Objective Insulin autoantibody (IAA) is known to exist in sera of type 1 diabetes mellitus (T1DM) patients and pre-T1DM individuals. The aim of this study was to establish a novel microtiter plate radioimmunoassay (RIA) for IAA and evaluate its clinical value. Methods Diluted 125Ⅰ-insulin was mixed with 5 ul serum samples in a 96-well microtiter plate and then incubated for 72 h on an orbital plate shaker (4℃). The immunocomplexes were transferred to another protein a coated Millipore plate, and then the plate was washed with Tri-Buffered Saline Tween-20 (TBT) buffer. Counts per minute (CPM) was measured with liquid scintillation and luminescence counter. The positive cut-off point of IAA index was defined as ≥0.06 based on the 99-percentile of the distribution in 317 healthy individuals. The specificity and sensitivity of the assay were calculated from the samples provided by the fourth Diabetes Autoantibodies Standardization Program (DASP 2005). The IAA levels were determined in 71 T1 DM and 551 newly diagnosed type 2 diabetes (T2DM) patients, and 317 healthy controls. The t test, non-parametric test, x2 test and linear correlation analysis were performed on the data using SPSS 11.5 software. The concordance rate was estimated with Kappa value. Results (1) The optimized testing condition was described as 2×104 CPM of 125Ⅰ-insulin, 5 ul serum sample and slowly horizontal shaking for 72 h. (2) The intra-assay CV was 4.8%-8.9% and inter-assay CV was 6.4%-10.5%. Based on DASP 2005 samples, the specificity and sensitivity of the assay were 97% (97/100) and 50% (25/50), respectively. Ninety-six serum samples with different IAA levels were selected and tested to compare between our new method and a domestic IAA RIA kit. The results showed that the IAA indices from the two methods were positively correlated (r= 0.678, P<0.001). The concordance rate was 72.9 %(Kappa value=0.402). There were 25 samples with discordant results, which were positive for IAA titer using the corresponding microtiter plate RIA but negative using the novel RIA kit. (3) In TIDM group the positive rate of IAA was 19.7% (16/71), higher than the healthy controls (0.9%, x2=54.36, P<0.001). The subgroup of T1DM children (with 0-9 years) showed the highest IAA positive rate (55.6% ,x2=4.85, P<0.05). In T2DM group the frequency of IAA was 1.5% (8/551), which had no significant difference comparing with that of healthy controls (x2= 0.95, P >0.05). Conclusions Our proposed microtiter plate RIA method for IAA is highly sensitive and specific, likely to be feasible for clinical application. The frequency of IAA is high in children with T1DM.

Objective The purpose of this study was to develop a high-throughput micro-plate radiobinding assay (RBA) of glutamic acid decarboxylase antibody (GAD-Ab) and to evaluate its clinical application. Methods 35labeled GAD65 antigen was incubated with sera for 24 h on a 96-well plate, and then transferred to the Millipore plate coated with protein A, which was washed with 4℃ PBS buffer, and then counted by a liquid scintillation counter. The GAD-Ab results were expressed by WHO standard unit (U/ml). A total of 224 healthy controls, 162 patients with type 1 diabetes mellitus(T1DM) and 210 patients with newly diagnosed type 2 diabetes (T2DM) were recruited. A total of 119 TI DM and healthy cases with gradually changing GAD-Ab levels were selected to compare the consistency of micro-plate RBA with conventional radioligand assay (RLA). Blood samples were obtained from the peripheral vein and finger tip in 32 healthy controls, 35 T1DM and 24 T2DM patients, and tested with micro-plate RBA and then compared with the conventional RLA to investigate the reliability of finger tip sampling. Linear correlation,student's t-test, variance analysis and receiver operating characteristic (ROC) curve were performed using SPSS 11.5. Results (1) The optimized conditions of micro-plate RBA included 2 μl serum incubated with3 ×104 counts/min 35S-GAD for 24 h under slow vibration, antigen-antibody compounds washed 10 times by 4℃ PBS buffer, and radioactivity counted with Optiphase Supermix scintillation liquid. (2)The intra-batch CV of the micro-plate RBA was 3.8%- 10.2%, and the inter-batch CV was 5.6%- 11.9%. The linearity analysis showed a good correlation when the GAD-Ab in serum samples ranged from 40.3 to 664 U/ml and the detection limit of measurement was 3.6 U/ml. The results from Diabetes Autoantibody Standardization Program (DASP) 2005 showed that the sensitivity and specificity for GAD-Ab were 78% (39 positive among 50 new-onset T1DM) and 98% (2 positive among 100 healthy controls). The results of GAD-Ab obtained with micro-plate RBA and RLA were closely correlated (r=0.915,P<0.001) with a high concordance level of 97.5% and a Kappa value of 0.95. (3)TI DM and T2DM patients showed higher positive rates for GAD-Ab than the healthy controls(46.9% and 5.2% vs 0.89% ,X2=123.5 and 10. 1 ,P <0.001 and <0.01, respectively). (4)The consistency of GAD-Ab measurement with RBA using finger tip blood and RLA measurement using venous blood was 96.7% (r =0.946,P <0.001, Kappa value: 0.905). Conclusions The micro-plate RBA of GAD-Ab has high sensitivity, specificity and reproducibility, and can be measured with finger tip blood sampling. It might be a better alternative for clinical practice.

Acute cerebral infarction(ACI)has the characteristics of onset nasty and high mortality,and thus the rapid determination of the occurrence and development of ACI plays a key role in the diagnosis,treatment and prognosis of ACI patients.It has shown that the serum level of human haptoglobin(Hp)is related to ACI.In this study,surface enhanced Raman scattering(SERS)combined with immune recognition was applied to establish a quantitative analysis method for serum Hp.Firstly,the SERS substrate of silver nanoparticles was prepared on silicon wafer,and 4-mercaptobenzoic Acid(MBA)was used as a Raman probe by forming Ag—S bond and connecting it on the surface of nanoparticles.The carboxyl group of MBA was linked to amino group of self-made high-affinity antibody through forming CO—NH structure thus forming a SERS self-assembled chip of Hp(Ag/MBA/anti-Hp).Hp in serum could be specifically captured by antibodies on SERS substrate,which caused the shift of SERS characteristic peak of MBA.The results showed that there was a good linear relationship between the logarithm of Hp concentration and the SERS characteristic peak shift of MBA.The detection range was 1-1000 ng/mL(R2=0.988).The Hp concentrations in serum of 90 ACI patients were determined by this method,and the results were consistent with those of ELISA method,which proved the practicability and accuracy of this method.This method was highly specific,simple and convenient,which could realize the specific recognition and quantitative analysis of serum Hp,so as to be an effective means for clinical detection of serum Hp,thus providing a reference for the treatment and prognosis of ACI.

Objective To observe distribution and morphological characteristics of symptomatic atherosclerotic plaques in the middle cerebral artery (MCA) using high?resolution magnetic resonance imaging (HR?MRI), and to investigate HR?MRI characteristics of atherosclerotic plaques in the MCA in patients with acute cerebral infarction. Methods A total of 57 symptomatic patients with MCA atherosclerotic plaques recruited in the Affiliated Hospital of Yangzhou University from January 2014 to January 2016 were imaged with diffusion weighted imaging (DWI), three dimensional time of flight magnetic resonance angiography (3D TOF?MRA) and HR?MRI scanning for plaque on a 3.0 T MRI scanner. According to the results of DWI examination, the 57 patients were divided into transient ischemic attack (TIA) group (27 cases) and acute cerebral infarction group (30 cases). The distribution of the narrowest lumen plaque was evaluated by cross?section division into four equal arcs (superior, inferior, ventral, dorsal arcs). For quantitative analysis, lumen area (LAMLN), vessel area (VAMLN) at maximal lumen narrow (MLN) and LAreference, VAreference were measured, then wall area (WA), plaque area (PA), percentage of plaque burden, rate of lumen stenosis and remodeling index (RI) were calculated. The data of each group were compared and analyzed. Results The location and morphological analysis of the 57 patients with symptomatic MCA atherosclerotic plaques revealed that plaques were located in the ventral wall in 19 cases (33.3%), the upper wall in 15 cases (26.3%), the dorsal wall in 10 cases (17.5%), and the lower wall in 13 cases (22.8%). For the location variations in ventral wall, upper wall, dorsal wall and lower wall, the TIA group was shown as six cases (22.2%), five cases (18.5%), seven cases (25.9%) and nine cases (33.3%), and the acute cerebral infarction group was shown as 13 cases (43.3%), 10 cases (33.3%), three cases (10.0%) and four cases (13.3%), respectively. There was no statistically significant difference in the distribution of each side wall between the two groups (P>0.05). VAreference, LAreference, VAMLN and RI of the TIA group and the acute cerebral infarction group were (19.89 ± 1.34) mm2, (15.19 ± 2.04) mm2, (20.78 ± 1.78) mm2, 1.09 ± 0.11 and (19.70 ± 1.34) mm2, (14.60 ± 2.33) mm2, (21.53 ± 2.34) mm2, 1.10 ± 0.11, respectively. There was no statistically significant difference between the two groups (P>0.05). The remodeling patterns of both groups were mainly positive remodeling, with a total of 44 cases (77.2%). In the TIA group and the acute cerebral infarction group, the WAMLN, PA, stenosis rate and plaque load percentages were (8.85±1.92) mm2, (4.00±3.00) mm2, 20.92%± 9.18%, 19.05% ± 14.93% and (11.10 ± 1.88) mm2, (6.00 ± 2.25) mm2, 28.56% ± 8.67%, 27.30% ± 7.69%, respectively. The differences between the two groups were statistically significant (t=-4.466, t=-2.865, t=-3.231, t=-2.580, P<0.01). There were eight patients (29.6%) with unsmooth plaque surface in the TIA group and 19 patients (63.3%) in the acute cerebral infarction group. The differences between the two groups were statistically significant (χ2=6.475, P<0.05). LAMLN in the TIA group and the acute cerebral infarction group was (11.93±1.59) mm2 and (10.43±2.08) mm2 respectively, and the difference between the two groups was statistically significant (t=3.033, P<0.01). Conclusions Symptomatic atherosclerotic plaques in MCA in the acute cerebral infarction group have higher plaque load, thicker vascular wall at the maximum stenosis and more unsmooth plaque surface. This indicates the characteristics of high?risk plaques to a certain extent.

OBJECTIVETo evaluate the effectiveness of dynamic SPECT (99m)Tc-galactosyl human serum albumin (GSA) scintigraphy on the assessment of reserve function of cirrhosis liver.

METHODSFrom January 2010 to December 2011, 55 patients with cirrhosis liver were enrolled in this study. The case numbers of male and female were 43 and 12 respectively and the age was (51 ± 9) years (ranging from 35 to 69 years). After routine biochemistry test, CT scan and (99m)Tc-GSA dynamic SPECT scan were performed in turn using a juxtaposed SPECT/CT system. Then the morphologic volume of liver parenchyma (MLV), functional liver volume (FLV) and the hepatic cell absorption rate constant (GSA-K) were calculated. The correlations between GSA-K and routine biochemistry test, Child-Pugh score, indocyanine green clearance rate (ICG-K) were analyzed. The patients were further divided into 3 groups according to whether there was occlusion or stenosis in the main branch of left portal vein (group 1, n = 5), right portal vein (group 2, n = 13) or not (group 3, n = 37) and the regional hepatic functions index of the 3 groups were compared.

RESULTSThe value of FLV of the whole, left and right liver was (594 ± 152) ml, (244 ± 119) ml and (356 ± 171) ml, respectively. There were correlations between GSA-K and total bilirubin, prothrombintime, Child-Pugh score and ICG-K (r = -0.730--0.298, P < 0.05). The FLV and MLV ratios of involved hemiliver to uninvolved hemiliver were 0.09 ± 0.06 and 0.30 ± 0.14 in group 1, 0.57 ± 0.43 and 1.08 ± 0.63 in group 2, 0.71 ± 0.30 and 0.71 ± 0.48 in group 3. The difference in MLV-FLV ratio was signifcant between group 1 and group 3, between group 2 and group 3 (P = 0.000).

CONCLUSIONSThe dynamic SPCECT (99m)Tc-GSA scintigraphy can not only assess the whole liver function of cirrhosis liver effectively, but also evaluate the variation of regional liver function accurately.

Adult ; Aged ; Female ; Humans ; Liver ; physiopathology ; Liver Cirrhosis ; physiopathology ; Liver Function Tests ; Male ; Middle Aged ; Technetium Tc 99m Aggregated Albumin ; metabolism ; Technetium Tc 99m Pentetate ; metabolism ; Tomography, Emission-Computed, Single-Photon

OBJECTIVETo establish a stable and reliable model of Sertoli-cell-only syndrome in mice.

METHODSWe randomly divided 60 NIH mice into two groups of equal number to receive intraperitoneal injection of busulfan (30 mg/kg) and 30 or 60 minutes of testis cooling. At 2, 4 and 8 weeks after treatment, we recorded the survival rate of the mice, weight of the testis and Johnsen scores, and conducted quantitative analysis on the degrees of spermatogenetic failure.

RESULTSThere were no significant differences in the baseline body weight and survival rate between the intervention and control groups (P > 0.05). At 4 and 8 weeks, the testis weight and Johnsen score were significantly lower in the intervention group than in the control ([0.04 +/- 0.01] g and [0.05 +/- 0.01] g vs [0.09 +/- 0.03] g and [0.11 +/- 0.02] g, P < 0.05; 3.86 +/- 0.50 and 2.70 +/- 0.67 vs 9.60 +/- 0.25 and 9.76 +/- 0.43, P < 0.01). At 2, 4 and 8 weeks, the testis weights were (0.07 +/- 0.02) g, (0.06 +/- 0.01) g and (0.09 +/- 0.01) g, respectively, in the 30-min cooling group and (0.05 +/- 0.01) g, (0.04 +/- 0.02) g and (0.04 +/- 0.02) g in the 60-min cooling group, significantly lower than in the control side at the same time points ([0.11 +/- 0.01] g, [0.11 +/- 0.01] g and [0.12 +/- 0.00] g) (P < 0.05), and the Johnsen scores were 4.70 +/- 0.67, 2.70 +/- 0.84 and 6.10 +/- 1.14 in the 30-min and 1.67 +/- 0.58, 1.20 +/- 0.45 and 1.00 +/- 0.00 in the 60-min cooling group, remarkably lower than in the control side (9.60 +/- 3.23, 9.60 +/- 0.55 and 9.70 +/- 0.45) (P < 0.01). Histopathological examination of the cooled testes revealed considerable atrophy of seminal tubules, necrosis of seminiferous epithelia and peritubular fibrosis.

CONCLUSIONAdministration of busulfan has no obvious influence on the survival of mice, and is a reliable method for constructing a mouse model of Sertoli-cell-only syndrome.

Animals ; Busulfan ; adverse effects ; Cold Temperature ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred Strains ; Organ Size ; Sertoli Cell-Only Syndrome ; chemically induced ; Sertoli Cells ; Testis

Through our pre-investigation and literature analysis, it was found that rhubarb could be categorized into two types, chrysophanol-type and rhein-type, based on the proportion of the two constituents in the total content of anthraquinones after acid hydrolysis. In this paper, the antimicrobial activities of chrysophanol-type and rhein-type rhubarbs against Staphylococcus aureus were compared with microcalorimetric analysis, in order to illustrate the bioactive differentiability between the two chemotypes. For the aim to display the distinction of chrysophanol and rhein percentage in total anthraquinones, the sampling volume was regulated to make the total anthraquinones equivalent, thus, the antimicrobial difference was only attributed to the difference of chemotypes. The results indicated that the antimicrobial difference between the two chemotypes was confirmable labeled at the biothermokinetic parameters of S. aureus growth affected by the rhubarb samples. The growth rate constant (k1) of the first exponential phase for the growth of S. aureus affected by the rhein-type rhubarb was significantly lower than that of chrysophanol-type (P<0.01), which suggested stronger antimicrobial activity of rhein-type rhubarb than that of chrysophanol-type. However, the antimicrobial activities of rhein-type rhubarbs were not positively correlated to the contents of rhein. It suggested that the antimicrobial activity of rhubarb might be related to some unknown components which were of same accumulating pattern of rhein. The findings in present study provided some experimental evidence on categorizing rhubarb into two chemotypes through the difference of antimicrobial activity on S. aureus by microcalorimetric analysis and, further, offered references to revision of the commercial specification of rhubarb from chemical view.
Anthraquinones ; isolation & purification ; pharmacology ; Anti-Infective Agents ; isolation & purification ; pharmacology ; Calorimetry ; Plants, Medicinal ; chemistry ; Rheum ; chemistry ; Staphylococcus aureus ; drug effects ; growth & development

In this article, canonical correlation analysis was used to explore the relationship between the toxicity-attenuating effect and the variation of chemical contents in rhubarb caused by processing. With quasi-acute toxicity test, the difference of hepatic and renal toxicity to mice with the processed materials of rhubarb was researched. The chemical contents of anthraquinones and tannins in rhubarb were measured by UV-vis spectrophotometry and high performance liquid chromatography. The results showed that there were toxic effects to liver and kidney in mice after repeated intragastric administration of rhubarb and its processed materials for 14 days at a dosage of 76 g x kg(-1). The toxic effect of processed materials was much lower than crude drug. With canonical correlation analysis, the sequence of the hepatic and renal toxicity of chemical contents in rhubarb were found as follows: total anthraquinone glycosides (AQGs) > tannins (Tns) > total anthraquinones (AQs); aloe-emodin (AE) > physcione (Ph) > rhein (Rn) > emodin (Ed) > chrysophanol (Ch) and AEG > PhG > ChG > EdG > RnG of glycosyl-anthraquinone. It could be concluded that processing would attenuate the toxicity of crude drug of rhubarb. The toxicity-attenuating effect might be correlated to the decline of the contents of both anthraquinone glycosides and tannins, especially the aloe-emodin glycoside and physcione glycoside. The results also suggested that the serum alanine aminotransferase (ALT) and creatine (CREA) would be useful to monitor the hepatic and renal toxicity of rhubarb.
Alanine Transaminase ; blood ; Animals ; Anthraquinones ; analysis ; Creatinine ; blood ; Kidney ; drug effects ; Liver ; drug effects ; Mice ; Plant Roots ; chemistry ; Proanthocyanidins ; analysis ; Rheum ; chemistry ; toxicity ; Technology, Pharmaceutical ; methods

To establish a new method to evaluate the COLD and HOT nature of Coptis & Evodia and their prescriptions Zuojinwan and Fanzuojinwan. Physical models of mice were established by diet restriction with cold-water swimming (weak model, WM) and fed with high protein animal feeds (strong model, SM). An instrument with cold and hot pads was used to investigate the variation of temperature tropism among SM and WM groups of mice affected by drugs. Meanwhile, the oxygen consumption and activity of adenosine triphosphatase (ATPase) were detected, in order to investigate the mechanism of energy metabolism which might be affected by these drugs. The results showed that the drug effects gradually changed in an order of "Coptis-->Zuojinwan--> Fanzuojinwan-->Evodia". In detail, Coptis increased the remaining rate (RR) of mice on hot pad, decreased oxygen consumption and ATPase activity (n=6, P < 0.01 or P < 0.05), while Evodia performed inversely; which indicated the COLD nature of Coptis and HOT nature of Evodia, and confirmed with their traditional definition in medicinal works. In conclusion, the methods applied in this work, can objectively and directly express the nature disparity between the two herbs and predict the tendency of changes of the nature of their combination, which brings a new approach in investigation of the nature theory of traditional Chinese medicine.
Animals ; Body Temperature ; Cold Temperature ; Coptis ; chemistry ; Diet ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Evodia ; chemistry ; Hot Temperature ; Medicine, Chinese Traditional ; Mice ; Oxygen Consumption ; drug effects ; Plants, Medicinal ; chemistry ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Swimming ; Tropism

Objective To verify the feasibility and accuracy of the quantitative evaluation of the volume of internal abdominal hemorrhage based on CT images.Methods The clinical data of 76 patients diagnosed as abdominal hemorrhage or hemoperitoneum and performed with emergency surgery in the Second Affiliated Hospital to Wenzhou Medical University from January 2009 to September 2016 were retrospectively analyzed by case-control study.The Noboru Oriuchi's formula was used to calculate the volume of abdominal hemorrhage based on CT images,and the results were compared and adjusted with the volume of actual abdominal hemorrhage recorded during the operation.SPSS 21.0 was used to statistically analyze the data.The linear regression was analyzed on the results measured by the two methods.Results The volume of abdominal hemorrhage measured by the CT calculation method ranged from 10 to 4 335 ml,while the corresponding volume measured by operational calculation method ranged from 200 ml to 4 490 ml.The absolute difference in the volume measured by these two methods ranged from 4.8 ml to 500 ml.The ratio of the absolute difference to the volume of abdominal hemorrhage by operational calculation method ranged from 0.2％ to 95.0％,the median of which was 4.5％ (2.8％,8.9％).When the exact volume of abdominal hemorrhage was ＜ 500 ml,the absolute difference in the exact volume ranged from 30.0％ to 95.0％,the median of which was 69.1％ (51.2％,78.6％).When the volume was less than 500 ml,the ratio ranged from 0.2％-13.6％,the median of which was 4.2％ (2.7％,6.4％).Analysis of the numbers of the two measuring methods with linear correlation method after eliminating the cases in which the bleeding volume was less than 500 ml showed that two methods presented a linear correlation (r =0.971,P ＜ 0.05).Conclusion After the conventional abdominal CT scanning,the Noboru Oriuchi's formula can be used to accurately calculate the volume of abdominal hemorrhage in patients with volume of abdominal hemorrhage more than 500 ml.