The roles of IL-2 and IL-10 enhance anti-CD45RBmAb immune inhibition in allograft skin.

As a new type of immune tolerance inducer, anti-CD45RB monoclonal antibodies (anti-CD45RBmAb) can prolong the graft survival time of animal organs or cell transplantation as well as induce stable immune tolerance. Both interleukin (IL)-2 and IL-10 have important roles in the induction and maintenance of immunological tolerance. However, whether these cytokines combined with anti-CD45RBmAb can promote immune tolerance is poorly understood. Therefore, we investigated the effect of IL-2 and IL-10 in vitro and in vivo on the tolerance induction by anti-CD45RBmAb. The changes of Treg and Th17 cells and Th1/Th2 cytokines in anti-CD45RBmAb induced prolongation of skin allograft survival in mice. The finding of a role for IL-2 is novel, of interest, IL-2 promoted anti-CD45RBmAb-induced CD4(+) T cell differentiation into Treg and Th2 cells and suppressed Th17 and Th1 cells. IL-2 enhanced the induction of immune tolerance by anti-CD45RBmAb and significantly prolonged skin graft survival time in vivo. In contrast, this effect should be demonstrated experimentally by neutralizing IL-2 and inhibition of the effect of anti-CD45RBmAb, and neutralizing IL-10 showed no effect for anti-CD45RBmAb-induced tolerance. These data reveal that IL-2 significantly enhances anti-CD45RBmAb-induced immune tolerance via up-regulated T regulatory (Treg) cells and the balance of Th1/Th2 shifts. Conversely, IL-10 showed no effect on anti-CD45RBmAb-induced tolerance.

Detection of circulating tumor cells in breast cancer with a refined immunomagnetic nanoparticle enriched assay and nested-RT-PCR.

Early detection of circulation tumor cells (CTCs) in breast cancer patients has great clinical relevance. Currently, immunomagnetic microparticles enriched assays require Fe3O4 inner cores, making it difficult to improve sensitivity. In this study, we prepared magnetic nanoparticles with carbon-coated pure iron (Fe@C) acted as the core, Conjugating with EpCAM monoclonal antibody for immunomagnetic nanoparticles(IMPs). IMPs were used in conjunction with immunocytochemistry (ICC) to develop a refined immunomagnetic nanoparticles enriched assay (IMPEA) for detection of circulating tumor cells (CTCs) in breast cancer patients. Compared with nested RT-PCR, this method achieved the same sensitivity, but with a significantly reduced false-positive rate. This method will help find hidden micrometastases, establish clinical stage, and guide individual treatment post-surgery, suggesting potentially significant value in the clinic. FROM THE CLINICAL EDITOR: This team of investigators prepared magnetic nanoparticles with carbon-coated pure iron as core and conjugated them with EpCAM monoclonal antibody to form immunomagnetic nanoparticles for circulating tumor cell (CTC) detection. Compared with nested RT-PCR, this method achieved the same sensitivity, but with a significantly reduced false-positive rate, paving the way to the development of a tool that enables enhanced detection of micrometastases and post-surgical treatment monitoring.

Protective effect of rat pancreatic progenitors cells expressing Pdx1 and nestin on islets survival and function in vitro and in vivo

To maintain islets survival and function is critical in successful pancreatic transplantation. Pancreatic progenitors cells (PPCs) with lineage potentials, giving rise to exocrine, endocrine, and duct cells, reside in developing and adult pancreas. As tissue-specific stem cells, they can produce pancreatic tissue-specific matrix factors to promote islets survival and function. The aim of our research was to investigate the protective effect of rat pancreatic-duodenal homeobox 1 (Pdx1)+/nestin+ PPCs on islets. In vitro, co-culturing islets with Pdx1+/nestin+ PPCs prolonged the former survival from 7 to 14 days. Furthermore, with high glucose (300.8 mg/dl) stimuli, the yield of insulin in co-cultures was significantly higher than that in control group (single islets group). In vivo, co-transplanting islets and Pdx1+/nestin+ PPCs for 3 days, the blood glucose of diabetic rat was significantly decreased to normal level and sustained for 2 weeks. Without Pdx1+/nestin+ PPCs in islets transplantation, hyperglycemia was reversed at day 7 and recovered at day 15. Pathology analysis showed that islets had remnants in co-transplantation at day 21, as complete graft rejection in alone islets transplantation. Our study showed that Pdx1+/nestin+ PPCs displayed the ability of preserving islets viability and function in vitro and prolonging their survival in vivo (AU)

Protective effect of rat pancreatic progenitors cells expressing Pdx1 and nestin on islets survival and function in vitro and in vivo.

To maintain islets survival and function is critical in successful pancreatic transplantation. Pancreatic progenitors cells (PPCs) with lineage potentials, giving rise to exocrine, endocrine, and duct cells, reside in developing and adult pancreas. As tissue-specific stem cells, they can produce pancreatic tissue-specific matrix factors to promote islets survival and function. The aim of our research was to investigate the protective effect of rat pancreatic-duodenal homeobox 1 (Pdx1)(+)/nestin(+) PPCs on islets. In vitro, co-culturing islets with Pdx1(+)/nestin(+) PPCs prolonged the former survival from 7 to 14 days. Furthermore, with high glucose (300.8 mg/dl) stimuli, the yield of insulin in co-cultures was significantly higher than that in control group (single islets group). In vivo, co-transplanting islets and Pdx1(+)/nestin(+) PPCs for 3 days, the blood glucose of diabetic rat was significantly decreased to normal level and sustained for 2 weeks. Without Pdx1(+)/nestin(+) PPCs in islets transplantation, hyperglycemia was reversed at day 7 and recovered at day 15. Pathology analysis showed that islets had remnants in co-transplantation at day 21, as complete graft rejection in alone islets transplantation. Our study showed that Pdx1(+)/nestin(+) PPCs displayed the ability of preserving islets viability and function in vitro and prolonging their survival in vivo.

A role for anti-CD45RB monoclonal antibody treatment upon dendritic cells.

Selective interference with CD45RB isoform by monoclonal antibody (anti-CD45RBmAb) reliably induces donor-specific tolerance. Dendritic cells (DCs) are the most potent antigen-presenting cells that are capable of activating naïve T cells. The purposes of the present study were to investigate the roles of anti-CD45RBmAb on the phenotypes and functioning of DCs and to further illustrate the mechanism of anti-CD45RBmAb-inducing immunologic tolerance. DCs from C57BL/6 mice were cultured and treated with various doses of anti-CD45RB monoclonal antibody. Cell phenotype, cycle and phagocytic ability were detected by flow cytometry. The production of IL-10 and IL-12 in the supernatants of mature DCs was measured with ELISA. Exosomes (Dex) were recovered from the supernatant of DCs cultured for 6 days in depleted medium, and effects of DCs and Dex on the ability of T-cell proliferation were detected by mixed lymphocyte culture. Anti-CD45RBmAb could inhibit DCs maturation in a dose-dependent manner, and the effects of exosomes (Dex) on DCs enhance or inhibition proliferation of T cells were also in a dose-dependent manner. Anti-CD45RBmAb could profoundly inhibit the maturation and functioning of DCs and generate tolerogenic dendritic cells (tDCs) as well as Dex, suggesting mechanistic contributions to tolerance development from the DCs through interactions with T cells.

Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells.

Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic ß cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas that expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic ß cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.

A rapid and highly sensitive protocol for the detection of Escherichia coli O157:H7 based on immunochromatography assay combined with the enrichment technique of immunomagnetic nanoparticles.

BACKGROUND: Escherichia coli O157:H7 (E. coli O157:H7) is an important pathogenic bacterium that threatens human health. A rapid, simple, highly sensitive, and specific method for the detection of E. coli O157:H7 is necessary. METHODS: In the present study, immunomagnetic nanoparticles (IMPs) were prepared with nanopure iron as the core, coated with E. coli O157:H7 polyclonal antibodies. These IMPs were used in combination with immunochromatographic assay (ICA) and used to establish highly sensitive and rapid kits (IMPs+ICA) to detect E. coli O157:H7. The kits were then used to detect E. coli O157:H7 in 150 food samples and were compared with conventional ICA to evaluate their efficacy. RESULTS: The average diameter of IMPs was 56 nm and the amount of adsorbed antibodies was 106.0 µg/mg. The sensitivity of ICA and IMPs+ICA was 10(5) colony-forming units/mL and 10(3) CFUs/mL, respectively, for purified E. coli O157:H7 solution. The sensitivity of IMPs+ICA was increased by two orders, and its specificity was similar to ICA. CONCLUSION: The kits have the potential to offer important social and economic benefits in the screening, monitoring, and control of food safety.

Detection of micrometastases in peripheral blood of non-small cell lung cancer with a refined immunomagnetic nanoparticle enrichment assay.

Fe(3)O(4) particles are currently used as the core of immunomagnetic microspheres in the immunomagnetic enrichment assay of circulating tumor cells (CTCs). It is difficult to further improve the sensitivity of CTC detection or to improve tumor cell-type identification and characterization. In the present study, we prepared immunomagnetic nanoparticles with nanopure iron as the core, coated with anti-cytokeratin 7/8 (CK7/8) monoclonal antibody. These immunomagnetic nanoparticles (IMPs) were used in conjunction with immunocytochemistry (ICC) to establish a refined immunomagnetic nanoparticle enrichment assay for CTC detection in non-small cell lung cancer (NSCLC). The assay was compared with nested reverse transcription polymerase chain reaction (RT-PCR) to detect CK19 mRNA and lung specific X protein (LUNX) mRNA. Human lung adenocarcinoma cell line A549 was used for sensitivity and specificity evaluation. Peripheral blood samples were collected from each group for CTC detection. The average diameter of the immunomagnetic nanoparticles was 51 nm, and the amount of adsorbed antibodies was 111.2 µg/mg. We could detect down to one tumor cell in 5 × 10(7) peripheral blood mononuclear cells. The sensitivity was consistent with that of nested RT-PCR; however, the false positive rate was significantly reduced. The modified assay combined with ICC did not differ from nested RT-PCR in sensitivity, but it had significantly increased specificity. This approach could, therefore, contribute to identification of micrometastases, re-defining clinical staging, and guiding individual postoperative treatments. The technique shows considerable potential clinical value and further clinical trials are warranted.

[The effect of co-transplantation of bone marrow-derived mesenchymal stem cells and islet on maturation and function of bone marrow-derived dendritic cells in recipient mice].

AIM: To investigate the effect of transplantation of bone marrow-derived mesenchymal stem cells (MSCs), or co-transplantation of islet and MSCs, on maturation and function of bone marrow-derived dendritic cells (DCs) in recipient mice. METHODS: Bone marrow MSCs were isolated from BALB/c mice and co-cultured with bone marrow mononuclear cells (BMCs) from C57BL/6 mice at indicated ratios. The co-cultures were treated with recombinant mice granulocyte-macro-phage colony-stimulating factor (rmGM-CSF) and recombinant mice IL-4 (rmIL-4) for 7 days to induce the differentiation of DCs, with lipopolysaccharide (LPS) favoring the maturation of DCs. The differentiation markers and antigen uptake capability of DCs were analyzed by FCM. Production of Interleukin-12 in the supernatants of the DC cultures was quantified by ELISA. BALB/c-derived MSCs and islet were co-transplanted to the capsule of kidney in allogeneic C57BL/c mice. The recipient mice were assayed for their tissue morphology, blood glucose level, and the in vitro differentiation ability of their BMC into mature and functional DCs. RESULTS: The transplantation of MSCs prevented BMC from differentiating into mature DCs, as shown by down-regulated surface markers of DCs including CD11c, CD83, CD86 and I-Ab; (P<0.05), impaired antigen uptake and decreased IL-12 secretion (P<0.01). Co-transplantation of MSCs and islet inhibited immune rejection in the allogeneic recipient mice. CONCLUSION: Transplantation of MSCs inhibits maturation and function of monocyte-derived dendritic cells in the recipient mice, resulting in immune tolerance for the allogeneic islet.

A novel sensitive immunoassay by nucleic acid barcode dot and its application in the detection of prostate-specific antigen.

BACKGROUND: The sensitivity and selectivity of traditional methods limits ultramicro detection of proteins. Bio-barcode amplification detection methods based on nanotechnology enables ultramicro detection of protein. However, bio-barcode amplification detection depends on the oligonucleotides being fixed on a glass chip. It also requires specialized equipment, which limits its application. We introduce a nano-nucleic acid barcode dot detection technology to determine ultramicro concentrations of protein. The method is simple, quick and accurate. METHODS: Magnetic probe (IgG-M) and dual-labeled gold nanoparticle bio-probe (IgG-Au-DNA) were prepared. Protein was captured using a sandwich assay technique and magnetic separation was used. The DNA barcode was released with dithiothreitol (DTT) and detected directly without the requirement for polymerase chain reaction (PCR). Serum prostate-specific antigen (PSA) from 135 patients was detected with this method and compared with enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). RESULTS: Each IgG-Au-DNA could be covered with 138+/-47 oligonucleotides and 11+/-3 antibodies. The IgG-M could bind 118 mug of antibody per mg. The sensitivity of nano-nucleic acid barcode dot detection technology might allow detection of 1 fg/mL. There were no significant differences in serum PSA from 135 patients when comparing the three methods (compared with ELISA, r=0.950; and with RIA, r=0.967). CONCLUSIONS: The nucleic acid barcode dot method does not require special equipment or complex procedures, but its detection limit is 2-3 orders of magnitude lower than ELISA.

Preparation of carboplatin-Fe@C-loaded chitosan nanoparticles and study on hyperthermia combined with pharmacotherapy for liver cancer.

BACKGROUND: Magnetic fluid hyperthermia is a kind of technology for treating tumors based on nanotechnology. It is suitable to various types of tumors. The purpose of this study was to prepare carboplatin-Fe@C-loaded chitosan nanoparticles with Fe@C as a magnetic core and to investigate efficacy of hyperthermia combined with chemotherapy for transplanted liver cancer in rats. METHODS: Fe@C nanopowder was treated with dilute hydrochloric acid to prepare Fe@C nanocage. Carboplatin-Fe@C-loaded chitosan nanoparticles were prepared by reverse microemulsion method with the nanocages as the magnetic cores, chitosan as the matrix. The shape, size, drug-loading rate, and in vitro cumulative release of the nanoparticles were observed and heat product under high frequency alternating electromagnetic field in vitro was explored. Eighty rats with transplanted liver cancer were randomly divided into 4 groups (group A: control group, group B: free carboplatin group, group C: nanoparticles with static magnetic field group, and group D: nanoparticles with static field and alternating magnetic field). Drug was injected into the hepatic artery. The therapeutic effect of hyperthermia combined with chemotherapy for tumor, toxicity and rat survival time were observed. RESULTS: Carboplatin-Fe@C-loaded chitosan nanoparticles were spherical in shape with an average size of (207 +/- 21) nm and high saturation magnetization. The drug-loading rate of the nanoparticles was 11.0 +/- 1.1%. The cumulative release percentage of carboplatin-Fe@C-loaded chitosan nanoparticles in vitro at different point time phase of 24 h, 48 h, 72 h, 96 h and 120 h were 51%, 68%, 80%, 87% and 91%, respectively. With an increase in carboplatin-Fe@C-loaded chitosan nanoparticle concentration and magnetic field strength, the heating rate and constant temperature of carboplatin-Fe@C-loaded chitosan nanoparticles dispersed in physiological saline were increased in an alternating magnetic field. In vivo experiments showed that after particle injection, tumor temperature reached 42.6 degrees +/- 0.2 degrees C within 10 min in the alternating magnetic field; and the temperatures in the right hepatic lobes and the rectum were significantly lower than in the tumor and the constant temperature could last up to 30 min. The inhibition ratio of tumor weight in group D was significantly enhanced, no obviously toxic and side-effect occurred and survival time was prolonged. CONCLUSION: Carboplatin-Fe@C-loaded chitosan nanoparticles possess good magnetic targeting and heat production properties. They can target liver cancer tissue by static magnetic field, and with the application of alternating magnetic field, effectively raise tumor tissue temperature and facilitate tumor apoptosis. The combination of chemotherapy and magnetic materials into nanoparticles as described herein demonstrates promising efficacy.

[Clinical significance of enrichment and detection of circulating tumor cells in NSCLC patients with immunomagnetic beads].

OBJECTIVE: To investigate the feasibility and clinical significance of detection of circulating tumor cells (CTCs) in peripheral blood of NSCLC patients by lung cancer cell immunomagnetic enrichment and detection kit. METHODS: Four groups of patients (Group A: 18 cases with stage I lung cancer; Group B: 33 cases with stage II - IV lung cancer; Group C: 20 cases with benign pulmonary diseases; Group D: 20 healthy volunteers) were enrolled for detection of CTCs using the lung cancer cell enrichment and detection kit developed by ourselves, and compared with the results obtained using simple ICC method and the detection of CK19 mRNA and LUNX mRNA using nested PCR as control. RESULTS: By using lung cancer cell enrichment and detection kit, it was revealed that the detection rates of CK positive cells were 33.3%, 60.6%, 0% and 0% in Group A, B, C and D, respectively. By using nested RT-PCR, the rates of positive expression of CK19 mRNA were 38.9%, 63.6%, 20.0% and 0% in Group A, B, C and D, respectively, and those of LUNX mRNA were 44.4%, 69.7%, 10.0% and 0%, respectively, while the detection rate of CTCs was negative in all groups using simple ICC. There was a significant difference between the results obtained by lung cancer cell enrichment and detection kit and simple ICC method (P < 0.05), while no significant difference was found between the results obtained by lung cancer cell enrichment and detection kit and nested RT-PCR (P > 0.05). There was a significant difference in the detection rates of CK positive cells between group A and groups B, C, D (P < 0.05). The micrometastasis in peripheral blood was closely related with pathological types, cell differentiation and TNM staging (P < 0.05). CONCLUSION: The lung cancer cell enrichment and detection kit developed by ourselves is a sensitive and reliable method to detect CTCs in patients with NSCLC. It may be helpful in diagnosis of NSCLC micrometastasis and circulating tumor cells in peripheral blood, re-determination of clinical stage and provide important information for cancer-therapy personalization.

Clinical characteristics of remote Zeus robot-assisted laparoscopic cholecystectomy: a report of 40 cases.

AIM: To summarize the performing essentials and analyze the characteristics of remote Zeus robot-assisted laparoscopic cholecystectomy. METHODS: Robot-assisted laparoscopic cholecystectomy was performed in 40 patients between May 2004 and July 2005. The operating procedures and a variety of clinical parameters were recorded and analyzed. RESULTS: Forty laparoscopic cholecystectomy procedures were successfully completed with Zeus robotic system. And there were no post-operative complications. Total operating time, system setup time and performing time were 100.3 +/- 18.5 min, 27.7 +/- 8.8 min and 65.6 +/- 18.3 min, respectively. The blood loss and post-operative hospital stay were 30.6 +/- 10.2 mL and 2.8 +/- 0.8 d, respectively. Camera clearing times and time used for operative field adjustment were 1.1+/- 1.0 min and 2.0 +/- 0.8 min, respectively. The operative error was 7.5%. CONCLUSION: Robot-assisted laparoscopic cholecystectomy following the principles of laparoscopic operation has specific performing essentials. It preserves the benefits of minimally invasive surgery and offers enhanced ability of controlling operation field, precise and stable operative manipulations.

Zeus robot-assisted laparoscopic cholecystectomy in comparison with conventional laparoscopic cholecystectomy.

BACKGROUND: The robotic surgical system overcomes many technological obstacles of conventional laparoscopic surgery, and possesses enormous clinical applied potential. The aim of this study was to compare the efficacy of Zeus robot-assisted laparoscopic cholecystectomy with conventional laparoscopic cholecystectomy. METHODS: Forty patients undergoing elective cholecystectomy were randomly divided into two groups. Patients in group A (n=20) underwent Zeus robot-assisted laparoscopic cholecystectomy, and patients in group B (n=20) received conventional laparoscopic cholecystectomy. The parameters on operative field, operative time, the number of actions, the rate of operative errors and minimal trauma were evaluated and compared between the two groups. RESULTS: The number of clearing camera (1.1+/-1.0 times) and the time of adjusting the operative field (2.2+/-0.7 minutes) in group A were significantly less than those (4.5+/-1.5 times) and (7.5+/-1.2 minutes) in group B. The number of dissection actions (337+/-86 times) and the rate of operative errors (10%) in group A were less than those (389+/-94 times), (25%) in group B. The total operation time (104.9+/-20.5 minutes) and setup time (29.5+/-9.8 minutes) in group A were significantly longer than those (78.6+/-17.1 minutes), (12.6+/-2.5 minutes) in group B. Blood loss and postoperative hospitalization were similar. No postoperative complications occurred in both groups, and open cholecystectomy was performed in each group. CONCLUSIONS: Zeus robot-assisted cholecystectomy inherits the benefits of minimally invasive surgery. The Zeus robotic surgical system is better than conventional laparoscopic technique in controlling the operative field and can be manipulated precisely and stably though it requires more operative time.

[Laparoscopic cholecystectomy with remote zeus surgical robotic system: report of 16 cases].

OBJECTIVE: To explore the methodology and operative essentials of laparoscopic cholecystectomy with remote Zeus surgical robotic system. METHODS: Based on strict training and successful experiment in animal model of swine, laparoscopic cholecystectomy using Zeus robotic system was performed on 16 patients with biliary diseases, including choledocholithiasis, cholelithiasis, polyposis of gallbladder, and chronic cholecystitis, 10 males and 16 females, aged 33 (14 approximately 27), 26 April to 31 August 2004. The general data, preoperative preparation time, operation time, amount of bleeding, complications, and hospitalization time were analyzed. RESULTS: All operations were performed without event. Along with the accumulation of experience the preoperative preparation time was shortened from 90 min to 30 min with an average of 41.7 min, and the operation time from 120 min to 30 min with an average of 64.4 min. The average amount of bleeding was 27.7 ml, and the average postoperative hospitalization time was 2.4 d. A telephone follow-up 30 days after operation showed no abnormality. CONCLUSION: Laparoscopic cholecystectomy with Zeus surgical robotic system is feasible and reliable with the advantages of clearer images in the field of operation, more precise handling, and remote surgery or education.