The MicroRNA-based Liquid Biopsy Improves Early Assessment of Lethal Acetaminophen Poisoning: A Case Report.

BACKGROUND Acetaminophen overdose is the most common cause of acute liver failure. Nevertheless, new biomarker approaches enabling early prediction of the outcome of the acetaminophen overdose are needed. Recently, using next-generation sequencing analysis of serum from human study participants we uncovered injury-specific signatures of circulating microRNAs (miRNAs) that represented underlying molecular mechanisms of toxicity. This case study is first to show the application of miRNA profiling to assess prognosis of acetaminophen poisoning. CASE REPORT The patient was admitted to the hospital following supra therapeutic acetaminophen ingestion. The patient showed elevated levels of biomarkers of hepatocellular injury alanine aminotransferase, aspartate transaminase, and glutamate dehydrogenase. Even though treatment with N-acetyl cysteine was initiated 24 hours post-ingestion, levels of alanine-aminotransferase and aspartate transaminase peaked at about 40 hours post ingestion of acetaminophen. We analyzed global circulating miRNA levels from 24 consecutive serum samples from this study participant covering the period from admission to time of death. CONCLUSIONS The resulting global miRNA profiles were compared with profiles from study participants with non-lethal acetaminophen poisoning and healthy controls. At the admission, the miRNA profiles of both lethal and non-lethal acetaminophen poisoning showed induction of cellular stress and oxidative damage. Later, the miRNA profiles of the lethal poisoning featured fibrosis and coagulation pathways while profiles of non-lethal cases resembled those of healthy study participants. Although additional confirmatory studies are needed, our case study is first to indicate that global miRNA profiles to be used as liquid biopsies have potential to facilitate the assessment of acetaminophen poisoning.

Evaluation of Potential Serum Biomarkers of Disease Activity in Diverse Forms of Vasculitis.

OBJECTIVE: We evaluated potential circulating biomarkers of disease activity in giant cell arteritis (GCA), Takayasu arteritis (TA), polyarteritis nodosa (PAN), and eosinophilic granulomatosis with polyangiitis (EGPA). METHODS: A panel of 22 serum proteins was tested in patients enrolled in the Vasculitis Clinical Research Consortium Longitudinal Studies of GCA, TA, PAN, or EGPA. Mixed models were used for most analyses. A J48 classification tree method was used to find the most relevant markers to differentiate between active and inactive GCA. RESULTS: Tests were done on 418 samples from 152 patients (60 GCA, 29 TA, 26 PAN, 37 EGPA), during both active vasculitis and remission. In GCA, these showed significant (p < 0.05) differences between disease states: B cell-attracting chemokine 1 (BCA)-1/CXC motif ligand 13 (CXCL13), erythrocyte sedimentation rate (ESR), interferon-γ-induced protein 10/CXC motif chemokine 10, soluble interleukin 2 receptor α (sIL-2Rα), and tissue inhibitor of metalloproteinase-1 (TIMP-1). In EGPA, these showed significant increases during active disease: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF, interleukin (IL)-6, IL-15, and sIL-2Rα. BCA-1/CXCL13 also showed such increases, but only after adjustment for treatment. In PAN, ESR and matrix metalloprotease (MMP)-3 showed significant differences between disease states. Differences in biomarker levels between diseases were significant for 11 markers and were more striking (all p < 0.01) than differences related to disease activity. A combination of lower values of TIMP-1, IL-6, interferon-γ, and MMP-3 correctly classified 87% of samples with inactive GCA. CONCLUSION: We identified novel biomarkers of disease activity in GCA and EGPA. Differences of biomarker levels between diseases, independent of disease activity, were more apparent than differences related to disease activity. Further studies are needed to determine whether these serum proteins have potential for clinical use in distinguishing active disease from remission or in predicting longer-term outcomes.

The association of serum interleukin-6 levels with clinical outcomes in antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: To investigate serum IL-6 (sIL-6) levels during active disease, complete remission (CR), and relapse in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), and to explore the association of changes in sIL-6 with clinical outcomes. METHODS: sIL-6 levels were measured at baseline and longitudinally over 18 months, in 78 patients with AAV enrolled in a randomized controlled trial comparing treatment with either rituximab (RTX) or cyclophosphamide (CYC)/azathioprine (AZA). Outcome variables included baseline clinical features, ANCA specificity, disease activity (active disease versus CR), time to relapse events, B cell repopulation, and ANCA titer increases. RESULTS: At baseline, sIL6 levels were detectable in 81% of patients; 73% (n = 57) of subjects were proteinase 3 (PR3)-ANCA positive, sIL-6 levels were higher in subjects with PR3-ANCAs and positively correlated with their levels (rs = 0.36,p < 0.01), but not with levels of myeloperoxidase (MPO)-ANCA (rs = -0.17,p = 0.47). Higher baseline sIL-6 levels were associated with PR3-ANCA positivity, fever, pulmonary nodules/cavities, conductive deafness, and absence of urinary red blood cell casts (p < 0.05). Baseline sIL6 levels did not predict CR at month 6 (p = 0.71), and the median sIL-6 level declined from baseline with induction therapy, regardless of CR achievement. An increase in sIL-6 during CR was a predictor for subsequent severe relapse in RTX-treated patients (hazard ratio (HR):7.24,p = 0.01), but not in CYC/AZA-treated patients (HR:0.62,p = 0.50). In contrast, a sIL-6 increase did not predict B cell repopulation or ANCA titer increase in either treatment arm (p > 0.05). CONCLUSION: At baseline, sIL-6 concentrations correlate with PR3-ANCA titers and are associated with specific clinical manifestations of AAV. Baseline sIL6 concentrations do not predict CR at 6 months, but the increase in sIL-6 concentrations during CR is associated with subsequent severe relapse among RTX-treated patients. Further investigation into the mechanistic role of IL6 in AAV might lead to identifying this pathway as a potential therapeutic target in this disease.

Brief Report: Circulating Cytokine Profiles and Antineutrophil Cytoplasmic Antibody Specificity in Patients With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: To evaluate circulating cytokine profiles in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV), classified by antineutrophil cytoplasmic antibody (ANCA) specificity (proteinase 3 ANCA [PR3-ANCA] versus myeloperoxidase ANCA [MPO-ANCA]) or by clinical diagnosis (granulomatosis with polyangiitis [GPA] versus microscopic polyangiitis [MPA]). METHODS: A panel of 29 cytokines was tested in 186 patients with active AAV at inclusion into the Rituximab in AAV trial. Cytokine concentrations were compared between groups within each classification system. Multivariable analyses adjusted for age, sex, and renal insufficiency were performed, with each biomarker as a dependent variable and ANCA specificity and clinical diagnosis as explanatory variables of interest. RESULTS: Levels of 9 circulating cytokines (interleukin-6 [IL-6], granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-15, IL-18, CXCL8/IL-8, CCL-17/thymus and activation-regulated chemokine [TARC], IL-18 binding protein [IL-18 BP], soluble IL-2 receptor α [sIL-2Rα], and nerve growth factor ß [NGFß]) were significantly higher in PR3-AAV than MPO-AAV, 4 cytokines (sIL6R, soluble tumor necrosis factor receptor type II [sTNFRII], neutrophil gelatinase-associated lipocalin [NGAL], and soluble intercellular adhesion molecule 1 [sICAM-1]) were higher in MPO-AAV than in PR3-AAV, 6 cytokines (IL-6, GM-CSF, IL-15, IL-18, sIL-2Rα, and NGFß) were higher in GPA than in MPA, and 3 cytokines (osteopontin, sTNFRII, and NGAL) were higher in MPA than in GPA (all P < 0.05). For nearly all cytokines, the difference between PR3-AAV and MPO-AAV was larger than that between GPA and MPA. The multivariate analysis showed that 8 cytokines (IL-15, IL-8, IL-18 BP, NGF-ß, sICAM-1, TARC, osteopontin, and kidney injury molecule 1 (P < 0.05) distinguished patients with AAV better (lower P values and larger effect sizes) when grouped by ANCA specificity than by clinical diagnosis. CONCLUSION: Distinct cytokine profiles were identified for PR3-AAV versus MPO-AAV and for GPA versus MPA. Differences in these circulating immune mediators are more strongly associated with ANCA specificity than with clinical diagnosis, suggesting that heterogeneity in the AAV subtypes extends beyond clinical phenotypes.

Monoclonal antibodies specific for oncofetal antigen--immature laminin receptor protein: Effects on tumor growth and spread in two murine models.

The oncofetal antigen - immature laminin receptor protein (OFA/iLRP) has been linked to metastatic tumor spread for several years. The present study, in which 2 highly-specific, high-affinity OFA/iLRP-reactive mouse monoclonal antibodies were examined for ability to suppress tumor cell growth and metastatic spread in the A20 B-cell leukemia model and the B16 melanoma model, provides the first direct evidence that targeting OFA/iLRP with exogenous antibodies can have therapeutic benefit. While the antibodies were modestly effective at preventing tumor growth at the primary injection site, both antibodies strongly suppressed end-organ tumor formation following intravenous tumor cell injection. Capacity of anti-OFA/iLRP antibodies to suppress tumor spread through the blood in the leukemia model suggests their use as a therapy for individuals with leukemic disease (either for patients in remission or even as part of an induction therapy). The results also suggest use against metastatic spread with solid tumors.

Performance of Biopsy Needle With Therapeutic Injection System to Prevent Bleeding Complications.

Renal disease is epidemic in the United States with approximately 8 × 106 people having chronic kidney disease. Renal biopsies are widely used to provide essential diagnostic information to physicians. However, the risk of bleeding complications possibly leading to life-threatening situations results in the contra-indication of biopsy in certain patient populations. Safer renal biopsies will allow more accurate diagnosis and better management of this epidemic health problem. We report the preclinical testing of a novel biopsy device called the therapeutic injection system (TIS). The device introduces a third stage to the standard two-stage side-cut percutaneous biopsy process. The third stage is designed to reduce bleeding complications by injecting a hemostatic plug at the time of biopsy. Laboratory evaluation and preliminary in vivo animal testing using an anticoagulated porcine model of the TIS and Bard Monopty® (Bard Medical, Covington, GA) control device were performed. The hemostatic material Gelfoam® (Pfizer, Brussels, Belgium) was selected as the active material comprising the hemostatic plugs. The performance of two composite plugs, one composed of polyvinyl alcohol (PVA) combined in 2:1 and 12:1 ratios with the hemostatic material, and one plug composed of 100[Formula: see text] hemostatic material were tested. Stroke sequence and hemostatic plug deployment were verified by sequential firing of the TIS biopsy needle into clear gelatin and ex vivo bovine kidney specimens. In vivo trials with porcine specimens revealed a significant reduction in blood loss (8.1 [Formula: see text] 3.9 ml, control versus 1.9 [Formula: see text] 1.6 ml, 12:1 PVA/hemostatic, TIS, [Formula: see text] = 0.01, [Formula: see text] = 6). The 100[Formula: see text] hemostatic plug showed a substantial and immediate reduction in blood loss (9.2 ml, control versus 0.0 ml, TIS, [Formula: see text] = 1). The prototype device was shown to work repeatedly and reliably in laboratory trials. Initial results show promise in this approach to control post biopsy bleeding. This solution maintains the simplicity and directness of the percutaneous approach, while not significantly changing the standard percutaneous biopsy procedure.

Serum proteins reflecting inflammation, injury and repair as biomarkers of disease activity in ANCA-associated vasculitis.

OBJECTIVE: To identify circulating proteins that distinguish between active anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and remission in a manner complementary to markers of systemic inflammation. METHODS: Twenty-eight serum proteins representing diverse aspects of the biology of AAV were measured before and 6 months after treatment in a large clinical trial of AAV. Subjects (n=186) enrolled in the Rituximab in ANCA-Associated Vasculitis (RAVE) trial were studied. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels were available for comparison. The primary outcome was the ability of markers to distinguish severe AAV (Birmingham Vasculitis Activity Score for Wegener's granulomatosis (BVAS/WG)≥3 at screening) from remission (BVAS/WG=0 at month 6), using areas under receiver operating characteristic (ROC) curve (AUC). RESULTS: All subjects had severe active vasculitis (median BVAS/WG=8) at screening. In the 137 subjects in remission at month 6, 24 of the 28 markers showed significant declines. ROC analysis indicated that levels of CXCL13 (BCA-1), matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) best discriminated active AAV from remission (AUC>0.8) and from healthy controls (AUC>0.9). Correlations among these markers and with ESR or CRP were low. CONCLUSIONS: Many markers are elevated in severe active AAV and decline with treatment, but CXCL13, MMP-3 and TIMP-1 distinguish active AAV from remission better than the other markers studied, including ESR and CRP. These proteins are particularly promising candidates for future studies to address unmet needs in the assessment of patients with AAV.

Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss.

Aprotinin has been used widely in surgery as an anti-bleeding agent but is associated with a number of side effects. We report that textilinin-1, a serine protease inhibitor from Pseudonaja textilis venom with sequence relatedness to aprotinin, is a potent but reversible plasmin inhibitor and has a narrower range of protease inhibition compared to aprotinin. Like aprotinin, textilinin-1 at 5 micromol/l gave almost complete inhibition of tissue plasminogen activator-induced fibrinolysis of whole blood clots. The activated partial thromboplastin time for plasma was markedly increased by aprotinin but unaffected by textilinin-1. In a mouse tail-vein bleeding model, intravenous textilinin-1 and aprotinin caused similar decreases in blood loss but time to haemostasis in the textilinin-treated animals was significantly shorter than in aprotinin-treated mice. Based on these data, textilinin-1 merits further investigation as a therapeutic alternative to aprotinin.

Role of interleukin-6 in a glucan-induced model of granulomatous vasculitis.

The role of interleukin-6 (IL-6) in granulomatous vasculitis is not well understood. To investigate its involvement in this type of vasculitis a model of glucan-induced pulmonary vasculitis employed interleukin-6 deficient (IL-6-/-) mice. Briefly, IL-6-/- mice and C57B/J6 wild type (IL-6+/+) mice were injected intravenously with a suspension of glucan isolated from the cell wall of bakers yeast which results in a granulomatous vasculitis primarily in the pulmonary vasculature. Histological examination demonstrated no significant difference in the number of infiltrating leukocytes between the IL-6+/+ and IL-6-/- glucan-injured mice. Similar numbers of granulomas were noted in both the IL-6+/+ and IL-6-/- injured animals, while no granulomas were seen in saline injected control mice. Cells recovered from the bronchoalveolar lavage (BAL) fluid were differentially stained and counted. While there was a significant increase in infiltrating leukocytes recovered from the BAL following glucan-induced injury, there was no significant difference between the IL-6+/+ and IL-6-/- mice. In addition, no difference was demonstrated in total protein content in the BAL fluid between IL-6+/+ and IL-6-/- mice. However, myeloperoxidase (MPO) activity in the lungs of the IL-6-/- mice was less than in their IL-6+/+ counterparts suggesting that these animals have a partial defect in their ability to recruit neutrophils in this model. Studies done to look for levels of other cytokines/chemokines in these animals to compensate for the loss of IL-6 revealed that only IL-10 in the sera (p<0.016) and BAL fluid (p<0.05) of IL-6-/- mice was significantly higher then their IL-6+/+-injured counterparts. These studies suggest that IL-6, while possibly involved in early neutrophil accumulation in this model does not appear critical to the development of the TH-2 mediated granulomatous vasculitis.

Generation of C5a in the absence of C3: a new complement activation pathway.

Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.

Development of an internally controlled antibody microarray.

Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also because antigen-antibody interactions are concentration-dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody-based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted, and the other color represents the amount of the antigen that is used to quantify the level of protein expression. When compared with median fluorescence intensity alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins interleukin (IL)-1beta, IL-5, IL-6, and macrophage inflammatory proteins 1alpha and 1beta. The levels of protein expression seen with the antibody microarray was compared with that obtained with Western blot analysis, and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as Western blot analysis, and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood.

Role of interleukin-6 in immune complex induced models of vascular injury.

Previous studies have suggested that Interleukin-6 (IL-6) acts as a marker of vasculitis. To determine the role of IL-6 in vasculitis we utilized two models of immune complex induced vascular injury (dermal Arthus and acute pulmonary alveolitis) in IL-6 deficient (IL-6(-/-)) and IL-6 sufficient (IL-6(+/+)) mice. Plasma and bronchoalveolar lavage (BAL) levels of IL-6 were elevated in the injured IL-6(+/+) mice with acute alveolitis and in the plasma of IL-6(+/+) mice with dermal Arthus vasculitis. While, IL-6 levels in IL-6(-/-) mice were near or below the levels of detection. Histological examination of the intensity of vascular injury response demonstrated no significant differences between IL-6(-/-) and IL6(+/+) mice. More specifically, lung permeability (total protein in the BAL) in the lung injury model in IL-6(-/-) mice was the same as injured IL-6(+/+) mice. As a corollary, assessment of vascular permeability in both models was the same in the IL-6(-/-) as the IL-6(+/+) mice. Quantification of leukocyte influx into the injured tissues in both models also revealed no differences between the IL-6(-/-) and IL-6(+/+) mice. These data demonstrate that while IL-6 is upregulated in acute vascular injury it does not appear to be critical in the development of the vascular inflammatory response.

Role of metalloelastase in a model of allergic lung responses induced by cockroach allergen.

Our laboratory and others have shown an important role of metalloelastase (MMP-12) in the pathogenesis of acute and chronic lung injury. Because chronic asthma is characterized by airway inflammation and alterations in the airway extracellular matrix, we explored the role of metalloelastase in a model of allergic airway inflammation induced by cockroach antigen (CRA). Using MMP-12-deficient mice we found a significant reduction in CRA-induced inflammatory injury, as evidenced by fewer peribronchial leukocytes, significantly less protein in the bronchoalveolar lavage (BAL) fluid, and a significant reduction in the number of infiltrating neutrophils, eosinophils, and macrophages, relative to wild-type mice. Although we did not find a significant reduction in the number of T cells in the injured MMP-12-deficient animals as compared to controls, levels of the chemotactic factors interleukin-5, macrophage inflammatory protein-1 alpha, monocyte chemoattractant protein-1, thymus activation regulated chemokine, and the proinflammatory cytokine tumor necrosis factor-alpha were significantly reduced in the bronchoalveolar lavage fluid of CRA-challenged MMP-12-deficient mice, relative to CRA-challenged control animals. These studies indicate that MMP-12 plays an important proinflammatory role in the development of allergic inflammation in the CRA model. Alterations in the levels of chemotactic factors and other proinflammatory cytokines in the MMP-12-deficient mice may underlie the decrease in leukocyte recruitment into inflamed lungs.

Matrix metalloproteinases in acute inflammation: induction of MMP-3 and MMP-9 in fibroblasts and epithelial cells following exposure to pro-inflammatory mediators in vitro.

Recent studies have demonstrated important pro-inflammatory roles for two matrix metalloproteinases (MMPs)-MMP-3 (stromelysin-1) and MMP-9 (gelatinase B)-in acute lung injury [Am. J. Respir. Cell Mol. Biol. 24 (2001) 1]. A role for MMP-3 in skin inflammation has also been demonstrated [Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 6885]. While leukocytes (neutrophils and macrophages) are known to elaborate these tissue-destructive enzymes, parenchymal cells are also capable of synthesizing MMPs. In the present study, we examined the production of MMP-3 and MMP-9 by rodent lung fibroblasts, type II epithelial cells, and vascular endothelial cells. Dermal fibroblasts were also examined. Cells were examined under control conditions and in response to agonists that induce acute inflammatory tissue injury (IgG-containing immune complexes and lipopolysaccharide [LPS]). In the absence of stimulation, MMP-3 and MMP-9 were not detected or were present at low level. However, upon stimulation with either of the two pro-inflammatory agonists, production of both enzymes occurred in fibroblasts and epithelial cells (though not in endothelial cells). The observation that resident cells in the tissue parenchyma can elaborate MMPs in direct response to pro-inflammatory stimuli provides insight into possible mechanisms by which tissue damage occurs in acute inflammation.

Attenuation of autoimmune disease in Fas-deficient mice by treatment with a cytotoxic benzodiazepine.

OBJECTIVE: Elimination of autoreactive cells relies on Fas-dependent activation-induced cell death mechanisms, an important component of peripheral tolerance. Defects in Fas or its cognate ligand lead to inefficient activation-induced cell death and are specific causes of lymphoproliferative and autoimmune diseases. The present study was undertaken to investigate a novel 1,4-benzodiazepine (Bz-423) that induces apoptosis and limits autoimmune disease in NZB/NZW mice, to determine its activity against lupus-like disease associated with defective Fas expression. We investigated the Fas-dependence of its cytotoxic actions, its therapeutic potential in mice deficient in Fas, and its therapeutic mechanism of action. METHODS: Primary lymphocytes isolated from Fas-deficient MRL/MpJ-Fas(lpr) (MRL-lpr) mice were tested for sensitivity to Bz-423. Bz-423 was administered to MRL-lpr mice for short (1-week) or long (14-week) periods, and its effects on cell survival were determined along with measures of nephritis, arthritis, antibody titers, and Th subpopulations. BALB/c mice were similarly treated to determine if Bz-423 alters normal immune functions in vivo. RESULTS: Administration of Bz-423 to MRL-lpr mice significantly reduced autoimmune disease including glomerulonephritis and arthritis. Treatment was associated with decreases in CD4+ T cells and an alteration in the Th1/Th2 balance. At the therapeutic dosage, Bz-423 did not interfere with normal T and B cell responses in BALB/c mice, suggesting that this agent is not globally immunosuppressive. CONCLUSION: Bz-423 is a novel immunomodulatory agent that is active against disease even in the context of defective Fas signaling. It is a leading compound for further investigation into the development of selective therapies for lupus.

Exogenous nitric oxide downregulates MIP-2 and MIP-1alpha chemokines and MAPK p44/42 after ischemia and reperfusion of the rat kidney.

The mechanisms by which nitric oxide (NO) exerts its protective effect in the ischemia/reperfusion (I/R) injury of the kidney have not been fully determined. The hypothesis of this study was based on the assumption that I/R upregulates some chemokines (MIP-2 and MIP-1alpha) as well as certain protein kinases (MAPK p44/42), and therefore we aimed in this work at recognizing if an exogenous NO donor would downregulate these effects in rat ischemic kidneys at the same time that it would offer functional protection as measured by serum creatinine. Sprague-Dawley rats were subjected to renal warm ischemia (75 min) and contralateral nephrectomy. Animals were divided into 3 groups (n = 8 per group): sham, ischemic control, and ischemic group treated with sodium nitroprusside (NaNP 5 mg/kg) given 15 min prior to reperfusion. Serum creatinine (SCr), serum chemokines (MIP-2 and MIP-1alpha), kidney tissue MAPK p44/42, kidney neutrophil infiltration determined by myeloperoxidase (MPO), and light histology were evaluated 4 h after reperfusion began. There were significant improvements in SCr and better histopathological features in the I/R-NaNP group compared with the I/R group. Similarly, the I/R-NaNP kidneys exhibited a downregulating effect of serum chemokines (MIP-2 and MIP-1alpha) and kidney tissue MAPK p44/42 that was not observed in the I/R group alone. The MPO levels were lower in the I/R-NaNP group compared with the I/R untreated group. We can conclude from these experiments that I/R of the rat kidney upregulated the production of MIP-2 and MIP-1alpha chemokines and the activation of MAPKp44/42. It also had a detrimental effect on the function and structure of the ischemic kidney. Exogenous NO had a temporal protective effect in organ function and histology and exerted a downregulating response in the production of MIP-2 and MIP-1alpha chemokines and the activation of MAPK p44/42 following I/R.

Expression and function of the C5a receptor in rat alveolar epithelial cells.

Although alveolar epithelial cells (AEC) form an important barrier for host defenses in the lung, there is limited information about ways in which AEC can directly participate in the lung inflammatory response. In the current studies, primary cultures of rat AEC (RAEC) have been shown to specifically bind recombinant rat C5a at high affinity and in a saturable manner. This binding was enhanced in a time-dependent manner by pre-exposure of RAEC to LPS, IL-6, or TNF-alpha, the increased binding of C5a being associated with increased levels of mRNA for the C5a receptor (C5aR). Exposure of RAEC to C5a also caused increased expression of mRNA for C5aR. As compared with exposure of RAEC to LPS or to C5a alone, exposure to the combination caused enhanced production of TNF-alpha, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant-1, as well as increased intracellular levels of IL-1beta. These data indicate that RAEC, when activated, have enhanced binding of C5a in association with increased mRNA for C5aR. The functional outcome is enhanced release of proinflammatory mediators. These data underscore the phlogistic potential of RAEC and the ability of C5a to enhance the phlogistic responses of RAEC.