Lactic Acid Dehydrogenase Levels Research Articles

Evaluation of Lactate Dehydrogenase and Gamma Glutamyl Transferase Among Pregnant Women with Hypertensive Disorders and Their Association with Disease Severity in Jimma Medical Center, Ethiopia

Background & Objective: Pregnancy-induced hypertensive disorders (PIHD) are the main reasons for maternal and perinatal mortality, as they complicate 10% of pregnancies worldwide. Serum lactic acid dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) are possible markers reflecting the occurrence of pregnancy-associated complications like preeclampsia and eclampsia. There is a paucity of data with conflicting results showing serum LDH and GGT on PIHD in Ethiopia. This investigation aimed to assess the serum LDH and GGT levels in pregnant women with PIHD along with their correlation with the severity of the disease at Jimma Medical Center (JMC), Ethiopia.Materials & Methods: This hospital-based comparative cross-sectional study was undertaken from August 03 to September 27, 2020, in JMC. A total of 97 study participants were recruited. Serum GGT and LDH levels were determined using a fully automated Roche Cobas 6000 chemistry analyzer. The data were analyzed using SPSS 25.0. One-way ANOVA and independent samples t-test were employed to compare serum GGT and LDH levels with categories of PIHD.Results: The significantly highest mean serum levels of LDH (580.9±193.8 U/L) and GGT (86.1±29.2 U/L) were observed in eclamptic women compared to gestational hypertensive (276.7±60.7 and 38.3±16.9 U/L) and preeclamptic patients (353±132.8 and 48.8±29.9 U/L), respectively. Both serum GGT and LDH levels were found to correlate with the severity of preeclampsia, respectively significantly.Conclusion: Serum LDH and GGT were found to be at the highest levels in eclamptic than preeclamptic and gestational hypertensive women. Blood pressure, gestational age, and severity of hypertensive disorders of pregnancy were predictor variables associated with serum GGT and LDH.

Association of the Transmembrane Serine Protease-2 (TMPRSS2) Polymorphisms with COVID-19.

SARS-CoV-2 uses the ACE2 receptor and the cellular protease TMPRSS2 for entry into target cells. The present study aimed to establish if the TMPRSS2 polymorphisms are associated with COVID-19 disease. The study included 609 patients with COVID-19 confirmed by RT-PCR test and 291 individuals negative for the SARS-CoV-2 infection confirmed by RT-PCR test and without antibodies anti-SARS-CoV-2. Four TMPRSS2 polymorphisms (rs12329760, rs2298659, rs456298, and rs462574) were determined using the 5′exonuclease TaqMan assays. Under different inheritance models, the rs2298659 (pcodominant2 = 0.018, precessive = 0.006, padditive = 0.019), rs456298 (pcodominant1 = 0.014, pcodominant2 = 0.004; pdominant = 0.009, precessive = 0.004, padditive = 0.0009), and rs462574 (pcodominant1 = 0.017, pcodominant2 = 0.004, pdominant = 0.041, precessive = 0.002, padditive = 0.003) polymorphisms were associated with high risk of developing COVID-19. Two risks (ATGC and GAAC) and two protectives (GAGC and GAGT) haplotypes were detected. High levels of lactic acid dehydrogenase (LDH) were observed in patients with the rs462574AA and rs456298TT genotypes (p = 0.005 and p = 0.020, respectively), whereas, high heart rate was present in patients with the rs462574AA genotype (p = 0.028). Our data suggest that the rs2298659, rs456298, and rs462574 polymorphisms independently and as haplotypes are associated with the risk of COVID-19. The rs456298 and rs462574 genotypes are related to high levels of LDH and heart rate.

Comparative Study Between Action Vitamin C vs. Action of Nitric Oxide in Prolonged Ventilation in Respiratory Failure Patients Due to ARDS

Introduction: Recent meta-analysis prove that vitamin C shorten the duration of mechanical ventilation and accelerate weaning from ventilator especially in prolonged ventilated patients. Nitric oxide considered a relatively old drug used for long time in this issue. Aim: To compare the effect of either vitamin C or nitric oxide on improvement of general condition of the patients, improvement lung mechanics, early weaning from prolonged ventilation and mortality rates. Material and Methods: This a prospective double-blind study done in King Abdul-Aziz specialist hospital between September 2020 and December 2021 in the intensive care unit on 60 patients had difficult weaning after ventilation for >10 days due to Acute Respiratory Distress Syndrome (ARDS) and still showing full criteria of ARDS. Allocated randomly in two groups 30 patients in each. All patients in both groups continued on the same protocol of ventilation. Group A received Nitric Oxide (NO) while group B received vitamin C intravenous. The duration of the study last 16 days. during this period, APCH II score, Hemodynamics, Chest X-ray, hypoxic index, lung compliance, Recruitment maneuver, arterial blood saturation, Lactic Acid Dehydrogenase (LDH), C-reactive protein used as indicator for improvement. Number of patients weaned from the ventilator and patients died also recorded. >Results: Showed significant improvement in the general condition between patients of group B compared to group A which monitored by number of patients had both APACH II score <10 and had normal core temperature. Significant improvement in lung condition between patients of group B compared to group A which monitored clinically by number of patients had arterial oxygen saturation >95, hypoxic index >300, better lung compliance, responder to recruitment maneuver. And monitored radiologically by significant higher number of patients in group B had parenchymatous lung infiltrate in chest X-ray less than one quadrant infiltration compared to group A. And monitored laboratory by significant higher number of patients had lower level of LDH from (100-200 U/L) and CRP (201-300) mg/L in group B compared to group A. There was significant higher number of patients weaned from the ventilator in group B compared to group A. There was significant lower morbidity between patients of group B compared to group A. No significant difference was found in mortality rates. Conclusion: Vitamin C significantly improve the general condition of the patients, improve all parameters of lung mechanics and accelerate weaning from ventilators in prolonged ventilated patients compared to nitric oxide.

OVERALL SURVIVAL EVALUATION OF PROSTATE CANCER PATIENTS TREATED WITH ANDROGEN DEPRIVATION THERAPY BY ESTIMATING FLUCTUANT PATTERNS OF METABOLIC FACTOR SERUM LEVELS

(Background) The effects of fluctuant patterns of serum alkaline phosphatase (ALP) and lactic acid dehydrogenase (LDH) levels on overall survival of patients with prostate cancer (PC) treated with androgen deprivation therapy (ADT) remain unclear. (Methods) We enrolled 236 patients with PC and divided into 3 cohorts by fluctuant patterns of serum levels of ALP and LDH between at baseline and at 1 year later, or at diagnosis of castration-resistant prostate cancer (CRPC): intermediate, within interquartile range (IQR) [I]; lower than IQR [L]; higher than IQR [H]. (Results) In the 1 year later ALP cohort, all parameters except age were significantly different. In the L cohort, 75% of patients had bone metastasis and > 50% developed CRPC or died. In the 1 year later LDH cohort, Eastern Cooperative Oncology Group-performance status (ECOG-PS) and clinical metastasis classification were significantly different among the cohorts. In the CRPC/ALP cohorts, baseline prostate-specific antigen values and clinical metastasis classification were significantly different among the cohorts, and all cases had metastasis in the L cohort. In the CRPC/LDH cohort, the L cohort had higher ECOG-PS and shorter time to CRPC. In the 1 year later ALP cohort, the hazard ratio (HR) for death of the L and H cohort to the I cohort was 3.77 and 2.27, respectively and both were significant. In the CRPC/LDH cohort, the HR for death of L cohort to I cohort was 1.99. (Conclusions) Larger fluctuations in serum ALP and LDH levels were a sign of poorer prognosis, especially for patients in the L cohort.

Analysis of Fungal and Bacterial Co-Infections in Mortality Cases among Hospitalized Patients with COVID-19 in Taipei, Taiwan.

Fungal or bacterial co-infections in patients with H1N1 influenza have already been reported in many studies. However, information on the risk factors, complications, and prognosis of mortality cases with coronavirus disease 2019 (COVID-19) are limited. We aimed to assess 36 mortality cases of 178 hospitalized patients among 339 patients confirmed to have had SARS-CoV-2 infections in a medical center in the Wenshan District of Taipei, Taiwan, between January 2020 and September 2021. Of these 36 mortality cases, 20 (60%) were men, 28 (77.7%) were aged >65 years, and the median age was 76 (54–99) years. Comorbidities such as hypertension, coronary artery disease, and chronic kidney disease were more likely to be found in the group with length of stay (LOS) > 7 d. In addition, the laboratory data indicating elevated creatinine-phosphate-kinase (CPK) (p < 0.001) and lactic acid dehydrogenase (LDH) (p = 0.05), and low albumin (p < 0.01) levels were significantly related to poor prognosis and mortality. The respiratory pathogens of early co-infections (LOS < 7 d) in the rapid progression to death group (n = 7 patients) were two bacteria (22.2%) and seven Candida species (77.8.7%). In contrast, pathogens of late co-infections (LOS > 7 d) (n = 27 patients) were 20 bacterial (54.1%), 16 Candida (43.2%), and only 1 Aspergillus (2.7%) species. In conclusion, the risk factors related to COVID-19 mortality in the Wenshan District of Taipei, Taiwan, were old age, comorbidities, and abnormal biomarkers such as low albumin level and elevated CPK and LDH levels. Bacterial co-infections are more common with Gram-negative pathogens. However, fungal co-infections are relatively more common with Candida spp. than Aspergillus in mortality cases of COVID-19.

Expression of a Crry/p65 is reduced in acute lung injury induced by extracellular histones.

Acute lung injury (ALI) occurs in patients with severe sepsis and has a mortality rate of 40%–60%. Severe sepsis promotes the release of histones from dying cells, which can induce platelet aggregation, activate coagulation and cause endothelial cell (EC) death. We previously reported that the expression of membrane complement receptor type 1‐related gene Y (Crry)/p65, which plays a principal role in defence against abnormal activation of complement in the blood, is reduced in response to peritoneal mesothelial cell injury, and we hence hypothesized that a similar mechanism occurs in pulmonary ECs. In this study, we examined the role of Crry/p65 in histone‐mediated ALI using an experimental animal model. In ALI model mice, exposure to extracellular histones induces lung injury and results in a decrease in Crry/p65 expression. The levels of lactic acid dehydrogenase (LDH), a marker of cell damage, were significantly increased in the serum of ALI model compared with vehicle mice. The significant inverse correlation between the expression of Crry/p65 and LDH levels in plasma revealed an association between Crry/p65 expression and cell damage. The levels of complement component 3a (C3a) were also significantly increased in the serum of the ALI model compared with vehicle mice. Notably, a C3a receptor antagonist ameliorated lung injury induced by histones. We hypothesize that extracellular histones induce complement activation via down‐regulation of Crry/p65 and that C3a might serve as a therapeutic target for the treatment of ALI.

DIAGNOSTIC UTILITY OF SERUM LACTATE DEHYDROGENASE LEVELS (LDL) IN DIFFERENTIATING MEGALOBLASTIC ANEMIA FROM MYELODYSPLASTIC SYNDROMES IN PAKISTAN

Objective: To study the diagnostic utility of lactate dehydrogenase levels in differentiating megaloblastic anemia from myelodysplastic anemia in Pakistan.&#x0D; Study Design: Comparative cross-sectional study.&#x0D; Place and Duration of Study: Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan from Feb, 2019 to Aug, 2019.&#x0D; Methodology: In this study, total 240 patients (18-75 years of age) males and females were selected by consecutive sampling technique and were equally divided into 3 groups; patients with megaloblastic anemia, patients with myelodysplastic syndromes and healthy control group. The clinical history and duration of anemia were recorded on special designed proforma. The laboratory investigations including lactate dehydrogenase levels were also noted. Both types of anemia were compared on basis of Lactate Dehydrogenase Levels.&#x0D; Results: The lactate dehydrogenase levels in megaloblastic group were more than 3000 IU/L in 58 out of 80 patients (72.5%). On other hand, myelodysplastic group had 79 out of 80 patients with lactic acid dehydrogenase levels below 450 IU/L (98.75%). The difference in lactic acid dehydrogenase levels between both groups was found to be statistically significant.&#x0D; Conclusion: Serum lactate dehydrogenase levels can be used to differentiate megaloblastic anemia from other anemia especially myelodysplastic syndromes before doing a bone marrow examination. High lactate dehydrogenase levels above 3000 IU/L in megaloblastic anemia can differentiate it from other anemia.

The ability of COVID-19 patients' blood to coagulate after withdrawn to EDTA tube: A case report

In the same family, the SARS-COV-2 virus is more virulent than the other virus, and it is known in many patients that it causes extreme symptoms ranging from high-grade fever, cough, dyspnea, to gastrointestinal and hematological symptoms. The extreme inflammatory state secondary to Covid‐19 may lead to a severe imbalance in hemostasis as found recently in a condition of disseminated intravascular coagulation (DIC) and consumption coagulopathy, identified as decreased platelet count, increased fibrin or fibrinogen and red blood cells destruction which growing ferritin. The virus has the ability to cause red blood cell hemolysis even if the blood is inside the EDTA tube, which reflects the virulent traits of the virus and its non-understandable pathogenesis, which is still suspected. The damage caused by the tissue may increase the level of lactic acid dehydrogenase and may be an indicator of the amount of damage caused by the disease in the organs of the body.

Predictive Markers of High-Grade or Serious Treatment-Emergent Infections with Daratumumab-Based Regimens in Newly Diagnosed Multiple Myeloma (NDMM)

Predictive Markers of High-Grade or Serious Treatment-Emergent Infections with Daratumumab-Based Regimens in Newly Diagnosed Multiple Myeloma (NDMM)

Relationship between various maternal conditions and lactic acid dehydrogenase activity in umbilical cord blood at birth.

Lactic acid dehydrogenase (LDH) is a valuable marker for some of the most important diseases in newborns and the plasma LDH activity in newborns correlates well with conditions such as asphyxia. If LDH should be considered as a useful tool also in obstetric care, key factors associated with maternal health before and during pregnancy which could affect umbilical cord LDH activity need to be known. The aims of this study were to explore relationships between selected maternal conditions and arterial lactic acid dehydrogenase activity (aLDH) in umbilical cord blood at delivery. A prospective observational study was conducted at Sodersjukhuset, Stockholm, Sweden. Included in the study were 1247 deliveries, and cord blood samples from each were analyzed for aLDH. Background, delivery and neonatal data were collected from the medical records. Higher median values of aLDH were found (P=0.001) among women with chronic disorders not related to pregnancy but there was no increased frequency of high aLDH levels (>612 μ/L, P=0.30). No difference in aLDH was identified between infants born to women with pregnancy-related disorders compared with healthy women, neither in median values, nor in high values (>612 μ/L, P=0.95). Newborn infants born to women with non-pregnancy-related chronic disorders had a somewhat higher median value of aLDH in cord blood at delivery. The influence of common maternal conditions and diseases on umbilical cord arterial LDH levels is small compared to the increase reported in fetal distress and several other critical conditions in the newborn.

MicroRNA‐181c‐5p exacerbates apoptotic cell death in H9C2 cardiomyocytes during hypoxia/reoxygenation

Myocardial ischemia is a leading cause of death all over the world. The most effective way to salvage ischemic myocardium is the restoration of blood flow, but reperfusion itself may cause additional tissue injury called “ischemia/reperfusion injury (I/RI)”. Activation of cell apoptosis is a major form of cell death during myocardial I/RI. Thus, examining ways to control cell apoptosis has important clinical significance for improving post‐ischemic recovery. MicroRNAs (miR), small non‐coding RNA molecules that regulate gene expression by inhibiting mRNA translation and/or inducing mRNA degradation, play a crucial role in various cellular and biological processes. The clinical evidence demonstrated that miR‐181c was significantly up‐regulated in the early phase of myocardial infarction. However, whether or not miR‐181c mediates cardiac I/RI through cell apoptosis pathway is unknown. Thus, the present study aimed to investigate the role of miR‐181c in hypoxia/reoxygenation (H/R, 6 hours hypoxia followed by6 hours reoxygenation) induced cell injury in H9C2 cardiomyocytes and its possible mechanism. The results showed that H/R significantly increased the expression of miR‐181c‐5p (P&lt;0.05 vs. Control) but not miR‐181c‐3p in H9C2 cells. In line with this, in an in vivo rat cardiac I/RI model (30 min left anterior descending coronary artery ligation followed by 2 hours reperfusion), miR‐181c‐5p expression was also significantly increased (P&lt;0.05 vs. Control), suggesting that miR‐181c‐5p may be involved in the pathogenesis of cardiac I/RI. Under the experimental setting, H/R significantly induced H9C2 cells injury, as evidenced by increased lactic acid dehydrogenase (LDH) leakage (cell injury marker, P&lt;0.05 vs. Control) and reduced cell viability (MTT assay, P&lt;0.05 vs. Control). Furthermore, H/R‐induced cell injury was accompanied by increased cleaved caspase‐3 expression and more TUNEL positive cells (markers of cell apoptosis, P&lt;0.05 vs. Control), but no significant changes in the expression of Bcl‐2 (anti‐apoptotic) and Bax (pro‐apoptotic), suggesting that H/R induces caspase‐3 dependent cell apoptosis, which was independent of the intrinsic mitochondrial pathway. To demonstrate the potential role of miR‐181c‐5p in H/R‐induced cell injury, H9C2 cells were transfected with the miR‐181c‐5pagomir or its negative control. The overexpression of miR‐181c‐5psignificantly aggravated H/R‐induced cell injury (increased LDH level and reduced cell viability, P&lt;0.05 vs.H/R) and exacerbated H/R‐induced cell apoptosis (greater cleaved caspases‐3 expression and more TUNEL positive cells, P&lt;0.05 vs.H/R). In contrast, in H9C2 cells transfected with miR‐181c‐5p antagomir, inhibition of miR‐181c‐5p significantly suppressed H/R‐induced enhancement of LDH level and cleaved caspase‐3 expression and apoptotic cell death(P&lt;0.05 vs.H/R). It is concluded that in H9C2 cells, H/R may induce cardiomyocytes apoptosis via enhanced miR‐181c‐5p expression, which may yield novel strategies to combat myocardial I/RI.Support or Funding InformationThe authors' research was supported by General Research Fund (17158616M and 17124614M, Research Grants Council of Hong Kong).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA‐17‐3p inhibits excessive post‐hypoxic autophagy and attenuates H9C2 cardiomyocytes reoxygenation injury via PTEN‐Akt‐mTOR signaling

BackgroundThe microRNA (miR)‐17~92 is one of the best‐characterized polycistronic miRNA clusters, which encodes six individual miRNAs and contributes to various cellular and biological processes. In particular, miR‐17‐3p (a passenger miRNA of miR‐17) has been shown to indirectly inhibit Phosphatase and tensin homolog (PTEN) and attenuate myocardial ischemia/reperfusion (I/R) injury, but the underneath mechanism is unclear. Inhibition of PTEN can upregulate Akt/mTOR signaling and therefore suppresses the excessive autophagy, which may be a mechanism that underlies myocardial I/R injury. We, thus, hypothesized that miR‐17‐3p may protect against hypoxia/reoxygenation (H/R) induced cell injury by inhibiting post‐hypoxic excessive autophagy in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.Methods and resultsIn rat H9C2 cardiomyocytes, H/R (6 hours hypoxia followed by 6 hours reoxygenation) significantly enhanced the expression of miR‐17‐3p (P &lt;0.05 vs. Control), which was concomitant by increased lactic acid dehydrogenase (LDH) leakage (cell injury marker, P&lt;0.05 vs. Control) and the levels of p62 and the ratio of LC3II/I (autophagy markers, P&lt;0.05 vs. Control), suggesting that miR‐17‐3p may be involved in the pathogenesis of H/R induced autophagic cell death. To explore the potential role of miR‐17‐3p in H/R‐induced cell injury, H9C2 cells were transfected with the miR‐17‐3p agomir or its negative control. The overexpression of miR‐17‐3p can significantly attenuate H/R‐induced cell injury (reduced LDH level, P &lt;0.05 vs. H/R) and inhibit H/R‐induced excessive autophagy (decreased levels of p62 and LC3II/I, P &lt;0.05 vs. H/R). Given the important role of PTEN‐Akt‐mTOR signaling in autophagy, the effects of miR‐17‐3p on the key events in the PTEN‐Akt‐mTOR signaling cascade during H/R were examined. As anticipated, miR‐17‐3p overexpression significantly down‐regulated PTEN expression and up‐regulated the levels of phosphorylated Akt1 (Thr308) and mTOR (P&lt;0.05 vs. H/R), indicating that the miR‐17‐3p may protect against excessive post‐hypoxic autophagic cell death in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.ConclusionUpregulating PTEN‐Akt‐mTOR axis and the subsequent inhibition of excessive autophagy may represent the major mechanism whereby miR‐17‐3p attenuates H/R injury in H9C2 cardiomyocytes.Support or Funding InformationStudied supported by RGC/GRF grants (17158616M, 17117217 M)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FOXD3 inhibits SCN2A gene transcription in intractable epilepsy cell models

The expression of sodium voltage-gated channel alpha subunit 2 (SCN2A) is closely related to the development of epilepsy. This study investigated regulatory element of the SCN2A gene involved in epilepsy. An intractable epilepsy cell model was constructed using hippocampal primary neurons and the SH-SY5Y cell line. SCN2A protein and gene expression in cells as well as the level of lactic acid dehydrogenase (LDH) in the cell culture supernatants was detected. Potential regulatory factors of SCN2A and its upstream regulatory elements were identified using the dual-luciferase reporter assay. Finally, the role of the hypothetical transcription factor in epilepsy was examined by using its small interfering RNA (siRNA). Results found that levels of LDH and expression of the hypothetical transcription factor, Forkhead box D3 (FOXD3), was both increased in the model cells, whereas that of SCN2A was decreased. The results of dual-luciferase reporter assays revealed that an upstream region of SCN2A gene spanning from nucleotides −1617 to −1470 was a transcription factor binding region and a trans-acting factor role of FOXD3 was identified in the core region (GGCAAAATTAT). Then the FOXD3 binding site was further verified by the chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA). After SH-SY5Y cells were transfected with FOXD3 siRNA, the release of LDH into culture supernatants and the LDH expression levels in cells were significantly decreased. SCN2A expression in model cells was increased by knockdown of FOXD3. Therefore, this study demonstrated that FOXD3 is a trans-acting factor of SCN2A, and this mechanism may play a role in cell injury after epilepsy.

The pharmacologically active components of oxytropis falcata bungereduce ischemic-reperfusion injury in the rat heart

Oxytropis falcata is used as the main material to prepare various Tibetan traditional medicine, which its activity was reported that anti-inflammation and analgesia. Flavonoids is the chemical basis for the anti-inflammatory and anti-oxidant efficacy. The influence of total flavonoids of Tibetan herb O. falcata on myocardial ischemic-reperfusion injury was investigated using ethanol, chloroform, and ethyl acetate extracts. Rats were pretreated with different concentrations of the extract for 7 days. The model of myocardial ischemic-reperfusion injury was established by ligating the left anterior descending (LAD) artery in the heart. Following reperfusion for 40 minutes, the enzyme activity of creatine kinase (CK), lactic acid dehydrogenase (LDH), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), and the concentration of malondialdehyde (MDA) in the rat serum were measured. The ultrastructural pathological change was observed under transmission electron microscopy (TEM). The serum levels of CK, LDH, and MDA were reduced, and the activity of SOD and GSH-Px were increased significantly, by chloroform and ethyl acetate extracts (P Less Than 0.01, PP Less Than 0.05). The ultrastructural pathological change in myocardial ischemic-reperfusion injury was alleviated by both extracts. The major components in chloroform extract were 7-hydroxy-flavonone and 2',4'-dihydroxy chalcone. 7-Hydroxy-flavanon naringenine naringetol and 2',4'-dihydroxy chalcone were the major components in ethyl acetate extract. Total flavonoids of Tibetan herb O. falcata might alleviate myocardial ischemic-reperfusion injury.

Study on mild hypothermia affecting the changes of proteome in damaged neurons

Objective To study the neuroprotection mechanism of mild hypothermia using a neuronal injury model and protein two-dimensional gel electrophoresis. Methods A neuron damage model was induced by the cell lysates soak assay. Neurons were cultured in temperature-controlled incubator at 33 ℃ and 37 ℃ respectively. The lactic acid dehydrogenase level in the culture supernatant was tested. TUNEL apoptosis assay was use to compare the cell apoptosis under the 2 culture temperatures. The total cellular protein was extracted. The protein two-dimensional gel electrophoresis technique was used to screen the differential protein spots. Mass spectrometry was used to complete the protein identification. Western blot was used to conduct protein confirmation. Statistical methods were used to analyze the test results. Results The impaired neuronal lactate dehydrogenase level and the cell numbers of occurring in situ apoptosis were significantly lower than those of the 37 ℃ culture group under the low temperature condition (P<0.05). A total of 14 differential protein spots were screened using the two-dimensional gel electrophoresis, 12 of them were identified effectively. They were involved in a variety of pathophysiological processes, including cellular energy metabolism, regeneration, and oxidative stress. Conclusion Mild hypothermia plays a certain neuroprotective effect by intervening and regulating multiple molecular events, including cellular energy metabolism, regeneration, and oxidative stress. Key words: Hypothermia, induced; Craniocerebral trauma; Immunoelectrophoresis, two-dimensional; Proteomics

Effects of lentivirus-mediated heat shock protein 70 gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia

To investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia, and the mechanism involved. PC12 cells at logarithmic phase were collected, and were divided into recombination lentivirus infection group (infected by lentivirus containing HSP70 and fluorescent gene), lentivirus control group (infected by lentivirus containing fluorescin without HSP70 gene) and non-infection group. HSP70 gene and protein expressions in PC12 cells were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot. After being challenged with ischemia and hypoxia for 4 hours, the viability of cells was detected by methyl thiazolyl tetrazolium (MTT), the levels of lactic acid dehydrogenase (LDH) in cell supernatant were determined by LDH measurement test kit. The concentration of intracelluer calcium ([Ca²⁺] i ) was assayed by flow cytometer. The activities of Na⁺ -K⁺ -ATPase, Ca⁺ -Mg⁺ -ATPase and total ATPase were measured by ATPase test kits. The expressions of exogenous HSP70 gene and protein were found by RT-PCR and Western Blot in the recombination lentivirus infection group. After being challenged with ischemia and hypoxia, the viability of cells in the recombination lentivirus infection group was increased significantly as compared with the lentivirus control group and non-infection group ( A value: 0.575 ± 0.020 vs. 0.395 ± 0.014, 0.363 ± 0.045, t1 = 17.996, t2= 10.600, both P < 0.001), the levels of LDH in culture medium and the concentration of [Ca⁺] i were decreased significantly [LDH ( U/L ): 743.46 ± 23.68 vs. 935.43±34.77, 962.89 ± 26.68, t1 = 11.179, t2 = 15.044, both P < 0.001; [ Ca²⁺] i relative fluorescence: ( 31.60 ± 2.43 )% vs. (41.48 ± 3.33)%, ( 40.40 ± 3.05 )%, t1 = 5.853, t2= 5.502, both P < 0.001], and the activities of Na⁺ -K⁺ -ATPase, Ca²⁺ -Mg²⁺ -ATPase and total ATPase were increased significantly [ Na⁺ -K⁺ -ATPase (μmol × mg⁻¹× h⁻¹): 8.608 ± 0.307 vs. 6.728 ± 0.173, 6.450 ± 0.091, t1 = 9.237, P1= 0.001, t2 = 11.675, P2 < 0.001; Ca²⁺ -Mg²⁺ -ATPase (μmol×mg⁻¹ × h⁻¹): 10.523 ± 0.036 vs. 7.910 ± 0.209, 8.064 ± 0.195, t1 = 9.718, P1 = 0.001, t2= 11.535, P2 < 0.001; total ATPase (μmol×mg⁻¹× h⁻¹): 17.041 ± 0.324 vs. 14.150 ± 0.182, 13.983 ± 0.085, t1= 16.113, t2 = 17.602, both P < 0.001]. There was no statistical difference in above indexes between lentivirus control group and non-infection group. HSP70 can maintain the PC12 cells calcium homeostasis, which may be one of the important mechanisms of anti-apoptosis.

Incidence and Clinical Features of Calreticulin mutation in Patients with Jak2V617F-Negative Essential Thrombocytosis and Primary Myelofibrosis from Singapore

Background: Slightly less than half of the patients with essential thrombocytosis (ET) and primary myelofibrosis (PMF) do not have specific gene mutations. Recent discovery of CALR mutation in JAK2V617F-negative ET and PMF patients have accounted for 20-25% of ET and PMF patients, and therefore redefining diagnostic approach to ET or PMF. Here we reported the incidence of CALR mutation and clinical features of clinically diagnosed Jak2V617F-negative ET and PMF in Singapore.Method: We retrospectively identified 140 cases of Jak2V617F-negative ET and PMF from two healthcare institutions in Singapore since 2006. Sanger sequencing was performed for the targeted detection of CALR exon 9 mutations. Clinicopathologic correlation was carried out by assessing relevant clinical data and the CALR mutation status, and compared with JAK2-positive cases (25 cases of ET and 9 cases of PMF).Results: The Jak2V617-negative ET/PMF patients were ethnic Chinese (76.2%), Malay (13.9%) and others (9.9%). CALR mutations were detected in 32.8% (40/122) of Jak2V617-negative ET and 55.6% (10/18) Jak2V617-negative PMF patients. Higher platelet count was observed in CALR-positive ET patients with median platelet count of 1056.5 x 109/L and 637.5 x 109/L for CALR-positive and -negative patients respectively (p<0.001). CALR-positive ET also required higher doses of hydroxyurea to maintain platelet count below 600 x 109/L (p=0.038). There was no difference in thrombotic events, presence of splenomegaly, presenting WBC count or hemoglobin (Hb) level between CALR-positive and -negative ET. However, when compared to Jak2-positive ET, Jak2-negative ET had a significantly lower incidence of thrombotic events (Jak2-negative ET, 8.2%; Jak2-positive ET, 50.0%, p<0.001), and lower Hb level (p<0.001). The incidence of thrombotic events in CALR-positive ET and PMF were 10.3% and 10.0% respectively. There was also no significant difference in presenting WBC count, platelet count, Hb level, presence of splenomegaly, or thrombotic event between CALR-positive vs -negative PMF. Lactic acid dehydrogenase (LDH) level at diagnosis however, was significantly higher in CALR-positive ET/PMF when compared to CALR-negative group (p=0.011). There was also significantly higher incidence of elevated peripheral blasts of >1% in CALR-positive ET/PMF (12% in CALR-positive and 3.3% in CALR-negative group, p=0.045). Blast transformation occurred in 6% of patients with CALR-positive PMF while only 1.1% in CALR-negative PMF. Besides the two commonly reported mutations (CALR52del, 30.8%; CALR5ins, 33.3%) in our screening of the CALR exon 9 in Jak2V617F-negative ET and PMF patients, 7 other novel mutations (c.1098_1137delinsTTTCTT, c.1102_1104delAAG, c.1122_1155delinsGGGCCAGGCACTTGTCG, c.1129_1153delinsCTTTGCGTTTCTTTT, c.1132_1155delinsTGTCG, c.1139delA, c.1141delG) were also found individually. All mutant CALR proteins possess an altered C-terminus with a longer peptide stretch caused by a disrupted reading frame due to these frameshift mutations.Conclusion:CALR mutation is common in Jak2V617F-negative PMF and ET in this multiracial Singaporean cohort, and has similar frequency when compared with Western report. It was associated with higher platelet count in ET, higher level of LDH and possibly a higher risk of blast transformation in PMF. 7 novel CALR mutations were identified and more subtypes of CALRmutation variants are expected to be uncovered in the near future. DisclosuresNo relevant conflicts of interest to declare.

The effects of early enteral nutrition with addition of probiotics on the prognosis of patients suffering from severe acute pancreatitis

To investigate the curative effect of early enteral nutrition (EN) supplemented with probiotics (bifidobacterium) in patients with severe acute pancreatitis (ASP). Seventy SAP cases admitted from January 2005 to October 2012 were randomly assigned into parenteral nutrition (PN) group (n=22), EN group (n=25) and bifidobacterium added EN (P+EN) group (n=23). In P+EN group, patients were given their nutrition the same as that of EN, and also probiotics (bifidobacterium, 4 capsules every 12 hours, given through nasal gastric tube, each capsule weighing 210 mg). The routine treatment including anti-infection and anti-acid agents, and that of inhibition of pancreatic secretion were given, except for the different nutritional interventions in all groups. The blood samples were collected for e same measurements of interleukin-8 (IL-8) and tumor necrosis factor (TNF-α) by enzyme linked immunosorbent assay (ELISA), and for the C-reactive protein (CRP), lactic acid dehydrogenase (LDH), white blood cell (WBC) count, amylase and lipase by biochemistry assay 1 day before intervention of nutrition, and 7 days and 14 days after intervention. Changes in organ function and outcome were also recorded at the same time points. The plasma levels of IL-8, TNF-α, CRP, LDH, WBC count, amylase and lipase were significantly reduced after nutritional intervention compared with their levels on day 1 before intervention in all three groups. The plasma IL-8, TNF-α, CRP, lipase, LDH at 14 days after intervention of nutrition in P+EN group were significantly lower than those in PN group and EN group (IL-8: 21.00 ± 7.07 μg/L vs. 48.00 ± 10.32 μg/L, 32.00 ± 9.30 μg/L; TNF-α: 44.3 ± 10.9 ng/L vs. 132.1 ± 34.1 ng/L, 67.8 ± 22.3 ng/L; CRP: 35.0 ± 12.4 mg/L vs. 103.2 ± 49.2 mg/L, 63.0 ± 29.2 mg/L; lipase: 269 ± 79 U/L vs. 670 ± 145 U/L, 310 ± 78 U/L; LDH: 21.8 ± 10.3 U/L vs. 78.1 ± 37.4 U/L, 37.9 ± 25.1 U/L, P<0.05 or P<0.01). The WBC count in P+EN group was significantly lower than that in PN group (5.9 ± 3.0 × 10⁹/L, 6.3 ± 3.2 × 10⁹/L vs. 9.6 ± 3.0 ×10⁹/L, both P<0.05), but there was no significant difference in amylase between P+EN group and PN group (211 ± 49 U/L, 236 ± 52 U/L vs. 298 ± 71 U/L, P>0.05). The gastrointestinal dysfunction score in P+EN, EN, PN groups 14 days after nutritional intervention was 0.28 ± 0.05, 0.43 ± 0.09, 0.71 ± 0.11, respectively, with statistically significant differences (all P<0.01). Compared with PN and EN groups, the incidence of upper gastrointestinal bleeding (1 vs. 9, 2), infection and abscess (2 vs. 12, 5) was lower (all P<0.01), and hospital day was significantly shortened in P+EN group (10.4 ± 3.9 days vs. 25.8 ± 6.4 days, 13.4 ± 5.2 days, both P<0.01). There was no significant statistical difference in mortality rate among three groups. Our results indicated that early EN with addition of probiotics (bifidobacterium) resulted in significant lowering of the level of pro-inflammatory cytokines, earlier restoration of gastrointestinal function, decrease of complications such as infection, and shortening of hospital day in patients with SAP.

Index of Suspicion

A 17-year-old girl presents with intermittent right upper extremity swelling and pain over the past 2 years. Her symptoms began after an episode of Bell’s palsy. She describes swelling and an “electrical shooting” pain in the right shoulder that radiates toward her hand, followed by a cool feeling of the hand and decreased range of motion of the arm. The episodes occur during both summer and winter and can last anywhere from 2 to 30 days. Initially, between these episodes, she regained normal function, but recently has noted residual weakness. She denies any history of trauma, fever, rash, or muscle weakness. On examination, there is swelling of the entire right arm. The nails are normal. The right arm is colder in comparison with the left and has decreased hair growth. Peripheral pulses and capillary refill were normal. Pinprick and temperature sensations are intact, without hyperesthesia. Range of motion of the right arm, forearm, and hand is compromised because of pain. Except for her right arm, she is warm, well perfused, and free of joint swelling. The muscle tone is normal, and strength in the other muscle groups is 5/5. Laboratory studies reveal an erythrocyte sedimentation rate of 15 mm/h, C-reactive protein <0.5 mg/dL, white blood cell count of 6.3 to 103/μL, and creatine phosphokinase level of 100 U/L. The results of additional laboratory studies, including antinuclear antibody, rheumatoid factor, antibody test for Borrelia burgdorferi , rapid plasma regain test for syphilis, urine-amplified DNA assay for Chlamydia and gonorrhea, hemoglobin electrophoresis, serum lactic acid dehydrogenase level, and uric acid level, are normal or negative. Radiographs of cervical spine, chest, right elbow, forearm, and hand as well as magnetic resonance imaging (MRI) of the brain, cervical spine, and entire arm are normal. Right upper extremity vascular duplex ultrasonography shows normal …

Predictive Factors for the Development of Brain Metastasis in Advanced Unresectable Metastatic Melanoma

Melanoma that metastasizes to distant sites is associated with a grave prognosis. The objectives of the study were (1) to identify predictive factors for the development of brain metastases from the time of diagnosis of stage III/IV disease, (2) to identify predictive factors for the development of central nervous system (CNS) metastases from the time of diagnosis of primary melanoma, and (3) to assess whether the incidence of brain metastasis is more frequent in patients who had no tumor response to systemic therapy for stage III/IV disease compared with those who had partial or complete response. We collected and retrospectively analyzed information of 740 patients with advanced metastatic melanoma treated at MD Anderson Cancer Center over 15 years. Three hundred and twenty-nine patients had CNS metastases. The characteristics of these patients in terms of median age, sex, primary site, Breslow thickness, stage at first visit, baseline serum parameters, and response to systemic therapy were compared with those of patients who did not develop CNS metastasis. Cox proportional hazards models were used to analyze the cause-specific hazard function for CNS metastasis and deaths without CNS metastasis. We identified that M-stage [stage M1b vs. stage III or M1a, hazard ratio (HR)=2.64; stage M1c vs. stage III or M1a, HR=2.13, P<0.0001] and lactic acid dehydrogenase (LDH) (elevated vs. normal LDH, HR=1.51, P<0.001) at diagnosis of unresectable stage III/IV disease can independently predict the risk of developing CNS metastasis from the time of diagnosis of stage III/IV disease. Older age (HR=1.01, P=0.076), chemoresistance (stable disease+progressive disease vs. complete response+partial response HR=2.91, P<0.0001), low level of albumin (vs. normal HR=2.87, P<0.0001), elevated LDH (vs. normal HR=1.55, P=0.0004), and M-stage (M1c disease vs. stage III or M1a HR=1.89, P<0.0001) can independently predict shorter time to death without CNS metastasis from the diagnosis of stage III/IV disease. The location (head and neck vs. limbs HR=1.56, P=0.028; trunk and abdomen vs. limbs HR=1.45, P=0.029; unknown site vs. limbs HR=8.43, P=0.036) and pathology [Clark level (CL)=3 and/or BR2 to 4 mm vs. CL≤2 and/or BR<2 mm HR=1.60, P=0.037; CL>3 and/or BR> 4 mm vs. CL≤2 and/or BR<2 mm HR=2.03, P=0.001) of the primary melanoma can independently predict CNS metastasis-free interval from the time of diagnosis of primaries. Age (HR=1.012, P=0.034) and pathology of the primary melanoma (CL>3 and/or BR>4 mm vs. CL≤2 and/or BR<2 mm HR=1.54, P=0.024) can independently predict time to death without CNS metastasis from primaries. We identified the predictive factors associated with the development of CNS metastasis in patients with unresectable metastatic melanoma.