The results indicate that EtFSC treatment can invariably decrease hepatic TG in hypercholesterolemic mice, as assessed by both preventive and therapeutic protocols, suggesting its potential use for fatty liver treatment.Effects of the ethanol extract of Fructus Schisandrae (EtFSC) on serum and liver lipid contents were investigated in mice fed with high fat/cholesterol (HFC) diet for 8 or 15 days. The induction of hypercholesterolemia by HFC diet caused significant increases in serum and hepatic total cholesterol (TC) levels  and hepatic triglyceride (TG) levels (up to 528%) in mice. EtFSC treatment  significantly decreased the hepatic TG level (down to 35%) and slightly increased the hepatic index (by 8%) in hypercholesterolemic mice. Whereas fenofibrate treatment  significantly lowered the hepatic TG level (by 61%), it elevated the hepatic index (by 77%) in hypercholesterolemic mice. Acute toxicity test showed that EtFSC was relatively non-toxic, with an LD Fatty liver, characterized by the buildup of fat in liver cells, is prevalent in modern societies. In USA as well as European and Asian countries, the incidence of fatty liver is 15–20% of the general population, and it is much higher in obese individuals . AlthougSchisandra chinensis, is a traditional Chinese herb (Wu-Wei-Zi) originally recorded in Shen Nong Ben Cao Jing . Its active components and related synthetic derivatives, such as schisandrin B, bifendate and bicyclol, have been shown to reduce the hepatotoxicity produced by chemicals in animals and to treat patients with chronic viral hepatitis in clinic conditions [Fructus Schisandrae (FSC), the fruit of nditions –14. The nditions . Our prenditions –20. In tmaterial medica market and authenticated by Dr H. Dong. FSC was ground into small pieces and extracted twice with five volumes of 80%  ethanol under reflux. The extracts were combined and filtered through a paper filter. The pooled extract was concentrated by rota-evaporation under reduced pressure until the removal of all ethanol. EtFSC was reconstituted in water (with 2 g of raw herb in 1 ml of extract) and stored at 4°C until use.FSC was obtained from the Anguo Chinese Cholesterol (certificate no. 041103) was obtained from Beijing Chemical Reagent Co. . Fenofibrate (certificate no. 0405030) was bought from Beijing Yongkang Medical Ltd. . Sodium carboxymethylcellulose  was obtained from Beijing Xudong Chemical Plant . Assay kits for TC and TG were bought from Zhongsheng Beikong Bio-technology and Science Inc. .Male ICR mice  2002–0003), weighing 18–20 g, were supplied by Vital River Lab Animal Co. Ltd . All animals were maintained on a 12 h (light on 7:00–19:00 h) light-dark cycle at 20-21°C, with a relative humidity of 50–55%. They were allowed free access to water and food. Experiments were performed when the animals had grown up to a body weight of 24–26 g. Hypercholesterolemia was induced by feeding high-fat diet  containing cholesterol . Non-hypercholesterolemic  animals were given the normal diet. In the drug treatment groups, EtFSC or fenofibrate (suspended in 0.5% CMC) was intragastrically administered at doses of 1–5 (based on raw herb) or 0.1 g/kg, respectively. The dosage of EtFSC adopted for the present pharmacological investigation was much higher than the recommended intake of FSC (1.5–5 g daily) for a human adult with an average body weight of 60 kg.The experimental design aimed to investigate the preventive and therapeutic effects of EtFSC treatment on liver steatosis. Mice were fed with HFC diet for 8 or 15 days. EtFSC (1–5 g/kg/day) or fenofibrate (0.1 g/kg/day) treatment was performed for 7 days (from Day 1 to 7 (preventive) or from Day 8 to 14 (therapeutic)). Drug-untreated animals received the vehicle (0.5% CMC) at 10 ml/kg. Animals were sacrificed 24 h after the last dosing with EtFSC or fenofibrate. Blood and liver tissue samples were obtained from ether-anesthetized animals which had been fasted for 6 h (from 6:00 to 12:00 h), and they were subjected to biochemical analyses. Hepatic index was estimated from the ratio of total liver weight to body weight. Experimental protocols were approved by the University Committee on Research Practice in Beijing University of Chinese Medicine.μl of the hepatic supernatants were used to determine the TG and TC levels, respectively.Serum samples were prepared by centrifuging the whole blood obtained from the orbital vein for 8 min at 2000 × g. The liver tissue sample was homogenized in 9 volumes of 0.9% (w/v) NaCl solution by two 10-s bursts of a tissue disintegrator and the homogenate was then centrifuged at 2000 g for 15 min to obtain the supernatants. Aliquots of 10 and 40 t-test and P < .05 was considered significant. Values are expressed as means ± SEM for 10 mice per group.Data were analyzed using Student's Feeding mice with HFC diet for 8 days significantly increased serum TC and TG levels , when compared with animals fed with normal diet . EtFSC aAs shown in Feeding mice with HFC diet for 8 days significantly increased hepatic TC and TG levels , when compared with animals fed with normal diet. EtFSC treatment  significantly decreased hepatic TG levels (by 26%), when compared with the drug-untreated hypercholesterolemic mice. Hepatic TC level was not affected by EtFSC or fenofibrate treatment in mice fed with HFC diet . Feeding mice with HFC diet for 15 days increased hepatic TC (by 165%) and TG (by 528%) levels, when compared with that of mice fed with normal diet. EtFSC treatment  produced dose-dependent decreases in hepatic TG levels (by 25–35%), when compared with the drug-untreated hypercholesterolemic mice. Fenofibrate significantly decreased both hepatic TG and TC levels , when compared with the drug-untreated hypercholesterolemic mice .50 value of EtFSC was estimated to be 35.63 ± 6.46 g/kg, using the Bliss method  and the mortality rate was determined within 24 h post-dosing in mice. LDs method . Fatty liver is caused by sedentary lifestyle, diet enriched with cholesterol and saturated fats, and medical conditions. Lifestyle contributors include obesity, deprivation of physical exercise, and excessive alcohol consumption . In the The acute treatment with EtFSC increased the serum TG level and at the same time, decreased hepatic TC level in normal mice. As expected, the hypertriglyceridemic action of EtFSC is suppressed by the co-administration of fenofibrate (data not shown). Fenofibrate, a broad-spectrum lipid-lowering drug of the fibrate class, inhibits the synthesis of cholesterol and triglycerides as well as enhances their elimination , 24. In β-oxidation of fatty acid in mitochondria [β-oxidation, or/and cholesterol absorption. The detail mechanism of action remains to be elucidated. In our previous study, the treatment of schisandrin B, bifendate (synthetic intermediate of schisandrin C) and bicyclol (synthetic dibenzocyclooctadiene derivative) [It has been reported that the hypolipidemic action of fenofibrate is attributed to the stimulation of lipolysis and the elimination of TG-rich particles from plasma through activating lipoprotein lipase and reducing the production of apolipoprotein C-III, as well as inducing enzymes for catalyzing chondria –27. In achondria . Therefoivative) –20 produα, is a useful therapeutic option for patients with dyslipidemias, particularly those associated with diabetes mellitus or coronary heart disease, and metabolic syndrome [Peroxisome proliferator-activated receptors (PPARs) play important roles in the regulation of lipid metabolism involved in inflammation, energy balance, atherosclerosis and non-alcoholic fatty liver disease –31. Fenosyndrome , 33. EtFIn conclusion, EtFSC treatment could prevent or ameliorate the degree of liver steatosis in hypercholesterolemic mice. While fenofibrate produced a more potent lipid-lowering action than that of EtFSC, it caused a larger extent of liver hypertrophy. Taken together with the relatively low toxicity, EtFSC may be used clinically for the management of fatty liver disease .Natural Science Foundation of Beijing City (Grant no. 7022017); Hong Kong Baptist University (Grant no. FGR/06-07/II-67).


Occurrence of hypoglycemia in diabetes patients is very rare. We report here a case of frequent hypoglycemic attacks caused by inappropriate endogenous hyperinsulinemia in a female patient with poorly controlled diabetes and protein-losing gastroenteropathy. The blood glucose profiles of the patient were unstable. Results of the fasting test performed to investigate the cause of hypoglycemia suggested endogenous hyperinsulinism. Repeated selective arterial calcium injection tests suggested that hyperinsulinemia might be extrapancreatic in origin. However, efforts to detect a responsible lesion such as insulinoma were unsuccessful. Octreotide was used for the treatment of hypoglycemia and protein-losing gastroenteropathy. After treatment, although her leg edema caused by hypoalbuminemia persisted, hypoglycemia almost disappeared. Hypoglycemia is a clinical syndrome with diverse causes characterized by low levels of plasma glucose . When itEndogenous hyperinsulinemic hypoglycemia such as insulinoma or nesidioblastosis is one of important causes of hypoglycemia and is suggested by a positive fasting test. To diagnose insulinoma, computed tomography (CT) scan, magnetic resonance imaging (MRI) scan, and endoscopic ultrasound were performed to localize the possible lesion. However, the diagnostic values of these tests are not high if the underlying insulinoma is a very tiny tumor or may be of extrapancreatic origin. Alternatively, if nesidioblastosis is the cause of hypoglycemia, the lesion is difficult to be depicted because it is characterized by diffuse or disseminated proliferation of islet cells arising from pancreatic ducts or ductules and is generally diagnosed only by pathology , 5. To dHere, we report an extremely rare case of endogenous hyperinsulinemic hypoglycemia without distinct mass in a patient with poorly controlled diabetes and protein-losing gastroenteropathy. Moreover, we discuss the unusual clinical presentation, especially relating to the results obtained with SACI tests, the pathophysiology, and effective management of such cases.A 52-year-old female with a history of admission to the hospital 5 times in the last 5 years because of frequent hypoglycemic attacks was admitted to our hospital.At the age of 36, her urine examination showed excessive glucose levels but she did not receive further medical treatment. The following year, she consulted a doctor due to general malaise and bilateral lower leg edema. She was then diagnosed with diabetes mellitus and hypoalbuminemia of unknown cause. Glibenclamide was administered for the treatment of diabetes at age of 40, but poor control persisted. She was referred to our hospital for the first time at the age of 43 because of exacerbation of her lower leg edema. Protein-losing gastroenteropathy (PLGE) due to idiopathic gastrointestinal lymphangiectasia was diagnosed by functional and histopathological examinations. She had been treated with elemental diet and steroid therapy for her PLGE, but those treatments were not effective. At the same time, insulin therapy was started because of the poorly controlled diabetes . After the age of 47, her average blood glucose levels were keeping consistently high (300 to 400 mg/dL) and even hypoglycemic attacks had been recognized. When she was hospitalized at the age of 48, the cause of the hypoglycemic attacks was unidentified (2004). Around the age of 50, the frequency of hypoglycemic attacks (defined as blood glucose level less than 70 mg/dL) had increased to about 40 times per month. The attacks occurred at postprandial and preprandial times. Her medication history suggested that hypoglycemia was not related to her insulin therapy. Precise examination of hypoglycemia was performed during the current hospitalization at the age of 52.2). Blood pressure was 128/78 mmHg and heart rate was 87. Physical examination revealed bilateral lower leg pitting edema, no Achilles tendon reflexes, and reduced sensation of vibration /4 s (lt.)). Chest X-ray revealed no pleural effusions and no cardiomegaly. Diabetic complications were confirmed, that is, bilateral proliferative retinopathy, nephropathy (stage 2), and peripheral neuropathy. She was treated with a combination of rapid-acting and NPH insulin injection for her diabetes. The results of laboratory tests were as follows: total protein: 4.0 g/dL; serum albumin: 2.3 g/dL; total cholesterol: 172 mg/dL; triglyceride: 104 mg/dL; HDL-c: 47.4 mg/dL; LDL-c: 116.7 mg/dL; creatinine: 0.49 mg/dL; HbA1c: 15.5%; urinary C-peptide: 125 μg/day; anti-GAD and anti-IA-2 antibodies: negative; urinary microalbumin: 73.8 mg/g·Cre; creatinine clearance: 109.4 mL/min/m2. Blood glucose profiles appeared unstable, with periodic (2 to 3 days) alternation between hyperglycemia (>300 mg/dL) and hypoglycemia (<70 mg/dL). Representative blood glucose profiles are shown in On admission, her body height was 137 cm and body weight was 44 kg , C-peptide level was 0.98 ng/mL (<0.6), and proinsulin level was 15.3 pmol/L (<5.0). Service's and Turner's criteria for diagnosing insulinoma were satisfied , Fajans 0.22 (>0.3), Turner 66.1 (>50), and Service 9.92 (>6)). Serum C-peptide and proinsulin levels at the time of hypoglycemia attack were significantly high, and the values satisfied the criteria for hyperinsulinism defined by Service [μg/dL and 27.45 μg/dL; growth hormone 0.33 ng/mL and 11.70 ng/mL; adrenaline <10 pg/mL and 583 pg/mL. Tests for both anti-insulin antibody and anti-insulin receptor antibody were negative. We excluded the specific hypoglycemic conditions induced by medicines, insulin autoimmunity, factitious hypoglycemia, and other conditions/diseases such as renal failure or liver cirrhosis. We did not measure plasma sulfonylurea concentration as we were aware that the patient had not been taking the medicine. To investigate the possible involvement of glucose-dependent insulinotropic polypeptide (GIP) or glucagon-like peptide 1 (GLP-1) in the hypoglycemia, we measured serum concentrations of total GIP  and total GLP-1  by enzyme-linked immunosorbent assays in her fasting, postprandial, and hypoglycemic state. Serum concentrations of total GIP were 7.6, 30.2, and 6.0 pg/mL, whereas those of total GLP-1 were 4.7, 4.2, and 4.1 ng/mL in fasting, postprandial, and hypoglycemic state, respectively. Taken together, these results suggested that inappropriate endogenous hyperinsulinemia was responsible for her hypoglycemia, possibly due to insulinoma, nesidioblastosis, or β cell dysfunction.Because the hypoglycemic attacks occurred at a time after cessation of insulin injections sufficiently longer than the effective time of the insulin, we were convinced that hypoglycemia was not related to the injections. Therefore, we suspected that it was induced by an endogenous cause. The result of a fasting test was positive . After a Service . Counter2+ 0.025 mEq/kg) were performed twice in 2004 (age 48) and 2008 (age 52). In keeping with other researchers, an increment in plasma immunoreactive insulin levels greater than 2-fold over prestimulation levels was considered indicative of a tumor or other responsive lesion in the vascular territory of the artery studied [Thus far, all efforts for obtaining diagnostic imaging of an insulinoma via CT scan, MRI scan, and endoscopic ultrasound were unsuccessful. SACI tests with calcium gluconate (Ca004 age 4 and 2008μg) of octreotide to evaluate its effects on serum insulin and glucose levels  for 2 weeks, monitoring its tolerability. Since almost no side effects except for controllable constipation were determined, we switched to octreotide LAR , which is a very long-acting octreotide. After using octreotide LAR for 4 months, although hyperglycemia was uncontrollable even with multiple insulin injections , a long-acting somatostatin analog, because this medicine suppresses insulin secretion  and has e levels . As we ojections , the frejections . This efTo the best of our knowledge, this is the first report of an extremely rare case with the following four characteristics: (1) frequent hypoglycemia induced by endogenous hyperinsulinemia derived from an unusual origin without distinct mass, (2) hypoglycemia in a patient with known diabetes, (3) coexistence with PLGE, and (4) amelioration of hypoglycemia by treatment with octreotide.The most interesting and important point in this case is that positive results from both SACI tests were obtained only via SMA stimulation. The SACI test is an important and useful method for localization of the lesion responsible for hyperinsulinism such as insulinoma. A 2-fold or greater stepup in hepatic vein insulin concentration from baseline after arterial calcium injection constitutes a positive response . Using tβ cell tumor responses. Taking the results of these reports into account, the delayed response observed in our case should be considered as significant.The delayed maximum reaction time (120 s) observed in both SACI tests was characteristic. In general, the maximum reaction time in the SACI test is observed 30–60 s after calcium injection. It has been reported that malignant insulinomas showed a slow increase with a peak observed 90–120 s after stimulation, depending on size and neovascularization . HoweverThe differential diagnosis of endogenous hyperinsulinemic hypoglycemia in this patient listed extrapancreatic insulinoma and nesidioblastosis. Filipi and Higgins reported that the frequency of ectopic insulinoma was 1.96% 20/1018) including the wall of the duodenum (5/20), near the tail of the pancreas (3/20), near the pancreas (5/20), gastrosplenic omentum (2/20), the paraduodenal region (2/20), and between the head of the pancreas and liver (1/20) . Another/1018 incThe development of endogenous hyperinsulinemic hypoglycemia in patients with known diabetes is extremely rare. Most of these cases have been due to benign insulinomas. In addition, islet hyperplasia and nesidioblastosis were reported in hypoglycemic patients with diabetes . The bloβ cells [The relationship between hyperinsulinemia and gut diseases including PLGE and intestinal lymphangiectasia remains unclear. Gut secretes incretins such as GIP and GLP-1, and both peptides stimulate insulin secretion from pancreatic β cells . In Caucβ cells , 26. Addβ cells , 28. Givβ cells . The racβ cells. However, approximately half of the patients administered diazoxide suffered from several adverse effects including edema [β cell type KATP channel have been developed recently [We considered a medication for appropriately raising her blood glucose levels and thus alleviating the symptom of hypoglycemia. The possible medicines listed were glucocorticoid, glucagon, diazoxide, somatostatin analog, and streptozotocin. Previously, we administered prednisolone for the treatment of PLGE but the treatment was not effective. Treatment of glucagon is difficult to continue for a patient because it needs to be administered intramuscularly or intravenously. Streptozotocin is an approved medicine for treatment of malignant islet cell tumor by destructing islet cells. Because the effect of the drug is irreversible to islet cells, thereby exacerbating diabetes inevitably, we could not select this medicine. Diazoxide was originally developed as an antihypertensive medicine but later found to inhibit insulin secretion by activating the potassium channel in ng edema . We worrrecently , and theOctreotide, a somatostatin analog, is effective against endogenous hyperinsulinemic hypoglycemia by inhibiting insulin secretion . FurtherRegarding the side effects of long-term use of octreotide, Vezzosi et al. reported that only one patient had a symptomatic biliary lithiasis that appeared to be caused by the medicine among 11 patients who received treatment with the medicine over 6 months . During To our knowledge, this is the first report of a diabetic patient with PLGE diagnosed with endogenous hyperinsulinemic hypoglycemia without distinct mass. We managed frequent hypoglycemic attacks with octreotide effectively.


Hericium erinaceus is a famous tonic in oriental medicine. The gastroprotective effects of aqueous extract of H. erinaceus against ethanol-induced ulcers in Sprague Dawley rats were investigated. The possible involvements of lipid peroxidation, superoxide dismutase, and catalase were also investigated. Acute toxicity study was performed. The effects of aqueous extract of H. erinaceus on the ulcer areas, ulcer inhibition, gastric wall mucus, gross and histological gastric lesions, antioxidant levels, and malondialdehyde (MDA) contents were evaluated in ethanol-induced ulcer in vivo. In acute toxicity study, a high dose of 5 g/kg did not manifest any toxicological signs in rats. The extract promoted ulcer protection as ascertained by a significant reduction of the ulcer area. Furthermore, it exhibited a significant protection activity against gastric mucosal injury by preventing the depletion of antioxidant enzymes. The level of MDA was also limited in rat stomach tissues when compared with the ulcer control group. Immunohistochemistry showed upregulation of HSP70 protein and downregulation of BAX protein in rats pretreated with the extract. The aqueous extract of H. erinaceus protected gastric mucosa in our in vivo model. It is speculated that the bioactive compounds present in the extract may play a major role in gastroprotective activity. Helicobacter pylori and nonsteroidal anti-inflammatory drugs account for the large majority of peptic ulcer disease [Peptic ulcer, including both gastric and duodenal ulcers, has represented a major threat to the world's population over the past two centuries. Chronic inflammation due to colonisation of  disease . In 2007 disease . Gastric disease , and dec disease , mucosal disease , mucosal disease . Treatme disease . Curcuma longa) [Flabellaria paniculata [Cassia sieberiana roots bark [Mucuna pruriens leaf [Phyllanthus niruri leaf [Polygonum chinense leaf [Ocimum suave leaf [Zingiber officinale Roscoe) [Hericium erinaceus fruiting body [As patients experience unpleasant side effects of the long-term use of commercially available drugs, the search for natural therapeutic agent to prevent gastric ulceration is still ongoing. Natural products from herbs, medicinal plants, spices, vegetables, and crude drug substances are considered to be potential sources to fight various diseases including gastric ulcers. Some of the medicinal plants such as turmeric (a longa) , Flabellniculata , Cassia ots bark , Mucuna ens leaf , Phyllanuri leaf , Polygonnse leaf , Ocimum ave leaf , ginger  Roscoe) , and Hering body  were repLentinula edodes (Berk.) Pegler, Pleurotus ostreatus (Jacq.) P. Kumm [Ganoderma lucidum (Fr.) P. Karst [H. erinaceus (Bull.:Fr.) Pers. (Aphyllophoromycetideae) is a temperate mushroom that has been domesticated and is commercially grown in Malaysia. The cultures of Hericium spp. or their extracts processed in tablets are used for curing gastric ulcer and chronic gastritis [H. erinaceus have not been reported. Thus, the present work aims to investigate the mechanisms of the gastroprotective effects of aqueous extract of H. erinaceus basidiocarp. The antioxidant parameters, that is, superoxide dismutase (SOD) and catalase (CAT), in the gastric tissue of rats were also studied. Further, the level of gastric mucosal malondialdehyde (MDA), which is thought to reflect free radical mediated cell membrane damage, was also accessed in the gastric tissues.Many mushrooms have a long history of applications in traditional oriental therapies. Modern clinical practice in countries such as Japan, China, and Korea continues to rely on mushroom-derived preparations. Mushrooms reported to possess anti-ulcer properties include  P. Kumm , and GanP. Karst . H. erinastritis , 21. Ourastritis . HoweverAssay kits for the activity of SOD and CAT were obtained from Cayman Chemical Company . All other chemicals used in this study were of analytical grade. Omeprazole was obtained from the University of Malaya Medical Centre (UMMC) Pharmacy.H. erinaceus were obtained from Highland Mushroom Farm, Genting Highlands, Pahang, Malaysia. The basidiocarps were freeze-dried and subjected to aqueous extraction, according to Wong et al. [Fresh basidiocarps (fruiting bodies) of g et al. .Sprague Dawley rats aged 6 to 8 weeks and weighed between 180 g and 200 g were used in the study. The female animals used were nulliparous and nonpregnant. Food and water were provided ad libitum. The animals were kept at 27°C (±2°C) and a 12 h light/dark cycle was maintained throughout the experimental period. Experimental protocols were approved by the ethical committee with Ethics number ISB/11/08/2011/WJY(R) under Laboratory Animal Science Centre, Faculty of Medicine, University of Malaya. All efforts were made to minimise both the number of animals used and unwanted stress or discomfort to the animals throughout the experiment. Healthy male and female n = 6), namely, normal control, low-dose, and high-dose groups. Normal control rats were administered orally with distilled water only, whereas low dose and high dose rats were fed orally with aqueous extract of H. erinaceus at dose levels of 2 g/kg and 5 g/kg body weight equivalent to a volume of 5 mL/kg body weight, according to OECD [For each gender, 18 rats were randomly divided into three groups . The ulcer area (UA) was calculated as described by Kauffman Jr. Grossman [I%) was calculated using the formulaStomach of each experimental animal was opened along the greater curvature and rinsed with distilled water to remove gastric contents. Grossly, gastric ulcers appeared as elongated bands on the gastric mucosa with hemorrhagic lesions being parallel to the long axis of the stomach. The mucosa was assessed for damage under dissecting microscope (1.8×) and a planimeter was used to measure the ulcers area (hemorrhagic lesions). The length and width of each lesion were measured and the sum of the area of all lesions for each stomach was expressed as the ulcer area  buffered formalin and processed to produce paraffin wax tissue sections. The stomach damage was assessed by examining 5 Tissue sections (prepared as given above) were heated at 60°C for 25 min in an oven  and then deparaffinized in xylene and rehydrated using graded alcohol. Antigen retrieval process was done in 10 mM sodium citrate buffer boiled in a microwave. Immunohistochemical staining was performed according to manufacturer's protocol . Positive findings of the immunohistochemical staining should be seen as brown stains under a light microscope.The glandular segments from stomach, which had been opened along their greater curvature, were weighed. The quantity of alcian blue recovered from the glandular tissue was then calculated.The gastric tissues were homogenised in 8 : 1 (w/v) phosphate buffer saline. After centrifugation at 12,000 ×g for 15 min at 4°C, the supernatant was stored at −80°C prior to analysis. The homogenates were used for the estimation of MDA level and SOD and CAT activities.μM 1,1,3,3-tetraethoxypropane equiv/g of wet tissue.The level of lipid peroxides in the gastric tissue homogenate was determined through the measurement of total thiobarbituric acid-(TBA-) reactive substance  at 532 nm . Each assay was carried out in triplicate. The results were expressed as μL) and samples (10 μL) were added to microplate wells. Reaction was initiated by adding 20 μL of diluted xanthine oxidase to the mixture followed by shaking for a few seconds to mix. The plate was then incubated on a shaker for 20 minutes at room temperature and absorbance was read at 440 nm using an ELISA reader.The SOD activity was assayed using SOD kit  following the manufacturer's instruction. Diluted radical detector solution (200 μL), methanol (30 μL), and sample (20 μL) were added to wells. Diluted hydrogen peroxide (20 μL) was then added to the mixture to initiate the reactions. The plate was covered and incubated on a shaker incubator for 20 min at room temperature. Diluted potassium hydroxide (30 μL) was then added to each well to terminate the reaction followed by 30 μL of catalase purpald (Chromagen). The plates were incubated for 10 min at room temperature on a shaker. Catalase potassium periodate (10 μL) was then added to each well and reincubated for 5 min at room temperature with agitation. The plate was read at 540 nm using an ELISA reader.For CAT assay , the diluted assay buffer (100 n = 6). Statistical analysis was done using SPSS version 17.0. The significant differences amongst groups were assessed using one-way ANOVA and Tukey's Multiple Comparison Test. A value of P < 0.05 was considered significant.All values reported are mean ± SEM  in the ulcer area was observed in group 3 animals which were pre-treated with omeprazole (the reference drug group) (285.90 ± 19.64 mm2). For the groups pre-treated with the mushroom extracts, the ulcer was significantly (P < 0.05) decreased in a dose-dependent manner when compared to group 2. The ulcer area was significantly (P < 0.05) reduced from 894.00 ± 36.69 mm2 (control rats) to 240.00 ± 17.31 mm2 in the rats pre-treated with 400 mg/kg of mushroom extracts. As shown in In ulcer prevention experiment, group 2 animals, the ulcer control animals, showed severe mucosal injury with ulcer area 894.00 ± 36.69 mm2 . A signiP < 0.05) than that of ulcer control group.Mucus production by gastric mucosa increased gradually in the experimental animals pre-treated with mushroom extracts in a dose-dependent manner . Group 7The rats in the ethanol-induced ulcer control group 2) showed extensive damage to the gastric mucosa, and necrotic lesions were found to penetrate deep into the mucosa. Extensive edema and leucocytes infiltration of the submucosal layer were also observed. Furthermore, the loss of normal glandular architecture of the stomach was observed Figures  and 2. I showed eH. erinaceus in ethanol-induced injury rats expressed high level of heat shock protein (HSP) 70 (The rats pre-treated with aqueous extract of (HSP) 70 . In cont(HSP) 70 . ImmunohH. erinaceus significantly increased (P < 0.05) the alcian blue binding capacity of gastric wall mucus when compared to ulcer control group (μg of mucus/g weight of stomach), followed by 100 mg/kg extract group (11.19 ± 0.45 μg of mucus/g weight of stomach). However, there was no significant difference between these two groups. The aqueous extract of ol group . SurprisH. erinaceus at 50 mg/kg did not significantly (P < 0.05) reduce SOD activity, it increased SOD activity significantly (P < 0.05) at the other doses  tested compared to the control. On the other hand, mushroom extract was at 100, 200, and 400 mg/kg were also found to prevent the depletion of CAT activities (P < 0.05) than that of group 3. As stipulated in μM TEP equivalent) in group 2 due to severe oxidative stress induced by ethanol. Mushroom extracts at all doses tested decreased the MDA level in gastric tissue dose dependently and significantly (P < 0.05) as compared to the control. As shown in tivities . The effH. erinaceus protected the gastric mucosa and inhibited leucocytes infiltration of gastric wall in rats with ethanol-induced gastric ulcer. Cheng and Koo [H. erinaceus may protect gastric mucosa viasuppression of neutrophil infiltration and antioxidant machinery.The present study showed that pre-treatment with the aqueous extract of  and Koo  and Hari and Koo  demonstr and Koo . Ethanol and Koo . Tsukimi and Koo  found thin vitro and in vivo [H. erinaceus protected the gastric tissues through the upregulation of HSP70.HSPs play important roles in normal condition as well as pathological situations involving both systemic and cellular stress . In the  in vivo . In acco in vivo –35, the H. erinaceus significantly increased the gastroprotective effect, with the free mucus being significantly (P < 0.05) increased when compared to the mucus barrier of ulcer control animals. The mucus was comprised of mucin-type glycoprotein, which was detected by a dye called alcian blue [H. erinaceus was able to trigger the gastric mucus barrier defence system. Gastric mucus barrier plays an important role in gastroprotection . The preian blue . Alcian ian blue . Neverthy P < 0.0 increaseian blue . Hence, 2−, one of the ROS responsible for lipid peroxidation [2O2−, which is also capable of producing more oxidative damage [2O2−. Treatment with the aqueous extract of H. erinaceus significantly (P < 0.05) decreased lipid peroxidation and increased SOD and CAT activities, indicating its efficacy in preventing free radical-induced damage by quenching free radicals in the gastric tissue of the ethanol-induced rats to exhibit gastroprotective activity. It is evident from the results of this study that antioxidant compounds mainly the phenolic compounds present in the aqueous extract of H. erinaceus, could play synergetic effect in experimental animals to protect against gastric tissue damage. In support of this, our previous study [H. erinaceus had potent antioxidant activities. Aqueous extracts of H. erinaceus contain mainly polysaccharides, phenolic compounds, and hericenones (unpublished data). Nevertheless, further studies determining the bioactive components responsible for the mechanism of anti-ulcer activities of H. erinaceus extracts are warranted.Arafa and Sayed-Ahmed  found thxidation . This ree damage . Catalasus study  had showH. erinaceus protected gastric mucosa against ethanol-induced injury. The protection was shown to be dose dependent according to the reduction of ulcer areas in the gastric wall and the reduction or inhibition of edema and leucocytes infiltration of submucosal layers, and protection was significant at the maximum dosage at 400 mg/kg. The extract also significantly increased the SOD and CAT activities and decreased the level of lipid peroxidation (as indicated by MDA level) in the gastric tissue homogenates. Immunohistochemistry analysis showed upregulation of HSP70 and downregulation of BAX protein in rats pre-treated with the mushroom extract. Further studies are required to determine the bioactive components responsible for the mechanism of antiulcer activities of H. erinaceus extracts indicated in this study.The aqueous extract of


Thus, we evaluated the effects of diazepam, intraperitoneally , intracerebroventricularly (ICV 10 μg), and concurrently with flumazenil (1 mg/kg) on cerulein-induced acute pancreatitis in mice. Interestingly, the pretreatment with diazepam (5 mg/kg i.p.) reduced significantly the inflammatory response of acute pancreatitis by ameliorating pancreatic edema, amylase and lipase serum levels, myeloperoxidase activity, pancreatic TNF-alpha, and pathological alteration compared to control group. Diazepam i.c.v. was ineffective, suggesting that central benzodiazepine receptors have no significant role in this property. These results demonstrate that pretreatment with diazepam exhibits anti-inflammatory property in cerulein-induced acute pancreatitis possibly through peripheral benzodiazepine receptors.Acute pancreatitis is a lethal inflammatory condition of pancreas with high mortality rate. There is a pressing need for research to explore active agents and novel mechanisms involving in the treatment of pancreatitis. Clinical studies have shown after the initial acinar cell injury plasma levels of pro-inflammatory cytokines are elevated in patients with acute pancreatitis and the degree of cytokine elevation correlates with disease severity. Diazepam may decrease interleukin release from macrophages, suppress neutrophil activities, and exhibit anti-inflammatory effects. So it is expected that  Acute pancreatitis is a potentially lethal disorder which is characterized by pancreatic tissue inflammation and elevation in serum level of digestive enzymes without specific therapy . The morThe early pathophysiology of the acute pancreatitis has not been well understood . But somDiazepam is a classical benzodiazepine (BDZ) with sedative, anxiolytic, and anticonvulsant properties. It has been found that diazepam binds with high affinity to peripheral benzodiazepine receptors (PBRs) which are distinct from the central benzodiazepine receptors (CBRs) . PBRs arIt has been shown that acute treatment with diazepam decreases interleukin release from macrophages and suppresses neutrophil activities . Some stα, and interleukin-6 by macrophages [It also has been determined that treatment of mice with PBRs agonist Ro5-4864 markedly reduces the production of inflammatory mediators such as interleukin-1, TNF-rophages . These data suggest that diazepam which acts on PBR as well as CBRs might possess protective effects against pancreatitis.The present study was performed to evaluate the protective effects of diazepam in an animal model of cerulein-induced acute pancreatitis in mice. To gain access to better insight into the mechanism(s) of actions of diazepam on pancreatitis, we have investigated the effects of diazepam administration, both peripherally and centrally, on pancreatic edema, leukocyte infiltration, amylase and lipase levels, TNF-alpha, and myeloperoxidase activity.Male mice weighting 25–30 g and bred in animal house  were used in this study. Animals were kept in uniform environment of temperature, humidity, and light/dark cycles (12/12 h) and allowed free access to pelleted rodent chow and tap water. Before initiation, the experiment animals were fasted over the night. The study was approved by the Ethics Committee for Animal Care and Uses, Isfahan University of Medical Sciences, Isfahan, Iran.μL) of 1% methylene blue into the site and then observing the distribution of the injected dye in the ventricular space. The dye-injected i.c.v. was found to be distributed in the ventricular spaces and ventral surface of the brain and in the upper cervical portion of the spinal cord [Mice were anesthetized with i.p. injection of a ketamine (50 mg/kg) and xylazine (10 mg/kg) mixture and positioned in a stereotaxic instrument . The guide cannula was positioned at stereotaxic coordinates of AP: −0.5 mm, relative to bregma; lateral: 0.8 mm; dorsal-ventral: −2.5 mm. At least seven days were allowed for recovery after the implantation of the ICV guide cannula before experimental protocols were begun. The injection sites were verified by injecting the same volume  with 1 h intervals according to the method which was previously demonstrated by Mazzon et al. [Acute pancreatitis was induced by five intraperitoneal (i.p.) injection of 50 n et al. . Cerulein = 6) on acute pancreatitis was studied. Diazepam was given 15 min before induction of pancreatitis. In the first series of experiments, effect of i.p. diazepam . In negative control groups, mice with acute pancreatitis were pretreated with normal saline (5 mL/kg and i.p).Control group received only vehicle  was administered with flumazenil (1 mg/kg i.p.) for blocking the central effect of diazepam. μg/mouse, n = 6) 15 min prior to induction of acute pancreatitis.In the third series, to distinguish between the central and peripheral effects of diazepam we also evaluated the supraspinal administration of diazepam (i.c.v) in pancreatitis. The drug was administered smoothly for 1 min through the injection cannula . The control group received vehicle  for histological examination. Besides, portions of this organ were promptly frozen in liquid nitrogen and stored at −70°C until assayed.Serum lipase and amylase activity were determined by using commercially available lipase and amylase kits  .MPO activity, an index of polymorphonuclear cell accumulation, was measured according to the modified method of Bradley et al., pancreas tissue was homogenized in 1 mL of 50 mM potassium phosphate buffer containing 0.5% HTAB (hexadecyltrimethylammonium bromide). Then, the homogenate was sonicated in an ice bath for 10 s, freeze thawed thrice with sonication between cycles. After that, the suspensions were centrifuged at 15,000 rpm for 15 min at 4°C and then the supernatant (0.1 mL) was allowed to react with 2.9 mL of 50 mM potassium phosphate buffer (pH 6.0) containing O-dianisidine dihydrochloride (0.167 mg/mL) and 0.005% hydrogen peroxide. The absorbance of the reaction mixture was measured at 450 nm using a UV-Vis spectrophotometer. MPO activity was expressed in units (U) per gram of wet tissue weight .α ELISA kit Glory Science Co., Ltd., Hong Kong). The cytokine levels were calculated after plotting the standard curves and expressed as pg/mL.Tissue TNF-alpha was measured using an enzyme-linked immunosorbent assay (ELISA) commercial kit according to the manufacturer's instructions (TNF-μm), stained with hematoxylin and eosin (H&E), and examined by coworker pathologist unaware of experimental protocol.Paraffin-embedded pancreas samples were sectioned . Leukocyte infiltration was also graded from 0 to 3  .U test. The minimal level of significance was considered at P < 0.05.Biochemical results are expressed as mean ± SEM. Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Nonparametric data was analyzed by Mann-Whitney Cerulein-induced pancreatitis in vehicle-treated mice was associated with significant rises in the serum levels of amylase and lipase. The increase in amylase and lipase was noticeably reduced in cerulein-treated mice which had been pretreated with diazepam in doses of 5 and 10 mg/kg by i.p. injection Figures .P < 0.05) in cerulean-treated mice which had been pretreated with diazepam in dose of 5 mg/kg by i.p. injection . Pancreas levels of TNF-alpha were significantly reduced (njection .P < 0.05)  .In normal saline treated mice, pancreas did not show any tissue injuries at light microscopic level (×10 magnification). Administration of cerulein induced acute edematous with severe leukocyte infiltration pancreatitis in all mice tested. Prominent interlobular and severe intralobular edema was also accompanied with moderate perivascular and abundant diffuse inflammatory infiltration. P < 0.05)  .The present data showed that pretreatment with diazepam before the induction of acute pancreatitis decrease the susceptibility to the disease and histological lesions.α, and interleukin-6 [β, and TNF-alpha mRNA concurs with the development of histologic lesions in pancreatitis and hyperamylasemia [Our results demonstrated that administration of diazepam (5 mg/kg i.p.) reduced amylase and lipase activity, TNF-alpha, MPO activity, and pathological alterations, which are markers of pancreatitis. Action of diazepam on PBRs was already reported as being related to its effects on acute inflammation . This isleukin-6 . Currentylasemia , 22.So it could be assumed that some beneficial effects of diazepam (5 mg/kg i.p.) on pancreatitis are because of anti-inflammatory effects of diazepam which mediate through PBRs. Also in this study to make distinguish between the central and peripheral effects of diazepam, we evaluated the supraspinal effect of diazepam (i.c.v) in pancreatitis. As results showed anti-inflammatory effect of diazepam (i.c.v.) was not prominent so the beneficial effects of diazepam (5 mg/kg i.p.) could be solely attributed to its actions on PBRs. Administration of diazepam increases plasma level of corticosterone with plasma peak after 90 minute of injection, via acting on PBRs on adrenal glands , 24. It κB) activation, thereby enhance cytokine expression [Despite our expectation, diazepam at higher test doses did not show any protection against cerulean-induced acute pancreatitis. It has been demonstrated that diazepam enhanced apoptosis in cancer cells in presence of cytotoxic agents, by mitochondrial transmembrane potential drop that facilitated release of apoptogenic factors such as cytochrome c and apoptosis-inducing factor in mitochondria , 26. Cerpression . Also hipression . AccordiFor determination the exact mechanisms of diazepam and role of PBRs in the pathogenesis of pancreatitis, it should be better to investigate the effect of PBRs selective agonists such as Ro5-4864 or PK 11195 on the severity of experimental pancreatitis. Therefore, we suggest further researches using selective PBRs agonists.  Here we demonstrated that pretreatment with diazepam showed anti-inflammatory effects against cerulein-induced acute pancreatitis in mice probably via acts on peripheral benzodiazepine receptor. But further experiments with selective PBRs agonists need to determine the principle mechanisms of diazepam in this situation.


A 60-year-old female who has a history significant for diabetes, depression, and rheumatoid arthritis presented with a progressively enlarging hematoma of the left upper extremity. She was found to have an enlarging hematoma and an isolated elevation of activated partial thromboplastin time (aPTT). Lab work-up revealed low factor VIII activity levels and inhibitor titers at 13.38 Bethesda units (BU). Dilute Russell's viper venom time (dRVVT) revealed a lupus anticoagulant. Hemostasis was achieved with factor VIII inhibitor bypassing activity (FEIBA) and inhibitor eradication with-rituxan after the failure of first-line treatment with cyclophosphamide and prednisone. Acquired hemophilia is a rare cause of aPTT elevation. It is associated with rheumatological conditions like systemic lupus erythematosus (SLE). Lupus anticoagulant (LA) may also increase the aPTT and be associated with many of the same rheumatological conditions. Both factor VIII inhibitors and LA can coexist in the same patient leading to a challenge in determining the etiology of an elevated aPTT.μL. Her ED course was complicated by multiple phlebotomies because of difficult venous access. She was subsequently discharged from the ED but three days later returned for progressive swelling, pain, numbness, and tingling in the left forearm, aggravated by wrist flexion and with minimal relief at rest. She denied any symptoms of infection, arm trauma, vigorous exercise of the upper extremity, or intravenous drug abuse. She noted that her symptoms began after multiple phlebotomy attempts in the ED. Her home medications included cyclobenzaprine, levothyroxine, trazodone, multivitamins, and nonsteroidal anti-inflammatory drugs (NSAIDS) as needed for RA. She denied the use of cytotoxic therapy, clopidogrel, aspirin or any other over the counter medications for the RA. Her physical exam revealed multiple ecchymosis of the left forearm extending from the elbow joint to wrist. She also was extremely tender upon deep palpation of the forearm. Her repeat lab work-up showed an Hct of 36, white blood cell count (WBC) of 12.6 × 103cells/μL, platelet count of 279 K/μL, and normal blood chemistry. Her prothrombin time (PT) was 12.6 seconds, and her international normalized ratio (INR) was 0.98; activated partial thromboplastin time (aPTT) was not measured during this episode. Roentgenography (X-ray) of the left forearm revealed the possibility of a compartment syndrome, and the patient was taken to surgery. Intraoperatively, a contused brachioradialis muscle was found without evidence of a significant hematoma or an arterial tear. A fasciotomy was performed with hemostasis. Several hours postoperatively, the dressing was blood-soaked and required two changes overnight. By the following postoperative day, her Hb had dropped from 12 to 8.3 gm/dL, and her Hct had dropped from 36% to 25%. The patient received fresh frozen plasma (FFP) but this failed to control the bleeding. Platelet function studies showed no significant abnormalities. Her coagulation panel revealed a PT of 13.8 sec, an INR of 1.06, an aPTT of 63 sec, a fibrinogen level of 317 mg/dL (170–470), and a d-dimer of 1.73 μg/L (<0.67 μg/mL). Given the isolated aPTT elevation along with the history of rheumatoid arthritis, acquired hemophilia was suspected. Factor VIII activity levels and von Willebrand activity levels were sent. Factor VIII activity levels came back less than 0.1, and inhibitor titers came back at 13.38 BU. Chromogenic factor VIII levels were not performed. Hematology service was consulted. Hemostasis was achieved with activated prothrombin complex concentrates (aPCC or FEIBA) at a dose of 100 units/kg twice daily. Prednisone was started at a dose of 1 mg/kg/day for inhibitor eradication. The hexagonal phospholipid neutralization assay was positive for the lupus anticoagulant (LA) that was confirmed by dilute Russell's viper venom time (dRVVT).A 60-year-old female with a medical history significant for seronegative rheumatoid arthritis (RA), depression, diabetes, and hypothyroidism presented to the emergency department (ED) for abdominal pain. A thorough evaluation failed to reveal any significant abnormalities. Her hemoglobin (Hb) was 11.8 gm/dL, hematocrit (hct) was 36%, and platelet count is 445 K/2 dose weekly for four weeks. After the first dose, inhibitor titers dropped to 21.8 BU, along with a decrease in aPTT. This drop in aPTT suggested that the initial aPTT value was secondary only to acquired hemophilia A (AHA). FEIBA which had been given once a day was discontinued after the drop in aPTT. The patient was discharged and followed with aPTT and inhibitor titers. Her postdischarge course was complicated by an episode of febrile neutropenia that was successfully treated.The patient's treatment response (prednisone) was monitored by following factor VIII activity levels and inhibitor titers. Subsequent consultation and evaluation by rheumatologist led to a diagnosis of systemic lupus erythematosus (SLE), and hydroxychloroquine was initiated. After one week on prednisone, the inhibitor titer increased from 13.38 to 21.76. Therefore, therapy with cyclophosphamide 2 mg/kg/day was initiated. Unfortunately, one week after starting cyclophosphamide, the inhibitor titers increased to 34.25 BU. Given her failure to respond to first-line treatment, rituximab was initiated at a 375 mg/mAcquired hemophilia A or acquired factor VIII inhibitor (AHA) is a rare autoimmune disorder that is secondary to development of autoantibodies to factor VIII. Prevalence of AHA is reported in one to four cases per million per year. It has a bimodal age distribution with the first peak in the second to third decades of life. This peak is primarily due to postpartum acquired hemophilia, and the second major peak is between 65 and 85 years .1-a1-A2-B-a3-A3-C1-C2. In acquired hemophilia (AH), IgG1 and IgG4 autoantibodies are developed against epitopes within the domains of A2, A3, and C2. The mechanism of action of antifactor (AF) VIII antibodies depends on target epitope location. Anti-A3 antibodies will prevent interaction of factor VIII with factor IXa. Anti-C2 antibody inhibits the binding of factor VIII to phospholipids and von Willebrand factor (VWF). Finally, anti-A2 & A3 antibodies interfere with factor VIII binding to factor X and factor IXa [Factor VIII has a domain structure of AThe clinical presentation of AHA is quite variable presenting as an incidental finding or as dramatic as a life-threatening hemorrhage. The most common presentations include easy bruisability, muscular hematomas, hematuria, intracerebral hemorrhage, gastrointestinal bleeding, epistaxis, or postpartum hemorrhage. In contrast to congenital hemophilia, hemarthrosis is not a part of the clinical presentation , 3, 4. A high index of clinical suspicion is needed to diagnose AHA. It is the most common of all acquired hemophilia, and an isolated elevation of aPTT in a bleeding patient should raise the suspicion of AHA. Once the diagnosis is suspected, mixing studies should be performed. The aPTT will not be corrected after mixing the patient's plasma with the plasma that contains 100% of clotting factors (1 : 1 ratio) in the presence of an inhibitor, while in factor deficiency it will normalize. Chromogenic factor VIII assays may also be performed once the diagnosis is suspected as these assays are not affected by inhibitors. In addition, acquired factor VIII antibodies follow second-order kinetics. Precipitation is both time and temperature dependent. It is very crucial to perform a timed inhibitor screen particularly in cases of low inhibitor titers –7. It isLupus anticoagulant (LA) also causes isolated elevation of aPTT; moreover, it also shares common associated conditions like SLE. As in this case, AHA and lupus anticoagulant can coexist in the same patient. LA can also decrease the plasma factor VIII levels and can make the diagnosis more challenging. Sometimes differentiation between these two conditions can be difficult as both conditions can cause an isolated aPTT elevation that will not correct on mixing studies. The clinical presentation is very helpful to differentiate between these two conditions. A patient presenting with a clinical picture of thrombosis will make LA more likely, while a presentation with bleeding should raise suspicion for AHA. During an isolated aPTT elevation without any clinical findings, the diagnosis can be more challenging. In these scenarios, serial dilutions of the patient's plasma might provide some insight. On serial dilutions, the clotting time will shorten and follow a nonlinear pattern when LA is present, while clotting time will follow a linear pattern that is parallel to the control if factor VIII inhibitor is present . Adding μg/kg intravenously every 2-3 hours or a continuous infusion at 8–50 mg/kg/h. Caution must be taken when administering rVIIa in patients with atherosclerotic disease, sepsis, and DIC. Both of these agents have equal efficacy, and the choice of agent to use is based on availability and physician's experience. In minor bleeding episodes such as mucosal hemorrhagesand with inhibitor titers <5 BU, desmopressin, and factor VIII concentrates can be used [The management of AHA has two steps. The first step is to achieve hemostasis. The treatment of choice depends on the clinical condition. In severe and life-threatening conditions, factor VIII bypassing agents should be used. These are activated prothrombin complex concentrates (aPCC or FEIBA) and recombinant factor VIIa (rVIIa). FEIBA dose ranges from 50 to 100 IU/Kg every 8–12 hours with caution for doses greater than 200 IU/KG/day as disseminated intravascular coagulation (DIC) may occur. An anamnestic response with increase in factor VIII titer has been reported. Also, antifibrinolytic agents should be avoided for 12 hours. Factor VIIa hemostasis can be achieved with a dose of 90  be used , 7. The second step is inhibitor eradication. All patients with AHA should be treated with steroids alone (1 mg/kg/day for 4–6 weeks) or steroids with cyclophosphamide (1-2 mg/kg/day for a maximum of 6 weeks). A benefit and risk assessment should be done before initiation of cytotoxic therapy. If the inhibitor titers remain significantly elevated after 3–6 weeks of first-line agents, alternative therapy with rituximab, immunoadsorption, other cytotoxic agents , or combination therapy should be considered .A complete response is defined as an undetectable inhibitor level and a normal factor VIII activity level, while a sustained response is defined as an undetectable inhibitor level and a factor VIII activity level greater than 50% after inhibitor eradication treatment. It is recommended to follow these patients with haemogram, aPTT, inhibitor titer, and factor VIII activity at least twice weekly while hospitalized and then once weekly as out-patients for 6 weeks. Once a complete response has been achieved, labs can be assessed monthly for the first 6 months and then every 2-3 months for the next 6 months. Patients should be monitored every 6 months for signs of relapse .Relapses can occur in 20% of the cases, the median time to relapse being about 7–9 months. A second complete remission can be achieved in at least half of the patients. In some patients, immunosuppressive therapy should be continued for life in order to prevent relapse .


Background. It was suggested that labor may influence the spread of intrathecal bupivacaine using combined spinal epidural (CSE) technique. However, no previous studies investigated this proposition. We designed this study to investigate the spinal block characteristics of plain bupivacaine between nonlaboring and laboring parturients using CSE technique. Methods. Twenty-five nonlaboring (Group NL) and twenty-five laboring parturients (Group L) undergoing cesarean delivery were enrolled. Following identification of the epidural space at the L3-4 interspace, plain bupivacaine 10 mg was administered intrathecally using CSE technique. The level of sensory block, degree of motor block, and hemodynamic changes were assessed. Results. The baseline systolic blood pressure (SBP) and the maximal decrease of SBP in Group L were significantly higher than those in Group NL . The median sensory level tested by cold stimulation was T6 for Group NL and T5 for Group L (P = 0.46). The median sensory level tested by pinprick was T7 for both groups (P = 0.35). The degree of motor block was comparable between the two groups (P = 0.85). Conclusion. We did not detect significant differences in the sensory block levels between laboring and nonlaboring parturients using CSE technique with intrathecal plain bupivacaine. Combined spinal epidural (CSE) anesthesia is commonly used for cesarean delivery. It has been suggested that nonlaboring parturients have a higher sensory block level than those in labor during CSE anesthesia . This prThis research was conducted at the West China Second Hospital of Sichuan University, Chengdu, Sichuan Province, China. With median two-segment dermatome blockade level difference to be clinically significant and variability (interquartile range) of two dermatomes in sensory block, 20 parturients in each group were required in each group to address a 2-segment difference with a power of 0.9 and a level of significance of 5%.Following the institutional research ethics board (IRB) approval and written informed consents, we enrolled two groups of parturients with 25 in each group. Group NL were 25 nonlaboring parturients undergoing elective cesarean delivery, and Group L were 25 parturients in spontaneous active labor  undergoing cesarean delivery for failure to progress. All parturients were between 20 and 40 years of age, ASA physical status I-II with a singleton pregnancy at greater than 36 weeks gestation, and received a lower segment transverse incision for cesarean delivery. The decision for cesarean delivery was made by the obstetric team, independent of the study investigators. Exclusion criteria included parturient refusal, having received any analgesic treatment, any contraindication to CSE or general anesthesia, height less than 150 cm or greater than 175 cm, body weight greater than 100 kg, obstetric and/or medical comorbidities such as preeclampsia, any cardiac, renal, neurologic, or other systemic diseases, unilateral block, or maximal pinprick sensory block level below T10 at 20 min postspinal injection.Each parturient was preloaded with 500 mL of lactated Ringer's solution intravenously (IV). On arrival in the operation room, baseline vital signs were recorded and the initial severity of labor pain for a laboring parturient was assessed on a 10 cm visual analog scale (VAS) before CSE anesthesia was placed. The degree of cervical dilations for laboring parturient was also recorded.One anesthesiologist performed all CSE procedures following previously published methodology , 3. Withμg was administrated for a decrease in systolic blood pressure (SBP) to less than 90 mmHg or a 30% reduction in SBP and was repeated as necessary. Bradycardia with less than 50 beats per minute was treated with IV atropine 0.3 mg. Sensory level of the block was assessed bilaterally at two-minute intervals using alcohol swab (sensory blockade level to cold ) and 25-gauge hypodermic needle (sensory blockade level to pinprick) in the cephalad direction. The degree of motor blockade was evaluated bilaterally using the modified Bromage scale [A second anesthesiologist recorded vital signs at two-minute intervals for 20 minutes. IV phenylephrine 100 Skin incisions were made 20 minutes after intrathecal injection. Prior to delivery of the fetuses, any reaction to surgical stimulation  was supplemented with IV ketamine at a dose of 0.5 mg/kg each time until adequate anesthesia was achieved. After childbirth, midazolam 0.03 mg/kg was administrated to all parturients, and IV pethidine 0.5 mg/kg each time was also utilized according to parturient's reaction to ensure adequate anesthesia. General anesthesia was reserved as a back-up plan. Time intervals from skin incision to uterine incision, from uterine incision to fetus delivery, and surgical time were collected. The Apgar scores and parturients' satisfaction with anesthesia care were recorded. All parturients were followed for three days postoperatively.t-test was used to examine the differences of quantitative data between groups. The Mann-Whitney U test was performed to analyze the ordered variables, such as median sensory levels, VAS scores, modified Bromage scores, and those skewed quantitative data. The incidences of hypotension, shivering, and nausea/vomiting between the two groups were compared with Fisher's exact test. The correlations between sensory blockade level and cervical dilation or labor pain scores were analyzed with Spearman's rank correlation. A P value <0.05 was considered statistically significant.Statistical analysis was performed using SPSS Version 13.0 software. One-sample Kolmogorov-Smirnov test was applied to analyze the distribution of the data. Student's unpaired All 50 parturients enrolled completed the study. No technical difficulty or inadvertent dural puncture was encountered. Demographical data including age, weight, height, and gestation weeks were presented in All parturients had similar bilateral sensory block level, and all the peak pinprick sensory block levels were above T10. Median peak dermatomal sensory block level to pinprick and cold sensation and the time taken to achieve it in 20 min after spinal injection were comparable between the two study groups .P = 0.81 and P = 0.10, resp.). In addition, Spearman's correlation coefficients for sensory blockade level to cold in relation with cervical dilation and labor pain scores were 0.22 and 0.17, respectively . During surgery, there were separately 14 nonlaboring parturients and 8 laboring parturients required rescue analgesic (P = 0.15). The median rescue ketamine dosage was 30 mg (IR: 0–50 mg) for nonlaboring parturients and 0 mg (IR: 0–40 mg) for laboring parturients (P = 0.21). The median rescue pethidine dosage was 30 mg (IR: 0–40 mg) for nonlaboring parturients and 0 mg (IR: 0–40 mg) for laboring parturients (P = 0.19). No conversion to general anesthesia was warranted.Spearman's correlation coefficients were 0.07 and −0.44, respectively, for sensory blockade level to pinprick in relation with cervical dilation and labor pain scores  in the present study was based on previous studies , 3, whicSelection bias was one of the concerns that could not be excluded completely from this study because of the non-randomized design. Laboring parturients were shorter in height (2.7 cm difference in average) and lighter in weight (3.6 kg difference in average) than those of nonlaboring parturients, which might in part explain the reason for their failure to progress resulting in cesarean delivery. However, it has been demonstrated that height, weight, and bodymass index did not influence the spread of sensory blockade after subarachnoidal injection of bupivacaine , 14. TheIn conclusion, our study could not demonstrate difference in the block level of spinal anesthesia between the laboring and nonlaboring parturients using plain bupivacaine injected with the CSE technique.


Supplementation with defined oligosaccharide fractions has been shown to attenuate allergic responses and enhance defensive immune responses. Despite the accumulating evidence for immunomodulatory effects, very limited information is available regarding the direct mechanism of action of oligosaccharides. This study aims to elucidate the effects of selected oligosaccharide fractions on the lipopolysaccharide (LPS) induced inflammatory response in equine peripheral blood mononuclear cells (PBMCs). We investigated three different products containing either galacto-oligosaccharides (GOS) alone, a combination of GOS with fructo-oligosaccharides (FOS), and a triple combination of GOS and FOS with acidic oligosaccharides (AOS), at different concentrations. These products have been used in an identical composition in various previously published in vivo experiments. As the selected oligosaccharide fractions were derived from natural products, the fractions contained defined amounts of mono- and disaccharides and minor amounts of endotoxin, which was taken into account in the design of the study and the analysis of data. Acquired data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses.Dietary supplementation with oligosaccharides has been proven to be beneficial for health in several mammalian species. Next to prebiotic effects resulting in a modulation of gut micro biota, immunomodulatory effects of oligosaccharides have been documented Exposing cultured PBMCs to either GOS or GOS/FOS fractions resulted in a substantial dose-dependent increase of tumour necrosis factor-α (TNF-α) production in LPS challenged PBMCs. In contrast, incubation with GOS/FOS/AOS resulted in a dose-dependent reduction of both TNF-α and interleukin-10 production following LPS challenge. In addition, incubation with GOS/FOS/AOS significantly increased the apparent PBMC viability, indicating a protective or mitogenic effect. Furthermore, mono- and disaccharide control fractions significantly stimulated the inflammatory response in LPS challenged PBMCs as well, though to a lesser extent than GOS and GOS/FOS fractions.We found distinct immunomodulating effects of the investigated standardised oligosaccharide fractions, which either stimulated or suppressed the LPS induced inflammatory response in PBMCs. Both scenarios require additional investigation, to elucidate underlying modulatory mechanisms, and to translate this knowledge into the clinical application of oligosaccharide supplements in foals and other neonates. The original goal of supplementing infant formulas with oligosaccharide fractions was to mimic prebiotic effects of human milk oligosaccharides in non-breastfed infants. Several oligosaccharide fractions were synthesised as possible surrogates of human milk oligosaccharides. Short chain galacto-oligosaccharides (GOS) are oligomers of lactose (degree of polymerization (dp) 2–6), produced by elongating lactose using β-galactosidase enzymes [During the past decade, numerous  enzymes . GOS is  enzymes . During  enzymes . The pre enzymes .in vivo. Dietary supplementation with these oligosaccharide fractions was shown to be beneficial for both allergic immune responses [. Eiwegger et al. [in vitro. In a more recent study by the same group, the in vitro incubation of unchallenged human cord blood mononuclear cells with low concentrations (10–100 μg/ml) of AOS or GOS combined with FOS did not result in an alteration of cytokine production, whereas incubation with similar concentrations of acidic human milk-derived oligosaccharides did significantly induce the production of interferon-γ and interleukin-10 [in vitro evidence for epithelial transport of prebiotic oligosaccharides, enabling direct contact between oligosaccharides and cells of the immune system.Moreover, immunomodulatory properties of GOS and combinations of both GOS/FOS and GOS/FOS/AOS have been documented esponses -10 and desponses -13. Furtesponses ,15. In mr et al.  reportedeukin-10 . The latin vivo nor in vitro.Neonatal foals possess limited defence mechanisms, in particular due to the impermeability of the equine placenta to maternal immunoglobulins. Consequently, newborn foals are strongly dependent on the transfer of immunoglobulins through the uptake of colostrum . MoreoveIn this study, we investigated the effects of defined oligosaccharide fractions on the inflammatory response in equine peripheral blood mononuclear cells (PBMCs) following a standardised lipopolysaccharide (LPS) challenge. We chose to use equine PBMCs as a readily available representative model of immune cells in this animal species.a. Blood samples were kept cooled and PBMC isolation was started within 1 hour after collection. All experimental procedures were approved by the committee of ethical considerations regarding animal experiments of Utrecht University (DEC Utrecht).Twelve healthy, adult, Dutch warm blood horses (K.W.P.N. studbook) were sampled in total during this study. PBMCs from five horses were used for experiments with oligosaccharide fractions, four horses were sampled for control experiments with glucose/lactose solutions, and PBMCs of three horses were used for cell viability assays. Experiments could not all be executed in the same horses, due to the large amounts of PBMCs required to investigate all fractions and concentrations in triplicate (at least). Adult horses had to be used as blood donors, as large volumes of blood were needed to isolate sufficient amounts of PBMCs for these first explorative experiments in the horse; withdrawing larger volumes of blood from neonatal foals is restricted by ethical concerns. From each horse, 250 ml of blood was collected by jugular venipuncture directly into sterile heparinised blood collection tubesb containing 2 mM EDTAc and subsequently layered over Ficoll- Paque™ plusd. After centrifugation , PBMCs were pipetted from the Ficoll layer and washed twice in PBS/EDTA. PBMCs were resuspended in RPMI 1640 Mediumb supplemented with 2 mM glutamineb, 100 IU/ml penicillinb, 100 μg/ml streptomycinb, and 10% horse serum (prepared in our own laboratory according to standard procedures). PBMCs were counted using trypan blue and resuspended to a density of 4*106 cells/ml medium. Following storage overnight at 4°C to attenuate possible stimulatory effects of the applied Ficoll, PBMCs were seeded in 24 well plates at a density of 4*106 cells/ml medium/well.Blood samples were diluted 1:1 in sterile PBS2. After this, the plates were centrifuged for 10 minutes at 400× g before refreshing the medium without removing PBMCs. The experiments were started by pre-incubating the PBMCs for 2 hours with supplemented RPMI containing different concentrations of oligosaccharide fractions . After pre-incubation, plates were centrifuged again and the medium was replaced with medium containing 0 or 1 μg/ml LPS (Escherichia coli O111:B4)c combined with different concentrations of oligosaccharide fractions (including blank controls). Plates were placed in the incubator for another 4 hours, after which samples for ELISA were collected and stored at −80°C. Thus, there was a total incubation time of 6 hours for all investigated conditions. All different incubations were performed in triplicate or in quadruplicate for each horse. Additional experiments were performed following the same protocol with different concentrations of glucose and lactose, to account for the fact that the oligosaccharide fractions- especially GOS- contain considerable amounts of these mono- and disaccharides.After seeding the PBMCs in 24 well plates, the plates were incubated for 2 hours at 37°C and 5% COin vivo, using oligosaccharide concentrations ranging from 0.5% to 2.0%. The exact composition of all 2% incubations is summarized in Table e, dp 2–6) consisted of approximately 45% GOS, 14% glucose, 16% lactose and 25% water. GOS was investigated separately, combined with FOS  in a 9:1 ratio, and combined with both FOS and AOSg  in a 9:1:2 ratio. Ratio’s and diluting factors were based on oligosaccharide contents of the used products. Thus, all 2% oligosaccharide solutions contained 20 mg/ml oligosaccharides and, in addition, defined amounts of mono- and disaccharides. The control incubations with glucosec and lactosec were chosen to correspond with the glucose and lactose concentrations of the GOS fraction, as this was the oligosaccharide fraction with the highest concentrations of mono- and disaccharides  and 156.25 pg/ml (IL-10), respectively.Protein levels of tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were measured by means of ELISA on the cell culture supernatants, using Duo set® ELISA Development System for equine TNF-α and equine IL-10c viability assays were performed according to manufacturer’s instructionsc. Prior to viability assessment, cell culture experiments were carried out identical to the experiments which are described above, with the exception that cells were seeded and incubated in 96 well plates .To investigate possible influence of the applied incubation mixtures on cell viability, CCK-8i. The selected standard curve (determining the range of detection) ranged from 0.1-1 Endotoxin Unit (EU)/ml.All applied solutions (as described in Table rjags (version 3–5) [R2jags (version 0.03-06) [Data were analysed by means of Bayesian hierarchical linear regression, assuming a normal distribution of the log-transformed ELISA data. A hierarchical approach was taken to account for variation between horses. PBMC responses were allowed to vary between horses with regard to general PBMC reactivity (random intercept), and PBMC reactivity specifically against incubation with LPS, GOS, FOS, and AOS (random slopes). Analyses were performed in a Bayesian framework, using JAGS, a program for analysis of Bayesian models using Markov Chain Monte Carlo (MCMC) simulation (version 3.2.0) . Simulation 3–5)  and R2ja0.03-06) . Posteri0.03-06) .Differences are stated significant, based on the calculated 95% BCI. In the results section, significant differences are quantified by percentages. Not all significant differences are quantified in the results section. An overview of all data and the corresponding 95% BCI’s is given in an Additional file In Table Production of IL-10 in unchallenged PBMCs was not significantly influenced by oligosaccharide fractions in comparison with blank controls.Table The IL-10 response in PBMCs incubated with LPS alone was significantly increased with 49% in comparison with blank controls. Whereas both GOS and GOS/FOS incubations did not significantly alter the IL-10 response following LPS stimulation, incubation with LPS and GOS/FOS/AOS 0.5% did increase IL-10 production significantly in comparison with incubation with LPS alone (35% increase). Incubation with higher concentrations of GOS/FOS/AOS resulted in a dose-dependent reduction of the LPS induced IL-10 response in PBMCs . Moreover, IL-10 production in LPS challenged PBMCs incubated with GOS/FOS/AOS 2% was significantly lower compared to incubation with LPS alone (35% reduction).To account for the mono- and disaccharide contents of the applied oligosaccharide preparations, effects of these oligosaccharide preparations were compared with effects of corresponding glucose/lactose concentrations in the same model. A comparison of the effects of oligosaccharide preparations and glucose/lactose controls on the TNF-α response in unchallenged PBMCs is given in Figure Incubation of PBMCs with medium containing glucose and lactose resulted in significantly higher TNF-α production in comparison with blank controls . However, no significant dose-dependent effects were detected among the glucose/lactose incubations.A comparison with the GOS incubations revealed that incubation of unchallenged PBMCs with 2% GOS resulted in significantly more TNF-α production compared to the corresponding glucose/lactose controls (52% increase), whereas effects of the 0.5% and 1% GOS and corresponding glucose lactose incubations did not differ significantly. For GOS/FOS and GOS/FOS/AOS, all effects on TNF-α production were significantly more profound compared to the effects of corresponding glucose/lactose controls.Effects of oligosaccharide preparations and glucose/lactose controls on the TNF-α response in LPS challenged PBMCs are compared in Figure Differences between effects of the applied oligosaccharide preparations and glucose/lactose controls were evident through the entire concentration range. For all investigated GOS and GOS/FOS concentrations, TNF-α production was significantly higher after incubation with the oligosaccharide fraction compared to the corresponding glucose/lactose concentrations. In contrast, for GOS/FOS/AOS a dose-dependent decrease in TNF-α production was evident. LPS challenged PBMCs incubated with GOS/FOS/AOS 2% produced significantly less TNF-α compared to corresponding glucose/lactose controls (71% reduction).A comparison of the effects of oligosaccharide preparations and corresponding glucose/lactose concentrations on IL-10 production could not be executed, as IL-10 concentrations which were measured in the glucose/lactose experiments were mostly below the detection limit of the ELISA.Results for the CCK-8 viability assays are illustrated in Figure According to the performed bioassay, all oligosaccharide solutions contained measurable concentrations of endotoxin. For the 2% solutions, the endotoxin concentrations  were estimated at 0.24 ± 0.02 EU/ml (GOS), 1.06 EU/ml  and 1.92 EU/ml . The glucose and lactose solutions did not contain measurable amounts of endotoxin (lower detection limit of the assay was 0.1 EU/ml).Based on our results, we can conclude that both GOS and GOS/FOS fractions dose-dependently enhanced the pro-inflammatory response by LPS challenged equine PBMCs. In contrast, after incubation with 2% GOS/FOS/AOS the LPS induced inflammatory response was dose-dependently reduced. This indicates that with increasing the AOS concentration in the incubations, the stimulatory effects of both GOS and GOS/FOS were eventually overruled by the suppressive effects of AOS in this model. Suppression of the cytokine response was not caused by cytotoxic properties of AOS. On the contrary, PBMC viability was significantly increased in all GOS/FOS/AOS incubations compared to blank incubations, suggesting a protective or mitogenic effect. In conclusion, the reduced cytokine production by PBMCs incubated with GOS/FOS/AOS was not the result of a reduced number of viable cells, as lower amounts of cytokines were produced by a higher number of cells.in vivo studies.We cannot exclude that a direct interaction between AOS and LPS influenced the LPS response in PBMCs incubated with GOS/FOS/AOS. Binding of LPS by AOS in the gut lumen might even contribute to the documented beneficial effects of oligosaccharide fractions containing AOS in Though we observed modulatory effects of mono- and disaccharides on the LPS induced inflammatory response in our model, these effects were less pronounced and overshadowed by the immunomodulating effects of the investigated oligosaccharide fractions. To our knowledge, literature regarding the underlying mechanism of action of mono- and disaccharides, which enhance the LPS induced inflammatory response, is lacking. However, our results are in line with a recently published study, which reported that high glucose conditions enhance the TNF-α and IL-6 response following LPS challenge .c of the applied LPS (1 ng LPS = 5–10 EU). We challenged the PBMCs with 1000 ng/ml LPS, as preliminary experiments with this PBMC model indicated that this concentration already results in a maximum TNF-α response after 4 hours of incubation. As 1.20- 19.20 ng/ml is almost negligible compared to a concentration of 1000 ng/ml, it can be assumed that this low endotoxin contamination cannot be held accountable for the enhancement of the LPS response by the oligosaccharide fractions in our model. Moreover, the enhancement of the TNF-α response in LPS challenged cells was not proportional to the mild stimulatory effects in unchallenged PBMCs.As PBMCs are extremely sensitive to bacterial endotoxin, it cannot be excluded that the significant increase of TNF-α production in unchallenged PBMCs after incubation with the applied oligosaccharide fractions was caused by the low endotoxin concentrations in these natural products. Although it is not possible to give an exact concentration based on the results obtained with the Limulus assay, it can be assumed that the degree of endotoxin contamination ranged from 1.20- 19.20 ng/ml for the 2% oligosaccharide solutions. This estimate is based on the information of the manufacturerHowever, endotoxin contamination might play a role in the reduction of the LPS response after incubation with 2% GOS/FOS/AOS. In this solution, the measured endotoxin concentration was the highest (approximately 10–19 ng/ml) and we cannot exclude that endotoxin tolerance was induced during the PBMC pre-incubation period, suppressing the response after the actual challenge . On the in vivo studies often ascribe immunomodulatory effects of dietary oligosaccharides to their prebiotic properties. However, the results of this study support the hypothesis of direct modulatory effects of oligosaccharides, independent of the intestinal micro biota. Though this notion is also supported by other in vitro studies [Previously performed  studies ,17, the  studies . In addi studies ,29. As kin vivo study in adult horses demonstrated beneficial effects of prebiotic short-chain fructo-oligosaccharides, preventing disruption of the intestinal flora in the equine hindgut in case of an abrupt change of diet composition [in vitro studies with human cell culture models [As of yet, research with oligosaccharides mainly involved direct intervention studies in human infants or in experimental (rodent) animal species as surrogates for human infants -15. One position . Concerne models ,17.The most notable finding was the divergence between the immuno-activation of PBMCs by GOS and GOS/FOS preparations and the apparent suppressive effect of AOS. This was an unexpected outcome and indicates that forthcoming experimental protocols should include conditions with AOS only. In the current experiments we applied the  combinations of oligosaccharides used in previous studies, and hence AOS was not tested separately.in vivo, depending on the clinical context. Future research into dietary supplementation of specific oligosaccharide fractions in foals and underlying immunomodulatory mechanisms could lead to the development of oligosaccharide supplements in foals, to support a rapid maturation of the immune competence in the first phase of life, hence protecting them from early, often life-threatening infections.In conclusion, this study provides evidence that oligosaccharides from different sources have distinct direct immunomodulatory properties. We showed that both GOS and FOS dose-dependently stimulated the LPS induced inflammatory response in equine PBMCs, and that AOS dose-dependently suppressed the production of both TNF-α and IL-10 following an LPS challenge. Such an activation or suppression of the immune system could be beneficial The data supporting the results of this article are included within the article and its Additional file aBD Vacutainer Systems, Plymouth, United KingdombLonza, Basel, SwitzerlandcSigma- Aldrich, St. Louis, USAdGE Healthcare, Waukesha, USAeFrieslandCampina Domo, Zwolle, The NetherlandsfOrafti, Wijchen, The NetherlandsgDanone Research, Wageningen, The NetherlandshR&D Systems, Minneapolis, USAiGenscript, Piscataway, USAAOS: Acidic oligosaccharides; dp: degree of polymerization; IL: Interleukin; FOS: Fructo-oligosaccharides; GOS: Galacto-oligosaccharides; LPS: Lipopolysaccharide; PBMC: Peripheral blood mononuclear cell; TLR: Toll- like receptor; TNF: Tumour necrosis factor.The authors declare that they have no competing interests.Conceived and designed the study: JF, JV. Collected materials and performed experiments: JV. Analysed the data: JV, LC. Wrote the manuscript: JV. Reviewed and revised the manuscript: JF, JV, LC. All authors read and approved the final manuscript.TNF-alpha protein: model parameter estimates and 95% Bayesian Credible Intervals. IL-10 protein: model parameter estimates and 95% Bayesian Credible Intervals. CCK-8 assay: model parameter estimates and 95% Bayesian Credible Intervals.Click here for file


N = 68) and DP (N = 73). Anthropometric parameters and symptom/sign index were measured. Metabolic profiling was performed using ultrahigh-performance liquid chromatography-mass spectrometry. The Ratio of subjects with slippery pulse was higher in DP pattern, but fine pulse was lower than that in non-DP pattern. As a result of metabolomics analysis, twenty-one metabolites displayed different levels between non-DP and DP patterns. Two were identified as lysophosphatidylcholines (LPCs), LPC(18:2), and LPC(20:3) having an unsaturated acyl chain and showed lower levels in DP pattern than in non-DP pattern . However, the saturated LPCs, LPC(18:0) and LPC(16:0), exhibited slight but statistically insignificant elevation in DP pattern. Our results demonstrated that plasma LPCs with polyunsaturated fatty acid groups were associated with DP pattern and suggest that variation of plasma lipid profiles may serve as potential biomarker for diagnosis of DP pattern.Traditional Korean Medicine classifies stroke into four subtype patterns according to symptomatic pattern identification: Qi deficiency (QD), Yin deficiency (YD), Dampness-phlegm (DP), and Fire and Heat (FH). This study investigated the difference in metabolic profiles of plasma comparing subjects displaying non-DP and DP patterns. A total of 141 patients with cerebral infarction enrolled in this study were distributed as non-DP ( Pattern Identification (PI) is a unique diagnosis system of traditional medicine practiced in East Asian countries including China, Korea, and Japan and provides information that guides appropriate treatment of patient disease. Previous reports described how traditional Korean medicine (TKM) categorizes stroke as four patterns according to their symptoms and sign: Qi deficiency (QD), Dampness-phlegm (DP), Yin deficiency (YD), and Fire and Heat (FH) . Among tHowever, the diagnosis of PI depends on symptoms/sign exhibited by patients, which may be interpreted incorrectly. Therefore, it is necessary to establish scientific evidence to aid accurate diagnosis based on PI. Previously, several studies showing relation between biological factors and PI were reported –13. In pMetabolomics provides a powerful approach for global and quantitative assessment of endogenous small molecule metabolites within biological fluids  and has In this study, we analyzed metabolic profiles in plasma of acute patients with cerebral infarction (CI) by ultrahigh-performance liquid chromatography/time-of-flight mass spectrometry (UHPLC-TOFMS) and searched for metabolites differing in levels between DP and non-DP patterns. This study was collected as parts of the project “The Fundamental Study for the Standardization and Objectification of PI in TKM for Stroke (SOPI-Stroke)” in the Korean Institute of Oriental Medicine (KIOM) . Patients with CI were admitted from 2009 to 2010 to the following Korean oriental medical hospitals participating in this study: Kyung Hee Oriental Medical Center (Seoul), Kyung Hee East-West Neo Medical Center (Seoul), Dong Guk International Hospital (Kyunggi-do), and Dae Jeon Oriental Medical Hospital (Daejeon). Subjects had to be enrolled as CI patients within three days of the onset, and their symptoms were confirmed by imaging diagnosis, using computerized tomography (CT), magnetic resonance imaging (MRI), or magnetic resonance angiography (MRA). CI subtypes were classified as large artery atherosclerosis (LAA), cardioembolism (CE), small vessel occlusion (SVO), stroke of other determined etiology (SOE), and stroke of undetermined etiology (SUE) subtype according to the trial of ORG 10172 in the Acute Stroke Treatment (TOAST) classification. Subjects with subarachnoid, subdural, or epidural hemorrhage were excluded in this study, and subjects with diabetes mellitus and stroke in history and with infectious diseases and liver diseases were also excluded. After obtaining written informed consent from all subjects, clinical data were collected. This study was approved by the Institutional Review Boards (IRB) of the KIOM and by each of the oriental medical hospitals. The symptoms and signs of subjects were collected using “stroke PI case report form” , and PI μL of plasma thawed on ice was thoroughly mixed with 600 μL methanol and 100 μL of internal standard solution and centrifuged at 15000 g for 15 min at 25°C. Supernatant was transferred and completely dried by using centrifugal vacuum evaporator over 4 hours. Dried samples were resolved by adding 100 μL of melting solution  and centrifuged at 15000 g for 15 min at 25°C. Supernatant was transferred into vial and used for UHPLC-MS analysis. Preparation of plasma from each subject for UHPLC-MS analysis was performed according to protocols provided by Human Serum Metabolome (HUSERMET) Consortium . BrieflyμL aliquot of the concentrated plasma extract was injected into an Ascentis Express C18 column  using a SIL-5000 autosampler . The solvent was delivered by LC-20AD pumps  at a flow rate of 0.4 mL/min, with solvent A being 0.15% aqueous formic acid and solvent B being acetonitrile/isopropanol (2 : 1). Gradient elution was performed and equilibrated with water containing 0.15% formic acid. Samples were eluted by gradient containing 0.15% formic acid for 30 min, and metabolites separated by UHPLC were assigned by MS . Separate injections were made for analyses conducted ESI-positive and ESI-negative modes. A 10 m/z 100–2000 with a scan time of 0.2 s. The accurate mass and composition for the metabolite ions were calculated by MassLynx 4.1  incorporated in the instrument.The voltage of capillary and sampling cone was, respectively, set at 3 kV and 10 V, and the desolvation flow was set to 350 L/h at a temperature of 350°C. The source temperature was set to 100°C. Collecting range of MS data was http://www.chemspider.com/) and Human Metabolome Database Version 2.5 (http://www.hmdb.ca/) or/and confirmed by external standards based on retention time and mass spectra.Results from UHPLC-MS analyses were processed using automated peak detection, integration, and retention time alignment using MarkerLynx 4.1 software , and MS data were assembled into a matrix of integrated extracted ion chromatogram peak areas, organized by mass and retention time. Parameters for MarkerLynx analysis were set as follows: peak width at 5% height: 15 s, intensity threshold: 20, mass window: 0.1 amu, retention time window: 0.5 min, noise elimination level: 6, mass tolerance: 0.1 Da. Normalization of MS spectra was performed by total normalization, and peaks not detected from over 2% of subjects were excluded in the following analysis. Metabolites were annotated using searches of the ChemSpider database . Normality of continuous variables in clinical data was decided by Kolmogorov-Smirnov test, and statistical difference was determined by P = 0.0489). In contrast, the fraction of subjects exhibiting weak pulse was significantly lower in DP pattern (P = 0.0073). The general characteristics between non-DP and DP were shown in m/z 520.366, 1039.724, and 1043.763, in positive-ion mode and m/z 590.335 in negative-ion mode were significantly lower in the DP pattern relative to the non-DP pattern. However, most differentiating metabolites were present at much lower levels or were removed by the abundance threshold of 20 ion counts. To find metabolite levels in plasma that could differentiate non-DP and DP patterns, we performed UHPLC-MS analysis . A list m/z 520.366 and 546.399, were 282 and 35 in DP pattern and were significantly lower than 304 and 41 in non-DP pattern . Additional LPCs with unsaturated fatty acid, LPC (20 : 4) and LPC (18 : 1), also showed a tendency to be lower in DP than non-DP pattern, but without significance . Otherwise, levels of LPCs without double bond, LPC (14 : 0), LPC (16 : 0), and LPC (18 : 0), were not different between two groups. To identify each differentiating metabolite, public databases were searched using ion masses determined using both positive-ion and negative-ion modes. Phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) lipids were identified from positive-ion mode data . For exam/z  590.335 was significantly lower in DP pattern (P = 0.017) and was identified as the formate ion adduct of LPC (20 : 3), confirming the result from positive-ion mode. Free fatty acids (FFA) and lysophosphatidylethanolamine (LPE), which were mainly detected in negative-ion mode analyses, were not different in the two patterns (P = 0.065).In the negative-ion mode data, a metabolite detected as patterns . Only LPPI presents a comprehensive system for the diagnosis of disease and is based on the patient's signs and symptoms. TKM doctors determine the cause, nature, and treatment of the illness depending on signs and symptoms in patients and use this information to provide individualized treatment to patients, who have the same disease . This apIn TKM, stroke is classified into four standard subtypes according to PI: QD, DP, YD, and FH based on position, internal or external etiology, or observations of affected organ. DP, which is classified by etiology, is a combination of phlegm and internal dampness causing disease. Major symptoms of the DP pattern include anorexia, dyspepsia, dizziness, headache, and heaviness . HoweverP = 0.010 and 0.034, resp.), and LPC (20 : 4) and LPC (18 : 1) showed a tendency to decrease in DP pattern (P = 0.070 and P = 0.077). Metabolomics provides powerful tools to measure and identify metabolic biomarkers from biofluids, and earlier studies to explain PI using the change in metabolite levels were performed in China , 22. In Lysophosphatidylcholines (LPCs), called lysolecithins, are a class of phospholipids derived from phosphatidylcholines and produced by two pathways. First is the result from partial hydrolysis of phosphatidylcholine, so that one of the fatty acids is removed by the action of phospholipase A2 . A seconUntil now, relationships between plasma levels of LPCs and DP pattern were not known, but earlier reports suggested the possibility of such correlations. It was known that DP pattern was related to obesity and hyperlipidemia , 8, 9, aThis study showed, for the first time, that some LPCs in plasma were associated with DP pattern in CI patient population. However, this study included several limitations. First, this is a simple cross-sectional study, and the subjects enrolled in this study were very acute patients. Second, this study does not have a sufficient sample size to generalize the relationship between plasma metabolites and DP pattern. Third, the PI was limited to stroke diagnoses. The lack of evidence on the accuracy of PI forced us to perform an observational study, not allowing a randomized controlled trial. Despite of these limitations, this study provided metabolic information about DP patterns, and further studies should be performed in subjects of another large population to generalize the conclusions of this study.