Intratracheal transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuates the hyperoxia-induced neonatal lung injury. The aim of this preclinical translation study was to optimize the dose of human UCB-derived MSCs in attenuating hyperoxia-induced lung injury in newborn rats. Newborn Sprague-Dawley rats were randomly exposed to hyperoxia (95% oxygen) or normoxia after birth for 14 days. Three different doses of human UCB-derived MSCs, 5 × 10(3) (HT1), 5 × 10(4) (HT2), and 5 × 10(5) (HT3), were delivered intratracheally at postnatal day (P) 5. At P14, lungs were harvested for analyses including morphometry for alveolarization, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, myeoloperoxidase activity, mRNA level of tumor necross factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and transforming growth factor-β (TGF-β), human glyceradehyde-3-phosphate dehydrogenase (GAPDH), and p47(phox), and collagen levels. Increases in TUNEL-positive cells were attenuated in all transplantation groups. However, hyperoxia-induced lung injuries, such as reduced alveolarization, as evidenced by increased mean linear intercept and mean alveolar volume, and increased collagen levels were significantly attenuated in both HT2 and HT3, but not in HT1, with better attenuation in HT3 than in HT2. Dose-dependent human GAPDH expression, indicative of the presence of human RNA in lung tissue, was observed only in the transplantation groups, with higher expression in HT3 than in HT2, and higher expression in HT2 than in HT1. Hyperoxia-induced inflammatory responses such as increased myeloperoxidase acitivity, mRNA levels of TNF-α, IL-1β, IL-6, and TGF-β of the lung tissue, and upregulation of both cytosolic and membrane p47(phox), indicative of oxidative stress, were significantly attenuated in both HT2 and HT3 but not in HT1. These results demonstrate that intratracheal transplantation of human UCB-derived MSCs with appropriate doses may attenuate hyperoxia-induced lung injury through active involvement of these cells in modulating host inflammatory responses and oxidative stress in neonatal rats.

Molecular hydrogen, which reacts with the hydroxyl radical, has been considered as a novel antioxidant. Here, we evaluated the protective effects of hydrogen-rich saline on the l-arginine (l-Arg)-induced acute pancreatitis (AP). AP was induced in Sprague-Dawley rats by giving two intraperitoneal injections of l-Arg, each at concentrations of 250mg/100g body weight, with an interval of 1h. Hydrogen-rich saline (>0.6mM, 6ml/kg) or saline (6ml/kg) was administered, respectively, via tail vein 15min after each l-Arg administration. Severity of AP was assessed by analysis of serum amylase activity, pancreatic water content and histology. Samples of pancreas were taken for measuring malondialdehyde and myeloperoxidase. Apoptosis in pancreatic acinar cell was determined with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL). Expression of proliferating cell nuclear antigen (PCNA) and nuclear factor kappa B (NF-kappaB) were detected with immunohistochemistry. Hydrogen-rich saline treatment significantly attenuated the severity of l-Arg-induced AP by ameliorating the increased serum amylase activity, inhibiting neutrophil infiltration, lipid oxidation and pancreatic tissue edema. Moreover, hydrogen-rich saline treatment could promote acinar cell proliferation, inhibit apoptosis and NF-kappaB activation. These results indicate that hydrogen treatment has a protective effect against AP, and the effect is possibly due to its ability to inhibit oxidative stress, apoptosis, NF-kappaB activation and to promote acinar cell proliferation.

To date, no study has evaluated the diversity of TH cell cytokine patterns of patients with chronic rhinosinusitis (CRS) among centers in different continents using identical methods.

We sought to assess TH cytokine profiles in patients with CRS from Europe, Asia, and Australia.

Patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP; n = 435) and control subjects (n = 138) were recruited from centers in Adelaide, Benelux, Berlin, Beijing, Chengdu, and Tochigi. Nasal mucosal concentrations of TH2, TH17, and TH1 cytokines; eosinophilic cationic protein (ECP); myeloperoxidase (MPO); IL-8; and tissue total and Staphylococcus aureus enterotoxin (SE)-specific IgE were measured by using identical tools.

Combinations of TH1/TH2/TH17 cytokine profiles in patients with CRSwNP varied considerably between regions. CRSwNP tissues from patients from Benelux, Berlin, Adelaide, and Tochigi were TH2 biased, whereas those from Beijing mainly demonstrated TH2/TH1/TH17 mixed patterns, and patients from Chengdu showed an even lower TH2 expression. Concentrations of IL-8 and tissue total IgE in patients with CRSwNP were significantly higher than those in control subjects in all regions. More than 50% of patients with CRSwNP in Benelux, Berlin, Adelaide, and Tochigi showed a predominantly eosinophilic endotype compared with less than 30% of patients in Beijing and Chengdu. SE-specific IgE was found in significantly greater numbers in patients with CRSwNP from Benelux, Adelaide, and Tochigi and significantly lower numbers in patients from Beijing and Chengdu. Moreover, the TH1/TH2/TH17 cytokine profiles in patients with CRSsNP showed diversity among the 6 regions.

TH cytokine levels, eosinophilic/neutrophilic patterns, and SE-specific IgE expressions show extreme diversity among patients with CRS from Europe, Asia, and Oceania.

Vasculitis is a rare complication of propylthiouracil therapy. Antineutrophil cytoplasmic antibodies (ANCA) have been described in association with several vasculitic disorders. We report detection of ANCA against human neutrophil elastase, proteinase 3, and myeloperoxidase in serum from six patients who developed evidence of vasculitis during propylthiouracil treatment of hyperthyroidism. On withdrawal of the drug ANCA concentrations fell and clinical symptoms resolved completely.

Ultra-endurance exercise, such as an Ironman triathlon, induces muscle damage and a systemic inflammatory response. As the resolution of recovery in these parameters is poorly documented, we investigated indices of muscle damage and systemic inflammation in response to an Ironman triathlon and monitored these parameters 19 days into recovery. Blood was sampled from 42 well-trained male triathletes 2 days before, immediately after, and 1, 5 and 19 days after an Ironman triathlon. Blood samples were analyzed for hematological profile, and plasma values of myeloperoxidase (MPO), polymorphonuclear (PMN) elastase, cortisol, testosterone, creatine kinase (CK) activity, myoglobin, interleukin (IL)-6, IL-10 and high-sensitive C-reactive protein (hs-CRP). Immediately post-race there were significant (P < 0.001) increases in total leukocyte counts, MPO, PMN elastase, cortisol, CK activity, myoglobin, IL-6, IL-10 and hs-CRP, while testosterone significantly (P < 0.001) decreased compared to prerace. With the exception of cortisol, which decreased below prerace values (P < 0.001), these alterations persisted 1 day post-race (P < 0.001; P < 0.01 for IL-10). Five days post-race CK activity, myoglobin, IL-6 and hs-CRP had decreased, but were still significantly (P < 0.001) elevated. Nineteen days post-race most parameters had returned to prerace values, except for MPO and PMN elastase, which had both significantly (P < 0.001) decreased below prerace concentrations, and myoglobin and hs-CRP, which were slightly, but significantly higher than prerace. Furthermore, significant relationships between leukocyte dynamics, cortisol, markers of muscle damage, cytokines and hs-CRP after the Ironman triathlon were noted. This study indicates that the pronounced initial systemic inflammatory response induced by an Ironman triathlon declines rapidly. However, a low-grade systemic inflammation persisted until at least 5 days post-race, possibly reflecting incomplete muscle recovery.

Dietary antioxidants may attenuate oxidative damage from strenuous exercise in various tissues. Beneficial effects of the antioxidant astaxanthin have been demonstrated in vitro, but not yet in vivo. We investigated the effect of dietary supplementation with astaxanthin on oxidative damage induced by strenuous exercise in mouse gastrocnemius and heart. C57BL/6 mice (7 weeks old) were divided into groups: rested control, intense exercise, and exercise with astaxanthin supplementation. After 3 weeks of exercise acclimation, both exercise groups ran on a treadmill at 28 m/min until exhaustion. Exercise-increased 4-hydroxy-2-nonenal-modified protein and 8-hydroxy-2'-deoxyguanosine in gastrocnemius and heart were blunted in the astaxanthin group. Increases in plasma creatine kinase activity, and in myeloperoxidase activity in gastrocnemius and heart, also were lessened by astaxanthin. Astaxanthin showed accumulation in gastrocnemius and heart from the 3 week supplementation. Astaxanthin can attenuate exercise-induced damage in mouse skeletal muscle and heart, including an associated neutrophil infiltration that induces further damage.

Multiple sclerosis is considered to be initiated by a deregulated, myelin-specific T cell response. However, the formation of inflammatory CNS lesions and the contribution of different leukocyte subsets in setting up these lesions are still incompletely understood. In this study, we show that, in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis, neutrophil granulocytes are important contributors in preparing CNS inflammation. Preclinical single-dose Ab-mediated depletion of neutrophils delayed the onset and continuous depletion attenuated the development of experimental autoimmune encephalomyelitis, whereas the generation of a myelin-specific T cell response remained unaffected. Neutrophil-related enzymes such as myeloperoxidase and neutrophil elastase did not contribute in mounting CNS inflammation, as analyzed by using respective knockout mice and inhibitors. CNS-infiltrating neutrophils secreted proinflammatory molecules and matured bone marrow-derived dendritic cells in vitro, which in turn enhanced their ability to restimulate myelin-specific T cells. This was mirrored in vivo, in which depletion of neutrophils specifically impaired maturation of microglia and macrophages into professional APCs, resulting in a diminished amplification of early CNS inflammation. Therefore, inside the CNS neutrophils provide local cofactors that are required for the maturation of myeloid cells into professional APCs representing an essential step for the local restimulation of myelin-specific T cells and the development of autoimmune disease.

The rapid evaluation of patients presenting with symptoms suggestive of an acute coronary syndrome is of great clinical relevance. Biomarkers have become increasingly important in this setting to supplement electrocardiographic findings and patient history because one or both can be misleading. Today, cardiac troponin is still the only marker used routinely in this setting due to its myocardial tissue specificity and sensitivity, as well as its established usefulness for therapeutic decision making. However, even current generation troponin assays have certain limitations such as insufficient sensitivity for diagnosing unstable angina. Novel high-sensitivity assays for cardiac troponin have the potential to overcome these limitations. Further studies are needed to answer some critical questions regarding the best cutoffs for diagnosis and risk assessment and the optimal work-up for rule-out of acute myocardial infarction. Other nonmyocardial tissue-specific markers might help in this setting. Myeloperoxidase, copeptin, and growth differentiation factor 15 reflect different aspects of the development of atherosclerosis or acute ischemia. Each has demonstrated impact in risk stratification of acute coronary syndromes. Limited data also show that copeptin may, when used together with cardiac troponin, improve the sensitivity for diagnosing acute myocardial infarction, and growth differentiation factor 15 may help in selection of patients that benefit from invasive therapy. Further evaluation is needed before these markers can be adopted routinely in clinical practice.

Despite clinical and experimental data suggesting a direct relationship between antineutrophil cytoplasmic antibodies (ANCAs) and disease activity in patients with microscopic polyangiitis (MPA), the causal relationship between perinuclear ANCAs specific for myeloperoxidase (MPO-ANCA) and disease manifestations has been controversial. We describe the case of a woman with a history of pulmonary-renal syndrome caused by MPA whose disease became clinically and serologically active during pregnancy. Forty-eight hours after delivery, the newborn developed pulmonary hemorrhage and abnormalities in renal function. The newborn's cord blood showed an immunoglobulin G MPO-ANCA level identical to that of the mother's serum, indicating passive transfer of the antibody to the neonate. Our findings represent the first human model supporting the interpretation that MPO-ANCAs were immunopathogenic.

Uropathogenic Escherichia coli (UPEC), the most frequent cause of urinary tract infection (UTI), is associated with an inflammatory response which includes the induction of cytokine/chemokine secretion by urothelial cells and neutrophil recruitment to the bladder. Recent studies indicate, however, that UPEC can evade the early activation of urothelial innate immune response in vitro. In this study, we report that infection with the prototypic UPEC strain NU14 suppresses tumor necrosis factor alpha (TNF-alpha)-mediated interleukin-8 (CXCL-8) and interleukin-6 (CXCL-6) secretion from urothelial cell cultures compared to infection with a type 1 piliated E. coli K-12 strain. Furthermore, examination of a panel of clinical E. coli isolates revealed that 15 of 17 strains also possessed the ability to suppress cytokine secretion. In a murine model of UTI, NU14 infection resulted in diminished levels of mRNAs encoding keratinocyte-derived chemokine, macrophage inflammatory peptide 2, and CXCL-6 in the bladder relative to infection with an E. coli K-12 strain. Furthermore, reduced stimulation of inflammatory chemokine production during NU14 infection correlated with decreased levels of bladder and urine myeloperoxidase and increased bacterial colonization. These data indicate that a broad phylogenetic range of clinical E. coli isolates, including UPEC, may evade the activation of innate immune response in the urinary tract, thereby providing a pathogenic advantage.

Transient Receptor Potential Melastatin-8 (TRPM8), a recently identified member of the transient receptor potential (TRP) family of ion channels, is activated by mild cooling and by chemical compounds such as the supercooling agent, icilin. Since cooling, possibly involving TRPM8 stimulation, diminishes injury-induced peripheral inflammation, we hypothesized that TRPM8 activation may also attenuate systemic inflammation. We thus studied the involvement of TRPM8 in regulating colonic inflammation using two mouse models of chemically induced colitis. TRPM8 expression, localized immunohistochemically in transgenic TRPM8(GFP) mouse colon, was up-regulated in both human- and murine-inflamed colon samples, as measured by real-time PCR. Wild-type mice (but not TRPM8-nulls) treated systemically with the TRPM8 agonist, icilin showed an attenuation of chemically induced colitis, as reflected by a decrease in macroscopic and microscopic damage scores, bowel thickness, and myeloperoxidase activity compared with untreated animals. Furthermore, icilin treatment reduced the 2,4,6-trinitrobenzenesulfonic acid-induced increase in levels of inflammatory cytokines and chemokines in the colon. In comparison with wild-type mice, Dextran Sodium Sulfate (DSS)-treated TRPM8 knockout mice showed elevated colonic levels of the inflammatory neuropeptide calcitonin-gene-related peptide, although inflammatory indices were equivalent for both groups. Further, TRPM8 activation by icilin blocked capsaicin-triggered calcitonin-gene-related peptide release from colon tissue ex vivo and blocked capsaicin-triggered calcium signaling in Transient Receptor Potential Vaniloid-1 (TRPV1) and TRPM8 transfected HEK cells. Our data document an anti-inflammatory role for TRPM8 activation, in part due to an inhibiton of neuropeptide release, pointing to a novel therapeutic target for colitis and other inflammatory diseases.

The mechanisms that facilitate remodeling of the cervix in preparation for and during parturition remain poorly understood. In the current study, we have evaluated the timing of inflammatory cell migration in cervix through comparisons between wild-type mice and steroid 5alpha-reductase type 1 null mice (Srd5a1-/-), which fail to undergo cervical ripening due to insufficient local progesterone metabolism. The timing of migration and distribution of macrophages, monocytes, and neutrophils were examined using cervices from wild-type and Srd5a1-/- mice before Day 15 (d15) and during cervical ripening (late d18), and postpartum (d19). Neutrophil numbers were quantitated by cell counts and activity was estimated by measurement of myeloperoxidase activity. The mRNA and/or protein expression of neutrophil chemoattractants, CXCL2 and CXCL1, and other proinflammatory and adhesion molecules, including IL1A, IL1B, TNF, CCL11, CCL5, CCL3, ITGAM, and ICAM1, were measured in cervices collected before, during, and after birth. The effect of neutrophil depletion on parturition was tested. Tissue macrophages, myeloperoxidase activity, and expression of proinflammatory molecules are not increased within the cervix until after birth. Neutrophil numbers do not change after birth and neutrophil depletion before term has no effect on timing or success of parturition. These results suggest that cervical ripening does not require neutrophils. Moreover, neutrophil activation and a general inflammatory response are not initiated within the cervix until shortly after parturition. The timing of inflammatory cell migration and activation in pregnant cervix suggest a role for these cells in postpartum remodeling of the cervix rather than in the initiation of cervical ripening at parturition.

We reported previously that treatment with antibody to transforming growth factor-beta (TGF-beta) caused a marked attenuation of bleomycin (BL)-induced lung fibrosis (LF) in mice. Decorin (DC), a proteoglycan, binds TGF-beta and thereby down-regulates all of its biological activities. In the present study, we evaluated the antifibrotic potential of DC in a three-dose BL-hamster model of lung fibrosis. Hamsters were placed in the following groups: (1) saline (SA) + phosphate-buffered saline (PBS) (SA + PBS); (2) SA + DC; (3) BL + PBS; and (4) BL + DC. Under pentobarbital anesthesia, SA (4 mL/kg) or BL was instilled intratracheally in three consecutive doses (2.5, 2.0, 1.5 units/kg/4 mL) at weekly intervals. DC (1 mg/mL) or PBS was instilled intratracheally in 0.4 mL/hamster on days 3 and 5 following instillation of each dose of SA or BL. In week 4, hamsters received three doses of either DC or PBS every other day. The hamsters were killed at 30 days following the first instillation, and their lungs were appropriately processed. Lung hydroxyproline levels in SA + PBS, SA + DC, BL + PBS, and BL + DC groups were 965, 829, 1854, and 1387 microg/lung, respectively. Prolyl hydroxylase activities were 103, 289, and 193% of SA + PBS control in SA + DC, BL + PBS, and BL + DC groups, respectively. The myeloperoxidase activities in the corresponding groups were 222, 890, and 274% of control (0.525 units/lung). Intratracheal instillation of BL caused significant increases in these biochemical markers, and instillation of DC diminished these increases in the BL + DC group. DC treatment also caused a significant reduction in the infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) of hamsters in the BL + DC group. However, DC treatment had little effect on BL-induced increases in lung superoxide dismutase activity and lipid peroxidation and leakage of plasma proteins in the BALF of the BL + DC group. Hamsters in the BL + PBS group showed severe multifocal fibrosis and accumulation of mononuclear inflammatory cells and granulocytes. In contrast, hamsters in the BL + DC group showed mild multifocal septal thickening with aggregations of mononuclear inflammatory cells. Hamsters in both control groups (SA + PBS and SA + DC) showed normal lung structure. Frozen lung sections following immunohistochemical staining revealed an intense staining for EDA-fibronectin and collagen type I in the BL + PBS group as compared with all other groups. It was concluded that DC potentially offers a novel pharmacological intervention that may be useful in treating pulmonary fibrosis.

Mucosal changes in inflammatory bowel disease (IBD) are characterized by ulcerative lesions accompanied by a prominent infiltrate of inflammatory cells including lymphocytes, macrophages, and neutrophils and alterations in 5-hydroxytryptamine (5-HT)-producing enterochromaffin (EC) cells. Mechanisms involved in recruiting and activating these cells are thought to involve a complex interplay of inflammatory mediators. Studies in clinical and experimental IBD have shown the upregulation of various chemokines including monocyte chemoattractant protein (MCP)-1 in mucosal tissues. However, precise information on the roles of this chemokine or the mechanisms by which it takes part in the pathogenesis of IBD are not clear. In this study, we investigated the role of MCP-1 in the development of hapten-induced experimental colitis in mice deficient in MCP-1. Our results showed a significant reduction in the severity of colitis both macroscopically and histologically along with a decrease in mortality in MCP-1-deficient mice compared with wild-type control mice. This was correlated with a downregulation of myeloperoxidase activity, IL-1beta, IL-12p40, and IFN-gamma production, and infiltration of CD3+ T cells and macrophages in the colonic mucosa. In addition, we observed significantly lower numbers of 5-HT-expressing EC cells in the colon of MCP-1-deficient mice compared with those in wild-type mice after dinitrobenzenesulfonic acid. These results provide evidence for a critical role of MCP-1 in the development of colonic inflammation in this model in the context of immune and enteric endocrine cells.

The concentration of myeloperoxidase, a neutrophil granule constituent, was measured in the perfusion fluid from sigmoid and rectal segments in patients with ulcerative colitis. The concentrations of myeloperoxidase were increased severalfold in the patients with ulcerative colitis compared with healthy controls pointing to an enhanced neutrophil activity. The release of myeloperoxidase correlated to an enhanced local release of the neutrophil activating peptide interleukin-8 (IL-8). Increased values of tumour necrosis factor (TNF-alpha) were also found during intestinal perfusion of the patients and correlated with those of IL-8. The results obtained are compatible with the hypothesis that local mucosal recruitment/activation of neutrophils in ulcerative colitis is mediated by an enhanced IL-8 synthesis. TNF-alpha may be one relevant factor as a stimulus to IL-8 synthesis.

A key cardioprotective effect of high-density lipoprotein involves the interaction of its major protein, apolipoprotein A-I (apoA-I) with ATP-binding cassette transporter A1 (ABCA1), a macrophage cholesterol exporter. ApoA-I is thought to remove cholesterol from macrophages by a cascade of events. First it binds directly to ABCA1, activating signaling pathways, and then it binds to and solubilizes lipid domains generated by ABCA1. HDL isolated from human atherosclerotic lesions and blood of subjects with established coronary artery disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of myeloperoxidase (MPO), a heme protein secreted by macrophages. Here we show that chlorination (but not nitration) of apoA-I by the MPO pathway impairs its ability to interact directly with ABCA1, to activate the Janus kinase 2 signaling pathway, and to promote efflux of cellular cholesterol. In contrast, oxidation of apoA-I has little effect on its ability to stabilize ABCA1 protein or to solubilize phospholipids. Our results indicate that chlorination of apoA-I by the MPO pathway selectively inhibits two critical early events in cholesterol efflux: (1) the binding of apoA-I to ABCA1 and (2) the activation of a key signaling pathway. Therefore, oxidation of apoA-I in the artery wall by MPO-generated chlorinating intermediates may contribute to atherogenesis by impairing cholesterol efflux from macrophages.

The involvement of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is controversial because of the absence of confirmatory data subsequent to the initial reports of their high prevalence in this disease. We characterized three assays for anti-hLAMP-2 antibodies: ELISA and Western blotting assays using unglycosylated recombinant hLAMP-2 expressed in Escherichia coli, and an indirect immunofluorescence assay using stably transfected ldlD cells that expressed glycosylated full-length hLAMP-2 on the plasma membrane. The assays detected autoantibodies to hLAMP-2 in human sera reproducibly and with comparable sensitivity and the assays gave the same results in 80.5% of the test panel of 40 selected positive and negative sera. In untreated patients at presentation, the frequencies of autoantibodies to LAMP-2 were 89%, 91%, and 80%, respectively, among three groups of patients with ANCA-associated vasculitis from Vienna, Austria (n=19); Groningen, the Netherlands (n=50) and Cambridge, United Kingdom (n=53). Prevalence of LAMP-2 autoantibodies was similar in both those with myeloperoxidase-ANCA and proteinase 3-ANCA. Furthermore, we detected LAMP-2 autoantibodies in two ANCA-negative patients. LAMP-2 autoantibodies rapidly became undetectable after the initiation of immunosuppressive treatment and frequently became detectable again during clinical relapse. We conclude that when robust assays are used, circulating autoantibodies to hLAMP-2 can be detected in most European patients with ANCA-associated vasculitis. Large-scale prospective studies are now needed to determine whether they are pathogenic or merely an epiphenomenon.

Inducible nitric oxide synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion (via intratracheal instillation of clodronate liposomes) and subsequent AM reconstitution (via intratracheal instillation of donor iNOS+/+ or iNOS-/- AM). Sepsis was induced by cecal ligation and perforation, and ALI was assessed at 4 h: protein leak by the Evans blue (EB) dye method, neutrophil infiltration via myeloperoxidase (MPO) activity, and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 +/- 0.1 vs. 1.4 +/- 0.1 microg EB.g lung(-1).min(-1); P < 0.05) and MPO activity (37 +/- 4 vs. 67 +/- 8 U/g lung; P < 0.05) compared with that shown in non-AM-depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9 +/- 0.3 microg EB.g lung(-1).min(-1)) but not with iNOS-/- donor AM. In iNOS-/- mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS-/- mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus septic pulmonary protein leak is absolutely dependent on the presence of functional AM and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.

Candida glabrata has emerged as an important fungal pathogen of humans, causing life-threatening infections in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, even with high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leads to fungal burdens that are transiently increased over those in immunocompetent mice. However, even immunocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immune responses to microabscesses were mild as monitored by clinical parameters, including blood enzyme levels, histology, myeloperoxidase, and cytokine levels. Furthermore, we investigated the suitability of amino acid auxotrophic C. glabrata strains for in vitro and in vivo studies of fitness and/or virulence. Histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitro growth in rich medium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parental wild-type strain during the first week of infection and was only mildly reduced 4 weeks after infection, suggesting that C. glabrata is capable of utilizing a broad range of host-derived nutrients during infection. These data suggest that C. glabrata histidine, leucine, or tryptophan auxotrophic strains are suitable for the generation of knockout mutants for in vivo studies. Notably, our work indicates that C. glabrata has successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts.

OBJECTIVE

To determine the effects of silencing Toll-like receptor (TLR) 9 signaling in Pseudomonas aeruginosa keratitis.

METHODS

Corneal TLR9 mRNA levels were tested by RT-PCR in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice and compared. The response of B6 mice to CpG DNA, which binds TLR9, was tested after subconjunctival injection of mice with control or CpG DNA; TLR9, IL-1beta, macrophage inflammatory protein (MIP)-2, IL-4, IL-10, IL-12, IL-18, and IFN-gamma levels were measured by RT-PCR. Langerhans cells (LCs) were stimulated with CpG DNA and treated with TLR9 or control siRNA, and mRNA levels of TLR9, IL-1beta, and MIP-2 were detected by RT-PCR. In addition, IL-1beta levels were tested by ELISA. Then B6 mice were injected subconjunctivally with control or TLR9 siRNA before infection and treated topically afterward. Slit lamp, clinical score, RT-PCR, ELISA, myeloperoxidase assay, and plate counts were performed.

RESULTS

TLR9 mRNA levels were sixfold higher in B6 than in BALB/c corneas the day after injection. B6 mice injected with CpG DNA exhibited an increase in corneal mRNA for TLR9, IL-1beta, MIP-2, IL-12, and IFN-gamma over controls. LCs stimulated with CpG DNA and treated with TLR9 siRNA exhibited reduced TLR9, IL-1beta, and MIP-2 levels compared with controls. Finally, B6 mice treated with TLR9 siRNA showed decreases in corneal opacity, polymorphonuclear leukocyte number, IL-12 and IFN-gamma mRNA, IL-1beta, and MIP-2 protein compared with those treated with control siRNA. Fewer corneas perforated in these mice, but bacterial loads were higher than in controls.

CONCLUSIONS

Signaling through TLR9 appears important in P. aeruginosa keratitis, and silencing TLR9 signaling reduces inflammation but likely contributes to decreased bacterial killing in the cornea.

OBJECTIVE

Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by visceral hypersensitivity and altered bowel motility. There is increasing evidence suggesting the role of inflammation in the pathogenesis of IBS, which addresses the possibility that formerly established rat model of colitis could be used as an IBS model after the inflammation subsided.

METHODS

Colitis was induced by intracolonic instillation of 4% acetic acid in male Sprague-Dawley rats. The extent of inflammation was assessed by histological examination and myeloperoxidase (MPO) activity assay. After subsidence of colitis, the rats were subjected to rectal distension and restraint stress, then the abdominal withdrawal reflex and the number of stress-induced fecal output were measured, respectively.

RESULTS

At 2 days post-induction of colitis, the colon showed characteristic inflammatory changes in histology and 8-fold increase in MPO activity. At 7 days post-induction of colitis, the histological features and MPO activity returned to normal. The rats at 7 days post-induction of colitis showed hypersensitive response to rectal distension without an accompanying change in rectal compliance, and defecated more stools than control animals when under stress.

CONCLUSIONS

These results concur largely with the characteristic features of IBS, visceral hypersensitivity and altered defecation pattern in the absence of detectable disease, suggesting that this animal model is a methodologically convenient and useful model for studying a subset of IBS.

Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO-DNA complexes and established a new HNE-DNA ELISA. We show that these methods reliably quantify MPO-DNA and HNE-DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired "early" and "late" bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways.

Arthritis of the knee is the most common type of joint inflammatory disorder and it is associated with pain and inflammation of the joint capsule. Few studies address the effects of the 810-nm laser in such conditions. Here we investigated the effects of low-level laser therapy (LLLT; infrared, 810-nm) in experimentally induced rat knee inflammation. Thirty male Wistar rats (230-250 g) were anesthetized and injected with carrageenan by an intra-articular route. After 6 and 12 h, all animals were killed by CO(2) inhalation and the articular cavity was washed for cellular and biochemical analysis. Articular tissue was carefully removed for real-time PCR analysis in order to evaluate COX-1 and COX-2 expression. LLLT was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity with 1, 3, and 6 J (Joules) of energy. This result was corroborated by cell counting showing the reduction of polymorphonuclear cells at the inflammatory site. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Both COX-1 and 2 gene expression were significantly enhanced by laser irradiation while PGE(2) production was inhibited. Low-level laser therapy operating at 810 nm markedly reduced inflammatory signs of inflammation but increased COX-1 and 2 gene expression. Further studies are necessary to investigate the possible production of antiinflammatory mediators by COX enzymes induced by laser irradiation in knee inflammation.

BACKGROUND

Staphylococcus aureus remains the predominant pathogen in diabetic foot infections and prevalence of methicillin resistant S.aureus (MRSA) strains further complicates the situation. The incidence of MRSA in infected foot ulcers is 15-30% and there is an alarming trend for its increase in many countries. Diabetes acts as an immunosuppressive state decreasing the overall immune functioning of body and to worsen the situation, wounds inflicted with drug resistant strains represent a morbid combination in diabetic patients. Foot infections caused by MRSA are associated with an increased risk of amputations, increased hospital stay, increased expenses and higher infection-related mortality. Hence, newer, safer and effective treatment strategies are required for treating MRSA mediated diabetic foot infections. The present study focuses on the use of lytic bacteriophage in combination with linezolid as an effective treatment strategy against foot infection in diabetic population.

METHODS

Acute hindpaw infection with S.aureus ATCC 43300 was established in alloxan induced diabetic BALB/c mice. Therapeutic efficacy of a well characterized broad host range lytic bacteriophage, MR-10 was evaluated alone as well as in combination with linezolid in resolving the course of hindpaw foot infection in diabetic mice. The process of wound healing was also investigated.

CONCLUSIONS

A single administration of phage exhibited efficacy similar to linezolid in resolving the course of hindpaw infection in diabetic animals. However, combination therapy using both the agents was much more effective in arresting the entire infection process (bacterial load, lesion score, foot myeloperoxidase activity and histopathological analysis). The entire process of tissue healing was also hastened. Use of combined agents has been known to decrease the frequency of emergence of resistant mutants, hence this approach can serve as an effective strategy in treating MRSA mediated foot infections in diabetic individuals who do not respond to conventional antibiotic therapy.