Increased frequency of CD14+HLA-DR-/low cells in type 2 diabetes patients with poor glycemic control.

AIMS: Type 2 diabetes (T2DM) is associated with alterations of the immune response and T2DM patients have an increased risk for infections and certain sorts of cancers. Although CD14+HLA-DR-/low cells have emerged as important mediators of immunosuppression in several pathologies, including cancer and non-malignant diseases, the presence of these cells in T2DM is not fully characterized. METHODS: In this study, we evaluated the frequency of CD14+HLA-DR-/low cells in non-obese T2DM patients and their association with glycemic control. Peripheral blood mononuclear cells were isolated from healthy controls (HC, n = 24) and non-obese T2DM patients (n = 25), the population was evaluated by flow cytometry, and an analysis of correlation between cell frequencies and clinical variables was performed. RESULTS: CD14+HLA-DR-/low monocytes were expanded in patients with T2DM compared to HC regardless of weight. Among the subjects with T2DM, the frequency of CD14+HLA-DR-/low was higher in patients with poor glycemic control (HbA1c > 9%) compared to those with better glycemic control (HbA1c < 9%) and, positively correlated with the years since the diagnosis of T2DM, the age of the patients and the glycemic index. CONCLUSIONS: An increased frequency of CD14+HLA-DR-/low cells in the blood of T2DM patients was recorded. The influence of hyperglycemia seems to be independent of obesity, but related to glycemic control and age.

Type 2 diabetes mellitus metabolic control correlates with the phenotype of human monocytes and monocyte-derived macrophages.

AIMS: Monocytes and macrophages express cell-surface markers indicative of their inflammatory and activation status. In this study, we investigated whether these markers are affected or correlated in non-obese T2D subjects, or glycemic/metabolic control variables. METHODS: Clinical data was recorded, and peripheral blood drawn from T2D patients (n = 28) and control subjects (n = 27). Isolated monocytes were evaluated by flow cytometry for the expression of CD14, CD16, and the phenotypic markers for the different states of activation spectrum, such as pro-inflammatory (M1) (HLA-DR, CD86), anti-inflammatory/pro-resolving (M2) (CD163, CD206, MERTK, PD-L1) and metabolically-activated (MMe) (CD36, ABCA-1). From a subset of individuals, monocytes-derived macrophages (MDM) were obtained and evaluated for phenotypic markers. A correlation analysis was performed between the clinical variables and the marker expression. RESULTS: The frequency of CD14++CD16- monocytes was lower in T2D patients and it correlates negatively with poor control in glycemic and metabolic variables. T2D monocytes expressed lower levels of HLA-DR, CD86, PD-L1, and CD163, which correlated negatively with poor metabolic control. In MDM from T2D patients, HLA-DR, CD86 and CD163 expression was lower and it inversely correlated with deficient glycemic or metabolic control parameters. CONCLUSION: The glycemic/metabolic control associated with T2D influences monocyte and MDM phenotypes toward an immune-suppressive phenotype.

Identification of putative miRNA biomarkers in early rheumatoid arthritis by genome-wide microarray profiling: A pilot study.

Despite the existing research, the etiology of rheumatoid arthritis (RA), an autoimmune disease remains poorly understood with early and accurate diagnosis difficult to achieve. MicroRNAs (miRNAs) play an important role in biological processes as modulators of transcription and translation. Previous studies have demonstrated a downregulation of several genes in early RA stages and in addition, miRNAs may serve as early biomarkers of subclinical changes in early RA. When comparing the four groups (ANOVA P < 0.01, fold change > 4), we found 253 differentially expressed miRNAs. Of these, 97 miRNAs were identified as overexpressed in early rheumatoid arthritis. The validation of miRNA microarray expression was performed in a set by RT-qPCR and showed strong agreement with microarray expression data. The putative targets of overexpressed microRNAs in early RA were significantly enriched in apoptosis, tolerance loss and Wnt pathways. Moreover, ROC analysis showed values of AUC 0.76 and P < 0.05 for miR 361-5p, identifying this miRNA as a potential biomarker of disease. We identified specific microRNAs associated with early rheumatoid arthritis and proposed them as early biomarkers of disease. Our results provide novel insight into immune disease physiopathology and describe unreported microRNAs in RA with potential for clinical use.

Dental, periodontal and salivary conditions in diabetic children associated with metabolic control variables and nutritional plan adherence.

AIM: Diabetes mellitus is a chronic disease that has manifestations other than alterations in endocrine regulation or in metabolic pathways. Several diseases of the oral cavity have been associated with diabetes mellitus type 1 and 2 in young people according to their evolution. Scarce information exists regarding the role of diabetes and its association with the oral health status in paediatric diabetic patients. The aims of the study were to assess the quality of saliva, saliva acidogenicity, dental caries experience, fluorosis and periodontal status in diabetic patients and to evaluate their relationship with metabolic control variables and nutritional plan adherence. MATERIALS AND METHODS: The study population consisted of 60 paediatric patients with both types of diabetes mellitus. Saliva testing included stimulated flow, pH (using pH indicator strips), buffer capacity and Snyder's Test. DMFT/dmft and dental caries experience were determined on the basis of ICDAS II codes. The periodontal status was assessed by PI and GI and fluorosis by FI. Nutritional plan adherence was established from the subscale "Dietary Control" of the Diabetes Self-Management Profile questionnaire. Medical Data was retrieved from the clinical registers in the Diabetic Clinic. RESULTS: We describe the main characteristics of the oral cavity related variables of our population that might guide the clinical practice in similar settings; we found a dmft/DMFT of 1.71 ± 1.74 and 0.64 ± 1.03, PI of 1.91 ± 0.75, GI of 0.50 ± 0.56 and a fluorosis prevalence of 61%. We identified several correlated variables, which indicate strong associations between the nutritional habits of the patients and co-occurrence of oral cavity physiopathological alterations. Several correlations were found between acidogenic activity of the saliva (Snyder Test) and the percentage of adherence to the nutritional plan and to the dmft index. Furthermore, a significant correlation between the buffering capacity of the saliva and the glycemic control of the participants was found. Neither an association nor a difference among means was found between treatment regime and the plaque index. CONCLUSION: The results of the present study concluded that there was a significant relationship between diabetes mellitus and an increased prevalence of oral cavity related diseases in the paediatric population. These are also associated with a poor adherence to the nutritional plan.

Transcriptional signature associated with early rheumatoid arthritis and healthy individuals at high risk to develop the disease.

BACKGROUND: Little is known regarding the mechanisms underlying the loss of tolerance in the early and preclinical stages of autoimmune diseases. The aim of this work was to identify the transcriptional profile and signaling pathways associated to non-treated early rheumatoid arthritis (RA) and subjects at high risk. Several biomarker candidates for early RA are proposed. METHODS: Whole blood total RNA was obtained from non-treated early RA patients with <1 year of evolution as well as from healthy first-degree relatives of patients with RA (FDR) classified as ACCP+ and ACCP- according to their antibodies serum levels against cyclic citrullinated peptides. Complementary RNA (cRNA) was synthetized and hybridized to high-density microarrays. Data was analyzed in Genespring Software and functional categories were assigned to a specific transcriptome identified in subjects with RA and FDR ACCP positive. Specific signaling pathways for genes associated to RA were identified. Gene expression was evaluated by qPCR. Receiver operating characteristic (ROC) analysis was used to evaluate these genes as biomarkers. RESULTS: A characteristic transcriptome of 551 induced genes and 4,402 repressed genes were identified in early RA patients. Bioinformatics analysis of the data identified a specific transcriptome in RA patients. Moreover, some overlapped transcriptional profiles between patients with RA and ACCP+ were identified, suggesting an up-regulated distinctive transcriptome from the preclinical stages up to progression to an early RA state. A total of 203 pathways have up-regulated genes that are shared between RA and ACCP+. Some of these genes show potential to be used as progression biomarkers for early RA with area under the curve of ROC > 0.92. These genes come from several functional categories associated to inflammation, Wnt signaling and type I interferon pathways. CONCLUSION: The presence of a specific transcriptome in whole blood of RA patients suggests the activation of a specific inflammatory transcriptional signature in early RA development. The set of overexpressed genes in early RA patients that are shared with ACCP+ subjects but not with ACCP- subjects, can represent a transcriptional signature involved with the transition of a preclinical to a clinical RA stage. Some of these particular up-regulated and down-regulated genes are related to inflammatory processes and could be considered as biomarker candidates for disease progression in subjects at risk to develop RA.

Regulatory T-cell subsets in response to specific Mycobacterium tuberculosis antigens in vitro distinguish among individuals with different QTF and TST reactivity.

Regulatory T cells (Tregs), a subset of CD4+ T cells related with immune regulation, have been associated with active and latent tuberculosis infection (LTBI). Treg frequencies were evaluated by multicolor flow cytometry (FC) in peripheral blood mononuclear cells (PBMCs) stimulated with mycobacterial antigens ESAT-6, CFP-10, and TB7.7 to assess their capacity to distinguish subjects with different reactivity to the QuantiFERON-TB® Gold In-Tube (QFT-IT) test and the tuberculin skin test (TST). Increased frequencies of CD4+CD25highCD39+ cells were found for the [TST+, QTF+] compared with the [TST+, QTF-] group. Also, higher frequencies were observed for the [TST+, QTF+] compared with the [TST+, QTF-] and [TST-, QTF-] groups in CD4+CD25highFoxp3+ and CD4+CD25highCD39+Foxp3+ populations. Receiver operating characteristics (ROC curve) analysis confirmed these discriminating results. QFT-IT and TST quantitative values correlated with several Treg population frequencies.

Production of antimicrobial peptides is preserved in aging.

There is an increased susceptibility to infections during elderly, mainly because of the decreased efficacy of adaptive immunity to contain microorganisms. Albeit most of the elderly adults develop this deficiency in adaptive immunity only a minor percentage of them developed recurrent infectious diseases, thus innate immunity represents an important barrier to avoid infections in this group of aged people. Since antimicrobial peptides are important molecules of innate immunity in the study we sought to determine whether healthy aging correlates with a proper antimicrobial production. Our results by ELISA and flow cytometry showed that healthy elder individuals produce significant amounts of both cathelicidin and ß-defensin-2 (hBD-2) comparable with those found in healthy young individuals. Our results suggest that during healthy aging the maintenance of the antimicrobial peptide innate immune response may be responsible for the protection against infectious diseases.

Induction of ß-defensins by l-isoleucine as novel immunotherapy in experimental murine tuberculosis.

Tuberculosis is a worldwide health problem, and multidrug-resistant (MDR) and extensively multidrug-resistant (XMDR) strains are rapidly emerging and threatening the control of this disease. These problems motivate the search for new treatment strategies. One potential strategy is immunotherapy using cationic anti-microbial peptides. The capacity of l-isoleucine to induce beta-defensin expression and its potential therapeutic efficiency were studied in a mouse model of progressive pulmonary tuberculosis. BALB/c mice were infected with Mycobacterium tuberculosis strain H37Rv or with a MDR clinical isolate by the intratracheal route. After 60 days of infection, when disease was in its progressive phase, mice were treated with 250 µg of intratracheal l-isoleucine every 48 h. Bacillary loads were determined by colony-forming units, protein and cytokine gene expression were determined by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively, and tissue damage was quantified by automated morphometry. Administration of l-isoleucine induced a significant increase of beta-defensins 3 and 4 which was associated with decreased bacillary loads and tissue damage. This was seen in animals infected with the antibiotic-sensitive strain H37Rv and with the MDR clinical isolate. Thus, induction of beta-defensins might be a potential therapy that can aid in the control of this significant infectious disease.

Kinetics and cellular sources of cathelicidin during the course of experimental latent tuberculous infection and progressive pulmonary tuberculosis.

In spite of advances in immunology on mycobacterial infection, there are few studies on the role of anti-microbial peptides in tuberculosis. The cathelin-related anti-microbial peptide (CRAMP) is the only cathelicidin isolated from mice. In this work we investigated the cellular sources and the production kinetics of this molecule during experimental tuberculosis, using two well-characterized models of latent or chronic infection and progressive disease. The lung of non-infected control mice expressed CRAMP at very low levels. In both models of experimental tuberculosis the main cells immunolabelled for CRAMP were bronchial epithelial cells, macrophages and pneumocytes types II and I. After intratracheal infection with a high bacilli dose (H37Rv strain) in Balb/c mice to produce progressive disease, a high CRAMP gene expression was induced showing three peaks: very early after 1 day of infection, at day 21 when the peak of protective immunity in this model is raised, and at day 28 when the progressive phase starts and the immunoelectronmicroscopy study showed intense immunolabelling in the cell wall and cytoplasm of intracellular bacilli, as well as in cytoplasmic vacuoles. Interestingly, at day 60 post-infection, when advanced progressive disease is well established, characterized by high bacillary loads and extensive tissue damage, CRAMP gene expression decreased but strong CRAMP immunostaining was detected in vacuolated macrophages filled with bacilli. Thus, cathelicidin is highly produced during experimental pulmonary tuberculosis from diverse cellular sources and could have significant participation in its pathogenesis.