Toll-like receptors-related genes in kidney transplant patients with chronic allograft nephropathy and acute rejection

Introduction: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. Methods: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. Results: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. Conclusion: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN. (C) 2008 Elsevier B.V. All rights reserved.

Low level laser therapy (LLLT) decreases pulmonary microvascular leakage, neutrophil influx and IL-1 beta levels in airway and lung from rat subjected to LPS-induced inflammation

Background and Objective. Low level laser therapy (LLLT) is a known anti-inflammatory therapy. Herein we studied the effect of LLLT on lung permeability and the IL-1 beta level in LPS-induced pulmonary inflammation. Study Design/Methodology. Rats were divided into 12 groups (n = 7 for each group). Lung permeability was measured by quantifying extravasated albumin concentration in lung homogenate, inflammatory cells influx was determined by myeloperoxidase activity, IL-1P in BAL was determined by ELISA and IL-1P mRNA expression in trachea was evaluated by RT-PCR. The rats were irradiated on the skin over the upper bronchus at the site of tracheotomy after LPS. Results. LLLT attenuated lung permeability. In addition, there was reduced neutrophil influx, myeloperoxidase activity and both IL-1 beta in BAL and IL-1 beta mRNA expression in trachea obtained from animals subjected to LPS-induced inflammation. Conclusion. LLLT reduced the lung permeability by a mechanism in which the IL-1 beta seems to have an important role.

Regular and moderate exercise before experimental sepsis reduces the risk of lung and distal organ injury

de Araujo CC, Silva JD, Samary CS, Guimaraes IH, Marques PS, Oliveira GP, do Carmo LGRR, Goldenberg RC, Bakker-Abreu I, Diaz BL, Rocha NN, Capelozzi VL, Pelosi P, Rocco PRM. Regular and moderate exercise before experimental sepsis reduces the risk of lung and distal organ injury. J Appl Physiol 112: 1206-1214, 2012. First published January 19, 2012; doi:10.1152/japplphysiol.01061.2011.-Physical activity modulates inflammation and immune response in both normal and pathologic conditions. We investigated whether regular and moderate exercise before the induction of experimental sepsis reduces the risk of lung and distal organ injury and survival. One hundred twenty-four BALB/c mice were randomly assigned to two groups: sedentary (S) and trained (T). Animals in T group ran on a motorized treadmill, at moderate intensity, 5% grade, 30 min/day, 3 times a week for 8 wk. Cardiac adaptation to exercise was evaluated using echocardiography. Systolic volume and left ventricular mass were increased in T compared with S group. Both T and S groups were further randomized either to sepsis induced by cecal ligation and puncture surgery (CLP) or sham operation (control). After 24 h, lung mechanics and histology, the degree of cell apoptosis in lung, heart, kidney, liver, and small intestine villi, and interleukin (IL)-6, KC (IL-8 murine functional homolog), IL-1 beta, IL-10, and number of cells in bronchoalveolar lavage (BALF) and peritoneal lavage (PLF) fluids as well as plasma were measured. In CLP, T compared with S groups showed: 1) improvement in survival; 2) reduced lung static elastance, alveolar collapse, collagen and elastic fiber content, number of neutrophils in BALF, PLF, and plasma, as well as lung and distal organ cell apoptosis; and 3) increased IL-10 in BALF and plasma, with reduced IL-6, KC, and IL-1 beta in PLF. In conclusion, regular and moderate exercise before the induction of sepsis reduced the risk of lung and distal organ damage, thus increasing survival.

Modulation of Lung Allergic Response by Renal Ischemia and Reperfusion Injury

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors. Copyright (C) 2012 S. Karger AG, Basel

In vitro generation and characterization of acute myeloid leukemia-reactive CD8 + cytotoxic T-lymphocyte clones from healthy donors

Donor-derived CD8+ cytotoxic T lymphocytes (CTLs) eliminating host leukemic cells mediate curative graft-versus-leukemia (GVL) reactions after allogeneic hematopoietic stem cell transplantation (HSCT). The leukemia-reactive CTLs recognize hematopoiesis-restricted or broadly expressed minor histocompatibility and leukemia-associated peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on recipient cells. The development of allogeneic CTL therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of current techniques for generating leukemia-reactive CTLs from unprimed healthy donors in vitro. In this work, a novel allogeneic mini-mixed lymphocyte/leukemia culture (mini-MLLC) approach was established by stimulating CD8+ T cells isolated from peripheral blood of healthy donors at comparably low numbers (i.e. 10e4/well) with HLA class I-matched primary AML blasts in 96-well microtiter plates. Before culture, CD8+ T cells were immunomagnetically separated into CD62L(high)+ and CD62L(low)+/neg subsets enriched for naive/central memory and effector memory cells, respectively. The application of 96-well microtiter plates aimed at creating multiple different responder-stimulator cell compositions in order to provide for the growth of leukemia-reactive CTLs optimized culture conditions by chance. The culture medium was supplemented with interleukin (IL)-7, IL-12, and IL-15. On day 14, IL-12 was replaced by IL-2. In eight different related and unrelated donor/AML pairs with complete HLA class I match, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs recognized neither lymphoblastoid B cell lines of donor and patient origin nor primary B cell leukemias expressing the corresponding HLA restriction element. CTLs expressed T cell receptors of single V-beta chain families, indicating their clonality. The vast majority of CTL clones were obtained from mini-MLLCs initiated with CD8+ CD62L(high)+ cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10e8-10e10 cells within six to eight weeks. The capability of mini-MLLC derived AML-reactive CTL clones to inhibit the engraftment of human primary AML blasts was investigated in the immunodeficient nonobese diabetic/severe combined immune deficient IL-2 receptor common γ-chain deficient (NOD/SCID IL2Rγnull) mouse model. The leukemic engraftment in NOD/SCID IL2Rγnull was specifically prevented if inoculated AML blasts had been pre-incubated in vitro with AML-reactive CTLs, but not with anti-melanoma control CTLs. These results demonstrate that myeloid leukemia-specific CTL clones capable of preventing AML engraftment in mice can be rapidly isolated from CD8+ CD62L(high)+ T cells of healthy donors in vitro. The efficient generation and expansion of these CTLs by the newly established mini-MLLC approach opens the door for several potential applications. First, CTLs can be used within T cell-driven antigen identification strategies to extend the panel of molecularly defined AML antigens that are recognizable by T cells of healthy donors. Second, because these CTLs can be isolated from the stem cell donor by mini-MLLC prior to transplantation, they could be infused into AML patients as a part of the stem cell allograft, or early after transplantation when the leukemia burden is low. The capability of these T cells to expand and function in vivo might require the simultaneous administration of AML-reactive CD4+ T cells generated by a similar in vitro strategy or, less complex, the co-transfer of CD8-depleted donor lymphocytes. To prepare clinical testing, the mini-MLLC approach should now be translated into a protocol that is compatible with good manufacturing practice guidelines.

Oral prednisolone induced acute generalized exanthematous pustulosis due to corticosteroids of group A confirmed by epicutaneous testing and lymphocyte transformation tests

BACKGROUND: Acute generalized exanthematous pustulosis (AGEP) is a rare cutaneous eruption which is often provoked by drugs. CASE REPORT: We report 2 cases of AGEP which showed rapidly spreading pustular eruptions accompanied by malaise, fever and neutrophilia after the administration of systemic prednisolone (corticosteroid of group A, hydrocortisone type). The histological examination showing neutrophilic subcorneal spongiform pustules was consistent with the diagnosis of AGEP. In both cases the rash cleared within a week upon treatment with topical steroids (corticosteroid of group D1, betamethasonedipropionate type and corticosteroid of group D2, hydrocortisone-17-butyrate type). Three months after recovery, the sensitization to corticosteroids of group A was confirmed by epicutaneous testing and positive lymphocyte transformation tests. CONCLUSION: These cases show that systemic corticosteroids can induce AGEP and demonstrate that epicutaneous testing and lymphocyte transformation tests may be helpful in identifying the causative drug. Our data support previous reports indicating an important role for drug-specific T cells in inducing neutrophil inflammation in this disease.

Double-lung transplantation in the rat: an acute, syngeneic in situ model

BACKGROUND. The high rate of reperfusion injury in clinical lung transplantation mandates significant improvements in lung preservation. Innovations should be validated using standardized and low-cost experimental models. METHODS. The model introduced here is analyzed by comparing global lung function after varying ischemic times (2, 4, 8, 16, and 24 hours). A rat double-lung block is flush-perfused, and the main pulmonary artery and left atrium are connected to the left pulmonary artery and vein of a syngeneic recipient using a T-shaped stent. With pressure side ports and incorporated flow crystals, measurement of vascular resistance and graft oxygenation can be performed. The transplant is ventilated separately, and compliance and resistance are determined. RESULTS. The increase in the ischemic interval from 2 to 24 hours caused an increase in the alveolar arterial oxygen difference from 220 +/- 20 to 600 +/- 34 mm Hg, pulmonary vascular resistance from 198 +/- 76 to 638 +/- 212 mm Hg.mL-1.min-1, and resistance to airflow from 274 +/- 50 to 712 +/- 30 cm H2O/L H2O, and a decrease in pulmonary compliance from 0.4 +/- 0.05 to 0.12 +/- 0.06 mL/cm H2O. CONCLUSIONS. This in situ, syngeneic rat lung transplantation model offers an alternative to large animal models for verification of lung preservation solutions and for modification of donor or recipient treatment regimens.

Serum IL-6: a candidate biomarker for intracranial pressure elevation following isolated traumatic brain injury.

BACKGROUND: Increased intracranial pressure (ICP) is a serious, life-threatening, secondary event following traumatic brain injury (TBI). In many cases, ICP rises in a delayed fashion, reaching a maximal level 48-96 hours after the initial insult. While pressure catheters can be implanted to monitor ICP, there is no clinically proven method for determining a patient's risk for developing this pathology. METHODS: In the present study, we employed antibody array and Luminex-based screening methods to interrogate the levels of inflammatory cytokines in the serum of healthy volunteers and in severe TBI patients (GCS RESULTS: Consistent with previous reports, we observed sustained increases in IL-6 levels in TBI patients irrespective of their ICP status. However, the group of patients who subsequently experienced ICP >or= 25 mm Hg had significantly higher IL-6 levels within the first 17 hours of injury as compared to the patients whose ICP remained 128 pg/ml correctly identified 85% of isolated TBI patients who subsequently developed elevated ICP, and values between these cut-off values correctly identified 75% of all patients whose ICP remained CONCLUSIONS: Our results suggest that serum IL-6 can be used for the differential diagnosis of elevated ICP in isolated TBI.

Innate immune cells express IL-17A/F in acute generalized exanthematous pustulosis and generalized pustular psoriasis.

Acute generalized exanthematous pustulosis (AGEP) and generalized pustular psoriasis (GPP) are rare pustular skin disorders with systemic involvement. IL-17A/F is a proinflammatory cytokine involved in various neutrophilic inflammatory disorders. Here we show that IL-17A/F is highly expressed by innate immune cells such as neutrophils and mast cells in both AGEP and GPP.

Influence of inspiration to expiration ratio on cyclic recruitment and derecruitment of atelectasis in a saline lavage model of acute respiratory distress syndrome*

OBJECTIVE Cyclic recruitment and derecruitment of atelectasis can occur during mechanical ventilation, especially in injured lungs. Experimentally, cyclic recruitment and derecruitment can be quantified by respiration-dependent changes in PaO2 (ΔPaO2), reflecting the varying intrapulmonary shunt fraction within the respiratory cycle. This study investigated the effect of inspiration to expiration ratio upon ΔPaO2 and Horowitz index. DESIGN Prospective randomized study. SETTING Laboratory investigation. SUBJECTS Piglets, average weight 30 ± 2 kg. INTERVENTIONS At respiratory rate 6 breaths/min, end-inspiratory pressure (Pendinsp) 40 cm H2O, positive end-expiratory pressure 5 cm H2O, and FIO2 1.0, measurements were performed at randomly set inspiration to expiration ratios during baseline healthy and mild surfactant depletion injury. Lung damage was titrated by repetitive surfactant washout to induce maximal cyclic recruitment and derecruitment as measured by multifrequency phase fluorimetry. Regional ventilation distribution was evaluated by electrical impedance tomography. Step changes in airway pressure from 5 to 40 cm H2O and vice versa were performed after lavage to calculate PO2-based recruitment and derecruitment time constants (TAU). MEASUREMENTS AND MAIN RESULTS In baseline healthy, cyclic recruitment and derecruitment could not be provoked, whereas in model acute respiratory distress syndrome, the highest ΔPaO2 were routinely detected at an inspiration to expiration ratio of 1:4 (range, 52-277 torr [6.9-36.9 kPa]). Shorter expiration time reduced cyclic recruitment and derecruitment significantly (158 ± 85 torr [21.1 ± 11.3 kPa] [inspiration to expiration ratio, 1:4]; 25 ± 12 torr [3.3 ± 1.6 kPa] [inspiration to expiration ratio, 4:1]; p < 0.0001), whereas the PaO2/FIO2 ratio increased (267 ± 50 [inspiration to expiration ratio, 1:4]; 424 ± 53 [inspiration to expiration ratio, 4:1]; p < 0.0001). Correspondingly, regional ventilation redistributed toward dependent lung regions (p < 0.0001). Recruitment was much faster (TAU: fast 1.6 s [78%]; slow 9.2 s) than derecruitment (TAU: fast 3.1 s [87%]; slow 17.7 s) (p = 0.0078). CONCLUSIONS Inverse ratio ventilation minimizes cyclic recruitment and derecruitment of atelectasis in an experimental model of surfactant-depleted pigs. Time constants for recruitment and derecruitment, and regional ventilation distribution, reflect these findings and highlight the time dependency of cyclic recruitment and derecruitment.

IL-33 promotes an innate immune pathway of intestinal tissue protection dependent on amphiregulin-EGFR interactions

The barrier surfaces of the skin, lung, and intestine are constantly exposed to environmental stimuli that can result in inflammation and tissue damage. Interleukin (IL)-33-dependent group 2 innate lymphoid cells (ILC2s) are enriched at barrier surfaces and have been implicated in promoting inflammation; however, the mechanisms underlying the tissue-protective roles of IL-33 or ILC2s at surfaces such as the intestine remain poorly defined. Here we demonstrate that, following activation with IL-33, expression of the growth factor amphiregulin (AREG) is a dominant functional signature of gut-associated ILC2s. In the context of a murine model of intestinal damage and inflammation, the frequency and number of AREG-expressing ILC2s increases following intestinal injury and genetic disruption of the endogenous AREG-epidermal growth factor receptor (EGFR) pathway exacerbated disease. Administration of exogenous AREG limited intestinal inflammation and decreased disease severity in both lymphocyte-sufficient and lymphocyte-deficient mice, revealing a previously unrecognized innate immune mechanism of intestinal tissue protection. Furthermore, treatment with IL-33 or transfer of ILC2s ameliorated intestinal disease severity in an AREG-dependent manner. Collectively, these data reveal a critical feedback loop in which cytokine cues from damaged epithelia activate innate immune cells to express growth factors essential for ILC-dependent restoration of epithelial barrier function and maintenance of tissue homeostasis.

Non-invasive Ventilation of Patients with ARDS: Insights from the LUNG SAFE Study

Background: Non-invasive ventilation (NIV) is increasingly used in patients with Acute Respiratory Distress Syndrome (ARDS). Whether, during NIV, the categorization of ARDS severity based on the PaO2/FiO2 Berlin criteria is useful is unknown. The evidence supporting NIV use in patients with ARDS remains relatively sparse.

Methods: The Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study described the management of patients with ARDS. This sub-study examines the current practice of NIV use in ARDS, the utility of the PaO2/FiO2 ratio in classifying patients receiving NIV and the impact of NIV on outcome.

Results: Of 2,813 patients with ARDS, 436 (15.5%) were managed with NIV on days 1 and 2 following fulfillment of diagnostic criteria. Classification of ARDS severity based on PaO2/FiO2 ratio was associated with an increase in intensity of ventilatory support, NIV failure, and Intensive Care Unit (ICU) mortality. NIV failure occurred in 22.2% of mild, 42.3% of moderate and 47.1% of patients with severe ARDS. Hospital mortality in patients with NIV success and failure was 16.1 % and 45.4%, respectively. NIV use was independently associated with increased ICU (HR 1.446; [1.159-1.805]), but not hospital mortality. In a propensity matched analysis, ICU mortality was higher in NIV than invasively ventilated patients with a PaO2/FiO2 lower than 150 mmHg.

Conclusions: NIV was used in 15% of patients with ARDS, irrespective of severity category. NIV appears to be associated with higher ICU mortality in patients with a PaO2/FiO2 lower than 150 mmHg.

Trial Registration: ClinicalTrials.gov NCT02010073

Primary neuroendocrine lung tumor presenting with acute ileal obstruction. Case report

The authors describe a clinical case of a patient with neuroendocrine carcinoma of the lung diagnosed after the onset of an intestinal obstruction from an ileal metastasis. A review of literature reveals that the incidence of symptomatic gastro-intestinal metastases from lung cancer has been estimated to be about 2-3% and is exceedingly rare that the intestinal symptoms may be the initial presentation of cancer of the lung. The authors emphasize the difficulty of preoperative diagnosis of gastro-intestinal metastases which is made, almost always, too late because of the lack of specific symptoms. In our case, on account of the computed tomography, we leaned towards the diagnosis of lymphoma because of the double mediastinal and abdominal localization. Furthermore, this diagnosis was supported by the fact that the pulmonary lesion did not have clear radiological features of a lung cancer. The prognosis is poor because once intestinal metastases occur, other metastatic sites, which would make surgery only a palliative measure, are already present. The review of the literature shows that the average survival rate of these patients is 136 days. In our case the patient survived 277 days.

Effect Of Atorvastatin On Wound Healing In Rats.

Skin-wound healing is a complex and dynamic biological process involving inflammation, proliferation, and remodeling. Recent studies have shown that statins are new therapeutical options because of their actions, such as anti-inflammatory and antioxidant activity, on vasodilation, endothelial dysfunction and neoangiogenesis, which are independent of their lipid-lowering action. Our aim was to investigate the effect of atorvastatin on tissue repair after acute injury in healthy animals. Rats were divided into four groups: placebo-treated (P), topical atorvastatin-treated (AT), oral atorvastatin-treated (AO), topical and oral atorvastatin-treated (ATO). Under anesthesia, rats were wounded with an 8-mm punch in the dorsal region. Lesions were photographed on Days 0, 1, 3, 7, 10, 12, and 14 post-injury and samples taken on Days 1, 3, 7, and 14 for protein-expression analysis of insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase (GSK)-3, endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), extracellular signal-regulated kinase (ERK), interleukin (IL)-10, IL-1β, IL-6, and tumor necrosis factor (TNF)-α. Upon macroscopic examination, we observed significant reductions of lesion areas in groups AT, AO, and ATO compared to the P group. Additionally, AT and AO groups showed increased expression of IRS-1, PI3K, Akt, GSK-3, and IL-10 on Days 1 and 3 when compared with the P group. All atorvastatin-treated groups showed higher expression of IRS-1, PI3K, Akt, GSK-3, IL-10, eNOS, VEGF, and ERK on Day 7. On Days 1, 3, and 7, all atorvastatin-treated groups showed lower expression of IL-6 and TNF-α when compared with the P group. We conclude that atorvastatin accelerated tissue repair of acute lesions in rats and modulated expressions of proteins and cytokines associated with cell-growth pathways.

Influence of Laser Photobiomodulation on Collagen IV During Skeletal Muscle Tissue Remodeling After Injury in Rats

Objective: The aim of the present study was to determine the effect of GaAlAs low-level laser therapy (LLLT) on collagen IV remodeling of the tibialis anterior (TA) muscle in rats after cryolesion. Background: Considerable interest exists in skeletal muscle regeneration in situations such as repair after exercise-induced muscle injury, after muscle transplantation, in muscular dystrophy, exercise-induced muscle injury, and the recovery of strength after atrophy due to disuse. A number of studies have demonstrated the potential of LLLT in facilitating the muscle-healing process; however, no consensus is found in the literature regarding the best laser-irradiation parameters. Methods: Adult male Wistar rats (n = 45) were used and randomly divided into three groups: control (n = 5); nontreated cryolesioned group (n = 20), and LLLT-cryolesioned group (n = 20). The cryolesioned groups were analyzed at 1, 7, 14, and 21 days after the injury procedure. Laser irradiation was performed 3 times per week on the injured region by using the GaAlAs laser (660 nm; beam spot of 0.04 cm(2), output power of 20 mW, power density of 500 mW/cm(2), and energy density of 5 J/cm(2), for 10 sec). The muscles were removed, frozen, cryosectioned, and then stained with hematoxylin-eosin for the visualization of general morphology or used for immunohistochemical analysis of collagen IV. Results: It was demonstrated that LLLT promotes an increase in collagen IV immunolabeling in skeletal muscle in the first 7 days after acute trauma caused by cryoinjury, but does not modify the duration of the tissue-repair process. Even with LLLT, the injured muscle tissue needs similar to 21 days to achieve the same state of organization as that in the noninjured muscle. Conclusion: The collagen IV content is modulated in regenerating skeletal muscle under LLLT, which might be associated with better tissue outcome, although the histologic analysis did not detect tissue improvement in the LLLT group.