Detection Of Inflammatory Markers Research Articles

518 - AUTOIMMUNE DEMENTIA – WHEN TO SUSPECT?

AbstractBackground:Autoimune dementias are underrecognized clinical entities, frequently misdiagnosed as neurodegenerative or prion disorders. However, the prognosis is vastly different since immunotherapy can treat these conditions and restore functionality.Research objective:To reflect on autoimmune dementias, briefly presenting the autoimmune syndromes, how to diagnose them and some clinical cues to be attentive of.Methods:Literature search on Pubmed and Google Scholar.Results:The incidence and prevalence of autoimmune dementias are unknown, but autoimmune and inflammatory causes account for 20% of dementia in patients younger than 45 years of age.Autoimmune dementias are classified according to syndromic presentation, specific serologic markers, or histopathologic findings.Patients with autoimmune dementias usually present with an acute or subacute disorder of memory, thinking, or behaviour. Clinical clues that can help clinicians identify autoimmune dementias include six of the following: (i) rapidly progressive or fluctuating course; (ii) multifocal and diverse clinical presentations; (iii) personal or family history of autoimmunity; (iv) detection of inflammatory markers in the cerebrospinal fluid; (v) presence of a neural-specific autoantibody and (vi) favourable response to a trial of immunotherapy. Also, unsuspected cancer, new or recurrent, may manifest neurologically as autoimmune dementia.In evaluating patients with dementia and autoimmune disease, clinicians should be aware of the possible coexistence of these disorders.Conclusions:Recognition of clinical and serologic clues to autoimmune dementia allows early and sustained treatment. Misdiagnosis of a potentially reversible condition as a progressive neurodegenerative disorder can have devastating consequences for the patient and family.

Flow cytometry detection of inflammatory markers in glial cells and synaptosomes derived from AD cortex

AbstractBackgroundPrevious research has indicated the involvement of nearly all cell types of the brain (i.e. neurons, microglia, etc.) in progression of Alzheimer Disease (AD). In the case of microglia, increased production of neuroinflammatory markers accompanies AD and has been implicated in the loss of synapses and neurons. In mouse models of AD, gene expression of many inflammatory markers is increased in microglial cells, including genetic risk factors LPL and TREM2, while homeostatic markers are diminished. Intact cells from post‐mortem AD tissue were isolated and used to better explore the relationship of inflammatory markers and AD‐specific pathological species. Further, microglial elements have been found to be associated with synaptosomes (resealed axon terminals) and were further explored to determine if specific inflammatory signals vary with amyloid and tau pathology.MethodPost‐mortem AD tissue (PMI<6 hours) from late AD patients (Braak V‐VI) and age‐matched controls was enzymatically dissociated, processed and labeled using fluorophore‐conjugated antibodies. The labeled cells were subsequently analyzed by an LSRII flow cytometer using FCS Express v5 software. Synaptosomes were prepared from the same AD patients by homogenizing brain tissue in 0.32 M sucrose with a cocktail of enzyme inhibitors. This preparation, known as P2, then underwent probing using western blotting (10‐20% Tris‐Glycine gel with PVDF membrane) and flow cytometry using the same instrument and software.ResultPreliminary results show a significant increase in the number of P2ry12+ (homeostatic) microglia in AD patients relative to controls. Further labeling of microglia with CD11b (myeloid marker) and TGFbeta1 (homeostatic marker) showed no significant difference between AD and controls, suggesting a more complex picture of microglial changes compared to mouse models. Preliminary results showed an increase in inflammatory markers TLR4 and IL‐6 in AD P2 samples relative to controls. Further, TLR4 was increased in individuals with at least one APOE4 allele.ConclusionMicroglial activation in AD mouse models differs from human AD. Human AD microglia demonstrate an increased expression of one homeostatic marker (P2ry12) but not another (TGFB1) while mice show a more uniform decrease of these markers. Further, microglial signals associated with synaptosomes suggests they contribute to neurotoxicity and synaptic loss, a parameter highly correlated with dementia.

The Detection of Bile Acids in the Lungs of Paediatric Cystic Fibrosis Patients Is Associated with Altered Inflammatory Patterns.

Background: Cystic fibrosis (CF) is a hereditary disorder in which persistent unresolved inflammation and recurrent airway infections play major roles in the initiation and progression of the disease. Little is known about triggering factors modulating the transition to chronic microbial infection and inflammation particularly in young children. Cystic fibrosis respiratory disease starts early in life, with the detection of inflammatory markers and infection evident even before respiratory symptoms arise. Thus, identifying factors that dysregulate immune responsiveness at the earliest stages of the disease will provide novel targets for early therapeutic intervention. Methods: We evaluated the clinical significance of bile acid detection in the bronchoalveolar lavage fluid of clinically stable preschool-aged children diagnosed with CF. Results: We applied an unbiased classification strategy to categorize these specimens based on bile acid profiles. We provide clear associations linking the presence of bile acids in the lungs with alterations in the expression of inflammatory markers. Using multiple regression analysis, we also demonstrate that clustering based on bile acid profiles is a meaningful predictor of the progression of structural lung disease. Conclusions: Altogether, our work has identified a clinically relevant host-derived factor that may participate in shaping early events in the aetiology of CF respiratory disease.

Amifostine reduces inflammation and protects against 5-fluorouracil-induced oral mucositis and hyposalivation.

Oral mucositis (OM) is a common and dose-limiting side effect of cancer treatment, including 5-fluorouracil (5-FU) and radiotherapy. The efficacy of the therapeutic measures to prevent OM is limited and disease prevention is not fully observable. Amifostine is a cytoprotective agent with a described anti-inflammatory potential. It is clinically used to reduce radiotherapy and chemotherapy-associated xerostomia. This study investigated the protective effect of amifostine on an experimental model of OM. Hamsters were divided into six groups: saline control group (5 mL/kg), mechanical trauma (scratches) of the right cheek pouch; 5-FU (60 and 40 mg/kg, ip, respectively, administered on days 1 and 2); amifostine (12.5, 25, or 50 mg/kg) + 5-FU + scratches. Salivation rate was assessed and the animals were euthanized on day 10 for the analysis of macroscopic and microscopic injury by scores. Tissue samples were harvested for the measurement of neutrophil infiltration and detection of inflammatory markers by ELISA and immunohistochemistry. 5-FU induced pronounced hyposalivation, which was prevented by amifostine (P<0.05). In addition, 5-FU injection caused pronounced tissue injury accompanied by increased neutrophil accumulation, tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) tissue levels, and positive immunostaining for TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS). Interestingly, amifostine prevented the inflammatory reaction and consequently improved macroscopic and microscopic damage (P<0.05 vs 5-FU group). Amifostine reduced inflammation and protected against 5-FU-associated oral mucositis and hyposalivation.

Detection of Increased Plasma Interleukin-6 Levels and Prevalence of Prevotella copri and Bacteroides vulgatus in the Feces of Type 2 Diabetes Patients.

Intestinal dysbiosis and metabolic endotoxemia have been associated with metabolic disorders, such as obesity, insulin resistance, and type 2 diabetes (T2D). The main goal of the present study was to evaluate the intestinal dysbiosis in Brazilian T2D patients and correlate these data with inflammatory cytokines and lipopolysaccharides (LPS) plasma concentrations. This study was approved by the Ethics Committees from Barretos Cancer Hospital and all individuals signed the informed consent form. Stool samples were required for DNA extraction, and the V3/V4 regions of bacterial 16S were sequenced using an Illumina platform. Peripheral blood was used to quantify inflammatory cytokines and plasma LPS concentrations, by CBA flex and ELISA, respectively. Statistical analyses were performed using Mann–Whitney and Spearman’s tests. Analysis of variance, diversity indexes, and analysis of alpha- and beta-diversity were conducted using an annotated Operational Taxonomic Unit table. This study included 20 patients and 22 controls. We observed significant differences (P < 0.01) in the microbiota composition (beta-diversity) between patients and controls, suggesting intestinal dysbiosis in Brazilian T2D patients. The prevalent species found in patients’ feces were the Gram-negatives Prevotella copri, Bacteroides vulgatus, Bacteroides rodentium, and Bacteroides xylanisolvens. The proinflammatory interleukin-6 (IL-6) was significantly increased (P < 0.05) in patients’ plasma and LPS levels were decreased. We find correlations between the proinflammatory interferon-gamma with Gram-negatives Bacteroides and Prevotella species, and a positive correlation between the LPS levels and P. copri reads. The P. copri and B. vulgatus species were associated with insulin resistance in previous studies. In this study, we suggested that the prevalence of Gram-negative species in the gut and the increased plasma IL-6 in patients could be linked to low-grade inflammation and insulin resistance. In conclusion, the P. copri and B. vulgatus species could represent an intestinal microbiota signature, associated with T2D development. Furthermore, the identification of these Gram-negative bacteria, and the detection of inflammatory markers, such as increased IL-6, could be used as diabetes predictive markers in overweight, obese and in genetically predisposed individuals to develop T2D.

MRI detection of endothelial cell inflammation using targeted superparamagnetic particles of iron oxide (SPIO)

BackgroundThere is currently no clinical imaging technique available to assess the degree of inflammation associated with atherosclerotic plaques. This study aims to develop targeted superparamagnetic particles of iron oxide (SPIO) as a magnetic resonance imaging (MRI) probe for detecting inflamed endothelial cells.MethodsThe in vitro study consists of the characterisation and detection of inflammatory markers on activated endothelial cells by immunocytochemistry and MRI using biotinylated anti-P-selectin and anti-VCAM-1 (vascular cell adhesion molecule 1) antibody and streptavidin conjugated SPIO.ResultsEstablished an in vitro cellular model of endothelial inflammation induced with TNF-α (tumor necrosis factor alpha). Inflammation of endothelial cells was confirmed with both immunocytochemistry and MRI. These results revealed both a temporal and dose dependent expression of the inflammatory markers, P-selectin and VCAM-1, on exposure to TNF-α.ConclusionThis study has demonstrated the development of an in vitro model to characterise and detect inflamed endothelial cells by immunocytochemistry and MRI. This will allow the future development of contrast agents and protocols for imaging vascular inflammation in atherosclerosis. This work may form the basis for a translational study to provide clinicians with a novel tool for the in vivo assessment of atherosclerosis.

Intratracheal therapy with autologous bone marrow-derived mononuclear cells reduces airway inflammation in horses with recurrent airway obstruction

This research evaluated the effects of bone marrow-derived mononuclear cells (BMMCs) on the inflammatory process in the equine recurrent airway obstruction (RAO). Eight horses in RAO clinical score were divided into cell therapy group (Gcel) treated with a single intratracheal dose of BMMCs, and dexamethasone group (Gdex) treated with 21days of oral dexamethasone. The horses were clinically revaluated on days 7 and 21, together with cytological evaluation of the BALF, and detection of inflammatory markers (interleukins [IL]-10, -4, and -17, and interferon γ and α). There were decreases in respiratory effort and clinical score on days 7 and 21(p<0.05) for both groups. The percentage of neutrophils decreased and macrophages increased on days 7 and 21 (p<0.005) in both groups. IL-10 levels increased in the Gcel group on day 21 compared to days 0 and 7 (p<0.05), but this was not observed in the Gdex group. The quantification of IL-4, IL-17, IFN-γ, and IFN-α did not change between evaluations in both groups. These preliminary results suggest that BMMCs may ameliorate the inflammatory response of RAO.

Kinetics of TH2 biomarkers in sputum of asthmatics following inhaled allergen

BackgroundAllergen-induced late airway response offers important pharmacodynamic targets, including T helper 2 (TH2) biomarkers. However, detection of inflammatory markers has been limited in dithiothreitol-processed sputum.ObjectivesTo test whether allergen-induced TH2 inflammatory markers can be reproducibly quantified by sensitive detection techniques in ultracentrifuged sputum and the effect of fluticasone (FP) on these endpoints.MethodsThirteen allergic asthmatics with dual allergen-induced airway responses, documented during a single-blind placebo run-in period, participated in a double-blind, two-period crossover study. Each period consisted of three consecutive days, separated by ≥3 weeks. Following randomization, subjects inhaled FP (500 µg bid, five doses total) or placebo. On Day 2 in each study period, allergen challenge was performed and airway response measured by forced expiratory volume in 1 sec (FEV1) until 7 h post-challenge. Sputum was induced 24 h pre-allergen and 7 and 24 h post-allergen. Sputum samples were split into two portions: TH2 biomarkers were quantified by Meso Scale multiplex platform following ultracentrifugation, and cell differentials were counted on Giemsa–May-Grünwald-stained cytospins. Allergen-induced changes in inflammatory endpoints were compared between FP and placebo using a mixed model ANCOVA.ResultsInhaled allergen induced dual airway responses in all subjects during both placebo periods with reproducible late asthmatic response (LAR) and increased sputum inflammatory biomarkers (IL-2, IL-4, IL-13, and eotaxin-1) and eosinophil counts. FP effectively blunted both the LAR and the inflammatory biomarkers.ConclusionsCombining novel, sensitive quantification methods with ultracentrifugation allows reproducible quantification of sputum biomarkers following allergen challenge, reversed by FP. This approach allows non-invasive identification of pharmacodynamic targets for anti-asthma therapies.

Analysis of serum inflammatory indicators before the onset of acute coronary syndrome and the incidence of infection

Objective To explore the changes of serum inflammatory markers in patients with acute coronary syndrome（ACS）,as well as the incidence of infection before the onset of the disease .Methods 83 patients with ACS were selected as the observation group ,and at the corresponding period of hospitalization ,71 patients with stable angi-na or coronary angiography negative were selected as the control group .The serum levels of C-reactive protein（CRP）, interleukin-6（IL-6） were measured.Results Before the onset of the disease,the total infection rate of the observa-tion group was 41.0%,which was significantly higher than 25.4%of the control group,the difference was statistically significant（χ2 =4.17,P〈0.05）.The types of infections including upper respiratory infections ,lung infections,biliary tract infections,intestinal infections,urinary tract infections,skin infections between the two groups had no statistical differences（χ2 =1.96,1.27,0.84,0.92,0.92,0.88,0.88,all P〉0.05）.The CRP,IL-6 levels of the observation group were （7.52 ±1.62）mg/L,（247.55 ±62.17）ng/L,respectively,which were significantly lower than [（2.76 ± 1.01）mg/L,（99.25 ±30.63）ng/L]of the control group（t=21.44,18.28,all P〈0.05）.Conclusion ACS pa-tients with acute onset prior to the onset of acute infection may be correlated ,and serum inflammatory markers in ACS patients were significantly increased ,suggesting that through the clinical detection of inflammatory markers in patients with ACS will help for further diagnosis . Key words: Acute coronary syndrome ; Inflammation mediators ; C-reactive protein

Detection of inflammatory markers in stools from patients with irritable bowel syndrome and collagenous colitis

Objective. Irritable bowel syndrome (IBS) and collagenous colitis (CC) share chronically recurring symptoms of altered bowel habits associated with abdominal pain or discomfort. The aims of the present study were to investigate whether inflammatory markers could be detected in faeces from patients with IBS and CC, and to elucidate whether such analyses could be used as non-invasive tools to distinguish between these disorders. Material and methods. Stool samples were obtained from 18 patients with CC, 46 patients with IBS and 20 healthy controls (HC). Eosinophil protein X (EPX), myeloperoxidase (MPO), tryptase, interleukin-1 beta (IL-1β) and tumour necrosis factor alpha (TNFα) were measured in supernatants from processed faeces using immunoassays. Results. EPX levels were enhanced in faeces from CC patients (median 3.8 µg/g (0.47–16.2)) compared to patients with IBS (0.44 µg/g (0.25–1.8)), p<0.001, and HC (0.46 µg/g (0.21–1.3)), p<0.001. In addition, MPO was increased in CC patients (11.7 µg/g (2.0–124)) compared to IBS patients (1.7 µg/g (0.81–5.2)), p<0.01, and HC (2.5 µg/g (1.1–6.3)), p<0.05. Tryptase was found in 9/18 patients with CC, 6/46 with IBS and 1/19 HC. IL-1β was only enhanced in 2/11 CC patients and TNFα was not detected in any sample. Conclusions. Increased levels of EPX, MPO and tryptase were observed in stools from collagenous colitis patients, whereas the levels in IBS patients did not differ from healthy controls. Our data suggest that faecal markers could be used as part of the clinical work-up to determine which patients should be biopsied and evaluated for collagenous colitis.

The effect of dithiothreitol on VLA-4 detection in peripheral blood and induced sputum cells

Induced sputum has been shown to be a reliable technique for investigating airway inflammation non-invasively. Flow cytometry could provide useful information in this area. However, the viscosity of the sample entails the use of a mucolytic agent. Dithiothreitol (DDT) is the most frequently used agent although it could affect detection of different inflammatory markers. To measure the effect of DDT on the detection of certain adhesion molecules in eosinophils and lymphocytes, sputum was induced from seven non-smoking asthmatic and non-asthmatic subjects treated with 0.1 M DDT. The samples were analyzed by flow cytometry. Whole blood samples from the same subjects were also processed with DTT and analyzed by flow cytometry. Very late activated antigen-4 (VLA-4) levels on eosinophils in intracellular and surface staining were much lower than expected. VLA-4 on lymphocytes was also altered but less so than on eosinophils. VLA-4 levels were also decreased on blood cells after DTT treatment. No abnormalities were found in the detection of CD29 on eosinophils and the beta7-chain in lymphocytes. Flow cytometry could be used as a complementary method to induced sputum in the investigation of airway inflammation. However, DTT could interfere with the detection of some inflammatory markers, as is the case with VLA-4.