Pleural cytokines MIF and MIP-3α as novel biomarkers for complicated parapneumonic effusions and empyema.

Patients with complicated parapneumonic effusion (CPPE)/empyema have high morbidity and mortality, particularly when adequate management is delayed. We aimed to investigate novel dysregulated cytokines that can be used as biomarkers for infectious pleural effusions, especially for CPPE/empyema. Expression of 40 cytokines in parapneumonic effusions (PPE) was screened in the discovery phase, involving 63 patients, using a multiplex immunobead-based assay. Six cytokines were subsequently validated by enzyme-linked immunosorbent assays (ELISAs). We then used ELISA to further evaluate the diagnostic values and cutoff values of these cytokines as potential biomarkers in an expanded group that included 200 patients with uncomplicated parapneumonic effusion (UPPE), CPPE, empyema, transudates, other exudates, and malignant pleural effusion (MPE). The pleural levels of four cytokines (MIF, MIP-3α, IL-1ß, ENA-78) were highest and significantly increased in CPPE/empyema compared with those in other etiologies. According to receiver operating characteristic curve analysis, the four cytokines (MIF, MIP-3α, IL-1ß, and ENA-78) had areas under the curve (AUCs) greater than 0.710 for discriminating parapneumonic pleural effusion from noninfectious pleural effusions. In a comparison of nonpurulent CPPE with UPPE, logistic regression analysis revealed that pleural fluid MIF ≥ 12 ng/ml and MIP-3α ≥ 4.3 ng/ml had the best diagnostic value; MIF also displayed the highest odds ratio of 663 for nonpurulent CPPE, with 97.5% specificity, 94.44% sensitivity, and an AUC of 0.950. In conclusion, our results show that elevated MIF and MIP-3α may be used as novel biomarkers for PPE diagnosis, particularly in patients with CPPE/empyema; the findings indicate that dysregulated cytokine expression may provide clues about the pathogenesis of pleural infection.

Expressions of IL-8 and CXCL5 in uterine endometrioid carcinomas which have frequent neutrophil infiltration and comparison to colorectal adenocarcinoma.

In endometrioid carcinomas (ECs) of the uterine corpus, neutrophil accumulation within the carcinoma cell clusters is a representative microscopic finding. Because this accumulation is active, some sort of transmitter ought to exist between the EC cells and neutrophils. Interleukin-8 (IL-8) and C-X-C motif chemokine ligand 5 (CXCL5) is a cytokine that attracts neutrophils in vivo. In this study, we investigated IL-8, CXCL5 and C-X-C motif chemokine receptor 2 (CXCR2) (their chemokine receptor) expressions in ECs by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). There are few reports on the relationship between these chemokines and ECs. For comparison, we enrolled samples of colorectal adenocarcinoma (CRAC), it is another representative tumor with neutrophil infiltration. We analyzed 30 ECs and 30 CRACs. We confirmed IL-8 expression (H-score ≥50 points) in 40% of EC and 7% of CRAC samples; CXCL5 expression in 7% of EC and 10% of CRAC samples; CXCR2 expression in 83% of EC and 53% of CRAC samples by immunohistochemistry. We examined each mRNA (IL-8 and CXCL5) expression of 3 representative EC and 3 CRAC samples. Finding IL-8 expression might indicate that this cytokine is important for the process of neutrophil accumulation, particularly within ECs. The participation of CXCL5 regarding neutrophil accumulation within their carcinoma cell clusters might be restrictive compared to IL-8.

Efficacy of bevacizumab-containing chemotherapy in metastatic colorectal cancer and CXCL5 expression: Six case reports.

BACKGROUND: In metastatic colorectal cancer (mCRC), the anti-vascular endothelial growth factor drug bevacizumab (BVZ) plus chemotherapy significantly improves progression-free survival compared to chemotherapy (CT) alone. This benefit is not, however, observed in all patients. While increased chemokine CXCL5 gene expression promoting angiogenesis has been proposed as a prognostic mCRC biomarker, few studies have examined its relationship with drug efficacy. This study sought to analyze tumor CXCL5 gene expression in six patients with different efficacy of BVZ-containing CT in terms of the tumor response to treatment. CASE SUMMARY: We report six cases of stage IV KRAS-mutated mCRC. Patients were given first line treatment with BVZ-containing chemotherapy in University Hospital of Fuenlabrada. The six patients differed in terms of primary tumor location (right/left side), tumor burden (mostly hepatic and peritoneal disease) and clinical disease course. Before treatment onset, total RNA was isolated from paraffinated tumor biopsy specimens and CXCL5 gene expression quantified through conventional RT-qPCR procedures. Our main finding was that CXCL5 expression levels were several times higher in three patients with lower progression free survival (under 6 mo) from the start of treatment. CONCLUSION: A higher expression of CXCL5 was observed in the three patients showing worse tumor response to treatment.

Impact of HIV-ART on the restoration of Th17 and Treg cells in blood and female genital mucosa.

The aim of this study was to evaluate the effectiveness of antiretroviral treatment (ART) on the proportion and functions of Th17 and Treg cells in peripheral blood and female genital tract (FGT) respectively. To this aim, samples from 41 HIV-neg, 33 HIV+ ART-naïve and 32 HIV+ ART+ subjects were obtained. In peripheral blood, altered Th17 and Th17/Treg proportions were normalized in HIV+ ART+, but certain abnormal Treg and activated T-cell proportions were still observed. In FGT, abnormal patterns of secretion for Th17-related cytokines were observed in cervical mononuclear cells (CMCs) from HIV+ women, even in those from HIV+ ART+, compared to the HIV-neg group. Moreover, these altered patterns of secretion were associated with diminished levels of CXCL5 and CXCL1 chemokines and with an immunoregulatory skew in the CCL17/CCL20 ratio in ectocervix samples of these women. Finally, ART did not restore proportions of Th17-precursor cells with gut-homing potential in PBMCs, and positive correlations between these cells and the levels of IL-17F and IL-21 production by CMCs may suggest that a better homing of these cells to the intestine could also imply a better restoration of these cells in the female genital tract. These results indicate that antiretroviral treatment did not restore Th17-related immune functions completely at the female mucosal level.

Development of a bifunctional nanobiosensor for screening and detection of chemokine ligand in colorectal cancer cell line.

Highly sensitive detection of chemokines in various biological matrices and its interaction with a natural receptor molecule has tremendous importance in cell signaling, medical diagnostics, and therapeutics. In this direction, we have designed the first bifunctional nanobiosensor for chemokine screening and detection in a single experimental setting. The sensor probe was fabricated by immobilizing CXCR2 on the gold nanoparticles (AuNPs) deposited 2,2':5',2''-terthiophene-3' (p-benzoic acid) (TBA) nanocomposite film. The interaction between CXCR2 and chemokines was studied using electrochemical impedance spectroscopy (EIS) and voltammetry. CXCL5 among three ligands showed the strongest affinity to CXCR2, which was further utilized to develop an amperometric CXCL5 biosensor. Analytical parameters, such as CXCR2 receptor concentration, temperature, pH, and incubation time were optimized to obtain the high sensitivity. A dynamic range for CXCL5 detection was obtained between 0.1 and 10ng/mL with the detection limit of 0.078 ± 0.004ng/mL (RSD < 4.7%). The proposed biosensor was successfully applied to detect CXCL5 in clinically relevant concentrations in human serum and colorectal cancer cells samples with high sensitivity and selectivity. Interference effect and the stability of the developed biosensor were also evaluated. Method verification was performed by comparing the results using commercially available ELISA kit for CXCL5 detection.

Ex vivo exposure of bone marrow from chronic kidney disease donor rats to pravastatin limits renal damage in recipient rats with chronic kidney disease.

INTRODUCTION: Healthy bone marrow cell (BMC) infusion improves renal function and limits renal injury in a model of chronic kidney disease (CKD) in rats. However, BMCs derived from rats with CKD fail to retain beneficial effects, demonstrating limited therapeutic efficacy. Statins have been reported to improve cellular repair mechanisms. METHODS: We studied whether exposing CKD rat BMCs ex vivo to pravastatin improved their in vivo therapeutic efficacy in CKD and compared this to systemic in vivo treatment. Six weeks after CKD induction, healthy BMCs, healthy pravastatin-pretreated BMCs, CKD BMCs or CKD pravastatin-pretreated BMCs were injected into the renal artery of CKD rats. RESULTS: At 6 weeks after BMC injection renal injury was reduced in pravastatin-pretreated CKD BMC recipients vs. CKD BMC recipients. Effective renal plasma flow was lower and filtration fraction was higher in CKD BMC recipients compared to all groups whereas there was no difference between pravastatin-pretreated CKD BMC and healthy BMC recipients. Mean arterial pressure was higher in CKD BMC recipients compared to all other groups. In contrast, 6 weeks of systemic in vivo pravastatin treatment had no effect. In vitro results showed improved migration, decreased apoptosis and lower excretion of pro-inflammatory Chemokine (C-X-C Motif) Ligand 5 in pravastatin-pretreated CKD BMCs. CONCLUSIONS: Short ex vivo exposure of CKD BMC to pravastatin improves CKD BMC function and their subsequent therapeutic efficacy in a CKD setting, whereas systemic statin treatment did not provide renal protection.

Matrix metalloproteinases and myeloperoxidase in gingival crevicular fluid provide site-specific diagnostic value for chronic periodontitis.

AIM: To identify the diagnostic accuracy of gingival crevicular fluid (GCF) candidate biomarkers to discriminate periodontitis from the inflamed and healthy sites, and to compare the performance of two independent matrix metalloproteinase (MMP)-8 immunoassays. MATERIALS AND METHODS: Cross sectional study. GCF (N = 58 sites) was collected from healthy, gingivitis and chronic periodontitis volunteers and analysed for levels of azurocidin, chemokine ligand 5, MPO, TIMP-1 MMP-13 and MMP-14 by ELISA or activity assays. MMP-8 was assayed by immunofluorometric assay (IFMA) and ELISA. Statistical analysis was performed using linear mixed-effects models and Bayesian statistics in R and Stata V11. RESULTS: MMP-8, MPO, azurocidin and total MMP-13 and MMP-14 were higher in periodontitis compared to gingivitis and healthy sites (p < 0.05). A very high correlation between MPO and MMP-8 was evident in the periodontitis group (r = 0.95, p < 0.0001). MPO, azurocidin and total levels of MMP-8, MMP-13 and MMP-14 showed high diagnostic accuracy (≥0.90), but only MMP-8 and MPO were significantly higher in the periodontitis versus gingivitis sites. MMP-8 determined by IFMA correlated more strongly with periodontal status and showed higher diagnostic accuracy than ELISA. CONCLUSIONS: MPO and collagenolytic MMPs are highly discriminatory biomarkers for site-specific diagnosis of periodontitis. The comparison of two quantitative MMP-8 methods demonstrated IFMA to be more accurate than ELISA.

Damaging role of neutrophilic infiltration in a mouse model of progressive tuberculosis.

Tuberculosis was studied using an experimental model based on the C3HeB/FeJ mouse strain, which mimics the liquefaction of caseous necrosis occurring during active disease in immunocompetent adults. Mice were intravenously infected with 2 × 10(4) Colony Forming Units of Mycobacterium tuberculosis and their histopathology, immune response, bacillary load, and survival were evaluated. The effects of the administration of drugs with anti-inflammatory activity were examined, and the C3H/HeN mouse strain was also included for comparative purposes. Massive intra-alveolar neutrophilic infiltration led to rapid granuloma growth and coalescence of lesions into superlesions. A central necrotic area appeared showing progressive cellular destruction, the alveoli cell walls being initially conserved (caseous necrosis) but finally destroyed (liquefactive necrosis). Increasing levels of pro-inflammatory mediators were detected in lungs. C3HeB/FeJ treated with anti-inflammatory drugs and C3H/HeN animals presented lower levels of pro-inflammatory mediators such as TNF-α, IL-17, IL-6 and CXCL5, a lower bacillary load, better histopathology, and increased survival compared with untreated C3HeB/FeJ. The observation of massive neutrophilic infiltration suggests that inflammation may be a key factor in progression towards active tuberculosis. On the basis of our findings, we consider that the C3HeB/FeJ mouse model would be useful for evaluating new therapeutic strategies against human tuberculosis.

Biomarkers from bronchoalveolar lavage fluid in systemic sclerosis patients with interstitial lung disease relate to severity of lung fibrosis.

OBJECTIVES: Decision on treatment of systemic sclerosis (SSc) related interstitial lung disease (ILD) largely relies on the findings on high resolution computed tomography (HRCT) and there is a need for improvement in assessment of the fibrotic activity. The objectives of this study were to study biomarkers in bronchoalveolar lavage fluid (BALF) from SSc patients with ILD and to relate the findings to the severity and activity of lung fibrosis. METHODS: Fifteen patients with early SSc and 12 healthy controls were subjected to BAL. Cell counts and analyses of CXCL5, CXCL8 and S100A8/A9 were performed in BALF and serum. COMP and KL-6 were measured in serum. HRCT of lungs was quantified for ground glass opacities (GGO), reticulation and traction bronchiectases. RESULTS: BALF concentrations of CXCL8 (p < 0.001), CXCL5 (p = 0.002) and S100A8/A9 (p = 0.016) were higher in patients than controls. Serum KL-6 (p < 0.001) was increased in SSc patients and correlated with BALF concentration of eosinophils (rS = 0.57, p = 0.027). Patients with more widespread GGO on HRCT were characterised in BALF by a higher eosinophil count (p = 0.002) and in serum by higher KL-6 (p = 0.008). Patients with more fibrosis were characterised in BALF by higher eosinophil count (p = 0.014), higher CXCL8 (p = 0.005) and S100A8A/A9 (p = 0.014) concentration and in serum by a higher serum COMP (p = 0.023). CONCLUSIONS: In SSc related ILD, biomarkers from BALF and serum correlate to findings on HRCT suggesting usefulness as markers of presence and extent of lung fibrosis.

Cytokine profile of bronchoalveolar lavage fluid in patients with combined pulmonary fibrosis and emphysema.

BACKGROUND AND OBJECTIVE: Combined pulmonary fibrosis and emphysema (CPFE) is characterized by upper lobe emphysema together with lower lobe fibrosis. The aim of this study was to examine whether cytokine levels in the alveolar space are associated with emphysematous changes superimposed on pulmonary fibrosis. METHODS: Consecutive patients (n = 102), diagnosed with pulmonary fibrosis were retrospectively evaluated. Cytokine levels and differential cell counts in bronchoalveolar lavage (BAL) fluid, pulmonary function, computed tomography (CT) scores and levels of serum markers were compared between patients with or without emphysema. RESULTS: Among the 102 patients (14 females, mean age 68 years), 38 (37%) had evidence of upper lobe emphysema on computed tomography (CT). Levels of epithelial neutrophil activating peptide 78 (ENA-78/CXCL5) and interleukin (IL)-8/CXCL8 in BAL fluid were significantly higher in patients with emphysema. Vital capacity (VC, % predicted) was greater, and ratio of forced expiratory volume in 1 s/forced vital capacity and diffusing capacity of carbon monoxide (DL(CO))/alveolar volume (V(A)) were lower in patients with emphysema. CXCL8 and CXCL5 levels were associated with percentage or absolute numbers of neutrophils in BAL fluid. In addition, CXCL8 levels were inversely correlated with VC and DL(CO)/V(A), and positively correlated with composite physiological index (CPI) and the extent of areas of low attenuation on CT. CONCLUSIONS: Increased CXC chemokine levels in the airspaces may be associated with emphysematous lung changes in patients with pulmonary fibrosis.

Neutrophil chemotaxis in granulomatosis with polyangiitis (Wegener's) and idiopathic pulmonary fibrosis.

The presence of antineutrophil cytoplasmic antibodies in granulomatosis with polyangiitis (Wegener's) (GPA) implicates the neutrophil as a key effector cell. Previous studies have reported elevated neutrophil counts in the lung, although the determinants of neutrophil chemotaxis in the GPA lung are unknown. Bronchoalveolar lavage fluid (BALF) cell counts, myeloperoxidase (MPO) and chemokines were measured in 27 patients with GPA, 20 disease controls with idiopathic pulmonary fibrosis (IPF) and six healthy controls. CXC chemokine ligand (CXCL)8, interleukin (IL)-1ß, epithelial neutrophil-activating protein 78, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor were measured by ELISA. The neutrophil chemotactic potential of BALF was investigated using the under-agarose method, and specific antibodies were used to examine the role of CXCL8 and IL-1ß. GPA BALF had an increased neutrophil percentage, and elevated MPO, CXCL8 and G-CSF concentrations compared with healthy controls. Chemotaxis of control neutrophils towards BALF from patients with active (p=0.006) and remission (p=0.077) GPA, and IPF (p=0.001) patients was increased compared with normal controls. BALF-induced chemotaxis correlated with BALF IL-1ß (r=0.761, p=0.001) and CXCL8 (r=0.640, p=0.012) in GPA, and was inhibited by anti-CXCL8 (85%; p<0.001) and anti-IL-1ß (69%; p<0.001). Our study confirms a neutrophilia and pro-inflammatory alveolar milieu that persists in clinical remission. CXCL8 and IL-1ß appear to play important roles in the neutrophil chemotactic response to BALF.

Serum perioperative profile of cytokines in patients with squamous cell carcinoma of the larynx.

OBJECTIVE: Altered immune, inflammatory, and angiogenesis responses have been noticed in head and neck cancer, and many of these responses have been associated with a poor clinical outcome. The objective of this study was to evaluate several immune mediators in the sera of patients with squamous cell carcinoma (SCC) of the larynx undergoing curative surgery in connection with clinicopathologic factors. METHODS: Multiplex analysis of cytokines (interleukin [IL]-6, IL-8, IL-10, tumour necrosis factor α [TNF-α], interferon-γ [IFN-γ]), chemokines (monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein 1α [MIP-1α], and epithelial neutrophil-activating protein 78 [ENA-78]), and growth factors (vascular endothelial growth factor and basic fibroblast growth factor) in the serum of patients with laryngeal cancer and healthy controls was performed using xMap technology. RESULTS: Patients with SCC presented an altered cytokine profile compared to healthy controls, both preoperatively (higher levels of IL-8 and IL-10) and postoperatively (higher values for IL-6, IL-8, IL-10, and TNF-α). Heavy smoking was associated with significantly lower levels of ENA-78 and higher levels of IL-8. CONCLUSION: Differences noticed in patients' immune mediator profiles seem to be attributable to both disease and treatment. Further longitudinal studies are necessary to elucidate the involvement of immune mediators in disease progression and clinical evolution.

CXCL5 into the upper airways of children with influenza A virus infection

BACKGROUND: Neutrophil infiltration is a major feature in the pathogenesis of influenza infection. The factors regulating the neutrophil influx are not fully understood. The chemokine CXCL5/ENA-78 is a potent neutrophil chemoattractant, that has been implicated in several inflammatory diseases. Our objectives was to study the release of CXCL5 in children with natural acquired influenza. METHODS: CXCL5 concentration was investigated by immunoenzyme assay in nasal aspirates of children (n = 18) in whom respiratory symptoms were precipitated predominantly by influenza A virus. RESULTS: There were increased CXCL5 levels in nasal aspirates when children were symptomatic as compared with samples from the same children when they had been asymptomatic for four weeks (medians 1850 pg/mL vs. 30 pg/mL, p < 0.005). We purified CXCL5 from these samples, and demonstrated biological neutrophil chemotactic activity. CONCLUSIONS: It is the first in vivo data that suggest an important role for CXCL5 in neutrophil influx in proven upper respiratory influenza infection. We suggest that CXCL5 might provide a target for therapeutic intervention in influenza induced respiratory diseases.

Angiostatic versus angiogenic chemokines in IPF and EAA.

BACKGROUND: Extrinsic allergic alveolitis (EAA) and idiopathic pulmonary fibrosis (IPF) share the presence of varying degree interstitial involvement and fibrosis. Vascular changes were often reported to accompany the development of fibrosis. OBJECTIVES: The aim of our study was to examine the differences in angiostatic and angiogenic chemokine milieu in both diseases. Correlations between chemokine levels in bronchoalveolar lavage fluid (BALF), expression of chemokine receptors on CD4+ T cells (CXCR2, CXCR3) in BALF and HRCT pattern of the diseases were investigated. METHODS: Sixteen patients with chronic EAA and 8 with IPF were enrolled to the study. Concentrations of interleukin (IL)-8, epithelial neutrophil activating protein (ENA)-78, interferon-gamma-inducible protein (IP)-10 and interferon-inducible T cell alpha chemoattractant (I-TAC) in BALF supernatants were quantified using Fluorokine MultiAnalyte profiling. RESULTS: There was no significant difference in the BALF chemokine levels between the EAA and IPF group. IL-8 BALF concentrations correlate with the extent of fibrosis in both EAA and IPF (p<0.01). The IP-10 BALF concentrations do not correlate either with the HRCT alveolar or interstitial score and should be evaluated in the relationship with the disease course. CONCLUSIONS: Both IL-8 and ENA-78 probably play a different role in IPF and chronic EAA pathogenesis. While we suggest ENA-78 as the marker of at least partial reversibility of the lung impairment in the EAA patients, IL-8 could be rather an indicator of continuous exposition to provoking agent in EAA patients. IL-8 might serve as a potential marker of early phase of IPF.

Disrupted expression of CXCL5 in colorectal cancer is associated with rapid tumor formation in rats and poor prognosis in patients.

PURPOSE: We isolated a subline (CC531M) from the CC531S rat colon carcinoma cell line, which grows and metastasizes much more rapidly than CC531S. We found, using RNA expression profiling, that one of the major changes in the CC531M cell line was a 5.8-fold reduction of the chemokine CXCL5. The purpose of this study was to determine the effect of CXCL5 expression on colorectal tumor growth and metastasis. EXPERIMENTAL DESIGN: CC531 clones were generated with either knockdown or restored expression of CXCL5. These clones were inoculated in the liver of rats. In addition, in two independent cohorts of colorectal cancer patients, the level of CXCL5 expression was determined and associated to clinical variables. RESULTS: Knockdown of CXCL5 expression in CC531S resulted in rapid tumor growth and increased number of metastasis, whereas restored expression of CXCL5 in CC531M resulted in a return of the "mild" tumor growth pattern of the parental cell line CC531S. In vitro, no difference was found in proliferation rate between clones with either high or low expression of CXCL5, suggesting that environmental interactions directed by CXCL5 determine tumor outgrowth. Finally, the importance of our findings was established for patients with colorectal cancer. We found that low expression of CXCL5 was significantly associated with poor prognosis for colorectal cancer patients. CXCL5 showed a trend (P = 0.05) for a positive correlation with intratumoral CD8(+) T-cell infiltration, suggesting a possible explanation for the observed poorer prognosis. CONCLUSIONS: Our results show that CXCL5 is important in growth and development of colorectal cancer, implicating a future role in both cancer therapy and diagnosis.