Macrophage Inflammatory Protein-1alpha Research Articles

CCR3, CCR2A and macrophage inflammatory protein (MIP)‐1α, monocyte chemotactic protein‐1 (MCP‐1) in the mouse hippocampus during and after pilocarpine‐induced status epilepticus (PISE)

To investigate protein and gene expressions of chemokine subtypes CCR3, CCR2A and their respective ligands macrophage inflammatory protein 1-alpha (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1) in the normal mouse central nervous system (CNS) and in the hippocampus at different time points during and after pilocarpine-induced status epilepticus (PISE). CCR3 and MIP-1alpha protein expressions were mapped in the mouse CNS. The protein and gene expressions of CCR3 and CCR2A and their respective ligands MIP-1alpha, MCP-1 in the hippocampus were studies by immunocytochemical and quantitative real-time RT-PCR during and after PISE. CCR3 and MIP-1alpha gene expression and immunopositive neurones were broadly distributed in the CNS. CCR3 and CCA2A gene and their protein expression were downregulated in the hippocampus at 1 h during PISE. The protein expression of MIP-1alpha, MCP-1 decreased but gene expression increased at 2 h during PISE. In the hilus of the dentate gyrus, significant reduction of the numbers of CCR3, CCR2A, MCP-1 immunopositive neurones occurred from 1 h during to 2 months after PISE, but the number of MIP-1alpha neurones reduced from 2 h during to 2 months after PISE. Induced expression of CCR3 at 1 week, CCR2A, MCP-1 or MIP-1alpha at 1 week and 2 months after PISE was found in reactive astrocytes. MCP-1 was also demonstrated in the blood vessels of the hippocampus at 2 months after PISE. CCR3 and MIP-1alpha may play important functional roles in the mouse brain. The downregulation of CCR3, CCR2A, MIP-1alpha and MCP-1 in the hippocampal neurones at the acute stage during and after PISE may weaken the neuroprotective mechanisms. However, induced expression of MCP-1 in hippocampal blood vessel may be related to changes in permeability of the blood-brain barrier during epileptogenesis.

Cytokine Gene Expression After Total Hip Arthroplasty: Surgical Site versus Circulating Neutrophil Response

After surgery, cytokines and chemokines are released at the surgical wound site, which can contribute to postoperative pain, local inflammation, and tissue repair. Multiple cell types are present that can release cytokines/chemokines at the wound site and, thus, the exact cellular source of these molecules is unclear. We sought to better understand the contribution of neutrophils to cytokine/chemokine gene expression at the surgical wound site during the initial postsurgery phase of total hip arthroplasty (THA). Hip drain fluid was collected at 24 h postsurgery from six patients undergoing standardized THA. In addition, venous blood was collected presurgery and 24 h postsurgery. Neutrophils were isolated, total RNA extracted, and a biotinylated cRNA probe generated. The probes were hybridized with a cDNA microarray containing approximately 100 oligonucleotide sequences representing various human cytokines/chemokines or receptor genes. Changes in gene expression seen in the microarray were verified by reverse transcription polymerase chain reaction. In the microarray analysis of hip drain neutrophils, interleukin-1 receptor antagonist (IL1RN), interleukin-18 receptor 1 (IL18R1), macrophage migration inhibitory factor (MIF), and macrophage inflammatory protein 3alpha (CCL20) were upregulated, whereas interleukin-8 receptor beta (IL8RB/CXCR2) was consistently downregulated, compared with presurgery blood neutrophils. All of these changes were confirmed by reverse transcription polymerase chain reaction. There is a distinct cytokine gene expression profile in neutrophils at the THA surgical wound site at 24 h postsurgery when compared with that found in presurgery circulating neutrophils. Understanding these changes may allow us to knowledgeably manipulate neutrophil activity to reduce postoperative pain and inflammation without impairing wound healing.

The non‐proteolytic house dust mite allergen Der p 2 induce NF‐κB and MAPK dependent activation of bronchial epithelial cells

House dust mites (HDM) are well-known as a source of indoor aeroallergens and for causing allergic airway diseases. Some proteolytic HDM allergens are known to activate respiratory epithelial cells to produce pro-inflammatory mediators, while there is limited knowledge regarding such activity among non-proteolytic HDM allergens. To investigate whether Der p 2, a major non-proteolytic allergen of Dermatophagoides pteronyssinus, activates respiratory epithelial cells to produce mediators involved in asthma pathogenesis and to elucidate the mechanism of such activation. The human bronchial epithelial cell line BEAS-2B, normal human bronchial epithelial (NHBE) cells and the alveolar epithelial cell line A549 were exposed to recombinant Der p 2. Following exposure, we analysed a panel of soluble mediators and cell adhesion receptors involved in asthma pathogenesis by promoting recruitment, survival and binding of inflammatory cells. The involvement of nuclear factor (NF)-kappaB and mitogen-activated protein kinases (MAPKs) was studied using specific inhibitors. Der p 2 activated bronchial BEAS-2B and NHBE cells, but not alveolar A549 cells. In BEAS-2B cells Der p 2 induced dose-dependent up-regulation in both mRNA level and protein secretion of granulocyte-macrophage colony-stimulating factor, IL-6, IL-8, monocyte-chemotactic protein-1 and macrophage inflammatory protein-3alpha. Secretion as well as surface expression of intercellular adhesion molecule (ICAM)-1 was also up-regulated, which was associated with increased adhesion of monocytes to the epithelial cells. The release of cytokines and chemokines was regulated by NF-kappaB and MAPK activation in different ways, while expression of ICAM-1 was solely dependent on NF-kappaB activation. These results show that Der p 2 activates respiratory epithelial cells, indicating that this non-proteolytic allergen, in addition to its immunogenic properties, can aggravate respiratory airway disease by adjuvant-like activation of the lung epithelium.

A multiplex immunoassay demonstrates reductions in gingival crevicular fluid cytokines following initial periodontal therapy

Cytokines and chemokines play an important role in the pathogenesis of periodontal diseases. The objective of this study was to quantitatively assess the effect of initial periodontal therapy on gingival crevicular fluid levels of a comprehensive panel of cytokines and chemokines, including several less extensively studied mediators. Clinical examinations were performed and gingival crevicular fluid samples obtained from six subjects with generalized severe chronic periodontitis prior to initial periodontal therapy and at re-evaluation (6-8 weeks). Four diseased and two healthy sites were sampled in each subject. Twenty-two gingival crevicular fluid mediators were examined using a multiplex antibody capture and detection platform. Statistical analyses were performed by fitting mixed effects linear models to log-transformed gingival crevicular fluid values. Gingival crevicular fluid interleukin (IL)-1alpha and IL-1beta were the only cytokines to differ in initially diseased vs. initially healthy sites. Following initial therapy, 13 of the 16 detectable cytokines and chemokines decreased significantly in diseased sites, including IL-1alpha, IL-1beta, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12 (p40), CCL5/regulated on activation, normally T cell expressed and secreted (RANTES), eotaxin, macrophage chemotactic protein-1, macrophage inflammatory protein-1alpha and interferon-gamma. At healthy sites, only three of the 16 mediators were significantly altered following therapy. This is the first study, to our knowledge, to evaluate such an extensive panel of gingival crevicular fluid mediators within the same sample prior to and following initial therapy. The results confirm that periodontal therapy effectively reduces pro-inflammatory cytokines and chemokines, including less well-described mediators that may be important in initiation and progression of periodontitis. The multiplex assay will prove useful for future gingival crevicular fluid studies.

Early kinetics of the transcriptional response of human leukocytes to staphylococcal superantigenic enterotoxins A and G

The severity of Staphylococcus aureus sepsis is positively associated with staphylococcal enterotoxin A (SEA) and negatively associated with the enterotoxin gene cluster ( egc), which encodes five staphylococcal enterotoxins (SE). It was recently demonstrated that SE can induce human leukocytes to release inflammatory mediators. Contrary to SEG (one of the five egc superantigens), SEA induces a strong proinflammatory/Th1 response, including tumor necrosis factor-alpha and macrophage inflammatory protein-1 alpha production. Here, we investigated the very early transcriptional response of human PBMC to these two SEs. We confirm that SEA is more potent than SEG. Importantly, our data also suggest that the early response to SE is likely induced more by T cells than by monocytes. In addition, negative feedback control is triggered at the same time as proinflammatory processes (inflammation and apoptosis). It confirms at the molecular level new models of sepsis pathophysiology as concomitant and opposite sides of a given mechanism participating to response to bacterial compound are both necessary. This preliminary study highlights the potential of transcriptional studies for unraveling the very early mechanisms of leukocyte responses to SE. Further studies are needed to understand the mechanisms underlying the putative synergy between SE and other bacterial components.

Expression of cytokines and chemokines and microvasculature alterations of the tongue from patients with chronic Chagas’ disease

Chagas' disease is caused by the protozoan Trypanosoma cruzi and continues to be a significant public health problem, since 10 million people are still infected in Latin America. The purpose of this study was to analyze the microvasculature alterations as well the expression of cytokines and chemokines in the tongues from patients with chronic Chagas' disease (CC; n = 18), comparatively with a non-chagasic group (NC; n = 22). We observed several vascular alterations in the tongue of CC such as a greater vascular diameter, increased vascular wall area, high density of the blood vessels, and increased thickening of the capillary basement membrane. The expression of cytokines interferon gamma and tumor necrosis factor alpha and chemokine macrophage inflammatory protein 1alpha were significantly down-regulated in the tongue of CC group. These results demonstrated that, in the tongue of chagasic patients, a microvascular abnormality and immunological impairment occurs, probably due to chronic inflammation evoked by T. cruzi antigens.

Innate Immune Processes Are Sufficient for Driving Cigarette Smoke–Induced Inflammation in Mice

The objective of this study was to characterize the impact of cigarette smoke exposure on lung immune and inflammatory processes. BALB/c and C57BL/6 mice were exposed to cigarette smoke for 4 days (acute) or at least 5 weeks (prolonged). Both mouse strains manifested an inflammatory response after acute smoke exposure, characterized by an influx of neutrophils and mononuclear cells. Multiplex analysis revealed a greater than twofold increase of the cytokines IL-1alpha, -5, -6, and -18, as well as the chemokines monocyte chemotactic protein-1 and -3, macrophage inflammatory protein-1alpha, -beta, and -gamma, -2, -3beta, macrophage defined chemokine, granulocyte chemotactic protein-2, and interferon-gamma-inducible protein-10. In BALB/c mice, neutrophilia persisted after prolonged exposure, whereas C57BL/6 showed evidence of attenuated neutrophilia both in the bronchoalveolar lavage and the lungs. In both mouse strains, cigarette smoke exposure was associated with an expansion of mature (CD11c(hi)/major histocompatibility complex class II(hi)) myeloid dendritic cells; we observed no changes in plasmacytoid dendritic cells. Lymphocytes in the lungs displayed an activated phenotype that persisted for CD4 T cells only after prolonged exposure. In BALB/c mice, T cells acquired T helper (Th) 1 and Th2 effector function after 5 weeks of smoke exposure, whereas, in C57BL/6 mice, neither Th1 nor Th2 cells were detected. In both mouse strains, cigarette smoke exposure led to an accumulation of FoxP3+ T regulatory cells in the lungs. Studies in RAG1 knockout mice suggest that these regulatory cells may participate in controlling smoke-induced inflammation. Acute and prolonged cigarette smoke exposure was associated with inflammation, activation of the adaptive immune system, and expansion of T regulatory cells in the lungs.

RANTES release contributes to the protective action of PACAP38 against sodium nitroprusside in cortical neurons.

Pituitary adenylate cyclase activating polypeptide (PACAP), a promising neuroprotective peptide, plays an important role during development of the nervous system and in regeneration after injury. PACAP directly promotes survival via multiple signaling systems in neurons. This neuropeptide also has immuno-modulatory properties and can regulate the expression of various inflammatory mediators such as chemokines in nonneuronal cells. Chemokines and their G protein-coupled receptors are widely distributed in the brain, suggesting important functions for these inflammatory proteins in the CNS. The ability of brain endothelial cells and glia to release chemokines has been well documented, whether neurons are also a source for these mediators is unclear. The objective of this study is to determine whether PACAP38 affects expression of regulated on activation normal T expressed and secreted (RANTES) and macrophage inflammatory protein 1-alpha (MIP-1alpha) in cultured neurons and if these chemokines contribute to the neuroprotective effect of PACAP38. The data show that incubation of neuronal cultures with both PACAP38 and sodium nitroprusside (SNP) reduces the neuronal cell death evoked by SNP alone. PACAP38 dose-dependently increases immunodetectable levels of both RANTES and MIP-1alpha released in the media by cultured neurons. Co-treatment with a neutralizing antibody to RANTES decreases the PACAP38-mediated protection against SNP. Although RANTES treatment of neurons increased MIP-1alpha levels in the media and MIP-1alpha supports neuronal survival in unstressed cultures, MIP-1alpha does not protect neurons from SNP-induced toxicity. Furthermore, co-treatment with a MIP-1alpha neutralizing antibody did not affect PACAP38-induced protection against SNP. These results show that the protective effect of PACAP38 on cultured neurons is mediated, in part, by release of RANTES. The ability of PACAP to directly enhance neuronal survival through multiple intracellular signaling pathways as well as via the release of neuroprotective mediators such as RANTES highlights its utility as a potential therapeutic agent for the treatment of neurodegenerative diseases.

Clinical Features, Adenovirus Types, and Local Production of Inflammatory Mediators in Adenovirus Infections

Adenovirus infection manifests in many ways, with respiratory and gastrointestinal symptoms predominating. We performed a retrospective chart review on children evaluated at our center who had a nasal wash culture positive for adenovirus. Archived nasal washes were retrieved. Polymerase chain reaction for 15 respiratory viruses was performed on these samples. Patients who were coinfected with another virus were excluded. Adenovirus typing was performed using polymerase chain reaction primers directed at the conserved hexon gene. Bead proteomics was used to measure concentrations of inflammatory mediators. Seventy-eight patients were infected only with adenovirus. The clinical diagnosis was upper respiratory infection in 60%, pneumonia in 18%, febrile seizure in 8%, and bronchiolitis in 6%. Subgroup-C and B1 infections were most common. Seventy percent of patients with upper respiratory infection and all 5 patients with bronchiolitis had a subgroup-C infection; pneumonia was caused by subgroup-B1 and C viruses. Compared with asymptomatic control patients, adenovirus infected patients had higher nasal wash concentrations of interleukin (IL)-1alpha, IL-6, inducible protein-10, macrophage inflammatory protein-1alpha, tumor necrosis factor alpha, monokine induced by gamma interferon, and interferon-alpha (P < 0.05). In addition, we found that IL-8 and IL-1alpha (P < 0.05) were higher in the nasal washes obtained from hospitalized patients than in nonhospitalized patients. Adenovirus infection causes an array of clinical disease and is associated with local production of several proinflammatory cytokines. The observation that nasal wash IL-8 and IL-1alpha concentrations were higher in patients requiring hospitalization suggests that these mediators contribute to disease severity.

Multiple osteolytic bone lesions with high serum levels of interleukin‐6 and CCL chemokines in a patient with adult T cell leukemia

A 37-year-old woman was diagnosed as having chronic adult T-cell leukemia (ATL) of the skin by a skin biopsy and human T-cell leukemia virus type-1 serology at our hospital in August 1992. The skin lesions of ATL were improved by treatment with psoralen ultraviolet ray A. She complained of severe pain in her bilateral forearms, hands and ankles, and X-ray examination in July 1999 revealed multiple punched-out lesions of the extremities. Serum levels of parathyroid hormone-related peptide, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha and total serum receptor activator of nuclear factor kappaB ligand were not elevated. However, serum levels of IL-6, CCL2 monocyte chemoattractant protein-1 (MCP-1), CCL3 [macrophage inflammatory protein-1alpha (MIP-1alpha)] and CCL4 (MIP-1beta) were markedly elevated. Here, we have discussed the possible mechanism underlying the onset of the osteolytic lesions.

Cytokine-induced killer cells are terminallydifferentiated activated CD8 cytotoxic T-EMRA lymphocytes

Cytokine-induced killer cells (CIK) are CD3(+)CD56(+) T cells with natural killer (NK)-like cytotoxic activity used for the immunotherapy of tumors. We aimed to fully characterize CIK cells and define their ontogeny. CIK were generated in vitro by stimulation of peripheral blood mononuclear cells or T-cell subsets with interferon-gamma, anti-CD3 and interleukin-2. They were fully characterized in terms of phenotype, cytotoxic activity, and gene expression with respect to circulating CD3(+)CD56(+) cells, NK cells, and CD56(-) T cells present in CIK cultures. We demonstrate that CIK are terminally differentiated CD8 T cells that derive from proliferating CD3(+)CD56(-)CD8(+) T cells. They express polyclonal T-cell receptor Vbeta chains and have acquired CD56, NKG2D, and large granular lymphocyte morphology, but lack expression of most NK-specific activating (NKp30, NKp44, NKp46) and inhibitory (KIR2DL1, KIR2DL2, KIR3DL1, NKG2A, CD94) receptors, and can kill K562 targets. Circulating CD3(+)CD56(+) cells are also CD8(+)CD16(-), but are oligoclonal, poorly cytotoxic for K562, and express lower levels of CD56 and NKG2D. Gene profiling of CIK, CD56(-) T and NK cells present at the end of culture shows that differences are much more limited between CIK and CD56(-) T compared to CIK and NK cells. Most of the genes upregulated in CIK cells compared to CD56(-) T cells are part of the tumor necrosis factor gene network. The CIK phenotype, that is CD45RA(+), CCR7(-), CD62L-weakly positive, CD11a(+), CD27(+), CD28(-), macrophage inflammatory protein 1alpha(+), perforin(+), Fas ligand(+) coincides almost exactly with that described for the T RA(+) effector memory CD27 single positive subset of terminally differentiated human memory T cells.

Effects of Pingchuan Recipe on airway remodeling in mice with bronchial asthma

To study the effects of Pingchuan Recipe, a compound traditional Chinese herbal medicine for treating bronchial asthma, on macrophage inflammatory protein-1alpha (MIP-1alpha) and immunoglobulin E (IgE) contents and CD86 expression in a mouse model of bronchial asthma, and to investigate the mechanism of Pingchuan Recipe in regulating airway remodeling in mice with bronchial asthma. Fifty BALB/c mice were randomly divided into normal control group, untreated group, dexamethasone group, low-dose Pingchuan Recipe group and normal-dose Pingchuan Recipe group. Bronchial asthma in mice was induced by intra-abdominal injection of mixed ovalbumin and stimulation by inhaling 5% ovalbumin. Mice in the normal control group and the untreated group did not receive treatment, but mice in the other groups were administered intragastrically with 50 g/ (kg * d) Pingchuan Recipe, 25 g/ (kg * d) Pingchuan Recipe and 0.75 mg/ (kg * d) dexamethasone, respectively. After consecutive treatment for 8 weeks, the MIP-1alpha contents in peripheral blood and bronchoalveolar lavage fluid (BALF) were measured by an avidin biotin complex-enzyme-linked immunosorbent assay (ABC-ELISA) system, and the IgE contents in serum and BALF were measured by ELISA method. Direct immunofluorescence-flow cytometry was used to detect the percentages of CD86(+) and CD3(-) CD86(+) cells in peripheral blood and BALF. General histological changes of the lung tissue were observed by HE staining and collagen fiber staining 12 weeks after activation. Compared with the normal control group, the contents of MIP-1alpha, the percentages of CD86(+) cells and CD3(-) CD86(+) cells in peripheral blood and BALF, and the contents of IgE in serum and BALF were raised significantly (P < 0.01). The contents of MIP-1alpha, the percentages of CD86(+) cells and CD3(-) CD86(+) cells in peripheral blood and BALF, and the contents of IgE in serum and BALF in the treated groups were lower than those in the untreated group (P < 0.01). The contents of MIP-1alpha in peripheral blood and BALF and the contents of IgE in serum and BALF had no significant differences among the treated groups (P > 0.05). The percentage of CD3(-) CD86(+) cells in peripheral blood in low-dose Pingchuan Recipe group was lower than that in the dexamethasone group. Compared with the untreated group, the collagen deposition in the wall of bronchiole in the treated groups was decreased. Pingchuan Recipe can decrease the contents of MIP-1alpha, IgE and the expressions of CD86 in peripheral blood and BALF, which may be one of its mechanisms in improving chronic airway inflammation and inhibiting airway remodeling.

In Vivo Imaging and Quantitative Analysis of Leukocyte Directional Migration and Polarization in Inflamed Tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1α (MIP-1α/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1gfp/gfp mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo.

Acetaminophen protects brain endothelial cells against oxidative stress

Increasing evidence suggests that acetaminophen has unappreciated anti-oxidant and anti-inflammatory properties. Drugs that affect oxidant and inflammatory stress in the brain are of interest because both processes are thought to contribute to the pathogenesis of neurodegenerative disease. The objective of this study is to determine whether acetaminophen affects the response of brain endothelial cells to oxidative stress. Cultured brain endothelial cells are pre-treated with acetaminophen and then exposed to the superoxide-generating compound menadione (25 μM). Cell survival, inflammatory protein expression, and anti-oxidant enzyme activity are measured. Menadione causes a significant ( p < 0.001) increase in endothelial cell death as well as an increase in RNA and protein levels of tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES. Menadione also evokes a significant ( p < 0.001) increase in the activity of the anti-oxidant enzyme superoxide dismutase (SOD). Pre-treatment of endothelial cell cultures with acetaminophen (25–100 μM) increases endothelial cell survival and inhibits menadione-induced expression of inflammatory proteins and SOD activity. In addition, we document, for the first time, that acetaminophen increases expression of the anti-apoptotic protein Bcl2. Suppressing Bcl2 with siRNA blocks the pro-survival effect of acetaminophen. These data show that acetaminophen has anti-oxidant and anti-inflammatory effects on the cerebrovasculature and suggest a heretofore unappreciated therapeutic potential for this drug in neurodegenerative diseases such as Alzheimer's disease that are characterized by oxidant and inflammatory stress.

Angiogenesis in Waldenström's Macroglobulinemia

Angiogenesis represents an essential step of disease progression in several hematological malignancies. In Waldenström's macroglobulinemia (WM) the bone marrow microvessel density is increased in 30%–40% of patients but seems to have no impact on survival. Angiogenic cytokines, such as angiogenin, vascular endothelial growth factor, and basic fibroblast growth factor are increased in the serum of WM or IgM-MGUS patients, while the ratio of angiopoietin-1/angiopoietin-2 is reduced in WM but not in IgM—monoclonal gammopathy of undetermined significance (MGUS). Angiogenin and angiopoietin-1/angiopoietin-2 ratio correlates with disease activity and clinical features of WM. Macrophage and mast-cell chemoattractants, such as macrophage inflammatory protein-1 alpha are also elevated in the serum of patients with WM, while both macrophages and mast cells that are increased in the WM microenvironment have angiogenic properties and participate in the angiogenesis process in several malignancies. This review summarizes all data available by November 2008 (end of literature search) for the role of angiogenesis in the biology of WM and its correlation with clinical and laboratory features of the disease.

Establishment of a Multi-Analyte Serum Biomarker Panel to Identify Lymph Node Metastases in Non-small Cell Lung Cancer

In non-small cell lung cancer (NSCLC), the presence of locoregional lymph node metastases remains the most important prognostic factor and significantly guides treatment regimens. Unfortunately, currently-available noninvasive staging modalities have limited accuracy. The objective of this study was to create a multianalyte blood test capable of discriminating a patient's true (pathologic) nodal status preoperatively. Pretreatment serum specimens collected from 107 NSCLC patients with localized disease were screened with 47 biomarkers implicated in disease presence or progression. Multivariate statistical algorithms were then used to identify the optimal combination of biomarkers for accurately discerning each patient's nodal status. We identified 15 candidate biomarkers that met our criteria for statistical relevance in discerning a patient's preoperative nodal status. A 'random forest' classification algorithm was used with these parameters to define a 6-analyte panel, consisting of macrophage inflammatory protein-1alpha, carcinoembryonic antigen, stem cell factor, tumor necrosis factor-receptor I, interferon-gamma, and tumor necrosis factor-alpha, that was the optimum combination of biomarkers for identifying a patient's pathologic nodal status. A Classification and Regression Tree analysis was then created with this panel that was capable of correctly classifying 88% of the patients tested, relative to the pathologic assessments. This value is in contrast to our observed 85% classification rate using conventional clinical methods. This study establishes a serum biomarker panel with efficacy in discerning preoperative nodal status. With further validation, this blood test may be useful for assessing nodal status (including occult disease) in NSCLC patients facing tumor resection therapy.

Reduced IL‐1Ra/IL‐1 ratio in ultraviolet B‐exposed skin of patients with polymorphic light eruption

Polymorphic light eruption (PLE) is a putative delayed-type allergic reaction to (solar) ultraviolet (UV) exposure. Inadequate immune suppression after UVB-induced sunburn appears to be associated with reduced trafficking of Langerhans cells (LCs) out of and neutrophils into the epidermis of patients sensitive to UVB provocation of PLE. Therefore, we investigated whether pro-inflammatory and chemotactic cytokines are differentially expressed in UVB-irradiated skin of UVB-provocable PLE patients (n = 6) and age- and gender-matched healthy controls (n = 6). Interstitial interleukin-1alpha (IL-1alpha), IL-1beta, IL-1Ra, IL-4, IL-8, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein 1-alpha (MIP-1alpha), MIP-1beta and monocyte chemotactic protein-1 (MCP-1) were measured in suction blister fluid raised 16 h after exposure to 0, three and six minimal erythemal UVB doses. In unirradiated skin, the IL-1Ra levels were significantly lower in the PLE patients than in controls (P < 0.05). IL-8 and TNF-alpha levels increased strongly upon UVB irradiation in both groups. No differential shifts in cytokine profiles were found that could explain a reduced trafficking of Langerhans cells and neutrophils in PLE patients. Dose-trend analyses showed that UVB irradiation caused significant increases in IL-1alpha in both groups, and that the levels of IL-1alpha and IL-1beta were on average twofold higher in the PLE group (P = 0.03 and P = 0.004, respectively.). Accordingly, the ratios of IL-1Ra over IL-1alpha and over IL-1beta were overall lower in the skin of PLE patients (P = 0.015 and P < 0.001, respectively.). This shift in cytokines in UVB-irradiated skin of PLE patients reveals an amplified early pro-inflammatory cytokine response, which may contribute to the allergic reaction to UVB radiation.

GABAergic activities enhance macrophage inflammatory protein‐1α release from microglia (brain macrophages) in postnatal mouse brain

Microglial cells (brain macrophages) invade the brain during embryonic and early postnatal development, migrate preferentially along fibre tracts to their final position and transform from an amoeboid to a ramified morphology. Signals by which the invading microglia communicate with other brain cells are largely unknown. Here, we studied amoeboid microglia in postnatal corpus callosum obtained from 6- to 8-day-old mice. These cells accumulated on the surface of acute brain slices. Whole-cell patch-clamp recordings revealed that the specific GABA(A) receptor agonist muscimol triggered a transient increase in conductance typical for inward rectifying potassium channels in microglia. This current increase was not mediated by microglial GABA(A) receptors since microglial cells removed from the slice surface no longer reacted and cultured microglia only responded when a brain slice was placed in their close vicinity. Muscimol triggered a transient increase in extracellular potassium concentration ([K(+)](o)) in brain slices and an experimental elevation of [K(+)](o) mimicked the muscimol response in microglial cells. Moreover, in adult brain slices, muscimol led only to a minute increase in [K(+)](o) and microglial cells failed to respond to muscimol. In turn, an increase in [K(+)](o) stimulated the release of chemokine macrophage inflammatory protein-1alpha (MIP1-alpha) from brain slices and from cultures of microglia but not astrocytes. Our observations indicate that invading microglia in early postnatal development sense GABAergic activities indirectly via sensing changes in [K(+)](o) which results in an increase in MIP1-alpha release.

The comparative proteomic analysis of serum cytokine expression in acute lung injury patients in peri-operative stage of liver transplantation

To investigate the characteristics of serum cytokine expression in acute lung injury (ALI) patients in peri-operative stage of liver transplantation with the aim of setting the basis for screening the early markers and treatment targets of ALI. Four male patients with ALI occurring in peri-operative stage of liver transplantation for hepatitis B liver cirrhosis, with no lung, renal, or brain abnormality, without difference in clinical findings (urine volume, blood loss, ascites, amount of blood transfusion, operation time, anhepatic time, the use of vaso-active drugs, diuretics and condition of circulation) were included for study. Blood was taken after anesthesia, 3 hours and 24 hours after new liver. RayBio human antibody array was used to analyze the cytokine expression. Compared with healthy people, in the patients with ALI in peri-operative stage of liver transplantation, upregulation of some cytokines appeared as early as after anesthesia, including interleukins (IL-3, IL-6, IL-12 p40, IL-12 p70), monocyte chemoattractant protein-2 (MCP-2), macrophage-colony stimulating factor (M-CSF), monokine induced by interferon-gamma (MIG), macro-phage inflammatory protein-1 alpha (MIP-1 alpha), soluble tumor necrosis factor receptor I (sTNFR I), especially sTNFR II which showed even stronger expression, while normal T cells expression and secretory factor (RANTES) and platelet-derived growth factor-BB (PDGF-BB) showed downregulation in expression. Some more cytokines showed upregulation in expression at neohepatic 3 hours, especially IL-12 p70, sTNFR I, sTNFR II showed upregulation, while RANTES, PDGF-BB and IL-1 alpha showed downregulation in expression. The number of cytokines showing upregulation was significantly increased at neohepatic 24 hours. Compared with those at neohepatic 3 hours, eotaxin, IL-1 alpha, IL-1 beta, IL-4, IL-15, MCP-2 showed significantly higher upregulation at neohepatic 24 hours, and among them IL-3 and IL-6 especially IL-2 showed even more upregulation in expression. There are changes in expression of different kinds of cytokines in various extent before operation, at neohepatic 3 hours and neohepatic 24 hours. Some of them may be considered as important early markers and treatment targets. Further researches with large samples would be necessary to elucidate the clinical implication.

Chemokines gene expression of RAW 264.7 cells by Actinobacillus actinomycetemcomitans lipopolysaccharide using microarray and RT-PCR analysis.

Actinobacillus actinoinycetemcomitans (A. actinomycetem-comitans) is an important pathogen casuing aggressive periodontitis. The present study was designed to investigate the chemokines expression regulated by A. actinomycetemcomitans lipopolysaccharide (LPS). Chemokines genes expression profiling was performed in Raw 264.7 cells by analyses of microarray and reverse transcription-polymerase chain reaction (RT-PCR). Microarray results showed that the induction of monocyte chemoattractant protein-1alpha (MCP-1alpha) and macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, MIP-1gamma, regulated upon activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein-2 (MIP-2), and interferon-gamma inducible protein 10 (IP 10) by A. actinomycetemcomitans LPS was increased to 12.5, 1.53, 9.09, 17.3, 2.82, 16.1, and 18.1 folds at 18 h, respectively. To check these chemokines expression by A. actinomycetemcomitans LPS, we examined gene expressions by RT-PCR, and found that the expression of MIP-1beta, MIP-1gamma, RANTES, MIP-2, and IP 10 was increased 107.1, 93.6, 106.8, 86.5, and 162.0 folds at 18 h, respectively. These results indicate that A. actinomycetemcomitans LPS stimulates the several chemokines expressions (MIP-1alpha, MIP-1beta, MIP-1gamma, RANTES, MIP-2, and IP 10) in Raw 264.7 cells.