Cloning and sequence analysis of cDNA for a human homolog of eubacterial ATP-dependent Lon proteases

AbstractOverlapping cDNA clones containing mRNA for a putative Lon protease (LonHS) were isolated from cDNA libraries prepared from human brain poly(A)+ RNA. The determined nucleotide sequence contains a 2814-bp open reading frame with two potential initiation codons (positions 62–64 and 338–340). The 5'-terminal 337-nucleotide fragment of LonHS mRNA is highly enriched with G and C nucleotides and could direct synthesis of the LonHS N-terminal domain. More likely this region promotes initiation of protein synthesis from the second AUG codon in a cap-independent manner. The amino acid sequence initiated at the second AUG codon includes 845 residues, over 30% of which are identical to those of eubacterial Lon proteases. Residues of the ‘A’ and ‘B’ motifs of NTP-binding pattern and a plausible catalytic serine residue are conserved in LonHS. Northern blot analysis revealed LonHS mRNA in lung, duodenum, liver and heart, but not in thymus cells

Insulin-induced lipohypertrophy: clinical and ultrasound characteristics

Background: Lipohypertrophy is primary dermal complication of insulin therapy. The data on the prevalence of lipohypertrophy in diabetic subjects are inconsistent, that may be due to the lack of sensitivity and subjectivity of palpation as diagnostic technique. Meanwhile, the reliability of lipohypertrophy detection can be increased by ultrasound. Aims: to compare clinical and ultrasound characteristics and to determine the risk factors of insulin-induced lipohypertrophy in diabetic subjects. Materials and methods: We observed 82 patients, including 26 individuals with type 1 diabetes and 56 subjects with type 2 diabetes. Duration of insulin therapy varied from 3 months to 37 years (median 14 years). The sites of insulin injections were assessed by palpation and ultrasound. Visualization protocol included gray-scale densitometry, strain elastography, and 3D Doppler power ultrasound. Scaled evaluation of ultrasound sings was applied. Insulin injection technique was assessed by questionnaire. Serum levels of insulin antibodies were determined by ELISA. Results: Lipohypertrophy was revealed by palpation and ultrasound in 57 and 80 patients (70% and 98%) respectively. Total lipohypertrophy area, acoustic density and total ultrasound score showed weak positive correlations with daily insulin dose (r=0.3, r=0.3 and r=0.35, respectively, all p0.006). Patients receiving insulin analogues had smaller area of abdominal lipohypertrophy than those on human insulin (p=0.03). A positive correlation was found between abdominal lipohypertrophy area and mean postprandial glucose (r=0.35, p=0.001). A rare needle change and injections in lipohypertrophy sites were the most common deviations in insulin injection technique (70 and 47 subjects, 85% and 53% respectively). The levels of insulin antibodies showed no association with lipohypertrophy parameters. Conclusions: Patients with type 1 and type 2 diabetes demonstrate high prevalence of lipohypertrophy in insulin injection sites. Ultrasonography is more sensitive method of diagnostics of lipohypertrophy compared with palpation. Insulin-induced lipohypertrophy is associated with errors in injection technique and higher insulin doses

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The relationships between bone turnover markers and bone mineral density in postmenopausal type 2 diabetic women

Aim. To determine the relationships between bone remodelling markers and bone mineral density (BMD), metabolic parameters and total body composition (TBC) in postmenopausal women with type 2 diabetes (T2D). Materials and methods. The study included 140 women who were diagnosed with T2D more than five years prior. The control group included 20 postmenopausal nondiabetic women with normal BMD. The BMD and TBC parameters were assessed by dual X-ray absorptiometry. Based on their T-scores, T2D women were divided into the following groups: normal BMD (n = 50), osteopenia (n = 50) and osteoporosis (n = 40). Serum levels of bone formation markers [osteocalcin and type 1 C-terminal collagen propeptide (CICP), osteoprotegerin (an inhibitor of bone resorption), parathyroid hormone (PHT) and urinary excretion of C-terminal telopeptides of type 1 collagen (alpha-CrossLaps, or CTX-I; a bone resorption marker)] were determined by ELISA. Results. Osteocalcin levels were decreased in all groups of T2D women (all P 0.0002), without any differences between groups. Osteoprotegerin levels were reduced in all patient groups but was significantly lower in diabetic women with osteoporosis and osteopenia compared to those with normal BMD (P = 0.003 and P = 0.01, respectively). Women with osteoporosis had higher urinary CTX-I excretion than control and diabetic women with normal BMD (P = 0.01 and P = 0.01, respectively). CICP levels did not differ between groups. PHT concentrations were increased in diabetic women (P 0.0001), without any differences between groups. After multiple regression analysis, BMI, age and CTX-I excretion were all associated with lumbar BMD (R2 = 0.38, P = 0.0007), whereas age, BMI, osteoprotegerin levels and CTX-I excretion were all predictive of BMD at the proximal femur (R2 = 0.44, P = 0.00003). There was no relationship between bone remodelling markers and HbA1c, lipid metabolism or TBC. Conclusions. In postmenopausal T2D women, osteoporosis is associated with decreased serum osteoprotegerin levels and enhanced urinary CTX-I excretion. The data do not support the existence of an interrelationship between bone remodelling markers, metabolic parameters and TBC in postmenopausal women with T2D

Effect of Cu-, Zn-containing complex based on a porous matrix on fibroblast proliferation, apoptosis, necrosis and nitric oxide production

A comparative study of the effect of a matrix based on porous aluminum and a silicon organic polymer polydimethylsiloxane (PDMS) (А12O3@PDMS) and a matrix complex with copper and zinc sorbed on its surface (Cu@Zn- А12O3@PDMS) on the functional properties of fibroblasts has been carried out. Material and methods. The effect of the Cu@Zn- А12O3@PDMS complex and the matrix on the cell proliferative potential (MTT test), apoptosis, necrosis and the production of stable nitric oxide (NO) metabolites were studied in an in vitro experiment with a human embryo fibroblast cell line (HEF-15). Results. The compared samples of Cu@Zn-A12O3@PDMS and Al2O3@PDMS are similar in their physico-chemical properties. Study of HEF-15 functional potential indicates a higher level of cell proliferation and ability to produce NO after contact with the complex. There was no significant increase in apoptosis and necrosis of HEF-15 in the presence of samples of the complex and the carrier. Conclusion. The absence of a significant negative effect of the tested samples on the functional status of cells of the FEH-15 line (proliferation, apoptosis, necrosis, NO secretion) in vitro allows the use of the Cu@Zn-A12O3@PDMS complex for further analysis of its safety in experimental models of skin defects in animals

Carotid Artery Disease in Subjects with Type 2 Diabetes: Risk Factors and Biomarkers

Carotid atherosclerosis (CA) and, especially, carotid artery stenosis (CAS), are associated with a high risk of cardiovascular events in subjects with type 2 diabetes (T2D). In this study, we aimed to identify risk factors and biomarkers of subclinical CA and CAS in T2D individuals. High-resolution ultrasonography of carotid arteries was performed in 389 patients. Ninety-five clinical parameters were evaluated, including diabetic complications and comorbidities; antihyperglycemic, hypolipidemic, and antihypertensive therapy; indices of glycemic control and glucose variability (GV); lipid panels; estimated glomerular filtration rate (eGFR); albuminuria; blood cell count; and coagulation. Additionally, serum levels of calponin-1, relaxin, L-citrulline, and matrix metalloproteinase-2 and -3 (MMP-2, -3) were measured by ELISA. In univariate analysis, older age, male sex, diabetes duration, GV, diabetic retinopathy, chronic kidney disease, coronary artery disease, peripheral artery disease, and MMP-3 were associated with subclinical CA. In addition to these factors, long-term arterial hypertension, high daily insulin doses, eGFR, and L-citrulline were associated with CAS. In multivariate logistic regression, age, male sex, BMI, GV, and eGFR predicted CA independently; male sex, BMI, diabetes duration, eGFR, and L-citrulline were predictors of CAS. These results can be used to develop screening and prevention programs for CA and CAS in T2D subjects

Effect of mesenchymal stem cells cultivation with erythropoietin on their morphofunctional properties

Mesenchymal stem cells (MSCs) are considered as a promising tool for the treatment of inflammatory and degenerative diseases, but their effectiveness is associated with the survival of cells in an unfavorable microenvironment. Erythropoietin exhibits an anti-apoptotic effect for non-hematopoietic cells of the human and animal body. The aim of the study was to evaluate the morphofunctional properties of MSCs cultured with erythropoietin. Material and methods. MSCs were obtained from bone marrow aspirate of patients with chronic heart failure. The primary culture of MSCs-EPO was obtained by growth MSCs of the 4th passage for 7-8 days with erythropoietin at a dose of 33 IU/mL. The phenotype, differentiation in the adipogenic and osteogenic directions, proliferation, migration, and production of nitric oxide of MSCs were studied. Results and discussion. The study revealed that the cultivation of MSCs with erythropoietin contributes to an increase in the expression of the main surface molecules that characterize the cells belonging to true MSCs, possess to differentiated of MSCs into adipocytes and osteoblasts, a change in the expression levels of adhesion molecules, a decrease in the level of apoptosis, increased cell migration during the induction of oxidative stress and hyperglycemia. Conclusion. Cultivation of MSCs with erythropoietin increases the resistance of cells to adverse microenvironment factors

Clinical and metabolic factors associated with chronic low-grade inflammation in type 2 diabetic patients

Aim. To identify the clinical and metabolic factors associated with serum concentration of high sensitivity C-reactive protein (hsCRP) and α1-acid glycoprotein (α1-AGP) in patients with type 2 diabetes. Material and methods. The study involved 210 patients with type 2 diabetes. Levels of hsCRP and α1-AGP were measured using ELISA and compared with those of the control (30 healthy normal individuals). Levels of acute-phase proteins, fat mass and glucose variability (GV) were compared among demographic, anthropometric, biochemical and haematological parameters. The fat mass was determined with Dual-energy X-ray absorptiometry (DEXA). GV parameters including mean amplitude of glycaemic excursions, continuous overlapping net glycaemic action (CONGA), J-index, M-value and mean absolute glucose change (MAG) were derived from continuous glucose monitoring. Results. Levels of hsCRP and α1-AGP significantly increased (p 0.0001) in patients with diabetes compared with controls. hsCRP level positively correlated with total, truncal and android fat (r = 0.34, r = 0.28 and r = 0.31; respectively, p 0.00004). α1-AGP level showed no relationship with fat mass but positively correlated with mean glucose, CONGA, M-value and MAG (r = 0.38, r = 0.36, r = 0.43 and r = 0.4; respectively, p 0.0001). Patients with the highest hsCRP levels (75 percentile) had a greater body mass index (p = 0.00009) as well as truncal and android fat mass (p = 0.04 and p = 0.03, respectively) than those with the lowest levels (25 percentile). High level of α1-AGP (75 percentile) was associated with urinary albumin/creatinine ratio (p = 0.01) and GV indices (M-value: p = 0.02, MAG: p = 0.04). Conclusions. Levels of acute-phase proteins (hsCRP and α1-AGP) increased in patients with type 2 diabetes. Levels of hsCRP were associated with fat mass; meanwhile, α1-AGP levels were associated with short-time GV in these patients. The results lend support to the notion that both obesity and enhanced GV are involved in the development of chronic low-grade inflammation associated with type 2 diabetes

Phenotype of bone-marrow mononuclear cells before and after short-time precondition with erythropoietin from patients with ischemic heart failure

Aim ― To reveal the results of the condition of the bone marrow mononuclear cells (BM-MNCs) with erythropoietin (Epo). Material and Methods ― There were 30-ty patients with coronary artery disease (CAD) enrolled in this study during 2016-2017 years. 95% were men. All were in angina NYHA class II-III. Hypertension presented in 90%, peripheral atherosclerosis in 60%. BM-MNCs were obtained by centrifugation of bone marrow aspirate on Ficoll-Paque, washed, and then preconditioned with Epo. Phenotype, cell cycle, cell death, proliferation, migration, and tube formation before, and after precondition BM-MNCs with Epo were done. Results ― In this study, we observed the presence in cellular graft of the hematopoietic stem cells (HSCs), and endothelial progenitor cells (EPCs) at the different stage of maturation/differentiation, and mesenchymal stem cells (MSCs). Precondition BM-MNCs with Epo increased number of HSCs carrying erythropoietin receptor (EpoR), and EPCs carrying CD184. Also, Epo detained СВ34+ cells in a rest phase of cell cycle (G0G1). Condition media from BM-MNCs treated with Epo augment tube formation and wound healing by EA.hy 929. Conclusion ― Epo in vitro increased number of the stem cells carrying EpoR and CD184, and increased accumulation of CD34+ cell in G0G1 phase of cell cycle, and induced production of proangiogenic factor by BM-MNCs. Further investigation is needed to assess the type of cytokines produced by BM-MNCs after condition with Epo

Reversed Corneal Fibroblasts Therapy Restores Transparency of Mouse Cornea after Injury

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury