Facilitation of IL-22 production from innate lymphoid cells by prostaglandin E2 prevents experimental lung neutrophilic inflammation.

Acute lung injury is a neutrophil-dominant, life-threatening disease without effective therapies and better understanding of the pathophysiological mechanisms involved is an urgent need. Here we show that interleukin (IL)-22 is produced from innate lymphoid cells (ILC) and is responsible for suppression of experimental lung neutrophilic inflammation. Blocking prostaglandin E2 (PGE2) synthesis reduces lung ILCs and IL-22 production, resulting in exacerbation of lung neutrophilic inflammation. In contrast, activation of the PGE2 receptor EP4 prevents acute lung inflammation. We thus demonstrate a mechanism for production of innate IL-22 in the lung during acute injury, highlighting potential therapeutic strategies for control of lung neutrophilic inflammation by targeting the PGE2/ILC/IL-22 axis.

Effector and central memory T helper 2 cells respond differently to peptide immunotherapy.

Peptide immunotherapy (PIT) offers realistic prospects for the treatment of allergic diseases, including allergic asthma. Much is understood of the behavior of naive T cells in response to PIT. However, treatment of patients with ongoing allergic disease requires detailed understanding of the responses of allergen-experienced T cells. CD62L expression by allergen-experienced T cells corresponds to effector/effector memory (CD62L(lo)) and central memory (CD62L(hi)) subsets, which vary with allergen exposure (e.g., during, or out with, pollen season). The efficacy of PIT on different T helper 2 (Th2) cell memory populations is unknown. We developed a murine model of PIT in allergic airway inflammation (AAI) driven by adoptively transferred, traceable ovalbumin-experienced Th2 cells. PIT effectively suppressed AAI driven by unfractionated Th2 cells. Selective transfer of CD62L(hi) and CD62L(lo) Th2 cells revealed that these two populations behaved differently from one another and from previously characterized (early deletional) responses of naive CD4(+) T cells to PIT. Most notably, allergen-reactive CD62L(lo) Th2 cells were long-lived within the lung after PIT, before allergen challenge, in contrast to CD62L(hi) Th2 cells. Despite this, PIT was most potent against CD62L(lo) Th2 cells in protecting from AAI, impairing their ability to produce Th2 cytokines, whereas this capacity was heightened in PIT-treated CD62L(hi) Th2 cells. We conclude that Th2 cells do not undergo an early deletional form of tolerance after PIT. Moreover, memory Th2 subsets respond differently to PIT. These findings have implications for the clinical translation of PIT in different allergic scenarios.

Filaggrin-null mutations are associated with increased maturation markers on Langerhans cells.

BACKGROUND: Mutations in the gene encoding filaggrin (FLG), an epidermal structural protein, are the strongest risk factor identified for the development of atopic dermatitis (AD). Up to 50% of patients with moderate-to-severe AD in European populations have FLG-null alleles compared with a general population frequency of 7% to 10%. OBJECTIVE: This study aimed to investigate the relationship between FLG-null mutations and epidermal antigen-presenting cell (APC) maturation in subjects with and without AD. Additionally, we investigated whether the cis isomer of urocanic acid (UCA), a filaggrin breakdown product, exerts immunomodulatory effects on dendritic cells. METHODS: Epidermal APCs from nonlesional skin were assessed by using flow cytometry (n = 27) and confocal microscopy (n = 16). Monocyte-derived dendritic cells from healthy volunteers were used to assess the effects of cis- and trans-UCA on dendritic cell phenotype by using flow cytometry (n = 11). RESULTS: Epidermal APCs from FLG-null subjects had increased CD11c expression. Confocal microscopy confirmed this and additionally revealed an increased number of epidermal CD83(+) Langerhans cells in FLG-null subjects. In vitro differentiation in the presence of cis-UCA significantly reduced costimulatory molecule expression on monocyte-derived dendritic cells from healthy volunteers and increased their ability to induce a regulatory T-cell phenotype in mixed lymphocyte reactions. CONCLUSIONS: We show that subjects with FLG-null mutations have more mature Langerhans cells in nonlesional skin irrespective of whether they have AD. We also demonstrate that cis-UCA reduces maturation of dendritic cells and increases their capacity to induce regulatory T cells, suggesting a novel link between filaggrin deficiency and immune dysregulation.

Combination peptide immunotherapy based on T-cell epitope mapping reduces allergen-specific IgE and eosinophilia in allergic airway inflammation.

Peptide immunotherapy using soluble peptides containing allergen-derived immunodominant T-cell epitopes holds therapeutic promise for allergic asthma. Previous studies in BALB/c mice using the immunodominant peptide epitope of chicken ovalbumin (p323-339) have been unable to demonstrate therapeutic effects in ovalbumin-induced allergic airway inflammation. We have previously shown that intravenous application of p323-339 can effectively tolerise p323-339-reactive T cells in a non-allergic model in C57BL/6 mice. This study aimed to assess the effects of using p323-339 immunotherapy in a C57BL/6 model of ovalbumin-induced allergic airway inflammation, identify any additional epitopes recognized by the ovalbumin-responsive T-cell repertoire in C57BL/6 mice and assess the effects of combination peptide immunotherapy in this model. Ovalbumin-reactive T-cell lines were generated from ovalbumin-immunized C57BL/6 mice and proliferative responses to a panel of overlapping peptides covering the ovalbumin sequence were assessed. Soluble peptides (singly or combined) were administered intravenously to C57BL/6 mice before the induction of ovalbumin-induced allergic airway inflammation. Peptide immunotherapy using the 323-339 peptide alone did not reduce the severity of allergic airway inflammation. An additional immunodominant T-cell epitope in ovalbumin was identified within the 263-278 sequence. Combination peptide immunotherapy, using the 323-339 and 263-278 peptides together, reduced eosinophilia in the bronchoalveolar lavage and ovalbumin-specific IgE, with apparent reductions in interleukin-5 and interleukin-13. Characterization of the T-cell response to a model allergen has allowed the development of combination peptide immunotherapy with improved efficacy in allergic airway inflammation. This model holds important potential for future mechanistic studies using peptide immunotherapy in allergy.

Kynurenine monooxygenase regulates inflammation during critical illness and recovery in experimental acute pancreatitis.

Kynurenine monooxygenase (KMO) blockade protects against multiple organ failure caused by acute pancreatitis (AP), but the link between KMO and systemic inflammation has eluded discovery until now. Here, we show that the KMO product 3-hydroxykynurenine primes innate immune signaling to exacerbate systemic inflammation during experimental AP. We find a tissue-specific role for KMO, where mice lacking Kmo solely in hepatocytes have elevated plasma 3-hydroxykynurenine levels that prime inflammatory gene transcription. 3-Hydroxykynurenine synergizes with interleukin-1ß to cause cellular apoptosis. Critically, mice with elevated 3-hydroxykynurenine succumb fatally earlier and more readily to experimental AP. Therapeutically, blockade with the highly selective KMO inhibitor GSK898 rescues the phenotype, reducing 3-hydroxykynurenine and protecting against critical illness and death. Together, our findings establish KMO and 3-hydroxykynurenine as regulators of inflammation and the innate immune response to sterile inflammation. During critical illness, excess morbidity and death from multiple organ failure can be rescued by systemic KMO blockade.

The role of macrophages in healing the wounded lung.

Acute tissue injury is often considered in the context of a wound. The host response to wounding is an orchestrated series of events, the fundamentals of which are preserved across all multicellular organisms. In the human lung, there are a myriad of causes of injury, but only a limited number of consequences: complete resolution, persistent and/or overwhelming inflammation, a combination of resolution/remodelling with fibrosis or progressive fibrosis. In all cases where complete resolution does not occur, there is the potential for significant ongoing morbidity and ultimately death through respiratory failure. In this review, we consider the elements of injury, resolution and repair as they occur in the lung. We specifically focus on the role of the macrophage, long considered to have a pivotal role in regulating the host response to injury and tissue repair.

Septic arthritis in an in vivo murine model induced by Staphylococcus aureus : a comparison between actions of the haemolysin toxin and the effects of the host immune response.

AIMS: Staphylococcus aureus is a major cause of septic arthritis, and in vitro studies suggest α haemolysin (Hla) is responsible for chondrocyte death. We used an in vivo murine joint model to compare inoculation with wild type S. aureus 8325-4 with a Hla-deficient strain DU1090 on chondrocyte viability, tissue histology, and joint biomechanics. The aim was to compare the actions of S. aureus Hla alone with those of the animal's immune response to infection. METHODS: Adult male C57Bl/6 mice (n = 75) were randomized into three groups to receive 1.0 to 1.4 × 107 colony-forming units (CFUs)/ml of 8325-4, DU1090, or saline into the right stifle joint. Chondrocyte death was assessed by confocal microscopy. Histological changes to inoculated joints were graded for inflammatory responses along with gait, weight changes, and limb swelling. RESULTS: Chondrocyte death was greater with 8325-4 (96.2% (SD 5.5%); p < 0.001) than DU1090 (28.9% (SD 16.0%); p = 0.009) and both were higher than controls (3.8% (SD 1.2%)). Histology revealed cartilage/bone damage with 8325-4 or DU1090 compared to controls (p = 0.010). Both infected groups lost weight (p = 0.006 for both) and experienced limb swelling (p = 0.043 and p = 0.018, respectively). Joints inoculated with bacteria showed significant alterations in gait cycle with a decreased stance phase, increased swing phase, and a corresponding decrease in swing speed. CONCLUSION: Murine joints inoculated with Hla-producing 8325-4 experienced significantly more chondrocyte death than those with DU1090, which lack the toxin. This was despite similar immune responses, indicating that Hla was the major cause of chondrocyte death. Hla-deficient DU1090 also elevated chondrocyte death compared to controls, suggesting a smaller additional deleterious role of the immune system on cartilage.Cite this article: Bone Joint Res 2022;11(9):669-678.

Immunity and vaccines against sexually transmitted Chlamydia trachomatis infection.

PURPOSE OF REVIEW: The aim is to review recent findings on immunity and vaccine development to Chlamydia trachomatis. RECENT FINDINGS: There is increasing knowledge on the interactions between C. trachomatis and infected host cells. During genital infection the organism avoids generating protective immunity but immune responses to a number of chlamydial proteins have been associated with reproductive tract pathology. Various vaccine and adjuvant preparations have been tried experimentally. Information generated by proteomics and complex studies of serological and T-lymphocyte immune responses points to novel vaccine candidates. SUMMARY: C. trachomatis, an obligate intracellular bacterium, is the commonest sexually transmitted infection worldwide and is associated with reproductive pathology. To develop rational vaccines it is necessary to understand the complex lifecycle of the organism, the host immune response to infection and how these relate to disease. Infection does not prevent re-infection and antibiotic treatment prevents antibody production at a population level. It remains unclear what type of immune response would be sufficient to prevent infection and/or re-infection. Although the prevalence and demographics of infection and the severity of disease associations suggest that it would be desirable, there is no vaccine currently available. A number of studies have identified novel vaccine candidates.

Oxidative damage and TGF-ß differentially induce lung epithelial cell sonic hedgehog and tenascin-C expression: implications for the regulation of lung remodelling in idiopathic interstitial lung disease.

Idiopathic interstitial lung diseases (iILDs) are characterized by inflammation, hyperplasia of Type-II alveolar epithelial cells (AECs) and lung remodelling often with progressive fibrosis. It remains unclear which signals initiate iILD and/or maintain the disease processes. Using real-time RT-PCR and immunohistochemistry on archival biopsies of three patterns of iILD (usual interstitial pneumonitis/UIP, non-specific interstitial pneumonitis/NSIP and cryptogenic organizing pneumonia/COP) we investigated whether hedgehog signalling (previously associated with lung damage and repair) was functional and whether the damage associated extracellular matrix protein tenascin-C was present in activated Type-II AECs in all three iILDs. Using tissue culture, protein and mRNA detection we also determined how two signals (oxidative damage and TGF-ß) associated with iILD pathogenesis affected Sonic hedgehog (SHH) and tenascin-C production by a Type-II AEC cell line. We report that SHH pathway and tenascin-C mRNA and proteins were found in UIP, NSIP and COP. SHH signalling was most active at sites of immature organizing fibrous tissue (fibroblastic foci) in UIP. In vitro Type-II AECs constitutively secrete SHH but not tenascin-C. Oxidative injury stimulated SHH release whereas TGF-ß inhibited it. TGF-ß and oxidative damage both upregulated tenascin-C mRNA but only TGF-ß induced synthesis and release of a distinct protein isoform. SHH signalling is active in Type-II AECs from three types of ILD and all three express tenascin-C.

Modified adenine (9-benzyl-2-butoxy-8-hydroxyadenine) redirects Th2-mediated murine lung inflammation by triggering TLR7.

Substitute adenine (SA)-2, a synthetic heterocycle chemically related to adenine with substitutions in positions 9-, 2-, and 8- (i.e., 9-benzyl-2-butoxy-8-hydroxyadenine), induces in vitro immunodeviation of Th2 cells to a Th0/Th1 phenotype. In this article, we evaluate the in vivo ability of SA-2 to affect Th2-mediated lung inflammation and its safety. TLR triggering and NF-kappaB activation by SA-2 were analyzed on TLR-transfected HEK293 cells and on purified bone marrow dendritic cells. The in vivo effect of SA-2 on experimental airway inflammation was evaluated in both prepriming and prechallenge protocols by analyzing lung inflammation, including tissue eosinophilia and goblet cell hyperplasia, bronchoalveolar lavage fluid cell types, and the functional profile of Ag-specific T cells from draining lymph nodes and spleens. SA-2 induced mRNA expression and production of proinflammatory (IL-6, IL-12, and IL-27) and regulatory (IL-10) cytokines and chemokines (CXCL10) in dendritic cells but down-regulated TGF-beta. Prepriming administration of SA-2 inhibited OVA-specific Abs and Th2-driven lung inflammation, including tissue eosinophilia and goblet cells, with a prevalent Foxp3-independent regulatory mechanism. Prechallenge treatment with SA-2 reduced the lung inflammation through the induction of a prevalent Th1-related mechanism. In this model the activity of SA-2 was route-independent, but adjuvant- and Ag dose-dependent. SA-2-treated mice did not develop any increase of serum antinuclear autoantibodies. In conclusion, critical substitutions in the adenine backbone creates a novel synthetic TLR7 ligand that shows the ability to ameliorate Th2-mediated airway inflammation by a complex mechanism, involving Th1 redirection and cytokine-mediated regulation, which prevents autoreactivity.

Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes.

BACKGROUND: Large production volumes of zinc oxide nanoparticles (ZnONP) might be anticipated to pose risks, of accidental inhalation in occupational and even in consumer settings. Herein, we further investigated the pathological changes induced by ZnONP and their possible mechanism of action. METHODS: Two doses of ZnONP (50 and 150 cm2/rat) were intratracheally instilled into the lungs of rats with assessments made at 24 h, 1 wk, and 4 wks after instillation to evaluate dose- and time-course responses. Assessments included bronchoalveolar lavage (BAL) fluid analysis, histological analysis, transmission electron microscopy, and IgE and IgA measurement in the serum and BAL fluid. To evaluate the mechanism, alternative ZnONP, ZnONP-free bronchoalveolar lavage exudate, and dissolved Zn2+ (92.5 µg/rat) were also instilled to rats. Acridine orange staining was utilized in macrophages in culture to evaluate the lysosomal membrane destabilization by NP. RESULTS: ZnONP induced eosinophilia, proliferation of airway epithelial cells, goblet cell hyperplasia, and pulmonary fibrosis. Bronchocentric interstitial pulmonary fibrosis at the chronic phase was associated with increased myofibroblast accumulation and transforming growth factor-ß positivity. Serum IgE levels were up-regulated by ZnONP along with the eosinophilia whilst serum IgA levels were down-regulated by ZnONP. ZnONP are rapidly dissolved under acidic conditions (pH 4.5) whilst they remained intact around neutrality (pH 7.4). The instillation of dissolved Zn2+ into rat lungs showed similar pathologies (eg., eosinophilia, bronchocentric interstitial fibrosis) as were elicited by ZnONP. Lysosomal stability was decreased and cell death resulted following treatment of macrophages with ZnONP in vitro. CONCLUSIONS: We hypothesise that rapid, pH-dependent dissolution of ZnONP inside of phagosomes is the main cause of ZnONP-induced diverse progressive severe lung injuries.

Cervical epithelial damage promotes Ureaplasma parvum ascending infection, intrauterine inflammation and preterm birth induction in mice.

Around 40% of preterm births are attributed to ascending intrauterine infection, and Ureaplasma parvum (UP) is commonly isolated in these cases. Here we present a mouse model of ascending UP infection that resembles human disease, using vaginal inoculation combined with mild cervical injury induced by a common spermicide (Nonoxynol-9, as a surrogate for any mechanism of cervical epithelial damage). We measure bacterial load in a non-invasive manner using a luciferase-expressing UP strain, and post-mortem by qPCR and bacterial titration. Cervical exposure to Nonoxynol-9, 24 h pre-inoculation, facilitates intrauterine UP infection, upregulates pro-inflammatory cytokines, and increases preterm birth rates from 13 to 28%. Our results highlight the crucial role of the cervical epithelium as a barrier against ascending infection. In addition, we expect the mouse model will facilitate further research on the potential links between UP infection and preterm birth.

Kynurenine 3-monooxygenase is a critical regulator of renal ischemia-reperfusion injury.

Acute kidney injury (AKI) following ischemia-reperfusion injury (IRI) has a high mortality and lacks specific therapies. Here, we report that mice lacking kynurenine 3-monooxygenase (KMO) activity (Kmonull mice) are protected against AKI after renal IRI. We show that KMO is highly expressed in the kidney and exerts major metabolic control over the biologically active kynurenine metabolites 3-hydroxykynurenine, kynurenic acid, and downstream metabolites. In experimental AKI induced by kidney IRI, Kmonull mice had preserved renal function, reduced renal tubular cell injury, and fewer infiltrating neutrophils compared with wild-type (Kmowt) control mice. Together, these data confirm that flux through KMO contributes to AKI after IRI, and supports the rationale for KMO inhibition as a therapeutic strategy to protect against AKI during critical illness.

Intrapulmonary Autoantibodies to HSP72 Are Associated with Improved Outcomes in IPF.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease, with high mortality. Currently, the aetiology and the pathology of IPF are poorly understood, with both innate and adaptive responses previously being implicated in the disease pathogenesis. Heat shock proteins (Hsp) and antibodies to Hsp in patients with IPF have been suggested as therapeutic targets and prognostic biomarkers, respectively. We aimed to study the relationship between the expression of Hsp72 and anti-Hsp72 antibodies in the BAL fluid and serum Aw disease progression in patients with IPF. METHODS: A novel indirect ELISA to measure anti-Hsp72 IgG was developed and together with commercially available ELISAs used to detect Hsp72 IgG, Hsp72 IgGAM, and Hsp72 antigen, in the serum and BALf of a cohort of IPF (n = 107) and other interstitial lung disease (ILD) patients (n = 66). Immunohistochemistry was used to detect Hsp72 in lung tissue. The cytokine expression from monocyte-derived macrophages was measured by ELISA. RESULTS: Anti-Hsp72 IgG was detectable in the serum and BALf of IPF (n = 107) and other ILDs (n = 66). Total immunoglobulin concentrations in the BALf showed an excessive adaptive response in IPF compared to other ILDs and healthy controls (p = 0.026). Immunohistochemistry detection of C4d and Hsp72 showed that these antibodies may be targeting high expressing Hsp72 type II alveolar epithelial cells. However, detection of anti-Hsp72 antibodies in the BALf revealed that increasing concentrations were associated with improved patient survival (adjusted HR 0.62, 95% CI 0.45-0.85; p = 0.003). In vitro experiments demonstrate that anti-Hsp72 complexes stimulate macrophages to secrete CXCL8 and CCL18. CONCLUSION: Our results indicate that intrapulmonary anti-Hsp72 antibodies are associated with improved outcomes in IPF. These may represent natural autoantibodies, and anti-Hsp72 IgM and IgA may provide a beneficial role in disease pathogenesis, though the mechanism of action for this has yet to be determined.

An Orally Active Galectin-3 Antagonist Inhibits Lung Adenocarcinoma Growth and Augments Response to PD-L1 Blockade.

A combination therapy approach is required to improve tumor immune infiltration and patient response to immune checkpoint inhibitors that target negative regulatory receptors. Galectin-3 is a ß-galactoside-binding lectin that is highly expressed within the tumor microenvironment of aggressive cancers and whose expression correlates with poor survival particularly in patients with non-small cell lung cancer (NSCLC). To examine the role of galectin-3 inhibition in NSCLC, we tested the effects of galectin-3 depletion using genetic and pharmacologic approaches on syngeneic mouse lung adenocarcinoma and human lung adenocarcinoma xenografts. Galectin-3-/- mice developed significantly smaller and fewer tumors and metastases than syngeneic C57/Bl6 wild-type mice. Macrophage ablation retarded tumor growth, whereas reconstitution with galectin-3-positive bone marrow restored tumor growth in galectin-3-/- mice, indicating that macrophages were a major driver of the antitumor response. Oral administration of a novel small molecule galectin-3 inhibitor GB1107 reduced human and mouse lung adenocarcinoma growth and blocked metastasis in the syngeneic model. Treatment with GB1107 increased tumor M1 macrophage polarization and CD8+ T-cell infiltration. Moreover, GB1107 potentiated the effects of a PD-L1 immune checkpoint inhibitor to increase expression of cytotoxic (IFNγ, granzyme B, perforin-1, Fas ligand) and apoptotic (cleaved caspase-3) effector molecules. In summary, galectin-3 is an important regulator of lung adenocarcinoma progression. The novel galectin-3 inhibitor presented could provide an effective, nontoxic monotherapy or be used in combination with immune checkpoint inhibitors to boost immune infiltration and responses in lung adenocarcinoma and potentially other aggressive cancers. SIGNIFICANCE: A novel and orally active galectin-3 antagonist inhibits lung adenocarcinoma growth and metastasis and augments response to PD-L1 blockade.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1480/F1.large.jpg.

Analysis of germline GLI1 variation implicates hedgehog signalling in the regulation of intestinal inflammatory pathways.

BACKGROUND: Ulcerative colitis (UC) and Crohn's disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis. METHODS AND FINDINGS: Using a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C-->G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09-1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1(+/lacZ) mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1(+/lacZ) mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model. CONCLUSIONS: HH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.

Commercially available recombinant sonic hedgehog up-regulates Ptc and modulates the cytokine and chemokine expression of human macrophages: an effect mediated by endotoxin contamination?

The Sonic hedgehog (Shh) signalling pathway plays an important role in developmental patterning and proliferation. Recent evidence suggests that Shh also plays a role in the development of the immune system. Here, we demonstrate that components of the Shh signalling pathway are expressed in human macrophages and that the receptor for Shh, Ptc, is up-regulated by a commercially available recombinant preparation of Shh (CArShh). Further, we report that the addition of CArShh up-regulates the production of IL-6, IL-8, MCP-1, IP-10, MIG and RANTES by macrophages, an effect enhanced by the presence of fetal calf serum in the culture medium. In contrast, TGF-beta, TNF-alpha, IL-1b, IL-12 and IL-10 production were not modulated by CArShh and VEGF was minimally up-regulated even in the presence of serum. The up-regulation of these cytokines and chemokines was abrogated by CD14 inhibition and polymixin B, but not reliably inhibited by the specific Shh pathway inhibitor cyclopamine. These results suggest that, although components of the Shh signalling pathway are expressed in macrophages, the modulation of macrophage cytokine and chemokine effector function seen in response to commercially available rShh results from low levels of endotoxin contained within the CArShh preparations employed to explore the effects of Shh in vitro.

Host chemokine signature as a biomarker for the detection of pre-cancerous cervical lesions.

BACKGROUND: The ability to distinguish which hrHPV infections predispose to significant disease is ever more pressing as a result of the increasing move to hrHPV testing for primary cervical screening. A risk-stratifier or "triage" of infection should ideally be objective and suitable for automation given the scale of screening. RESULTS: CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL12 emerged as the strongest, candidate biomarkers to detect underlying disease [cervical intraepithelial neoplasia grade 2 or worse (CIN2+)]. For CIN2+, CCL2 had the highest area under the curve (AUC) of 0.722 with a specificity of 82%. A combined biomarker panel of six chemokines CCL2, CCL3, CCL4, CXCL1, CXCL8, and CXCL12 provides a sensitivity of 71% and specificity of 67%. CONCLUSION: The present work demonstrates that the levels of five chemokine-proteins are indicative of underlying disease. We demonstrate technical feasibility and promising clinical performance of a chemokine-based biomarker panel, equivalent to that of other triage options. Further assessment in longitudinal series is now warranted. METHODS: A panel of 31 chemokines were investigated for expression in routinely taken archived and prospective cervical liquid based cytology (LBC) samples using Human Chemokine Proteomic Array kit. Nine chemokines were further validated using Procartaplex assay on the Luminex platform.