Admission Circulating Cell-Free DNA Levels as a Prognostic Factor in Pediatric Burns.

Background: Burn injuries in children are a major physical and psychological trauma, often a severe condition with long-term consequences. Current methods of assessing the extent of burn injuries on admission are inaccurate. Circulating cell-free DNA (cfDNA) is a potential marker of tissue damage that may be useful in burn care. Objective: To explore the use of cfDNA admission levels as a prognostic marker of pediatric burn severity and outcome. Methods: cfDNA levels of 38 pediatric burn patients (otherwise healthy) and 12 matched pediatric controls (minor elective surgery patients) admitted to our center were quantified by a direct fluorometric assay. Results: We found significantly higher admission cfDNA levels in the patient group (median 724 ng/ml, range 44-4405), compared to the control group (median 423 ng/ml, range 206-970, Mann-Whitney, P = 0.03) and a significant difference between cfDNA levels of partial-thickness burns (median 590 ng/ml, range 44-2909) and full-thickness burns (median 2394 ng/ml, range 528-4405, Mann-Whitney, P = 0.01). We also found significant correlations between cfDNA levels and hospitalization duration (Spearman, R = 0.42, P < 0.01) and undergoing surgical procedures (Spearman, R = 0.40, P < 0.01). PICU admission did not correlate to cfDNA levels (Spearman, R = 0.14, P = NS). Discussion. Admission cfDNA levels may be a valuable objective tool for assessing the severity of pediatric burn injuries on admission, including correlations with the length of hospitalization and surgical burden. Conclusion: Admission cfDNA levels may be a promising novel pediatric burn assessment method. Further investigation of cfDNA levels in healthy children standardized to age and larger cohorts are needed to establish cfDNA as a valuable prognostic factor for pediatric burn injury.

Cell-free DNA and telomere length among women undergoing in vitro fertilization treatment.

PURPOSE: The current research is aimed at finding potential non-invasive bio-markers that will help us learn more about the mechanisms at play in failed assisted reproduction treatment. This exploratory pilot study examined the relationship between cell-free DNA (CFD) in plasma and telomere length in lymphocytes among women undergoing in vitro fertilization (IVF) and compared telomere length and CFD levels to a healthy control group. METHODS: Blood of 20 women undergoing IVF was collected at three time points during the IVF cycle. We assessed the relationship between CFD and telomere length as well as controlling for morning cortisol levels. We also collected blood of 10 healthy controls at two time points (luteal and follicular phases of the menstrual cycle) and compared mean telomere length, CFD, and cortisol levels between the IVF patients and healthy controls. RESULTS: The results revealed an inverse relationship between CFD levels and telomere lengths at several time points that remained significant even after controlling for cortisol levels. Women undergoing IVF had statistically significant higher levels of CFD and shorter telomeres compared to healthy controls. CONCLUSIONS: The relationship between telomere length and CFD should be further explored in larger studies in order to uncover potential mechanisms that cause both shortened telomere length and elevated CFD in women undergoing IVF.

Adenosine in burn blister fluid.

BACKGROUND: Burn wound blister fluid is known to sustain suppressive effects on various components of the immune system. Damaged tissues cause an increase of adenosine concentrations. Since adenosine is a potent anti-inflammatory agent we hypothesized that burn blister fluid contains high concentrations of this nucleoside. METHODS: Burn blister fluid was drawn from eleven patients who suffered a second degree burn injury. Adenosine concentrations were determined using high performance liquid chromatography (HPLC). RESULTS: Elevated adenosine levels were detected in 6 of the 11 patients (54.5%), with an overall mean of 1.13+/-0.52 mM. CONCLUSIONS: This is the first documented data showing increased concentrations of adenosine in burn blister fluid.

Adenosine is upregulated during peritonitis and is involved in downregulation of inflammation.

Loss of function of the peritoneal membrane is associated with peritonitis. Adenosine levels in sites of inflammation were shown to increase and exhibit immunoregulatory effects. Our aim was to elucidate the regulatory role of adenosine during peritonitis and to test the involvement of peritoneal mesothelial cells (PMC) in adenosine regulation. In a mice model of Escherichia coli peritonitis, the adenosine A(2A) receptor (A(2A)R) agonist (CGS21680) prevented leukocyte recruitment and reduced tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) levels. Peritonitis induced the elevation of adenosine with a peak at 24 h. Analysis of adenosine receptor levels on peritoneum showed that A(1) receptor (A(1)R) protein levels peak at 12 h after inoculation and then return to baseline at 24 h, whereas high affinity A(2A)R protein levels peak at 24 h concomitantly with the peak of adenosine concentration. Low affinity A(2B) receptor (A(2B)R) levels elevated slowly, remaining elevated up to 48 h. In human PMC (HPMC), the early cytokines, IL-1-alpha, and TNF-alpha upregulated the A(2B) and A(2A) receptors. However, interferon-gamma (IFN-gamma) upregulated the A(2B)R and decreased A(2A)R levels. Treatment with the A(2A)R agonist reduced IL-1-dependent IL-6 secretion from HPMC. In conclusion, the kinetics of adenosine receptors suggest that at early stage of peritonitis, the A(1)R dominates, and later its dominance is replaced by the G stimulatory (Gs) protein-coupled A(2A)R that suppresses inflammation. Early proinflammatory cytokines are an inducer of the A(2A)R and this receptor reduces their production and leukocyte recruitment. Future treatment with adenosine agonists should be considered for attenuating the damage to mesothelium during the course of acute peritonitis.

Interleukin-15 is the main mediator of lymphocyte proliferation in cultures mixed with human kidney tubular epithelial cells.

BACKGROUND: Interleukin- (IL) 15 is a potent non-T cell-derived cytokine with IL-2-like activities. Elevated levels of IL-15 expression were observed in biopsies of acutely rejected human kidney grafts. We tested the role of IL-15 in mixed lymphocyte kidney reaction (MLKR) and the effects of immunosuppressive drugs on this reaction. METHODS: Primary cultures of human tubular epithelial cells (TEC) were stimulated by interferon-gamma and treated with cyclosporin A (CsA, 10-1000 ng/ml), rapamycin (Rapa, 2.5-10 ng/ml), and dexamethasone (Dex, 10-10-10-7 M). IL-15 levels were measured by ELISA. To induce MLKR, we seeded OKT3-prestimulated allogenic human peripheral blood mononuclear cells (PBMC) on a monolayer of TEC in the presence of CsA (25-250 ng/ml), Rapa (0.25-1 ng/ml), and Dex (10-10-10-7 M). PBMC proliferation was quantified by 3H-thymidine incorporation. RESULTS: CsA, Dex, and Rapa had no effect on IL-15 production by TEC. The presence of TEC induced marked proliferation of PBMC. Pretreatment of TEC with IFN-gamma enhanced MLKR in direct correlation with the increased IL-15 levels. MLKR was blocked by anti-IL-15, but not significantly by anti-IL-2 monoclonal antibody. Contrary to Rapa and Dex, CsA failed to inhibit MLKR CONCLUSIONS: IL-15 is a major mediator of lymphocyte proliferation in MLKR. Its production by TEC was unaffected by CsA, Rapa, and Dex. However, IL-15 activity is effectively inhibited by Rapa and Dex but not by CsA. The diversity in the effects of the various drugs is probably related to the different mechanisms. Our results support the possible involvement of renal IL-15 in graft rejection and suggest that resistance to CsA treatment is related to its failure to decrease IL-15 activity.

Regulation of TNF-alpha by 1alpha,25-dihydroxyvitamin D3 in human macrophages from CAPD patients.

BACKGROUND: We have previously reported that 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] accumulates in the dialysis fluid of uremic patients treated by continuous ambulatory peritoneal dialysis (CAPD). It has been reported that this metabolite regulates the production of cytokines by monocytes/macrophages. Since tumor necrosis factor-alpha (TNF-alpha) initiates an inflammatory cascade during peritonitis, the aim of the present study was to investigate the effect of 1alpha, 25(OH)2D3 on the production of TNF-alpha by human peritoneal macrophages (HPMs). METHODS: HPMs were obtained from patients on CAPD. Cells were incubated with various concentrations of 1alpha, 25(OH)2D3, 1alpha,24(S) dihydroxyvitamin D2 [1alpha,24(S)(OH)2D2] or 25-hydroxyvitamin D3 (25-OH-D3) for 16 hours. This was followed by lipopolysaccharide (LPS; 1 microg/mL) incubation for 2.5 to 6 hours. TNF-alpha protein production was determined by enzyme-linked immunosorbent assay. TNF-alpha mRNA was assayed by the reverse transcriptase-polymerase chain reaction procedure, using internal synthetic mRNA standards for quantitative results. RESULTS: Incubation of HPMs with 1alpha,25(OH)2D3 prior to stimulation with LPS dose dependently inhibited the expression of TNF-alpha on both mRNA and protein levels. Similar results were obtained with the less calcemic vitamin D2 analogue 1alpha,24(S)(OH)2D2. Incubation of HPMs with 25-OH-D3 also revealed a down-regulation of TNF-alpha expression. Since this down-regulatory effect was blocked by ketoconazole, it is likely that this effect was caused by the conversion of 25-OH-D3 into 1alpha,25(OH)2D3 by HPMs. CONCLUSIONS: 1alpha,25(OH)2D3 has a potent inhibitory effect on the production of TNF-alpha by LPS-activated HPMs. We hypothesize that 1alpha, 25(OH)2D3 may constitute a regulatory mechanism that, by controlling the intensity of the inflammatory response of the peritoneum, will moderate tissue damage during peritonitis.

Gel clot LAL assay in the initial management of peritoneal dialysis patients with peritonitis: a retrospective study.

BACKGROUND: Indiscriminate use of broad-spectrum antibiotic treatment of peritonitis in peritoneal dialysis patients may have either unwanted side-effects or contribute to the development of antibiotic resistance. This may be avoided by improved diagnosis at presentation. The Limulus amoebocyte lysate assay is a convenient test detecting bacterial endotoxins or fungal beta glucans. This study evaluates a qualitative Limulus amoebocyte lysate test as a diagnostic tool used at presentation of a peritoneal dialysis patient with peritonitis. METHODS: One-hundred and eleven episodes of peritonitis in peritoneal dialysis patients have been analysed retrospectively. Limulus amoebocyte lysate results at presentation were compared with culture results. A Limulus amoebocyte lysate assay was performed using a commercial kit by incubating a mixture of dialysate effluent and Limulus amoebocyte lysate reagent at 37 degrees C. The development of a stable solid clot was considered positive. The specificity and sensitivity of the test were calculated. RESULTS: The specificity of the Limulus amoebocyte lysate assay was found to be 98% and the sensitivity 74%. Limulus amoebocyte lysate assay was false-negative in 13 cases of Gram-negative peritonitis (22%). Limulus amoebocyte lysate was positive in three of seven cases of fungal peritonitis. The study included one case each with false-positive Limulus amoebocyte lysate and with culture-negative peritonitis. CONCLUSIONS: The Limulus amoebocyte lysate assay is a convenient and valuable diagnostic tool for excluding Gram-positive peritonitis in peritoneal dialysis patients. This allows more specific antibiotic treatment at presentation and may avoid the development of bacterial resistance. A negative Limulus amoebocyte lysate test is not reliable for the exclusion of Gram-negative peritonitis. In the absence of a positive culture result 48 h after presentation, accompanied by a delayed response to treatment, a positive Limulus amoebocyte lysate assay may indicate the presence of fungus. This justifies early empiric antifungal treatment before definitive culture results are made available. Routine Limulus amoebocyte lysate assay of dialysate effluent from continuous ambulatory peritoneal dialysis patients presenting with peritonitis is recommended.

Accessory role of human peritoneal mesothelial cells in antigen presentation and T-cell growth.

BACKGROUND: To assess the role of human peritoneal mesothelial cells (HPMCs) in the generation of an immune response during peritonitis, we tested their ability to activate T-cells by antigen presentation (AP) and by the secretion of interleukin-15 (IL-15). IL-15 is a potent leukocyte activator that stimulates the proliferation of CD4+, CD8+, and B and natural killer (NK) cells. METHODS: HPMCs and mononuclear cells were derived from six volunteer patients who underwent elective abdominal surgery. Flow cytometry was used to analyze human lymphocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1), and B7 molecules on HPMCs. Affinity-purified CD4 cells were used for AP assays. We used a specific enzyme-linked immunosorbent assay to detect interferon-gamma (IFN-gamma), IL-2, and IL-15 protein and reverse transcription-polymerase chain reaction for mRNA analysis. RESULTS: HPMCs expressed HLA-DR molecules following IFN-gamma treatment. ICAM-1 molecules were expressed at high levels, and B7-1 and B7-2 molecules could not be detected. The accessory function of HPMCs was assayed by T-cell stimulation using anti-CD3 antibodies (OKT3). HPMCs were essential for a significant OKT3-induced T-cell proliferation. Anti-ICAM-1 antibodies blocked OKT3-induced proliferation. HPMCs served as effective antigen-presenting cells when Tetanus toxoid (TT) or Staphylococcus aureus-alpha-toxin were used as antigens. IFN-gamma, IL-2, and IL-15 accumulated during AP reactions. We found that IL-15 is produced by HPMCs, and IFN-gamma up-regulated its mRNA levels and protein secretion in a dose-dependent manner. We also detected IL-15 in the peritoneal effluent of patients undergoing continuous peritoneal dialysis treatment. In patients suffering from peritonitis, IL-15 levels were elevated (35.0 +/- 6.0 pg/mL, N = 10) as compared with noninfected patients (16.2 +/- 4.0 pg/mL, N = 7). CONCLUSIONS: HPMCs participate in the peritoneal immune response against invading pathogens by AP. For this process, ICAM-1 is the major accessory molecule. In addition, HPMCs may contribute to T-cell activation by secretion of IL-15.

Raised interleukin-6 levels in obese patients.

OBJECTIVE: Obese patients demonstrate a variety of biochemical, metabolic, and pulmonary abnormalities. Inflammatory mediators such as tumor necrosis factor-alpha and interleukin-6 (IL-6) may have a direct effect on glucose and lipid metabolism. Hypoxemia in itself induces release of IL-6. The aim of this study was to examine the relationship between IL-6 levels in healthy volunteers (control group) and three different groups of obese patients: patients without obstructive sleep apnea syndrome (OSAS), patients with OSAS, and patients with obesity hypoventilation syndrome (OHS) (daytime baseline oxygen saturation of <93%). RESEARCH METHODS AND PROCEDURES: We measured serum IL-6 levels in 25 obese patients (body mass index of >35 kg/m2) and 12 healthy women. RESULTS: The results demonstrate statistically significant differences in serum IL-6 levels between the control group (1.28 +/- 0.85 pg/mL) and obese patients without OSAS (7.69 +/- 5.06 pg/mL, p < 0.05) and with OSAS (5.58 +/- 0.37 pg/mL, p < 0.0005). In the patients with OHS, IL-6 concentrations were highest (43.13 +/- 24.27 pg/mL). DISCUSSION: We conclude that serum IL-6 is increased in obese patients. The highest IL-6 levels were found in the patients with OHS.