Innate Immunity Alterations in Type 2 Diabetes Mellitus: Understanding Infection Susceptibility.

Diabetes is a chronic disease characterized by marked alterations in the metabolism of glucose and by high concentrations of glucose in the blood due to a decreased insulin production or resistance to the action of this hormone in peripheral tissues. The International Diabetes Federation estimates a global incidence of diabetes of about 10% in the adult population (20 - 79 years old), some 430 million cases reported worldwide in 2018. It is well documented that people with diabetes have a higher susceptibility to infectious diseases and therefore show higher morbidity and mortality compared to the non-diabetic population. Given that the innate immune response plays a fundamental role in protecting against invading pathogens through a myriad of humoral and cellular mechanisms, the present work makes a comprehensive review of the innate immune alterations in patients with type 2 diabetes mellitus (T2D) as well as a brief description of the molecular events leading or associated to such conditions. We show that in these patients a compromised innate immune response increases susceptibility to infections.

Methotrexate reduces keratinocyte proliferation, migration and induces apoptosis in HaCaT keratinocytes in vitro and reduces wound closure in Skh1 mice in vivo.

The aim of the present work was to evaluate MTX treatment (0.1, 1 and 10 µg mL-1) in vitro in order to characterize its effects on cell proliferation alterations in cell cycle of HaCaT keratinocytes and wound healing in a Skh1 mice treated with MTX (low doses 30 mg kg-1, high doses 200 mg kg-1 and repeated doses at 1.5 mg kg-1). We analyzed the cytotoxic effect of methotrexate by a resazurin assay. The effects in the proliferation, cell cycle and apoptosis of HaCaT cells were analyzed by flow cytometry. The effects of MTX on wound healing in vivo were also analyzed. A trend toward reduction in the resazurin assay was found (p > 0.05). Reduced proliferation was also identified in a clonogenic assay and a CFSE assay (p < 0.05) due to the MTX treatment. A reduction in the G2/M and S phases was observed accompanied by apoptosis induction with increased sub G0 phase and annexin V FITC staining. Effect of MTX was evidenced in vivo on the wound closure process after day 10 (p < 0.05) with alterations in tissue architecture and remodeling. There is a marked effect of MTX on wound healing in vivo in Skh1 mice with implications for long-term therapy and surgical interventions.

Lipid Metabolism Alterations in a Rat Model of Chronic and Intergenerational Exposure to Arsenic.

Chronic exposure to arsenic (As), whether directly through the consumption of contaminated drinking water or indirectly through the daily intake of As-contaminated food, is a health threat for more than 150 million people worldwide. Epidemiological studies found an association between chronic consumption of As and several pathologies, the most common being cancer-related disorders. However, As consumption has also been associated with metabolic disorders that could lead to diverse pathologies, such as type 2 diabetes mellitus, nonalcoholic fatty liver disease, and obesity. Here, we used ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization/quadrupole time-of-flight mass spectrometry (ESI-QToF) to assess the effect of chronic intergenerational As exposure on the lipid metabolism profiles of serum from 4-month-old Wistar rats exposed to As prenatally and also during early life in drinking water (3 ppm). Significant differences in the levels of certain identified lysophospholipids, phosphatidylcholines, and triglycerides were found between the exposed rats and the control groups, as well as between the sexes. Significantly increased lipid oxidation determined by the malondialdehyde (MDA) method was found in exposed rats compared with controls. Chronic intergenerational As exposure alters the rat lipidome, increases lipid oxidation, and dysregulates metabolic pathways, the factors associated with the chronic inflammation present in different diseases associated with chronic exposure to As (i.e., keratosis, Bowen's disease, and kidney, liver, bladder, and lung cancer).

Medicinal Plant Extracts and Their Use As Wound Closure Inducing Agents.

Skin insult and damage start a complex healing process that involves a myriad of coordinated reactions at both the cellular and molecular level occurring simultaneously. These processes can be divided into that of cell migration and tissue remodeling of the wound. In addition, it is well known that deep wounds that derive from surgical procedures need a multidisciplinary approach to have a successful wound healing process. Recently, there has been a renowned interest in the identification of active compounds derived from ornamental, edible, and wild plants being used in the cosmetic and skin product industry. Recent reports suggest that active components of several plants such as Propolis and Aloe vera could be used to induce the process of wound healing and tissue regeneration and reducing therefore the time to complete wound closure. Other plant species such as Achillea millefolium or Salvia officinalis have anti-inflammatory properties and promote cellular proliferation contributing to faster tissue regeneration. It has been described that Malva sylvestris influences the formation of fibrosis-free granulation tissue in the skin. Recent observations suggest that Casearia sylvestris induces the angiogenic process. These effects have been evaluated in cell lines, different animal models, and some in randomized clinical trials. In this review we summarize the evidence of plant extracts and their active components (when known) in the acceleration of the wound closure process and tissue repair.

Myeloid-Derived Suppressor Cells Show Different Frequencies in Diabetics and Subjects with Arterial Hypertension.

Type 2 diabetes mellitus (DM2) is strongly associated with other comorbidities such as obesity, atherosclerosis, and hypertension. Obesity is associated with sustained low-grade inflammatory response due to the production of proinflammatory cytokines. This inflammatory process promotes the differentiation of some myeloid cells, including myeloid-derived suppressor cells (MDSCs). In this study, two groups of individuals were included: DM2 patients and non-DM2 individuals with similar characteristics. Immunolabeling of CD15+ CD14- and CD33+ HLA-DR-/low was performed from whole peripheral blood, and samples were analyzed by flow cytometry, and frequencies of MDSCs and the relationship of these with clinical variables, cytokine profile (measured by cytometric bead array), and anthropometric variables were analyzed. The frequency of CD33+ HLA-DR-/low MDSCs (that produce IL-10 and TGF-ß, according to an intracellular detection) is higher in patients with DM2 (P < 0.05), and there is a positive correlation between the frequency of CD15+ CD14- and CD33+ HLA-DR-/low MDSC phenotypes. DM2 patients have an increased concentration of serum IL-5 (P < 0.05). Also, a negative correlation between the frequency of CD15+ CD14- MDSCs and LDL cholesterol was found. Our group of DM2 patients have an increased frequency of mononuclear MDSC CD33+ HLA-DR-/low that produce TGF-ß and IL-10. These cytokines have been associated with immune modulation and reduced T cell responses. DM2 and non-DM2 subjects show a similar cytokine profile, but the DM2 patients have an increased concentration of IL-5.

Correction: Metformin Induces Cell Cycle Arrest, Reduced Proliferation, Wound Healing Impairment In Vivo and Is Associated to Clinical Outcomes in Diabetic Foot Ulcer Patients.

[This corrects the article DOI: 10.1371/journal.pone.0150900.].

Metformin Induces Cell Cycle Arrest, Reduced Proliferation, Wound Healing Impairment In Vivo and Is Associated to Clinical Outcomes in Diabetic Foot Ulcer Patients.

BACKGROUND: Several epidemiological studies in diabetic patients have demonstrated a protective effect of metformin to the development of several types of cancer. The underlying mechanisms of such phenomenon is related to the effect of metformin on cell proliferation among which, mTOR, AMPK and other targets have been identified. However, little is known about the role that metformin treatment have on other cell types such as keratinocytes and whether exposure to metformin of these cells might have serious repercussions in wound healing delay and in the development of complications in diabetic patients with foot ulcers or in their exacerbation. MATERIAL AND METHODS: HaCaT Cells were exposed to various concentrations of metformin and cell viability was evaluated by a Resazurin assay; Proliferation was also evaluated with a colony formation assay and with CFSE dilution assay by flow cytometry. Cell cycle was also evaluated by flow cytometry by PI staining. An animal model of wound healing was used to evaluate the effect of metformin in wound closure. Also, an analysis of patients receiving metformin treatment was performed to determine the effect of metformin treatment on the outcome and wound area. Statistical analysis was performed on SPSS v. 18 and GraphPad software v.5. RESULTS: Metformin treatment significantly reduces cell proliferation; colony formation and alterations of the cell cycle are observed also in the metformin treated cells, particularly in the S phase. There is a significant increase in the area of the wound of the metformin treated animals at different time points (P<0.05). There is also a significant increase in the size and wound area of the patients with diabetic foot ulcers at the time of hospitalization. A protective effect of metformin was observed for amputation, probably associated with the anti inflammatory effects reported of metformin. CONCLUSIONS: Metformin treatment reduces cell proliferation and reduces wound healing in an animal model and affects clinical outcomes in diabetic foot ulcer patients. Chronic use of this drug should be further investigated to provide evidence of their security in association with DFU.

Novel approaches to tuberculosis prevention: DNA vaccines.

It is estimated that there are approximately eight million new cases of active tuberculosis (TB) worldwide annually. There is only 1 vaccine available for prevention: bacillus Calmette-Guérin (BCG). This has variable efficacy and is only protective for certain extrapulmonary TB cases in children, therefore new strategies for the creation of novel vaccines have emerged. One of the promising approaches is the DNA vaccine, used as a direct vaccination or as a prime-boost vaccine. This review describes the experimental data obtained during the design of DNA vaccines for TB.

Prime-boost BCG vaccination with DNA vaccines based in ß-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model.

The World Health Organization (WHO) has estimated that there are about 8 million new cases annually of active Tuberculosis (TB). Despite its irregular effectiveness (0-89%), the Bacillus Calmette-Guérin) BCG is the only vaccine available worldwide for prevention of TB; thus, the design is important of novel and more efficient vaccination strategies. Considering that ß-defensin-2 is an antimicrobial peptide that induces dendritic cell maturation through the TLR-4 receptor and that both ESAT-6 and Ag85B are immunodominant mycobacterial antigens and efficient activators of the protective immune response, we constructed two DNA vaccines by the fusion of the gene encoding ß-defensin-2 and antigens ESAT6 (pDE) and 85B (pDA). After confirming efficient local antigen expression that induced high and stable Interferon gamma (IFN-γ) production in intramuscular (i.m.) vaccinated Balb/c mice, groups of mice were vaccinated with DNA vaccines in a prime-boost regimen with BCG and with BCG alone, and 2 months later were challenged with the mild virulence reference strain H37Rv and the highly virulent clinical isolate LAM 5186. The level of protection was evaluated by survival, lung bacilli burdens, and extension of tissue damage (pneumonia). Vaccination with both DNA vaccines showed similar protection to that of BCG. After the challenge with the highly virulent Mycobacterium tuberculosis strain, animals that were prime-boosted with BCG and then boosted with both DNA vaccines showed significant higher survival and less tissue damage than mice vaccinated only with BCG. These results suggest that improvement of BCG vaccination, such as the prime-boost DNA vaccine, represents a more efficient vaccination scheme against TB.

Pharmacological inhibition of N-methyl d-aspartate receptor promotes secretion of vascular endothelial growth factor in müller cells: effects of hyperglycemia and hypoxia.

PURPOSE: To characterize the effect of glutamate receptor activation/inhibition on the secretion of vascular endothelial growth factor (VEGF) in retina-specific glial (Müller) cells under experimental conditions of hyperglycemia and hypoxia, two intrinsic pathologic conditions of diabetic retinopathy. METHODS: Purified rat Müller cells were grown in normoglycemic or diabetic-like, hyperglycemic (5.6 or 25 mM glucose, respectively) culture media under normoxic or chemically-induced hypoxic conditions. After treatments, cells were incubated with glutamate receptor agonists and antagonists and VEGF secretion was determined by ELISA. Cell viability was determined by Lactate Dehydrogenase (LDH) secretion-assay and Ki67 immunocytochemistry. Activation of the Akt signal transduction pathway was assessed by western blot using antibodies against phosphorylated Akt. The bio-activity of the secreted VEGF was analyzed by western blot with a phospho-VEGF receptor 2 specific antibody and an in vitro endothelial cell proliferation assay. RESULTS: In control (normoglycemic/normoxic) conditions, N-methyl-D-aspartate receptor (NMDA-R) antagonists MK801 and AP-5 increased secretion of VEGF from Müller cells, and this was not observed after AMPA/kainate receptor blockade. VEGF secretion after NMDA-R antagonists was independent of cell proliferation or cell lysis and it was maintained in cultures grown in hyperglycemia or hypoxia. However, under hyperglycemic and hypoxic conditions, the observed phenomenon was impaired. We also determined that NMDA-R blockade causes a rapid and sustained increase on Akt phosphorylation, a signaling molecule that has been previously linked to VEGF expression. Müller cell-derived VEGF was capable of promoting VEGF receptor 2 phosphorylation and proliferation of endothelial cells. CONCLUSIONS: Our results show that NMDA-R exert a tonic inhibition on VEGF secretion in cultures of rat purified Müller cells and indicate that in healthy retina, glutamatergic stimulation could potentially contribute to the protective antiangiogenic role of Müller glia. We suggest that conditions present on diabetic retinopathy could cause malfunction of control points on VEGF synthesis on Müller cells.