Randomized comparison of unfractionated heparin with corticosteroids in severe active inflammatory bowel disease

BACKGROUND: Heparin therapy may be effective in steroid resistant inflammatory bowel disease.

AIM: A randomized pilot study, to compare unfractionated heparin as a first-line therapy with corticosteroids in colonic inflammatory bowel disease.

METHODS: Twenty patients with severe inflammatory bowel disease (ulcerative colitis, n=17; Crohn's colitis, n=3) were randomized to either intravenous heparin for 5 days, followed by subcutaneous heparin for 5 weeks (n=8), or high-dose intravenous hydrocortisone for 5 days followed by oral prednisolone 40 mg daily, reducing by 5 mg per day each week (n=12). After 5 days, non-responders in each treatment group were commenced on combination therapy. Response to therapy was monitored by: clinical disease activity (ulcerative colitis: Truelove and Witt Index; Crohn's colitis: Harvey and Bradshaw Index), stool frequency, serum C-reactive protein and alpha1 acid glycoprotein, endoscopic and histopathological grading.

RESULTS: The response rates were similar in both treatment groups: clinical activity index (heparin vs. steroid; 75% vs. 67%; P=0.23), stool frequency (75% vs. 67%; P=0.61), endoscopic (75% vs. 67%; P=0.4) and histopathological grading (63% vs. 50%; P=0.67). Both treatments were well-tolerated with no serious adverse events.

CONCLUSION: Heparin as a first line therapy is as effective as corticosteroids in the treatment of colonic inflammatory bowel disease. Large multicentre randomized comparative studies are required to determine the role of heparin in the management of inflammatory bowel disease.

Glucocorticoid receptor and Klf4 co-regulate anti-inflammatory genes in keratinocytes

The glucocorticoid (GC) receptor (GR) and Kruppel-like factor Klf4 are transcription factors that play major roles in skin homeostasis. However, whether these transcription factors cooperate in binding genomic regulatory regions in epidermal keratinocytes was not known. Here, we show that in dexamethasone-treated keratinocytes GR and Klf4 are recruited to genomic regions containing adjacent GR and KLF binding motifs to control transcription of the anti-inflammatory genes Tsc22d3 and Zfp36. GR- and Klf4 loss of function experiments showed total GR but partial Klf4 requirement for full gene induction in response to dexamethasone. In wild type keratinocytes induced to differentiate, GR and Klf4 protein expression increased concomitant with Tsc22d3 and Zfp36 up-regulation. In contrast, GR-deficient cells failed to differentiate or fully induce Klf4, Tsc22d3 and Zfp36 correlating with increased expression of the epithelium-specific Trp63, a known transcriptional repressor of Klf4. The identified transcriptional cooperation between GR and Klf4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases.

Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration

BACKGROUND: Reactive microglia are commonly seen in retinal degenerative diseases, and neurotoxic microglia responses can contribute to photoreceptor cell death. We and others have previously shown that translocator protein (18 kDa) (TSPO) is highly induced in retinal degenerations and that the selective TSPO ligand XBD173 (AC-5216, emapunil) exerts strong anti-inflammatory effects on microglia in vitro and ex vivo. Here, we investigated whether targeting TSPO with XBD173 has immuno-modulatory and neuroprotective functions in two mouse models of acute retinal degeneration using bright white light exposure.

METHODS: BALB/cJ and Cx3cr1 (GFP/+) mice received intraperitoneal injections of 10 mg/kg XBD173 or vehicle for five consecutive days, starting 1 day prior to exposure to either 15,000 lux white light for 1 h or 50,000 lux focal light for 10 min, respectively. The effects of XBD173 treatment on microglia and Müller cell reactivity were analyzed by immuno-stainings of retinal sections and flat mounts, fluorescence-activated cell sorting (FACS) analysis, and mRNA expression of microglia markers using quantitative real-time PCR (qRT-PCR). Optical coherence tomography (OCT), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings, and morphometric analyses were used to quantify the extent of retinal degeneration and photoreceptor apoptosis.

RESULTS: Four days after the mice were challenged with bright white light, a large number of amoeboid-shaped alerted microglia appeared in the degenerating outer retina, which was nearly completely prevented by treatment with XBD173. This treatment also down-regulated the expression of TSPO protein in microglia but did not change the TSPO levels in the retinal pigment epithelium (RPE). RT-PCR analysis showed that the microglia/macrophage markers Cd68 and activated microglia/macrophage whey acidic protein (Amwap) as well as the pro-inflammatory genes Ccl2 and Il6 were reduced after XBD173 treatment. Light-induced degeneration of the outer retina was nearly fully blocked by XBD173 treatment. We further confirmed these findings in an independent mouse model of focal light damage. Retinas of animals receiving XBD173 therapy displayed significantly more ramified non-reactive microglia and more viable arrestin-positive cone photoreceptors than vehicle controls.

CONCLUSIONS: Targeting TSPO with XBD173 effectively counter-regulates microgliosis and ameliorates light-induced retinal damage, highlighting a new pharmacological concept for the treatment of retinal degenerations.

TH2 and TH17 inflammatory pathways are reciprocally regulated in asthma

Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms. In a cross-sectional study of asthmatics of varying severity (n = 51), endobronchial tissue gene expression analysis revealed three major patient clusters: TH2-high, TH17-high, and TH2/17-low. TH2-high and TH17-high patterns were mutually exclusive in individual patient samples, and their gene signatures were inversely correlated and differentially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) and TH17 signatures, we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in a preclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells and neutrophilic inflammation in the lung. However, neutralization of IL-13 and IL-17 protected mice from eosinophilia, mucus hyperplasia, and airway hyperreactivity and abolished the neutrophilic inflammation, suggesting that combination therapies targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes.

Targeting the complement system for the management of retinal inflammatory and degenerative diseases

The retina, an immune privileged tissue, has specialized immune defense mechanisms against noxious insults that may exist in diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), uveoretinitis and glaucoma. The defense system consists of retinal innate immune cells (including microglia, perivascular macrophages, and a small population of dendritic cells) and the complement system. Under normal aging conditions, retinal innate immune cells and the complement system undergo a low-grade activation (parainflammation) which is important for retinal homeostasis. In disease states such as AMD and DR, the parainflammatory response is dysregulated and develops into detrimental chronic inflammation. Complement activation in the retina is an important part of chronic inflammation and may contribute to retinal pathology in these disease states. Here, we review the evidence that supports the role of uncontrolled or dysregulated complement activation in various retinal degenerative and angiogenic conditions. We also discuss current strategies that are used to develop complement-based therapies for retinal diseases such as AMD. The potential benefits of complement inhibition in DR, uveoretinitis and glaucoma are also discussed, as well as the need for further research to better understand the mechanisms of complement-mediated retinal damage in these disease states.

Fluid strategies and outcomes in patients with acute respiratory distress syndrome, systemic inflammatory response syndrome and sepsis: a protocol for a systematic review and meta-analysis

Background
Fluid administration to critically ill patients remains the subject of considerable controversy. While intravenous fluid given for resuscitation may be life-saving, a positive fluid balance over time is associated with worse outcomes in critical illness. The aim of this systematic review is to summarise the existing evidence regarding the relationship between fluid administration or balance and clinically important patient outcomes in critical illness.

Methods
We will search Medline, EMBASE, the Cochrane Central Register of Controlled Trials from 1980 to the present and key conference proceedings from 2009 to the present. We will include studies of critically ill adults and children with acute respiratory distress syndrome (ARDS), sepsis and systemic inflammatory response syndrome (SIRS). We will include randomised controlled trials comparing two or more fluid regimens of different volumes of fluid and observational studies reporting the relationship between volume of fluid administered or fluid balance and outcomes including mortality, lengths of intensive care unit and hospital stay and organ dysfunction. Two independent reviewers will assess articles for eligibility, data extraction and quality appraisal. We will conduct a narrative and/or meta-analysis as appropriate.

Discussion
While fluid management has been extensively studied and discussed in the critical care literature, no systematic review has attempted to summarise the evidence for post-resuscitation fluid strategies in critical illness. Results of the proposed systematic review will inform practice and the design of future clinical trials.

Systematic review registration
PROSPERO CRD42013005608. (http://​www.​crd.​york.​ac.​uk/​PROSPERO/​)

Nonsteroidal Anti-Inflammatory Drug Use is Not Associated With Reduced Risk of Barrett’s Esophagus

OBJECTIVES: Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced risk of esophageal adenocarcinoma. Epidemiological studies examining the association between NSAID use and the risk of the precursor lesion, Barrett’s esophagus, have been inconclusive.

METHODS: We analyzed pooled individual-level participant data from six case-control studies of Barrett’s esophagus in the Barrett’s and Esophageal Adenocarcinoma Consortium (BEACON). We compared medication use from 1474 patients with Barrett’s esophagus separately with two control groups: 2256 population-based controls and 2018 gastroesophageal reflux disease (GERD) controls. Study-specific odds ratios (OR) and 95% confidence intervals (CI) were estimated using multivariable logistic regression models and were combined using a random effects meta-analytic model.

RESULTS: Regular (at least once weekly) use of any NSAIDs was not associated with the risk of Barrett’s esophagus (vs. population-based controls, adjusted OR = 1.00, 95% CI = 0.76–1.32; I2=61%; vs. GERD controls, adjusted OR = 0.99, 95% CI = 0.82–1.19; I2=19%). Similar null findings were observed among individuals who took aspirin or non-aspirin NSAIDs. We also found no association with highest levels of frequency (at least daily use) and duration (≥5 years) of NSAID use. There was evidence of moderate between-study heterogeneity; however, associations with NSAID use remained non-significant in “leave-one-out” sensitivity analyses.

CONCLUSIONS: Use of NSAIDs was not associated with the risk of Barrett’s esophagus. The previously reported inverse association between NSAID use and esophageal adenocarcinoma may be through reducing the risk of neoplastic progression in patients with Barrett’s esophagus.

Synthesis and biological activities of polyphenolic hybrids - evaluation of anti-cancer, anti-inflammatory and antioxidant activities

Os compostos polifenólicos constituem uma classe de metabolitos secundários de plantas, mas existe também uma enorme quantidade de derivados sintéticos ou semi-sintéticos contendo múltiplas unidades fenólicas. Estes compostos apresentam importantes características biológicas, que dependem das suas estruturas básicas. Certos derivados desta família de compostos, tais como flavonoides, cromonas e cumarinas contribuem para os benefícios da dieta humana, e partilham o núcleo de benzopiran-(2 e 4)-ona ou benzofuran-3-ona. A presente dissertação inclui uma introdução geral e três capítulos que descrevem as novas rotas sintéticas estabelecidas para a preparação de novos híbridos de diversos compostos polifenólicos, assim como a sua elucidação estrutural e termina com a presentação dos resultados da avaliação biológica desses mesmos compostos. No segundo capítulo discute-se a preparação de híbridos de pirimidina- e imidazolidina-polifenóis, especialmente a síntese diastereoseletiva de novos híbridos benzofuran-3-ona-hidantoína e derivados de uracilo. A rota sintética envolve a ação de carbodiimidas sobre os ácidos cromona-(2- e 3)-carboxílicos num só passo ou em dois passos sequenciais, catalisada por uma base orgânica ou inorgânica. O terceiro capítulo descreve reações do tipo adições conjugadas 1,4 - hetero-ciclisações em cascata de compostos 1,3-dicarbonílicos em ácido cromona-3-carboxílico catalisadas por uma base orgânica, que originaram novas cromonas, cromanonas e flavonas polissubstituídas. As bispiranonas [bispiran-2 e 4)-onas] foram elaboradas numa reacção de acoplamento da 4-hidroxicumarina ou da lactona do ácido triacético com o ácido cromona-3-carboxílico ou precursores formil-funcionalizados (ω-formil-2’-hydroxy acetofenonas e cromona-3-carbaldeídos) utilizando organocatálise básica. Finalmente, alargou-se o estudo das adições conjugadas 1,4 para uma variedade de 4-hidroxipiran-2-onas e cetonas α,β-insaturadas para originar novos análogos de warfarina. Obteve-se uma variedade de estruturas complexas por hibridação das unidades de 4-hidroxicumarina ou da lactona do ácido triacético com os novos derivados de cromonas polissubstituídas. Todos as reações foram executadas em condições suaves e ambientalmente favoráveis, utilizando a 4-pirrolidinopiridina como organocatalisador básico. As estruturas dos novos híbridos polifenólicos foram caracterizados por técnicas espectroscópicas de alta resolução, incluindo espectroscopia de ressonância magnética nuclear (1D e 2D) e por difractometria de raios-X, que nos permitiram resolver o complexidade estrutural dos compostos sintetizados. O quarto capítulo apresenta os resultados da avaliação biológica obtidos com os híbridos polifenólicos sintetizados neste trabalho, mostrando a possibilidade de seu envolvimento na terapia do cancro. A maioria dos compostos foram avaliados quanto ao seu efeito sobre a citotoxicidade e proliferação de células leucémicas e ao seu envolvimento na regulação de via pró-inflamatória NF-kB, na qual, os híbridos de biscumarinas exibiram actividades elevadas (IC50 = 6-19 μM para inibição de NF-kB depois de 8 horas de incubação e IC50 = 15-39 μM para efeitos citotóxicos em células cancerosas, após 24 horas de incubação). Uma inibição moderada das enzimas HDAC e Cdc25 foi induzida pelos derivados de benzofuran-3-ona-hidantoína. Catorze dos novos derivados polifenólicos polissubstituídos, tendo como estrutura básica a benzopiran-4-ona, foram avaliados pela sua actividade quimiopreventiva do cancro mediada pela indução de sinalização citoprotectora Nrf2 (fator 2 relacionado com o fator nuclear da proteína E2) e capacidade para inibir a proliferação das células de cancro da mama. Os derivados da classe das cromanonas foram identificados como os indutores mais potentes da actividade Nrf2. As concentrações necessárias para aumentar a actividade de luciferase em 10 vezes (C10) foram de 2,8-21,3 μM. Todos os novos híbridos polifenólicos que apresentam atividade citotóxica e anti-proliferativa não afectam o crescimento de células saudáveis periféricas do sangue (PBMC) (IC50 > 50 μM), indicando a sua seletividade para as células cancerosas e sugerindo que alguns deles são estruturalmente interessantes para posteriores análises. A avaliação da atividade antioxidante utilizando os testes do radical livre DPPH e o poder redutor do ião férrico FRAP foram realizados em algumas estruturas híbridas polifenólicas.

Detection of non-steroidal anti-inflammatory drugs in aqueous effluents using ionic liquids

Significant improvements in human health have been achieved through the increased consumption of pharmaceutical drugs. However, most of these active pharmaceutical ingredients (APIs) are excreted by mammals (in a metabolized or unchanged form) into the environment. The presence of residual amounts of these contaminants was already confirmed in aqueous streams since treatment processes either wastewater treatment plants (WWTPs) or sewage treatment plants (STPs) are not specifically designed for this type of pollutants. Although they are present in aqueous effluents, they are usually at very low concentrations, most of the times below the detection limits of analytical equipment used for their quantification, hindering their accurate monitoring. Therefore, the development of a pre-concentration technique in order to accurately quantify and monitor these components in aqueous streams is of major relevance. This work addresses the use of liquid-liquid equilibria, applying ionic liquids (ILs), for the extraction and concentration of non-steroidal anti-inflammatory drugs (NSAIDs) from aqueous effluents. Particularly, aqueous biphasic systems (ABSs) composed of ILs and potassium citrate were investigated in the extraction and concentration of naproxen, diclofenac and ketoprofen from aqueous media. Both the extraction efficiency and concentration factor achievable by these systems was determined and evaluated. Within the best conditions, extraction efficiencies of 99.4% and concentration factors up to 13 times were obtained.

A RG-II type polysaccharide purified from Aconitum coreanum and their anti-inflammatory activity

Korean mondshood root polysaccharides (KMPS) isolated from the root of Aconitum coreanum (Lévl.) Rapaics have shown anti-inflammatory activity, which is strongly influenced by their chemical structures and chain conformations. However, the mechanisms of the anti-inflammatory effect by these polysaccharides have yet to be elucidated. A RG-II polysaccharide (KMPS-2E, Mw 84.8 kDa) was isolated from KMPS and its chemical structure was characterized by FT-IR and NMR spectroscopy, gas chromatography–mass spectrometry and high-performance liquid chromatography. The backbone of KMPS-2E consisted of units of [→6) -β-D-Galp (1→3)-β-L-Rhap-(1→4)-β-D-GalpA-(1→3)-β-D-Galp-(1→] with the side chain →5)-β-D-Arap (1→3, 5)-β-D-Arap (1→ attached to the backbone through O-4 of (1→3,4)-L-Rhap. T-β-D-Galp is attached to the backbone through O-6 of (1→3,6)-β-D-Galp residues and T-β-D-Ara is connected to the end group of each chain. The anti-inflammatory effects of KMPS-2E and the underlying mechanisms using lipopolysaccharide (LPS) - stimulated RAW 264.7 macrophages and carrageenan-induced hind paw edema were investigated. KMPS-2E (50, 100 and 200 µg/mL) inhibits iNOS, TLR4, phospho-NF-κB–p65 expression, phosphor-IKK, phosphor-IκB-α expression as well as the degradation of IκB-α and the gene expression of inflammatory cytokines (TNF-α, IL-1β, iNOS and IL-6) mediated by the NF-κB signal pathways in macrophages. KMPS-2E also inhibited LPS-induced activation of NF-κB as assayed by electrophorectic mobility shift assay (EMSA) in a dose-dependent manner and it reduced NF-κB DNA binding affinity by 62.1% at 200µg/mL. In rats, KMPS-2E (200 mg/kg) can significantly inhibit carrageenan-induced paw edema as ibuprofen (200 mg/kg) within 3 h after a single oral dose. The results indicate that KMPS-2E is a promising herb-derived drug against acute inflammation.

Development of CO-releasing molecular for the treatment of inflammatory diseases

Dissertation presented to obtain a Ph.D. degree (Doutoramento) in Chemistry at the Instituto de Tecnologia Quimica e Biol6gica da Universidade Nova de Lisboa