LDH Measurements Research Articles

Abstract 469: Systematic analysis of circulating tumor DNA in melanoma patients to uncover mechanisms of resistance and disease clonality

Abstract Recent advances in targeted and immunotherapies have unlocked potent treatment options for malignant melanoma patients. However, targeted therapies are associated with limited response durations and only a fraction of patients benefit from immunotherapies. The analysis of circulating tumour DNA (ctDNA) provides a powerful tool for the continuous assessment of treatment responses in melanoma patients. We have established a robust pipeline for the serial, prospective analysis of ctDNA in melanoma patients. By combining next generation sequencing (NGS)- and droplet digital PCR (ddPCR)-based approaches we make ideal use of currently available technologies. To date, plasma samples from over 120 individuals have been collected, with follow-up times surpassing one year in many cases. For routine monitoring of patients commencing BRAF-mutant targeted therapies, pre-treatment DNA, derived from tumour biopsy or plasma, is tested to confirm the exact BRAF mutation and rule out pre-existing resistance-associated mutations using a custom NGS sequencing panel. Timely follow-up throughout treatment is achieved by measuring mutant BRAF fractions in ctDNA using ddPCR. When increasing BRAF variant allele frequencies are detected, additional ddPCR-based profiling for NRAS mutations and NGS-based re-sequencing for other resistance-associated mutations is initiated. We demonstrate superiority of ctDNA monitoring over LDH measurements and present a strategy to detect emerging resistant disease ahead of clinical scans in a cost-effective, timesaving and low technology way. In addition to the basic characterization of BRAF-mutant cases undergoing targeted therapy, we have used the analysis of ctDNA to uncover clonally distinct tumour sub-populations resulting in complex responses to treatment. For instance, we monitored a case of metastatic vaginal mucosal melanoma undergoing sequential targeted, immuno- and chemotherapy. Despite the presence of a KIT mutation in the primary tumour, response to KIT inhibitor imatinib was mixed. In the absence of tumour biopsies, we used whole exome-wide NGS of ctDNA to study the underlying mechanisms. This strategy revealed a KIT-mutant tumour sub-clone that responded to imatinib and a second sub-clone including an SF3B1 mutation that did not respond. The sub-clones also responded differentially to immunotherapies, but both responded to chemotherapy. The analysis of ctDNA is a powerful approach to monitor responses to systemic therapies in melanoma. By providing early warning of resistance and crucial information of clonal composition/evolution of the disease, ctDNA analysis has the potential to revolutionize the implementation of precision medicine. Citation Format: Gabriela Gremel, Rebecca J. Lee, Maria R. Girotti, Grace Garner, Amit K. Mandal, Sally Wood, Alberto Fusi, Sara Valpione, Patricio Serra-Bellver, Nathalie Dhomen, Paul Lorigan, Richard Marais. Systematic analysis of circulating tumor DNA in melanoma patients to uncover mechanisms of resistance and disease clonality. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 469.

Organelles and chromatin fragmentation of human umbilical vein endothelial cell influence by the effects of zeta potential and size of silver nanoparticles in different manners

Recently, it has been disclosed that silver nanoparticles (AgNPs) have the potential to inhibit infection and cancerous cells and eventually penetrate through injected site into the capillary due to their small size. This study focuses on the effect of size and zeta potential of bare and citrate-coated AgNPs on human umbilical vein endothelial cells (HUVECs) as main capillary cells. AgNPs with high and low concentrations and no citrate coating were synthesized by using simple wet chemical method and named as AgNP/HC, AgNP/LC, and AgNP, respectively. Citrate coated particles showed larger zeta potential of −22 mV and AgNp/HC showed the smallest size of 13.2 nm. UV-Visible spectroscopy and dynamic light scattering (DLS) were performed to evaluate particle size and hydrodynamic diameter of NPs in water and cell culture media. Results indicated that higher concentrations of citrate decreased hydrodynamic diameter and NP agglomeration. reactive oxygen species (ROS) production of all AgNPs was similar at 28 ppm although it was significantly higher than control group. Their effects on cell membrane and chromosomal structure were studied using LDH measurement and 4′,6-diamidino-2-phenylindole (DAPI) staining, as well. Results demonstrated that AgNP/LC was less toxic to cells owing to higher value of IC50, minimum inhibitory concentration (MIC), and less release of LDH. Cancerous (Human Caucasian neuroblastoma) and immortal cells (Mouse embryonic fibroblast cell line) were about twice more sensitive than HUVECs to toxic effects of AgNPs. DAPI staining results showed that AgNP and AgNP/HC induced highest and lowest breaking of chromosome. Overall results suggest that viability of HUVECs will be higher than 90% when viability of cancerous cells is 50% in AgNPs chemotherapy.

What is the acceptable hemolysis index for the measurements of plasma potassium, LDH and AST?

Hemolysis is a cause of variability in test results for plasma potassium, LDH and AST and is a non-negligible part of measurement uncertainty. However, allowable levels of hemolysis provided by reagent suppliers take neither analytical variability (trueness and precision) nor the measurand into account. Using a calibration range of hemolysis, we measured the plasma concentrations of potassium, LDH and AST, and hemolysis indices with a Cobas C501 analyzer (Roche Diagnostics(®), Meylan, France). Based on the allowable total error (according to Ricós et al.) and the expanded measurement uncertainty equation we calculated the maximum allowable bias for two concentrations of each measurand. Finally, we determined the allowable hemolysis indices for all three measurands. We observed a linear relationship between the observed increases of concentration and hemolysis indices. The LDH measurement was the most sensitive to hemolysis, followed by AST and potassium measurements. The determination of the allowable hemolysis index depends on the targeted measurand, its concentration and the chosen level of requirement of allowable total error.

The acute inflammatory response induced in mice by the venom of the giant ant Dinoponera quadriceps involves macrophage and interleukin-1β

Dinoponera quadriceps (Hymenoptera, Formicidae, Ponerinae) is a primitive and endemic ant of Northeastern Brazil, that uses its sting and associated venom gland to capture preys and for defense. Venom of Dinoponera is of potential clinical importance, since it causes intense local pain, accompanied by erythema and edema, when injected by the sting. With other hymenopteran venoms, inflammatory effects are also reported. The aim of this study was to evaluate the inflammatory activity of D. quadriceps venom (DqV) in mice. Acrylamide electrophoresis of DqV revealed five main protein bands varying between 15 and 100 kDa, confirming the proteinous nature of DqV. DqV subplantar injection elicited edema at 5 μg/kg (3 fold), 50 μg/kg (4 fold) or 500 μg/kg (7 fold) from zero to 360 min compared to saline. DqV (50 μg/kg) increased vascular permeability (4 fold) in the first hour after induction. The paw tissue histology showed moderate inflammatory focus caused by DqV (50 μg/kg) in the first hour of paw edema, but severe tissue changes (edema, inflammatory infiltrate and focal areas of hemorrhage) in the third hour. Intraperitoneal injection of DqV (50 μg/kg) stimulated neutrophil (7 fold) and mononuclear (1.4 fold) migration vs saline. DqV edematogenic effect was inhibited by dexamethasone (92%), thalidomide (82%), cyproheptadine (62%), AA861 (58%), celecoxib (34%) or l-NAME (34%), but the neutrophil migration was only by dexamethasone (57%). DqV-elicited neutrophil migration at 50 μg/kg was potentiated 1.7 fold by the animals pre-treatment with 3% thioglycolate. DqV injection increased the levels of interleukin-1 beta (IL-1β) in peritoneal cavities. DqV (50, 100 and 200 μg/mL) increased phospholipase activity (A425nm) from 10 min to 40 min. Raw 267 macrophages incubated with DqV (from 3.12 to 50 mg/mL) showed no significant decrease in cell viability or LDH measurements and at 35 μg/mL induced increase in IL-1β (from 3 to 6 h). This study demonstrated, in mice, the inflammatory effect of D. quadriceps venom, characterized by edema, increase in vascular permeability and neutrophil migration, implying the participation of resident macrophages and IL-1β, among other inflammatory mediators.

Role of Autophagy in Capsaicin-Induced Apoptosis in U251 Glioma Cells.

In recent years, the role of capsaicin in cancer prevention and treatment has gained people's attention. However, the mechanism of anti-glioma cells by capsaicin has not been elucidated. Here, we discuss the mechanism of capsaicin in U251 cells. Cell viability was detected by MTT and extracellular LDH measurements, while immunofluorescence was performed to measure changes of LC3 in U251 cells. The expressions of LC3II, Puma-α, Beclin1, P62, Procaspase-3, and P53 were observed by immunoblotting. The cell viability decreased and the punctate patterns of LC3 in U251 cells were observed after Capsaicin treatment. Meanwhile, the expressions of Beclin1, P62, and Puma-α increased. After using 3-MA, the expressions of Beclin1 and Procaspase-3 were reduced while those of P53 and Puma-α increased. The expression of LC3II was increased after Pifithrin-α treatment. Therefore, we believed that capsaicin could induce apoptosis in U251 cells, and the inhibition of autophagy could contribute to apoptosis.

Distinct kinetics and pathways of apoptosis in influenza A and B virus infection

Annual influenza epidemics are associated with high incidence and mortality rates, and are an important cause of work absenteeism and productivity losses. For successful replication, influenza viruses have evolved as to counteract and/or take a part on host defense mechanisms. Manipulation of apoptosis is one of such mechanisms that have been subject of attention, particularly in relation to influenza type A viruses over the past years. However, this knowledge has not been extended to include influenza type B viruses. In this study, MDCK-SIAT1 cells were infected with influenza A and B strains and the kinetics and pathways of apoptosis post infection were studied, through LDH measurements, Hoechst dye staining, caspase activity assays and protein expression analysis. The resulting data points to a difference in induction of apoptosis profiles between influenza A and B strains. While influenza A strain induced apoptosis later in the course of infection and mainly by the intrinsic pathway, influenza B strain induced apoptosis early in infection by both intrinsic and extrinsic pathways. Also, data revealed the IκB/NF-κB pathway as the major contributor for the observed differences. The study of the virus–host interactions, particularly those that could have an impact on viral replication, are essential in both influenza A and B viruses, as they will allow the identification of viral/host targets common to both influenza types, which could affect viral replication. This information may prove useful for vaccine and antiviral research.

Can diffusion weighted MRI differentiate between inflammatory-infectious and malignant pleural effusions?

To assess exudative pleural effusions with diffusion-weighted magnetic resonance imaging (DW-MRI) in order to determine non-invasive differentiation criteria for inflammatory-infectious and malignant effusions. Thirty-two patients with pleural effusions underwent DW-MRI with 4 different b values (10, 500, 750 and 1000 s/mm2). ADC maps were generated automatically. Signal intensity and ADC values were measured. Following MRI, pleural fluid of 10-15 ml was obtained and analyzed. AUC values were compared for different diffusion levels of ADC and SI measurements. The relationship between ADC values and pleural effusion LDH and total protein levels was examined. The cut-off values obtained from signal intensity and ADC measurements to differentiate exudates with malignant pathology were not found to be statistically significant. In the inflammatory-infectious group, a significant negative correlation was observed between ADC values and pleural fluid LDH measurements in all b values. In the malignant group, a significant positive correlation was observed between ADC values and pleural fluid total protein measurements in b values of 500 and 1000. Infectious/inflammatory and malignant effusions overlap strongly and cannot therefore be differentiated using DW MRI.

Usefulness of lactate dehydrogenase concentration in thoracic and abdominal fluid of dogs

The aims of this study were to investigate the usefulness of LDH measurement in effusions in dogs to classify the fluid as exudate or transudate and to classify the fluid based on the pathophysiological mechanism. In thoracic (n = 107) and abdominal (n = 199) fluid of dogs cell count, protein and LDH concentrations were measured. The fluid was retrospectively categorized into exudate (group A), protein-poor (B) or protein-rich transudate (C) as well as based on pathophysiology into the following five groups (group 1 - 5): hemorrhagic, chylous, inflammatory, oncotic and congestive. In thoracic and abdominal fluid LDH concentrations were significantly higher in group A compared to group B and C. There was a significant difference of LDH concentration between the groups 1 to 5 in both thoracic and abdominal fluid, however there was a large overlap between the five groups. While fluid LDH measurement in dogs is helpful to distinguish exudate from transudate it is only of little help to elucidate the pathophysiological cause.

Prognostic Value Of sFLC Ratio At Baseline On Response After Induction Therapy In Patients With Multiple Myeloma In The GMMG MM5 Trial

Studies in newly diagnosed multiple myeloma (MM) described a prognostic value of the serum free light chain ratio (sFLCr) on progression free survival (PFS) and overall survival (OS). The GMMG MM5 phase III trial compares VCD (bortezomib, cyclophosphamide, dexamethasone) and PAd (bortezomib, adriamycin, dose-reduced dexamethasone) for induction therapy followed by stem cell mobilization and harvest, high-dose therapy and a lenalidomide-based consolidation/maintenance therapy for 2 years vs. lenalidomide until complete response (CR). First primary end point of the ongoing study was response after induction, second primary end point was progression-free survival. To analyse if the sFLCr has a prognostic value on the response after induction therapy with respect to achieve a very good partial response (VGPR) or better, we investigated patients with newly diagnosed MM which were included in the MM5 trial.504 patients between 18-70 years with newly diagnosed MM were included in the MM5 trial between July 2010 and October 2012. Patient serum samples were collected and sent to a central laboratory for analysis of the free light chains (sFLC) in serum prior to treatment and after induction therapy. For the sFLC measurement the Freelite® assay (The Binding Site) was used.The analysis was based on the intention-to-treat (ITT) population (502 patients evaluable). The sFLC ratio (sFLCr) was available for n=498 patients at baseline. After induction treatment, there were 179 patients achieving CR and VGPR (VGPR+ group) and 318 patients not achieving at least VGPR (PR- group). There was no significant difference in the response between the PAd and the VCD (reported separately), so we analysed the data irrespective of the treatment arm.We examined in univariate and multivariate analyses the association of response VGPR+ after induction treatment with clinical variables (age, gender, ISS, CRAB criteria, IgA vs non-IgA type MM, WHO status), LDH measurement, cytogenetic factors (deletion 17p13, gain 1q21, translocation (4;14) ) and sFLCr at baseline. sFLCr was analysed either as categorized factor for values within the range [1/32, 32] or outside this range, as proposed by Snozek et al. (2008), or as continuous factor, where the absolute value of log2 transformed sFLCr is used for analyses. Further, LDH values were log2 transformed for analyses. In univariate analysis, the groups of VGPR+ and PR- showed no significant difference in age, gender, ISS, presence of anemia or bone disease, WHO state or the cytogenetic abnormalities del17p13, gain 1q21 or translocation t(4;14), but were significantly associated with elevated Calcium (p=0.07), renal insufficiency (p=0.004), presence of IgA type MM (p<0.001) and LDH measurement (p<0.001). There was no significant difference between patients with sFLCr at baseline within or outside the range set at 1/32 – 32 in achieving VGPR+ (p=0.23) whereas the absolute value of log2 transformed values was significantly associated with response (p=0.02).VGPR+ was further analysed by multivariate logistic regression models which were adjusted for the clinical and cytogenetic variables, for LDH and for sFLCr (either as categorical or continuous factor). sFLCr revealed as significant prognostic factor when considered as continuous variable (p = 0.03) but failed significance as categorized factor. Patients with extreme sFLCr values showed a better chance of VGPR+, as did IgA myeloma patients (p=0.03) and patients with renal insufficiency (p=0.04).Cut point analyses showed that in our trial patients with a baseline sFLCr < 1/1097 and >1097 had a significantly better chance to achieve VGPR+ after induction compared to patients with a sFLCr within this range. There were 44 patients below the range (IgA 22.7%, IgG 34.1%, IgD 6.8%, light chain MM (LCMM) 36.4%), 412 patients in the range (IgA 20.9%,IgG 63.8%, IgD 1%, IgM 0.2%, LCMM 14.1%) and 42 patients above the range (IgA 19.1%, IgG 42.9%, LCMM 38.1%).In conclusion, the cut point for baseline sFLCr published by Snozek et al. did not define patient groups with significantly different response in the MM5 trial. This cut point was set for the outcome OS and PFS and could not be replicated for response after induction treatment in our trial. The cut point which showed a significant difference in achieving VGPR+ was much higher in our population which might be explained by the higher proportion of light chain MM below and above the cut point range. Disclosures:Salwender:Janssen Cilag: Honoraria; Celgene: Honoraria. Duerig:Janssen Cilag: Honoraria; Celgene: Honoraria. Schmidt-Wolf:Janssen: Honoraria; Novartis: Honoraria. Weisel:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Scheid:Janssen Cilag: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Goldschmidt:Chugai: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen Cilag: Consultancy, Honoraria, Research Funding; The Binding Site: Provision of materials, Provision of materials Other.

LDH as a predictor of in-hospital and late mortality in acute pulmonary embolism

Purpose: Acute pulmonary embolism (PE) is a common cardiovascular emergency, which can be acute life-threatening and can have a dismal long-term prognosis. Prognostic assessment of these patients is based mainly on hemodynamic status, echocardiography findings and biochemical markers of myocardial injury. However, LDH measurement may have a role in the risk stratification of the patients. The aim of this study was to assess the value of LDH as a mortality predictor in acute PE. Methods: Retrospective, observational study including 165 patients with acute PE (age 72.8±16.4; 76 males), diagnosed by pulmonary CT angiography in the emergency department, between January 2010 and December 2010. The studied endpoints were in-hospital mortality and follow-up all-cause mortality. A receiver operating characteristics (ROC) curve was used to define the best cut-off between two groups [group 1 (LDH > 310 U/L, n=53) and group 2 (LDH ≤ 310 U/L, n=112)], regarding all-cause mortality. Mean follow-up time was 415±358 days. Results: No differences were found between the 2 groups regarding demographic data, symptoms and cardiovascular risk factors. Area under the ROC curve (AUC) was 0.626. The best cut off value was 310 U/L (sensitivity 54.5%, specificity 71.3%). In univariate regression analysis, a LDH higher than 310 U/L was found to be strongly associated with worse outcome [Hazard-ratio: 2.841 (95% CI, 1.226 – 6.584), p=0.015]. Patients from group 1 had a significantly higher in-hospital (18.2% vs. 7.4%, p<0.05) and long-term mortality (22.6% vs. 8.9%, Log-rank p<0.02). ![Figure][1] Kaplan-Meier survival analysis Conclusions: LDH is a predictor of both in-hospital and all-cause mortality at follow-up after acute PE. Its addition to current risk stratifying systems may be of interest. [1]: pending:yes

Studies on the use of BD Vacutainer® SST II™ and RST™ in general practice: investigation of artefactual hyperkalaemia

Current sampling and transport conditions of samples in general practice can result in pseudohyperkalaemia. This study was undertaken to determine, in a general practice setting, whether there is any difference in haemolysis obtained when using BD Vacutainer® Rapid Serum Tubes (BD RST) compared with using BD Vacutainer® SST™ II Advance Blood Collection Tubes (BD SSTII). Blood was collected from 353 patients requiring blood sampling who were attending 31 general practitioner practices in Belgium. For each patient, two BD SSTII tubes and two BD RST tubes were drawn in a randomized order. One of each pair of tubes was inverted five times, the other was not. Serum potassium concentration, serum LDH activity and haemolysis index were measured in each sample. There was no significant difference in measured potassium concentration according to tube type (P = 0.16). Measured LDH activities were 1.7% higher in serum collected into BD SSTII tubes compared to BD RST tubes (P = 0.02). When comparing serum from unmixed BD RST with BD SSTII tubes, there was a slight reduction in the haemolysis index but no significant difference in measured potassium concentration or LDH activity. Risk of hyperkalaemia was 4.8 times higher in serum from tubes that were incompletely filled compared to those that were filled with the correct amount of blood. Both types of blood tubes are suitable for the measurement of serum potassium and LDH in patients from general practice. Tube inversion does not improve the accuracy of either serum potassium or LDH measurement. Blood tubes should be filled to the level recommended by the manufacturer to avoid artefactual increases in measured serum potassium concentration and LDH activity.

Identification of Clinical Biomarkers for Pre-Analytical Quality Control of Blood Samples

Pre-analytical conditions are key factors in maintaining the high quality of biospecimens. They are necessary for accurate reproducibility of experiments in the field of biomarker discovery as well as achieving optimal specificity of laboratory tests for clinical diagnosis. In research at the National Biobank of Korea, we evaluated the impact of pre-analytical conditions on the stability of biobanked blood samples by measuring biochemical analytes commonly used in clinical laboratory tests. We measured 10 routine laboratory analytes in serum and plasma samples from healthy donors (n = 50) with a chemistry autoanalyzer (Hitachi 7600-110). The analyte measurements were made at different time courses based on delay of blood fractionation, freezing delay of fractionated serum and plasma samples, and at different cycles (0, 1, 3, 6, 9) of freeze-thawing. Statistically significant changes from the reference sample mean were determined using the repeated-measures ANOVA and the significant change limit (SCL). The serum levels of GGT and LDH were changed significantly depending on both the time interval between blood collection and fractionation and the time interval between fractionation and freezing of serum and plasma samples. The glucose level was most sensitive only to the elapsed time between blood collection and centrifugation for blood fractionation. Based on these findings, a simple formula (glucose decrease by 1.387 mg/dL per hour) was derived to estimate the length of time delay after blood collection. In addition, AST, BUN, GGT, and LDH showed sensitive responses to repeated freeze-thaw cycles of serum and plasma samples. These results suggest that GGT and LDH measurements can be used as quality control markers for certain pre-analytical conditions (eg, delayed processing or repeated freeze-thawing) of blood samples which are either directly used in the laboratory tests or stored for future research in the biobank.

S-100B: A Stronger Prognostic Biomarker than LDH in Stage IIIB–C Melanoma

In melanoma patients with nodal macrometastases, the distinction between good and poor prognosis is based on the presence of primary melanoma ulceration or metastatic involvement of 4 or more lymph nodes in the 7th edition of the American Joint Committee on Cancer (AJCC) classification. We hypothesized that biomarkers would increase the accurateness of staging in these patients. The aim was to assess and compare the prognostic impact of biomarkers S-100B and LDH and to determine the best timing of their measurement in stage IIIB-C melanoma. A total of 119 patients underwent therapeutic lymph node dissection (TLND) for nodal macrometastases with serum S-100B and LDH level measurements preoperatively. In 75 of them, S-100B and LDH were also measured on postoperative days 1 and 2. S-100B and LDH levels on days 0, 1, and 2 were compared for their association with disease-free survival (DFS) and disease-specific survival (DSS). At a median follow-up of 17 (range 1-89) months, S-100B levels at all time points were associated with DFS. In multivariable analysis, preoperative S-100B and S-100B measured on day 2 showed the strongest association with DFS (hazard ratio [HR] 2.55, P=0.007 and HR 3.80, P=0.01). For DSS, the preoperative S-100B level was the strongest independent predictor (HR 2.81, P=0.01). LDH measurements showed a significant association with DSS in univariate analysis only when measured preoperatively (HR 2.46, P=0.01). In multivariable analysis, LDH measurement was not associated with melanoma prognosis. The S-100B level measured preoperatively is, in contrast to LDH, one of the most important independent predictors of melanoma prognosis in patients undergoing TLND for nodal macrometastases.

Comparative study of respiratory tract immune toxicity induced by three sterilisation nanoparticles: Silver, zinc oxide and titanium dioxide

Silver, zinc oxide, and titanium dioxide nanoparticles are used as sterilisation materials to enhance the performance of disinfectants. We investigated the respiratory tract immune toxicity (“immunotoxicity”) of these nanoparticles in vivo and in vitro, and we explored the relationships between particle size, particle shape, chemical composition, chemical stability and the toxicological effects of these typical nanoparticles in rats. In vivo, the rats were exposed to nanoparticles by intratracheal instillation. Exposure to nanoparticles caused an increase in oxidative injury to the lungs and disorders in regulating the cytokine network, which were detected in the bronchoalveolar lavage fluid, suggesting that oxidative stress might be important for inducing the respiratory immunotoxicity of nanoparticles. In vitro, the phagocytic function of alveolar macrophages (AMs) was dose-dependently reduced by nanoparticles, and ZnO nanoparticles induced greater cytotoxicity than the silver and titanium-dioxide nanoparticles, which were coincident with the results of multiple measurements, such as a cell viability assay by WST-8 and LDH measurements. Comparative analyses demonstrated that particle composition and chemical stability most likely had a primary role in the biological effects of different nanoparticles.

Ischemia Modified Albumin Can Predict Necrosis at Incarcerated Hernias

Purpose. To evaluate the predictive effect of IMA in incarcerated hernias. Methods. Three groups (n = 7) of rats were operated. Group I aimed to mimic incarceration, group II aimed the strangulation, and group III was the sham group. IMA and LDH measurements were made. Results. IMA levels were significantly higher in strangulation mimicking group and IMA levels were normal at postoperative 6th hour in incarceration mimicking group. LDH levels were significantly higher in both incarceration and strangulation mimicking groups. Conclusion. IMA seems to be an effective marker in incarcerated hernias to predict necrosis. But we need further studies to generalise this hypothesis.

Photodynamic activities of protoporphyrin IX and its dopamine conjugate against cancer and bacterial cell viability

The photoinactivation of three different human cancer cell lines, HeLa, neuroblastoma and U87 and antimicrobial activities against Gram-positive (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) by protoporphyrin IX and protoporphyrin–dopamine conjugate were examined. The antimicrobial activities were screened by optical density measurement at 600 nm. Also, the cytotoxicity and proliferation assays were performed with LDH measurements at 490 nm and WST-1 assays at 450 nm, respectively, with and without irradiation.

The clinical features of patients with the homozygous 235delC and the compound-heterozygous Y136X/G45E of the GJB2 mutations (Connexin 26) in cochlear implant recipients

This study aimed to investigate the prevalence of GJB2 gene for the 235delC mutations, the clinical features and the outcomes of patients who had undergone cochlear implantation. We have sequenced the coding region of GJB2 gene for 135 patients with sensorineural deaf from September 2000 to May 2009. Of the 135 patients, the patients with the homozygous 235delC and the compound-heterozygous Y136X/G45E were detected and were investigated clinically. The GJB2 gene for the 235delC mutations was found in 39 alleles of 270 alleles (14.4%), especially for the homozygous of 235delC was detected in 26 alleles (9.6%), the single heterozygous of 235delC was 1 allele (0.4%), the compound heterozygous of 235delC was found in 12 alleles (4.4%). Of 16 subjects (29 alleles) with the homozygous 235delC and the compound-heterozygous Y136X/G45E, 2 subjects (4 alleles) were found to have complications. All of the subjects were found to show severe hearing loss and some of them have indicated progressive hearing loss. However, they showed better performance regarding the thresholds after implantation. The subjects with complications, although, suggested poorer performance in the auditory speech performance. The findings of poorer outcomes might depend on complications and brain functions. In addition, considering the blood test parameters, an independent elevated LDH and ChE at diagnosis were found to be associated with hereditary enzyme's metabolic disease. Therefore, the value of LDH measurements in patients might be a helpful predictive parameter in hereditary diseases.

Correlation between prior therapeutic dendritic cell vaccination and the outcome of patients with metastatic melanoma treated with ipilimumab

e20006 Background: Ipilimumab (Ipi) is an anti-cytotoxic T lymphocyte associated antigen 4 (CTLA4) IgG1 monoclonal antibody active against metastatic melanoma. Ipi acts by reinforcement of CTL-activation trough B7/CD28 receptor stimulation. A pre- existing anti-tumor CTL repertoire, induced by dendritic cell (DC) vaccination, could influence the therapeutic effect of Ipi. The influence of prior DC-vaccination on the outcome of melanoma patients (pts) treated with Ipi at a single center was analyzed to evaluate this hypothesis. Methods: Data were obtained from the medical files of pts treated at the UZ Brussel with Ipi in protocols CA184–022, -025, and the BMS Ipi medical need program. Ipi (0.3, 3 or 10 mg/kg) was administered iv q3 wks x4 q12 wks thereafter. Results: 20 pts (10M/10F; med age 51y, range 28–72) were identified. Prior therapy: autologous DC-vaccine loaded with melanoma associated antigens (14 pts) ± IFN-a2b (9/14 pts), cytotoxic agents (17 pts). Baseline characteristics: AJCC st IV melanoma (20 pts); baseline LDH &gt; ULN (16 pts). Ipi dose: 0.3 mg/kg (4 pts), 3 mg/kg (2 pts), 10 mg/kg (14 pts). Serial LDH measurements were available for 17 pts; 4 types of LDH-response following Ipi administration were observed: increase (11 pts), decrease (1 pt), fluctuation (4 pts), stable (1 pt). Objective response according to mWHO or irWHO: 3 PR, 1 SD, 16 PD. One pt received 0.3 mg/kg of Ipi (CA184–022) and had SD but no LDH response; at PD she received DC-vaccine and subsequently 10 mg/kg Ipi (medical need program) resulting in a fluctuating LDH-response and regression of an orbita-metastasis. Increasing LDH values following Ipi were less often observed in pts with prior DC-vaccination (5/6 pts (83%) without prior DC-vaccination had increasing LDH-values vs 6/12 pts (50%) with prior vaccination). DC- vaccination also correlated with regression of metastases and disease control (3/15 pts (20%) who were vaccinated had a PR/SD vs 1/6 (17%) PR for those who had not received prior DC-vaccinations). Conclusions: These data represent a small sample size from a single institution; however, our results suggests a potential correlation between prior therapeutic DC vaccination and outcomes in advanced melanoma patients treated with ipilimumab. [Table: see text]

Commutable Calibrator with Value Assigned by the IFCC Reference Procedure to Harmonize Serum Lactate Dehydrogenase Activity Results Measured by 2 Different Methods

The availability of commutable calibrator materials may ease considerably the task of harmonizing assay results and ensuring their traceability to reference procedures. We sought to verify the commutability of potential calibrator materials and evaluate their effectiveness in harmonizing LDH results by 2 measurement methods. We measured LDH in 109 serum samples and 31 materials, including frozen serum pools (with either normal or abnormal isoenzyme patterns), commercial stabilized materials, and the ERM-AD453/IFCC reference material. We assayed LDH activity with the IFCC reference procedure and with 2 commercial methods, 1 using the lactate-to-pyruvate (LP) reaction, and the other the pyruvate-to-lactate (PL) reaction. We selected a commutable material, with LDH value assigned by the reference procedure, as a calibrator for recalculating the results for patient sera by both LP and PL, thereby making them traceable to the IFCC reference procedure. Original values for patient sera (n = 109) by the 2 commercial methods showed a mean (SD) PL/LP ratio of 1.97 (0.03); this ratio changed to 1.06 (0.02) after recalculation of results. Linear regression of PL vs LP recalibrated values gave y = 1.108x - 9.7. At the clinically important concentration of 250 U/L (upper reference limit), the systematic difference between methods was 6.8%, which met our proposed quality specifications for inaccuracy and total error. By properly selecting the calibrator, the results of serum LDH measurement by 2 different methods may be harmonized and made traceable to the selected highest (reference) metrological level.

Influence of cyclodextrins on the proliferation of HaCaT keratinocytes in vitro

Safety and efficacy of pharmaceutical agents can be greatly improved by encapsulation within, or covalent attachment to, a biomaterial carrier. Drug delivery systems must deliver the necessary amount of drug to the targeted site for a necessary period of time, both efficiently and precisely. Various kinds of high-performance biomaterials are being constantly developed for this purpose. Cyclodextrins are potential candidates for such a role, because of their ability to alter physical, chemical, and biological properties of guest molecules through the formation of inclusion complexes. The alpha-, beta-, and gamma-cyclodextrins are widely used natural cyclodextrins, consisting of six, seven, and eight D-glucopyranose residues, respectively, linked by -1,4 glycosidic bonds into a macro cycle. Each cyclodextrin has its own ability to form inclusion complexes with specific guests, an ability, which depends on a proper fit of the guest molecule into the hydrophobic cyclodextrin cavity. The most common pharmaceutical application of cyclodextrins is to enhance the solubility, stability, and bioavailability of drug molecules. Such kinds of ligand-receptor complexes can be used for different applications, e.g., for a transdermal therapeutic system (TTS) or in biofunctional textiles. The aim of this study was the investigation of the influence of the different cyclodextrins on the cell proliferation using HaCaT keratinocytes as an in vitro test system. Moreover, the study was performed to find harmless and nontoxic cyclodextrin concentrations for dermal applications. By means of different independent in vitro tests could be confirmed that alpha-, beta-, and gamma-cyclodextrins in concentrations up to 0.1% (w/v) do not show any antiproliferative influence on HaCaT keratinocytes. Sometimes even proliferative effects could be found. However, all used cyclodextrins (besides gamma-cyclodextrin and its derivatives) in concentrations of 0.5 and 1% (w/v), respectively, exert a cytotoxic influence on the proliferation of HaCaT keratinocytes. On the basis of these findings, the following rank order of cyclodextrins regarding their cytotoxicity was proposed: M-beta-CD > beta-CD > HP-beta-CD > alpha-CD > (gamma-CD). It could be confirmed that beta-CD and M-beta-CD trigger the activity of the effectors caspases -3 and -7. A significant increase of LDH release could be found for beta-CD and methyl-beta-CD in concentrations of 0.5 and 1% (w/v). The calculated cytotoxicity amounted 45 and 79%, respectively. The measurements of the interleukins IL-6 and IL-8 confirmed the findings of the proliferation assays as well as the LDH measurements. These findings may provide further rationale to the use of CDs in topical formulations for dermal and transdermal drug delivery or as raw material to functionalize textiles for medical applications.