Deficiency of leukocyte-specific protein 1 (LSP1) alleviates asthmatic inflammation in a mouse model.

BACKGROUND: Asthma is a major cause of morbidity and mortality in humans. The mechanisms of asthma are still not fully understood. Leukocyte-specific protein-1 (LSP-1) regulates neutrophil migration during acute lung inflammation. However, its role in asthma remains unknown. METHODS: An OVA-induced mouse asthma model in LSP1-deficient (Lsp1-/-) and wild-type (WT) 129/SvJ mice were used to test the hypothesis that the absence of LSP1 would inhibit airway hyperresponsiveness and lung inflammation. RESULTS: Light and electron microscopic immunocytochemistry and Western blotting showed that, compared with normal healthy lungs, the levels of LSP1 were increased in lungs of OVA-asthmatic mice. Compared to Lsp1-/- OVA mice, WT OVA mice had higher levels of leukocytes in broncho-alveolar lavage fluid and in the lung tissues (P < 0.05). The levels of OVA-specific IgE but not IgA and IgG1 in the serum of WT OVA mice was higher than that of Lsp1-/- OVA mice (P < 0.05). Deficiency of LSP1 significantly reduced the levels of IL-4, IL-5, IL-6, IL-13, and CXCL1 (P < 0.05) but not total proteins in broncho-alveolar lavage fluid in asthmatic mice. The airway hyper-responsiveness to methacholine in Lsp1-/- OVA mice was improved compared to WT OVA mice (P < 0.05). Histology revealed more inflammation (inflammatory cells, and airway and blood vessel wall thickening) in the lungs of WT OVA mice than in those of Lsp1-/- OVA mice. Finally, immunohistology showed localization of LSP1 protein in normal and asthmatic human lungs especially associated with the vascular endothelium and neutrophils. CONCLUSION: These data show that LSP1 deficiency reduces airway hyper-responsiveness and lung inflammation, including leukocyte recruitment and cytokine expression, in a mouse model of asthma.

Atopy risk among school-aged children in relation to early exposures to a farm environment: A systematic review.

BACKGROUND AND OBJECTIVES: Childhood atopy is a complex condition with both a genetic and an environmental component. This systematic review will explore the current understanding of the importance of early life exposures to a farm in the development of atopy measured by objective markers of skin prick testing, and specific IgE measurements in school age children. METHODS: A systematic review was performed. RESULTS: Among 7285 references identified, 14 studies met the inclusion criteria (13 cross-sectional studies and 1 case-control study). The results were fairly consistent in that early farm-related exposures can protect children from becoming atopic at school age. In general, there was heterogeneity in the assessment of outcomes and exposures. CONCLUSIONS: Early-life farm exposures are associated with a protective effect on childhood atopy as assessed by objective markers. Future work should focus on understanding specific farm exposures that may important in these associations between atopy and farm exposures in children.

CD40 signaling augments IL-10 expression and the tolerogenicity of IL-10-induced regulatory dendritic cells.

CD40 expressed on stimulatory dendritic cells (DC) provides an important accessory signal for induction of effector T cell responses. It is also expressed at lower levels on regulatory DC (DCreg), but there is little evidence that CD40 signaling contributes to the tolerogenic activity of these cells. Indeed, CD40 silencing within DCreg has been reported to induce T cell tolerance in multiple disease models, suggesting that CD40 is superfluous to DC-induced tolerance. We critically assessed whether CD40 does have a role in tolerance induced by IL-10-differentiated DC (DC10) by using DC10 generating from the bone marrow of wild-type (w.t.) or CD40-/- donor mice, or IL-10-complemented CD40-/- DC10 to treat asthmatic mice. Wild-type DC10 ablated the OVA-asthma phenotype via induction of Foxp3+ Treg responses, but CD40-/- DC10 had no discernible effects on primary facets of the phenotype (e.g., IL-5, IL-9, IL-13 levels, IgE & IgG1 antibodies; p>0.05) and were ≤40% effective in reversal of others. Foxp3+ T cells from the lungs of CD40-/- DC10-treated mice expressed reduced levels of a panel of six Treg-specific activation markers relative to Treg from w.t. DC10-treated mice. Coculture with effector T cells from asthmatic mice induced a marked upregulation of cell surface CD40 on w.t. DC10. While untreated CD40-/- and w.t. DC10 secreted equally low levels of IL-10, stimulation of w.t. DC10 with anti-CD40 for 72 h increased their expression of IL-10 by ≈250%, with no parallel induction of IL-12. Complementing IL-10 expression in CD40-/- DC10 by IL-10 mRNA transfection fully restored the cells' abilities to suppress the asthma phenotype. In summary, CD40 signaling in DC10 contributes importantly to their expression of IL-10 and to a robust induction of tolerance, including activation of induced Treg.

Regulatory Dendritic Cells, T Cell Tolerance, and Dendritic Cell Therapy for Immunologic Disease.

Dendritic cells (DC) are antigen-presenting cells that can communicate with T cells both directly and indirectly, regulating our adaptive immune responses against environmental and self-antigens. Under some microenvironmental conditions DC develop into anti-inflammatory cells which can induce immunologic tolerance. A substantial body of literature has confirmed that in such settings regulatory DC (DCreg) induce T cell tolerance by suppression of effector T cells as well as by induction of regulatory T cells (Treg). Many in vitro studies have been undertaken with human DCreg which, as a surrogate marker of antigen-specific tolerogenic potential, only poorly activate allogeneic T cell responses. Fewer studies have addressed the abilities of, or mechanisms by which these human DCreg suppress autologous effector T cell responses and induce infectious tolerance-promoting Treg responses. Moreover, the agents and properties that render DC as tolerogenic are many and varied, as are the cells' relative regulatory activities and mechanisms of action. Herein we review the most current human and, where gaps exist, murine DCreg literature that addresses the cellular and molecular biology of these cells. We also address the clinical relevance of human DCreg, highlighting the outcomes of pre-clinical mouse and non-human primate studies and early phase clinical trials that have been undertaken, as well as the impact of innate immune receptors and symbiotic microbial signaling on the immunobiology of DCreg.

Soluble Low-density Lipoprotein Receptor-related Protein 1 in Juvenile Idiopathic Arthritis.

OBJECTIVES: This study aimed to expand knowledge about soluble low-density lipoprotein receptor-related protein 1 (sLRP1) in juvenile idiopathic arthritis (JIA) by determining associations of sLRP1 levels in nonsystemic JIA patients with clinical and inflammatory biomarker indicators of disease activity. METHODS: Plasma sLRP1 and 44 inflammation-related biomarkers were measured at enrollment and 6 months later in a cohort of 96 newly diagnosed Canadian patients with nonsystemic JIA. Relationships between sLRP1 levels and indicators of disease activity and biomarker levels were analyzed at both visits. RESULTS: At enrollment, sLRP1 levels correlated negatively with age and active joint counts. Children showed significantly higher levels of sLRP1 than adolescents (mean ranks: 55.4 and 41.9, respectively; P = 0.02). Participants with 4 or fewer active joints, compared to those with 5 or more active joints, had significantly higher sLRP1 levels (mean ranks: 56.2 and 40.7, respectively; P = 0.006). At enrollment, considering the entire cohort, sLRP1 correlated negatively with the number of active joints (r = -0.235, P = 0.017). In the entire cohort, sLRP1 levels at enrollment and 6 months later correlated with 13 and 6 pro- and antiinflammatory biomarkers, respectively. In JIA categories, sLRP1 correlations with inflammatory markers were significant in rheumatoid factor-negative polyarticular JIA, oligoarticular JIA, enthesitis-related arthritis, and psoriatic arthritis at enrollment. Higher sLRP1 levels at enrollment increased the likelihood of absence of active joints 6 months later. CONCLUSION: Plasma sLRP1 levels correlate with clinical and biomarker indicators of short-term improvement in JIA disease activity, supporting sLRP1 as an upstream biomarker of potential utility for assessing JIA disease activity and outcome prediction.

Prospective Analysis of the Effects of Maternal Immune Activation on Rat Cytokines during Pregnancy and Behavior of the Male Offspring Relevant to Schizophrenia.

Influenza during pregnancy is associated with the development of psychopathology in the offspring. We sought to determine whether maternal cytokines produced following administration of viral mimetic polyinosinic-polycytidylic acid (polyI:C) to pregnant rats were predictive of behavioral abnormalities in the adult offspring. Timed-pregnant Sprague Dawley rats received a single intravenous injection of 4-mg/kg polyI:C or saline on gestational day (GD)15. Blood was collected 3 h later for serum analysis of cytokine levels with ELISA. Male offspring were tested in a battery of behavioral tests during adulthood and behavior was correlated with maternal cytokine levels. Maternal serum levels of CXCL1 and interleukin (IL)-6, but not tumor necrosis factor (TNF)-α or CXCL2, were elevated in polyI:C-treated dams. PolyI:C-treated dams experienced post-treatment weight loss and polyI:C pups were smaller than controls at postnatal day (PND)1. Various behavior alterations were seen in the polyI:C-treated offspring. Male polyI:C offspring had enhanced MK-801-induced locomotion, and reduced sociability. PolyI:C offspring failed to display crossmodal and visual memory, and oddity preference was also impaired. Set-shifting, assessed with a lever-based operant conditioning task, was facilitated while touchscreen-based reversal learning was impaired. Correlations were found between maternal serum concentrations of CXCL1, acute maternal temperature and body weight changes, neonatal pup mass, and odd object discrimination and social behavior. Overall, while the offspring of polyI:C-treated rats displayed behavior abnormalities, maternal serum cytokines were not related to the long-term behavior changes in the offspring. Maternal sickness effects and neonatal pup size may be better indicators of later effects of maternal inflammation in the offspring.

CXCR1/CXCR2 antagonist G31P inhibits nephritis in a mouse model of uric acid nephropathy.

The prevalence of gout is relatively high worldwide, and many gout patients suffer from uric acid nephropathy (UAN) concomitantly. ELR-CXC chemokines such as CXCL8 and CXCL1 have a elevated expression in UAN. In this research, a mouse UAN model was established for a 12 week duration, and uric acid-related crystals were observed. CXCL8(3-72)K11R/G31P (G31P) is a mutant protein of CXCL8/interleukin 8 (IL-8), which has been reported to have therapeutic efficacy in both inflammatory diseases and malignancies for it acts as a selective antagonist towards CXCR1/CXCR2. In this study, G31P-treated mice showed declined production of the blood urea nitrogen (BUN) level and urine volume in UAN mice compared with G31P-untreated UAN counterparts. In addition, G31P effectively improved renal fibrosis, and reduced uric acid accumulation and leukocyte infiltration in UAN kidneys. Furthermore, the expressions of CXCL1 and CXCL2 were reduced and the activation of NOD-like receptors protein 3 (NLRP3) was inhibited by G31P treatment. This study has demonstrated that G31P attenuates inflammatory progression in chronic UAN, and plays a renoprotective function.

IL-8 analogue CXCL8 (3-72) K11R/G31P, modulates LPS-induced inflammation via AKT1-NF-kß and ERK1/2-AP-1 pathways in THP-1 monocytes.

IL-8 is elevated during inflammation, and it initiates cascade of down-stream reactions. Its antagonist, CXCL8 (3-72) K11R/G31P (G31P), represses inflammatory reactions via competitive binding to CXC chemokine family, preferentially G protein-couple receptors (GPCRs) CXCR1/2. This study reports the effect of G31P on the transcription profile of lipopolysaccharide (LPS) induced inflammation in THP-1 monocytes ex-vivo. LPS (1 µg/ml) induced elevation of IL-8 was significantly reduced by G31P (20 µg/ml and 30 µg/ml), with relatively increased inhibition of CXCR2 than CXCR1. Transcription of IL-1ß, IL-6, and TNF-α were significantly inhibited, while IL-10 remained relatively unchanged. G31P treatment also had repressing effect on the inflammatory associated enzymes COX-2, MMP-2, and MMP-9. Significant restriction of c-Fos, and NF-kß mRNA expression was observed, while that of c-Jun was marginally elevated. Conversely, SP-1 mRNA expression was seen to increase appreciably by G31P treatment. While the translation of pAKT, pERK1/2, and p65- NF-kß were down-regulated by the G31P following THP-1 cells stimulation with LPS, reactive oxygen species (ROS) expression was on the positive trajectory. Collectively, the IL-8 analogue, G31P, modulates the inflammatory profile of LPS induced inflammation in THP-1 monocytes via AKT1-NF-kß and ERK1/2-AP-1 pathways.

Cytotoxic effect of interleukin-8 in retinal ganglion cells and its possible mechanisms.

AIM: To investigate the effect of interleukin-8 (IL-8) on neural retinal ganglion cells (RGCs) and whether it can be alleviated by G31P. METHODS: RGC-5 cells were exposed to IL-8 with or without its specific receptor antagonist G31P for 24h, and the cell viability was assessed by Cell Counting Kit 8 (CCK-8). Apoptosis was measured by examining nuclear morphology and quantifying with flow cytometry. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot were used to investigate the expression of apoptosis-related genes. RESULTS: CCK-8 assay showed that IL-8 significantly inhibits the viability of RGC-5 cells in a dose-dependent manner. Cell apoptosis assays exhibited higher apoptotic rate in IL-8 treatment group compared to control group. We further found that IL-8 could promote Bax and caspase-3 expressions, but decrease the level of Bcl-2 in the aspect of mRNA and protein. However, pre-treatment with G31P partly attenuated these effects in RGC-5 cells (P<0.05). CONCLUSION: These results indicate that anti-proliferation effects of IL-8 through induction of cell apoptosis regulated by Bcl-2, Bax and caspase-3 expressions, can be ameliorated by G31P.

IL-8 antagonist, CXCL8(3-72)K11R/G31P coupled with probiotic exhibit variably enhanced therapeutic potential in ameliorating ulcerative colitis.

Inflammatory bowel disease (IBD) remains a major health challenge due in part to unsafe and limited treatment options, hence there is the need for alternatives. CXCL8/interleukin 8 (IL-8) is elevated in inflammation, and binds preferentially to G protein-couple receptors (GPCRs) CXCR1/2 of the CXC chemokine family to initiate cascades of downstream inflammatory signals. A mutant CXCL8 protein, CXCL8(3-72)K11R/G31P (G31P), competitively and selectively binds to CXCR1/2, making CXCL8 redundant. We explore the therapeutic potential of G31P in dextran sulfate sodium (DSS) induced ulcerative colitis (UC), and the corresponding effect if G31P treatment is augmented with Lactobacillus acidophilus (LACT). The treatment options administered significantly reduced TNF-α, IFN-γ, IL-1ß, IL-6, and IL-8, but maintained elevated levels of IL-10. CD68 and F4/80 expressions were down-regulated and showed restricted infiltration to inflamed colon, while IL-17F levels were insignificantly different from the DSS treated mice. Also, we observed up-regulation of IL-17A in G31P + LACT but not G31P treated mice if compared with Control group. The treatments ameliorated colonic fibrosis by reducing VEGF, TGF-ß, MMP-2 and MMP-9. In addition, we observed elevated levels of E-cadherin, and marginal up-regulation of occludin, suggesting the role of the treatments in regulating tight intestinal junction and adherence proteins. Mechanism-wise, G31P interferes with AKT and ERK signaling pathways. Our study suggests that G31P confers protection in IBD, particularly UC, and when G31P treatment is augmented with Lactobacillus acidophilus, the protection is variably enhanced.

Contributions of direct versus indirect mechanisms for regulatory dendritic cell suppression of asthmatic allergen-specific IgG1 antibody responses.

IL-10-differentiated dendritic cells (DC10) can reverse the asthma phenotype in mice, but how they suppress the asthmatic B cell response is unclear. Herein we assessed the mechanism(s) by which DC10 and DC10-induced Treg affect IgG1 production in asthma. We observed a rapid decline in lung-resident OVA-specific IgG1-secreting B cells on cessation of airway allergen challenge, and intraperitoneal DC10 therapy did not amplify that (p>0.05). It did however increase the loss of IgG1-B cells from the bone marrow (by 45+/-7.2%; p≤0.01) and spleen (by 65+/-17.8%; p≤0.05) over 2 wk. Delivery of OVA-loaded DC10 directly into the airways of asthmatic mice decreased the lung IgG1 B cell response assessed 2 dy later by 33+/-9.7% (p≤0.01), while their co-culture with asthmatic lung cell suspensions reduced the numbers of IgG1-secreting cells by 56.5+/-9.7% (p≤0.01). This effect was dependent on the DC10 carrying intact allergen on their cell surface; DC10 that had phagocytosed and fully processed their allergen were unable to suppress B cell responses, although they did suppress asthmatic Th2 cell responses. We had shown that therapeutic delivery of DC10-induced Treg can effectively suppress asthmatic T and B cell (IgE and IgG1) responses; herein CD4+ cells or Treg from the lungs of DC10-treated OVA-asthmatic mice suppressed in vitro B cell IgG1 production by 52.2+/-8.7% (p≤0.001) or 44.6+/-12.2% (p≤0.05), respectively, but delivery of DC10-induced Treg directly into the airways of asthmatic mice had no discernible impact over 2 dy on the numbers of lung IgG1-secreting cells (p≥0.05). In summary, DC10 treatment down-regulates OVA-specific B cell responses of asthmatic mice. While DC10 that carry intact allergen on their cell surface can dampen this response, DC10-induced Treg are critical for full realization of this outcome. This suggests that infectious tolerance is an essential element in regulatory DC control of the B cell response in allergic asthma.

Metformin inhibits the development, and promotes the resensitization, of treatment-resistant breast cancer.

Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original treatment, and provide evidence that metformin may function through a mechanism involving post-translational histone modifications via an indirect histone deacetylase inhibitor (HDACi) activity. We find that metformin, at low physiological concentrations, reduces the expression of multiple classic protein markers of MDR in vitro and in preliminary in vivo models. Our demonstration that metformin can prevent MDR development and resensitize MDR cells to chemotherapy in vitro, provides important medical relevance towards metformin's potential clinical use against MDR cancers.

Effects of K11R and G31P Mutations on the Structure and Biological Activities of CXCL8: Solution Structure of Human CXCL8(3-72)K11R/G31P.

The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), the expression levels of which are elevated in many inflammatory diseases, binds to both the CXCR1 and CXCR2 receptors with high affinity. Recently, an analogue of human CXCL8, CXCL8(3-72)K11R/G31P (hG31P) has been developed. It has been demonstrated that hG31P is a high affinity antagonist for both the CXCR1 and CXCR2. Herein, we have determined the solution structure and the CXCR1 N-terminal peptide binding sites of hG31P by NMR spectroscopy. We have found that the displacement within the tertiary structure of the 30 s loop and the N-terminal region and more specifically change of the loop conformation (especially H33), of hG31P may affect its binding to the CXCR1 receptor and thereby inhibit human neutrophil chemotactic responses induced by ELR-CXC chemokines. Our results provide a structural basis for future clinical investigations of this CXCR1/CXCR2 receptor antagonist and for the further development of CXCL8 based antagonists.

CXCL8 Antagonist Improves Diabetic Nephropathy in Male Mice With Diabetes and Attenuates High Glucose-Induced Mesangial Injury.

Inflammation is recognized as a crucial contribution to diabetic nephropathy (DN). CXCL8 binds to its CXC chemokine receptors (CXCR1 and CXCR2) for recruiting neutrophil infiltration and initiates tissue inflammation. Therefore, we explored the effect of CXCR1 and CXCR2 inhibition on DN. This was achieved by CXCL8(3-72)K11R/G31P (G31P), an antagonist of CXCL8 that has exhibited therapeutic efficacy in inflammatory diseases and malignancies. In this study, we found that renal leukocyte accumulation and rapid increases of CXCL8 occurred in high-fat diet/streptozocin-induced diabetic mice. G31P effectively reduced urine volume, urine albumin/creatinine ratio, blood urea nitrogen, and creatinine clearance rate in mice with diabetes. In addition, renal histopathologic changes including mesangial expansion, glomerulosclerosis, and extracellular matrix deposition were partially moderated in G31P-treated diabetic mice. Furthermore, G31P attenuated renal inflammation and renal fibrosis of diabetic mice by inhibiting proinflammatory and profibrotic elements. G31P also inhibited high glucose-induced inflammatory and fibrotic factor upregulation in human renal mesangial cells. At the molecular level, G31P inhibited activation of CXCR1/2 downstream signaling JAK2/STAT3 and ERK1/2 pathways in in vitro and in vivo experiments. Our results suggest blockade of CXCR1/2 by G31P could confer renoprotective effects that offer potential therapeutic opportunities in DN.

Therapeutic reversal of food allergen sensitivity by mature retinoic acid-differentiated dendritic cell induction of LAG3+CD49b-Foxp3- regulatory T cells.

BACKGROUND: Anaphylaxis is a life-threatening condition for which we have limited therapeutic options. Although specific immunotherapy for food allergies is becoming more effective, it is still laborious and carries substantial risk of adverse events. On the other hand, regulatory dendritic cell (DC) therapy is effective in mouse models of allergic disease and has been shown to work with TH2 cells from atopic asthmatic patients. OBJECTIVE: We assessed whether DC immunotherapy could reverse food allergen sensitivity in mouse models to provide proof of concept relating to their use in the clinic. METHODS: We generated and characterized mature retinoic acid-skewed dendritic cells (DC-RAs) and assessed their abilities to reverse ovalbumin or peanut allergies in mouse models, as well as their operative mechanisms. RESULTS: DC-RAs displayed a mature yet tolerogenic phenotype, expressing IL-10, TGF-ß, IL-27, and aldehyde dehydrogenase 1A2 but not IL-12 or IL-35; IL-10 and TGF-ß together drove their suppression of TH2 cell proliferation. Delivery of specific allergen-presenting DC-RAs to half-maximally sensitized mice with ovalbumin or peanut allergy reduced anaphylactic responses to oral allergen challenge by 84% to 90%, as well as diarrhea, mast cell activation, and TH2 cytokine responses and serum allergen-specific IgE/IgG1 levels. DC-RA expression of IL-27 was important to their induction of CD25+ lymphocyte activation gene 3 (LAG3)+, CD49b-, forkhead box P3 (Foxp3)- regulatory T cells in vitro, such that ß subunit of IL-27 (Ebi)-/- (ie, IL-27-incompetent) DC-RAs were ineffective in inducing food allergen tolerance. CONCLUSION: Our data indicate that regulatory DC immunotherapy can be effective for food allergies and suggest that induction of Foxp3- regulatory T cells might be a useful strategy for tolerance induction in this context.

ELR-CXC chemokine antagonism is neuroprotective in a rat model of ischemic stroke.

Inflammation-related cerebral damage mediated by infiltrating neutrophils following reperfusion plays a role in reperfusion-induced brain damage subsequent to a stroke event. The ELR-CXC family of chemokines are CXCR1 and CXCR2 agonists that are known to drive neutrophil migration and activation. The present study demonstrated the benefit of anti-inflammatory therapy in the treatment of ischemic stroke with the administration of the competitive ELR-CXC chemokine antagonist, CXCL8(3-72)K11R/G31P (G31P). Male Sprague-Dawley rats were anaesthetized, and the middle cerebral artery (MCA) was occluded for 30 min followed by 5.5 h of reperfusion. Pretreatment with G31P resulted in a significant, dose-dependent (approximately 61-72%) decrease in infarct volumes compared to vehicle-treated animals, but neuroprotection was also observed when G31P (0.5 mg/kg) was administered 1 or 3 h following the start of reperfusion. The neuroprotection observed following the administration of this competitive CXCR1/CXCR2 antagonist may present therapeutic opportunities for addressing reperfusion-induced inflammatory damage in patients presenting with transient ischemic episodes.

CXCR1/2 antagonism with CXCL8/Interleukin-8 analogue CXCL8(3-72)K11R/G31P restricts lung cancer growth by inhibiting tumor cell proliferation and suppressing angiogenesis.

CXCR1 and CXCR2 together with cognate chemokines are significantly upregulated in a number of cancers, where they act as key regulators of tumor cell proliferation, metastasis, and angiogenesis. We have previously reported a mutant protein of CXCL8/Interleukin-8, CXCL8(3-72)K11R/G31P (G31P), which can act as a selective antagonist towards CXCR1/2 with therapeutic efficacy in both inflammatory diseases and malignancies. In this study, we investigated the effect of this ELR-CXC chemokine antagonist G31P on human non-small cell lung cancer cells and lung tumor progression in an orthotopic xenograft model. We report increased mRNA levels of CXCR1 and CXCR2 in human lung cancer tissues compared to normal counterparts. Expression levels of CXCR1/2 cognate ligands was determined by ELISA. CXCR1/2 receptor antagonism via G31P leads to decreased H460 and A549 cell proliferation and migration in a dose-dependent manner. G31P also enhanced apoptosis in lung cancer cells as determined by elevated levels of cleaved PARP, Caspase-8, and Bax, together with a reduced expression of the anti-apoptotic protein Bcl-2. In an in vivo orthotopic xenograft mouse model of human lung cancer, G31P treatment suppressed tumor growth, metastasis, and angiogenesis. At the molecular level, G31P treatment was correlated with decreased expression of VEGF and NFкB-p65, in addition to reduced phosphorylation of ERK1/2 and AKT. Our results suggest that G31P blockage of CXCR1 and CXCR2 can inhibit human lung cancer cell growth and metastasis, which offers potential therapeutic opportunities.

Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane.

F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization.

Les Escherichia coli entérotoxinogénique (ETEC) positif pour F4ac doivent s'attacher à la muqueuse intestinale pour causer la diarrhée chez les porcelets. L'empêchement de l'attachement bactérien à la muqueuse intestinale est le moyen de défense le plus efficace contre la diarrhée induite par les ETEC. Les membranes de globules de gras de lait porcin (MFGM) ont été montré comme étant capable d'inhiber l'attachement des ETEC à la bordure en brosse intestinale; toutefois, les composantes spécifiques des MFGM porcines qui inhibaient l'attachement des ETEC aux entérocytes ne furent pas identifiées. Ainsi, le but de la présente étude était d'identifier les protéines des MFGM liant F4ac par immunobuvardage par superposition et chromatographie d'affinité. Le protéome des MFGM porcine fut caractérisé et les protéines liant F4as suivantes furent détectées par immunobuvardage par superposition et chromatographie d'affinité : lactadhérine, butyrophiline, adipophiline, acyl-CoA synthétase 3, et la protéine 3 liant les acides gras. La fonction biologique de ces protéines ne fut pas étudiée mais il est possible que leur interaction avec les fimbriae F4ac interfère avec l'attachement bactérien et la colonisation.(Traduit par Docteur Serge Messier).

Behavioral alterations in rat offspring following maternal immune activation and ELR-CXC chemokine receptor antagonism during pregnancy: implications for neurodevelopmental psychiatric disorders.

Research suggests that maternal immune activation (MIA) during pregnancy increases the risk of neurodevelopmental disorders including schizophrenia and autism in the offspring. Current theories suggest that inflammatory mediators including cytokines and chemokines may underlie the increased risk of these disorders in humans. For example, elevated maternal interleukin-8 (IL-8) during pregnancy is associated with increased risk of schizophrenia in the offspring. Given this association, the present experiments examined ELR-CXC chemokines CXCL1 and CXCL2, rodent homologues of human IL-8, and activation of their receptors (CXCR1 and CXCR2) in an established rodent model of MIA. Pregnant Long Evans rats were treated with the viral mimetic polyinosinic-polycytidylic acid (polyI:C; 4 mg/kg, i.v.) on gestational day 15. Protein analysis using multiplex assays and ELISA showed that polyI:C significantly increased maternal serum concentrations of interleukin-1ß, tumor necrosis factor, and CXCL1 3h after administration. Subsequent experiments tested the role of elevated maternal CXCL1 on behavior of the offspring by administering a CXCR1/CXCR2 antagonist (G31P; 500 µg/kg, i.p.; 1h before, 48 and 96 h after polyI:C treatment). The male offspring of dams treated with polyI:C demonstrated subtle impairments in prepulse inhibition (PPI), impaired associative and crossmodal recognition memory, and altered behavioral flexibility in an operant test battery. While G31P did not completely reverse the behavioral impairments caused by polyI:C, it enhanced PPI during adolescence and strategy set-shifting and reversal learning during young adulthood. These results suggest that while polyI:C treatment significantly increases maternal CXCL1, elevations of this chemokine are not solely responsible for the effects of polyI:C on the behavior of the offspring.

G31P, CXCR1/2 inhibitor, with cisplatin inhibits the growth of mice hepatocellular carcinoma and mitigates high­dose cisplatin-induced nephrotoxicity.

Cisplatin (DDP), a cytotoxic antitumor drug, functions in a dose-dependent manner. However, the pursuit for high­dose therapeutic effects leads to more serious side effects including kidney toxicity. Nephrotoxicity caused due to endothelial cell dysfunction and neutrophils infiltration in kidneys. Interleukin-8 (IL-8) is an ELR+ chemokine binds with CXCR1/2 receptors and its role is primarily in neutrophils recruitment and also involved in invasion, angiogenesis and metastasis of different solid tumors including liver cancer. G31P, a CXCR1/2 antagonist, binds with CXCR1/2 with high affinity, and acts as an anti-inflammatory and antitumor agent. In the present study, we examined the antitumor effects of G31P and DDP on mouse liver cancer cells, and the effects exerted by G31P on cisplatin-induced renal injury. In vitro, effects of the G31P and DDP regimen on H22 cell proliferation were investigated by MTT assay. In vivo BALB/c mice were inoculated subcutaneously with 1x106 H22 cells and treated after one week with a high single dose of DDP with and without G31P on alternative days until the experiment was terminated. On the 15th day the mice were sacrificed, dissected and kidney tissues were analyzed using H&E staining. Myeloperoxidase (MPO) activity was assessed and RT-PCR was performed to detect inflammatory cytokines. Solid tumors were weighed for tumor growth and performed pathological examination, immunohistochemistry and western blotting were performed to detect tissue-related protein expressions in tumor tissue. The tumor inhibitory rate of DDP, G31P and DDP+G31P groups was 38.40, 40.74 and 74.80%, respectively, and the general state of mice in the DDP+G31P group was significantly improved as compared to the DDP group. The results indicated that G31P with DDP significantly inhibited the proliferation while the growth of H22 cell carnimona in vitro and in vivo enhanced the efficacy of cisplatin in cancer treatment with reduced side effects on acute renal failure.