Meta-Inflammation and Metabolic Reprogramming of Macrophages in Diabetes and Obesity:The Importance of Metabolites

Diabetes mellitus type II and obesity are two important causes of death in modern society. They are characterized by low-grade chronic inflammation and metabolic dysfunction (meta-inflammation), which is observed in all tissues involved in energy homeostasis. A substantial body of evidence has established an important role for macrophages in these tissues during the development of diabetes mellitus type II and obesity. Macrophages can activate into specialized subsets by cues from their microenvironment to handle a variety of tasks. Many different subsets have been described and in diabetes/obesity literature two main classifications are widely used that are also defined by differential metabolic reprogramming taking place to fuel their main functions. Classically activated, pro-inflammatory macrophages (often referred to as M1) favor glycolysis, produce lactate instead of metabolizing pyruvate to acetyl-CoA, and have a tricarboxylic acid cycle that is interrupted at two points. Alternatively activated macrophages (often referred to as M2) mainly use beta-oxidation of fatty acids and oxidative phosphorylation to create energy-rich molecules such as ATP and are involved in tissue repair and downregulation of inflammation. Since diabetes type II and obesity are characterized by metabolic alterations at the organism level, these alterations may also induce changes in macrophage metabolism resulting in unique macrophage activation patterns in diabetes and obesity. This review describes the interactions between metabolic reprogramming of macrophages and conditions of metabolic dysfunction like diabetes and obesity. We also focus on different possibilities of measuring a range of metabolites intra-and extracellularly in a precise and comprehensive manner to better identify the subsets of polarized macrophages that are unique to diabetes and obesity. Advantages and disadvantages of the currently most widely used metabolite analysis approaches are highlighted. We further describe how their combined use may serve to provide a comprehensive overview of the metabolic changes that take place intracellularly during macrophage activation in conditions like diabetes and obesity

Biomarker Discovery In Chronic Obstructive Pulmonary Disease (COPD) Using Epithelial Lining Fluid:A Proteomic Approach

RATIONALE Chronic Obstructive Pulmonary Disease (COPD) is the third most frequent disease worldwide with increasing mortality. Cigarette smoking is the principle risk factor and 15-20% of smokers develop COPD. Epithelial Lining Fluid (ELF) covers the internal part of the airways and can be collected during bronchoscopy. ELF appears to be well-suited for proteomic analysis, since it contains a higher concentration of proteins (150-300 μg /mL) than other lung fluids and can be obtained from different locations of the lungs. No comprehensive proteomic analysis of human ELF has been performed to date, which makes ELF a highly interesting fluid for biomarker discovery in COPD. AIM To discover proteins that change in abundance in ELF from COPD patients versus healthy controls using a quantitative proteomics approach. METHODS The ELF proteome from COPD patients and healthy controls was studied by 1D polyacrylamide gel electrophoresis in the presence of SDS followed by in-gel tryptic digestion to establish the methodology and assess the feasibility of such an approach. Approximately 40 gel slices were obtained from each lane of the gel (corresponding to one patient). Digested samples were analyzed by nanoChip-LC-MS/MS using an ion trap. We performed a quantitative pilot study of ELF from 4 COPD patients and 4 healthy controls (table 1) to test for statistically significant differences in protein levels. ELF samples were digested by trypsin, labeled with stable isotope-containing reagents (iTRAQ®, 8-plex) and processed by strong cation-exchange chromatography followed by nanoLC-MS/MS. In order to validate the results, a second quantitative analysis of an independent sample set (4 COPD vs 4 healthy) using the same methodological approach was done. RESULTS The 1D electrophoretic approach resulted in more than 300 identified proteins. Most of the identified proteins were present in both COPD and healthy samples, although some proteins were only identified either in healthy control or in COPD samples. The quantitative studies showed that a number of proteins was significantly different between ELF of COPD patients and controls, including 4 up-regulated proteins in common in both studies. CONCLUSIONS This is the first study in ELF of COPD patients and healthy controls in which such a large number of proteins has been identified. The obtained results show the feasibility of this proteomic approach and the possibility to discover proteins that are differentially expressed in ELF of COPD patients and controls. We are currently validating these proteins further by western blot and immunohistochemistry

Proteomic alterations in early stage cervical cancer

Laser capture microdissection (LCM) allows the capture of cell types or welldefined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, n = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue (n = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by "all-ornothing" analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer (n = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive "all-or-nothing" way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction

High Abundance Proteins Depletion vs Low Abundance Proteins Enrichment: Comparison of Methods to Reduce the Plasma Proteome Complexity

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol

West Nile Fever in the Rostov Region: Ecological and Epidemiological Peculiarities of the Outbreak in 2010

This paper describes the outbreak of West Nile fever in the Rostov Region in 2010 and evaluates its ecological and epidemiological peculiarities. From 15th of July till 22nd of September 2010, detected were the 64 cases (1, 4800/0000) of the disease, which were characterized by vector-born mechanism of transmission. Peak of morbidity coincided with mass breeding of Culicidae, increase in the number of Culex mosquitoes, and reoccurring growth of Aedes mosquito population. Diffuse type of the epidemiological process, higher rates of the cases among urbanites, infected in the country-side area, were the characteristic features of that outbreak. West Nile virus antigen was detected by means of IFA in samples taken from An. maculipennis and Cx. pipiens mosquitoes, wild and synanthropic birds, Rh. rossicus ticks, house and wood mice, which facilitates identification of the core factors for the agent circulation and West Nile fever natural focus formation

Susceptibility to COPD:Differential Proteomic Profiling after Acute Smoking

Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as "susceptible individuals". Here we perform unbiased analyses of proteomic profiles to assess how "susceptible individuals" differ from age-matched "non-susceptible individuals" in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms that induce COPD. Our data suggest that SerpinB3 and Uteroglobin could be interesting proteins in understanding the processes leading to COPD