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.

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.

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.

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.

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.

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.

CXCL4 mediates tumor regrowth after chemotherapy by suppression of antitumor immunity.

The recurrence of colorectal cancer after chemotherapy is the leading cause of its high mortality. We propose that elucidating the mechanisms of tumor regrowth after chemotherapy in tumor-bearing mice may provide new insights into tumor relapse in cancer patients. We firstly report the identification of a chemokine, CXCL4, that plays an important role in the molecular mechanism of cancer regrowth after chemotherapy. A syngenic transplantation tumor model was established with murine colon cancer CT26 cells and treated with 5-FU. Genome-wide gene expression analysis determined that CXCL4 was transiently upregulated in the tumor model. Systemic overexpression of CXCL4 accelerated cancer growth in vivo, but not in vitro. Conversely, the anti-CXCL4 monoclonal antibody (CXCL4-mab) retarded tumor-regrowth after 5-FU treatment in immune-competent mice, but not nude mice. The CXCL4-mab treatment increased the local expression levels of IFN-γ and Gran-b genes in the tumor-bed, and elevated the function of CTLs against CT26 cells. Thus, the colon cancer cells in responding to the cytotoxic stress of 5-FU produce a high level of CXCL4, which suppresses antitumor immunity to confer the residual cancer cells an advantage for regrowth after chemotherapy. Our findings provide a novel target for developing therapeutics aiming to increase antitumor immunity after chemotherapy.

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.

Recombinant interleukin-1 receptor antagonist attenuates the severity of chronic pancreatitis induced by TNBS in rats.

Chronic pancreatitis (CP) is a common disease in the department of gastroenterology, with the main symptoms of exocrine and/or endocrine insufficiency and abdominal pain. The pathogenic mechanism of CP is still not fully clarified and the aims of treatment now are to relieve symptoms. In this study, we attempted to find a connection between interleukin-1ß (IL-1ß) and interleukin-1 receptor antagonist (IL-1Ra) in trinitrobenzene sulfonic acid (TNBS)-induced chronic pancreatitis, and then the therapeutic effect of recombinant IL-1Ra was also detected in the CP model. Chronic pancreatitis was induced by intraductal infusion of TNBS in SD rats followed by a consecutive administration of rIL-1Ra, and the histological changes and collagen content in the pancreas were measured, as well as the abdominal hypersensitivity. We found that rhIL-1Ra could attenuate the severity of chronic pancreatic injury, modulate the extracellular matrix secretion, focal proliferation and apoptosis, and cellular immunity in TNBS-induced CP. Interestingly, rIL-1Ra could also block the pancreatitis-induced referred abdominal hypersensitivity. In conclusion, IL-1Ra may play a protective role in CP and rIL-1Ra would be a potential therapeutic target for the treatment of CP, while its possible mechanisms and clinical usage still need further investigation.

The chemokine CXCL9 exacerbates chemotherapy-induced acute intestinal damage through inhibition of mucosal restitution.

PURPOSE: Acute intestinal damage induced by chemotherapeutic agent is often a dose-limiting factor in clinical cancer therapy. The aim of this study was to investigate the effect of chemokine CXCL9 on the intestinal damage after chemotherapy and explore the therapeutic potential of anti-CXCL9 agents. METHODS: In vitro cell proliferation assay was performed with a non-tumorigenic human epithelial cell line MCF10A. Multiple pathway analysis was carried out to explore the pathway that mediated the effect of CXCL9, and the corresponding downstream effector was identified with enzyme-linked immunosorbent assays. Chemotherapy-induced mouse model of intestinal mucositis was prepared by a single injection of the chemotherapeutic agent 5-fluorouracil (5-FU). In vivo expression of cxcl9 and its receptor cxcr3 in intestinal mucosa after chemotherapy was determined by quantitative real-time PCR. Therapeutic treatment with anti-CXCL9 antibodies was investigated to confirm the hypothesis that CXCL9 can contribute to the intestinal epithelium damage induced by chemotherapy. RESULTS: CXCL9 inhibited the proliferation of MCF10A cells by activating phosphorylation of p70 ribosomal S6 kinase (p70S6K), which further promotes the secretion of transforming growth factor beta (TGF-ß) as the downstream effector. A blockade of phospho-p70S6K with inhibitor abolished the effect of CXCL9 on MCF10A cells and reduced the secretion of TGF-ß. The expression levels of cxcl9 and cxcr3 were significantly up-regulated in intestinal mucosa after 5-FU injection. Neutralizing elevated CXCL9 with anti-CXCR9 antibodies successfully enhanced reconstitution of intestinal mucosa and improved the survival rate of mice that received high-dose chemotherapy. CONCLUSIONS: CXCL9 inhibits the proliferation of epithelial cells via phosphorylation of p70S6K, resulting in the excretion of TGF-ß as downstream mediator. CXCL9/CXCR3 interaction can exacerbate chemotherapeutic agent-induced intestinal damage, and anti-CXCL9 agents are potential novel therapeutic candidates for promoting mucosal restitution.

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.

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.

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.

Energy stress regulates hippo-YAP signaling involving AMPK-mediated regulation of angiomotin-like 1 protein.

Hippo signaling is a tumor-suppressor pathway involved in organ size control and tumorigenesis through the inhibition of YAP and TAZ. Here, we show that energy stress induces YAP cytoplasmic retention and S127 phosphorylation and inhibits YAP transcriptional activity and YAP-dependent transformation. These effects require the central metabolic sensor AMP-activated protein kinase (AMPK) and the upstream Hippo pathway components Lats1/Lats2 and angiomotin-like 1 (AMOTL1). Furthermore, we show that AMPK directly phosphorylates S793 of AMOTL1. AMPK activation stabilizes and increases AMOTL1 steady-state protein levels, contributing to YAP inhibition. The phosphorylation-deficient S793Ala mutant of AMOTL1 showed a shorter half-life and conferred resistance to energy-stress-induced YAP inhibition. Our findings link energy sensing to the Hippo-YAP pathway and suggest that YAP may integrate spatial (contact inhibition), mechanical, and metabolic signals to control cellular proliferation and survival.

Activation of p38-MAPK by CXCL4/CXCR3 axis contributes to p53-dependent intestinal apoptosis initiated by 5-fluorouracil.

Chemotherapy-induced mucositis (CIM) is a major does limiting side-effect of chemoagents such as 5-fluorouracil (5-FU). Molecules involved in this disease process are still not fully understood. We proposed that the homeostatically regulated genes during CIM may participate in the disease. A cluster of such genes were previously identified by expression gene-array from the mouse jejunum in 5-FU-induced mucositis model. Here, we report that CXCL4 is such a homeostatically regulated gene and serves as a new target for the antibody treatment of CIM. CXCL4 and its receptor CXCR3 were confirmed at both the gene and protein levels to be homeostatically regulated during 5-FU-induced mucositis. Using of CXCL4 neutralizing monoclonal antibody (CXCL4mab) decreased the incidence, severity, and duration of the chemotherapy-induced diarrhea, the major symptom of CIM, in a 5-FU mouse CIM model. Mechanistically, CXCL4mab reduced the apoptosis of the crypt epithelia by suppression of the 5-FU-induced expression of p53 and Bax through its receptor CXCR3. The downstream signaling pathway of CXCL4 in activation of the epithelial apoptosis was identified in an intestinal epithelial cell line (IEC-6). CXCL4 activated the phosphorylation of p38 MAPK, which mediated the stimulated expression of p53 and Bax, and resulted in the ultimate activation of Caspase-8, -9, and -3. Taken together, activation of CXCL4 expression by 5-FU in mice participates in 5-FU-induced intestinal mucositis through upregulation of p53 via activation of p38-MAPK, and CXCL4mab is potentially beneficial in preventing CIM in the intestinal tract.

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.

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.

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.

Recombinant human interleukin-1 receptor antagonist reduces acute lethal toxicity and protects hematopoiesis from chemotoxicity in vivo.

Cyclophosphamide (CY), targeting to fast dividing cells, results in bone marrow (BM) suppression, which is the most common side effect of cancer chemotherapy. Interleukin-1 receptor antagonist (IL-1Ra), activated by variety of chemotherapeutic drugs, is a natural inhibitor of interleukin-1 (IL-1) and blocks the functional IL-1 receptor signaling. Our previous studies showed the protective effect of recombinant murine IL-1Ra on hematopoiesis in mice after treatment with chemotherapeutic agent 5-fluorouracil. In this report, we demonstrate that the pretreatment use of recombinant human IL-1Ra (rhIL-1Ra) significantly alleviated chemotherapy-induced peripheral blood injury in mice, and reduced the incidence and severity of neutropenia in beagle dogs. Moreover, acute lethal toxicity in single and repeated CY treatment was markedly reduced by rhIL-1Ra administration. The chemoprotective role of rhIL-1Ra is attributed to the attenuated BM damage, accelerated recovery of BM cells, and enhanced survival of hematopoietic progenitor cells which expressed high level of aldehyde dehydrogenase and IL-1 receptor type I. Thus, our data strongly suggest that the prophylactic use of exogenous rhIL-1Ra renders BM primitive hematopoietic cells resistant to chemotherapy, which provides novel strategies to prevent BM suppression in clinical settings.

Characterization of activin A in the culture of primitive human umbilical cord blood hematopoietic cells.

Transforming growth factor-ß (TGF-ß) superfamily controls many physiological processes such as cell proliferation and differentiation, immune responses, wound repair and various endocrine activities. As a member of TGF-ß, activin A can maintain the pluripotency of embryonic stem cells. We report here that activin A exhibited cell type-dependent function of expanding the human primitive hematopoietic cells isolated from umbilical cord blood (UCB). However, the multipotency of the cells pretreated with activin A was exhausted in the sequential dilution culture. In conclusion, activin A may not be a key factor, but a regulator, in the multipotency maintenance of primitive hematopoietic cells and the application of activin A in the hematopoietic stem/progenitor cells (HS/PCs) culture expansion remains a significant challenge.