Tumour inhibitory activity on pancreatic cancer by bispecific nanobody targeting PD-L1 and CXCR4.

BACKGROUND: Antibodies and derivative drugs targeting immune checkpoints have been approved for the treatment of several malignancies, but there are fewer responses in patients with pancreatic cancer. Here, we designed a nanobody molecule with bi-targeting on PD-L1 and CXCR4, as both targets are overexpressed in many cancer cells and play important roles in tumorigenesis. We characterized the biochemical and anti-tumour activities of the bispecific nanobodies in vitro and in vivo. METHODS: A nanobody molecule was designed and constructed. The nanobody sequences targeting PD-L1 and CXCR4 were linked by the (G4S)3 flexible peptide to construct the anti-PD-L1/CXCR4 bispecific nanobody. The bispecific nanobody was expressed in E. coli cells and purified by affinity chromatography. The purified nanobody was biochemically characterized by mass spectrometry, Western blotting and flow cytometry to confirm the molecule and its association with both PD-L1 and CXCR4. The biological function of the nanobody and its anti-tumour effects were examined by an in vitro tumour cell-killing assay and in vivo tumour inhibition in mouse xenograft models. RESULTS: A novel anti-PD-L1/CXCR4 bispecific nanobody was designed, constructed and characterized. The molecule specifically bound to two targets on the surface of human cancer cells and inhibited CXCL12-induced Jurkat cell migration. The bispecific nanobody increased the level of IFN-γ secreted by T-cell activation. The cytotoxicity of human peripheral blood mononuclear cells (hPBMCs) against pancreatic cancer cells was enhanced by the molecule in combination with IL-2. In a human pancreatic cancer xenograft model, the anti-PD-L1/CXCR4 nanobody markedly inhibited tumour growth and was superior to the combo-treatment by anti-PD-L1 nanobody and anti-CXCR4 nanobody or treatment with atezolizumab as a positive control. Immunofluorescence and immunohistochemical staining of xenograft tumours showed that the anti-tumour effects were associated with the inhibition of angiogenesis and the infiltration of immune cells. CONCLUSION: These results clearly revealed that the anti-PD-L1/CXCR4 bispecific nanobody exerted anti-tumour efficacy in vitro and inhibited tumour growth in vivo. This agent can be further developed as a therapeutic reagent to treat human pancreatic cancer by simultaneously blocking two critical targets.

IL-1Ra protects hematopoietic cells from chemotoxicity through p53-induced quiescence.

The protection of constantly proliferating gut epithelia and hematopoietic tissues from cytotoxicity could improve conventional chemotherapy efficacy and widen its therapeutic window. Previously, we reported that, in mouse models, pretreatment of recombinant human IL-1 receptor antagonist (rhIL-1Ra) protected both types of vulnerable tissues from chemotherapeutics. Here, we showed that rhIL-1Ra treatment up-regulated the protein levels of phosphorylated p38, p53, and p21 and induced transient hematopoietic stem/progenitor cell (HS/PC) quiescence. Knockout of IL-1 receptor I (IL-1RI), p53, or p21 alleles and pharmacological inactivation of p38 mapped the rhIL-1Ra pathway in the induction of HS/PC quiescence. Therefore, rhIL-1Ra administration before but not after chemotherapy alleviated 5-fluorouracil-induced neutropenia. In addition, in vivo and in vitro cell proliferation assays revealed that the rhIL-1Ra treatment did not affect cancer cell proliferation or chemosensitivity. Lastly, we propose an IL-1/IL-1Ra pathway (IL-1RI → p38 → p53 → p21), which regulates HS/PC quiescence. The rhIL-1Ra may provide a new route for p53-based cyclotherapy, which spares normal cells but kills cancer cells during chemotherapy.-Ye, H., Qian, L., Zhu, S., Deng, S., Wang, X., Zhu, J., Chan, G. L., Yu, Y., Han, W. IL-1Ra protects hematopoietic cells from chemotoxicity through p53-induced quiescence.

Exogenous IL-1Ra attenuates intestinal mucositis induced by oxaliplatin and 5-fluorouracil through suppression of p53-dependent apoptosis.

Chemotherapy-induced intestinal mucositis (CIM) is a major dose-limiting side effect of many chemoagents, resulting in weight loss, diarrhea, and even death. The current treatments for CIM are palliative and have limited benefit. Interleukin-1 receptor antagonist is a natural antagonist of interleukin-1. Our previous studies showed the protective effect of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) on the intestine in mice after 5-fluorouracil chemotherapy. In this study, we further evaluated rhIL-1Ra in the treatment of CIM induced by different chemoagents and their combination. Normal as well as tumor-bearing mice were administered oxaliplatin (L-OHP), 5-fluorouracil, or their combination to induce intestinal mucositis and mortality. rhIL-1Ra administered after the chemotherapy, but not after the onset of diarrhea, significantly improved mouse survival, attenuated body weight loss, and reduced the incidence, severity, and duration of diarrhea. Histological examination showed that rhIL-1Ra-treated mice had a relatively intact mucosa structure, more proliferating crypt cells, and higher acid mucin content than the vehicle-treated mice. rhIL-1Ra suppressed crypt apoptosis by reducing the levels of proapoptotic proteins in wild-type, but not in IL-1RI or p53 mice. In addition, rhIL-1Ra was as effective as octreotide acetate in the treatment of chemotherapy-induced diarrhea, but with the advantage of reducing the epithelial apoptosis, the major cause of CIM. Importantly, the tumor sensitivity to chemotherapy was not affected by rhIL-1Ra. Thus, our data strongly suggest that rhIL-1Ra may be useful for the treatment of intestinal mucositis and improving the quality of life for cancer patients on chemotherapy.

Activated expression of the chemokine Mig after chemotherapy contributes to chemotherapy-induced bone marrow suppression and lethal toxicity.

Alterations in gene expression after chemotherapy may potentially help to identify mediators that induce suppression or regeneration in bone marrow. This paper reports our observation that the expression of the chemokine monokine induced by IFN-γ (Mig) and its receptor CXCR3 was significantly activated in mice after treatment with the chemotherapeutic agent 5-fluorouracil (5-FU). The neutralization of antibodies against the activated Mig increased the survival rate and accelerated BM recovery after chemotherapy. In addition, elevation of Mig plasma levels after 5-FU treatment corresponded with increased mortality. The cell cycle-inhibiting effect of the prophylactic administration of Mig protected hematopoietic progenitor cells (HPCs) from 1-ß-d-arabinofuranosylcytosine in spleen colony assays and enhanced the irradiated recipients' survival. In CXCR3(-/-) mice, Mig did not propagate BM suppression, indicating that the suppressive effect of Mig is dependent on CXCR3. On the one hand, Mig stimulated p70 S6K and Erk1/2 pathways in mesenchymal stroma cells, inhibiting mesenchymal stroma cell-dependent HPC expansion. Moreover, Mig suppressed the STAT5 pathway in HPCs, inhibiting leukocyte differentiation. Our results strongly suggest that Mig contributes to the acute lethal toxicity arising from 5-FU administration. Neutralization of Mig may offer new strategies to alleviate BM toxicity with potentially dramatic implications for chemotherapy.

IL-1Ra selectively protects intestinal crypt epithelial cells, but not tumor cells, from chemotoxicity via p53-mediated upregulation of p21(WAF1) and p27(KIP1.).

Chemotherapy-induced intestinal mucositis (CIM) is a major dose-limiting side effect, resulting from the nonspecific cytoablative actions of chemoagents, including 5-fluorouracil (5-FU) and irinotecan (CPT-11). Preventive strategies are urgently needed for the predictable CIM. Previously, we have demonstrated an important role of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) in the prevention of cyclophosphamide-induced mucositis in mice. In this study, the preventive role of rhIL-1Ra was further evaluated in 5-FU- and CPT-11-induced mucositis mouse models. rhIL-1Ra pretreatment reduced the incidence, severity, and duration of chemotherapy-induced diarrhea, through attenuating crypt apoptosis and improving crypt survival in wild-type mice, but not in IL-1RI(-/-), p53(-/-), and p21(-/-) mice. Further studies demonstrated that rhIL-1Ra promoted the cell cycle arrest of intestinal crypt epithelia (ICE) through elevating the cellular level of p21(WAF1) and p27(KIP1), which was abolished in IL-1RI(-/-) and p53(-/-) mice, and in p21(WAF1) and p27(KIP1) silenced IEC-6 cells. Importantly, the tumor growth and sensitivity to chemotherapy were not affected by rhIL-1Ra in cultures of tumor cell lines and in a syngeneic tumor-transplantation mouse model. The present study demonstrated that rhIL-1Ra effectively and specifically protected ICE from chemotoxicity through reversible reduction of the basal level of IL-1 signaling to promote normal cell cycle arrest, but not tumor cells. Our findings support the clinical development of rhIL-1Ra in the prevention of CIM.

The therapeutic effect of rhMK on osteoarthritis in mice, induced by destabilization of the medial meniscus.

Osteoarthritis (OA) is a worldwide disease in aged people, causing not only physical suffering to the patients themselves, but also a great burden on their families and on society. Here we used a mouse OA model induced by destabilization of the medial meniscus (DMM), and studied the therapeutic effect of recombinant human midkine (rhMK) on this OA model. Our results indicated that the DMM surgery induced mechanical allodynia and locomotor activity obstacles, together with cartilage injury in the C57BL/6 mice. The rhMK treatment mitigated the OA related mechanical allodynia, improved locomotor activity capacity, and prevented degradation of the cartilage. Considering the safety issue of rhMK used as a biologic, we also inspected the main organs in the rhMK treated mice throughout the process and found no pathological change. These results suggest that rhMK could be used as a biologic to treat OA or OA related pain.

Functional receptors and intracellular signal pathways of midkine (MK) and pleiotrophin (PTN).

Midkine (MK) and pleiotrophin (PTN) belong to the subfamily of heparin binding growth factors. They have ca. 50% structural homology, with similar C- and N-domains as well as comparable binding affinity to heparin, glycoproteins and proteoglycans. Both MK and PTN have diverse functions, such as mitogenicity, inflammation, angiogenesis, oncogenesis and stem cell self-renewal. The high expression of MK and PTN in many kinds of cancers makes them excellent as cancer biomarkers and targets for anticancer drug development. In addition, the important roles of MK and PTN in the regeneration of tissues, such as myocardium, cartilage, neuron, muscle, and bone, make them attractive candidates for the treatment of degenerative diseases such as myocardiac and cerebral infarction, Alzheimer's disease, Parkinson's disease and skeletal muscle injury. As a result, there has been a growing interest in the mechanisms of MK and PTN function, including the diverse receptors on the cell membrane and complex signal pathways in the cytoplasm. This work reviews the structures of MK and PTN, as well as the receptors and the intracellular signal pathways involving MK and PTN which will pave the way for future development of MK and PTN therapeutics.

Antifibrotic role of chemokine CXCL9 in experimental chronic pancreatitis induced by trinitrobenzene sulfonic acid in rats.

Chemokines have been shown to play an important role in the pathogenesis of pancreatitis, but the role of chemokine CXCL9 in pancreatitis is poorly understood. The aim of this study was to investigate whether CXCL9 was a modulating factor in chronic pancreatitis. Chronic pancreatitis was induced in Sprague-Dawley rats by intraductal infusion of trinitrobenzene sulfonic acid (TNBS) and CXCL9 expression was assessed by immunohistochemistry, Western blot analysis and enzyme linked immunosorbent assay (ELISA). Recombinant human CXCL9 protein (rCXCL9), neutralizing antibody and normal saline (NS) were administered to rats with chronic pancreatitis by subcutaneous injection. The severity of fibrosis was determined by measuring hydroxyproline in pancreatic tissues and histological grading. The effect of rCXCL9 on activated pancreatic stellate cells (PSCs) in vitro was examined and collagen 1α1, TGF-ß1 and CXCR3 expression was assessed by Western blot analysis in isolated rat PSCs. Chronic pancreatic injury in rats was induced after TNBS treatment and CXCL9 protein was markedly upregulated during TNBS-induced chronic pancreatitis. Although parenchymal injury in the pancreas was not obviously affected after rCXCL9 and neutralizing antibody administration, rCXCL9 could attenuate fibrogenesis in TNBS-induced chronic pancreatitis in vivo and exerted antifibrotic effects in vitro, suppressing collagen production in activated PSCs. In conclusion, CXCL9 is involved in the modulation of pancreatic fibrogenesis in TNBS-induced chronic pancreatitis in rats, and may be a therapeutic target in pancreatic fibrosis.

Traffic conflicts in the lane-switching sections at highway reconstruction zones.

INTRODUCTION: There are designated sections for lane-shifting in several highway reconstruction and expansion zones. Similar to the bottleneck sections of highways, these sections are characterized by poor pavement surface conditions, disorderly traffic flow, and high safety risk. This study examined the continuous track data of 1,297 vehicles collected using an area tracking radar. METHOD: The data from the lane shifting sections were analyzed in contrast with the regular section data. Further, the single-vehicle attributes, traffic flow factors, and the respective road characteristics in the lane-shifting sections were also taken into account. In addition, the Bayesian network model was established to analyze the uncertain interaction between the various other influencing factors. The K-Fold cross validation method was used to evaluate the model. RESULTS: The results showed that the model has a high reliability. The analysis of the model revealed that the significant influencing factors in decreasing order of their influence on the traffic conflict are: the curve radius, cumulative turning angle per unit length, standard deviation of the single-vehicle speed, vehicle type, average speed, and the standard deviation of the traffic flow speed. The probability of traffic conflicts is estimated to be 44.05% when large vehicles pass through the lane- shifting section while it is 30.85% for small vehicles. The probabilities of traffic conflict are 19.95%, 34.88%, and 54.79% when the turning angles per unit length are 0.20 °/m, 0.37 °/m, and 0.63 °/m, respectively. PRACTICAL APPLICATIONS: The results support the view that the highway authorities help reduce traffic risks on lane change sections by diverting large vehicles, implementing speed limits on road sections, and increasing the turning angle per unit length of vehicles.

Expression and purification of recombinant human Mig in Escherichia coli and its comparison with murine Mig.

Monokine induced by IFN-γ (Mig) is a member of CXC-chemokines and recruits T-lymphocytes to activate the immune response. In recent years, it has raised much interest in the areas of autoimmune disease and allograft rejection, as the production of recombinant human Mig (rHuMig) would be of considerable significance for both research and potential clinical use. Here we report the expression, preparation and characterization of non-tagged recombinant human Mig (rHuMig) using a prokaryotic expression system. Following expression in Escherichia coli (E. coli) BL21, the 103 amino acid residue of rHuMig was purified from bacteria inclusion bodies with a one-step S-Sepharose cation exchange chromatography. The product was immunologically characterized via Western blot and its purity was determined via SDS-PAGE and silver staining to be above 99%, with an endotoxin level <0.5EU/µg via a chemotaxis assay, rHuMig demonstrated chemotactic activity on mouse spleen lymphocytes with an ED50 of 15 ng/mL. Additionally, using a proliferation assay, rHuMig significantly inhibited proliferation of the human bladder cell line T24. In vivo experiments revealed that rHuMig could inhibit mouse bone marrow mononuclear cells cycling into the S-phase and reduced intestinal cell proliferation. Our results demonstrate that rHuMig is fully functional in the mouse model.

Reg4 protects against acinar cell necrosis in experimental pancreatitis.

Background and aims Reg4 is a recently discovered member of the regenerating gene family with distinctive expression profiles in primary cancers. To date, the physiological function of Reg4 is poorly understood. Previously, the authors found that Reg4 was markedly upregulated during acute pancreatitis (AP). The aim of this study was to investigate the role of Reg4 in experimental pancreatitis. Methods AP was induced in C57BL/6 mice by administration of either l-arginine or caerulein, and Reg4 expression was assessed by immunofluorescence, reverse transcriptase (RT)-PCR and western blot analyses. Recombinant human Reg4 protein (rReg4), heat-inactivated Reg4, neutralising antibody and vehicle were also administered to mice by subcutaneous injection. The severity of AP was determined by measuring amylase and lipase activities in the serum and histological grading. The effect of rReg4 on cell death was examined and epidermal growth factor receptor (EGFR), p-EGFR, Akt, p-Akt, Bcl-2 and Bcl-xL expression were assessed by western blot analysis of isolated murine acinar cells treated with l-arginine. Results Reg4 mRNA and protein were markedly upregulated during arginine-induced pancreatitis. Reg4 was widely expressed in residual acinar cells around the islets and regenerating metaplastic epithelium. rReg4 could protect against arginine-induced necrosis of acinar cells both in vivo and in vitro. This protective effect was also confirmed in the caerulein-induced murine model of AP. It was shown that arginine induced expression of Bcl-2 and Bcl-xL, while rReg4 upregulated Bcl-2 and Bcl-xL expression by activating the EGFR/Akt pathway. The upregulation of Bcl-xL correlated inversely with cell necrosis in isolated pancreatic acinar cells. Conclusions The data suggest that Reg4 may protect against acinar cell necrosis in experimental pancreatitis by enhancing the expression of Bcl-2 and Bcl-xL via activation of the EGFR/Akt signalling pathway.

Purification of a bioactive recombinant human Reg IV expressed in Escherichia coli.

Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily. Reg IV is highly expressed in the gastrointestinal tract and markedly up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel disease. However, the physiological and pathological functions of Reg IV are largely unknown, partly due to the limited access of the bioactive protein. We report here the first expression and purification of Reg IV proteins using a prokaryotic system. Human Reg IV was expressed in Escherichia coli as an insoluble protein which was identified in the fraction of inclusion body after ultrasonication of the bacteria. After the protein aggregate was solubilized by guanidine-HCl, it was refolded by sucrose and arginine-assisted procedures and purified using cation-exchange chromatography. The protein identity and purity of the final preparation were confirmed by analysis of the protein mass and immune specificity in SDS-PAGE, Western blotting, and HPLC assay. The biological activity of the protein was determined by the HCT116 and HT29 cell proliferation assays. The highly purified bioactive human Reg IV should aid in further characterization of its physiological and pathological functions.

Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli.

Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/microg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays.

Effects of peripheral transverse line markings on drivers' speed and headway choice and crash risk in car-following: A naturalistic observation study.

Rear-end crashes are closely related to car-following situation of vehicles. Speeding and insufficient headway are the major reasons as the drivers have not enough time to react to a sudden brake from the leading vehicle. Perceptual countermeasures, like speed reduction markings, are widely used in practice for accident prevention, and are verified with substantial effectiveness. However, compared with its practical application, the perceptual countermeasures are rarely analyzed in depth from the perspective of drivers' visual perception where the meaning of "perceptual" actually dwells. In addition, its effect on drivers' headway (distance) choice is almost ignored in previous research. Given this, the present study explored the effects of a certain type of perceptual treatment, i.e., the peripheral transverse line markings (PTLMs), on drivers' choice of speed and headway (distance) in car-following by a series of on-road experiments. In the on-road experiments, temporary line markings were installed on a real-world freeway in China to shape the PTLMs. The intersection angle (α) and the longitudinal spacing (λ) of the PTLMs were manipulated to attempt to associate the line markings with drivers' visual perception. Results of general and sectional relative differences of time headway (ηh, θh), speed (ηv, θv), and distance (ηd, θd) suggests that 1) the speed was reduced, the distance and time headway were increased significantly after the installation of PTLMs when compared with the original condition; 2) a larger intersection angle (α) and a smaller longitudinal spacing (λ) of PTLMs could lead to a greater variations in speed and headway (distance); in particular, the PTLMs in a form of α=150°, λ=2m resulted in 0.44 s increase in time headway, 1.33 m/s reduction in speed, and 4.07 m increase in distance in maximum; 3) the real-time crash risk variations under the influence of PTLMs were evaluated by two modified and extended surrogate safety indicators. The effects of PTLMs were discussed and explained considering the influences of optical illusion on lane width narrowing, edge rate on speed and "discontinuity effect" on distance, respectively. The findings of this study provide theoretical support for the perceptual countermeasures and suggest comparative advantages of PTLMs in dealing with rear-end crashes by intervening drivers' speed and headway choice.

Expression and purification of bioactive high-purity human midkine in Escherichia coli.

Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation. The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important growth factor. In the present work, rhMK was expressed in Escherichia coli (E. coli) BL21 (DE3). The expression of midkine was efficiently induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). After sonication, midkine was recovered in an insoluble form, and was dissolved in guanidine hydrochloride buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose ion-exchange chromatography. The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC). The purified rhMK enhanced the proliferation of NIH3T3 cells.

Structural equations modeling of real-time crash risk variation in car-following incorporating visual perceptual, vehicular, and roadway factors.

In this study, we attempted to explain drivers' crash risk variation in car-following for crash avoidance considering the effects of drivers' visual perception, vehicle type, and horizontal curves, with a structural equations model. The model was built by incorporating drivers' speed risk perception and distance risk perception as latent variables. A series of on-road experiments was conducted on the curved segments of a freeway in China to collect naturalistic driving data to approximate the model. The results indicate that (1) the amount of variance in speed risk perception accounted for by the temporal and spatial frequency and the following vehicle type was 21%; (2) the amount of variance in distance risk perception accounted for by the temporal and spatial frequency, leading vehicle type, stopping sight distance (SSD), horizontal sightline offset (HSO), and radius was 29%; and (3) speed risk perception and distance risk perception explained 27% of the total variance in crash risk variation, which is significantly higher than previous similar results that were commonly limited to 10%. The results were explained from the perspective of the effect of line markings, vehicle type (size), and curves on driving behaviors, respectively. In addition, the difference between the effect of speed risk perception and distance perception on crash risk variation was discussed considering the direct and indirect origins of risk in driving. The findings suggests that the incorporation of visual perceptual, vehicular, and roadway factors and its relevant speed risk perception and distance risk perception can better explain the crash risk in car-following. This study also emphasized the possibility and the need of applying the line markings as a visual intervention to prevent the drivers from rear-end crashes on curves, which may provide new insights and be a new solution for roadway safety.

Antagonist effect of Interleukin 1 receptor on normal thymopoiesis and thymus toxicity of 5-azacytidine in mouse.

Thymopoiesis is essential and significant for development and maintenance of the robust and healthy immune system. The acute suppression of thymopoiesis induced by 5-Azacytidine (5-Aza) is an intractable clinical problem complicating chemotherapy. Interleukin 1 receptor antagonist (IL-1Ra) is a cytokine that competitively blocks binding of interleukin 1 (IL-1) to its receptor. This study aims to investigate the effects of the IL-1Ra on the thymus toxicity of 5-Aza in mouse. In this study, we treated the mice with the 5-Aza (100 mg/kg per mouse). The GeneChip methodology developed by Affymetrix was used to monitor global gene expression during mouse thymus regeneration induced by a single injection of 5-Aza. The total thymocytes were counted using a hemocytometer. Cell cycle of samples were analyzed on a Becton Dickinson FACScan. Cells surfaces were labeled with anti-CD4, anti-CD8 and anti-CD45RA antibodies, and detected by flow cytometry. BrdU incorporation was detected by flow cytometry. The results indicated that administering exogenous IL-1Ra to normal mice inhibited cell cycle progress of thymocytes in a dosage-dependent manner. Proliferation of immature CD4(-)CD8(-) double negative (DN) and CD4(+)CD8(+) double positive (DP) thymocytes were both inhibited. The pretreatment of normal mice with exogenous IL-1Ra reduced acute toxicity on thymus and immune suppression induced by 5-Aza. Furthermore, thymus reconstitution after 5-Aza treatment was accelerated by IL-1Ra. In conclusion, interleukin 1 receptor antagonist could inhibit normal thymopoiesis and reduce thymus toxicity of 5-azacytidine in mouse. Pretreatment with IL-1Ra would offer a new and promising strategy to alleviate immunotoxicity of chemotherapy in clinical.

Investigation on road traffic safety of the mountain areas in southwest China based on Wulong County, Chongqing Municipality.

OBJECTIVE: To study the present situation, characteristics, and main problems of the traffic safety in the mountain areas of Southwest China. METHODS: A study was done on the traffic status of Wulong County, Chongqing Municipality, by means of document review, field investigation, and inquiry. RESULTS: The traffic crashes and deaths in Wulong County were increasing since 1999. From 1997 to 2001, there occurred 126 severe and very severe traffic crashes resulting in 175 deaths and 411 serious injuries, which accounted for 9.4% of total crashes, 96.1% of total deaths, and 48.4% of total serious injuries, respectively. The severe and very severe traffic crashes were highest in 2000. One part for 83 km in length accounting for 3.0% of No. 319 National Highway was responsible for 54.5% of very severe traffic crashes, which closely related to conditions of the road itself and its environment and defective execution of traffic laws. CONCLUSIONS: Wulong County, one of the mountain counties in Southwest China, is now facing a complex and serious road traffic situation and serious traffic safety, with a high incidence of severe and very severe road traffic crashes. The crashes closely relate to imperfect road design, defective execution of traffic laws, imperfect management, and environmental factors.

Clickable analogue of cerulenin as chemical probe to explore protein palmitoylation.

Dynamic palmitoylation is an important post-translational modification regulating protein localization, trafficking, and signaling activities. The Asp-His-His-Cys (DHHC) domain containing enzymes are evolutionarily conserved palmitoyl acyltransferases (PATs) mediating diverse protein S-palmitoylation. Cerulenin is a natural product inhibitor of fatty acid biosynthesis and protein palmitoylation, through irreversible alkylation of the cysteine residues in the enzymes. Here, we report the synthesis and characterization of a "clickable" and long alkyl chain analogue of cerulenin as a chemical probe to investigate its cellular targets and to label and profile PATs in vitro and in live cells. Our results showed that the probe could stably label the DHHC-family PATs and enable mass spectrometry studies of PATs and other target proteins in the cellular proteome. Such probe provides a new chemical tool to dissect the functions of palmitoylating enzymes in cell signaling and diseases and reveals new cellular targets of the natural product cerulenin.

Generation and Characterization of a New Monoclonal Antibody Against CXCL4.

CXCL4 plays important roles in numerous disease processes, which makes the CXCL4 signaling pathway a potential therapeutic target. In this study, we aimed to develop a neutralizing antibody against both human and mouse CXCL4. Rats were immunized with recombinant human CXCL4 (rhCXCL4). Hybridoma clones were created by fusion of the immunized rat spleen cells with mouse myeloma SP2/0 cells and screened using recombinant mouse CXCL4 (rmCXCL4) and rhCXCL4. The CXCL4 monoclonal antibody (CXCL4 MAb) produced by the 16D6-3 hybridoma clone was sequenced and characterized by Western blot and Biacore assays. It recognized both human and mouse CXCL4 with high affinity and neutralized the effect of rhCXCL4 in vitro. Thus, the antibody may be used in the studies of CXCL4 in murine disease models and as a template in the antibody humanization for clinical developments.