BACKGROUND

Combined pegylated interferon-α and ribavirin therapy has sustained virological response (SVR) rates of 54% to 61%. Pretreatment predictors of SVR to interferon therapy have not been fully investigated yet. The current study assesses a group of chemokines that may predict treatment response in Egyptian patients with chronic HCV infection.

METHODS

CXCL5, CXCL9, CXCL11, CXCL12, CXCL 13, CXCL 16 chemokines and E-Cadherin were assayed in 57 chronic HCV patients' sera using quantitative ELISA plate method. All studied patients were scheduled for combined pegylated interferon alpha and ribavirin therapy (32 patients received pegylated interferon α 2b, and 25 patients received pegylated interferon α 2a). Quantitative hepatitis C virus RNA was done by real time RT-PCR and HCV genotyping by INNOLIPAII.

RESULTS

There was no significant difference (p>> 0.05) in baseline HCV RNA levels between responders and non-responders to interferon. A statistically significant difference in CXCL13 (p = 0.017) and E-Cadherin levels (P = 0.041) was reported between responders and nonresponders at week 12. Significant correlations were found between changes in the CXCL13 levels and CXCL9, CXCL16, E-cadherin levels as well as between changes in E-cadherin levels and both CXCL16 and ALT levels that were maintained during follow up. Also, significant changes have been found in the serum levels of CXCL5, CXCL13, and CXCL16 with time (before pegylated interferon α 2 a and α 2 b therapy, and at weeks 12 and 24) with no significant difference in relation to interferon type and response to treatment.

CONCLUSIONS

Serum levels of CXCL13 and E-Cadherin could be used as surrogate markers to predict response of combined PEG IFN-α/RBV therapy, especially at week 12. However, an extended study including larger number of patients is needed for validation of these findings.

UNASSIGNED

NCT01758939.

OBJECTIVE

The IL-23/IL-17 axis is central to the pathogenesis of psoriatic arthritis (PsA). We aimed to identify Th17 signalling genes that are dysregulated in synovial fluid of PsA compared to osteoarthritis (OA) patients and to determine if differences in peripheral blood can distinguish PsA from psoriasis patients and controls.

METHODS

Synovial fluid cells (SFCs) from 14 PsA and 9 OA patients were obtained and stored in TRIzol reagent. RNA was isolated by phenol-chloroform extraction and purified with RNeasy miniprep kits. Total RNA was extracted from PAXgene whole blood from 20 PsA, 20 psoriasis without arthritis (PsC) and 11 controls. Quantitative RT-PCR arrays were used to profile expression of 84 genes related to the Th17 regulatory network. Fold change differences were compared by Mann-Whitney U-test with false discovery rate (FDR) correction (FDR<0.05).

RESULTS

In PsA compared to OA SFCs, a total of 33 genes were up-regulated and 27 genes were down-regulated. Signalling molecules (such as STAT3, FOXP3) were highly expressed in PsA SFCs, while cytokines (such as IL17F, IL6) were more predominant in OA SFCs after non-supervised hierarchal clustering. Nine genes (MMP3, CCL1, IL17C, CCL20, IL17F, IL3, CXCL5, IL6 and CX3CL1) had concordant expression in SFCs and in peripheral blood cells (PBCs) of PsA compared to PsC and/or controls.

CONCLUSIONS

We identified expression differences in Th17 signalling genes in PsA compared to OA SFCs, with an elevation of signalling molecules and attenuation of cytokine expression in PsA. A subset of genes was concordant in PBCs; these may thus be potential biomarkers of PsA.

Introduction: C-X-C motif chemokine 5 is primarily chemotactic for neutrophils and previously shown to increase in the bronchoalveolar lavage fluid of patients with chronic obstructive pulmonary disease. However, whether C-X-C motif chemokine 5 levels correlate with lung function decline in patients or mouse model of chronic obstructive pulmonary disease was not clear. Methods: The mouse model was induced by cigarette smoke exposure. Plasma/serum and bronchoalveolar lavage fluid were obtained from patients and mouse model of chronic obstructive pulmonary disease; C-X-C motif chemokine 5 levels were assessed and correlated with lung functions and granulocyte-colony stimulating factor levels, respectively. Results: The C-X-C motif chemokine 5 levels increased and correlated to granulocyte-colony stimulating factor levels in both plasma/serum and bronchoalveolar lavage fluid obtained from patients and mouse model of chronic obstructive pulmonary disease. Circulating levels of C-X-C motif chemokine 5 correlated to lung functions decline in patients and mouse model. Conclusions: Granulocyte-colony stimulating factor might coordinate with C-X-C motif chemokine 5 in the pathogenesis of neutrophilic inflammation in chronic obstructive pulmonary disease. Circulating C-X-C motif chemokine 5 might serve as a potential blood-based biomarker to add additional modest predictive value on the preliminary screening and diagnosis of chronic obstructive pulmonary disease. Key messages Circulating C-X-C motif chemokine 5 might serve as a potential blood-based biomarker to add additional modest predictive value on the preliminary screening and diagnosis of COPD. Granulocyte-colony stimulating factor might coordinate with C-X-C motif chemokine 5 in the pathogenesis of neutrophilic inflammation in chronic obstructive pulmonary disease.

The current study was undertaken to explore the correlation of adjuvanticity and local inflammatory response elicited in the murine vagina and the draining lymph nodes following local administration of two candidate vaginal adjuvants, Toll like receptor (TLR) 9 agonist CpG ODN, and a non-TLR targeting molecule alpha-galactosylceramide (alpha-GalCer). Using real-time PCR array analysis, we could show that a group of 13 common cytokine genes are activated in the vagina within 24h after vaginal administration of these adjuvants, including Ccl2, Ccl7, Ccl12, Ccl19, Ccl20, Ccl22, Cxcl1, Cxcl5, Il10 and the Th1-inducing molecules Ifng, Cxcl9, Cxcl10 and Cxcl11. A high degree of inflammation in and damage to the epithelium was exclusively observed in the vagina of the CpG ODN treated mice, which was reversed within 48h. These results indicate that there is a group of common genes that correlate with the adjuvanticity of CpG ODN and alpha-GalCer in the vagina, and that alpha-GalCer induces less of local inflammatory reactions in the murine vagina compared to CpG ODN.

OBJECTIVE

Intestinal adaptation involves villus lengthening, crypt deepening, and increased capillary density following small bowel resection (SBR). Mice lacking the proangiogenic chemokine CXCL5 have normal structural adaptation but impaired angiogenesis. This work evaluates the impact of incomplete adaptive angiogenesis on the functional capacity of the intestine after SBR.

METHODS

CXCL5 knockout (KO) and C57BL/6 wild-type (WT) mice underwent 50% SBR. Magnetic resonance imaging measured weekly body composition. Intestinal absorptive capacity was evaluated through fecal fat analysis. Gene expression profiles for select macronutrient transporters were measured via RT-PCR. Postoperative crypt and villus measurements were assessed for structural adaptation. Submucosal capillary density was measured through CD31 immunohistochemistry.

RESULTS

Comparable postoperative weight gain occurred initially. Diminished weight gain, impaired fat absorption, and elevated steatorrhea occurred in KO mice after instituting high-fat diet. Greater postoperative upregulation of ABCA1 fat transporter occurred in WT mice, while PEPT1 protein transporter was significantly downregulated in KO mice. KO mice had impaired angiogenesis but intact structural adaptation.

CONCLUSIONS

After SBR, KO mice display an inefficient intestinal absorption profile with perturbed macronutrient transporter expression, impaired fat absorption, and slower postoperative weight gain. In addition to longer villi and deeper crypts, an intact angiogenic response may be required to achieve functional adaptation to SBR.

We established a model of immune-mediated bone marrow (BM) failure in C57BL/6 (B6) mice with 6.5 G total-body irradiation followed by the infusion of 4-10 × 10(6) lymph node (LN) cells/recipient from Friend leukemia virus B/N (FVB) donors. Forty-three percent of animals succumbed, with surviving animals showing marked declines in blood neutrophils, red blood cells, platelets and total BM cells at 8 to 14 days following LN cell infusion. Lowering the total-body irradiation dose to 5 G or altering the LN source from FVB to BALB/cBy donors failed to produce BM destruction. Affected animals showed significant expansion and activation of CD8 T lymphocytes in both the blood and BM; cytotoxic T cells had elevated Fas ligand expression and were oligoclonal, mainly displaying Vβ7 and Vβ17 T cell receptors. There were significant increases in blood plasma interferon γ and tissue necrosis factor α in affected animals. Chemokine ligands CCL3, CCL4, CCL5, CCL20, CXCL2, and CXCL5 and hematopoietic growth factors G-CSF, M-CSF, GM-CSF, VEGF were also elevated. In B6 mice carrying a Fas gene mutation, BM failure was attenuated when they were infused with FVB LN cells. Our model establishes a useful platform to define the roles of individual genes and their products in immune-mediated BM failure.

Hypersensitivity pneumonitis (HP) is an immune mediated lung disease induced by the repeated inhalation of a wide variety of antigens. Bird-related hypersensitivity pneumonitis (BRHP) is one of the most common forms of HP in human and results from the inhalation of avian antigens. The findings of a recent clinical analysis suggest that in addition to Th1 factors, the levels of interleukin(IL)-17 and IL-17-associated transcripts are increased in the setting of HP, and that both IL-17A and neutrophils are crucial for the development of pulmonary inflammation in murine models of HP. Our objectives were to investigate the roles of IL-17A and neutrophils in granuloma-forming inflammation in an acute HP model. We developed a mouse model of acute BRHP using pigeon dropping extract. We evaluated the process of granuloma formation and the roles of both IL-17A and neutrophils in a model. We found that the neutralization of IL-17A by the antibody attenuated granuloma formation and the recruitment of neutrophils, and also decreased the expression level of chemokine(C-X-C motif) ligand 5 (CXCL5) in the acute HP model. We confirmed that most of the neutrophils in the acute HP model exhibited immunoreactivity to the anti-IL-17 antibody. We have identified the central roles of both IL-17A and neutrophils in the pathogenesis of granuloma formation in acute HP. We have also assumed that neutrophils are an important source of IL-17A in an acute HP model, and that the IL-17A-CXCL5 pathway may be responsible for the recruitment of neutrophils.

BACKGROUND

Myelodysplastic syndromes (MDS) are characterized by bone marrow failure due to disturbed bone marrow maturation. MDS is associated with increased risk of transformation to acute myeloid leukemia (AML) and features of immunological dysregulation.

METHODS

Serum levels of 47 soluble immune mediators were examined in samples derived from 49 MDS patients (35 low-risk and 14 high-risk) and 23 healthy adults. Our patients represent an unselected population-based cohort. The mediators included cytokines, soluble adhesion proteins, matrix metalloproteases, and tissue inhibitors of proteases. Levels were determined using Luminex assays. Patients were classified as low- and high-risk based on the international prognostic scoring system (IPSS) score.

RESULTS

When comparing the serum levels of single mediators the MDS patients showed a relatively wide variation range for several mediators compared with healthy adults, especially interleukin 6 (IL-6), IL-8/CXCL8, CCL3, and CCL4. The high-risk patients had lower levels of epidermal growth factor (EGF), cluster of differentiation 40 ligand (CD40L), CCL5, CCL11, CXCL5, matrix metalloproteinase 1 (MMP-1), MMP-9, and tissue inhibitor of metalloproteinases 2 (TIMP-2) compared with low-risk patients. Unsupervised hierarchical cluster analysis visualized marked serum mediator profile differences between MDS patients; based on this analysis three patient subsets could be identified. The healthy adults were also included in this analysis and, as expected, they formed their own separate cluster, except for one outlier. Both low- and high-risk patients showed considerable heterogeneity with regard to serum profile, and this heterogeneity seems stable over time (one year follow-up). Finally, very few mediators differed between low- and high-risk patients, but hierarchical clustering based both on all mediators, as well as five selected mediators (EGF, CCL11, TIMP-2, MMP-1, and MMP-9) identified subsets of patients with significantly increased frequency of high-risk disease (χ-square test p = 0.0158 and p = 0.0148).

Lipocalin 2 (Lcn2) is a bacteriostatic factor produced during the innate immune response to bacterial infection. Whether Lcn2 has a function in viral infection is unknown. We investigated the regulation and function of Lcn2 in the central nervous system (CNS) of mice during West Nile virus (WNV) encephalitis. Lcn2 mRNA and protein were induced in the brain by day 5, and this induction increased further by day 7 postinfection but was delayed compared with the induction of the toll-like receptor 3 (TLR3) gene, retinoic acid-inducible gene 1 (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) gene. The Lcn2 mRNA and protein were both found at high levels in the choroid plexus, vascular endothelium, macrophage/microglia, and astrocytes. However, some neuronal subsets contained Lcn2 protein but no detectable mRNA. In Lcn2 knockout (KO) mice, with the exception of CXC motif chemokine 5 (CXCL5), which was significantly more downregulated than in wild-type (WT) mice, expression levels of a number of other host response genes were similar in the two genotypes. The brain from Lcn2 and WT mice with WNV encephalitis contained similar numbers of infiltrating macrophages, granulocytes, and T cells. Lcn2 KO and WT mice had no significant difference in tissue viral loads or survival after infection with different doses of WNV. We conclude that Lcn2 gene expression is induced to high levels in a time-dependent fashion in a variety of cells and regions of the CNS of mice with WNV encephalitis. The function of Lcn2 in the host response to WNV infection remains largely unknown, but our data indicate that it is dispensable as an antiviral or immunoregulatory factor in WNV encephalitis.

The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 μM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.

Melanoma is the most deadly type of skin cancer, constituting annually ∼ 75% of all cutaneous cancer-related deaths due to metastatic spread. Currently, because of metastatic spread, there are no effective treatment options for late-stage metastatic melanoma patients. Studies over the past two decades have provided insight into several complex molecular mechanisms as to how these malignancies evade immunological control, indicating the importance of immune escape or suppression for tumor survival. Thus, it is essential to develop innovative cancer strategies and address immune obstacles with the goal of generating more effective immunotherapies. One important area of study is to further elucidate the role and significance of myeloid-derived suppressor cells (MDSCs) in the maintenance of the tumor microenvironment. These cells possess a remarkable ability to suppress immune responses and, as such, facilitate tumor growth. Thus, MDSCs represent an important new target for preventing tumor progression and escape from immune control. In this study, we investigated the role of MDSCs in immune suppression of T cells in an antigen-specific B16 melanoma murine system utilizing a novel synthetic tyrosinase (Tyr) DNA vaccine therapy in both prophylactic and therapeutic models. This Tyr vaccine induced a robust and broad immune response, including directing CD8 T-cell infiltration into tumor sites. The vaccine also reduced the number of MDSCs in the tumor microenvironment through the downregulation of monocyte chemoattractant protein 1, interleukin-10, CXCL5 and arginase II, factors important for MDSC expansion. This novel synthetic DNA vaccine significantly reduced the melanoma tumor burden and increased survival in vivo, due likely, in part, to the facilitation of a change in the tumor microenvironment through MDSC suppression.

Butyrate (NaBu), a product of intestinal microbial metabolism, has been proposed as an anti-inflammatory agent for treating inflammatory bowel diseases. However, the molecular mechanisms implicated in the modulation of intestinal epithelial cell inflammatory response to NaBu remain unknown. Here, microarray analysis performed on nontransformed human crypt intestinal epithelial cells (HIEC) shows that NaBu regulated specifically the short-term IL-1beta -dependent induction of different inflammatory genes. While NaBu significantly increased the IL-1beta -induction of genes like SAA2, C3, and IL-1alpha , other inflammatory genes like CXCL5, CXCL11, and IL-1beta were decreased. Induction of various genes such as CXCL8, CCL20, and IL-6 was unaffected by NaBu. We show that, compared to genes that are upregulated or downregulated by NaBu, genes that are unaffected by NaBu were induced more rapidly after IL-1beta treatment and contained a higher concentration of transcription factor binding sites in their promoter region. In addition, transient treatment with IL-1beta was sufficient for subsequent induction of NaBu-upregulated and NaBu-unaffected classes of genes, while a continuous presence of IL-1beta was required for NaBu-downregulated gene expression. In conclusion, our results suggest that fundamental differences predispose inflammatory genes to specific regulation by NaBu in intestinal epithelial cells, thereby allowing precise control of inflammation.

The inflammatory potential and molecular mechanisms underscoring inflammatory responses of lung cells to compounds from fungi that grow on damp building materials is poorly understood in vitro. In this study we evaluated the effect of pure fungal compounds on potentiating acute inflammatory response in primary mouse alveolar macrophages (AMs) and tested the hypothesis that AM responses to low molecular weight fungal compounds exhibit temporal and compound specificity that mimic that observed in the whole lung. Transcriptional responses of 13 inflammation/respiratory burst-associated genes (KC=Cxcl1, Cxcl2, Cxcl5, Cxcl10, Ccl3, Ccl112, Ccl20, IL-1β, Il-6, ifi27 Tnfα, iNOS and Blvrb) were evaluated in mouse AMs exposed to a 1ml (10(-8)mol) dose of either pure atranone C, brevianimide, cladosporin, curdlan, LPS, neoechinulin A & B, sterigmatocystin or TMC-120A for 2h, 4h and 12h PE using customized reverse transcription (RT)-PCR based arrays. Multianalyte ELISA was used to measure expression of 6 pro-inflammatory cytokines common to the transcriptional assays (Cxcl1, Cxcl10, Ccl3, IL1β, Ifn-λ and Tnf-α) to determine whether gene expression corresponded to the transcription data. Compared to controls, all of these compounds induced significant (≥2.5-fold or ≤-2.5-fold change at p≤0.05) time- and compound-specific transcriptional gene alterations in treatment AMs. The highest number of transcribed genes were in LPS treatment AMs at 12h PE (12/13) followed by neoechinulin B at 4h PE (11/13). Highest fold change values (>30) were associated with KC, Cxcl2, Cxcl5 and IL1β genes in cells exposed to LPS. Compound exposures also induced significant (p≤0.05) time- and compound-specific pro-inflammatory responses manifest as differentially elevated Cxcl1, Cxcl10, Ccl3, Ifn-λ and Tnf-α concentrations in culture supernatant of treatment AMs. Dissimilarity in transcriptional responses in AMs and our in vivo model of lung disease is likely attributable to whole lung vs. isolated cell responsive and dose differences between the two studies. The results not only indicate that low molecular weight compounds from fungi that grow in damp built environments are potently pro-inflammatory in vitro, it further highlights the important role AMs play in innate lung defence, and against exposure to low molecular weight fungal compounds. These observations further support our position that exposure to low molecular weight compounds from indoor-associated fungi may provoke some of the inflammatory health effects reported from humans in damp building environments. They also open up a hypothesis building process that could explain the rise of non-atopic asthma associated with fungi.

Limitations of current treatments for skin loss caused by major injuries leads to the use of skin substitutes. It is assumed that secretion of wound healing mediators by these skin substitutes plays a role in treating skin loss. In our previous study, single layer keratinocytes (SK), single layer fibroblast (SF) and bilayer (BL; containing keratinocytes and fibroblasts layers) skin substitutes were fabricated using fibrin that had shown potential to heal wounds in preclinical studies. This study aimed to quantify the secretion of wound healing mediators, and compare between single and bi-layer skin substitutes. Skin samples were digested to harvest fibroblasts and keratinocytes, and expanded to obtain sufficient cells for the construction of skin substitutes. Acellular fibrin (AF) construct was used as control. Substitutes i.e. AF, SK, SF and BL were cultured for 2 days, and culture supernatant was collected to analyze secretion of wound healing mediators via multiplex ELISA. Among 19 wound healing mediators tested, BL substitute secreted significantly higher amounts of CXCL1 and GCSF compared to SF and AF substitute but this was not significant with respect to SK substitute. The BL substitute also secreted significantly higher amounts of CXCL5 and IL-6 compared to other substitutes. In contrast, the SK substitute secreted significantly higher amounts of VCAM-1 compared to other substitutes. However, all three skin substitutes also secreted CCL2, CCL5, CCL11, GM-CSF, IL8, IL-1α, TNF-α, ICAM-1, FGF-β, TGF-β, HGF, VEGF-α and PDGF-BB factors, but no significant difference was seen. Secretion of these mediators after transplantation may play a significant role in promoting wound healing process for the treatment of skin loss.

BACKGROUND

Roflumilast is the first phosphodiesterase-4 (PDE4) inhibitor to have been approved for the treatment of COPD. The anti-inflammatory profile of PDE4 inhibitors has not yet been explored in human lung tissues. We investigated the effects of roflumilast and its active metabolite roflumilast-N-oxide on the lipopolysaccharide (LPS)-induced release of tumor necrosis factor-alpha (TNF-α) and chemokines by human lung parenchymal explants. We also investigated roflumilast's interaction with the long-acting β2-agonist formoterol.

METHODS

Explants from 25 patients undergoing surgical lung resection were incubated with Roflumilast, Roflumilast-N-oxide and formoterol and stimulated with LPS. Levels of TNF-α, chemokines (in the culture supernatants) and cyclic adenosine monophosphate (in tissue homogenates) were determined with appropriate immunoassays.

RESULTS

Roflumilast and Roflumilast-N-oxide concentration-dependently reduced the release of TNF-α and chemokines CCL2, CCL3, CCL4, CXCL9 and CXCL10 from LPS-stimulated human lung explants, whereas CXCL1, CXCL5 and CXCL8 release was not altered. Formoterol (10 nM) partially decreased the release of the same cytokines and significantly increased the inhibitory effect of roflumilast on the release of the cytokines.

CONCLUSIONS

In human lung parenchymal explants, roflumilast and roflumilast-N-oxide reduced the LPS-induced release of TNF-α and chemokines involved in the recruitment of monocytes and T-cells but not those involved in the recruitment of neutrophils. Addition of formoterol to roflumilast provided superior in vitro anti-inflammatory activity, which may translate into greater efficacy in COPD.

OBJECTIVE

To investigate the influence of cigarette smoking on plasma epithelial cell-derived neutrophil-activating peptide-78 (CXCL5/ENA-78) and interleukin-6 (IL-6) in supportive therapy periodontitis patients.

METHODS

Plasma concentrations of CXCL5/ENA-78 and IL-6 were evaluated in 167 systemically healthy subjects (54 smokers and 113 non-smokers) divided into four groups: non-smokers with periodontitis (n=90), smokers with periodontitis (n=49), healthy non smokers (n=23) and healthy smokers (n=5).

RESULTS

Clinical probing depth (CPD) of smokers with periodontitis were significantly greater than those of non-smoking patients (p<0.05). Although clinical attachment loss (CAL) and the number of deep sites affected were greater in the smokers with periodontitis, these differences were not significant. Periodontitis patients had significantly higher plasma IL-6 and ENA-78 than healthy subjects (p<0.05). There was no significant difference in IL-6 between smokers and non-smokers with periodontitis but CXCL5/ENA-78 concentrations were significantly greater in smokers with periodontitis (p=0.006). Plasma CXCL5/ENA-78 correlated with CPD, CAL and tobacco consumption (all p<0.05).

CONCLUSIONS

Plasma CXCL5/ENA-78 concentrations are a good systemic indicator of the inflammatory process and disease severity in subjects with periodontitis and in addition are potential indicator of inflammatory effects of cigarette smoking. Further studies are required to elucidate the biological mechanisms underlining this increase in CXCL5/ENA-78.

The aim of this study was to examine the expression of CD44v6, CD54, Cdx2, CXCL5, Cyclin B1, MMP-7, nm23, RCAS1 and Survivin in primary gastric cancer and to investigate whether these molecules were useful in predicting the lymph node status. They were selected as candidates for indicators of lymph node metastasis from various kinds of cancer-associated genes reported previously. In 135 cases of radically resected primary gastric adenocarcinoma, we investigated the association between the expression of these molecules and clinocopathologic factors by immunohistochemistry. The results revealed that the expression of CD44v6 and MMP-7 were significantly associated with lymph node status. By contrast, nuclear Cdx2 expression was found to be inversely correlated with lymph node metastasis. Moreover, multivariate analysis demonstrated that CD44v6, MMP-7 and nuclear Cdx2 were independent predictors for lymph node status. In conclusion, our results suggest that positive expression of both CD44v6 and MMP-7, and negative expression of nuclear Cdx2 may serve as powerful predictors of lymph node metastasis in gastric cancer. Combined evaluation of these markers could be further useful to predict lymph node status clinically.

BACKGROUND

The medical care of malaria is a clinical emergency because it may develop into severe malaria, which has a high risk of complications and death. One of the major complications of Plasmodium falciparum infections is cerebral malaria (CM), which is responsible for at least 175,000 deaths worldwide each year and has long-term neurological sequelae. Moreover, treatment for CM is only partially effective. Statins are now known to have anti-inflammatory action, to attenuate sepsis and to have neuroprotective effects. In vitro, atorvastatin (AVA) has an anti-malarial activity and has improved the activity of quinine (QN), mefloquine (MQ), and dihydroartemisinin (DHA).

OBJECTIVE

This study had two objectives. First, the ability of AVA to enhance DHA efficacy by improving the survival rate for CM and also decreasing signs of CM was evaluated in a murine model of experimental cerebral malaria (ECM), which was designed in C57BL6/N mice. Second, the inflammatory biomarkers were assessed at D6 and D10 in mice treated by DHA and in untreated mice in which clinical signs of CM appear rapidly and death occurs before D12. Both experiments were designed with seven days of treatment with 40 mg/kg AVA combined with five days of 3 mg/kg DHA administered intraperitoneally.

RESULTS

AVA in combination with DHA in a therapeutic scheme leads to a significant delay in mouse death, and it has an effect on the onset of CM symptoms and on the level of parasitaemia. Evaluation of the biomarkers highlights the significant difference between treated and control mice for five cytokines and chemokines (Eotaxin-CCL11, IL-13, LIX-CXCL5, MIP1b-CCL4 and MIP2) that are known to have a role in chemotaxis.

CONCLUSIONS

The combination of DHA and AVA seems to be effective as a therapeutic scheme for improving mouse survival but less effective for cytokine modulation, which is associated with protection against CM. These results call for clinical trials of AVA as an adjuvant with anti-malarial therapy, especially with artemisinin-based combination therapy, in CM treatment or prevention.

In this study, Trypanosoma brucei was naturally transmitted to mice through the bites of infected Glossina morsitans tsetse flies. Neutrophils were recruited rapidly to the bite site, whereas monocytes were attracted more gradually. Expression of inflammatory cytokines (il1b, il6), il10 and neutrophil chemokines (cxcl1, cxcl5) was transiently up-regulated at the site of parasite inoculation. Then, a second influx of neutrophils occurred that coincided with the previously described parasite retention and expansion in the ear dermis. Congenital and experimental neutropenia models, combined with bioluminescent imaging, indicate that neutrophils do not significantly contribute to dermal parasite control and elicit higher systemic parasitemia levels during the infection onset. Engulfment of parasites by neutrophils in the skin was rarely observed and was restricted to parasites with reduced motility/viability, whereas live parasites escaped phagocytosis. To our knowledge, this study represents the first description of a trypanosome infection promoting role of early innate immunological reactions following an infective tsetse fly bite. Our data indicate that the trypanosome is not hindered in its early development and benefits from the host innate responses with the neutrophils being important regulators of the early infection, as already demonstrated for the sand fly transmitted Leishmania parasite.

Urinary tract infections (UTIs) are among the most common infections worldwide. Diagnosing UTIs in older adults poses a significant challenge as asymptomatic colonization is common. Identification of a non-invasive profile that predicts likelihood of progressing from urine colonization to severe disease would provide a significant advantage in clinical practice. We monitored colonization susceptibility, disease severity, and immune response to two uropathogens in two mouse strains across three age groups to identify predictors of infection outcome. Proteus mirabilis caused more severe disease than Escherichia coli, regardless of mouse strain or age, and was associated with differences in IL-1β, IFN-β, CXCL5 (LIX), CCL5 (RANTES), and CCL2 (MCP-1). In comparing the response to infection across age groups, mature adult mice were better able to control colonization and prevent progression to kidney colonization and bacteremia than young or aged mice, regardless of mouse strain or bacterial species, and this was associated with differences in IL-23, CXCL1, and CCL5. A bimodal distribution was noted for urine colonization, which was strongly associated with bladder CFUs and the magnitude of the immune response but independent of age or disease severity. To determine the value of urine cytokine and chemokine levels for predicting severe disease, all infection datasets were combined and subjected to a series of logistic regressions. A multivariate model incorporating IL-1β, CXCL1, and CCL2 had strong predictive value for identifying mice that did not develop kidney colonization or bacteremia, regardless of mouse genetic background, age, infecting bacterial species, or urine bacterial burden. In conclusion, urine cytokine profiles could potentially serve as a non-invasive decision-support tool in clinical practice and contribute to antimicrobial stewardship.

Chemokines mold the tumor microenvironment by recruiting distinct immune cell populations, thereby strongly influencing disease progression. Previously, we showed that CXCL5 expression is upregulated in advanced stages of primary melanomas, which correlates with the presence of neutrophils in the tumor. The analysis of neutrophil populations in various tissues revealed a distinct phenotype of tumor-associated neutrophils. Tumor-associated neutrophils expressed PD-L1, CXCR4, CCR5, Adam17, and Nos2 and were immunosuppressive in a T-cell proliferation assay. To investigate the impact of CXCL5 and neutrophils in vivo, we established a syngeneic mouse tumor transplantation model using CXCL5-overexpressing and control melanoma cell lines. Growth behavior or vascularization of primary tumors was not affected by CXCL5 expression and neutrophils alone. However, in combination with Poly(I:C), tumor-associated neutrophils were able to attenuate induced antitumoral T-cell responses. CXCL5-overexpressing tumors had reduced lung metastasis compared with control tumors. Neutrophil depletion reversed this effect. In vitro, unstimulated lung-derived neutrophils had higher levels of reactive oxygen species compared with tumor-associated neutrophils, and CXCL5 stimulation further increased reactive oxygen species levels. In summary, in melanoma, neutrophils play a context-dependent role that is influenced by local or systemic factors, and interfere with therapies activating the acquired immune system. Actively switching neutrophils into antitumorigenic mode might be a new therapeutic strategy.

The emerging virus SARS-CoV-2 has caused a global pandemic, and understanding its pathogenesis and host antiviral immunity is critical for the development of vaccines and antivirals. Mice are frequently used to generate animal models of infectious diseases due to their convenience and ability to undergo genetic manipulation. However, normal adult mice are not susceptible to SARS-CoV-2. Here, we developed a viral receptor (human angiotensin-converting enzyme 2, hACE2) pulmonary transfection mouse model to rapidly establish SARS-CoV-2 infection in the mouse lung. Based on the model, the virus successfully infected the mouse lung after 2 days of transfection. Viral RNA/protein, innate immune cell infiltration, inflammatory cytokine expression, and pathological changes in the infected lung were observed after infection. Further studies indicated that neutrophils were the first and most abundant leukocytes that infiltrated the infected lung soon after viral infection. In addition, using infected CXCL5-knockout mice, the chemokine CXCL5 was found to be responsible for neutrophil recruitment. CXCL5 knockout decreased lung inflammation without diminishing viral clearance, suggesting a potential target for controlling pneumonia.

Keywords: ACE2; CXCL5; Lung infection; Mouse model; Neutrophil; SARS-CoV-2.

Ozone pollution is associated with adverse effects on respiratory health in adults and children but its effects on the neonatal lung remain unknown. This study was carried out to define the effect of acute ozone exposure on the neonatal lung and to profile the transcriptome response. Newborn mice were exposed to ozone or filtered air for 3h. Total RNA was isolated from lung tissues at 6 and 24h after exposure and was subjected to microarray gene expression analysis. Compared to filtered air-exposed littermates, ozone-exposed newborn mice developed a small but significant neutrophilic airway response associated with increased CXCL1 and CXCL5 expression in the lung. Transcriptome analysis indicated that 455 genes were down-regulated and 166 genes were up-regulated by at least 1.5-fold at 6h post-ozone exposure (t-test, p < .05). At 24h, 543 genes were down-regulated and 323 genes were up-regulated in the lungs of ozone-exposed, compared to filtered air-exposed, newborn mice (t-test, p < .05). After controlling for false discovery rate, 50 genes were identified as significantly down-regulated and only a few (RORC, GRP, VREB3, and CYP2B6) were up-regulated at 24h post-ozone exposure (q < .05). Gene ontology enrichment analysis revealed that cell cycle-associated functions including cell division/proliferation were the most impacted pathways, which were negatively regulated by ozone exposure, an adverse effect that was associated with reduced bromo-deoxyuridine incorporation. These results demonstrate that acute ozone exposure alters cell proliferation in the developing neonatal lung through a global suppression of cell cycle function.

BACKGROUND

Murine Staphylococcus aureus-mediated brain abscess comprises 2 major phases, an initial phase of cerebritis, followed by a healing phase characterized by capsule formation.

METHODS

C57BL/6 wild-type (WT) and IL-12p35(-/-) mice were intracerebrally infected with S. aureus to induce brain abscesses. Clinical disease activity and bacterial load were monitored. The cell populations that were involved, as well as their specific mediators, were analyzed by immunohistochemistry, quantitative real-time polymerase chain reaction, and flow cytometry.

RESULTS

In the acute phase, IL-12p35(-/-) mice were protected from disease. This was associated with enhanced recruitment of granulocytes, accompanied by upregulated expression of Il17a, Csf2 (which encodes granulocyte-macrophage colony-stimulating factor), Cxcl1, and Cxcl5, as well as increased expression of proinflammatory mediators, including Nos2 (which encodes inducible nitric oxide synthase), Ptgs2 (which encodes cyclooxygenase 2), and Tnf, that were primarily produced by granulocytes and activated microglia/macrophages. Furthermore, mechanisms associated with beneficial wound healing, including an accelerated formation of a fibrous capsule, were demonstrated by prominent VEGF-A production and collagen deposition driven by an earlier onset of T-helper 2 immunity in the absence of interleukin 12 (IL-12).

CONCLUSIONS

Brain abscess development is orchestrated by IL-12 at different stages of disease. Our data indicate that IL-12 has a nonprotective role in the acute phase and that IL-12 deficiency results in the accelerated formation of a protective capsule during the healing phase, which we consider crucial for early recovery from disease.