Blockade of sodium-glucose co-transporters improves peritoneal ultrafiltration in uraemic rodent models.

BACKGROUND: The most used PD fluids contain glucose as a primary osmotic agent. Glucose peritoneal absorption during dwell decreases the osmotic gradient of peritoneal fluids and causes undesirable metabolic consequences. Inhibitors of sodium-glucose co-transporter (SGLT) type 2 are wildly used for the treatment of diabetes, heart and kidney failure. Previous attempts to use SGLT2 blockers in experimental peritoneal dialysis yielded contrasting results. We studied whether peritoneal SGLTs blockade may improve ultrafiltration (UF) via partial inhibition of glucose uptake from dialysis fluids. METHODS: Kidney failure was induced in mice and rats by bilateral ureteral ligation, and dwell was performed by injection of glucose-containing dialysis fluids. The effect of SGLT inhibitors on glucose absorption during fluid dwell and UF was measured in vivo. RESULTS: Diffusion of glucose from dialysis fluid into the blood appeared to be sodium-dependent, and blockade of SGLTs by phlorizin and sotagliflozin attenuated blood glucose increment thereby decreasing fluid absorption. Specific SGLT2 inhibitors failed to reduce glucose and fluid absorption from the peritoneal cavity in a rodent kidney failure model. CONCLUSIONS: Our study suggests that peritoneal non-type 2 SGLTs facilitate glucose diffusion from dialysis solutions, and we propose that limiting glucose reabsorption by specific SGLT inhibitors may emerge as a novel strategy in PD treatment to enhance UF and mitigate the deleterious effects of hyperglycaemia.

A prospective cohort study of dynamic cell-free DNA elevation during cardiac surgery with cardiopulmonary bypass.

Cardiac surgery and cardiopulmonary bypass (CPB) are associated with a systemic inflammatory reaction that occasionally induces a life-threatening organ dysfunction caused by the dysregulated host response to the damage-associated molecular patterns (DAMPs). In severe inflammation, cell-free DNA (cfDNA) and histones are released by inflammatory cells and damaged tissue and act as DAMPs. We sought to characterize the changes in circulating cell-free DNA (cfDNA) levels during CPB. Primary outcomes were renal failure, ventilation time (>18 hr), length of stay (LOS) in the intensive care unit (ICU) (>48hr), hospital LOS (>15 days), and death. We looked for associations with blood tests and comparison to standard scores. In a prospective cohort study, we enrolled 71 patients undergoing non-emergent coronary artery bypass grafting. Blood was drawn at baseline, 20 and 40 minutes on CPB, after cross-clamp removal, and 30 minutes after chest closure. cfDNA was measured by our fast fluorescent method. Baseline cfDNA levels [796 (656-1063) ng/ml] increased during surgery, peaked after cross-clamp removal [2403 (1981-3357) ng/ml] and returned to baseline at recovery. The difference in cfDNA from 20 to 40 minutes on CPB (ΔcfDNA 40-20) inversely correlated with peripheral vascular disease (PVD), longer ventilation time, and longer ICU and hospital length of stay (LOS). Receiver operating characteristic (ROC) curve of ΔcfDNA 40-20 for long ICU-LOS (>48hr) was with an area under the curve (AUC) of 0.738 (p = 0.022). ROC AUC of ΔcfDNA 40-20 to long Hospital LOS (>15 days) was 0.787 (p = 0.006). Correction for time on CPB in a multivariate logistic regression model improved ROC-AUC to 0.854 (p = 0.003) and suggests that ΔcfDNA 40-20 is an independent risk factor. To conclude, of measured parameters, including STS and Euroscore, the predictive power of ΔcfDNA 40-20 was the highest. Thus, measurement of ΔcfDNA 40-20 may enable early monitoring of patients at higher risk. Further studies on the mechanism behind the negative association of ΔcfDNA 40-20 with PVD and outcomes are warranted.

Cord Blood Cell-Free DNA Concentration: A Novel Marker for Neonatal Wellbeing.

OBJECTIVE: Cord gas values and Apgar scores, currently used as markers for newborn wellbeing and postpartum complications, provide rough estimates, and their use remains elusive. Circulating cell-free DNA (cfDNA) may better represent newborn status at birth and the effect of parturition on the fetus. This pilot study investigates the association between cord blood (CB) cfDNA and neonatal outcomes. STUDY DESIGN: In a prospective cohort study, cfDNA concentration was measured in immediately following delivery collected CB sera of newborns using our rapid fluorescent assay. RESULTS: During the study period, blood samples from umbilical cords of 100 newborns were collected. Vaginal delivery was associated with a higher median CB cfDNA than cesarean delivery (277 [95% confidence interval [CI] 199-377] vs. 100 [95% CI 43-265] ng/mL, p < 0.01). cfDNA levels were significantly associated with gestational age at delivery (rho = 0.308, p = 0.002) and CB base deficit (BD, r = 0.252, p = 0.017). According to maternal and fetal complications, CB cfDNA was elevated in fetuses with category II of heart rate tracing (p < 0.05), with maternal positive vaginal culture (p < 0.01), and with premature rupture of membranes (PROM, p < 0.001). Logistic regression models of CB cfDNA fourth quartiles demostrate a double odds ratio for elevated BD (>3mmol/L) and for worse heart rate tracing category. CONCLUSION: Serum CB cfDNA concentration reflects the newborn's status and hazards with an excellent association with CB BD, fetal heart rate category, and maternal risk factors for infection (positive vaginal culture and PROM). This preliminary observation suggests that cfDNA can serve as a point of care biomarker for newborn status at the time of delivery. KEY POINTS: · CB cfDNA levels correlated with newborn's BD.. · CB cfDNA levels reflect parturition stress and inflammation.. · cfDNA serve as a diagnostic and prediction tool for the identification of newborns at risk for morbidity..

Circulating Cell-free DNA in Patients With Acute Biliary Pancreatitis: Association With Disease Markers and Prolonged Hospitalization Time-A Prospective Cohort Study.

OBJECTIVE: To evaluate cfDNA as an indicator of pancreatitis severity. BACKGROUND: Acute pancreatitis severity scores have limited proficiency, and are complex and challenging to use clinically. Elevation of circulating cfDNA concentration has been shown to be associated with hospital length of stay (LOS) and mortality. METHODS: In a prospective study, cfDNA concentration was measured by a simple fluorometric test, at admission and for 2 consecutive days, in patients with acute biliary pancreatitis (ABP). Ranson and APACHE II scores were used as measures of pancreatitis severity. Hospital LOS and mortality were used as outcome measures. RESULTS: Seventy-eight patients were included. Patients with severe disease according to Ranson's Criteria (n = 24) had elevated median admission cfDNA compared to patients with mild disease (n = 54, 2252ng/ml vs 1228 ng/ml, P < 0.05 ). Admission cfDNA levels correlated with Ranson and APACHE II scores and markers of bile duct obstruction. LOS did not differ between patients with mild and severe disease according to Ranson and APACHE II scores. Patients with cfDNA at 24 hours concentrations above the cutoff value of healthy patients (>850 ng/ml) had a significantly longer LOS compared to those with normal cfDNA levels ( P < 0.001 ). CONCLUSIONS: cfDNA, measured by a rapid simple assay, proved a valuable early marker of severity in ABP with clear advantages for prediction of LOS over Ranson and APACHE II. Measurement of cfDNA has the potential to be an effective practical approach to predict the course of ABP and should be further evaluated in larger trials.

Quick cell-free DNA testing for the prediction of postconcussion syndrome: a single-center prospective pilot trial.

OBJECTIVE: Mild traumatic brain injury (mTBI) is a major cause of emergency room (ER) admission. Thirty percent of mTBI patients have postconcussion syndrome (PCS), and 15% have symptoms for over a year. This population is underdiagnosed and does not receive appropriate care. The authors proposed a fast and inexpensive fluorometric measurement of circulating cell-free DNA (cfDNA) as a biomarker for PCS. cfDNA is a proven, useful marker of a variety of acute pathological conditions such as trauma and acute illness. METHODS: Thirty mTBI patients were recruited for this prospective single-center trial. At admission, patients completed questionnaires and blood was drawn to obtain cfDNA. At 3-4 months after injury, 18 patients returned for cognitive assessments with questionnaires and the Color Trails Test (CTT). The fast SYBR Gold assay was used to measure cfDNA. RESULTS: Seventeen men and 13 women participated in this trial. The mean ± SD age was 50.9 ± 13.9 years. Of the 18 patients who returned for cognitive assessment, one-third reported working fewer hours, 4 (22.2%) changed their driving patterns, and 5 (27.7%) reduced or stopped performing physical activity. The median cfDNA level of the mTBI group was greater than that of the matched healthy control group (730.5 vs 521.5 ng/ml, p = 0.0395). Admission cfDNA concentration was negatively correlated with performance on the CTT1 and CTT2 standardized tests (r = -0.559 and -0.599), meaning that greater cfDNA level was correlated with decreased cognitive performance status. The performance of the patients with normal cfDNA level included in the mTBI group was similar to that of the healthy participants. In contrast, the increased cfDNA group (> 800 ng/ml) had lower scores on the CTT tests than the normal cfDNA group (p < 0.001). Furthermore, patients with moderate/severe cognitive impairment according to CTT1 results had a greater median cfDNA level than the patients with scores indicating mild impairment or normal function (1186 vs 473.5 ng/ml, p = 0.0441, area under the receiver operating characteristic curve = 0.8393). CONCLUSIONS: The data from this pilot study show the potential to use cfDNA, as measured with a fast test, as a biomarker to screen for PCS in the ER. A large-scale study is required to establish the value of cfDNA as an early predictor of PCS.

A1 and A2A adenosine receptors play a protective role to reduce prevalence of autoimmunity following tissue damage.

Adenosine is a potent modulator that has a tremendous effect on the immune system. Adenosine affects T cell activity, and is necessary in maintaining the T helper/regulatory T cell (Treg ) ratio. Adenosine signalling is also involved in activating neutrophils and the formation of neutrophil extracellular traps (NETs), which has been linked to autoimmune disorders. Therefore, adenosine, through its receptors, is extremely important in maintaining homeostasis and involved in the development of autoimmune diseases. In this study, we aim to evaluate the role of adenosine A1 and A2A receptors in involvement of autoimmune diseases. We studied adenosine regulation by NETosis in vitro, and used two murine models of autoimmune diseases: type I diabetes mellitus (T1DM) induced by low-dose streptozotocin and pristane-induced systemic lupus erythematosus (SLE). We have found that A1 R enhances and A2A R suppresses NETosis. In addition, in both models, A1 R-knock-out (KO) mice were predisposed to the development of autoimmunity. In the SLE model in wild-type (WT) mice we observed a decline of A1 R mRNA levels 6 h after pristane injection that was parallel to lymphocyte reduction. Following pristane, 43% of A1 R-KO mice suffered from lupus-like disease while WT mice remained without any sign of disease at 36 weeks. In WT mice, at 10 days A2A R mRNA levels were significantly higher compared to A1R-KO mice. Similar to SLE, in the T1DM model the presence of A1 R and A2A R was protective. Our data suggest that, in autoimmune diseases, the acute elimination of lymphocytes and reduction of DNA release due to NETosis depends upon A1 R desensitization and long-term suppression of A2A R.

Elevated Circulating Cell-Free DNA in Hemodialysis-Treated Patients Is Associated with Increased Mortality.

BACKGROUND: Predicting the mortality risk of patients un-dergoing hemodialysis (HD) is challenging. Cell-free DNA (cfDNA) is released into circulation from dying cells, and its elevation is predictive of unfavorable outcome. In a pilot study, we found post-HD cfDNA level to be a predictor of all-cause mortality. Thus, the aim of this study was to confirm the prognostic power of cfDNA in a larger prospective cohort study conducted at 2 medical centers. METHODS: CfDNA levels were measured by a rapid fluorometric assay on sera obtained before and after 1 HD session. One hundred fifty-three patients were followed up to 46 months for mortality during which time 47 patients died. We compared the predictive value of cfDNA to age, comorbidities, and standard blood tests. RESULTS: Examining standard blood tests, only post-HD cfDNA levels were elevated in the non-survivor group compared to survivors (959 vs. 803 ng/mL, p = 0.04). Pre- and post-HD cfDNA levels correlated with age and diabetes. Patients with elevated cfDNA (>850 ng/mL) showed lower survival than those with normal levels. A Cox proportional hazard regression model demonstrated a significant hazard ratio of 1.92 for post-HD cfDNA levels. Logistic regression models showed that post-HD cfDNA was a significant predictor of mortality at 1-3 years with odd ratios of 4.61, 4.36, and 6.22, respectively. CONCLUSIONS: Post-HD cfDNA level was superior to standard blood tests and could serve as a biomarker to assist in decision-making for HD-treated patients.

Cell-free DNA concentration in patients with clinical or mammographic suspicion of breast cancer.

Mammography has a crucial role in the detection of breast cancer (BC), yet it is not limitation-free. We hypothesized that the combination of mammography and cell-free DNA (cfDNA) levels may better discriminate patients with cancer. This prospective study included 259 participants suspected with BC before biopsy. Blood samples were taken before biopsy and from some patients during and at the end of treatment. cfDNA blood levels were measured using our simple fluorescent assay. The primary outcome was the pathologic diagnosis of BC, and the secondary aims were to correlate cfDNA to severity, response to treatments, and outcome. Median cfDNA blood levels were similar in patients with positive and negative biopsy: 577 vs. 564 ng/ml (p = 0.98). A significant decrease in cfDNA blood level was noted after the following treatments: surgery, surgery and radiation, neo-adjuvant chemotherapy and surgery, and at the end of all treatments. To conclude, the cfDNA level could not be used in suspected patients to discriminate BC. Reduction of tumor burden by surgery and chemotherapy is associated with reduction of cfDNA levels. In a minority of patients, an increase in post-treatment cfDNA blood level may indicate the presence of a residual tumor and higher risk. Further outcome assessment for a longer period is suggested.

Circulating cell-free DNA (cfDNA) levels in BRCA1 and BRCA2 mutation carriers: A preliminary study.

BACKGROUND: Female carriers of BRCA1 or BRCA2 germline mutations are at a substantially increased risk for developing breast and ovarian cancer. The lack of effective early detection schemes for ovarian cancer, mandate surgical removal of adnexa at age 35-40 years in these high-risk women. The role of circulating cell-free DNA (cfDNA) levels as a marker for early detection in high-risk women has rarely been reported. OBJECTIVE: To quantify cfDNA levels in BRCA1BRCA2 carriers. METHODS: Serum cfDNA levels, measured by direct fluorometric assay in cancer-free female BRCA1BRCA2 mutation carriers were compared with cancer-free controls recruited from among women undergoing breast biopsy or routine colonoscopy. RESULTS: Overall, 10 BRCA1 (185delAG) and 10 BRCA2 (6174delT) mutation carriers, 20 breast biopsy controls, and 20 colonoscopy controls participated. cfDNA levels [Median (95% CI)], were 472 (317-589) ng/ml and 525 (339-621) ng/ml in breast biopsy and colonoscopy controls, respectively. Median levels of cfDNA in BRCA1 and BRCA2 mutation carriers combined were 921 (835-1087) ng/ml, significantly higher than in both controls (P< 0.0001). CONCLUSIONS: cfDNA levels are significantly higher in BRCA1 and BRCA2 mutation carriers compared with non-carriers. This finding, if validated, may facilitate development of early detection breast/ovarian cancer biomarker in high-risk women.

Pharmacological preconditioning with adenosine A1 receptor agonist induces immunosuppression and improves graft survival in novel allogeneic transplantation models.

Adenosine is widely known as a potent modulator of innate and acquired immunity. It is released during transplants, and acts on four subtype receptors. In previous studies, we demonstrated that pharmacological preconditioning (PPC), pre-administration of the selective A1 receptor (A1R) agonist led to A1R desensitization, is followed by upregulation of the adenosine A2A receptor. This immunosuppressive effect resulted in lymphopenia, and it reduced T-cell reactivity. The aim of the current study was to challenge the immunosuppressive effects of A1R-PPC in models of allogeneic grafts. PPC mice were treated by intraperitoneal injection using specific adenosine A1R agonist 24 h and 12 h before starting any procedure. We challenged our method in novel allogeneic muscle and skin grafts models. Mice and grafts were assessed by complete blood counts, MLR from PPC splenocytes, and pathological evaluation. We found a significant reduction in WBC and lymphocyte counts in PPC-treated mice. Two-way MLR with splenocytes from PPC grafted mice showed decreased proliferation and anergy. Histology of PPC allogeneic grafts revealed profoundly less infiltration and even less muscle necrosis compared to vehicle treated allografts. Similar results observed in PPC skin transplantation. To conclude, PPC moderated graft rejection in separate allogeneic challenges, and reduced lymphocytes infiltration and ischemic damage.

Circulating cell-free DNA as a potential marker in smoke inhalation injury.

Failure in evaluation of smoke inhalation injury (SII) is related to increased morbidity and mortality. Prognostic biomarkers that reflect the injury are undoubtedly needed. Cell-free DNA (CFD) concentrations are associated to the extent of tissue damage and inflammation in various pathologies. We have developed a simple assay for CFD quantification and previously found it prognostic in various pathologies including burns, lung disease, and sepsis. The aim of this study was to evaluate admission CFD as an injury severity marker in patients with SII.In a prospective study, we measured admission CFD levels in 18 SII patients and matched control subjects. Daily CFD levels were also performed in 4 hospitalized patients. Serum CFD levels were measured by our direct rapid fluorometric assay.Admission CFD levels of SII patients were significantly higher than those of healthy controls, 879 (236-3220) ng/mL vs. 339 (150-570) ng/mL, [median (range)], P < .0001. Admission CFD levels of hospitalized patients were significantly higher than those of nonhospitalized patients, 1517 (655-3220) ng/mL vs. 675 (236-1581) ng/mL, P < .05. Admission CFD positively correlated with hospitalization time (Rho = 0.578, P < .05) and was in linear correlation with CO poisoning (carboxyhemoglobin (COHb) levels, R = 0.621, P < .0001). Additionally, along with the recovery of hospitalized patients, we observed a matched reduction of CFD levels.CFD appears to be a potentially valuable marker for severity and follow-up of SII. We believe this rapid assay can help introduce the routine use of CFD measurement into daily practice.

Maternal total cell-free DNA in preeclampsia and fetal growth restriction: Evidence of differences in maternal response to abnormal implantation.

OBJECTIVES: Preeclampsia and fetal growth restriction are obstetrical syndromes associated with abnormal placental implantation and changes in the activation status of maternal leukocytes. This study is aimed to determine by a simple, rapid fluorescent assay the changes in maternal serum total cell-free DNA (t-cfDNA) concentrations in women with preeclampsia and those with fetal growth restriction (FGR). STUDY DESIGN: A cross-sectional study was conducted measuring maternal serum t-cfDNA concentrations. Women were classified into the following groups: 1) patients with preeclampsia (n = 21); 2) FGR-estimated fetal weight below the 10thpercentile (n = 28); and 3) normal pregnancy (n = 39). Serum samples were directly assayed for t-cfDNA using a rapid fluorescent SYBR Gold assay. Elevated maternal serum t-cfDNA concentrations were defined as a cutoff>850ng/ml. Nonparametric statistics were used for analysis. RESULTS: Women with preeclampsia had a higher median maternal serum concentration (802 ng/ml, 400-2272 ng/ml) than women with a normal pregnancy (499 ng/ml, 0-1892 ng/ml, p = 0.004) and those with FGR (484 ng/ml, 72-2187 ng/ml, p = 0.012). Moreover, even patients with FGR <5th percentile and abnormal Doppler had a lower median maternal serum t-cfDNA than those with preeclampsia (median 487 ng/ml, 144-1971 ng/ml, p = 0.022). The median concentration of t-cfDNA did not differ between women with a normal pregnancy and those with FGR (p = 0.54), as well as those with fetuses <5th percentile and abnormal Doppler (p = 0.7). Women with preeclampsia had a higher proportion of elevated t-cfDNA than those with a normal pregnancy (p = 0.015) and patients with FGR (p = 0.025). CONCLUSIONS: Preeclampsia is associated with higher maternal serum t-cfDNA concentration than normal pregnancy or FGR. This observation may reflect an increased systemic activation of the maternal inflammation, rather than placental; this assumption is supported by the fact that we did not observe a significant change in the maternal serum t-cfDNA in patients with placental-mediated FGR.

Elevated Neutrophil Gelatinase Lipocalin Levels Are Associated With Increased Oxidative Stress in Hemodialysis Patients.

BACKGROUND: Administration of intravenous iron is an essential treatment of anemia in hemodialysis patients, but it may lead to oxidative stress and increased morbidity and mortality. There is evidence that neutrophil gelatinase-associated lipocalin (NGAL) is protective against oxidative stress and thus the aim of the present study was to investigate the relationship between plasma NGAL and advanced oxidative protein products (AOPP) in hemodialysis patients treated with intravenous iron. METHODS: In a prospective study, 47 hemodialysis patients (mean age 63 years, SD = 13.6; 40% women) were enrolled from two separate hospitals. Oxidative stress was induced by an intravenous administration of 100 mg iron saccharate 0.5 h after the start of dialysis. Blood samples were drawn at the beginning of the dialysis, 0.5 h after iron administration and at the end of dialysis. NGAL levels were measured from the first blood sample, AOPP levels were measured from all blood samples. RESULTS: Our results showed that higher NGAL and AOPP levels at the beginning of the dialysis, prior to iron administration, significantly predicted higher levels of AOPP toward the end of dialysis, (ß = 0.355, SE = 0.054, P = 0.035; ß = 0.297, SE = 0.159, P = 0.043, respectively). CONCLUSIONS: Our results suggest that higher level of NGAL is a risk factor for oxidative stress, as measured by AOPP levels, in dialysis patients receiving intravenous iron. Our findings could identify dialysis patients who are at higher risk from iron supplementation via measurement of NGAL levels.

Circulating Cell-Free DNA Levels in Patients with Metastatic Renal Cell Carcinoma.

BACKGROUND: Limited data about biomarkers are available to predict the outcomes of targeted therapy in metastatic renal cell carcinoma (mRCC). Circulating cell-free DNA (CFD) is elevated in various cancers. PATIENTS AND METHODS: We performed a prospective study of patients with mRCC who received targeted therapy in the Soroka Medical Center between 2013 and 2015. CFD levels were measured using a simple fluorometric assay. Blood samples for CFD were collected before treatment and at weeks 1, 4, 12, 18, and 24 of treatment. The normal cut-off level of CFD was defined as 800 ng/ml. The association of CFD with objective response, progression-free survival (PFS), and overall survival was tested, with adjustment for known confounding risk factors. RESULTS: A total of 23 patients were included; 18 were treated with first-line therapy and 5 with second- and third-line therapies. Patients with normal pretreatment CFD level had a better PFS versus patients with increased levels (p = 0.023). In multivariate analysis, factors associated with PFS were pretreatment CFD levels (p = 0.020) and Heng risk (p = 0.006). CONCLUSIONS: Elevated pretreatment CFD levels measured using a simple fluorometric assay may be associated with a worse PFS in patients with mRCC. A larger prospective study is warranted in order to validate our observation.

Cell-free deoxyribonucleic acid as a prognostic marker of bowel ischemia in patients with small bowel obstruction.

BACKGROUND: Patients with strangulation small bowel obstruction are at a high risk for serious morbidity and mortality due to ischemic bowel. Measuring serum, cell-free deoxyribonucleic acid levels could help recognize early cell death. Our hypothesis was that small bowel ischemia or necrosis is associated with increases in serum cell-free deoxyribonucleic acid and that recovery is associated with a decrease in cell-free deoxyribonucleic acid levels. METHODS: A prospective cohort study in addition to standard treatment of patients admitted with a diagnosis of small bowel obstruction. The participants were divided into groups depending on the presence of ischemic or necrotic bowel according to operative and clinical outcome. Clinical data and serum-based cell-free deoxyribonucleic acid levels were compared. Cell-free deoxyribonucleic acid levels from these 2 groups also were compared with a third group of healthy controls. RESULTS: In the study, 58 patients were enrolled, and 18 patients (31%) underwent operation. During the operative procedure, ischemic or necrotic bowel was found in 10 cases (17%). Serum levels of cell-free deoxyribonucleic acid at the time of admission in the ischemic/necrotic bowel group were increased compared with patients with well perfused or spontaneously recovered bowel (P = .03). Cell-free deoxyribonucleic acid levels decreased on the day after admission in 88% of the nonoperated patients. No significant differences were found in demographics, medical background, imaging performed, and cause of obstruction nor in clinical admission data. CONCLUSION: Surgeons currently rely on imprecise clinical parameters, including degree of pain, abdominal tenderness, leukocytosis etc to decide when operative intervention is needed. The association of cell-free deoxyribonucleic acid with small bowel obstruction, ischemia, and recovery supports our hypothesis and suggests that this biomarker is a potential surrogate of small bowel perfusion.

Systemic inflammatory response syndrome-related lymphopenia is associated with adenosine A1 receptor dysfunction.

SIRS is associated with lymphopenia, and prolonged lymphopenia of septic patients has been associated with increased mortality risk. We hypothesize that elevated adenosine during SIRS down-regulates Gi-coupled A1R, which signals an effect that sensitizes a cAMP-dependent lymphotoxic response. In this study, we evaluate the role of adenosine in SIRS-mediated lymphopenia and impaired IL-15 production. Cecal ligation and puncture was used to induce sepsis-associated SIRS in mice. BMDCs were cultured and used to measure the effect of adenosine on IL-15. We found that A1R mRNA levels were significantly down-regulated and A1R-dependent Gi activity was abolished in T cells of septic mice. In accordance, cAMP was elevated in isolated T cells from cecal ligation and puncture compared with sham-treated mice. Similar to septic mice, leukopenia was evident in sham A1R-KO mice, after treatment with the A1R antagonist (8-cyclopentyl-1,3-dipropylxanthine), or after A1R desensitization. In contrast, A2AR-KO mice were protected from leukopenia. In addition, we observed that septic A1R-KO mice exhibited low IL-15 levels. Cultured BMDC agonists of A2AR and A2BR inhibited IL-15 production and adenosine blocked IL-15-dependent proliferation of cytotoxic T cells that were cocultured with stimulated BMDCs. To conclude, we suggest that SIRS-associated lymphopenia is initiated by A1R desensitization and adenosine-mediated inhibition of IL-15 production is part of the mechanism that accounts for the delay in leukopenia recovery in patients with severe sepsis. Interference with adenosine signaling may thus be potentially beneficial for septic patients with leukopenia.

Prognostic utility of admission cell-free DNA levels in patients with chronic obstructive pulmonary disease exacerbations.

BACKGROUND: Chronic obstructive pulmonary disease exacerbations (COPDEs) are associated with increased morbidity and mortality. Cell-free DNA (cfDNA) is a novel biomarker associated with clinical outcomes in several disease states but has not been studied in COPD. The objectives of this study were to assess cfDNA levels during a COPDE, to evaluate the association of cfDNA with clinical parameters and to explore the prognostic implications of cfDNA levels on long-term survival. METHODS: This was an observational study that assessed cfDNA levels in patients admitted to hospital for a COPDE. Plasma cfDNA levels of COPDE patients were compared to those of matched stable COPD patients and healthy controls. Multivariable and Cox regression analyses were used to assess the association of cfDNA levels with blood gas parameters and long-term survival. RESULTS: A total of 62 patients (46 males, forced expiratory volume in 1 second [FEV1] 38%±13%) were included. The median cfDNA levels on admission for COPDE patients was 1,634 ng/mL (interquartile range [IQR] 1,016-2,319) compared to 781 ng/mL (IQR 523-855) for stable COPD patients, matched for age and disease severity, and 352 ng/mL (IQR 209-636) for healthy controls (P<0.0001, for both comparisons). cfDNA was correlated with partial arterial pressure of carbon dioxide (PaCO2, r=0.35) and pH (r=-0.35), P=0.01 for both comparisons. In a multivariable analysis, PaCO2 was the only independent predictor of cfDNA. Using a cfDNA level of 1,924 ng/mL (threshold for abnormal PaCO2), those with high levels had a trend for increased 5-year mortality risk adjusted for age, sex and FEV1% (hazard ratio 1.92, 95% confidence interval 0.93-3.95, P=0.08). CONCLUSION: Plasma cfDNA might offer a novel technique to identify COPD patients at increased risk of poor outcomes, but the prognostic utility of this measurement requires further study.

Elevated Cell-Free DNA Measured by a Simple Assay Is Associated With Increased Rate of Colorectal Cancer Relapse.

OBJECTIVES: For patients with early stage colorectal cancer (CRC), markers of high-risk relapse are needed. In a previous study on 38 randomly selected patients with CRC, we found good correlation between presurgery cell-free DNA (CFD) concentrations and standard prognostic factors. In the current study, we revisited the same patients at 5-year survival, aiming to evaluate the predictive power of presurgery CFD levels. METHODS: We revisited 38 patients with CRC previously analyzed for 5-year outcome. CFD was measured using a simple fluorescent assay that we developed. RESULTS: All recurrent patients and patients who had died of cancer within 5 years were shown to have presurgery CFD values above 800 ng/mL. The negative predictive value for cancer-related disease was 100%. Cox regression analysis for disease-free survival showed a hazard ratio of 6.03 (P = .003) for CFD, which was higher than the ratio of the disease stage, 1.9 (P = .006). The survival-free curve of stage I and II patients with elevated CFD was significantly different from patients with normal levels (P = .0136); 5 (41.7%) of 12 patients had died of cancer or had experienced a recurrence. CONCLUSIONS: CFD may possibly be a decisive criterion to identify patients with local disease who might benefit from adjuvant chemotherapy.

Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor.

Epidemiologic studies depict a negative correlation between caffeine consumption and incidence of tumors in humans. The main pharmacological effects of caffeine are mediated by antagonism of the adenosine receptor, A2AR. Here, we examine whether the targeting of A2AR by caffeine plays a role in anti-tumor immunity. In particular, the effects of caffeine are studied in wild-type and A2AR knockout (A2AR(-/-)) mice. Tumor induction was achieved using the carcinogen 3-methylcholanthrene (3-MCA). Alternatively, tumor cells, comprised of 3-MCA-induced transformed cells or B16 melanoma cells, were inoculated into animal footpads. Cytokine release was determined in a mixed lymphocyte tumor reaction (MLTR). According to our findings, caffeine-consuming mice (0.1% in water) developed tumors at a lower rate compared to water-consuming mice (14% vs. 53%, respectively, p=0.0286, n=15/group). Within the caffeine-consuming mice, tumor-free mice displayed signs of autoimmune alopecia and pronounced leukocyte recruitment intocarcinogen injection sites. Similarly, A2AR(-/-) mice exhibited reduced rates of 3-MCA-induced tumors. In tumor inoculation studies, caffeine treatment resulted in inhibition of tumor growth and elevation in proinflammatory cytokine release over water-consuming mice, as depicted by MLTR. Addition of the adenosine receptor agonist, NECA, to MLTR resulted in a sharp decrease in IFNγ levels; this was reversed by the highly selective A2AR antagonist, ZM241385. Thus, immune response modulation through either caffeine or genetic deletion of A2AR leads to a Th1 immune profile and suppression of carcinogen-induced tumorigenesis. Taken together, our data suggest that the use of pharmacologic A2AR antagonists may hold therapeutic potential in diminishing the rate of cancer development.