The extracellular domain of Staphylococcus aureus LtaS binds insulin and induces insulin resistance during infection.

Insulin resistance is a risk factor for obesity and diabetes and predisposes individuals to Staphylococcus aureus colonization; however, the contribution of S. aureus to insulin resistance remains unclear. Here, we show that S. aureus infection causes impaired glucose tolerance via secretion of an insulin-binding protein extracellular domain of LtaS, eLtaS, which blocks insulin-mediated glucose uptake. Notably, eLtaS transgenic mice (eLtaS trans ) exhibited a metabolic syndrome similar to that observed in patients, including increased food and water consumption, impaired glucose tolerance and decreased hepatic glycogen synthesis. Furthermore, transgenic mice showed significant metabolic differences compared to their wild-type counterparts, particularly for the early insulin resistance marker α-hydroxybutyrate. We subsequently developed a full human monoclonal antibody against eLtaS that blocked the interaction between eLtaS and insulin, which effectively restored glucose tolerance in eLtaS trans and S. aureus-challenged mice. Thus, our results reveal a mechanism for S. aureus-induced insulin resistance.

[Characterization of the size variants of a recombinant humanized monoclonal antibody (rhumAb1)].

The composition and potency of the high temperature (40 ℃) stress induced size variants of a recombinant humanized monoclonal antibody(rhumAb1) were characterized by means of SEC-HPLC, non- reduced CE-SDS, liquid chromatography coupled with mass spectrometry (LC-MS) and antibody dependent cell-mediated cytotoxicity (ADCC) assay. The molecular masses of the four size variants (SEC-1-SEC-4) separated by SEC-HPLC and seven size variants(NR-1-NR-7) detected by non-reduced CE-SDS were all characterized by LC-MS. The major low molecular weight variants were generated due to the hinge region fragmentation of heavy chain. The hinge region cleavage was found mainly in the Ser221-Cys-Asp-Lys-Thr- His-Thr-Cys228 sequence, in which C222-D223 and H226-T227 were the major cleavage sites. The size variants of rhumAb1, namely dimer and fragments, have significantly reduced ADCC activity in comparison with the intact rhumAb1 drug product. This study provided insights into the stability profiling for rhumAb1 drug product. The study protocols presented here may be applicable to the analytical characterization of other monoclonal antibody-based therapeutic products.

Generation of a recombinant West Nile virus stably expressing the Gaussia luciferase for neutralization assay.

West Nile virus (WNV) is a neurotropic human pathogen that has caused increasing infected cases over recent years. There is currently no licensed vaccine or effective drug for prevention and treatment of WNV infection in humans. To facilitate antiviral drug discovery and neutralizing antibody detection, a WNV cDNA clone containing a luciferase reporter gene was constructed through incorporating Gaussia luciferase (Gluc) gene within the capsid-coding region of WNV genome. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated luciferase reporter WNV (WNV-Gluc) and the stable WNV-Gluc with high titers (>10(7)PFU/ml) was obtained through plaque purification. Luciferase activity was used to effectively quantify the viral production of WNV-Gluc. Using the reporter virus WNV-Gluc, we developed a luciferase based assay in a 12-well format for evaluating neutralizing antibodies. The reporter virus could be a powerful tool for epidemiological investigation of WNV, vaccine evaluation, antiviral drug screening, and the study of WNV replication and pathogenesis.

An optimized B lymphocyte stimulator (BLyS) antagonist peptide inhibits the interaction of BLyS with BCMA.

B lymphocyte stimulator (BLyS) antagonists are new therapeutic reagents for treating the autoimmune diseases. Peptibodies can inhibit the bioactivity of BLyS, the same as other BLyS antagonists: decoyed BLyS receptors and anti-BLyS antibodies. In this study, a new optimized BLyS antagonist peptide was designed according to our previous work by the computer-aided homology modeling. Competitive ELISA showed that the peptide at 100 µg/ml could inhibit 54 % of the BCMA-Fc binding to BLyS. To maintain its stability and spatial conformation, the peptide was fused to human IgG1 Fc to form a peptide-Fc fusion protein-a novel peptibody by gene engineering. ELISA indicated that the peptibody could bind with BLyS in dosage-dependent manner as BCMA-Fc did. This study highlights the possibility of designing and optimizing BLyS antagonist peptides with high biopotency by the computer-aided design. Thus, these peptides could neutralize BLyS activity and be potential antagonists to treat autoimmune diseases related with BLyS overexpression.

[Influence of excessive complement activation on pathological process of acute graft versus host disease in mice].

This study was aimed to explore the influence of excessive complement activation on the pathological process of acute graft-versus-host disease (aGVHD) in mice. A murine model with aGVHD was established by injecting cell mixture containing splenocytes and bone marrow cells at 2:1 ratio from donor C57BL/6(H-2K(b)) mice into recipient BALB/c (H-2K(d)) mice within 4-6 hours after 8 Gy (60)Co γ-ray total body irradiation. The mice received syngeneic bone marrow transplantation were used as control group. After transplantation, the mice were monitored daily for body weight and mortality. At day 14, all mice were sacrificed and each liver was freshly dissociated for histological analysis. The hepatic mRNA abundance for complement components C3a and C5a as well as receptors for these two anaphylatoxin were tested by real-time quantitative PCR method. And the levels of C3a and C5a production in liver were detected by ELISA. The deposition of complement C3 in liver was determined by immunofluorescence staining using frozen section. The results indicated that as compared with syngeneic bone-marrow transplantation control group, experimental animals underwent aGVHD characterized by weight loss, depilation, diarrhea and lassitude. Interestingly, the hepatic mRNA expression for complement anaphylatoxin family member C3a and C5a as well as their receptors C3aR and C5aR1 in mice with aGVHD were significantly up-regulated in comparison with control group (p < 0.05). Consistently, the content of C3a and C5a in liver increased markedly in mice with aGVHD (p < 0.01). For animals ongoing aGVHD, complement component C3 depositions were observed in hepatic portal areas, around which massive inflammatory cell infiltration was also observed. It is concluded that in aGVHD animals, excessive complement activation occurs, and the activated complement components participate in pathological process of the aGVHD.

CD3 mAb treatment ameliorated the severity of the cGVHD-induced lupus nephritis in mice by up-regulation of Foxp3+ regulatory T cells in the target tissue: kidney.

Teff/Treg imbalance orchestrated the onset and the progression of the lupus nephritis in a DBA/2→B6D2F1 murine model with cGVHD. In this paper, we first used 145-2C11 Ab to treat these human SLE-like diseased animals. The results showed that short-term low-dose anti-CD3 antibody treatment induced a significant remission of established proteinuria, production of autoantibodies, immune complex deposition and renal parenchyma lesions in lupus nephritic mice. Of note, we found a robust up-regulation of Foxp3 mRNA expression in the target tissue: kidney from mice with anti-CD3 antibody treatment compared to those with control IgG treatment. Likewise, an increased renal mRNA abundance for IL-10 was also observed in anti-CD3 antibody treated mice. In contrast, genes associated with inflammation and fibrosis as well as cytokines related to effector T cell responses were down-regulated by anti-CD3 mAb treatment. These findings suggested that short-term low-dose anti-CD3 antibody treatment might induced an IL-10-secreting Foxp3(+) regulatory T cells in this cGVHD target tissue: kidney, that suppressed the activation of effector T cells (Th1, Th2 and Th17), thus ameliorating the severity of the lupus nephritis in mice.

[Establishment and evaluation of experimental sepsis mouse model].

After treating with chemotherapy or immunosuppressant, malignant diseases of hematopoietic system such as leukemia, malignant lymphoma and aplastic anemia usually induced severe infection such as sepsis. Sepsis which is hard to be diagnosed causes high death rate. This study was purposed to establish an experimental sepsis mouse model so as to provide a basis for pathogenesis and intervention study. A classic caecal ligation and puncture (CLP) was used to establish experimental sepsis model. ELISA was used to detect levels of C5a, IL-6, TNFalpha, and IFN-gamma. Flow Cytometry was applied to measure apoptosis of lymphocytes in thymus and mesentery. The pathologic changes of thymus and spleen were confirmed by HE staining. The results showed that almost 70%-80% mice died at 72 hours after CLP. Only approximate 20% animal survived during finite time, mice in CLP group had significant weight lose. Meanwhile large release of different inflammatory mediators which are related with sepsis (C5a, IL-6, TNF-alpha, and IFN-gamma) was observed after CLP. Apoptosis of lymphocytes in thymus and mesentery lymphonodus was enhanced markedly after CLP. Significantly pathologic injury was also observed in thymus and spleen. It is concluded that a mouse model of experimental sepsis was successfully established by caecal ligation and puncture which can well mimic the clinical symptom of sepsis. The experimental sepsis mouse model provides an excellent tool for exploring the pathogenesis and intervention ways for sepsis accompanied with complicated malignant hematological diseases in vivo.

Inhibition of IgE Activity to Bind its High Affinity Receptor (FcεRIα) by Mouse Anti-IgE Cε3∼4 Monoclonal Antibody (QME5).

Using computer-guided homology modeling method, the 3-D structure of the Fv fragment of a functional anti-IgE antibody (MAE11) was constructed and the spatial structure of E24-MAE11 complex was modeled based on the crystal structure of IgE-Fc (abbr. E24) and molecular docking method. Then the identified epitope of IgE was determined theoretically, which showed the key role of IgE-Cɛ3 in interacting with both FcɛRIα and MAE11. By normal protocols, we immunized mice with purified protein E34 and screened six anti-E34 monoclonal antibodies. Purified antibodies could identify E34 by Western blot; furthermore, all of them could bind IgE by ELISA, in which QME5 seemed to be the best. Flow cytometry analysis displayed that only QME5 could bind membrane IgE and it could compete with membrane FcɛRIα to bind soluble IgE. Meanwhile, QME5 couldn't bind FcɛRIα-attached IgE, which suggested no hypersensitivity in triggering the target cells (mast cells or basophils) by crosslinking or inducing the release of a variety of chemical mediators.

[Immunoregulation effects in vitro of the xenoprotein in combination with recombinant human granulocyte-macrophage colony stimulating factor and bacillus Calmette-Guerin].

This study was aimed to investigate the effects of xenogeneic antigen neu-Fc in combination with the recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and Bacillus Calmette-Guerin (BCG) on the regulation of Th1 and Th2 immune response in vitro. The rat neu L2-S2 domain was engineered as a chimeric protein with human IgG Fc. The eukaryotic expression vector was constructed. The recombinant protein was stably expressed in CHO cells and purified by rProtein A Sepharose Fast Flow column. The recombinant protein was identified by SDS-PAGE and Western blot. Peripheral blood mononuclear cells (PBMNCs) were obtained by means of standard Ficoll separation from the blood of healthy donors. Neu-Fc-induced PBMNC proliferation was tested by MTT. The production of IL-12 and IL-10 was measured by ELISA. The results showed that the level of IL-12 decreased and IL-10 increased after PBMNCs were incubated with MCF-7 cultural supernatant. 10 nmol/L neu-Fc strongly induced the cell proliferation. Compared with neu-Fc or GM-CSF or BCG treatment alone, neu-Fc in combination with GM-CSF and BCG significantly stimulated IL-12 production and inhibited IL-10 production (p < 0.01). It is concluded that the neu-Fc can stimulate the proliferation activity of PBMNCs. neu-Fc, GM-CSF and BCG costimulation efficiently induces Th1 immune response.

The characteristic of an anti-human DR5 antibody A6.

The efficacy of many cancer treatments is due to their ability to induce apoptosis. DR5 can activate apoptosis pathway after binding with its natural ligand, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L). Both TRAIL and agonistic anti-DR5 monoclonal antibody are currently being explored for cancer therapy. The mechanisms of cytotoxicity of our previously prepared monoclonal antibody A6 against DR5 were investigated here. A6 could cause viability loss of Jurkat cells in both time- and dose-dependent manner which could be attributed to the activation of apoptosis pathway. Caspases 3, 8 and 9 were activated in Jurkat cells and the caspase specific inhibitors, such as broad caspases inhibitor Z-VAD-FMK, caspase 8 specific inhibitor Z-IETD-FMK and caspase 9 specific inhibitor Z-LEHD-FMK could recover the viability loss caused by A6. The function and molecular mechanism of TRAIL-mediated apoptosis were also investigated and compared with those of A6. Although A6 and TRAIL recognize a different epitope, they could induce a similar reaction in Jurkat cells.

Autoantibody profiling of Chinese patients with autoimmune hepatitis using immunoproteomic analysis.

In the present study, immunoproteomic analysis was utilized to systemically characterize global autoantibody profiles in autoimmune hepatitis (AIH). Sera from 21 patients with AIH and 15 healthy controls were analyzed for the antibody reactivity against the protein antigens of HepG2, a human hepatoma cell line. The lysates of HepG2 cells were separated by two-dimensional electrophoresis and then immunoblotted with each serum sample. Matrix-assisted laser desorption/ionization mass spectrometry or/and nanoelectrospray ionization MS/MS were then used to identify antigens, among which a bifunctional enzyme in mitochondrial, fumarate hydratase (FH), was further analyzed by ELISA using recombinant FH as a coating antigen. A total of 18 immunoreactive spots were identified as 13 proteins, 8 of which have not been reported in AIH. Immune reactivity to FH was detected in 66.67% of patients with AIH, 19.35% of patients with primary biliary cirrhosis (PBC), 12.31% of patients with chronic hepatitis B (CHB), 6.35% of patients with chronic hepatitis C (CHC), 11.32% of patients with systemic lupus erythematosus (SLE), and 3.57% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P<0.001). These data demonstrate the serological heterogeneity in AIH and suggest the diversity of the mechanisms underlying AIH. FH, recognized mainly in AIH rather than in viral hepatitis and other autoimmune diseases, may have utility in improved diagnosis of AIH.

[Transfer RNAs inhibit the growth of L929 cells in vitro].

AIM: To explore the effects of tRNA on the growth of mammalian cells. METHODS: L929, NIH3T3, MCF-7 and PC12 cells were seeded in 96 well culture plate individually, and incubated at 37 degrees C in 5% CO2 for 4 h, the tRNAs from different species were added to the culture media individually. After certain time of incubation, the viability of the cells was evaluated by the MTT methods. Sub-confluent L929 cells were incubated with 200 microg/ml ytRNA for different times, then the cells were pooled and analyzed with flow cytometry assay. RESULTS: tRNA specifically inhibited the growth of L929 cells in a dose-dependent manner. The sizes of tRNA-treated cells showed larger sizes and longer processes than those of untreated cells. Flow cytometric analysis further showed that most of tRNA-treated cells were arrested in S phase of the cell cycle. CONCLUSION: The cell growth inhibitory effects of tRNAs were caused mainly by their degraded fragments. The results suggested that tRNA or its degraded fragments might play important roles in regulation of cell proliferation.

J2 prolongs the corneal allograft survival through inhibition of the CD4+ T cell-mediated response in vivo.

In our previous report, we described a novel non-peptidic organic ligand of CD4 D1, designated J2, as a potential inhibitor of CD4 D1 and thus CD4-dependent T cell responses in vitro. In this work, we further used a murine model of corneal allograft rejection to determine its in vivo immunosuppressive activities. To mimic the situation in high-risk human eyes, the recipient mice corneas were all induced by intrastromal sutures to serve as neovascularized graft beds. J2 was administrated by mouth 3 h before transplantation and thereafter on consecutive 12 days. The results showed that J2 could significantly prolong the median survival time of the corneal allografts, compared to the untreated control group. And the subsequent functional assays, including T cell phenotype analysis, delayed-type hypersensitivity (DTH) and enzyme-linked immunospot (ELISPOT) assays revealed that the immunosuppressive activity of J2 was associated with its inhibitory effects on the CD4(+) T cells and these cells-mediated responses. All these results suggest that J2 is a potential lead for the development of new immunosuppressive agents to prevent the corneal allograft rejection.

Efficient growth inhibition of ErbB2-overexpressing tumor cells by anti-ErbB2 ScFv-Fc-IL-2 fusion protein in vitro and in vivo.

AIM: To investigate the antitumor activities of an anti-ErbB2 scFv-Fc-interleukin 2 (IL-2) fusion protein (HFI) in vitro and in vivo. METHODS: Fusion protein HFI was constructed. The efficacy of HFI in mediating tumor cell lysis was determined by colorimetric lactate dehydrogenase release assays. The antitumor activity of HFI was evaluated in tumor xenograft models. RESULTS: The fusion protein was folded as a homodimer formed by covalently linking Fc portions and it retained ErbB2 specificity and IL-2 biological activity. HFI mediated antibody-dependent cell-mediated cytotoxicity (ADCC) at low effector-to-target ratios in vitro and improved the therapeutic efficacy of IL-2 in experiments in vivo. CONCLUSION: The genetically-engineered anti-ErbB2 scFv-Fc-IL-2 fusion protein exhibited high efficiency both in mediating ADCC in vitro and significant antitumor activity in tumor xenograft models.

[Gene expression and primary functional characterization of human DR5 extracellular fragment].

AIM: To acquire human DR5 extracellular fragment with bioactivity. METHODS: Total RNA was prepared from Jurkat cells by Trizol. Human DR5 extracellular fragment gene was amplified by RT-PCR, cloned into pGEM-T Easy vector, and confirmed by sequence analysis. Then the gene was subcloned into expression vector pET30a with a His-tag at the amino terminus and expressed in E.coli BL21 (DE3). The products was purified by Ni-NTA chromatography column and identified by SDS-PAGE and Western blot. ELISA method was used to detect its binding activity to anti-DR5 monoclonal antibody (mAb) mDRA-6. RESULTS: Human DR5 extracellular fragment gene was successfully amplified and high level expression was obtained in E.coli BL21 (DE3) induced by 0.1 mmol/L IPTG. The DR5 extracellular fragment protein was identified by SDS-PAGE and Western blot analysis. ELISA results showed that the purified DR5 could be recognized by mDRA-6. CONCLUSION: The extracellular region of DR5 with bioactivity has been successfully expressed and purified, which lay the foundation for further study.

A5, a new small-molecule inhibitor of CD4 D1 obtained from a computer-aided screening method, contributes to the inhibition of CD4+ T-cell function.

In this study, the authors apply a computer-based strategy to screen thousands of small-molecule, nonpeptidic organic compounds in the Available Chemicals Directory database and to select a series of potential candidates as ligands of the proposed CD4 D1 surface pocket. Then, several cell-based models are used to determine the actual biological functions of these compounds. A small molecule designated A5 (N-((pyridine-4-yl)methylene)thiophene-2-carbohydrazide) was obtained by a virtual screening followed by 3 cell-based functional assays. The results show that A5 could specifically block the CD4-major histocompatibility complex II binding in a rosetting assay, inhibit the mixed lymphocyte reaction-induced T-cell proliferation in a concentration-dependent manner, and reduce the PMA plus ionomycin-stimulated interleukin-2 secretion from peripheral blood mononuclear cells.

Human IgG1 Cgamma1 domain is crucial for the bioactivity of the engineered anti-CD20 antibodies.

In this study, we discussed the necessity of human IgG1 Cgamma1 domain for recombinant antibody using computer-aided homology modeling method and experimental studies. The heavy (VH) and light (VL) chain variable regions of 1-28, a murine IgM-type anti-CD20 mAb, were ligated by linker peptide (Gly4Ser)3 to form the single-chain Fv fragment (scFv). Then, the engineered antibody (LH1-3) was generated by fusing scFv with the entire IgG1 heavy constant regions. The 3-D structure of LH1-3 was modeled using computer-aided homology modeling method and the binding activity of LH1-3 was evaluated theoretically. Compared to the 3-D structure of the Fv fragment of the parent antibody, the conformation of the active pocket of LH1-3 was remained because of the rigid support of Cgamma1. Further experimental results of flow cytometry showed that the engineered anti-CD20 antibody possessed specifically binding activity to CD20-expressing target cells. The anti-CD20 antibody fragments could also mediate complement-dependent cytotoxicity (CDC) of human B-lymphoid cell lines. Our study highlights some interests and advantages of a methodology based on the homology modeling and analysis of molecular structural properties.

CUE domain containing 2 regulates degradation of progesterone receptor by ubiquitin-proteasome.

Accumulated evidence indicates that progesterone receptors (PR) are involved in proliferation of breast cancer cells and are implicated in the development of breast cancer. In this paper, a yeast two-hybrid screen for PR led to the identification of CUE domain containing 2 (CUEDC2), whose function is unknown. Our results demonstrate that CUEDC2 interacts with PR and promotes progesterone-induced PR degradation by the ubiquitin-proteasome pathway. The inhibition of endogenous CUEDC2 by siRNA nearly abrogated the progesterone-induced degradation of PR, suggesting that CUEDC2 is involved in progesterone-induced PR ubiquitination and degradation. Moreover, we identify the sumoylation site Lys-388 of PR as the target of CUEDC2-promoted ubiquitination. CUEDC2 decreases the sumoylation while promoting ubiquitination on Lys-388 of PRB. We also show that CUEDC2 represses PR transactivation, inhibits the ability of PR to stimulate rapid MAPK activity, and impairs the effect of progesterone on breast cancer cell growth. Therefore, our results identify a key post-translational mechanism that controls PR protein levels and for the first time provide an important insight into the function of CUEDC2 in breast cancer proliferation.

An efficient and targeted gene integration system for high-level antibody expression.

Random integration linking genomic amplification has been used to generate desired cell lines for stable and high-level expressing recombinant antibodies. But this technique is laborious, and the expression level is unpredictable due to position effects. Here, we have constructed a cell-vector system for high-level antibody expression using an FRT/FLP strategy to overcome position effects. The key is to target the FRT sequence to chromosomal locations where there is a high rate of transcription and gene amplification, and the amplified genes can be maintained. To screen desired loci with high transcriptional activity and amplifiable capacity, dual weakened markers (selectable galactosidase and amplifiable dihydrofolate reductase, DHFR) and the FRT sequence were synchronously cloned into a plasmid. After transfection of a Chinese hamster ovary host cell line with this plasmid, we selected 20 candidate cell lines from 721 individual clones. An antibody gene-targeting vector carrying an FRT-fused hygromycin gene was constructed to target antibody genes into the chromosomal FRT site by FLP recombinase. Three out of 20 cell lines can be used as host cells for site-specific recombination. By using southern blot and fluorescence in situ hybridization (FISH), a candidate engineered cell line, number 37, was chosen. It contains a single FRT-tagged locus in its genome. FISH analysis indicated that the antibody genes were all located at the original FRT-tagged locus in the genome of the gene-targeted and gene-amplified cell lines. Three kinds of recombinant antibodies were successfully expressed in candidate cell line 37. The highest producers produced more than 200 mug/ml of the antibody in 6 days of continuous culture in a spinner flask.