Phase I evaluation of the safety and pharmacokinetics of a single-dose intravenous injection of a murine monoclonal antibody against Hantaan virus in healthy volunteers.

Anti-Hantaan virus monoclonal antibody (AHM) is a murine monoclonal antibody against Hantaan virus being developed for the treatment of hemorrhagic fever with renal syndrome. The purpose of the present study was to describe the tolerance and pharmacokinetics of an intravenously administered single ascending dose of AHM in Chinese healthy volunteers. Four cohorts of 22 healthy subjects received AHM at 2.5 to 20 mg, and the results indicated that AHM was well tolerated. We established a highly sensitive, rapid, and accurate immunoassay for the kinetic analysis of AHM in serum. Serial blood samples were obtained after intravenous administration for up to 17 days. A one-compartment model was determined to best describe the disposition of AHM. The maximal level in serum and the area under the serum concentration-time curve were proportional to the doses. The mean clearance, the half-life, and the volume of distribution were constant, irrespective of the dose. AHM was slowly cleared and had a half-life of approximately 110 h. These data support the use of a treatment regimen in which AHM is given only once intravenously.

CTLA4-Ig-modified dendritic cells inhibit lymphocyte-mediated alloimmune responses and prolong the islet graft survival in mice.

The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In this study, we transfected CTLA4-Ig gene into dendritic cells (DCs), and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on pancreatic islet allograft in mice. An IDDM C57BL/6 murine model induced by streptozotocin is as model mouse. The model mice were transplanted of the islet cells isolated from the BALB/c mice to their kidney capsules, and injected of CTLA4-Ig modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation and induce its apoptosis; whereas, unmodified DCs (umDCs) promoted the murine lymphocyte proliferation. Compared with injection of umDCs and IgG1 modified DCs, the injection of mDCs prolonged IDDM mice's allograft survival, and normalized their plasma glucose (PG) levels within 3 days and maintained over 2 weeks. The level of IFN-gamma was lower and the level of IL-4 was higher in mDCs treated recipient mice than that in control mice, it indicated that mDCs led to Th1/Th2 deviation. After 7 days of islet transplantation, HE stain of the renal specimens showed that the islets and kidneys were intact in structure, and islet cells numbers are increased in mDCs treated mice. Our studies suggest that DCs expressing CTLA4-Ig fusion protein can induce the immune tolerance to islet graft and prolong the allograft survival through the inhibition of T cell proliferation in allogeneic mice.

Uric acid enhances T cell immune responses to hepatitis B surface antigen-pulsed-dendritic cells in mice.

AIM: To study the induction of T cellular immune responses in BALB/c mice immunized with uric acid and dendritic cells (DCs) pulsed with hepatitis B virus surface antigen (HBsAg). METHODS: DCs were generated from bone-marrow cells of BABL/c mice, and then pulsed or unpulsed with HBsAg protein (HBsAg-pulsed-DCs or unpulsed-DCs) in vitro. BABL/c mice were immunized with HBsAg-pulsed-DCs (1 x 10(6)) and uric acid, injected through the tail vein of each mouse. The mice in control groups were immunized with HBsAg-pulsed-DCs alone, unpulsed-DCs alone or 200 microg uric acid alone or PBS alone. The immunization was repeated 7 d later. Cytotoxic T lymphocytes (CTLs) in vivo were determined by the CFSE labeled spleen lysis assay. Spleen cells or spleen T cells were isolated, and re-stimulated in vitro with HBsAg for 120 h or 72 h. Production of IFN-gamma and IL-4 secreted by spleen cells were determined by ELISA method; proliferation of spleen T cells were detected by flow cytometry. RESULTS: The cytotoxicities of HBsAg-specific-CTLs, generated after immunization of HBsAg-pulsed-DCs and uric acid, were 68.63% +/- 11.32% and significantly stronger than that in the control groups (P < 0.01). Compared with control groups, in mice treated with uric acid and HBsAg-pulsed-DCs, the spleen T cell proliferation to HBsAg re-stimulation was stronger (1.34 +/- 0.093 vs 1.081 +/- 0.028, P < 0.01), the level of IFN-gamma secreted by splenocytes was higher (266.575 +/- 51.323 vs 135.223 +/- 32.563, P < 0.01) , and IL-4 level was lower (22.385 +/- 2.252 vs 40.598 +/- 4.218, P < 0.01). CONCLUSION: Uric acid can strongly enhance T cell immune responses induced by HBsAg-pulsed-DCs vaccine. Uric acid may serve as an effective adjuvant of DC vaccine against HBV infection.

Roles of TGF-ß signaling pathway in endoplasmic reticulum stress in endothelial cells stimulated with cigarette smoke extract.

To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress (ER stress) in endothelial cells stimulated with cigarette smoke extract (CSE). Human umbilical vein endothelial cells (HUVECs) were cultured and divided into 3 groups: CSE-stimulated group, CSE-stimulated with 4-PBA group, and negative control group. HUVECs were cultured and stimulated with CSE at concentrations of 5%, 10% and 20%, respectively, mRNA of CXCL-8 and GRP78 was detected by real-time PCR. ELISA was performed to test the expression of CXCL-8 protein, and neutrophils migration was detected by Transwell board test. The NF-κB, ERK, p38MAPK and transforming growth factor beta (TGF-ß) were detected by flow cytometry. The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs (P<0.05). Furthermore, it was concentration-dependent. 4-PBA significantly reduced the expression of CXCL-8 protein (P<0.05) and neutrophil migration (P<0.05). The TGF-ß, rather than the NF-κB, ERK and P38MAPK pathway might be involved in ER stress stimulated by CSE. CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells. ER stress might play a role in the effect of neutrophils migration stimulated with CSE, and TGF-ß pathway may contribute to the ER stress in HUVECs.

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase.

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase (AP). METHODS: The VH-linker-VL, namely scFv gene, was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by Sfi I and Not I, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and Not I restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

[The construction of transferrin receptor- mediated HSV-TK gene transfer system and its effect on human hepatocellular carcinoma cells in vitro].

OBJECTIVE: To construct the localization system involving anti-TfR monoclonal antibody (McAb) and AFP promoters and assess its effect on human hepatoma cell lines. METHODS: The conjugate of anti-TfR McAb and polylysine (PLL) was made by SPDP and purified by molecular screen chromatography. DNA blocking test determined that the ratio of one pEBAF/tk to six Ab-PLL was the most suitable to couple them. The pEBAF/tk recombinant plasmid bearing HSV-TK gene was coupled to Ab-PLL by noncovalent bond. The pEBAF/tk was transferred into human hepatoma cell line HepG2, SMMC7721 and pulmonary cancer cell line A549 by receptor-mediated gene delivery (Ab-PLL-DNA) and liposome procedure. The growth inhibitory rates of HepG2, SMMC7721 and A549 cells were measured by MTT assay. RESULTS: The inhibitory rates of HepG2/tk in 100 mg/L and 1 mg/L of GCV were 60.5% and 24.3%, respectively. The inhibitory rate of GCV to SMMC7721 was 23.2% in 3 days. The pulmonary cancer cell A549, A549/tk (Ab) and A549 /tk (lipo) could not be inhibited by the addition of GCV. CONCLUSION: The localization system employed in this paper has high specificity, effectiveness and safety for gene therapy. It would be a promising strategy for gene therapy.

Roles of TGF-β Signaling Pathway in Endoplasmic Reticulum Stress in Endothelial Cells Stimulated with Cigarette Smoke Extract / 华中科技大学学报（医学）（英德文版）

To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress (ER stress) in endothelial cells stimulated with cigarette smoke extract (CSE).Human umbilical vein endothelial cells (HUVECs) were cultured and divided into 3 groups:CSE-stimulated group,CSE-stimulated with 4-PBA group,and negative control group.HUVECs were cultured and stimulated with CSE at concentrations of 5％,10％ and 20％,respectively,mRNA of CXCL-8 and GRP78 was detected by real-time PCR.ELISA was performed to test the expression of CXCL-8 protein,and neutrophils migration was detected by Transwell board test.The NF-κB,ERK,p38MAPK and transforming growth factor beta (TGF-β) were detected by flow cytometry.The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs (P＜0.05).Furthermore,it was concentration-dependent.4-PBA significantly reduced the expression of CXCL-8 protein (P＜0.05) and neutrophil migration (P＜0.05).The TGF-β,rather than the NF-κB,ERK and P38MAPK pathway might be involved in ER stress stimulated by CSE.CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells.ER stress might play a role in the effect of neutrophils migration stimulated with CSE,and TGF-β pathway may contribute to the ER stress in HUVECs.

Interleukin(IL)-4 promotion of CXCL-8 gene transcription is mediated by ERK1/2 pathway in human pulmonary artery endothelial cells.

Interleukin-4 is central to allergic pulmonary inflammatory responses, but its contribution to airway neutrophilia remains controversial. The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. However, its response to IL-4 is reported to either increase or decrease the production of neutrophil chemotactic factors. We hypothesized that these conflicting findings may be due to the tissue and the size of the vessels from which endothelial cells have been derived. The expression of CXCL-8 by human primary culture umbilical veins endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and human pulmonary microvascular endothelial cells (HPMECs) when stimulated with recombinant human IL-4 (rhIL-4) was studied. The chemoattractant property of the cells' supernatants for neutrophils was evaluated using Boyden chambers. The role of the nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPK) in IL-4-induced HPAECs was studied using Western blotting and electrophoretic mobility shift assay (EMSA). We demonstrated that IL-4 increased the mRNA expression and the protein production of CXCL-8 in HPAECs, but not in HUVECs and HPMECs. The supernatants of HAPECs stimulated by IL-4 significantly promoted neutrophils migration in a dose-dependent manner, and was significantly attenuated by an inhibitor of CXCL-8. We also found that extracellular-regulated protein kinase1/2 (ERK1/2) is activated by IL-4 in HPAECs, but not JUN-N-terminal protein kinase (JNK) or p38 MAPK pathway. Furthermore, NF-κB-DNA binding activity, phosphorylation of IκBα and p65 levels were not affected by rhIL-4 in HAPECs. These findings indicate marked functional differences in the response of micro and macro-ECs to IL-4. ERK1/2, rather than NF-κB, JNK and p38 MAPK signaling, plays a role in IL-4 induced chemokine activation. Our results suggest that inhibition of ERK1/2 may be a possible target for airway neutrophilia in allergic lung diseases.

[Efficacy and safety of reduced osmolarity oral rehydration salts in treatment of dehydration in children with acute diarrhea--a multicenter, randomized, double blind clinical trial].

OBJECTIVE: To assess the efficacy and safety of reduced osmolarity oral rehydration salts (ROORS) in treatment of mild to moderate dehydration caused by acute diarrhea in children. METHODS: A multicenter, randomized, double-blind, positive drug controlled clinical trial was conducted in 125 cases aged 1 to 17 years. These children with acute diarrhea and signs of dehydration were randomly assigned to receive either ROORS (trial group, n = 62) or oral rehydration salts II (ORS II) (control group, n = 63). The volume of intravenous infusion were recorded. The improvements of systemic symtoms and signs, diarrhea, dehydration and total scores were compared between the two groups. The adverse events and changes of electrolyte and other laboratory tests during treatment were also observed and analyzed. RESULTS: The overall effective rates in trial group and control group were 96.8% and 96.8%, respectively. The recovery of systemic symptoms, dehydration signs and diarrhea occurred in 96%, 97% and 78% patients in trial groups, and 96%, 98% and 85% patients in control group. The scores of symptoms and signs in both groups decreased significantly after treatment. All the above parameters and the number of cases who needed intravenous infusion (41 vs. 39) were not statistically different between two groups. However, the average volume of intravenously infused fluids in trial group was (450.98 +/- 183.07) ml, 24.5% less than that in the control group (597.30 +/- 343.37) ml (P < 0.05). The mean serum Na(+) concentration elevated from (137.48 +/- 4.55) mmol/L to (139.52 +/- 3.25) mmol/L (P < 0.01) in control group after treatment, but the change was not statistically significant in trail group. Serum K(+), Cl(-), HCO(3)(-) and other laboratory result did not change significantly after treatment. The total scores in both groups decreased obviously after treatment, but no significant difference was demonstrated between two groups (P > 0.05). A case in trial group had mild abdominal distention and recovered spontaneously. CONCLUSION: ROORS was shown to be effective and safe in the treatment of mild and moderate dehydration induced by acute diarrhea. Compared to ORS II, ROORS could decrease the intravenous supplement of fluid and lower the risk of hypernatremia.