Immune cell profiles of patients with interstitial cystitis/bladder pain syndrome.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a disorder characterized by bladder pain upon filling which severely affects quality of life. Clinical presentation can vary. Local inflammatory events typify the clinical presentation of IC/BPS patients with Hunner lesions (IC/BPS-HL). It has previously been proposed that B cells are more prevalent in HL, but understanding their exact role in this environment requires a more complete immunological profile of HL. We characterized immunological dysfunction specifically in HL using immunohistochemistry. We detected significantly more plasma cells (50× increase, p < 0.0001), B cells (28× increase, p < 0.0001), T cells (3× increase, p < 0.0001), monocytes/macrophages (6× increase, p < 0.0001), granulocytes (4× increase, p < 0.0001), and natural killer cells (2× increase, p = 0.0249) in IC/BPS patients with HL than in unaffected controls (UC). Patients with IC/BPS-HL also had significantly elevated urinary levels of IL-6 (p = 0.0054), TNF-α (p = 0.0064) and IL-13 (p = 0.0304) compared to patients with IC/BPS without HL (IC/BPS-NHL). In contrast, IL-12p70 levels were significantly lower in the patients with HL than in those without these lesions (p = 0.0422). Different cytokines were elevated in the urine of IC/BPS patients with and without HL, indicating that different disease processes are active in IC/BPS patients with and without HL. Elevated levels of CD138+, CD20+, and CD3+ cells in HL are consistent B and T-cell involvement in disease processes within HL.

Coronary vasodilation mediated by T cells expressing choline acetyltransferase.

CD4+ T cells expressing choline acetyltransferase (ChAT) have recently been shown to cause a drop in systemic blood pressure when infused into mice. The aim of this study was to determine if ChAT-expressing T cells could regulate coronary vascular reactivity. Preconstricted segments of epicardial and intramyocardial porcine coronary arteries relaxed in response to Jurkat T cells (JT) that overexpressed ChAT (JTChAT cells). The efficacy of the JTChAT cells was similar in epicardial and intramyocardial vessels with a maximum dilator response to 3 × 105 cells/mL of 38.0 ± 6.7% and 38.7 ± 7.25%, respectively. In contrast, nontransfected JT cells elicited a weak dilator response, followed by a weak contraction. The response of JTChAT cells was dependent on the presence of the endothelial cells. In addition, the response could be significantly reduced by Nω-nitro-l-arginine methyl ester (l-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the presence of indomethacin. JTChAT cells, but not JT cells, increased the expression of phosphorylated endothelial nitric oxide synthase (eNOS). JTChAT cells contained significantly greater levels of acetylcholine compared with JT cells; however, the nonselective muscarinic antagonist atropine and the M1 receptor antagonist pirenzepine both failed to block the dilator effect of JTChAT cells. Exogenously added acetylcholine induced only a weak relaxation (∼10%) at low concentrations, which became a contractile response at higher concentrations. These data illustrate the capacity for cells that express ChAT to regulate coronary vascular reactivity, via mechanisms that are dependent on interaction with the endothelium and in part mediated by the release of nitric oxide.NEW & NOTEWORTHY This study shows ChAT-expressing T cells can induce vasodilation of the blood vessel in the coronary circulation and that this effect relies on a direct interaction between T cells and the coronary vascular endothelium. The study establishes a potential immunomodulatory role for T cells in the coronary circulation. The present findings offer an additional possibility that a deficiency of ChAT-expressing T cells could contribute to reduced coronary blood flow and ischemic events in the myocardium.

Contribution of Connexin Hemichannels to the Pathogenesis of Acute Lung Injury.

Connexin (Cx) family members form hemichannels (HCs) and gap junctions (GJs). Biological functions of Cx HCs have not been adequately characterized due to the inability to selectively target HCs or GJs. Recently, we developed a 6-mer peptide mimetic (P5) of the first extracellular loop of Cx43 and showed that it can block the permeability of HCs but not GJs formed by Cx43. In this study, we further characterized the HC blocking property of P5 and investigated the role of Cx HCs in acute lung injury (ALI). We found that P5 administration decreased HC permeability, in pulmonary microvascular endothelial cells, HepG2 cells, and even Cx43-deficient astrocytes, which express different sets of Cxs, suggesting that P5 is a broad spectrum Cx HC blocker. In addition, P5 reduced HC permeability of alveolar cells in vivo. Moreover, P5 decreased endotoxin-induced release, by vascular endothelial cells in vitro, of high mobility group box protein 1 (HMGB1), a critical mediator of acute lung injury (ALI), and reduced HMGB1 accumulation in bronchoalveolar lavage fluid (BALF) of mice subjected to intratracheal endotoxin instillation. Furthermore, P5 administration resulted in a significant decrease in the concentrations of ALT, AST, and LDH in the BALF, the accumulation of leukocytes in alveoli, and the mortality rate of mice subjected to ALI. Wright-Giemsa staining showed that P5 caused similar reductions of both neutrophils and monocytes in BALF of ALI mice. Together, these results suggest that Cx HCs mediate HMGB1 release, augment leukocyte recruitment, and contribute to ALI pathology.

Correction: MIF inhibition enhances pulmonary angiogenesis and lung development in congenital diaphragmatic hernia.

In the original version of this article, the name of the author "Kamesh Ayasolla" was incorrectly given as "Kamesh Ayyasola". This has now been corrected to "Kamesh Ayasolla" in both the PDF and HTML versions of the article.

MIF inhibition enhances pulmonary angiogenesis and lung development in congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a complex birth anomaly with significant mortality and morbidity. Lung hypoplasia and persistent pulmonary hypertension (PPHN) limit survival in CDH. Macrophage migration inhibitory factor (MIF), a key regulator of innate immunity, is involved in hypoxia-induced vascular remodeling and PPHN. We hypothesized that antenatal inhibition of MIF in CDH fetuses, would reduce vascular remodeling, and improve angiogenesis and lung development. METHODS: Pregnant rats were randomized into three groups: Control, nitrofen, and nitrofen + ISO-92. Lung volumes of pups were measured by CT scanning. Right ventricular systolic pressure (RVSP) and vascular wall thickness (VWT) were measured together with MIF concentration, angiogenesis markers, lung morphometry, and histology. RESULTS: Prenatal treatment with ISO-92, an MIF inhibitor, improved normalization of static lung volume, lung volume-to-body weight ratio, decreased alveolar septal thickness, RVSP and VWT and improved radial alveolar count as compared to the non-treated group. Expression of MIF was unaffected by ISO-92; however, ISO-92 increased p-eNOS and VEGF activities and reduced arginase 1, 2 and Sflt-1. CONCLUSION: Prenatal inhibition of MIF activity in CDH rat model improves angiogenesis and lung development. This selective intervention may be a future therapeutic strategy to reduce the morbidity and mortality of this devastating condition.

Development of a biomarker mortality risk model in acute respiratory distress syndrome.

BACKGROUND: There is a compelling unmet medical need for biomarker-based models to risk-stratify patients with acute respiratory distress syndrome. Effective stratification would optimize participant selection for clinical trial enrollment by focusing on those most likely to benefit from new interventions. Our objective was to develop a prognostic, biomarker-based model for predicting mortality in adult patients with acute respiratory distress syndrome. METHODS: This is a secondary analysis using a cohort of 252 mechanically ventilated subjects with the diagnosis of acute respiratory distress syndrome. Survival to day 7 with both day 0 (first day of presentation) and day 7 sample availability was required. Blood was collected for biomarker measurements at first presentation to the intensive care unit and on the seventh day. Biomarkers included cytokine-chemokines, dual-functioning cytozymes, and vascular injury markers. Logistic regression, latent class analysis, and classification and regression tree analysis were used to identify the plasma biomarkers most predictive of 28-day ARDS mortality. RESULTS: From eight biologically relevant biomarker candidates, six demonstrated an enhanced capacity to predict mortality at day 0. Latent-class analysis identified two biomarker-based phenotypes. Phenotype A exhibited significantly higher plasma levels of angiopoietin-2, macrophage migration inhibitory factor, interleukin-8, interleukin-1 receptor antagonist, interleukin-6, and extracellular nicotinamide phosphoribosyltransferase (eNAMPT) compared to phenotype B. Mortality at 28 days was significantly higher for phenotype A compared to phenotype B (32% vs 19%, p = 0.04). CONCLUSIONS: An adult biomarker-based risk model reliably identifies ARDS subjects at risk of death within 28 days of hospitalization.

D-dopachrome tautomerase predicts outcome but not the development of acute kidney injury after orthotopic liver transplantation.

BACKGROUND: Elevated concentrations of D-dopachrome tautomerase (D-DT) were associated with adverse outcome in various clinical settings. However, no study assessed D-DT concentrations in patients requiring orthotopic liver transplantation (OLT). The aim of this observational study was to measure serum D-DT concentrations in patients undergoing OLT and associate D-DT with survival and acute kidney injury (AKI). METHODS: Forty-seven adults with end-stage liver disease undergoing OLT were included. Areas under the receiver operating curves (AUC) were calculated to assess predictive values of D-DT for outcome and AKI after OLT. Survival was analyzed by Kaplan-Meier curves. RESULTS: Serum D-DT concentrations were greater in non-survivors than in survivors prior to OLT (86 [50-117] vs. 53 [31-71] ng/ml, P = 0.008), and on day 1 (357 [238-724] vs. 189 [135-309] ng/ml, P = 0.001) and day 2 (210 [142-471] vs. 159 [120-204] ng/ml, P = 0.004) following OLT. Serum D-DT concentrations predicted lethal outcome when measured preoperatively (AUC = 0.75, P = 0.017) and on postoperative day 1 (AUC = 0.75, P = 0.015). One-year survival of patients with preoperative D-DT concentrations >85 ng/ml was 50%, whereas that of patients with preoperative D-DT concentrations <85 ng/ml was 83% (Chi2 = 5.83, P = 0.016). In contrast, D-DT was not associated with AKI after OLT. CONCLUSION: In patients undergoing OLT, serum D-DT might predict outcome after OLT.

Graft-derived macrophage migration inhibitory factor correlates with hepatocellular injury in patients undergoing liver transplantation.

Experimental studies suggest that macrophage migration inhibitory factor (MIF) mediates ischemia/reperfusion injury during liver transplantation. This study assessed whether human liver grafts release MIF during preservation, and whether the release of MIF is proportional to the extent of hepatocellular injury. Additionally, the association between MIF and early allograft dysfunction (EAD) after liver transplantation was evaluated. Concentrations of MIF, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatine kinase (CK) were measured in effluents of 38 liver grafts, and in serum of recipients. Concentrations of MIF in the effluent were greater than those in the recipients' serum before and after reperfusion (58 [interquartile range, IQR:23-79] µg/mL vs 0.06 [IQR:0.03-0.07] µg/mL and 1.3 [IQR:0.7-1.8] µg/mL, respectively; both P<.001). Effluent MIF concentrations correlated with effluent concentrations of the cell injury markers ALT (R=.51, P<.01), AST (R=.51, P<.01), CK (R=.45, P=.01), and LDH (R=.56, P<.01). Patients who developed EAD had greater MIF concentrations in effluent and serum 10 minutes after reperfusion than patients without EAD (Effluent: 80 [IQR:63-118] µg/mL vs 36 [IQR:20-70] µg/mL, P=.02; Serum: 1.7 [IQR:1.2-2.5] µg/mL vs 1.1 [IQR:0.6-1.7] µg/mL, P<.001). CONCLUSION: Human liver grafts release MIF in proportion to hepatocellular injury. Greater MIF concentrations in effluent and recipient's serum are associated with EAD after liver transplantation.

Age-Related Changes in Immunological and Physiological Responses Following Pulmonary Challenge.

This review examines the current status of knowledge of sepsis and pneumonia in the elderly population and how the dynamics of the pulmonary challenge affects outcome and consequences. Led by an unprecedented shift in demographics, where a larger proportion of the population will reach an older age, clinical and experimental research shows that aging is associated with certain pulmonary changes, but it is during infectious insult of the lungs, as in the case of pneumonia, that the age-related differences in responsiveness and endurance become obvious and lead to a worse outcome than in the younger population. This review points to the neutrophil, and the endothelium as important players in understanding age-associated changes in responsiveness to infectious challenge of the lung. It also addresses how the immunological set-point influences injury-repair phases, remote organ damage and how intake of drugs may alter the state of responsiveness in the users. Further, it points out the importance of considering age as a factor in inclusion criteria in clinical trials, in vitro/ex vivo experimental designs and overall interpretation of results.

Regulation of angiopoietin-2 secretion from human pulmonary microvascular endothelial cells.

INTRODUCTION: Sepsis is characterized by dysregulated systemic inflammation and cytokine storm. Angiopoietin-2 (Ang-2) is known to closely correlate with severity of sepsis-related acute lung injury and mortality. The aim of this study was to clarify the mechanisms involved in Ang-2 secretion to better understand the pathophysiology of sepsis. MATERIALS AND METHODS: The concentration of Ang-2 was assessed in culture medium of pulmonary microvascular endothelial cells in the presence or absence of Gram-positive bacteria cell wall components [lipoteichoic acid (LTA) and peptidoglycan (PGN)] stimulation at different time points ranging from 15 minutes to 24 hours. Constitutive and LTA-PGN-stimulated Ang-2 level changes were also assessed after cells were pretreated with different pathway inhibitors for 1 hour. RESULTS: Two distinctive mechanisms of Ang-2 secretion, constitutive and stimulated secretion, were identified. Constitutive secretion resulted in slow but continuous increase in Ang-2 in culture medium over time. It was regulated by 3'5'-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-Ca2+ and nitric oxide (NO)-3'5'-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG)-Ca2+ pathways and partially regulated by N-ethyl-maleimide-sensitive factor-Ca2+ pathways. LTA-PGN stimulation caused rapid and potent increase followed by gradual decrease of Ang-2. It was partially regulated by both Ral A-phospholipase D and NSF-Ca2+ pathways. CONCLUSIONS: We demonstrated characteristics and involved pathways for two distinctive secretory mechanisms, constitutive and stimulated, of Ang-2 in pulmonary microvascular endothelial cells. Considering the close correlation of Ang-2 with sepsis outcomes, our findings provide a better understanding of an important mechanism associated with sepsis pathophysiology and identify possible therapeutic targets to improve outcomes in the potentially lethal disease.

Hypothesis: Neuroendocrine Mechanisms (Hypothalamus-Growth Hormone-STAT5 Axis) Contribute to Sex Bias in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a disease with high morbidity and mortality. The prevalence of idiopathic pulmonary arterial hypertension (IPAH) and hereditary pulmonary arterial hypertension (HPAH) is approximately two- to four-fold higher in women than in men. Paradoxically, there is an opposite male bias in typical rodent models of PH (chronic hypoxia or monocrotaline); in these models, administration of estrogenic compounds (for example, estradiol-17ß [E2]) is protective. Further complexities are observed in humans ingesting anorexigens (female bias) and in rodent models, such as after hypoxia plus SU5416/Sugen (little sex bias) or involving serotonin transporter overexpression or dexfenfluramine administration (female bias). These complexities in sex bias in PH remain incompletely understood. We recently discovered that conditional deletion of signal transducer and activator of transcription 5a/b (STAT5a/b) in vascular smooth muscle cells abrogated the male bias in PH in hypoxic mice and that late-stage obliterative lesions in patients of both sexes with IPAH and HPAH showed reduced STAT5a/b, reduced Tyr-P-STAT5 and reduced B-cell lymphoma 6 protein (BCL6). In trying to understand the significance of these observations, we realized that there existed a well-characterized E2-sensitive central neuroendocrine mechanism of sex bias, studied over the last 40 years, that, at its peripheral end, culminated in species-specific male ("pulsatile") versus female ("more continuous") temporal patterns of circulating growth hormone (GH) levels leading to male versus female patterned activation of STAT5a/b in peripheral tissues and thus sex-biased expression of hundreds of genes. In this report, we consider the contribution of this neuroendocrine mechanism (hypothalamus-GH-STAT5) in the generation of sex bias in different PH situations.

Deletion of STAT5a/b in vascular smooth muscle abrogates the male bias in hypoxic pulmonary hypertension in mice: implications in the human disease.

Chronic hypoxia typically elicits pulmonary hypertension (PH) in mice with a male-dominant phenotype. There is an opposite-sex bias in human PH, with a higher prevalence in women, but greater survival (the "estrogen paradox"). We investigated the involvement of the STAT5a/b species, previously established to mediate sexual dimorphism in other contexts, in the sex bias in PH. Mice with heterozygous or homozygous deletions of the STAT5a/b locus in vascular smooth muscle cells (SMCs) were generated in crosses between STAT5a/b(fl/fl) and transgelin (SM22α)-Cre(+/+) parents. Wild-type (wt) males subjected to chronic hypoxia showed significant PH and pulmonary arterial remodeling, with wt females showing minimal changes (a male-dominant phenotype). However, in conditional STAT5(+/-) or STAT5(-/-) mice, hypoxic females showed the severest manifestations of PH (a female-dominant phenotype). Immunofluorescence studies on human lung sections showed that obliterative pulmonary arterial lesions in patients with idiopathic pulmonary arterial hypertension (IPAH) or hereditary pulmonary arterial hypertension (HPAH), both male and female, overall had reduced STAT5a/b, reduced PY-STAT5 and reduced endoplasmic reticulum (ER) GTPase atlastin-3 (ATL3). Studies of SMCs and endothelial cell (EC) lines derived from vessels isolated from lungs of male and female IPAH patients and controls revealed instances of coordinate reductions in STAT5a, STAT5b and ATL3 in IPAH-derived cells, including SMCs and ECs from the same patient. Taken together, these data provide the first definitive evidence for a contribution of STAT5a/b to the sex bias in PH in the hypoxic mouse and implicate reduced STAT5 in the pathogenesis of the human disease.

Brain region-specific alterations in the gene expression of cytokines, immune cell markers and cholinergic system components during peripheral endotoxin-induced inflammation.

Inflammatory conditions characterized by excessive peripheral immune responses are associated with diverse alterations in brain function, and brain-derived neural pathways regulate peripheral inflammation. Important aspects of this bidirectional peripheral immune-brain communication, including the impact of peripheral inflammation on brain region-specific cytokine responses, and brain cholinergic signaling (which plays a role in controlling peripheral cytokine levels), remain unclear. To provide insight, we studied gene expression of cytokines, immune cell markers and brain cholinergic system components in the cortex, cerebellum, brainstem, hippocampus, hypothalamus, striatum and thalamus in mice after an intraperitoneal lipopolysaccharide injection. Endotoxemia was accompanied by elevated serum levels of interleukin (IL)-1ß, IL-6 and other cytokines and brain region-specific increases in Il1b (the highest increase, relative to basal level, was in cortex; the lowest increase was in cerebellum) and Il6 (highest increase in cerebellum; lowest increase in striatum) mRNA expression. Gene expression of brain Gfap (astrocyte marker) was also differentially increased. However, Iba1 (microglia marker) mRNA expression was decreased in the cortex, hippocampus and other brain regions in parallel with morphological changes, indicating microglia activation. Brain choline acetyltransferase (Chat ) mRNA expression was decreased in the striatum, acetylcholinesterase (Ache) mRNA expression was decreased in the cortex and increased in the hippocampus, and M1 muscarinic acetylcholine receptor (Chrm1) mRNA expression was decreased in the cortex and the brainstem. These results reveal a previously unrecognized regional specificity in brain immunoregulatory and cholinergic system gene expression in the context of peripheral inflammation and are of interest for designing future antiinflammatory approaches.

Age influences inflammatory responses, hemodynamics, and cardiac proteasome activation during acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a significant source of morbidity and mortality in critically ill patients. Age is a major determinant of clinical outcome in ALI. The increased ALI-associated mortality in the older population suggests that there are age-dependent alterations in the responses to pulmonary challenge. The objective of this observational study was to evaluate age-dependent differences in the acute (within 6 hours) immunological and physiological responses of the heart and lung, to pulmonary challenge, that could result in increased severity. METHODS: Male C57Bl/6 mice (young: 2-3 months, old: 18-20 months) were challenged intratracheally with cell wall components from Gram-positive bacteria (lipoteichoic acid and peptidoglycan). After 6 hours, both biochemical and physiological consequences of the challenge were assessed. Alveolar infiltration of inflammatory cells and protein, airspace and blood cytokines, cardiac function and myocardial proteasome activity were determined. RESULTS: In young mice, there was a dose-dependent response to pulmonary challenge resulting in increased airspace neutrophil counts, lung permeability, and concentrations of cytokines in bronchoalveolar lavage fluid and plasma. A midrange dose was then selected to compare the responses in young and old animals. In comparison, the old animals displayed increased neutrophil accumulation in the airspaces, decreased arterial oxygen saturation, body temperatures, plasma cytokine concentrations, and a lack of myocardial proteasome response, following challenge. CONCLUSIONS: Age-dependent differences in the onset of systemic response and in maintenance of vital functions, including temperature control, oxygen saturation, and myocardial proteasome activation, are evident. We believe a better understanding of these age-related consequences of ALI can lead to more appropriate treatments in the elderly patient population.

Thyroxine is a potential endogenous antagonist of macrophage migration inhibitory factor (MIF) activity.

Abnormally low plasma concentrations of thyroid hormones during sepsis often occur in the absence of thyroidal illness; however, the mechanisms involved in the "euthyroid sick syndrome" remain poorly understood. Here, we describe a previously unrecognized interaction between the thyroid hormone thyroxine (T(4)) and the proinflammatory cytokine macrophage migration inhibitory factor (MIF), together with its clinical relevance in sepsis. We found that in both patients with severe sepsis, and our rodent model, low plasma T(4) concentrations were inversely correlated with plasma MIF concentrations. The MIF molecule contains a hydrophobic pocket that is important for many of its proinflammatory activities. Binding of L-T(4) (or its hormonally inert isomer D-T(4)) significantly, and dose-dependently, inhibited the catalytic activity of this pocket. Moreover, administration of exogenous D-T(4) significantly improved survival in mice with severe sepsis. To examine the specificity of the MIFT(4) interaction, wild-type and MIF knockout mice were subjected to the carrageenan-air pouch model of inflammation and then treated with D-T(4) or vehicle. D-T(4) significantly inhibited leukocyte infiltration in wild-type mice but not in MIF knockout mice, providing evidence that in vivo T(4) may influence MIF-mediated inflammatory responses via inhibition of its hydrophobic proinflammatory pocket. These findings demonstrate a new physiological role for T(4) as a natural inhibitor of MIF proinflammatory activity. The data may also, in part, explain the low plasma T(4) concentrations in critically ill, euthyroid patients and suggest that targeting the imbalance between MIF and T(4) may be beneficial in improving outcome from sepsis.

Macrophage migration inhibitory factor (MIF) and thyroid hormone alterations in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine known to be released from lymphocytes, macrophages and endothelial cells and also in animal models shown to be inducible with glucocorticoids (GC). In contrast, thyroxine seems to antagonize MIF activity. To investigate whether MIF is increased in active antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and possible correlations with GC dosing and thyroid hormone levels, 27 consecutive patients with active AAV were studied and followed prospectively. Disease activity was assessed using Birmingham Vasculitis Activity Score 2003 (BVAS) at baseline and at follow-up at 3 and 6 months, along with MIF, thyroid hormones free triiodothyronine (fT3) and free thyroxine (fT4), C-reactive protein (CRP) and creatinine. MIF was elevated significantly at baseline compared with follow-up at 3 and 6 months (8,618 pg/mL versus 5,696 and 6,212 respectively; P < 0.002) but did not correlate to CRP, GC dose, creatinine or organ involvement. fT3 was depressed significantly at baseline compared with follow-up (1.99 pg/mL versus 2.31 and 2.67 respectively; P = 0.01) and correlated inversely to the BVAS score at baseline. We found a significant correlation between the MIF/fT4 ratio at baseline versus MIF/fT4 ratio at 6 months (ρ = 0.52, P < 0.005) and a trend between the baseline MIF/fT3 ratio versus MIF/fT3 ratio at 6 months (ρ = 0.39, P = 0.05). These results suggest a possible role for MIF and thyroid status in AAV. Further studies could reveal whether the association between AAV and thyroid hormone levels in the context of elevated MIF may present a link as well as a target of treatment.

Extracellular superoxide dismutase overexpression can reverse the course of hypoxia-induced pulmonary hypertension.

Hypoxia leads to free radical production, which has a pivotal role in the pathophysiology of pulmonary hypertension (PH). We hypothesized that treatment with extracellular superoxide dismutase (EC-SOD) could ameliorate the development of PH induced by hypoxia. In vitro studies using pulmonary microvascular endothelial cells showed that cells transfected with EC-SOD had significantly less accumulation of xanthine oxidase and reactive oxygen species than nontransfected cells after hypoxia exposure for 24 h. To study the prophylactic role of EC-SOD, adult male wild-type (WT) and transgenic (TG) mice, with lung-specific overexpression of human EC-SOD (hEC-SOD), were exposed to fraction of inspired oxygen (FiO(2)) 10% for 10 d. After exposure, right ventricular systolic pressure (RVSP), right ventricular mass (RV/S + LV), pulmonary vascular wall thickness (PVWT) and pulmonary artery contraction/relaxation were assessed. TG mice were protected against PH compared with WT mice with significantly lower RVSP (23.9 ± 1.24 versus 47.2 ± 3.4), RV/S + LV (0.287 ± 0.015 versus 0.335 ± 0.022) and vascular remodeling, indicated by PVWT (14.324 ± 1.107 versus 18.885 ± 1.529). Functional studies using pulmonary arteries isolated from mice indicated that EC-SOD prevents hypoxia-mediated attenuation of nitric oxide-induced relaxation. Therapeutic potential was assessed by exposing WT mice to FiO(2) 10% for 10 d. Half of the group was transfected with plasmid containing cDNA encoding human EC-SOD. The remaining animals were transfected with empty vector. Both groups were exposed to FiO(2) 10% for a further 10 d. Transfected mice had significantly reduced RVSP (18.97 ± 1.12 versus 41.3 ± 1.5), RV/S + LV (0.293 ± 0.012 versus 0.372 ± 0.014) and PVWT (12.51 ± 0.72 versus 18.98 ± 1.24). On the basis of these findings, we concluded that overexpression of EC-SOD prevents the development of PH and ameliorates established PH.

Macrophage migration inhibitory factor mediates hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) is a devastating disease leading to progressive hypoxemia, right ventricular failure, and death. Hypoxia can play a pivotal role in PH etiology, inducing pulmonary vessel constriction and remodeling. These events lead to increased pulmonary vessel wall thickness, elevated vascular resistance and right ventricular hypertrophy. The current study examined the association of the inflammatory cytokine macrophage migration inhibitory factor (MIF) with chronic lung disease and its role in the development of hypoxia-induced PH. We found that plasma MIF in patients with primary PH or PH secondary to interstitial lung disease (ILD) was significantly higher than in the control group (P = 0.004 and 0.007, respectively). MIF involvement with hypoxia-induced fibroblast proliferation was examined in both a human cell-line and primary mouse cells from wild-type (mif⁺/⁺) and MIF-knockout (mif⁻/⁻) mice. In vitro, hypoxia-increased MIF mRNA, extracellular MIF protein accumulation and cell proliferation. Inhibition of MIF inflammatory activity reduced hypoxia-induced cell proliferation. However, hypoxia only increased proliferation of mif⁻/⁻ cells when they were supplemented with media from mif⁺/⁺ cells. This growth increase was suppressed by MIF inhibition. In vivo, chronic exposure of mice to a normobaric atmosphere of 10% oxygen increased lung tissue expression of mRNA encoding MIF and accumulation of MIF in plasma. Inhibition of the MIF inflammatory active site, during hypoxic exposure, significantly reduced pulmonary vascular remodeling, cardiac hypertrophy and right ventricular systolic pressure. The data suggest that MIF plays a critical role in hypoxia-induced PH, and its inhibition may be beneficial in preventing the development and progression of the disease.

Inhibition of high-mobility group box 1 protein (HMGB1) enhances bacterial clearance and protects against Pseudomonas Aeruginosa pneumonia in cystic fibrosis.

Pulmonary infection with Pseudomonas aeruginosa and neutrophilic lung inflammation significantly contribute to morbidity and mortality in cystic fibrosis (CF). High-mobility group box 1 protein (HMGB1), a ubiquitous DNA binding protein that promotes inflammatory tissue injury, is significantly elevated in CF sputum. However, its mechanistic and potential therapeutic implications in CF were previously unknown. We found that HMGB1 levels were significantly elevated in bronchoalveolar lavage fluids (BALs) of CF patients and cystic fibrosis transmembrane conductance regulator (CFTR )(-/-) mice. Neutralizing anti-HMGB1 monoclonal antibody (mAb) conferred significant protection against P. aeruginosa-induced neutrophil recruitment, lung injury and bacterial infection in both CFTR(-/-) and wild-type mice. Alveolar macrophages isolated from mice treated with anti-HMGB1 mAb had improved phagocytic activity, which was suppressed by direct exposure to HMGB1. In addition, BAL from CF patients significantly impaired macrophage phagocytotic function, and this impairment was attenuated by HMGB1-neutralizing antibodies. The HMGB1-mediated suppression of bacterial phagocytosis was attenuated in macrophages lacking toll-like receptor (TLR)-4, suggesting a critical role for TLR4 in signaling HMGB1-mediated macrophage dysfunction. These studies demonstrate that the elevated levels of HMGB1 in CF airways are critical for neutrophil recruitment and persistent presence of P. aeruginosa in the lung. Thus, HMGB1 may provide a therapeutic target for reducing bacterial infection and lung inflammation in CF.

Synergistic protection against hyperoxia-induced lung injury by neutrophils blockade and EC-SOD overexpression.

BACKGROUND: Oxygen may damage the lung directly via generation of reactive oxygen species (ROS) or indirectly via the recruitment of inflammatory cells, especially neutrophils. Overexpression of extracellular superoxide dismutase (EC-SOD) has been shown to protect the lung against hyperoxia in the newborn mouse model. The CXC-chemokine receptor antagonist (Antileukinate) successfully inhibits neutrophil influx into the lung following a variety of pulmonary insults. In this study, we tested the hypothesis that the combined strategy of overexpression of EC-SOD and inhibiting neutrophil influx would reduce the inflammatory response and oxidative stress in the lung after acute hyperoxic exposure more efficiently than either single intervention. METHODS: Neonate transgenic (Tg) (with an extra copy of hEC-SOD) and wild type (WT) were exposed to acute hyperoxia (95% FiO2 for 7 days) and compared to matched room air groups. Inflammatory markers (myeloperoxidase, albumin, number of inflammatory cells), oxidative markers (8-isoprostane, ratio of reduced/oxidized glutathione), and histopathology were examined in groups exposed to room air or hyperoxia. During the exposure, some mice received a daily intraperitoneal injection of Antileukinate. RESULTS: Antileukinate-treated Tg mice had significantly decreased pulmonary inflammation and oxidative stress compared to Antileukinate-treated WT mice (p < 0.05) or Antileukinate-non-treated Tg mice (p < 0.05). CONCLUSION: Combined strategy of EC-SOD and neutrophil influx blockade may have a therapeutic benefit in protecting the lung against acute hyperoxic injury.