Modulation of the Wnt pathway through inhibition of CLK2 and DYRK1A by lorecivivint as a novel, potentially disease-modifying approach for knee osteoarthritis treatment.

OBJECTIVES: Wnt pathway upregulation contributes to knee osteoarthritis (OA) through osteoblast differentiation, increased catabolic enzymes, and inflammation. The small-molecule Wnt pathway inhibitor, lorecivivint (SM04690), which previously demonstrated chondrogenesis and cartilage protection in an animal OA model, was evaluated to elucidate its mechanism of action. DESIGN: Biochemical assays measured kinase activity. Western blots measured protein phosphorylation in human mesenchymal stem cells (hMSCs), chondrocytes, and synovial fibroblasts. siRNA knockdown effects in hMSCs and BEAS-2B cells on Wnt pathway, chondrogenic genes, and LPS-induced inflammatory cytokines was measured by qPCR. In vivo anti-inflammation, pain, and function were evaluated following single intra-articular (IA) lorecivivint or vehicle injection in the monosodium iodoacetate (MIA)-induced rat OA model. RESULTS: Lorecivivint inhibited intranuclear kinases CDC-like kinase 2 (CLK2) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Lorecivivint inhibited CLK2-mediated phosphorylation of serine/arginine-rich (SR) splicing factors and DYRK1A-mediated phosphorylation of SIRT1 and FOXO1. siRNA knockdowns identified a role for CLK2 and DYRK1A in Wnt pathway modulation without affecting ß-catenin with CLK2 inhibition inducing early chondrogenesis and DYRK1A inhibition enhancing mature chondrocyte function. NF-κB and STAT3 inhibition by lorecivivint reduced inflammation. DYRK1A knockdown was sufficient for anti-inflammatory effects, while combined DYRK1A/CLK2 knockdown enhanced this effect. In the MIA model, lorecivivint inhibited production of inflammatory cytokines and cartilage degradative enzymes, resulting in increased joint cartilage, decreased pain, and improved weight-bearing function. CONCLUSIONS: Lorecivivint inhibition of CLK2 and DYRK1A suggested a novel mechanism for Wnt pathway inhibition, enhancing chondrogenesis, chondrocyte function, and anti-inflammation. Lorecivivint shows potential to modify structure and improve symptoms of knee OA.

A small-molecule inhibitor of the Wnt pathway (SM04690) as a potential disease modifying agent for the treatment of osteoarthritis of the knee.

OBJECTIVES: Osteoarthritis (OA) is a degenerative disease characterized by loss of cartilage and increased subchondral bone within synovial joints. Wnt signaling affects the pathogenesis of OA as this pathway modulates both the differentiation of osteoblasts and chondrocytes, and production of catabolic proteases. A novel small-molecule Wnt pathway inhibitor, SM04690, was evaluated in a series of in vitro and in vivo animal studies to determine its effects on chondrogenesis, cartilage protection and synovial-lined joint pathology. DESIGN: A high-throughput screen was performed using a cell-based reporter assay for Wnt pathway activity to develop a small molecule designated SM04690. Its properties were evaluated in bone-marrow-derived human mesenchymal stem cells (hMSCs) to assess chondrocyte differentiation and effects on cartilage catabolism by immunocytochemistry and gene expression, and glycosaminoglycan breakdown. In vivo effects of SM04690 on Wnt signaling, cartilage regeneration and protection were measured using biochemical and histopathological techniques in a rodent acute cruciate ligament tear and partial medial meniscectomy (ACLT + pMMx) OA model. RESULTS: SM04690 induced hMSC differentiation into mature, functional chondrocytes and decreased cartilage catabolic marker levels compared to vehicle. A single SM04690 intra-articular (IA) injection was efficacious in a rodent OA model, with increased cartilage thickness, evidence for cartilage regeneration, and protection from cartilage catabolism observed, resulting in significantly improved Osteoarthritis Research Society International (OARSI) histology scores and biomarkers, compared to vehicle. CONCLUSIONS: SM04690 induced chondrogenesis and appeared to inhibit joint destruction in a rat OA model, and is a candidate for a potential disease modifying therapy for OA.

Acetylation changes at lysine 5 of histone H4 associated with lytic gene promoters during reactivation of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a pathogenic agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease in humans. Similarly to other gammaherpesviruses such as Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS), KSHV displays two alternative life cycles, latent and lytic one. The transactivation from latency to the lytic phase is the result of transcriptional changes in the KSHV genome caused by the replication and transcriptional activator (RTA). During KSHV reactivation, epigenetic modifications of histone protein on the viral genome occur, which regulate the transcriptional activation of a number of lytic genes. The reactivation of EBV from latency to lytic cycle, induced by an immediate-early Zta protein, was shown to be accompanied by acetylation of specific lysines in histone H4. Accordingly, we hypothesized that the RTA-induced transactivation of KSHV could also be accompanied by histone acetylation. To validate this hypothesis, we assayed alterations of acetyl-histone H4-lysine 5 (acH4K5) during the RTA-mediated KSHV reactivation. While the modified histone protein in a total cell lysate was not distinguished between control and RTA-expressed cells, upregulated acH4K5 was detected on several lytic gene promoter regions during KSHV reactivation. Our results clearly indicate that this epigenetic change is related to transcription of genes expressed in the lytic cycle of KSHV.

Comparison of transnasal small-caliber vs. peroral conventional esophagogastroduodenoscopy for evaluating varices in unsedated cirrhotic patients.

BACKGROUND AND STUDY AIMS: We aimed to evaluate the accuracy of transnasal small-caliber esophagogastroduodenoscopy (TNSC-EGD) compared with peroral conventional EGD (POC-EGD) for evaluating varices in unsedated patients with liver cirrhosis. The success rate, safety, endoscopist satisfaction, and patient tolerability of TNSC-EGD were also addressed. PATIENTS AND METHODS: One hundred patients with liver cirrhosis participated in this randomized crossover trial, and 84 subjects completed both procedures. Of the 84 patients, 28 had marked bleeding diathesis (platelet count ≤ 50000/mm (3) and/or prothrombin time ≥ 1.7 INR). Endoscopists and patients answered questionnaires using a 100-mm visual analog scale about, respectively, their satisfaction and their tolerance of the procedure. RESULTS: The success rate of TNSC-EGD was comparable to that of POC-EGD (96% vs. 99%). Nasal mucosal hemorrhages induced by TNSC-EGD occurred in 5 patients (6%), but were easily controlled. Compared to the POC-EGD reference test, diagnostic accuracies of TNSC-EGD for detecting esophageal varices, gastric varices, and red color signs were 98%, 98%, and 96%, respectively. Concordance rates on grading esophageal varices and gastric varices were excellent at 93% (κ = 0.85) and 96% (κ = 0.87). Endoscopist satisfaction was not significantly different between TNSC-EGD and POC-EGD, whereas patient tolerance of TNSC-EGD was significantly greater than that of POC-EGD (79.0 ± 14.4 vs. 69.5 ± 16.1; P = 0.001). CONCLUSION: TNSC-EGD without sedation was found to be feasible, safe, and accurate for evaluating esophageal varices, gastric varices, and red color signs in patients with cirrhosis - even in those with marked bleeding diathesis. Furthermore, it was significantly better tolerated by patients, without altering endoscopist satisfaction. Our findings indicate that TNSC-EGD without sedation might be viewed as a potential alternative to POC-EGD for evaluation of varices.

Percutaneous aspiration thrombectomy for the treatment of acute lower extremity deep vein thrombosis: is thrombolysis needed?

AIM: To assess the technical feasibility and initial success of aspiration thrombectomy as a potential alternative to lytic therapy in initial endovascular management of acute lower extremity deep vein thrombosis (DVT). MATERIALS AND METHODS: From July 2004 to October 2007, a retrospective analysis of 27 patients (male:female 5:22; mean age 59 years) with acute iliofemoral or femoropopliteal DVT of less than 2 weeks was performed. All patients underwent sonography of the lower extremities, and 13 patients underwent computed tomography (CT) venography. All patients received an inferior vena cava (IVC) filter and were initially treated with aspiration thrombectomy using the pullback technique with or without basket thrombus fragmentation. If persistent stenotic portions (>50% luminal narrowing) were noted, balloon angioplasty or stent placement was performed. Successful recanalization was defined as successful restoration of antegrade flow in the treated vein with elimination of any underlying obstructive lesion. RESULTS: The mean procedure time was 65 min (range 40-100 min). Successful initial recanalization was achieved in 24 patients (88.9%) without complications. Urokinase was required for three patients (11.1%) due to a hard thrombus remaining in the iliac vein. Of the 27 patients, 23 had residual venous stenosis in the common iliac vein or external iliac vein. Therefore, balloon angioplasty (n=23) and stent placement (n=22) was performed. The remaining four patients were treated using only aspiration thrombectomy without angioplasty or stent placement. CONCLUSION: Aspiration thrombectomy without catheter-directed thrombolysis is a safe and effective treatment for acute DVT of the lower extremities, and minimizes the risk of haemorrhagic complications.

Diagnosis and treatment of obscure gastrointestinal bleeding using combined capsule endoscopy and double balloon endoscopy: 1-year follow-up study.

BACKGROUND AND STUDY AIMS: Several studies have shown the value of capsule endoscopy and double balloon endoscopy (DBE) in small-intestinal bleeding. The purpose of this study was to evaluate the impact of capsule endoscopy results on subsequent DBE examination, and the 1-year clinical outcome of this combined approach in patients with obscure gastrointestinal bleeding (OGIB). PATIENTS AND METHODS: A total of 45 consecutive patients with OGIB underwent capsule endoscopy. Patients with positive capsule endoscopy results underwent DBE for biopsy or therapy, and those with negative results underwent further assessment for possible diagnostic misses on capsule endoscopy. Tumors, ulcerations, and vascular lesions were considered as sources of bleeding. Diagnoses of OGIB lesions and clinical outcome were assessed 1 year after these examinations. RESULTS: Responsible lesions were found in 22 patients (49 %): 19 lesions in 18/45 patients (40 %) undergoing capsule endoscopy, and 18/36 patients (50 %) undergoing subsequent DBE. In all, 10 tumors, nine vascular lesions, and four ulcerations were found. In two patients, vascular lesions were only later diagnosed by conventional methods (4 %). Capsule endoscopy results guided our choice of the proper DBE model for successful therapeutic intervention in five patients. Re-bleeding rates were low during 1-year follow-up of the entire group (mean follow-up, 18.8 months): 5 % in cases with positive diagnoses on capsule endoscopy and/or DBE, and 12 % in negative cases. CONCLUSIONS: A combined approach using capsule endoscopy followed by DBE proves valuable in the diagnosis and treatment of patients with OGIB, leaves a low rate of undiagnosed bleeding sources, and has a good long-term outcome.

Effects of intracerebroventricular infusions of arginine vasopressin in sheep.

In sheep, neither the in vivo effect of vasopressin administered by a method other than systemic infusion nor the central effects on behavior from the perspective of stress regulation has been fully elucidated in an intact animal. We examined changes in behavioral, adrenocorticotropic, and autonomic nervous functions after intracerebroventricular infusions of arginine vasopressin (AVP) to elucidate its central role. Intracerebroventricular infusions of AVP (0, 0.12, 1.2 and 12 microg/500 microl/30 min) evoked a dose-related increase in plasma cortisol concentration. There were significant treatment-related effects on the total duration of sham-chewing (Friedman's test, X2=12.75, p=.0052), on the total duration of bar-biting (Friedman's test, X2=15.0, p=.0018), and on the total duration of rubbing (Friedman's test, X2=12.0, p=.0074). AVP 12 microg treatment induced a greater degree of sham-chewing and bar-biting than the other three treatments did (Nemenyi multiple comparisons: p<0.1). These findings indicate, together with our previous findings, that AVP has the same corticotropic potential as corticotropin-releasing hormone infused intracerebroventricularly in equal molar concentrations. Although the degree to which central stress signaling pathways are involved in these responses remains speculative, the relationships between stereotypies and central AVP are of particular interest.

Detection of aflatoxin B1 in serum samples of male Japanese subjects by radioimmunoassay and high-performance liquid chromatography.

Aflatoxin B1 (AFB1) was detected in serum samples of healthy Japanese males by radioimmunoassay and high-performance liquid chromatography. Blood samples were obtained from 20 subjects after fasting and from 80 subjects after lunch. The subjects ranged in age from 20 to 63 years of age. Measurement of AFB1 in the samples was performed by radioimmunoassay and was confirmed by high-performance liquid chromatography using a mu Porasil column and a C18-mu Bondapak column after the conversion of AFB1 to its water adduct AFB2a. AFB1 was detected in 5 of 20 fasting blood samples [20 to 56 pg/ml of serum; 33.6 +/- 14.6 (S.D.)] and in 29 of 80 serum samples taken after lunch (20 to 1169 pg/ml of serum; 218.1 +/- 268.3). Mass spectral analysis of the material obtained after high-performance liquid chromatography separation from serum samples confirmed the presence of AFB1.

Characterization of alpha 2-macroglobulin receptor low density lipoprotein receptor-related protein (alpha 2 MR/LRP) in White Carneau pigeon peritoneal macrophages: its role in lipoprotein metabolism.

White Carneau pigeons develop atherosclerosis naturally, and at an accelerated rate with cholesterol feeding. Macrophages play a central role in the pathogenesis of atherosclerosis in pigeons, as they do in man. The purpose of this study was to determine whether pigeon macrophages express the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) and whether this receptor would recognize beta-VLDL, the major cholesterol-transporting lipoprotein in cholesterol-fed pigeons. The binding of 125I-methylamine-treated alpha 2M (125I-alpha 2 M+) at 4 degrees C was saturable (> 10 nM), specific, Ca2+ dependent, was competed for by the receptor-associated protein (RAP), and had a Kd of binding of 1-5.6 nM, similar to mouse peritoneal macrophages studied simultaneously. At 37 degrees C the bound 125I-alpha 2 M+ was rapidly internalized and degraded in lysosomes. The binding of alpha 2 M+ was not down-regulated with cholesterol loading, as is the LDL receptor on pigeon macrophages. At 4 degrees C there was no competition for binding of 125I-alpha 2 M+ by either pigeon or rabbit beta-VLDL, nor was binding of 125I-pigeon or rabbit beta-VLDL competed for by alpha 2 M+. Stimulation of cholesterol esterification by rabbit or pigeon beta-VLDL was unaffected by RAP, lactoferrin, or alpha 2 M+. Metabolism of 125I-pigeon or rabbit beta-VLDL was not competed by RAP, lactoferrin, or alpha 2 M+ even in the presence of lipoprotein lipase. Pigeon macrophages, and a 500 kDa membrane protein isolated from them, were recognized by several antihuman alpha 2 MR/LRP monoclonal antibodies. The 500 kDa membrane protein also bound 45Ca. These data suggest considerable sequence homology with the human alpha 2 MR/LRP. This is the first study to characterize a functional alpha 2 MR/LRP on peritoneal macrophages from an avian species. There was no evidence, however, that the alpha 2 MR/LRP mediates uptake of beta-VLDL by pigeon macrophages.

Identification of glucokinase mutations in subjects with gestational diabetes mellitus.

Recent studies have shown that mutations in the glucokinase gene on chromosome 7 can cause an autosomal dominant form of NIDDM with a variable clinical phenotype and onset during childhood. The variable clinical phenotype includes mild fasting hyperglycemia (i.e., a plasma glucose value of > 110 mg/dl, a value that is at least 2-3 SDs above normal), impaired glucose tolerance, gestational diabetes mellitus, as well as overt NIDDM as defined using National Diabetes Data Group or World Health Organization criteria. Because gestational diabetes mellitus was a clinical feature associated with glucokinase mutations, we have screened a group of women with gestational diabetes who also had a first-degree relative with diabetes mellitus for the presence of mutations in this gene. Among 40 subjects, we identified two mutations, suggesting a prevalence of approximately 5% in this group. Extrapolating from this result, the prevalence of glucokinase-deficient NIDDM among Americans may be approximately 1 in 2500.

Human GLUT4/muscle-fat glucose-transporter gene. Characterization and genetic variation.

Four overlapping DNA fragments spanning 32 kb containing the human GLUT4 facilitative glucose-transporter gene were isolated and characterized. The sequence of the GLUT4 gene (approximately 6.3 kb) and 2.0 kb of the promoter region was determined. The sequence of the promoter revealed potential binding sites for transcription factors known to regulate gene expression in muscle cells and adipocytes. However, transfection of constructs including 2 kb of the GLUT4 promoter fused to the bacterial CAT gene into 3T3-L1 adipocytes displayed only weak promoter activity. Because insulin resistance plays a prominent role in the development of NIDDM, genetic variation in the sequence of GLUT4 also was evaluated. Oligonucleotide primer pairs were selected that allowed the protein-coding region of the human GLUT4 gene to be amplified by PCR. The sequence of the protein-coding region of the GLUT4 gene and all intron-exon junctions was determined for a single diabetic Pima Indian and was identical to that of the cloned gene and cDNA. SSCP analysis was used to screen patients with diabetes mellitus and normal, healthy nondiabetic individuals for mutations at the GLUT4 locus. In addition to the silent substitution in the codon for Asn130 (AAC or AAT) and a Val383 (GTC)-->Ile(ATC) replacement described previously, two new variants were identified. One was a T-->A substitution in intron 1 that was found in 1 of 36 NIDDM patients who were typed for this variant. The second was a Ile385(ATT)-->Thr(ACT) replacement that occurred in 1 normal individual and was not found in any of 676 other normal and diabetic subjects. A large and racially diverse group of normal and diabetic individuals also was screened for the Ile383 polymorphism. It occurred in both diabetic and nondiabetic subjects. There is no indication from our data that these polymorphisms are associated with NIDDM.

Integration of reverse transcriptase loop-mediated isothermal amplification with an immunochromatographic strip on a centrifugal microdevice for influenza A virus identification.

A novel centrifugal microdevice which could perform reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and immunochromatographic strip (ICS) based amplicon detection was demonstrated for simple and cost-effective influenza A virus identification. The proposed centrifugal microdevice consists of the sample and running buffer loading reservoirs, the RT-LAMP chamber, and the ICS for detecting gene expression. The entire process could be completed sequentially and automatically by simply controlling the rotation speed and by optimizing the microfluidic design. Monoplex and multiplex RT-LAMP reactions targeting H1 and/or M gene were executed at 66 °C for 40 min, and the resultant amplicons were successfully analysed on the ICS within 15 min. Influenza A H1N1 virus was subtyped by detecting H1 and M gene on the ICS even with 10 copies of viral RNAs. Highly specific and multiplex viral typing of the integrated RT-LAMP-ICS microdevice was also demonstrated. The combination of the rapid isothermal amplification with the simple colorimetric detection on a strip in a single centrifugal microdevice will provide an advanced genetic analysis platform in the field of on-site pathogen diagnostics.

Anticonvulsant activity of novel derivatives of 2- and 3-piperidinecarboxylic acid in mice and rats.

The relative ability of derivatives of 2-piperidinecarboxylic acid (2-PC; pipecolic acid) and 3-piperidinecarboxylic acid (3-PC; nipecotic acid) to block maximal electroshock (MES)-induced seizures, elevate the threshold for electroshock-induced seizures and be neurotoxic in mice was investigated. Protective index (PI) values, based on the MES test and rotorod performance, ranged from 1.3 to 4.5 for 2-PC benzylamides and from < 1 to > 7.2 for 3-PC derivatives. PI values based on elevation of threshold for electroshock-induced seizures and rotorod performance ranged from > 1.6 to > 20 for both types of derivatives. Since preliminary data indicated that benzylamide derivatives of 2-PC displace [3H]1-[1-(2-thienyl)-cyclohexyl]piperidine (TCP) binding to the phencyclidine (PCP) site of the N-methyl-D-aspartate (NMDA) receptor in the micromolar range and such low affinity uncompetitive antagonists of the NMDA receptor-associated ionophore have been shown to be effective anticonvulsants with low neurological toxicity, the 2-PC derivatives were evaluated in rat brain homogenates for binding affinity to the PCP site. Although all compounds inhibited [3H]TCP binding, a clear correlation between pharmacological activity and binding affinity was not apparent. Select compounds demonstrated minimal ability to protect against pentylenetetrazol-, 4-aminopyridine- and NMDA-induced seizures in mice. Corneal and amygdala kindled rats exhibited different sensitivities to both valproic acid and the nonsubstituted 2-PC benzylamide, suggesting a difference in these two models. Enantiomers of the alpha-methyl substituted benzylamide of 2-PC showed some ability to reduce seizure severity in amygdala kindled rats.

Coronary arterial perfusion during venoarterial extracorporeal membrane oxygenation.

The effects of venoarterial extracorporeal membrane oxygenation on left ventricular performance have not been studied in detail. Coronary arterial flow obtained by direct measurement with an electromagnetic flowmeter and blood gas analysis from the aortic root were tabulated during venoarterial extracorporeal membrane oxygenation 14 puppies, and these parameters were evaluated with respect to changes in the venoarterial extracorporeal membrane oxygenation flow. Unique automatic blood pumps generating pulsatile flow were used for the venoarterial extracorporeal membrane oxygenation bypass. Coronary arterial flow decreased as the extracorporeal membrane oxygenation flow increased (106 +/- 26 ml/min per 100 gm of left ventricle at 20 ml x min(-1) x kg bypass flow to 71 +/- 17 ml/min per 100 gm of left ventricle at 100 ml x min(-1) x kg bypass flow, p < 0.01). There were no significant changes in the mean or diastolic pressures in the ascending aorta despite changes in the extracorporeal membrane oxygenation flow. Arterial oxygen tension in the ascending aorta was not increased even under high-flow venoarterial extracorporeal membrane oxygenation. This result indicates that oxygenated blood from the extracorporeal membrane oxygenation circuit does not pass in a retrograde fashion into the aortic root and thus does not perfuse the coronary arteries. The diastolic aortic pressure did not correlate with the changes in extracorporeal membrane oxygenation flow. The decrease in coronary arterial flow is therefore predominantly caused by increased coronary arterial resistance. Tension-time index, an indicator of myocardial oxygen consumption, did not decrease with venoarterial extracorporeal membrane oxygenation. In conclusion, high-flow venoarterial extracorporeal membrane oxygenation causes undesirable hemodynamic effects on the left ventricle.

Radiologic anatomy of the rabbit liver on hepatic venography, arteriography, portography, and cholangiography.

UNLABELLED: Seo TS, Oh JH, Lee DH, et al. Radiologic anatomy of the rabbit liver on hepatic venography, arteriography, portography, and cholangiography. Invest Radiol 2001;36:186-192. RATIONALE AND OBJECTIVES: The radiologic anatomy of rabbit liver has received little study but is important in many experimental investigations. METHODS: Twenty-four rabbits were studied by using hepatic venograms, aortograms, hepatic arteriograms, cholangiograms, and portograms. RESULTS: In all cases, the right, middle, and left hepatic veins drained into the inferior vena cava just below the diaphragm, and the caudate lobe hepatic vein drained more inferiorly. The proper hepatic artery was a branch of the common hepatic artery in 96%. The first branch of the proper hepatic artery was the caudate lobe artery. The remaining main hepatic artery was divided into the right and left hepatic arteries. The left hepatic artery was further divided into the medial and lateral segmental branches in 95%. The anatomy of the portal vein or bile duct was the same as the hepatic artery in 100% of cases. CONCLUSIONS: Knowledge of the normal patterns and variations of the vessels and bile duct will be helpful for experiments of the rabbit liver in future studies.

Effects of bicarbonate ion on serotonin binding to rat frontal cortex membranes.

Several factors are known to regulate ligand binding to 5-hydroxytryptamine (5-HT) receptors. In the present experiments we have investigated the mechanism by which bicarbonate ion modify central 5-HT receptor sensitivity in rats. Mn(2+) (10(?6)-10(?3)M) increased specific [(3)H]5-HT binding to 5-HT(1) receptor sites (+60-70%), this effect being further enhanced by the addition of HCO(?)(3) (+300-400%), while the binding of [(3)H]spiperone binding to 5-HT(2) receptor sites was not affected by Mn(2+) and HCO(?)(3). The effect of other divalent cations, Mg(2+), Cu(2+), Ca(2+) and Fe(2+), however, were not enhanced by the addition of HCO(?)(3). Scatchard analysis indicated that the effect of bicarbonate ion was associated with increase in the number of high affinity binding sites and appearance of low affinity binding sites. This effect of bicarbonate ion was characterized by decreased dissociation rate of the specific binding, was temperature-dependent, reduced by N-ethylmaleimide and iodoacetamide, and was completely inhibited by ascorbate, dithiothreitol and 2-mercaptoethanol. The effect was not influenced by GTP or GppNHp but it was significantly inhibited by ATP. Pretreatment of membranes with Triton X-100 (0.1%) increased the effect of bicarbonate ion. From these results, it is suggested that bicarbonate ion specifically interacts with Mn(2+) and selectively increases [(3)H]5-HT binding.

New neonatal extracorporeal membrane oxygenation circuit with a self-regulating blood pump.

BACKGROUND: Because a roller pump used in a conventional extracorporeal membrane oxygenation (ECMO) circuit with a roller pump cannot change the output automatically according to the venous return, ECMO management requires considerable personnel to prevent serious mechanical complications. An automatic blood pump will make ECMO less laborious and safer. METHODS: Takagi's self-regulating blood pump was modified for neonatal ECMO. The new ECMO circuit was tested in a simulation circuit, in puppies, and in two neonates clinically. Self-regulation of the pump was studied in response to various hemodynamic conditions. RESULTS: The priming volume including a membrane lung and a heat exchanger was about 90 ml. The maximum flow was 700 ml/min in the simulation circuit, 101 ml/min/kg in puppies, and 113 and 135 ml/min/kg in two newborns, respectively. ECMO flow was self-regulating and stable in response to hemodynamic changes. The blood pumps remained functional for more than 400 hours in puppies and 67 and 149 hours in the two newborns, respectively. CONCLUSIONS: The new automatic ECMO circuit is more reliable and requires less personnel than a conventional ECMO circuit.

Aggressive preoperative management and extended surgery for inflammatory pseudotumor involving the hepatic hilum in a child.

Inflammatory pseudotumors involving the hepatic hilum are rare. Only 14 cases have been reported (Table). Liver transplantation has been required when the pseudotumor has invaded extensively into the right and left lobes. (1,2) However, transplantation is associated with the lifelong use of immunosuppressants. This is particularly problematic in children. we report a case of a 6-year-old boy with an inflammatory pseudotumor extensively invading the hepatic hilum who was treated with aggressive surgical excision using the techniques devised for the treatment of hilar cholangiocarcinoma. (3)

Treatment of hepatoblastoma: less extensive hepatectomy after effective preoperative chemotherapy with cisplatin and adriamycin.

BACKGROUND: Although the prognosis of hepatoblastoma was improved by the introduction of cisplatin and doxorubicin (Adriamycin) for adjuvant chemotherapy, extensive hepatectomy continues to be the usual practice. We retrospectively reviewed our recent experience with hepatoblastoma to determine whether the new modality of intensive chemotherapy could change the resectability, extent of hepatectomy, operative complications, and prognosis. METHODS: The clinical features of 15 children with hepatoblastoma treated between 1985 and 1995 were reviewed. Intensive chemotherapy was added before surgical resection not only when a tumor was unresectable but also when it was large enough to increase the risk of operative morbidity. RESULTS: There was 100% resectability, and the overall mortality rate was only 6.7%. Fourteen patients have been free of disease for 2 to 12 years. Preoperative chemotherapy enabled resection of six previously unresectable hepatoblastomas. Moreover, hepatic resection tended to be less invasive in several patients whose tumors had been much reduced after preoperative chemotherapy. Intraoperative and postoperative complications were minimal, with a short operative time and small amount of blood loss, especially in the group with delayed primary operation. CONCLUSIONS: The preoperative administration of cisplatin and Adriamycin reduced the tumor size so that a safe hepatectomy could be performed with less blood loss and minimal technical complications. Unnecessary sacrifice of the normal hepatic tissue was avoided by performing the less extensive hepatectomy.

Congenital stenosis of the intrahepatic bile duct associated with choledochal cysts.

BACKGROUND: There are few studies that report on stenosis of the intrahepatic bile ducts associated with choledochal cysts. We investigated the presence and clinical significance of stenosis of the intrahepatic bile ducts associated with choledochal cysts. STUDY DESIGN: We examined intrahepatic bile ducts in patients with choledochal cysts using cholangiography (93 patients), endoscopy and direct observation during surgery (19 patients), and histologic examination (12 patients). RESULTS: Stenosis of the intrahepatic bile duct was present in 75 (80.6 percent) of 93 patients with choledochal cysts. Endoscopic and direct observation detected membranous stenosis, which consisted of a diaphragm, in 11 of 19 patients. Septal stenosis, which consisted of a bridge-like septum, was present in eight of 19 patients. Stenoses consisted of mucosal and fibromuscular layers. Intrahepatic calculi developed in eight patients with stenosis following resection of the choledochal cysts. CONCLUSIONS: Membranous or septal stenosis of the intrahepatic bile duct is a characteristic feature of choledochal cysts. Our findings suggest that these stenoses should be treated surgically because of the risk of intrahepatic calculi.