B cells assist allograft rejection in the deficiency of protein kinase c-theta.

We have previously shown that mice deficient in protein kinase C theta (PKCθ) have the ability to reject cardiac allografts, but are susceptible to tolerance induction. Here we tested role of B cells in assisting alloimmune responses in the absence of PKCθ. Mouse cardiac allograft transplantations were performed from Balb/c (H-2d) to PKCθ knockout (PKCθ(-/-)), PKCθ and B cell double-knockout (PBDK, H-2b) mice and wild-type (WT) C57BL/6 (H-2b) mice. PBDK mice spontaneously accepted the allografts with the inhibition of NF-κB activation in the donor cardiac allograft. Anti-B cell antibody (rituximab) significantly delayed allograft rejection in PKCθ(-/-), but not in WT mice. Co-transfer of PKCθ(-/-) T plus PKCθ(-/-) B cells or primed sera triggered allograft rejection in Rag1(-/-) mice, and only major histocompatibility complex class II-enriched B cells, but not class I-enriched B cells, were able to promote rejection. This, together with the inability of PKCθ(-/-) and CD28(-/-) double-deficient (PCDK) mice to acutely reject allografts, suggested that an effective cognate interaction between PKCθ(-/-) T and B cells for acute rejection is CD28 molecule dependent. We conclude that T-B cell interactions synergize with PKCθ(-/-) T cells to mediate acute allograft rejection.

Assessment of different bariatric surgeries in the treatment of obesity and insulin resistance in mice.

OBJECTIVE: To assess the effects of different bariatric surgical procedures on the treatment of obesity and insulin resistance in high fat diet-induced obese (DIO) mice. BACKGROUND: Bariatric surgery is currently considered the most effective treatment for morbid obesity and its comorbidities; however, a systematic study of their mechanisms is still lacking. METHODS: We developed bariatric surgery models, including gastric banding, sleeve gastrectomy, Roux-en-Y gastric bypass (RYGB), modified RYGB (mRYGB) and biliopancreatic diversion (BPD), in DIO mice. Body weight, body fat and lean mass, liver steatosis, glucose tolerance and pancreatic beta cell function were examined. RESULTS: All bariatric surgeries resulted in significant weight loss, reduced body fat and improved glucose tolerance in the short term (4 weeks), compared with mice with sham surgery. Of the bariatric surgery models, sleeve gastrectomy and mRYGB had higher success rates and lower mortalities and represent reliable restrictive and gastrointestinal (GI) bypass mouse bariatric surgery models, respectively. In the long term, the GI bypass procedure produced more profound weight loss, significant improvement of glucose tolerance and liver steatosis than the restrictive procedure. DIO mice had increased insulin promoter activity, suggesting overactivation of pancreatic beta cells, which was regulated by the mRYGB procedure. Compared with the restrictive procedure, the GI bypass procedure showed more severe symptoms of malnutrition following bariatric surgery. DISCUSSIONS: Both restrictive and GI bypass procedures provide positive effects on weight loss, fat composition, liver steatosis and glucose tolerance; however, in the long term, the GI bypass shows better results than restrictive procedures.

Quantitative analysis of the secretome of TGF-beta signaling-deficient mammary fibroblasts.

Transforming growth factor beta (TGF-beta) is a master regulator of autocrine and paracrine signaling pathways between a tumor and its microenvironment. Decreased expression of TGF-beta type II receptor (TbetaRII) in stromal cells is associated with increased tumor metastasis and shorter patient survival. In this study, SILAC quantitative proteomics was used to identify differentially externalized proteins in the conditioned media from the mammary fibroblasts with or without intact TbetaRII. Over 1000 proteins were identified and their relative differential levels were quantified. Immunoassays were used to further validate identification and quantification of the proteomic results. Differential expression was detected for various extracellular proteins, including proteases and their inhibitors, growth factors, cytokines, and extracellular matrix proteins. CXCL10, a cytokine found to be up-regulated in the TbetaRII knockout mammary fibroblasts, is shown to directly stimulate breast tumor cell proliferation and migration. Overall, this study revealed hundreds of specific extracellular protein changes modulated by deletion of TbetaRII in mammary fibroblasts, which may play important roles in the tumor microenvironment. These results warrant further investigation into the effects of inhibiting the TGF-beta signaling pathway in fibroblasts because systemic inhibition of TGF-beta signaling pathways is being considered as a potential cancer therapy.

Multi-ATOM: Ultrahigh-throughput single-cell quantitative phase imaging with subcellular resolution.

A growing body of evidence has substantiated the significance of quantitative phase imaging (QPI) in enabling cost-effective and label-free cellular assays, which provides useful insights into understanding the biophysical properties of cells and their roles in cellular functions. However, available QPI modalities are limited by the loss of imaging resolution at high throughput and thus run short of sufficient statistical power at the single-cell precision to define cell identities in a large and heterogeneous population of cells-hindering their utility in mainstream biomedicine and biology. Here we present a new QPI modality, coined multiplexed asymmetric-detection time-stretch optical microscopy (multi-ATOM) that captures and processes quantitative label-free single-cell images at ultrahigh throughput without compromising subcellular resolution. We show that multi-ATOM, based upon ultrafast phase-gradient encoding, outperforms state-of-the-art QPI in permitting robust phase retrieval at a QPI throughput of >10 000 cell/sec, bypassing the need for interferometry which inevitably compromises QPI quality under ultrafast operation. We employ multi-ATOM for large-scale, label-free, multivariate, cell-type classification (e.g. breast cancer subtypes, and leukemic cells vs peripheral blood mononuclear cells) at high accuracy (>94%). Our results suggest that multi-ATOM could empower new strategies in large-scale biophysical single-cell analysis with applications in biology and enriching disease diagnostics.

A high-throughput all-optical laser-scanning imaging flow cytometer with biomolecular specificity and subcellular resolution.

Image-based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single-cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all-optical ultrafast laser-scanning imaging technique, called free-space angular-chirp-enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s-1 ) 1 to 2 orders of magnitude higher than the camera-based imaging flow cytometers. It also has 2 critical advantages over optical time-stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular-specific cellular assay and (2) it enables high-throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single-cell image analysis which reveals cellular heterogeneity, for example, in the cell-death processes demonstrated in this work-the information generally masked in non-imaging flow cytometry. Therefore, this platform empowers not only efficient large-scale single-cell measurements, but also detailed mechanistic analysis of complex cellular processes.

Multi-MHz laser-scanning single-cell fluorescence microscopy by spatiotemporally encoded virtual source array.

Apart from the spatial resolution enhancement, scaling of temporal resolution, equivalently the imaging throughput, of fluorescence microscopy is of equal importance in advancing cell biology and clinical diagnostics. Yet, this attribute has mostly been overlooked because of the inherent speed limitation of existing imaging strategies. To address the challenge, we employ an all-optical laser-scanning mechanism, enabled by an array of reconfigurable spatiotemporally-encoded virtual sources, to demonstrate ultrafast fluorescence microscopy at line-scan rate as high as 8 MHz. We show that this technique enables high-throughput single-cell microfluidic fluorescence imaging at 75,000 cells/second and high-speed cellular 2D dynamical imaging at 3,000 frames per second, outperforming the state-of-the-art high-speed cameras and the gold-standard laser scanning strategies. Together with its wide compatibility to the existing imaging modalities, this technology could empower new forms of high-throughput and high-speed biological fluorescence microscopy that was once challenged.

[Distribution of micrometastatic nodules of low rectal cancer in mesorectum: a pathological study using whole-mount sections].

OBJECTIVE: To investigate the regional spread of micrometastatic nodules in the mesorectum from low rectal cancer, and provide further pathological evidence to optimize radical resection procedure for rectal cancer. METHODS: A total of 62 patients with low rectal cancer underwent low anterior resection and total mesorectal excision (TME) was included in this study. Surgical specimens were sliced transversely and serial embedded blocks were made at 2.5 mm interval, and paraffin sections were stained with hematoxylin and eosin. The mesorectum on whole-mount sections was divided into three regions: outer region of mesorectum (ORM), middle region of mesorectum (MRM) and inner region of mesorectum (IRM). Microscopic spread were examined microscopically on the sections for the distribution in different mesorectal regions, frequency, types, involvement of lymphatic system and correlation with the primary tumor. RESULTS: Microscopic spread of the tumor in mesorectum and ORM was observed in 38.7% (24/62) and 25.8% (16/62) of the patients, respectively. Circumferential resection margin (CRM) involved by microscopic tumor foci occurred in 6.5% (4/62) of the patients, and distal mesorectum (DMR) involvement was recorded in 6.5% (4/62) with a spread extent within 3 cm of distal border of the main lesions. Most (20/24) of the patients with microscopic spread in mesorectum were in TNM stage III. CONCLUSION: Results of the present study support that complete excision of mesorectum without destruction of the ORM is essential for surgical management of low rectal cancer, and an optimal DMR clearance resection margin should not be less than 4 cm.

[Autologous versus allogeneic stem cell transplantation in adult patients with acute lymphoblastic leukemia].

OBJECTIVE: To explore the efficacy of autologous or allogeneic stem cell transplantation in adult patients with acute lymphoblastic leukemia (ALL) and investigate its relevant prognostic factors. METHODS: A total of 96 adult patients with ALL who had admitted to our hospital from November 1986 to June 2004 were followed up till February 28, 2005. They were divided into autologous stem cell transplantation (Auto-SCT) group (n = 56) and allogeneic stem cell transplantation (Allo-SCT) group (n = 40). Auto-SCT group was further divided to treated subgroup, in which patients received graft-purified transplantation and (or) maintenance immunotherapy or chemotherapy after transplantation (n = 26), and non-treated subgroup (n = 30). Clinical characteristics of these groups were retrospectively analyzed. Survival date were analyzed by the Kaplan-Meier method and the prognostic factors were analyzed with the COX regression model. RESULTS: The 1-, 3-, and 5-year leukemia-free-survival (LFS) were not significantly different between the auto-SCT group and the allo-SCT group. The 3-and 5-year LFS of auto-SCT treated subgroup, auto-SCT non-treated subgroup and allo-SCT group were [(73.0 +/- 8.7)%, (69.2 +/- 9.0)%], [(42.2 +/- 10.1)%, (35.1 +/- 10.0)%], and [(50.9 +/- 8.2)%, (50.9 +/- 8.2)%], respectively, which showed statistical significance (P < 0.05). CONCLUSIONS: The long-term LFS is similar after auto-SCT and after allo-SCT. Purified graft and maintain immunotherapy or chemotherapy post-transplantation may decrease the relapse rate after auto-SCT and improve survival.

Chronic hepatitis C virus infection and post-liver transplantation diabetes mellitus.

Patients with chronic hepatitis C virus (HCV) infection have a significantly increased prevalence of type 2 diabetes mellitus compared to controls or HBV-infected patients. Moreover, the incidence rate of post-liver transplantation diabetes mellitus (PTDM) also appears to be higher among patients with HCV infection. PTDM is often associated with direct viral infection, autoimmune disorders, and immunosuppressive regimen. Activation of tumor necrosis factor-alpha may be the link between HCV infection and diabetes. In this article, we reviewed the epidemiologic association between HCV infection and PTDM, highlighting the most recent pathophysiologic insights into the mechanisms underlying this association.

Hepatocytes targeting of cationic liposomes modified with soybean sterylglucoside and polyethylene glycol.

AIM: In this study, a hepatocyte-specific targeting technology was developed by modifying cationic liposomes with soybean sterylglucoside (SG) and polyethylene glycol (PEG) (C/SG/PEG-liposomes). METHODS: The liposomal transfection efficiencies in HepG(2) 2.2.15 cells were estimated with the use of fluorescein sodium (FS) as a model drug, by flow cytometry. The antisense activity of C/SG/PEG-liposomes entrapped antisense oligonucleotides (ODN) was determined as HBsAg and HBeAg in HepG(2) 2.2.15 cells by ELISA. The liposome uptake by liver and liver cells in mice was carried out after intravenous injection of (3)H-labeled liposomes. RESULTS: C/SG-liposomes entrapped FS were effectively transfected into HepG(2) 2.2.15 cells in vitro. C/SG/PEG-liposomes entrapped ODN, reduced the secretion of both HBsAg and HBeAg in HepG(2) 2.2.15 cells when compared to free ODN. After in vivo injection of (3)H-labeled C/SG/PEG-liposomes, higher radiation accumulation was observed in the hepatocytes than non-parenchymal cells of the liver. CONCLUSION: C/SG/PEG-liposomes mediated gene transfer to the liver is an effective gene-delivery method for hepatocytes-specific targeting, which appears to have a potential for gene therapy of HBV infections.

Pituitary adenylate cyclase activating-peptide and its receptor antagonists in development of acute pancreatitis in rats.

AIM: Pituitary adenylate cyclase activating-peptide (PACAP) is a late member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family of brain-gut peptides. It is unknown whether PACAP takes part in the development of acute pancreatitis and whether PACAP or its antagonists can be used to suppress the progression of acute pancreatitis. We investigated the actions of PACAP and its receptor antagonists in acute pancreatitis on rats. METHODS: Acute pancreatitis was induced in rats with caerulein or 3.5% sodium taurocholate. The rats were continuously infused with 5-30 microg/kg PACAP via jugular vein within the first 90 min, while 10-100 microg/kg PACAP6-27 and (4-Cl-D-Phe6, Leu17) VIP (PACAP receptor antagonists) were intravenously infused for 1 h. Biochemical and histopathological assessments were made at 4 h after infusion. Pancreatic and duodenal PACAP concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Chinese ink-perfused pancreas was fixed, sectioned and cleared for counting the functional capillary density. RESULTS: PACAP augmented caerulein-induced pancreatitis and failed to ameliorate sodium taurocholate-induced pancreatitis. ELISA revealed that relative concentrations of PACAP in pancreas and duodenum were significantly increased in both sodium taurocholate- and caerulein-induced pancreatitis compared with those in normal controls. Unexpectedly, PACAP6-27 and (4-Cl-D-Phe6, Leu17) VIP could induce mild acute pancreatitis and aggravate caerulein-induced pancreatitis with characteristic manifestations of acute hemorrhagic/necrotizing pancreatitis. Functional capillary density of pancreas was interpreted in the context of pancreatic edema, and calibrated functional capillary density (calibrated FCD), which combined measurement of functional capillary density with dry weight/wet weight ratio, was introduced. Hyperemia or congestion, rather than ischemia, characterized pancreatic microcirculatory changes in acute pancreatitis. CONCLUSION: PACAP may take part in the pathogenesis of acute pancreatitis in rats. The two PACAP receptor antagonsits might act as partial agonists. Calibrated functional capillary density can reflect pancreatic microcirculatory changes in acute pancreatitis.

Differences in platelet endothelial cell adhesion molecule-1 expression between peripheral circulation and pancreatic microcirculation in cerulein-induced acute edematous pancreatitis.

AIM: To investigate the changes of platelet endothelial cell adhesion molecule-1 (PECAM-1) expression on polymorphonuclear leukocytes (PMNs) in peripheral circulation and pancreatic microcirculation in cerulein-induced acute edematous pancreatitis (AEP). METHODS: Fifty Wistar rats were randomly divided into control group (n = 10) and AEP group (n = 40). A model of AEP was established by subcutaneous injection of cerulein 5.5 and 7.5 mug/kg at 0 and 1 h after the beginning of experiment respectively. PECAM-1 expression on PMNs from splenic vein and inferior vena cava was determined by RT-PCR at mRNA level and determined by flow cytometry at protein level. RESULTS: In experimental rats, an increased PECAM-1 mRNA expression was seen from 4 to 8 h of AEP in peripheral circulation (0.77+/-0.25%, 0.76+/-0.28%, 0.89+/-0.30%, 1.00+/-0.21%), while in pancreatic microcirculation, expression decreased from 2 h and reached the lowest level at 6 h of AEP (0.78+/-0.29%, 0.75+/-0.26%, 0.62+/-0.28%, 0.66+/-0.20%). There were significant differences at 8-h time point of AEP between peripheral circulation and pancreatic microcirculation (1.00+/-0.21% vs 0.66+/-0.20%, P<0.05). Meanwhile, the difference at protein level was also found. CONCLUSION: A reverse expression of PECAM-1 on PMNs was found between peripheral circulation and pancreatic microcirculation, suggesting that inhibition of PECAM-1 expression may improve the pathological change of AEP.

Characteristics and biodistribution of soybean sterylglucoside and polyethylene glycol-modified cationic liposomes and their complexes with antisense oligodeoxynucleotide.

A novel cationic liposome modified with soybean sterylglucoside (SG) and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) as a carrier of antisense oligodeoxynucleotide (ODN) for hepatitis B virus (HBV) therapy was constructed. Characteristics of the cationic liposomes modified with SG and PEG (SG/PEG-CL) and their complexes with 15-mer phosphorothioate ODN (SG/PEG-CL-ODN complex) were investigated by incorporation efficiency, morphology, electrophoresis, zeta potentials, and size analysis. Antisense activity of the liposomes and ODN complexes was determined as hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in HepG2 2.2.15 cells by ELISA. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. The complexes gained high incorporation efficiency and intact vesicular structure with mean size at approximately 200 nm. The SG/PEG-CL-ODN complexes enhanced the inhibition of both HBsAg and HBeAg expression in the cultured HepG2 2.2.15 cells relative to free ODN. The uptake of SG/PEG-CL and nonmodified cationic liposomes (CL) was primarily by liver, spleen, and lung. Furthermore, the concentration of SG/PEG-CL was significant higher than that of CL in hepatocytes at 0.5 hr postinjection. The biodistribution of SG/DSPE-CL-ODN complex compare with free ODN showed that liposomes enhanced the accumulation of ODN in the liver and spleen, while decreasing its blood concentration. SG/PEG-CL-mediated ODN transfer to the liver is an effective gene delivery method for cell-specific targeting, which has a potential for gene therapy of HBV infections. SG and PEG-modified cationic liposomes have proven to be an alternative carrier for hepatocyte-selective drug targeting.

[Effects of analogs of pituitary adenylate cyclase activating polypeptide on acute pancreatitis in rats].

OBJECTIVE: To examine the effects of analogs of pituitary adenylate cyclase activating polypeptide (PACAP)--PACAP6-27 (10, 100 microg/kg) and (4-Cl-D-Phe6, Leu17)VIP (10, 100 microg/kg) on the pancreata of normal rats and on the development of experimental acute pancreatitis. METHODS: Male Wistar rats were allocated into normal control groups, experimental acute pancreatitis groups and PACAP analog intervention groups. Acute pancreatitis was induced with s.c. cerulein and intraductal sodium taurocholate; PACAP analogs were infused intravenously immediately after pancreatitis induction. Pancreatic morphology was observed at 4 h, and serum amylase, pancreatic water content and PACAP contents were measured. RESULTS: It was found that PACAP6-27 induced pancreatic edema, inflammatory cell infiltration, and elevation of serum amylase [(1464.33 +/- 265.6)-(1692.17 +/- 312.18)] IU/L vs (520.8 +/- 163.27) IU/L of control, P < 0.05); that PACAP6-27 aggravated vacuolization of pancreatic acinar cells in cerulein-induced pancreatitis with hemorrhage and fatty and parenchymal necrosis; and that the pathological changes of cerulein plus 100 microg/kg PACAP group were similar to those of sodium taurocholate-induced pancreatitis. Pancreatic hemorrhage, vacuolization of acinar cells and parenchymal necrosis in sodium taurocholate-induced pancreatitis were worsened by PACAP6-27. (4-Cl-D-Phe6, Leu17)VIP had similar effects. ELISA showed that pancreatic and duodenal levels of PACAP were increased in cerulein- and sodium taurocholate-induced pancreatitis. CONCLUSION: PACAP6-27 and (4-Cl-D-Phe6, Leu17)VIP could induce mild pancreatitis and aggravate experimental acute pancreatitis. PACAP probably plays a role in the pathogenesis of acute pancreatitis.

Role of COX-2 in microcirculatory disturbance in experimental pancreatitis.

AIM: To elucidate the role of COX-2 in the development of capillary leakage in rats with acute interstitial pancreatitis. METHODS: Rats with acute interstitial pancreatitis were induced by caerulein subcutaneous injection. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene expression of COX-2 in pancreatic tissues, spectrophotometry was used to assay the parameters of acute pancreatitis such as the serum amylase and plasma myeloperoxidase, and determination of capillary permeability in the pancreas by quantifying the permeability index (PI) assisted response of pancreatic microvascular via intravital fluorescence microscope video image analysis system. RESULTS: A significant increase of COX-2 expression, elevation of serum amylase, and plasma myeloperoxidase were detected in rats with acute edematous pancreatitis compared with control rats. The changes of pancreatic microvascular after caerulein injection were as following: (a) the decrease of pancreatic capillary blood flow (4th h, 0.56+/-0.09 nL/min, P<0.05; 8 th h, 0.34+/-0.10 nL/min, P<0.001); (b) reduction of functional capillary density (4 th h, 381+/-9 cm(-1), P>0.05; 8th h, 277+/-13 cm(-1), P<0.001); (c) irregular and intermittent capillary perfusion was observed at the 8th h and these vessels were also prone to permeation. CONCLUSION: COX-2 plays an important role in mediating capillary permeability in pancreatitis, thereby contributing to capillary leakage.

CTLA-4 gene A/G polymorphism associated with diabetes mellitus in Han Chinese.

OBJECTIVE: To investigate the association of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) gene A/G polymorphism with susceptibility to diabetes mellitus in Han Chinese. METHODS: An A/G transition at position 49 of exon 1 was analyzed in 31 patients with type 1 diabetes, 31 patients with type 2 diabetes, and 36 controls were analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: A highly significant increase in the frequency of the G allele was seen in patients with type 1 diabetes compared with controls (66.1 % vs. 34.7%, respectively; P < 0.0005; OR = 3.670) . This reflected an increase in the GG genotype in patients (48.4% vs. 22.2%, respectively; P =0.025; OR =3.281) and a significant decrease in the AA genotype (16.1 % vs. 52.8%, respectively; P = 0.002). The allele frequencies of A and G in patients with type 2 diabetes were not significantly different from controls(A/G, 50.0/50.0% vs. 65.3/34.7%; P = not significant) . The distribution of genotype, however, differed significantly. This difference reflected an increase in the AG genotype in patients (54.8% vs.25.0%, respectively; P=0.012; OR=3.643) and a decrease in the AA genotype (22.6% vs. 52.8%, respectively; P=0.011). CONCLUSIONS: CTLA-4 49 AA is protective from diabetes mellitus, whereas, CTLA-4 49 G allele (both as homozygotes and as heterozygotes ) confers an increased risk of diabetes mellitus.

Effect of inducible cyclooxygenase expression on local microvessel blood flow in acute interstitial pancreatitis.

OBJECTIVE: To investigate the role of inducible cyclooxygenase (COX-2) mRNA expression in local microvessels in rats with acute interstitial pancreatitis (AIP) induced by caerulein injection. METHODS: The reverse transcription polymerase chain reaction (RT-PCR) was used to detect COX-2 gene expression in pancreatic tissue. Parameters of acute pancreatitis, such as serum amylase (AMS) and plasma myeloperoxidase (MPO) activities, were assayed using spectrophotometry. Intravital fluorescence microscopy with fluorescein isothiocyanate-labelled erythrocytes was used to study the pancreatic microvessels of rats with AIP and normal control rats. RESULTS: Highly significant increases in COX-2 expression and AMS and MPO activity were seen in rats with AIP compared with controls. After caerulein injection, pancreatic capillary blood flow was decreased (4 hours, p > 0.05; 8 hours, p < 0.001), functional capillary density was reduced (4 hours, p > 0.05; 8 hours, p < 0.001), and there was irregular and intermittent capillary perfusion at 8 hours. There was also a positive correlation between the level of COX-2 expression and MPO activity (plasma, r = 0.5449, p < 0.05; tissue, r = 0.5698, p < 0.05). CONCLUSIONS: The correlations between increased COX-2 expression and decreased capillary perfusion and blood flow and increased oedema following AIP may show that COX-2 expression can induce neutrophil sequestration to the pancreas, which may be one of the cascading inflammatory factors in the development of AIP.

[Studies on pulsatile release tablets of diltiazem hydrochloride in explosion way].

AIM: To investigate the preparation of pulsatile release tablets, the release of the drug in vitro and the pharmacokinetics in vivo. METHODS: Diltiazem hydrochloride (DIL) was used as model drug. The pulsatile release tablets were prepared by film-coated method using ethylcellulose and Eudragit L. The effect of formulation on pulsatile release of diltiazem hydrochloride was investigated under release rate test. The mechanism of pulsatile release of drug was proved by the test of water-uptake. The pharmacokinetic and bioavailability study in eight human subjects was performed by HPLC method. RESULTS: The release of diltiazem hydrochloride effected by the formulation of the core tablets and the composition and thickness of the coating film. In vitro, the delayed-release time T10 was 4.4 h, the maximum release time Trm was 8.0 h and the pulsed-release time Trm-10 was 3.6 h. In vivo, the delayed-release time Tlag was 4.9 h, the peak time was 8.0 h and the pulsed-release time was 3.1 h. The relative bioavailability was 105%. CONCLUSION: The release of drug from pulsatile release tablets of diltiazem hydrochloride was shown to be in pulsed way both in vitro and in vivo.

[Biodistribution and hepatocytes targeting of cationic liposomes surface-modified with sterylglucoside and golyethylene glycol].

AIM: To investigate the biodistribution and the hepatocytes targeting of cationic liposome containing 3beta[N-( N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and surface-modified liposomes with sterylglucoside (SG) and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE). METHODS: Cationic liposomes (CL) composed of DC-Chol and dipalmitoylphosphatidylcholine (DPPC), SG/PEG modified cationic liposome (SG/PEG-CL), both contained trace 3H-cholesterol (3H-Chol) as radiolabel, were prepared. The liposomes encapsulating 125I-labled antisense oligodeoxynucleotide (125I-asODN) (SG/PEG-CL-asODN) were also prepared. The biodistribution of CL, SG/PEG-CL, SG/PEG-C2-asODN as well as 125I-asODN solution, were studied. The radioactivities in hepatocytes and non-hepatocytes after administration of CL and SG/PEG-CL were determined by infuseing method. RESULTS: CL and SG/PEG CL significantly aggregated in liver. The distribution of SG/PEG CL was significantly higher in hepatocytes (P < 0.01) and lower in non-hepatocytes (P < 0.01) than that of CL. The concentrations of SG/PEG-CL-asODN in liver and spleen were significantly higher than that of asODN solution (P < 0.01). CONCLUSION: Cationic liposome modified with SG/PEG changed the distribution of asODN. Cationic liposome can target hepatocytes more effective after being modified with SG.

[Cationic lipid and polyethylene glycol enhance liposomes-mediated cell transfection and increase the fluidity of liposomes membranes].

AIM: To prepare fluorescein sodium (FS) cationic liposomes and investigate the influence of cationic lipid (DC-chol) and polyethylene glycol (PEG) with different molecule weight (MW) on cationic liposome incorporation efficiency, cellular delivery and fluidity of liposome membrane. METHODS: Using FS as a model material for encapsulation, the liposomes were prepared and separated (by sephadex G-50 1 cm x 20 cm column), and the liposome incorporation efficiencies was measured. The interaction between the FS and cationic liposomes was investigated by measuring the change of fluorescent spectrum. The cellular uptake of different liposome forms by choosing HepG2 2.2.15 as an in vitro cell culture assay model, and the influence of PEG on the fluidity of liposome membrane with the technique of fluorescence polarization were investigated. RESULTS: Cationic lipid and different PEGs showed great effects on increasing liposome incorporation efficiency (from 0.64% to 86.57%), cellular uptake (from 2.18% to 48.46%) and fluidity of liposome membrane. The effect of PEG was MW dependent, and with the increase of MW, the incorporation efficiency and transfection was improved, and the fluidity of liposome membrane increased. CONCLUSION: Addition of cationic lipid and high MW PEG into cationic liposomes can enhance the cellular delivery and fluidity of cationic liposomes. Also, they can improve the incorporation efficiency of cationic liposomes.