectomy (OVX). METHODS: A total of 110 female Wistar rats were randomized into normal control (n=10), chronic constrictive injury (CCI, n=10), CCI+EA (n=30), OVX+CCI (n=30), and OVX+CCI+EA (n=30) groups. Each of the latter 3 groups was further divided into 2 days (2 d), 2 weeks (2 W) and 3 weeks (3 W) subgroups, respectively (n=10 in each subgroup). The CCI pain model was established by ligature of the right sciatic nerve, and the memory impairment model duplicated by OVX. The paw withdrawal latency (PWL, pain threshold) of the bilateral footplates was detected by radiant heat irradiation, and the bilateral difference in PWL (PWLD) was used to evaluate changes in the pain reaction. Morris water maze test was conducted for evaluating the rats' learning-memory ability. EA was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) for 2 d, 2 W and 3 W, respectively. Pituitary and hypothalamic beta-endorphin (EP) and adrenocorticotrophic hormone (ACTH) contents were detected by immunoradioassay. RESULTS: Compared with the CCI group, PWLD of the CCI+EA-3 W group decreased significantly (P<0.05). Compared with the OVX+CCI group, PWLD of the OVX+CCI+EA-3 W group was lowered considerably (P<0.05), but the value was markedly higher than its basal value and those of the normal control and CCI+EA groups (P<0.05). In comparison with the sham-OVX group, the escape latency, swimming distance (SD) in the target quadrant and total SD were increased remarkably in the OVX group (P<0.05), while the number of target platform crossings was decreased significantly (P<0.05), suggesting an impairment of the OVX rats' learning-memory ability. In simple CCI rats, both beta-EP and ACTH contents of the pituitary increased markedly (P<0.05), and those of the hypothalamus decreased obviously compared to the normal control group (P<0.05). After EA, pituitary and hypothalamic ACTH levels were significantly lowered at 2 d and hypothalamic ACTH and beta-EP contents increased obviously at 3 W in comparison with the CCI group (P<0.05). In OVX+CCI rats, following EA, pituitary beta-EP contents at 2 d, 2 W and 3 W, and hypothalamic beta-EP and ACTH contents at 2 W and hypothalamic ACTH levels at 3 W increased significantly (P<0.05), but hypothalamic beta-EP level at 3W decreased markedly (P<0.05). The effects of repeated EA in lowering pituitary ACTH and raising hypothalamic beta-EP and ACTH levels disappeared after OVX+CCI. CONCLUSIONS: Repeated EA has a cumulative analgesic effect, which is closely associated with its effects in regulating pituitary and hypothalamic beta-EP and ACTH levels. OVX may weaken the analgesic effect of EA by affecting hypothalamic-pituitary axis activity.

Persistent effect of IFNAR-1 genetic polymorphism on the long-term pathogenesis of chronic HBV infection.

Genetic polymorphism of IFNAR-1 plays a large role in determining the clearance or chronicity after hepatitis B virus (HBV) exposure. However, it is not clear whether type I interferon receptor-1 (IFNAR-1) variations continuously exert their effects to influence the final outcomes following HBV chronicity, including acute-on-chronic hepatitis B liver failure (ACLF-HBV), chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Here we report that these four potential outcomes of chronic HBV infection are strongly associated with IFNAR-1 polymorphisms through a hospital-based case-control study of 663 cases. ACLF-HBV and HCC were each compared with CHB+LC. In comparison with the G/G genotype, the C/G and C/C genotypes at both single-nucleotide polymorphism (SNP) sites (rs1012335 and rs2257167) showed significant susceptibility to ACLF-HBV (the highest odds ratio [OR] reached 2.374; 95% CI = 1.488, 3.788; p < 0.001 for the C/G genotype at rs2257167), as well as HCC (OR = 2.475; 95% CI = 1.435, 4.426; p < 0.001 for the C/C genotype at rs1012335). The C allele at both loci was a susceptibility allele for ACLF-HBV and HCC, with the highest ORs reaching 1.653 (95% CI = 1.233, 2.216; p < 0.01 at rs1012335) in the ACLF-HBV group, and 1.659 (95% CI = 1.274, 2.159; p < 0.01 at rs1012335) in the HCC group. A strongly linked disequilibrium was also found within these two alleles (p < 0.001). Our research indicates that genetic polymorphisms of IFNAR-1 not only contribute to the determination of clearance or chronicity in the early stages of HBV exposure, but they also persistently influence pathogenesis over the long-term process of chronic HBV infection.

Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination.

AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40Tag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.

Combined en bloc liver/pancreas transplantation in two different patients.

Combined en bloc liver/pancreas transplantation (CLPT) was used primarily in the treatment of otherwise non-resectable upper abdominal malignancy. In fact, a more appropriate indication is in patients with liver disease and insulin-dependent diabetes mellitus (IDDM). Here, we report on two successful cases of CLPT at our hospital. One was a patient with non-resectable advanced liver cancer. The recipient survived for 23 mo and finally died of recurrent tumor. The other was a patient with severe biliary complication after orthotopic liver transplantation and preoperative IDDM. We performed CLPT with a modified surgical technique of preserving the native pancreas. He is currently liver-disease- and insulin-free more than 27 mo post-transplant. Based on our experience in two cases of abdominal cluster transplantation, we describe the technical details of CLPT and a modification of the surgical procedure.