Evaluation of mobilized peripheral stem cells according to CD34 and aldehyde dehydrogenase expression and effect of SSClo ALDHbr cells on hematopoietic recovery

Background aimsWe evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte–colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m² once daily, intravenously on day 1). We also investigated the relationship between the number of SSC^lo CD45^dim CD34^hi cells, SSC^lo ALDH^br cells and engraftment.MethodsThirty patients (20 males and 10 females), who were candidates for autologous peripheral blood stem cell transplantation, were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n% = 14), Hodgkin's lymphoma (n% = 7), non-Hodgkin's lymphoma (n% = 2), acute myloid leukemia (n% = 2), chronic lymphocytic leukemia (n% = 1) and germ cell testis tumor (n% = 1).ResultsNumbers of SSC^lo CD45^dim CD34^hi cells and SSC^lo ALDH^br cells were highly correlated in both peripheral blood and apheresis products (P < 0.001). We could not find a relationship between the transplanted SSC^lo CD45^dim CD34^hi cell dose or SSC^lo ALDH^br cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC^lo CD45^dim CD34^hi cells were 5.40 × 10⁶/kg for neutrophil recovery and 7.22 × 10⁶/kg for platelet recovery. The optimal thresholds for SSC^lo ALDH^br cells were 6.53 × 10⁶/kg for neutrophil recovery and 8.72 × 10⁶/kg platelet recovery.ConclusionsAccording to our data, numbers of SSC^lo ALDH^br cells are in very good agreement with numbers of SSC^lo CD45^dim CD34^hi cells and can be a predictor of stem cell mobilization.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use

Background aimsThe manufacture of multipotential stromal cell (MSC)-based products is costly; therefore, a rapid evaluation of bone marrow (BM) ‘quality’ with respect to MSC content is desirable. The aim of this study was to develop a rapid single-platform assay to quantify MSC in BM aspirates.MethodsAspirated MSC were enumerated using the CD45^?/low CD271^bright phenotype and AccuCheck counting beads and compared with a classic colony-forming unit–fibroblast (CFU-F) assay. The phenotype of CD45^?/low CD271^bright cells was defined using a range of MSC (CD73, CD105, CD90) and non-MSC (CD31, CD33, CD34, CD19) markers. The effect of aspirated BM volume on MSC yield was also determined.ResultsCD45^?/low CD271^bright cells had a classic MSC phenotype (CD73⁺ CD105⁺ CD90⁺ ). Their numbers correlated positively with CFU-F counted manually (R = 0.81, P < 0.001) or using automatic measurements of surface area occupied by colonies (R = 0.66, P < 0.001). Simultaneous enumeration of CD34 ⁺ cells revealed donor variability ranges compatible with standard International Society of Hematotherapy and Graft Engineering (ISHGE) protocols. Aspirating larger marrow volumes gave a significant several-fold reduction in the frequency of CFU-F and CD45^?/low CD271^bright cells per milliliter. Therefore aspirated MSC yields can be maximized through a standardized, low-volume harvesting technique.ConclusionsAbsolute quantification of CD45^?/low CD271^bright cells was found to be a reliable method of predicting CFU-F yields in BM aspirates. This rapid (< 40 min) procedure could be suitable for intra-operative quality control of BM aspirates prior to volume reduction/direct injection in orthopedics. In the production of culture-expanded MSC, this assay could be used to exclude samples containing low numbers of MSC, resulting in improved consistency and quality of manufactured MSC batches.     

Viability and engraftment of hematopoietic progenitor cells after long-term cryopreservation: effect of diagnosis and percentage dimethyl sulfoxide concentration

Background aimsWe carried out a retrospective analysis of viability by diagnosis and dimethyl sulfoxide (DMSO) concentration in patients who had undergone autologous transplants using hematopoietic progenitor cells (HPC) after long-term storage (up to 17.8 years).MethodsViability was tested using flow cytometry for HPC that were harvested and preserved using a controlled rate freezer and 5% or 10% DMSO with human serum albumin, then stored in liquid nitrogen. Data from 262 samples were analyzed (249 myeloma patients and 13 other diagnoses): 100 consecutively thawed samples with a storage time of <1 year (all 10% DMSO), 50 consecutive samples stored for 1–4.9 years (10% DMSO), 50 samples stored for 5–9 years (5% DMSO) and all samples stored and used for transplant after >9 years (60 samples, 5% DMSO; two samples, 10% DMSO).ResultsNo statistically significant difference in viability between the 5% DMSO and 10% DMSO groups was observed (P = 0.08), so the 1–4.9 years and 5–9 years were combined and the three groups (<1 year, 1–9 years and >9 years) were compared using an anova test. There was no difference in viability based on cryostorage period (P = 0.23) or between myeloma and other diagnoses (P = 0.45). No difference was seen in time to White blood cell (WBC) engraftment (P = 0.10) or to platelet engraftment between groups (P = 0.52).ConclusionsThese data suggest that long-term storage in 5% DMSO and human serum albumin is safe.     

Induction of abortion with aglepristone significantly changed the expression of progesterone and estrogen receptors in canine endometrial stromal cells

In the present study, resorption/abortion was induced between days 25 and 45 of gestation with aglepristone (group IRA, n = 10). The aim was to observe the change in the distribution of progesterone (PR) and estrogen receptors (ER), in comparison to a group of spontaneous resorptions/abortions (group SRA, n = 5), and a further group of normal healthy pregnant animals, ovariohysterectomized between days 25 and 45 of gestation (controls, n = 7). The receptors were assessed by means of immunohistochemistry (IHC) and RT-PCR, at the placental and interplacental sites of the uterine horn as well as in the corpus uteri. Significant differences were observed between the controls on one side and the groups of resorption/abortion on the other side. The total scores of the progesterone receptors (TPR) in the placental and interplacental part of the uterine horn, was significantly lower in the endometrial stromal cells (ESC) of the control group than in those of the SRA- and IRA-group, respectively (placenta: 5.8 vs. 6.5 and 6.7, p < 0.01; interplacental sites: 5.6 vs. 6.6 and 6.6, p < 0.05). In contrary, the total scores of the estrogen receptors (TER) at interplacental sites and the corpus uteri, respectively, was significantly higher in the myometrial smooth muscle cells (MSMC) and the ESC (p < 0.05) of the controls. We therefore conclude, that the here observed differences between groups point to an up-regulation of TPR- and a down-regulation of TER-scores in endometrial stromal cells at different uterine sites during resorption/abortion, which indicates a special role of these cells.     

Investigating micronucleus assay applicability for prediction of normal tissue intrinsic radiosensitivity in gynecological cancer patients

BackgroundPelvic organs morbidity after irradiation of cancer patients remains a major problem although new technologies have been developed and implemented. A relatively simple and suitable method for routine clinical practice is needed for preliminary assessment of normal tissue intrinsic radiosensitivity. The micronucleus test (MNT) determines the frequency of the radiation induced micronuclei (MN) in peripheral blood lymphocytes, which could serve as an indicator of intrinsic cell radiosensitivity.AimTo investigate a possible use of the micronucleus test (MNT) for acute radiation morbidity prediction in gynecological cancer patients.Materials and methodsForty gynecological cancer patients received 50 Gy conventional external pelvic irradiation after radical surgery. A four-field “box” technique was applied with 2D planning. The control group included 10 healthy females.Acute normal tissue reactions were graded according to NCI CTCAE v.3.0. From all reaction scores, the highest score named “summarized clinical radiosensitivity” was selected for a statistical analysis.MNT was performed before and after in vitro irradiation with 1.5 Gy. The mean radiation induced frequency of micronuclei per 1000 binucleated cells (MN/1000) and lymphocytes containing micronuclei per 1000 binucleated cells (cells with MN/1000) were evaluated for both patients and controls.An arbitrary cut off value was created to pick up a radiosensitive individual: the mean value of spontaneous frequency of cells with MN/1000 ± 2SD, found in the control group.ResultsBoth mean spontaneous frequency of cells with MN/1000 and MN/1000 were registered to be significantly higher in cancer patients compared to the control group (t = 2.46, p = 0.02 and t = 2.51, p = 0.02). No statistical difference was registered when comparing radiation induced MN frequencies between those groups.Eighty percent (32) of patients developed grade 2 summarized clinical radiosensitivity, with great variations in MNT parameters. Only three patients with grade 2 “summarized clinical radiosensitivity” had values of cells with MN/1000 above the chosen radiosensitivity threshold.ConclusionThe present study was not able to confirm in vitro MNT applicability for radiosensitivity prediction in pelvic irradiation.     

Neoadjuvant chemotherapy with or without oxaliplatin after short-course radiotherapy in high-risk rectal cancer: A subgroup analysis from a prospective study

AimTo evaluate the role of oxaliplatin in neoadjuvant chemotherapy delivered after short-course irradiation.BackgroundUsing oxaliplatin in the above setting is uncertain.Patients and methodsA subgroup of 136 patients managed by short-course radiotherapy and 3 cycles of consolidation chemotherapy within the framework of a randomised study was included in this post-hoc analysis. Sixty-seven patients received FOLFOX4 (oxaliplatin group) while oxaliplatin was omitted in the second period of accrual in 69 patients because of protocol amendment (fluorouracil-only group).ResultsGrade 3+ acute toxicity from neoadjuvant treatment was observed in 30% of patients in the oxaliplatin group vs. 16% in the fluorouracil-only group (p = 0.053). The corresponding proportions of patients having radical surgery or achieving complete pathological response were 72% vs. 77% (odds ratio [OR] = 0.88; 95% confidence interval [CI]: 0.39–1.98; p = 0.75) and 15% vs. 7% (OR = 2.25; 95% CI: 0.83–6.94; p = 0.16), respectively. The long-term outcomes were similar in the two groups. Overall and disease-free survival rates at 5 years were 63% vs. 56% (p = 0.78) and 49% vs. 44% (p = 0.59), respectively. The corresponding numbers for cumulative incidence of local failure or distant metastases were 33% vs. 38% (hazard ratio [HR] = 0.89; 95% CI: 0.52–1.52; p = 0.68) and 33% vs. 33% (HR = 0.78; 95% CI: 0.43–1.40; p = 0.41), respectively.ConclusionOur findings do not support adding oxaliplatin to three cycles of chemotherapy delivered after short-course irradiation.     

Harvesting human adipose tissue-derived adult stem cells: resection versus liposuction

BackgroundAdipose tissue is an abundant source of mesenchymal stem cells (MSC), which can be used for tissue-engineering purposes. The aim of our study was to determine the more suitable procedure, surgical resection or liposuction, for harvesting human adipose tissue-derived stem cells (hASC) with regard to viability, cell count and differentiation potential.MethodsAfter harvesting hASC, trypan blue staining and cell counting were carried out. Subsequently, hASC were cultured, analyzed by fluorescence-activated cell sorting (FACS) and differentiated under adipogenic, osteogenic and chondrogenic conditions. Histologic and functional analyzes were performed at the end of the differentiation period.ResultsNo significant difference was found with regard to the cell counts of hASC from liposuction and surgically resected material (P = 0.086). The percentage of viable cells was significantly higher for liposuction aspirates than for resection material (P = 0.002). No significant difference was found in the adipogenic differentiation potential (P = 0.179). A significantly lower number of cultures obtained from liposuction material than from resection material could be differentiated into osteocytes (P = 0.049) and chondrocytes (P = 0.012).DiscussionEven though some lineages from lipoaspirated hASC can not be differentiated as frequently as those from surgically resected material, liposuction may be superior for some tissue-engineering purposes, particularly because of the less invasive harvesting procedure, the higher percentage of viable cells and the fact that there is no significant difference between lipoaspirated and resected hASC with regard to adipogenic differentiation potential.     

CFU-F circulating in cord blood

CFU-F (colony forming units-fibroblast) were studied from cord blood and, as controls, from normal bone marrow of older children and adults. Numbers of CFU-F in cord blood buffy coat cells are lower by a factor of 10 in comparison to bone marrow CFU-F. Cytomorphology and staining with monoclonal antibody identify the progeny cells of CFU-F as fibroblasts. Cord blood CFU-F derived fibroblasts have properties supporting hematopoiesis: They produce CSF (colony stimulating factor) to which fresh cord blood CFU-GM (colony forming units-granulocytic, monocytic) react by colony formation in a dose-response manner. In addition, fibroblast colonies discharge clonogenic round cells into the medium forming CFU-GM and CFU-F colonies in secondary methyl cellulose cultures. We conclude that fetal blood contains clonogenic stromal cells (CFU-F) that give rise to fibroblasts with properties of hematopoietic support.     

Reliable isolation of human mesenchymal stromal cells from bone marrow biopsy specimens in patients after allogeneic hematopoietic cell transplantation

Isolation of mesenchymal stromal cells (MSCs) from pretreated, hematologic patients is challenging. Especially after allogeneic hematopoietic cell transplantation (HCT), standard protocols using bone marrow aspirates fail to reliably recover sufficient cell numbers. Because MSCs are considered to contribute to processes that mainly affect the outcome after transplantation, such as an efficient lymphohematopoietic recovery, extent of graft-versus-host disease as well as the occurrence of leukemic relapse, it is of great clinical relevance to investigate MSC function in this context. Previous studies showed that MSCs can be isolated by collagenase digestion of large bone fragments of hematologically healthy patients undergoing hip replacement or knee surgeries. We have now further developed this procedure for the isolation of MSCs from hematologic patients after allogeneic HCT by using trephine biopsy specimens obtained during routine examinations. Comparison of aspirates and trephine biopsy specimens from patients after allogeneic HCT revealed a significantly higher frequency of clonogenic MSCs (colony-forming unit–fibroblast [CFU-F]) in trephine biopsy specimens (mean, 289.8 ± standard deviation 322.5 CFU-F colonies/1 × 10⁶ total nucleated cells versus 4.2 ± 9.9; P < 0.0001). Subsequent expansion of functional MSCs isolated from trephine biopsy specimen was more robust and led to a significantly higher yield compared with control samples expanded from aspirates (median, 1.6 × 10⁶; range, 0–2.3 × 10⁷ P0 MSCs versus 5.4 × 10⁴; range, 0–8.9 × 10⁶; P < 0.0001). Using trephine biopsy specimens as MSC source facilitates the investigation of various clinical questions.     

GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion

Background aimsBone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque? PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.MethodsBM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.ResultsNo differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45⁺ fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.ConclusionsBoth GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.     

Tumor necrosis factor-related apoptosis-inducing ligand as an independent predictor of mortality in hemodialysis patents

BackgroundTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was originally isolated as an inducer of apoptosis in transformed cells. In addition to tumor surveillance, recent findings suggest that TRAIL and its receptor system have a protective role against infection and cardiovascular disease (CVD). Patients undergoing hemodialysis have a high mortality rate with a unique risk factor profile. Considering that the leading causes of death in these patients are infection and CVD, TRAIL represents an attractive candidate for predicting mortality in this population. We therefore investigated whether TRAIL predicted mortality in hemodialysis patients.MethodsThe study was a retrospective observational cohort design of 45-month duration in 149 male hemodialysis patients. The subjects were divided into two groups according to their baseline TRAIL level measured by ELISA (low or high TRAIL group). The main outcome was all-cause mortality.ResultsDuring the follow-up period, 33 patients died, mostly because of CVD (n = 11) or infection (n = 9). Crude survival analyses showed that a low TRAIL level was a powerful predictor of all-cause (p = 0.011) and infectious mortality (p = 0.048). The predictive power of TRAIL remained after adjustment for various confounding factors.ConclusionsThe serum TRAIL level may be a novel biomarker for predicting prognosis in hemodialysis patients.     

Prediction of all-cause mortality with copeptin in cardio-cerebrovascular patients: A meta-analysis of prospective studies

BackgroundMeasurement of the biomarker copeptin may help identify disease severity and risk of mortality for a various diseases. This study sought to determine the relationship between copeptin and all-cause mortality of patients with cardio-cerebrovascular disease.MethodsDatabase of Medline and Web of Science were searched for studies with data involving the baseline copeptin levels and subsequent all-cause mortality outcomes. The pooled HRs of all-cause mortality were calculated and presented with 95%CIs. Subgroup analysis and sensitivity analysis were conducted to explore the possible sources of heterogeneity.ResultsData from 14,395 participants were derived from 28 prospective studies. Higher copeptin significantly increased the risk of all-cause mortality (per unit copeptin: HR = 1.020, 95%CI = 1.004–1.036; log unit copeptin: HR = 2.884, 95%CI = 1.844–4.512; categorical copeptin: HR = 3.371, 95%CI = 2.077–5.472). Subgroup analysis indicated that the risk of all-cause death was higher in cerebrovascular patients (per unit copeptin: HR = 2.537, 95%CI = 0.956–6.731; log unit copeptin: HR = 3.419, 95%CI = 2.391–4.888) than cardiovascular patients (per unit copeptin: HR = 1.011, 95%CI = 1.002–1.020; log unit copeptin: HR = 2.009, 95%CI = 1.119–3.608).ConclusionCopeptin is associated with all-cause mortality of patients with cardiovascular and cerebrovascular disease. Our study suggests that copeptin seems to be a promising novel biomarker for prediction of mortality in cardio-cerebrovascular patients, especially for cerebrovascular patients.     

Punto de corte de espesor de tejido adiposo epicárdico para predecir síndrome metabólico en población venezolana

ObjectiveTo define an echocardiographically-assessed cut-off point for epicardial adipose tissue (EAT) thickness associated to metabolic syndrome (MS) components in Venezuelan subjects.MethodsFifty-two subjects aged 20-65 years diagnosed with MS according to International Diabetes Federation criteria and 45 sex- and age-matched controls were selected. Blood glucose and plasma lipids were tested; EAT thickness and left ventricular mass were measured by echocardiography.ResultsNo significant age and sex differences were found between the two groups. Body weight, body mass index, waist circumference, and systolic and diastolic blood pressure were significantly higher (P = .0001) in the MS group. This group showed significantly higher levels of fasting blood glucose (P = .0001), total cholesterol (P = .002), LDL-C (P = .007), non-HDL-C (P = .0001), triglycerides (P = .0001), Tg-HDL-C ratio (P = .0001), and lower HDL-C levels (P = .0001) as compared to the control group. EAT thickness (P = .0001) and left ventricular mass (P = .017) were significantly higher in the MS group. The ROC curve showed an AUC of 0.852 (P = .0001) with a power of the test of 0.99. A 5-mm EAT thickness showed a sensitivity of 84.62% (95% CI: 71.9-93.1) and a specificity of 71.11% (95% CI: 55.7-83.6) for predicting MS. The odds ratio of this population for experiencing MS due to an EAT ≥ 5 mm was 8.25 (95% CI: 3.15-21.56; P = .0001).ConclusionAn EAT value ≥ 5 mm has good sensitivity and specificity for predicting MS in the Venezuelan population.     

Association between selenium nutritional status and metabolic risk factors in men with visceral obesity

Background and aimPrevious evidence has suggested an association between selenium and cardiovascular disease, which is main outcome of metabolic syndrome. The aim of this study was to examine possible correlation between selenium nutritional status and metabolic risk factors in men with visceral obesity.MethodsPlasma samples were collected from 123 Indonesian men with visceral obesity. Their metabolic risk factors and selenium nutritional status were analyzed. The eligible subjects (n = 78) were stratified according to the International Diabetes Federation: obese, obese plus one component, and obese plus two components or more. Obese plus two components or more were diagnostic criteria of metabolic syndrome. Pearson's correlation was performed to examine the correlation in each group.ResultsIn the obese group, selenium positively correlated with high-density lipoprotein (HDL) cholesterol (r = 0.390, P < 0.05) and with fatty acid binding protein-4 (FABP4) (r = 0.474, P < 0.05); glutathione peroxidase-3 (GPx3) activity was inversely correlated with FABP4 (r = ?467, P < 0.05). In the obese plus one component group, GPx3 activity positively correlated with HDL cholesterol (r = 0.413, P < 0.05). In the metabolic syndrome group, selenium negatively correlated with monocytes chemoattractant protein (MCP)-1 (r = ?0.429, P < 0.05).ConclusionsThese results show that the association between selenium nutritional status and metabolic risk factors is limited to particular group of obese men with or without metabolic syndrome.     

Biomarkers in critically ill patients with systemic inflammatory response syndrome or sepsis supplemented with high-dose selenium

ObjectiveLow levels of selenium (Se) and glutathione peroxidase (GSHPx), a key selenoenzyme, were documented in systemic inflammatory response syndrome (SIRS) and sepsis, both associated with high mortality. Se supplementation had mixed effects on outcome. We hypothesized that Se supplementation could have a different impact on biomarkers and 28-day mortality in patients with SIRS vs. sepsis.MethodsAdult patients with SIRS or sepsis were randomized to either high-dose (Se+, n = 75) or standard-dose (Se−, n = 75) Se supplementation. Plasma Se, whole blood GSHPx activity, C-reactive protein (CRP), procalcitonin (PCT), prealbumin, albumin and cholesterol levels were measured serially up to day 14.ResultsThere was no difference in mortality between Se− (24/75) vs. Se+ group (19/75; p = 0.367) or between SIRS and septic patients (8/26 vs. 35/124; p = 0.794). There was a trend to reduced mortality in SIRS patients in the Se+ vs. Se− group (p = 0.084). Plasma Se levels increased in the Se+ group only in patients with sepsis but not in patients with SIRS. Plasma Se levels correlated with GSHPx. In SIRS/Se+ group, Se correlated only with GSHPx. In SIRS/Se− group, Se correlated with cholesterol but not with other biomarkers. In sepsis patients, Se levels correlated with cholesterol, GSHPx and prealbumin. Cholesterol levels were higher in survivors in the Se− group.ConclusionsSe levels correlated with GSHPx activity and other nutritional biomarkers with significant differences between SIRS and sepsis groups. High-dose Se supplementation did not affect mortality but a strong trend to decreased mortality in SIRS patients warrants further studies in this population.     

Neutrophil-to-lymphocyte ratio is not associated with risk of sarcopenia in elderly COVID-19 patients

BackgroundTo assess the existence of association between neutrophil to lymphocyte ratio (NLR) and the risk of sarcopenia in COVID-19 patients.MethodsA retrospective cross-sectional study was conducted in a university hospital with patients with an active COVID-19 infection admitted to the nursing ward or intensive care unit (ICU) between September to December 2020. Sarcopenia risk was assessed using the Strength, Assistance for walking, Rise from a chair, Climb stairs and Falls (SARC-F). Biochemical analyses were assessed by circulating of C-reactive protein, D-dimer, neutrophils, lymphocytes count and NLR. Sixty-eight patients were evaluated and divided into tertiles of NLR values and the association between NLR and sarcopenia risk were tested using the linear regression analyses and p < 0.05 were considered as significant.ResultsSixty-eight patients were evaluated and divided in NLR tertiles being the 1st (men = 52.2%; 71.1 ± 9.0 y; NLR: 1.1–3.85), 2nd (women = 78.3%; 73.2 ± 9.1 y; NLR: 3.9–6.0) and 3rd (men = 72.7%; 71.7 ± 10.4 y; NLR: 6.5–20.0). There was a difference between the tertiles in relation to the first to the biochemical parameters of total neutrophils count (p = 0.001), C-reactive protein (p = 0.012), and D-dimer (p = 0.012). However, no difference was found in linear regression analysis between tertiles of NLR and SARC-F, if in total sample (p = 0.054) or divided by sex, if men (p = 0.369) or women (p = 0.064).ConclusionIn elderly patients hospitalized with COVID-19, we do not find an association between the risk of sarcopenia and NLR.     

Diagnostic performance of salivary cortisol and serum osteocalcin measurements in patients with overt and subclinical Cushing's syndrome

ObjectiveThe cut-off value for salivary cortisol measurement for the diagnosis of Cushing's syndrome (CS) may depend both on the severity of the disease and the composition of control group. Therefore, we examined the utility of midnight salivary cortisol measurements in patients who were evaluated for signs and symptoms of CS or because they had adrenal incidentalomas. Because serum osteocalcin (OC) is considered as a sensitive marker of hypercortisolism, we also investigated whether OC could have a role in the diagnosis of CS.Patients and methodsEach of the 151 patients was included into one of the following groups: (A) overt CS (n = 23), (B) subclinical CS (n = 18), (C) inactive adrenal adenomas (n = 40), (D) patients without HPA disturbances (n = 70). Patients (C + D) were used as controls. Serum, salivary and urinary cortisol, and OC were measured by electrochemiluminescence immunoassay.ResultsGroup A had suppressed OC as compared to both group B and group (C + D). Serum and salivary cortisol concentrations showed strong negative correlations with OC in patients with overt CS. The areas under the curves of salivary and serum cortisol at 24:00 h (0.9790 and 0.9940, respectively) serum cortisol after low dose dexamethasone test (0.9930) and OC (0.9220) obtained from ROC aanalysis for the diagnosis of overt CS were not statistically different.ConclusionThis study confirms the usefulness of midnight salivary cortisol measurements in the diagnosis of overt CS in the everyday endocrinological praxis. Our results suggest that OC may have a role in the diagnosis of overt CS.     

A let-7 microRNA polymorphism in the KRAS 3′-UTR is prognostic in oropharyngeal cancer

IntroductionThis study aimed to investigate the effect of genetic polymorphisms in miRNA sequences, miRNA target genes and miRNA processing genes as additional biomarkers to HPV for prognosis in oropharyngeal squamous cell carcinoma (OPSCC) patients. Secondarily, the prevalence of HPV-associated OPSCC in a European cohort was mapped.MethodsOPSCC patients (n = 122) were genotyped for ten genetic polymorphisms in pre-miRNAs (pre-mir-146a, pre-mir-196a2), in miRNA biosynthesis genes (Drosha, XPO5) and in miRNA target genes (KRAS, SMC1B). HPV status was assessed by p16 immunohistochemistry (IHC) and high-risk HPV in situ hybridization (ISH) or by p16 IHC and PCR followed by enzyme-immunoassay (EIA). Overall and disease specific survival were analysed using Kaplan–Meier plots (log-rank test). Cox proportional hazard model was used to calculate hazard ratios (HR).ResultsThe overall HPV prevalence rate in our Belgian/Dutch cohort was 27.9%. Patients with HPV⁺ tumours had a better 5-years overall survival (78% vs. 46%, p = 0.001) and a better 5-years disease specific survival (90% vs. 70%, p = 0.016) compared to patients with HPV⁻ tumours. In multivariate Cox analysis including clinical, treatment and genetic parameters, HPV negativity (HR = 3.89, p = 0.005), advanced T-stage (HR = 1.81, p = 0.050), advanced N-stage (HR = 5.86, p = 0.001) and >10 pack-years of smoking (HR = 3.45, p = 0.012) were significantly associated with reduced overall survival. The variant G-allele of the KRAS-LCS6 polymorphism was significantly associated with a better overall survival (HR = 0.40, p = 0.031).ConclusionsOur results demonstrate that OPSCC patients with the KRAS-LCS6 variant have a better outcome and suggest that this variant may be used as a prognostic biomarker for OPSCC.