Creatine Kinase BB Research Articles

Cerebrospinal Creatine Kinase BB Isoenzyme: A Biomarker for Predicting Outcome After Cardiac Arrest.

Cerebrospinal fluid creatine kinase BB isoenzyme (CSF CK-BB) after cardiac arrest (CA) has been shown to have a high positive predictive value for poor neurological outcome, but it has not been evaluated in the setting of targeted temperature management (TTM) and modern CA care. We aimed to evaluate CSF CK-BB as a prognostic biomarker after CA. We performed a retrospective cohort study of patients with CA admitted between 2010 and 2020 to a three-hospital health system who remained comatose and had CSF CK-BB assayed between 36 and 84h after CA. We examined the proportion of patients at hospital discharge who achieved favorable or intermediate neurological outcome, defined as Cerebral Performance Category score of 1-3, compared with those with poor outcome (Cerebral Performance Category score 4-5) for various CSF CK-BB thresholds. We also evaluated additive value of bilateral absence of somatosensory evoked potentials (SSEPs). Among 214 eligible patients, the mean age was 54.7 ± 4.8years, 72% of patients were male, 33% were nonwhite, 17% had shockable rhythm, 90% were out-of-hospital CA, and 83% received TTM. A total of 19 (9%) awakened. CSF CK-BB ≥ 230 U/L predicted a poor outcome at hospital discharge, with a specificity of 100% (95% confidence interval [CI] 82-100%) and sensitivity of 69% (95% CI 62-76%). When combined with bilaterally absent N20 response on SSEP, specificity remained 100% while sensitivity increased to 80% (95% CI 73-85%). Discordant CK-BB and SSEP findings were seen in 13 (9%) patients. Cerebrospinal fluid creatine kinase BB isoenzyme levels accurately predicted poor neurological outcome among CA survivors treated with TTM. The CSF CK-BB cutoff of 230 U/L optimizes sensitivity to 69% while maintaining a specificity of 100%. CSF CK-BB could be a useful addition to multimodal neurological prognostication after CA.

Surveilling brain damage using brain biomarkers in hypoglycemic neonatal calves with diarrhea.

Hypoglycemia is a condition associated with neonatal diarrhea in calves, leading to increased mortality and neurological clinical signs. The aim of the present study was to determine the development of brain damage in hypoglycemic calves with neonatal diarrhea and the diagnostic and prognostic significance of these biomarkers. Ten healthy and 50 hypoglycemic calves with diarrhea were included in the study. Clinical examination, blood gases and complete blood count were performed at admission. Blood serum calcium-binding protein B (S100B), neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolysis isoenzyme-1 (UCHL-1), activitin A (ACT), adrenomodullin (AM) concentrations, and creatine kinase-BB (CK-BB) enzyme activity were measured using commercial bovine-specific ELISA kits to assess brain damage. Of the hypoglycemic calves enrolled in the study, 13 (26%) survived and 37 (74%) died. In addition, 32 (64%) of the calves had severe acidosis and 24 (48%) had sepsis. S100B, GFAP, UCHL-1, CK-BB (p < 0.001) and NSE (p < 0.05) concentrations were significantly higher in hypoglycemic calves compared to healthy calves, while ACT concentrations were lower. Blood glucose concentration was negatively correlated with serum S100B, GFAP, UCHL-1, and CK-BB enzyme activity and positively correlated with ACT in hypoglycemic calves (p < 0.01). Brain injury biomarkers were not predictive of mortality (p > 0.05). Morever, severe hypoglycemia, severe acidosis and sepsis variables were not found to have sufficient capacity to predict mortality when considered alone or together (p > 0.05). In conclusion, brain damage may develop as a consequence of hypoglycemia in calves. S100B, NSE, GFAP, UCHL-1, ACT, and CK-BB concentrations can be used to diagnose brain damage in hypoglycemic calves. However, the variables of severe hypoglycemia, severe acidosis, and sepsis together with the biomarkers of brain injury have a limited value in predicting the prognosis of neonatal calves with diarrhea.

Diagnostic Competence of Creatine Kinase BB, in Mild Traumatic Brain Injury and its Prognostic Value.

Due to the very high and increasing prevalence, essential complications, and risk factors for psychiatric disorders, it is necessary to introduce screening tests for diagnosing and predicting mild traumatic brain injury (mTBI) prognosis. After completing the consent form and recording information and examination findings of patients with mild trauma, venous blood samples were taken from these patients. The samples were measured by observing the cold chain. After 3 months from mTBI, the post concussion symptoms questionnaire (PCSQ) and the short form 36 (SF-36) questionnaire for physical and mental evaluations were performed. Statistical tests analyzed the relationship between different variables and serum Creatine kinase BB (CKBB) levels. Statistic analyses showed no relation between CKBB level of serum and age, gender, level of consciousness, PCSQ, and SF 36 scale, and the interval between trauma and arrival to the hospital. Further, there is a significant correlation between CK-BB levels and intracranial damage based on Fisher's exact test. This study and following more significant considerations can introduce a serum-based biomarker panel that can accurately differentiate patients with complicated mTBI from those with uncomplicated.

Abstract WP113: Cerebrospinal Creatine Kinase BB Isoenzyme: A Biomarker For Predicting Never Awakening In Patients Treated With Therapeutic Hypothermia After Cardiac Arrest

Introduction: Creatine kinase BB isoenzyme (CK-BB) levels in CSF after cardiac arrest (CA) have been shown to predict never awakening though have not been evaluated in patients treated with therapeutic hypothermia (TH). Clinically, CSF CK-BB is tested in those who have not already awakened and are suspected of having a poor prognosis. We aimed to evaluate CSF CK-BB as a prognostic biomarker in the era of TH. Methods: We performed a retrospective cohort study at a single institution of CA patients admitted between 2010 and 2020 and with CSF CK-BB assayed between 36 and 84 hours after CA. We examined the proportion of patients who awakened at hospital discharge and latest follow up in the electronic health record for various CSF CK-BB thresholds. We examined concordance and additive value somatosensory evoked potentials (SSEP). Awakening was defined as following commands or having comprehensible speech. Results: Among 214 eligible patients, mean age was 55 ± 15 years, 72% were male, 33% were non-white, 17% had initial ventricular fibrillation or tachycardia, 90% were out-of-hospital CA, and 83% had TH. A total of 19 (9%) awoke at a mean of 22 ± 28 days. Two patients in a vegetative state at discharge later awoke at 73 (CSF CK-BB 163 U/L) and 101 days (228 U/L). A CSF CK-BB cut-off of ≥ 230 U/L predicted never awakening with a specificity of 100% (binomial 95% CI 82-100%) and a sensitivity of 69% (95% CI 62-76%). When combined with bilaterally absent SSEP, the specificity remained 100% while the sensitivity increased to 80% (95% CI 73-85%). Results were similar after excluding 165 (77%) who had withdrawal of life-sustaining treatment. Discordant findings among 149 patients who had both studies were seen in 13 (9%) with intact SSEP bilaterally but CSF CK-BB ≥ 230 U/L and in 20 (13%) with absent SSEP bilaterally but CSF CK-BB &lt; 230, none of whom awoke. Conclusion: CSF CK-BB levels accurately predict never awakening among CA survivors treated with TH. As part of multimodal prognostic testing with SSEP, the CSF CK-BB cutoff of 230 can optimize sensitivity while maintaining a specificity of 100%. These results are susceptible to a self-fulfilling prophecy, and specificity has a large confidence interval because only a small number of eligible patients awakened.

Screening for Fatal Traumatic Brain Injuries in Cerebrospinal Fluid Using Blood-Validated CK and CK-MB Immunoassays.

A single, specific, sensitive biochemical biomarker that can reliably diagnose a traumatic brain injury (TBI) has not yet been found, but combining different biomarkers would be the most promising approach in clinical and postmortem settings. In addition, identifying new biomarkers and developing laboratory tests can be time-consuming and economically challenging. As such, it would be efficient to use established clinical diagnostic assays for postmortem biochemistry. In this study, postmortem cerebrospinal fluid samples from 45 lethal TBI cases and 47 controls were analyzed using commercially available blood-validated assays for creatine kinase (CK) activity and its heart-type isoenzyme (CK–MB). TBI cases with a survival time of up to two hours showed an increase in both CK and CK–MB with moderate (CK–MB: AUC = 0.788, p < 0.001) to high (CK: AUC = 0.811, p < 0.001) diagnostic accuracy. This reflected the excessive increase of the brain-type CK isoenzyme (CK–BB) following a TBI. The results provide evidence that CK immunoassays can be used as an adjunct quantitative test aid in diagnosing acute TBI-related fatalities.

Chronotoxicity of Semen Strychni is associated with circadian metabolism and transport in mice.

We aimed to determine the circadian responses of mice to Semen Strychni and to investigate the role of pharmacokinetics in generating chronotoxicity. Total extract of Semen Strychni was administered by oral gavage to wild-type (WT) and Bmal1-/- (a circadian clock-deficient model) mice at different circadian time points for toxicity (including survival) and pharmacokinetic characterization. Nephrotoxicity and neurotoxicity were evaluated by measuring plasma creatinine and creatine kinase BB (CK-BB), respectively. Drug metabolism and transport assays were performed using liver/intestine microsomes and everted gut sacs, respectively. Semen Strychni nephrotoxicity and neurotoxicity as well as animal survival displayed significant circadian rhythms (the highest level of toxicity was observed at ZT18 and the lowest level at ZT2 to ZT6). According to pharmacokinetic experiments, herb dosing at ZT18 generated higher plasma concentrations (and systemic exposure) of strychnine and brucine (two toxic constituents) compared with ZT6 dosing. This was accompanied by reduced formation of both dihydroxystrychnine and strychnine glucuronide (two strychnine metabolites) at ZT18. Bmal1 ablation sensitized mice to Semen Strychni-induced toxicity (with increased levels of plasma creatinine and CK-BB) and abolished the time dependency of toxicity. Metabolism of Semen Strychni (strychnine and brucine) in the liver and intestine microsomes of WT mice was more extensive at ZT6 than at ZT18. These time differences in hepatic and intestinal metabolism were lost in Bmal1-/- mice. Additionally, the intestinal efflux transport of Semen Strychni (strychnine and brucine) was more extensive at ZT6 than ZT18 in WT mice. However, the time-varying transport difference was abolished in Bmal1-/- mice. Circadian responses of mice to Semen Strychni are associated with time-varying efflux transport and metabolism regulated by the circadian clock (Bmal1). Our findings may have implications for optimizing phytotherapy with Semen Strychni via timed delivery.

Structure and function of resistance arteries from BB-creatine kinase and ubiquitous Mt-creatine kinase double knockout mice

Increasing evidence indicates that the enzyme creatine kinase (CK) is intimately involved in microvascular contractility. The mitochondrial isoenzyme catalyses phosphocreatine synthesis from ATP, while cytoplasmic CK, predominantly the BB isoenzyme in vascular tissue, is tightly bound near myosin ATPase, where it favours ATP production from phosphocreatine to metabolically support vascular contractility. However, the effect of CK gene inactivation on microvascular function is hitherto unknown. We studied functional and structural parameters of mesenteric resistance arteries isolated from 5 adult male mice lacking cytoplasmic BB-CK and ubiquitous mitochondrial CK (CK-/-) vs 6 sex/age-matched controls. Using a Mulvany Halpern myograph, we assessed the acute maximum contractile force with 125mMK+ and 10-5M norepinephrine, and the effect of two inhibitors, dinitrofluorobenzene, which inhibits phosphotransfer enzymes (0.1μM), and the specific adenylate kinase inhibitor P1, P5-di(adenosine 5') pentaphosphate (10-6 to 10-5M). WT and CK-/- did not significantly differ in media thickness, vascular elasticity parameters, or acute maximum contractile force. CK-/- arteries displayed greater reduction in contractility after dinitrofluorobenzene 38%; vs 14% in WT; and after AK inhibition, 14% vs 5.5% in WT, and displayed abnormal mitochondria, with a partial loss of the inner membrane. Thus, CK-/- mice display a surprisingly mild phenotype in vascular dysfunction. However, the mitochondrial abnormalities and greater effect of inhibitors on contractility may reflect a compromised energy metabolism. In CK-/- mice, compensatory mechanisms salvage energy metabolism, as described for other CK knock-out models.

Serum and Saliva Concentrations of Biochemical Parameters in Men with Prostate Cancer and Benign Prostate Hyperplasia.

To find suitable biomarkers for diagnosis of prostate cancer (PC) in serum and saliva; also, to evaluate the diagnostic efficacy of saliva in patients with PC. This case-control study included 20 patients with PC and 20 patients with benign prostatic hyperplasia (BPH). Blood and saliva were collected from the participants and centrifuged. Serum and supernatant saliva were used for biochemical analysis. We evaluated serum and salivary levels of urea, creatinine, prostate-specific antigen (PSA), creatine kinase BB (CK-BB), zinc, β-2 microglobulin (B2M), and melatonin. Also, we used Mann-Whitney U testing, Spearman correlation coefficients, and receiver operating characteristic (ROC) analysis to evaluate the data. Serum and salivary concentrations of urea, creatinine, PSA, CK-BB, zinc, and B2M were significantly higher in patients with PC, compared with the BPH group (P <.05). However, serum and salivary concentrations of melatonin were significantly lower in patients with PC, compared with BPH group (P <.05). In both groups, salivary concentrations of all markers were lower (P <.05), compared with those values in serum. We observed positive correlation between serum and salivary concentrations of all markers studied (P <.05). From the data, we conclude that investigation using saliva specimens is a noninvasive, simple, and effective tool for screening of biochemical parameters.

Oxidative Stress and Serum Creatine Kinase BB Levels can Help Mark Severity and Stage of Brain Tumor

The exact cause of brain tumor is still unknown, and it is a multi-factorial disease. Brain tumor can originate from brain cells or secondary from other organs via metastasis. Generally, tumor cells show aberrant metabolism of free radicals. The current study aims at evaluating creatine kinase BB (CK-BB) and total oxidative stress (TOS) levels among brain tumor patients for diagnosis, prognosis, and treatment strategy. Hospital-based comparative cross-sectional study was conducted on a total of 90 participants (50 brain tumor patients and 40 healthy controls) from April 2018 to October 2018. Venous blood samples were collected for TOSand CK-BB activity measurement. Purposive sampling technique was implemented to select study participants in the hospital. Catalytic activity of CK-BB and TOS were significantly increased in the serum of brain tumor patients as compared to controls. TOS levels in patients with malignant brain tumor were significantly higher than those with benign tumor, but CK-BB levels were not significantly different between benign and malignant types of brain tumor. Both serum levels of TOS and CK-BB were significantly associated with brain tumor. The CK-BB activity was significantly associated with brain tumor and can be used as markers for diagnosis. Participants with increased TOS had significantly higher risk of brain tumor development compared to healthy controls. In conclusion, oxidative stress status and CK-BB level may be used as possible brain tumor markers.

High-Intensity Interval Training Is Associated With Alterations in Blood Biomarkers Related to Brain Injury.

Purpose: Blood biomarkers are a useful tool to study concussion. However, their interpretation is complicated by a number of potential biological confounds, including exercise. This is particularly relevant in military and athletic settings where injury commonly occurs during physical exertion. The impact of high-intensity interval training (HIIT) on putative brain injury biomarkers remains under-examined. The purpose of this study was to observe the effects of HIIT on a panel of blood biomarkers associated with brain injury.Methods: Eleven healthy, recreationally active males (median age = 29.0, interquartile range = 26.0–31.5) performed HIIT on a bicycle ergometer (8-12 × 60-s intervals at 100% of peak power output, interspersed by 75-s recovery at 50 W) three times/week for 2 weeks. Peripheral blood samples were collected before and immediately after HIIT during the first and last training sessions. Plasma concentrations of s100 calcium-binding protein beta (S100B), glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), brain-derived neurotrophic factor (BDNF), neurogranin (NRGN), peroxiredoxin (PRDX)-6, creatine kinase-BB isoenzyme (CKBB), visinin-like protein (VILIP)-1, von Willebrand factor (vWF), monocyte chemoattractant protein (MCP)-1, matrix metalloproteinase (MMP)-9, and total tau (T-tau) were quantitated by high-sensitivity MULTI-SPOT® immunoassay, on the MesoScale Diagnostics electrochemiluminescence detection platform. Differences in biomarker concentrations in response to HIIT were evaluated by partial least squares discriminant analysis (PLSDA) within a repeated-measures bootstrapped framework.Results: Ten of 12 biomarkers were increased pre-to-post HIIT; VILIP-1 remained unchanged, and GFAP was not statistically evaluated due to insufficient detectability. After 2 weeks of HIIT, T-tau was no longer significantly elevated pre-to-post HIIT, and significant attenuation was noted in the acute responses of NRGN, PRDX-6, MMP-9, and vWF. In addition, compared to session 1, session 6 pre-exercise concentrations of NSE and VILIP-1 were significantly lower and higher, respectively.Conclusion: Blood biomarkers commonly associated with brain injury are significantly elevated in response to a single bout of HIIT. After a 2-week, six-session training protocol, this response was attenuated for some, but not all markers. While biomarkers continue to provide promise to concussion research, future studies are necessary to disentangle the common biological sequelae to both exercise and brain injury.

Ultrasonic characteristics and clinical significance of umbilical cord blood flow in acute fetal distress

ObjectiveTo study ultrasonic characteristics of umbilical cord blood flow in acute fetal distress and its correlation with umbilical artery blood gas parameters, oxidative stress parameters, neonatal brain injury and myocardial injury. MethodsThe pregnant women delivered in Department of Obstetrics of our hospital were chosen during the period from May 2012 to August 2015. The pregnant women with acute fetal distress were included in the distress group, and the healthy pregnant women with no acute fetal distress were included in the control group. The resistance index (RI), pulsatility index (PI) and systolic/diastolic (S/D) ratio of umbilical artery were measured at 24–30 weeks, 31–36 weeks and 37–41 weeks of pregnancy. After delivery, umbilical artery blood was taken for analysis of blood gas and determination of oxidative stress parameters. The venous blood of newborns was taken to measure the myocardial injury and brain injury parameters. ResultsAt 24–30 weeks, 31–36 weeks and 37–41 weeks of pregnancy, RI, S/D and PI in pregnant women of distress group were significantly higher than those in control group. The pH, contents of arterial partial pressure of oxygen, vitamin C, vitamin E, superoxide dismutase and glutathione peroxidase in umbilical artery blood in pregnant women of distress group was significantly lower than those in control group and negatively correlated with the umbilical artery RI, PI and S/D. The contents of partial pressure of carbon dioxide in artery, lactic acid and malondialdehyde in pregnant women of distress group were significantly higher than those in control group and positively correlated with the umbilical artery RI, PI and S/D. The contents of lactate dehydrogenase, hydroxybutyrate dehydrogenase, creatine kinase, creatine kinase-MB, S100B, neuron-specific enolase, creatine kinase-BB and Tau in newborns' venous blood in distress group were significantly higher than those in control group and positively correlated with the umbilical artery RI, PI and S/D. ConclusionsThe ultrasonic characteristics of umbilical cord blood flow in patients with acute fetal distress are increase of the resistance, reduction of blood flow, and significant reduction of ultrasonic parameters of RI, PI and S/D. The degree of hypoxia, oxidative stress, myocardial injury and brain injury can also be evaluated.

Biomarkers of injury to neural tissue in veterinary medicine

Abstract There are numerous biomarkers of central and peripheral nervous system damage described in human and veterinary medicine. Many of these are already used as tools in the diagnosis of human neurological disorders, and many are investigated in regard to their use in small and large animal veterinary medicine. The following review presents the current knowledge about the application of cell-type (glial fibrillary acidic protein, neurofilament subunit NF-H, myelin basic protein) and central nervous system specific proteins (S100B, neuron specific enolase, tau protein, alpha II spectrin, ubiquitin carboxy-terminal hydrolase L1, creatine kinase BB) present in the cerebrospinal fluid and/or serum of animals in the diagnosis of central or peripheral nervous system damage in veterinary medicine.

Resistance Artery Creatine Kinase mRNA and Blood Pressure in Humans

Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6-1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature's pressor responses.

Mixtures of Receiver Operating Characteristic Curves

Receiver operating characteristic (ROC) curves are ubiquitous in the analysis of imaging metrics as markers of both diagnosis and prognosis. While empirical estimation of ROC curves remains the most popular method, there are several reasons to consider smooth estimates based on a parametric model. A mixture model is considered for modeling the distribution of the marker in the diseased population motivated by the biological observation that there is more heterogeneity in the diseased population than there is in the normal one. It is shown that this model results in an analytically tractable ROC curve which is itself a mixture of ROC curves. The use of creatine kinase-BB isoenzyme in diagnosis of severe head trauma is used as an example. ROC curves are fit using the direct binormal method, ROCKIT software, and the Box-Cox transformation as well as the proposed mixture model. The mixture model generates an ROC curve that is much closer to the empirical one than the other methods considered. Mixtures of ROC curves can be helpful in fitting smooth ROC curves in datasets where the diseased population has higher variability than can be explained by a single distribution.

Effects of chronic tramadol exposure on the zebrafish brain: A proteomic study

Tramadol hydrochloride (TH), has become the most prescribed opioid worldwide. However, its neurotoxicity and abuse potential are not well documented. In the present study, TH administration induced abnormal behavior and body and brain mean weight loss. Two principal metabolites O- and N-desmethyltramadol were detected in the brain tissue, and N-desmethyltramadol was the main metabolite produced. A total of 30 differential protein spots were identified using semi-quantitative 2D-PAGE and proteomic analyses, and classified into 13 categories, in which subtypes of 14-3-3 proteins, creatine kinase, ATP synthase beta chain, and tubulin were identified at the separated location on the gels 3, 3, 4, and 11 times respectively. Many TH responsive proteins have functions related to oxidative stress, including 14-3-3 proteins, creatine kinase BB, ubiquitin carboxy-terminal hydrolase L-1, ATP synthase, synaptosome-associated protein, tubulin and actin. Irrespective of oxidative damage, other pathways affected include apoptosis, energy metabolism, signal disorders, and cytoskeletal structure. Ultrastructural observation of mitochondria showed a series of morphological changes in the case of TH exposure.

Death Is Associated with Complement C3 Depletion in Cerebrospinal Fluid of Patients with Pneumococcal Meningitis

ABSTRACTPneumococcal meningitis can lead to death or serious neurological sequelae as a result of the host inflammatory response. We investigated the association between host response protein expression and outcome in patients with pneumococcal meningitis. Cerebrospinal fluid (CSF) was obtained from 80 patients with pneumococcal meningitis (40 nonsurvivors and 40 survivors) and 10 normal controls. Candidate proteins were analyzed for an association with survival. Complement C3 levels were 5-fold lower in nonsurvivors than in survivors (P < 0.05). This C3 reduction was not associated with lower levels in serum, indicating a compartmentalized CSF response. Transferrin levels were significantly higher in CSF (but not serum) from nonsurvivors than in CSF from survivors, suggestive of blood-brain barrier damage. Classical apoptosis proteins caspase 3 and apoptosis-inducing factor were not present in CSF. Expression of creatine kinase BB in clinically infected CSF suggested neuronal necrosis, but there was no clear association between level of expression and clinical outcome. Increased blood-brain barrier permeability and complement C3 depletion may have a role in determining outcome from bacterial meningitis. Therapeutic use of citicoline or caspase inhibitors is unlikely to have beneficial effects in patients with meningitis.

Subcellular Distribution and Relative Expression of Fibrocyte Markers in the CD/1 Mouse Cochlea Assessed by Semiquantitative Immunogold Electron Microscopy

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.

Sex specific response of cultured human bone cells to ERα and ERβ specific agonists by modulation of cell proliferation and creatine kinase specific activity

We have previously reported that human cultured bone cells (hObs) respond to estradiol-17β (E2) by stimulating DNA synthesis, creatine kinase BB specific activity (CK) and other parameters sex-specifically. We now investigate the sex specificity of the response of these hObs to estrogen receptor (ER) α and ERβ specific agonists. Real time PCR revealed that all cells express mRNA for both ERs. ERα mRNA but not ERβ mRNA was stimulated by all estrogenic compounds in both pre- and post-menopausal hObs with no effect in male hObs. Cells treated with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4′,4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris–phenol (PPT; ERα specific agonist) showed increased DNA synthesis and CK in all female but not male hObs. Raloxifene (Ral), a specific ERα antagonist, inhibited the stimulation of DNA synthesis and CK by E2 or PPT, but not by DPN. DPN and PPT like E2 modulated the expression of both 12 and 15 lipooxygenase (LO) mRNA in both female but not male hObs. 12 and 15 HETE production was modulated only by DPN and PPT in these cells. The LO inhibitor baicaleine inhibited only E2 and PPT but not DPN effects in both female hObs. In conclusion, we provide herein evidence for the separation of age- and sex-dependent mediation via both ERα and ERβ pathways in the effects of estrogens on hObs, with a yet unknown mechanism.

The effects of estrogen receptors α‐ and β‐specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.

Identification of Five Candidate Lung Cancer Biomarkers by Proteomics Analysis of Conditioned Media of Four Lung Cancer Cell Lines

Detection of lung cancer at an early stage is necessary for successful therapy and improved survival rates. We performed a bottom-up proteomics analysis using a two-dimensional LC-MS/MS strategy on the conditioned media of four lung cancer cell lines of different histological backgrounds (non-small cell lung cancer: H23 (adenocarcinoma), H520 (squamous cell carcinoma), and H460 (large cell carcinoma); small cell lung cancer: H1688) to identify secreted or membrane-bound proteins that could be useful as novel lung cancer biomarkers. Proteomics analysis of the four conditioned media allowed identification of 1,830 different proteins (965, 871, 726, and 847 from H1688, H23, H460, and H520, respectively). All proteins were assigned a subcellular localization, and 38% were classified as extracellular or membrane-bound. We successfully identified the internal control proteins (also detected by ELISA), kallikrein-related peptidases 14 and 11, and IGFBP2. We also identified known or putative lung cancer tumor markers such as squamous cell carcinoma antigen, carcinoembryonic antigen, chromogranin A, creatine kinase BB, progastrin-releasing peptide, neural cell adhesion molecule, and tumor M2-PK. To select the most promising candidates for validation, we performed tissue specificity assays, functional classifications, literature searches for association to cancer, and a comparison of our proteome with the proteome of lung-related diseases and serum. Five novel lung cancer candidates, ADAM-17, osteoprotegerin, pentraxin 3, follistatin, and tumor necrosis factor receptor superfamily member 1A were preliminarily validated in the serum of patients with lung cancer and healthy controls. Our results demonstrate the utility of this cell culture proteomics approach to identify secreted and shed proteins that are potentially useful as serological markers for lung cancer.