Distinct blood and visceral adipose tissue regulatory T cell and innate lymphocyte profiles characterize obesity and colorectal cancer

Visceral adipose tissue (VAT) is a main site where metabolic and immunologic processes interplay to regulate, at local and systemic level, the inflammatory status and immune response. Obesity-associated inflammation and immune dysfunctions are inextricably linked to tumor but, in spite of intense efforts, the mechanisms underpinning this asso- ciation remain elusive. In this report, we characterized the profile of VAT-associated and circulating innate lymphocyte and regulatory T (T reg ) cell subsets underlying inflammatory conditions, such as obesity and colorectal cancer (CRC). Analysis of NK, NKT-like, γδ T, and T reg cell populations in VAT and blood of healthy lean subjects revealed that CD56 hi NK and OX40 + T reg cells are more abundant in VAT with respect to blood. Conversely, CD56 dim NK and total T reg cells are most present in the circulation, while γδ T lymphocytes are uniformly distributed in the two compartments. Interestingly, a reduced frequency of circulating activated T reg cells, and a concomitant preferential enrichment of OX40- expressing T reg cells in VAT, were selectively observed in obese (Ob) subjects, and directly correlated with body mass index. Likewise, CRC patients were characterized by a specific enrichment of VAT-associated NKT-like cells. In addition, Ob and CRC-affected individuals shared a significant reduction of the V γ 9V δ 2/ γδ T cell ratio at systemic level. The alterations in the relative proportions of T reg and NKT-like cells in VAT were found to correlate with the content of pro- and anti-inflammatory polyunsaturated fatty acids (PUFA), respectively. Overall, these results provide evidence for distinct alterations of the immune cell repertoire in the periphery with respect to the VAT microenvironment that uniquely characterize or are shared by different inflammatory conditions, such as obesity and CRC, and suggest that VAT PUFA composition may represent one of the factors that contribute to shape the immune phenotypes

Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

BACKGROUND:Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). METHODOLOGY/PRINCIPAL FINDINGS:Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2)O(2); 0.1-0.5 mM range). Results show that A) Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2)O(2) concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2)O(2) treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase. CONCLUSION/SIGNIFICANCE:Apoptosis can be modulated by "on/off" switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein

Dietary Fatty Acids at the Crossroad between Obesity and Colorectal Cancer: Fine Regulators of Adipose Tissue Homeostasis and Immune Response

Colorectal cancer (CRC) is among the major threatening diseases worldwide, being the third most common cancer, and a leading cause of death, with a global incidence expected to increase in the coming years. Enhanced adiposity, particularly visceral fat, is a major risk factor for the development of several tumours, including CRC, and represents an important indicator of incidence, survival, prognosis, recurrence rates, and response to therapy. The obesity-associated low-grade chronic inflammation is thought to be a key determinant in CRC development, with the adipocytes and the adipose tissue (AT) playing a significant role in the integration of diet-related endocrine, metabolic, and inflammatory signals. Furthermore, AT infiltrating immune cells contribute to local and systemic inflammation by affecting immune and cancer cell functions through the release of soluble mediators. Among the factors introduced with diet and enriched in AT, fatty acids (FA) represent major players in inflammation and are able to deeply regulate AT homeostasis and immune cell function through gene expression regulation and by modulating the activity of several transcription factors (TF). This review summarizes human studies on the effects of dietary FA on AT homeostasis and immune cell functions, highlighting the molecular pathways and TF involved. The relevance of FA balance in linking diet, AT inflammation, and CRC is also discussed. Original and review articles were searched in PubMed without temporal limitation up to March 2021, by using fatty acid as a keyword in combination with diet, obesity, colorectal cancer, inflammation, adipose tissue, immune cells, and transcription factors

New method for bacterial load measurement during hydro-debridement of cutaneous ulcer

New method for bacterial load measurement during hydro-debridement of cutaneous ulcer. Apperti M, Di Lucia A, Goffredi L, Federico P, Mattera E, Masella A, Della Rocca MD, Apperti S. SourceSeconda Università degli Studi di Napoli, Dipartimento di Scienze Anestesiologiche, Chirurgiche e dell'Emergenza. Abstract AIM OF THE STUDY: Based on their experience using Versajet for debridement of chronic wounds, the Authors set up a study protocol to verify whether the hydro-surgical cleansing could offer the possibility of taking tissue specimens suitable for diagnostic microbiological evaluation. The aims of the study were the following: MAIN PURPOSE: To evaluate the efficacy of hydro-surgery in detecting the presence of microorganisms and measuring their load, as an alternative to conventional tissue sampling methods; SECONDARY PURPOSE: To set up an easier and less invasive diagnostic modality than surgical biopsy, even though likewise significant. RESULTS: The results of this study show that tissue specimen collection by hydro-aspiration using Versajet is comparable to biopsy sampling (and in some cases it can be even more reliable); moreover, it is not more time-consuming and is certainly less invasive. Compared to surgical biopsy, with such a method a greater amount of tissue may be collected; moreover, tissue specimens can be taken from a broader surface or, depending on the needs, from a more focused area on the margin or at the bottom of the woun

Proposed role of Bcl-x_L deamidation and methylation in apoptosis.

<p>Deamidation-isomerization of two critical Asn residues of Bcl-x_L abolishes its antiapoptotic function. Methylation of the same residues can restore the functional integrity of Bcl-x_L through repair of the isopeptide bonds. Regulation of apoptosis toward antiapoptosis is gained through fine balancing of Bcl-x_L deamidation and methylation.</p

Quantitation of the extent of protein deamidation in cells lysates after oxidative stress.

<p>Cells transfected with <i>PCMT</i> wild type (sense), antisense <i>PCMT</i> (anti), PCMT mutants [Asp₈₃ →Phe₈₃ (Phe) and Asp₈₃→Val₈₃ (Val)] and endothelial cells transfected with void plasmid (PAE) were stressed with 0.3 mM of H₂O₂. Deamidated proteins were quantitated in each lysate preparation using recombinant PCMT. Standard deviation error bars are included for each analysis.</p

Identification of PCMT substrates.

<p>Panel A: <u>Experimental strategy for isolation and characterization of PCMT substrates</u>. Step 1: human recombinant PCMT, purified to homogeneity, was immobilized onto sulfoSBED by <i>N</i>-hidroxysuccinimide chemistry; Step 2: cell extracts as a source of substrates were added and Step 3: proteins interacting with PCMT were immobilized upon UV photoactivation; Step 4: PCMT was released and biotin “transferred” onto the methyltrasferase substrate (Label Transfer Method); Step 5: purification was accomplished by exploiting streptavidin-biotin interactions. Panel B: <u>2D gel electrophoresis imaging of comparative proteomics</u>. HUVEC were infected with antisense <i>PCMT</i> carrying retrovirus and then stressed with 0.3 mM of H₂O₂. Cells lysates were reacted with Sulfo-SBED previously cross-linked with recombinant PCMT (Panel B). Arrows indicate the protein spots which have been characterized as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003258#pone-0003258-t001" target="_blank">Table 1</a>. Background noise due to aspecific binding was subtracted by comparison with the 2D image obtained from a parallel sample reacted with non-cross-linked Sulfo-SBED.</p

Effect of PCMT expression levels and mutants on apoptosis induced by oxidative stress on PAE cells.

<p>Column A: <u>Apoptotic DNA ladder in cells overexpressing PCMT constructs and subject to oxidative treatment</u>. Apoptotic DNA ladder patterns, of transfected endothelial cells stimulated with different concentrations of H₂O₂, were detected after transfection with plasmid void (control) or carrying <i>PCMT wild type</i> (sense); antisense <i>PCMT</i>; <i>PCMT</i> Asp₈₃ →Phe₈₃ Mut and PCMT Asp₈₃ →Val₈₃ Mut. Column B: <u>Caspase-3 activation and PARP cleavage in cells overexpressing PCMT constructs and subject to oxidative treatment</u>. Immunoblot developed with a polyclonal antibody against caspase-3 and PARP (a final effector of various apoptotic pathways) on transfected endothelial cells stimulated with different concentration of H₂O₂ after transfection with plasmid void (control) or carring PCMT <i>wild type</i> (sense), antisense PCMT, PCMT Asp₈₃ →Phe₈₃ Mut and PCMT Asp₈₃ →Val₈₃ Mut. CP is a positive control obtained by treating a parallel cell sample with cisplatin. Column C: <u>Flow cytometry analysis of cells overexpressing PCMT constructs and subject to oxidative treatment</u>. Cells stimulated with 0.3 mM of H₂O₂ after transfection with void plasmid (control), <i>PCMT wild type</i> (sense), Antisense <i>PCMT</i>, PCMT Asp₈₃ →Phe₈₃ Mut. PCMT Asp₈₃ →Val₈₃ Mut. PI: propidium iodide.</p

Chronic dietary intake of plant-derived anthocyanins protects the rat heart against ischemia-reperfusion injury.

International audienceConsumption of flavonoid-rich foods and beverages is thought to reduce the risk of cardiovascular diseases. Whereas the biological activities of flavonoids have been characterized in vitro, there are no clear experimental data demonstrating that chronic dietary intake and intestinal absorption of flavonoids actually protects the heart against ischemia-reperfusion injury. We tested whether long-term consumption of specific flavonoids (anthocyanins) included in normal food could render the heart of rats more resistant to myocardial infarction. Maize kernels that differed specifically in their accumulation of anthocyanins were used to prepare rodent food in which anthocyanins were either present or absent. Male Wistar rats were fed the anthocyanin-rich (ACN-rich) or the anthocyanin-free (ACN-free) diet for a period of 8 wk. Anthocyanins were significantly absorbed and detected in the blood and urine of only rats fed the ACN-rich diet. In Langendorff preparations, the hearts of rats fed the ACN-rich diet were more resistant to regional ischemia and reperfusion insult. Moreover, on an in vivo model of coronary occlusion and reperfusion, infarct size was reduced in rats that ate the ACN-rich diet than in those that consumed the ACN-free diet (P < 0.01). Cardioprotection was associated with increased myocardial glutathione levels, suggesting that dietary anthocyanins might modulate cardiac antioxidant defenses. Our findings suggest important potential health benefits of foods rich in anthocyanins and emphasize the need to develop anthocyanin-rich functional foods with protective activities for promoting human health