Additionally, polymerase chain reaction, fluorescence in situ hybridization, capillary electrophoresis with laser-induced fluorescence, and mass spectrometry using matrix-assisted laser absorption-deionization time-of-flight and surface-enhanced laser desorption/ionization time-of-flight techniques are being tested for in vitro assessment of the non-cellular biomarkers in CSF. For in vivo detection of cancer in the CSF, research techniques include certain quantum dot platforms as well as magnetic iron oxide nanoparticles. As systemic therapies for cancer improve, the CNS is becoming a more common site of disease recurrence. This increases the importance of effective detection methods in the CSF, since early intervention can maximize therapeutic benefit. Furthermore, many cell-based detection methods can be combined with therapeutic agents to serve multiple medical functions through a common targeting system.The spread of cancer into the central nervous system is a serious problem leading to neurological symptoms and rapid mortality. The current tools available for detecting the spread of cancer into the cerebrospinal fluid (CSF) are cytology, neurologic examination, and neuroimaging. All three of these methods can be applied in concert to reach a diagnosis, but they all suffer from a lack of sensitivity, leading to delays in treatment in many cases. An overview of research tools in the field of CSF cancer detection reveals a variety of promising technologies that can be used to answer questions about the biology of metastatic cancer and to develop more powerful clinical detection methods. Methods currently under investigation include new immunocytochemistry methods and flow cytometry for the  The ability to detect and characterize malignant cells in the CSF derived from primary neural and extraneural cancers may allow us to answer fundamental questions about the biology of metastatic spread through identification and characterization of the cancer cell populations capable of infiltrating the CSF. Cancer may reach the CSF through hematogenous spread, direct extension from the tumor itself, or by migration along perineural or perivascular spaces ,2. ImproA prior review by Chamberlain and colleagues described the three primary methods of detecting leptomeningeal metastasis and the spread of tumor cells into the CSF: CSF cytology, neurologic examination, and radiographic imaging of the neuraxis . While aCSF cytology, in which CSF is prepared and examined under a microscope to look for cells, is currently considered the gold standard for diagnosis of leptomeningeal metastasis . While iThe second standard method of detecting cancer in the CSF is by clinical findings including headache, mental status changes, diplopia, weakness, and sensory loss . This meThe third primary means of detecting cancer in the CSF is through neuroimaging. Among the neuroimaging modalities, gadolinium-enhanced MRI of the neuraxis is the method of choice . MRI hasin vitro analysis of CSF and in vivo imaging and spectroscopy techniques.Because the clinically utilized methods for detecting the presence of cancer cells in the CSF suffer from multiple limitations, a wide array of alternatives are being developed to better answer clinical and basic science questions. Broadly, these efforts focus on both improved As far back as 1984, the use of monoclonal antibodies like UJ13A for neuroectodermal cells, LE61 for cytokeratin, and 2D1 for leucocytes to detect and diagnose cancer in the CSF, showed advantages over conventional cytology . Combinein vitro [The arrival of quantum dots coated for biomedical applications using polymer coating, lipid encapsulation, or ligand exchange provides an ideal fluorescent tag for identifying cancer cells in vitro -23. Orgain vitro . In contin vitro . Outsidein vitro .Quantum dots are now proving successful at improving cellular detection using a variety of antibody and peptide ligands . As new P = 0.002). Impressively, flow cytometry required a neoplastic clone to make up only 0.2% of all CSF lymphocytes in order to be detected. For comparison, conventional cytology generally requires 5% tumor cells to find a positive result. The same study showed higher sensitivity in relapsed CNS lymphoma, although the sample size was smaller [P = 0.05). This is encouraging for the future of this technique, since occult levels of neoplastic cells in the CSF appear to have a bearing on patient risk. The fact that only positive cytology showed a significant relationship with overall survival suggests a continuum of severity in which increased cell number corresponds to poorer prognosis and greater need for intervention.Another promising method for the detection of cancer cells in the CSF is flow cytometry. This has been attempted mostly for hematologic tumors (leukemias and lymphomas) and has shown promise as a complementary method to cytology . In work smaller . One con smaller . More re smaller . In the  smaller . They foOne caveat to the use of flow cytometry on CSF samples, however, is that the inherently low cell number and suboptimal cell environment mean that cancer cells decay rapidly following removal from patient, even more so when centrifuged repeatedly . Other pIn-Situ Hybridization (FISH). In one study, 13 of 15 CSF samples with cells from leptomeningeal cancer patients showed numerical chromosomal abnormalities compared to no chromosomal abnormalities in the 10 control samples [One method that has been used to identify genetic changes in cancer cells of the CSF is Fluorescence  samples . The stu samples . SpecifiIn another notable study, FISH was performed on tumor samples and CSF cytology slides of 16 patients with metastatic breast cancer to the leptomeninges to compare the primary tumor's HER2 status to that of the metastatic cells . Using tAnother approach to improving the sensitivity of cell detection is to use polymerase chain reaction (PCR) methods to amplify DNA specific for cancer cells. One example is the amplification of IgH rearrangements in the CSF of patients with CNS lymphomas . More coP < 0.0002 using ganglioside GD3 [In addition to detecting cancer cells in the CSF through direct methods, a considerable amount of effort has focused on indirectly detecting CSF malignancy through changes in expression patterns of proteins and metabolites in CSF samples. The proteomic/metabolomic approach could theoretically be more sensitive because even a few cells could alter the chemical milieu through disruption of the blood-brain barrier or through direct shedding of detectable molecules. Examples of CSF biomarkers for cancer are many. In an early study, carcinoembryonic antigen (CEA) and the β subunit of human chorionic gonadotropin (β-HCG) were found to correlate with leptomeningeal infiltration of the CSF by carcinoma . Anotherside GD3 . An imposide GD3 .Detection of amino acid profiles in the CSF using capillary electrophoresis followed by laser-induced fluorescence (CE-LIF) detection has been performed in 3 patients with acute lymphoblastic leukemia . This stMass spectrometry using matrix-assisted laser absorption-deionization time-of-flight (MALDI-TOF) and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) techniques has also been applied to the CSF to detect malignancy. Studies of MALDI-TOF and SELDI-TOF MS in serum have demonstrated high sensitivity and specificity for detecting protein biomarkers related to a variety of cancers including prostate and ovarian, and have been used in both blood and CSF -54. CSF-Using another potentially useful approach, zymography for matrix metalloproteinases, particularly MMP2, and pMMP9 reportedly detect the presence of primary and metastatic brain tumors . InteresFinally, trying to detect the fewest number of cells possible leads to a need either to improve sensitivity directly through more powerful targeting systems or to enrich target population of cells. While RT-PCR is one approach for amplifying rare molecules, another method that deserves some consideration for increasing the cell number is through culture of the CSF samples. While it may prove impossible to meet with routine success with this method, any successful cultures derived from CSF could help answer important questions about metastatic behavior. To date, only a few cases of successful isolation of cell lines have been documented, and their properties have not been examined through the lens of understanding the process of CSF metastasis ,65. Althin vitro approaches suffer from the collection limitations mentioned above. To be able to examine the entire CSF pool, in vivo methods are necessary (possibly with multiple access points in the event of an obstruction of flow due to solid tumor or other changes in the CNS anatomy). Therefore, multiple modalities of in vivo CSF screening for cancer are under development.For all their advantages, all Iron oxide, is a potentially useful targeted imaging agent because of its ability to function as a T2 contrast agent for MRI . In cellin vivo applications. In animal models, quantum dots have been successfully targeted to a variety of tumors [in vivo as part of multimodal diagnostic and therapeutic agents targeted to tumors with antibodies and peptide ligands. To create an imaging platform of this nature, Erogbogbo and colleagues combined quantum dots with magnetic iron oxide nanoparticles and packed the combination into micelles for in vivo delivery. Combining both particles in one platform allowed for both optical and magnetic resonance imaging using targeted agents [The desirable luminescent properties of quantum dots have led to multiple investigations of their f tumors . The crif tumors . Recentld agents .in vitro assessments and in vivo surveillance, although further studies must be done to establish their true clinical potential. In addition to the methods under investigation, the literature appears to lack any published efforts in some other potentially useful techniques, including multiplexed cytokine analysis, which has been useful in neurodegenerative diseases such as ALS, and magnetic resonance spectroscopy, which is a common method of assessing neural biomarkers noninvasively [As therapies for extra-neural cancers become more successful, the CNS in general and the CSF in particular are increasingly becoming the most common sites of disease recurrence -73. Emervasively ,75.The ultimate goal of diagnostics in CSF is to detect the earliest meaningful sign of neoplastic presence in the CSF to enable clinicians to react with the most relevant therapy. Currently, three key methods exist that are fairly specific but lack the sensitivity early in the disease course that will enable the most timely therapeutic response. Future therapies must overcome the challenges of detecting few cells in a comparatively large space either directly or by profiling soluble biomarkers that herald their arrival. In the past decade, advances in clinical imaging and targeting ligand development have improved our ability to detect leptomeningeal involvement. Based on the state of the field, flow cytometry appears the most promising among the emerging technologies detailed here, particularly for aiding in the diagnosis of hematologic malignancies. Profiling of the soluble markers of the CSF is likely to become important in the future, but it is too early to say which method will prove most reliable in attaining that end.With the CSF more clinically relevant than ever, methods for surveillance are becoming increasingly important . The effThe authors declare that they have no competing interests.CLW drafted the manuscript, performed revisions, and provided the final manuscript draft. MJG provided clinical perspective on the article, offered criticism, provided revisions, and gave final approval to the submitted manuscript. JRC established the concept of the article, offered criticism, provided revisions, and gave final approval to the submitted manuscript. All authors have read and approved the final version of the manuscript.


The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers.Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data.A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival.Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer. Epithelial ovarian cancer (EOC) remains one of the deadliest gynaecological cancers with more than 204 000 new cases being diagnosed worldwide each year . The canMCPH5 encoding abnormal spindle-like microcephaly-associated protein (ASPM) and MCPH1 encoding microcephalin have been shown to be associated with autosomal primary microcephaly, a developmental brain disorder characterised by an architecturally normal but small brain and associated mental retardation (http://www.proteinatlas.org)). We hypothesised that as mitosis- and DNA repair-associated proteins, ASPM and microcephalin, might have a role in the tumourigenesis of cancers with highly aneuploid tumours, such as high-grade EOC. Recently, both ASPM and microcephalin have been shown to be deregulated in a variety of cancers , all obtained from Cancer Research UK, and cells from 40 primary cultures of EOC derived from ovarian ascites samples collected from patients receiving treatment at St James's Hospital Institute of Oncology. Also included were primary cultures established from the peritoneal washings of two patients who were found to have benign epithelial neoplasms . Ethical approval was obtained for all clinical samples involved in this study from the local Research Ethics committee of the Leeds Teaching Hospitals NHS Trust, Leeds.2 flasks in a 5% CO2 incubator at 37°C. Once cells were confluent they were trypsinised and passaged at a 1/3 dilution. Passage levels were kept low to minimise phenotypic changes due to adaptation to cell culture.On receipt, ascites samples were processed aseptically as described previously  were used throughout this study. Although information on the specificity of these antibodies has been previously published  and by s2 flasks until confluent. The cells were washed with PBS. Fresh PBS (10 ml) was added and the cells were detached using cell scrapers. The cells were pelleted by centrifugation for 5 min at 1000 r.p.m., resuspended in 1 ml of ice-cold RIPA buffer ) and transferred to Eppendorf tubes. To promote lysis the cells were passed repeatedly through a 19-gauge needle after which the samples were placed on ice for 15 min before being transferred to a microcentrifuge and spun for 30 min at 13 000 r.p.m. at 4°C. The supernatants were carefully removed and aliquots were stored at −20°C until further use. Protein estimation was performed using the Modified Bradford method . Samples with a total protein concentration of 0.5 mg ml−1 or more were analysed by slot blotting including 4 cell lines, 1 benign sample and 32 primary cultures.To compare ASPM protein levels in established cancer cell lines, benign samples and primary cultures, a slot blotting technique was applied. Both primary cultures and cell lines were grown in 75 cm−1 of protein and heated to 95°C for 5 min. A volume of 100 μl of the diluted cell lysate solution was loaded onto nitrocellulose supported by a pre-wetted Whatman filter paper  contained within a slot blot apparatus  according to the manufacturers’ instructions. Lysate protein was transferred onto the nitrocellulose in individual slots by applying a vacuum of 0.8 atmospheres for 2 min. All slots were washed with 100 μl of PBS with pressure reapplied for 2 min. The nitrocellulose was removed from the slot blotter apparatus and blocked with 1% non-fat dried milk in PBS for 30 min on a rocker at room temperature. Rabbit anti-ASPM antibody 217-2  at 1/3000 was added to the nitrocellulose and incubated for 1 h at room temperature. After three washes for 5 min each in PBS-Tween (0.1%) the nitrocellulose was incubated in the secondary antibody solutions, anti-rabbit or anti-rat IgG-horseradish peroxidase  at 1/50 000 in non-fat dried milk in PBS for another hour on a rocker at room temperature. The nitrocellulose was washed three times for 5 min in PBS-Tween and once in PBS only. The Pierce SuperSignal West Femto ECL reagent (Pierce) was added to the nitrocellulose membrane and incubated for 5 min. Signals were recorded using a Bio-Rad ChemiDoc XRS system (Bio-Rad). The slot blot images from three repeats for each sample were scanned and band densities analysed to determine relative levels of ASPM and of tyrosinated α-tubulin using Bio-Rad Quantity 1 software (Bio-Rad). After background levels were subtracted the resulting values were examined as a ratio of ASPM/α-tubulin for each sample, with high values representing the samples with highest ASPM protein levels. Cutoff points were determined to classify samples as having low, medium or high ASPM levels.For slot blotting, protein lysates were diluted in RIPA buffer to a concentration of 0.1 mg mlα-tubulin antibody  were prepared in blocking solution (0.5% non-fat dried milk in PBS). They were spun for 5 min in a microfuge at 13 000 r.p.m. before use. Coverslips were inverted over a 200 μl drop of this supernatant on parafilm in a humidified chamber for 1 h at room temperature, then returned to the 6-well dish for washing. After three washes in PBS for 5 min each, the coverslips were incubated in secondary antibody solution containing a mixture of goat anti-rabbit IgG Alexa fluor 488 , goat anti-rat IgG Alexa fluor 568  and DAPI (5 μg ml−1) diluted with the blocking solution and spun for 5 min at 13 000 r.p.m. before introduction to the cells. After a further incubation for 1 h in a humidified chamber at room temperature, the coverslips were washed four times in PBS for 5 min each and then mounted onto microscope slides using Mowiol (Sigma). Slides were allowed to set overnight and viewed using a Zeiss Axiovert 200 fluorescence microscope  with a × 63 Plan Apo oil immersion lens. Images were captured and processed using a Hamamatsu Orca ER cooled CCD camera  and Andor IQ imaging software .Primary cultures and cell lines were grown on sterile coverslips in 6-well tissue culture dishes  until they reached at least 75% confluency. The cells were washed in PBS and fixed with ice cold 100% methanol at −20°C for 2 min. After removal of the methanol, the cells were incubated in 0.5% non-fat dried milk in PBS for 10 min to block non-specific antibody-binding sites. The rabbit anti-ASPM antibody 216-1 (1/500) (n=22) and microcephalin immunofluorescence (n=36) in the cell lines and in the primary cultures for comparison with the associated clinical data. Five hundred to one thousand cells were observed for staining profiles and quantification of fluorescence intensities was performed for each sample in each of 20 interphase cells and at least 20 mitotic cells using Andor IQ software. Samples containing <20 mitotic cells per 1000 cells were not included in the mitotic analysis. For microcephalin, nuclei were identified by DAPI staining and the fluorescence within the nucleus measured and divided by the area to give the integrated intensity per unit area. The same method was applied for establishing nuclear ASPM and cytoplasmic fluorescence levels. For ASPM levels in mitotic cells, the fluorescence intensity within a defined region covering each spindle pole at metaphase was recorded and the sum of intensities for both spindle poles was determined. All recorded data were examined and subsequently presented as weak, medium or strong after establishing logical cutoff points for each category.We determined the levels and localisation of ASPM  and the non-parametric Kruskall–Wallis and Wilcoxon–Mann–Whitney test for continuous variables. The association between ASPM or microcephalin and survival data was evaluated using the Kaplan–Meier method of estimating survival functions and Cox proportional hazards regression. All analysis was undertaken in the R Environment for Statistical Computing .Clinical data was available for 40 ovarian cancer patients from which ascitic fluid was obtained to generate the primary cultures. The mean age of the patient group was 62.2 years with a range of 26–85 years. In all, 35 out of 40 (87.5%) of the patients were over 50 years of age and 5 out of 40 (12.5%) were under 50 years of age. Among the ovarian cancer patients, 3 out of 40 (7.5%) presented with stage 1 ovarian cancer, 1 out of 40 (2.5%) stage 2, 30 out of 40 (75%) stage 3 and 6 out of 40 (15%) stage 4 disease. Grading and tumour typing was performed on histological material according to the FIGO grading system identifying 8 out of 40 (20%) of patients with grade 1 tumours, 11 out of 40 (27.5%) with grade 2 tumours and 19 out of 40 (47.5%) with grade 3 tumours. One sample was a borderline case (2.5%). One patient (2.5%) had been given chemotherapy based on ascitic fluid cytology together with elevated tumour markers  and radiological features consistent with stage 3c primary peritoneal carcinoma. It was not possible to grade this tumour based on the cytological preparation. The majority of the carcinomas were high-grade serous adenocarcinoma , with the remaining samples comprising low-grade serous cystadenocarcinoma , endometrioid and mucinous type  and a further group assigned serous carcinoma , which were post-chemotherapy samples. The median survival times from diagnosis for the patients was 39 months with a range of 2.7–112.5 months.et al, 2005; P=0.331). In addition, our slot blot analysis demonstrated that there were significantly fewer deaths among patients with high ASPM levels (1 out of 11 9%) when compared with patients with low levels (7 out of 13 53.8% P=0.036).Previous publications have indicated that ASPM levels are upregulated in various cancers at the RNA level . The association indicates that patients with a weak score have an increased hazard of death, which correlates with the loss of nuclear staining and increase in cytoplasmic staining seen in the higher tumour grades , r grades .We also examined ASPM and microcephalin expression using IHC in 23 paraffin-embedded primary tumour samples from patients whose ascites were used to generate primary cultures. Importantly, staining patterns and intensities in these samples were similar to those detected in the corresponding primary cultures , confirmMCPH gene products, ASPM and microcephalin, were deregulated in EOC at the protein level. We also aimed to investigate if there was an association between tumourigenesis and ASPM and microcephalin protein levels in EOC, because ovarian cancers exhibit a high degree of aneuploidy and these proteins are known to be involved in mitotic processes and DNA repair. There has recently been an increased interest in proteins associated with chromosomal instability and aneuploidy in cancer. High expression of the mitotic kinase BubR1 was demonstrated to be associated with aggressive clinico-pathological parameters including advanced stage of disease, serous histology and high-grade tumours in EOC  according to p53 (high-grade carcinomas; MCPH1 mutations caused the localisation of microcephalin to change from nuclear to cytoplasmic, providing a plausible explanation for our latter observations (BRCA1 in EOC have been shown to be responsible for a shift from nuclear to cytoplasmic localisation, inhibiting nuclear DNA repair and transcription function (In our study, we utilised primary cultures of EOC derived from ascites. Malignant cells derived from ascitic fluids have recently been used for the study of drug response and novel treatment regimes . In the function , but we In future studies it will be interesting to investigate associations between microcephalin and ASPM levels and chemoresistance to paclitaxel/carboplatin. Carboplatin reacts with DNA inhibiting DNA replication and causing DNA breaks, whereas paclitaxel is a mitosis-specific drug, binding specifically to microtubules and inhibiting mitosis. Both of these modes of action could potentially be impacted by changes in the functional activity of microcephalin or ASPM. In conclusion, we have shown that ASPM and microcephalin have variable expression levels in primary cultures of malignant cells derived from ovarian tumour ascites and that there are distinctive localisation patterns for both proteins. Our data indicate that ASPM and microcephalin may be considered as potential prognostic markers in ovarian cancer and further large-scale studies are warranted.


Elevations of cardiac enzymes are commonly used to indicate myocardial ischemia, but they can be elevated due to other conditions. Different forms of Troponin , can cause cross-reactivity in the Troponin T assay, leading to false positives. This report describes a patient with polymyositis who had elevated Troponin T, but no cardiac abnormalities. The purpose is to show that Troponin T, which is believed to be solely from cardiac muscle breakdown, can be seen in inflammatory muscle disease, so Troponin I should be used instead.This is a case report of a 70-year-old woman with a history of diabetes, hypertension, gout and polymyositis, who presented with one-day history of lightheadedness and abdominal pain. To rule out myocardial ischemia, cardiac enzyme testing was ordered which showed elevated CK, CK-MB, and Troponin T. A full cardiac workup was performed which showed no signs of ischemia. Troponin I was <0.05 ng/mL, .In inflammatory myositis, there are elevations in many cardiac markers due to non-cardiac causes, which could be related to muscle regeneration and gene expression. This is not seen certain isoforms of Troponin I, specifically cardiac Troponin I.In patients with history of diabetes and other comorbidities, silent myocardial ischemias should be ruled out. Non-cardiac elevations in Troponin T can be seen in patients with inflammatory, so Troponin I should be ordered to get an accurate interpretation. Patients with inflammatory myopathies can have elevations in CK, CK-MB, and Troponin T, but not Troponin I. Currently, elevations in cardiac enzymes like creatine kinase- cardiac muscle (CK-MB) and Troponin T are commonly used as indications of myocardial ischemia, but these enzymes can be falsely elevated  in many other pathophysiological constellations as well. Troponin T is considered to be a highly sensitive marker of myocardial injury, but it can be falsely elevated in conditions like renal failure, subarachnoid hemorrhage, myasthenia gravis, rhabdomyolysis, rheumatoid arthritis and inflammatory muscle diseases like dermatomyositis and polymyositis , 6. In pA 70 year old woman with history of diabetes type 2, hypertension, gout and polymyositis was admitted to our hospital. She presented to the ED with symptoms of light-headedness, dizziness and pre-syncopal episode earlier in the morning, along with a sudden onset of generalized body weakness. She denied any loss of consciousness or palpitations before presentation to the ED. She also complained of sharp pain in the upper right abdominal quadrant, verbally stated as 10/10 in pain, non-radiating, for 4 days duration, with associated nausea. Patient recently had right kidney surgery with a nephrostomy tube draining 5-10 ml of blood as well for the past 4 days. She had past history of nephrolithiasis one year ago, leading to severe right-sided hydronephrosis  of the abdomen showed a small right kidney with reduced cortex thickness and with small amount of perinephric fluid accumulation with no signs of hydronephrosis. Patient was started on Imipenem to cover for pyelonephritis, and the right upper quadrant pain resolved in 4 days with stable vital signs.To assess if the symptoms of lightheadedness, sudden generalized weakness and pre-syncopal episode were due to a cardiac cause  or transient ischemic attack, cardiac evaluation, along with doppler ultrasound of the carotid arteries and CT of the brain, were performed. CT showed no signs of ischemia or hemorrhage, and an electrocardiogram (ECG) from the day of admission showed left axis deviation and signs of possible old anterior infarction with associated ST-T/ T wave abnormalities, unchanged compared to the ECG from a year ago. Carotid doppler was normal with no signs of stenosis. Cardiac enzymes results are shown in Table From patient’s admission one year ago, CK was 484 U/L, and CK-MB was 12.3 mg/mL, indicating elevated CK and CK-MB were due to polymyositis muscle breakdown. Further laboratory findings showed myoglobin 186.2 ng/mL , haptoglobin 337 mg/dL , with normal blood uria nitrogen (BUN), creatinine and thyroid function. Though there were no acute changes on ECG, the patient is at increased risk of coronary artery disease with a stress test pretest probability of 10%-90% risk due to her age and history of diabetes and hypertension. To further assess causes of the abnormal ECG, 2D echocardiogram of the heart was obtained which showed left ventricle ejection fraction of 65% to 70% with normal atria and ventricles. There was mild mitral regurgitation and mild tricuspid regurgitation with right ventricle systolic pressure of 43 mm, which was consistent with mild pulmonary hypertension. To ensure there were no signs of myocardial ischemia a nuclear stress test was performed instead of the traditional stress test due to the patient’s muscle weakness. The nuclear stress test measures blood flow to the myocardium at rest and during exercise, simulated with the use of medications. The study showed normal myocardial perfusion with normal left ventricular centrality at rest, and an ejection fraction of 54%, all within normal limits. Though all the tests performed indicated there were no signs of myocardial ischemia, we decided to obtain a Troponin I level, which was <0.05 ng/mL . By performing all these tests we had concluded that there was no cardiac cause for the increased CK-MB and Troponin T, reflecting that patient’s Troponin T elevations must have been due to polymyositis.Polymyositis is an inflammatory muscle disorder caused by auto-antibodies against skeletal myositis, which results in proximal muscle weakness and muscle disorder. Treatment for this systemic disease requires the use of immune-suppressants such as corticosteroids, azathioprine or methotrexate, but other organs can also be affected by this disease. Patients with inflammatory myositis are at increased risk of cardiovascular complications including arrhythmias, myocardial infarction and myocarditis because of antibody cross-reactivity with the cardiac muscle . The mosAs seen in this case report, in patients with polymyositis CK and CK-MB are usually elevated due to the constant muscle breakdown and rebuilding secondary to immune mediated destruction of skeletal muscle. CK is an enzyme used to carry and release energy, and is found in various tissues in the body. It has 3 different isoenzymes, CK-BB (brain), CK-MM , and CK-MB (cardiac muscle). As the skeletal muscle begins to regenerate, it has been shown that there is an increase in CK-MB in the new skeletal muscles due to re-expression of embryonic genes, and this increase in CK-MB can represent anywhere from 5-10% of total CK , 10, as Based on the impairment and regeneration of skeletal muscle, this tissue also releases Troponin T. Troponin as a regulatory protein is responsible to stabilize and activate actin for muscle contraction, and is composed of three subunits: Troponin T (TnT), Troponin I (TnI), and Troponin C (TnC). Troponin T binds to tropomyosin to generate a complex, Troponin I binds to actin to block its active site, and Troponin C binds to calcium and induces enzyme activation. Skeletal muscle shows specific Troponin groups , which are released in case of alterations focusing on skeletal muscle; cardiac forms of Troponin T (cTnT), Troponin I (cTnI) and Troponin C (cTnC) also exist, which are released when myocardial damage occurs . As the no correlation between cTnI and CK-MB  have elevated levels of CK, CK-MB, sTnT and cTnT that arise due to the the continuous impairment and regeneration of skeletal muscle. The antibodies used to detect cTnT in the “Troponin T assay” cross-react with the various other subtypes of Troponin T, and elevated levels of these proteins are often misinterpreted as Troponin T (cTnT) . To deli


Nonalcoholic steatohepatitis (NASH) is a stage of nonalcoholic fatty liver disease (NAFLD), and, in most patients, it is associated with obesity and metabolic syndrome with progression to end-stage liver disease in about 20% of patients (McCullough (2004); Matteoni et al. (1999); Liou and Kowdley (2006)). It has been estimated that between 20 and 30% of patients with end-stage cirrhosis referred for liver transplantation (LT) evaluation and 30 to 70% of LT recipients exhibit some degree of obesity (Muñoz and ElGenaidi (2005)). Management of obesity in chronic liver disease patients is not only difficult but also preludes them from undergoing major bariatric surgery due to associated high morbidity and mortality. Here, we present a case report of a morbidly obese patient who underwent laparoscopic sleeve gastrectomy followed by single-lobe living donor liver transplantation (LDLT) with a successful outcome. We believe that this is the first report of successful LDLT following planned weight loss to facilitate LDLT. He was diagnosed to have cirrhosis with persistently high bilirubin which fluctuated between 2 and 5 mg/dL. His Child's score at presentation was B9 with a MELD score of 14. Extensive evaluation for the etiology of liver disease including viral markers and autoimmune markers and iron and copper studies were all negative. There was no history of alcohol intake; hence, in view of morbid obesity and dyslipidemia, the diagnosis of nonalcoholic steatohepatitis for cirrhosis was considered. His gastroduodenoscopy showed small varices with portal hypertensive gastropathy, and a triple-phase CT scan of the abdomen showed a shrunken liver with splenomegaly, multiple portosystemic collaterals, and no ascites. He was listed for deceased donor liver transplantation and advised weight reduction with dietary and behavioral measures. However, he did not show any significant improvement over the next three months. Since he was morbidly obese and had failed medical therapy for weight reduction, he was planned for bariatric surgery after counseling for worsening of liver disease in the postoperative period and a possible requirement for immediate liver transplantation in that eventuality. A prospective living donor was identified in the family and completely worked up in case the patient showed signs of worsening liver disease following bariatric surgery. He underwent laparoscopic sleeve gastrectomy and had a smooth postoperative course. Following bariatric surgery, he had a significant reduction of weight from 160 kg to 102 kg in six-month period. However he developed worsening of his liver disease in the form of progressive jaundice, ascites, and recurrent episodes of hepatic encephalopathy. His Child's score worsened from B9 to C12, and MELD score increased to 24 over the next six months. In the absence of availability of cadaveric donor, living donor liver transplantation was considered as his weight had now decreased substantially. He underwent a modified right-lobe LDLT from his sister-in-law as the donor with a graft weight of 837 grams and an acceptable GRBWR of 0.81. The postoperative period was smooth except for basal atelectasis, which required intense physiotherapy and intermittent BIPAP for a few days.A 29-year-old young male law graduate, morbidly obese since adolescence, has been symptomatic since the last 7 years with fatigue, generalized weakness, and fluctuating jaundice. He was 160 kg at the time of presentation with a BMI of 55.36 kg/mLiver transplant in obese patients has been associated with increased morbidity and mortality in the postoperative period , 2. OptiInadequate GRBWR is a problem encountered in obese patients. Cadaveric transplant is ideal; or else, dual-lobe transplant would need to be considered in societies where cadaveric donation is uncommon. Dual-graft living donor liver transplantation can overcome small-for-size syndrome in large recipients and ensure donor safety by leaving sufficient remnant liver volume in the donor. However, dual-lobe transplant is often not possible and is also technically demanding. This patient had a successful outcome by a planned, preoperative bariatric surgery and single-right-lobe graft. To the best of our knowledge, this is the first report of successful bariatric surgery for weight loss and planned living-donor-related liver transplantation in a morbidly obese patient. However, we suggest that such technically demanding procedures should be undertaken only in large-volume centers with adequate surgical expertise and critical care management.Bariatric surgery in end-stage liver disease is risky and may be associated with poor outcome. However, in certain special circumstances and centers with backup for living donor liver transplantation, such surgeries may be considered in improving the outcome of morbidly obese cirrhotic patients.


P<0.0001), a longer period of severe neutropenia , longer treatment with intravenous antibiotics  and longer hospital stay . Although the cost of G-CSF was lower in the DGD group (mean $308 vs $2467), the additional hospitalization raised the median total cost of ASCT in this group by 17%. There was, however, no adverse effect of deferred dosing on the rate of febrile neuropenic episodes or Day 100 survival, so that deferred dosing of G-CSF may be suitable for patients receiving ASCT as outpatients, for whom longer hospital stay would not be an offsetting cost.Routine administration of G-CSF following autologous hematopoietic SCT (ASCT) expedites ANC recovery and reduces hospitalization by 1–2 days; it has no impact on febrile neutropenia, infections, morbidity, mortality, event-free survival or OS. To determine whether delayed G-CSF dosage could result in equivalent ANC recovery and thereby improve cost effectiveness, we deferred the administration of G-CSF until WBC recovery had begun. A total of 117 patients with multiple myeloma received ASCT from January 2005 to September 2012. Of these, 52 were in the conventional dosing group (CGD) and received G-CSF from Day +7 for a median of five doses. In the deferred dosing group (DGD), 65 patients received G-CSF from median day 14 post transplant for a median of zero doses. There was no difference between groups in the incidence or duration of febrile neutropenia, duration of ⩾grade III mucositis, weight gain, rash, engraftment syndrome or early death (100 days). The DGD group had a significantly longer time to neutrophil engraftment than the CGD group (15 days vs 12 days;  When administered from day +7 until ANC recovery >1000/μL, G-CSF expedites ANC recovery and reduces hospitalization by 1–2 days.10 Standard of care prior to transplantation now uses agents with less myelosuppressive activity than oral melphalan, the agent most widely used in the past. Moreover, CD-34+ cell collection techniques have improved11 and alternative agents such as plerixafor are now available to assist mobilization where G-CSF/chemotherapy alone has failed.12 As a consequence, the median number of infused CD-34+ cells/kg has significantly increased over the past decade at our own center (3.79 (2.61–9.42) vs 4.49 .ASCT is an established treatment option for patients with multiple myeloma.Given this change, together with the improvements in post-transplant supportive care, it may no longer be appropriate to rely on earlier studies that showed reduced hospital stay for patients routinely receiving G-CSF to dictate the current standard of practice. We therefore stopped the routine use of G-CSF in January 2010 and adapted an alternative dosing schedule in which G-CSF was given only to accelerate neutrophil recovery once this had begun (>200/μL), and if subsequent increases to levels required for discharge (>500/μL) did not follow within 48 h. G-CSF optionally administered in this fashion was labeled deferred G-CSF dosing (DGD). We have now compared the outcomes of 65 patients who received autologous stem cell treatment for multiple myeloma in the DGD group with the outcomes in 52 patients with the disease who routinely received G-CSF daily from day 7 . We have compared each group for engraftment, complication rates, day of discharge and overall costs.P=0.003). More patients in the DGD group received plerixafor (55% vs 12% P=<0.0001) and patients in the DGD group received a higher CD-34+ cell dose .Following Institutional Review Board approval, we retrospectively analyzed data from 117 consecutive patients with multiple myeloma treated with ASCT between January 2005 to September 2012. Fifty-two patients were in the CGDgroup and 65 in the deferred G-CSF dosing (DGD) group. Patient, disease and transplant-related variables are described in 2. Patients with co-morbid conditions including renal failure received a reduced dose of melphalan . All patients received supportive care following institutional standard operating procedures.In 83 patients, melphalan was administered intravenously at 200 mg/m+ cell counts of less than 10 μL on the fourth day of mobilization received plerixafor  at 24 mg/day, which was continued as necessary to completion of collection. Patients with creatinine clearance <60 mL/h received a reduced plerixafor dose (16 mg/day). Twenty-two of 117  patients received chemotherapy-assisted mobilization using cyclophosphamide at 2–4 g/m2 followed by G-CSF (Patients received G-CSF (filgrastim) at 10 μg/kg/day, which was continued until the completion of stem cell collection. Patients with CD-34by G-CSF . PBSCs w+ cells/kg was 3.79 × 106/kg (2.61–9.42) in the CGD group and 4.49 × 106/kg (2.49–10.2) in the DGD patients. Patients in the CGD group received daily doses of 5 μg/kg G-CSF beginning day +7 and those in the DGD group received 5 μg/kg doses at the physician's discretion following WBC recovery. DGD at physician's discretion is a optional alternative dosing schedule in which G-CSF is given only to accelerate WBC recovery once this had begun (>200/μL), and if subsequent increases to levels required for discharge (ANC >500/μL) did not follow within 48 hrs.The median dose of CD 34n=42), those receiving >5.0 × 106/kg CD 34+ cells (n=37) and those receiving no G-CSF in the DGD group (n=36).The primary end point was neutrophil engraftment (day post transplant on which ANC>500/μL × 3 consecutive days). Duration of severe neutropenia, time to platelets recovery to 20 000/μL and that to 50 000/μL, episodes of febrile neutropenia, regimen-related toxicity, duration of hospitalization and cost analysis were secondary end points . SubgrouNeutropenia was defined as an ANC of <500/μL, and temperature of 100.5 °F, defined as neutropenic fever. Severe neutropenia was defined as ANC of <100/μL. Platelet engraftment was defined as platelet counts of >20 000/μL for 3 consecutive days in the absence of platelet transfusions. Cost analysis included cost of G-CSF, cost of intravenous antibiotics and hospital charges for room and board .Our comprehensive cost analysis included an assessment of costs for the transplant admission, room and board, and drugs including antimicrobials and G-CSF. Total admission costs were calculated by combining costs from all cost centers including room and board, pharmacy, radiology, laboratory, central supply, blood bank and physician's fees. Costs for room and board were extracted from the admission cost for each patient. Drug costs reported for antimicrobials and G-CSF represent the average wholesale price (AWP) for each medication. Admission costs, including room and board and drug costs were compiled from The Methodist Hospital's centralized computer billing system. All reported costs represent actual costs for the administration of patient care as determined by the individual departmental finance sections.2 or Fisher's exact test for categorical variables. Statistical significance was defined as P<0.05. All analyses were conducted using SAS 9.3.Descriptive statistics were calculated to describe baseline patient characteristics, transplant outcomes and cost assessment. Comparisons between the CGD and DGD groups were performed using the Wilcoxon rank-sum test for continuous variables and the χP<0.0001) than patients in the DGD; indeed 36/65 (55%) patients in the DGD group received no G-CSF at all. The median post transplant day when G-CSF administration started in the DGD group was 14 days (range=9–18 days). The median total WBC counts and neutrophil counts at the first dose of G-CSF in the DGD group were 1080/μL (range=200–2540) and 148/μL (range=0–1168), respectively. The median ANC rise with the first dose of G-CSF in this group was 2203/μL (range=0–16 257/μL). Patients in the CGD group received more doses of G-CSF  and a longer period of severe neutropenia (<100/μL), at 8 days vs 6 days (P<0.0001). There were no differences between groups in time to platelet engraftment or in their requirement for platelet transfusions.As shown in n=75). As illustrated in P<0.0001), duration of severe neutropenia was improved , duration neutropenia was significantly superior  patients received plerixafor to facilitate CD-34 mobilization. The outcomes of these patients were compared with those who did not receive plerixafor , G-CSF+chemotherapy (n=8) and G-CSF+plerixafor (n=15).The outcomes in these patients were compared with those receiving <5.0 × 106 CD-34 cells/kg (n=80). Interestingly, duration of neutropenia , duration of severe neutropenia  and duration of hospitalization  were superior in patients receiving less than 5.0=106 CD-34 cells/kg. Of the 37 patients who received more than 5.0=106 CD34 cells/kg, 22 patients did not receive G-CSF+plerixafor mobilization. Compared to G-CSF+plerixafor mobilization, patients mobilized with G-CSF alone (n=14) or G-CSF+chemotherapy (n=8) had shorter duration of neutropenia (median 8 days), shorter duration of severe neutropenia (median 7 days) and shorter duration of hospitalization (median 16 days).A total of 37 (31.6%) patients received >5.0 × 10P=0.702); the duration of febrile neutropenia (P=0.759); total number of antimicrobial drugs given ; or the incidence of cultures from any site positive for fungus or bacteria (P=0.338). However, the days of treatment with i.v. antibiotics was longer in the DGD group  (P=0.016) .There were no deaths (by day 100) in either treatment group, and no significant difference in the incidence or duration of mucositis, weight gain, rash or bone pain.P=<0.0001). Despite the offsetting cost of additional G-CSF, the median total cost of ASCT for the CGD group was $61 547 (25 715–122 819) compared with $74 107 (33 104.27–112 213.44) for the DGD patients, a median difference of 17%.The duration of hospital stay was 2 days shorter in the CGD group , without increasing morbidity or hospital stay, and thereby improve the cost-effectiveness of the approach. Our results indicate that routine administration of G-CSF given from day +7 onward to patients receiving auto-SCT for myeloma results in faster engraftment than a deferred dose schedule, and also shortens the duration of severe neutropenia. These effects result in significant cost savings because of a median 2-day reduction in length of hospital stay. Nonetheless, there was no difference between the two groups in the incidence of febrile neutropenia or its duration, in time to platelet recovery or the number of platelet transfusions, infections, mucositis, or in day 100 mortality. Our observation of greater antibiotic utilization in the DGD group even in the absence of any difference in rates of infection likely reflects the slower kinetics of neutophil recoveryPatients who received plerixafor-assisted mobilization had delayed engraftment, longer neutropenia and longer hospitalization compared with those who did not require plerixafor. As we offer plerixafor only to sub optimal mobilizers, the outcomes here likely select a population of patients who are 'hard to mobilize‘ and likely have accompanying slower engraftment kinetics.14 and retrospective analyses19 in which G-CSF after ASCT accelerated engraftment and reduced both the administration of intravenous antibiotics and the duration of hospitalization. Most of these studies also demonstrated that early administration of G-CSF (filgrastim or its pegylated preparation) had no effect on the incidence of febrile neutropenia, infection rates, platelet recovery or transfusion, red cell recovery or transfusion, reduction in regimen-related toxicity or mortality. Our results are, however, different from Schmitz et al.,4 who reported reduced rate of infections in a randomized controlled trial of 192 patients (91% infection rate in the placebo group vs 67% in the G-CSF group). Unlike Bensinger et al.,20 we saw no evidence for a ‘platelet steal' phenomenon in recipients of G-CSF, as the recovery in both the CGD and DGD groups were identical.Several earlier studies have compared the routine administration of G-CSF post transplant  with placebo but a comparison of the efficacy and safety with a deferred dose regimen has not been made. Although our study of conventional vs deferred G-CSF administration is a retrospective analysis, the data are derived from a single center in which a relatively homogenous patient cohort has been treated following clinical standard operating procedures that were consistent over the study period (January 2005–September 2012), thereby minimizing the bias related to patient and treatment heterogeneity. Our results are similar to previous randomized placebo-controlled trials13Interestingly, receiving >5.0 × 106 CD34+ cells/kg did not facilitate engraftment. The duration of neutropenia, duration of severe neutropenia and duration of hospitalization was significantly superior in patients receiving less than 5.0 × 106 CD34+ cells/kg. The reason for this phenomenon is unclear. However, 15/37 (40%) patients have had mozobil mobilization, thus selecting a population of patients as discussed above. Although our patient size is small, these results are in contrast to a large trial studying CD34 cell dose-based engraftment kinetics.We recognize the retrospective nature of the study and its inherent shortcomings. Since we do not perform out patient transplants at our institution, we are not able to provide cost analysis for in patient vs out patient ASCT and its bearing on cost analysis for CGD or DGDAlthough our results showed an overall cost benefit in recipients of CGD, the benefit was almost entirely attributable to the shorter period of hospitalization, which more than offset the savings from reduced G-CSF administration. As there was no adverse effect of deferred dosing on the rate of febrile neuropenic episodes or Day 100 survival, deferred dosing of G-CSF may be an attractive option for patients receiving ASCT on an outpatient basis in whom additional days of hospital stay would not be an offsetting factor.


A number of 13 metabolites have been identified and quantified from cerebrospinal fluid collected from a group of 10 mix breed healthy dogs. The biological variability as resulting from the relative standard deviation of the physiological concentrations of the identified metabolites had a mean of 18.20% (range between 9.3% and 44.8%). The reported concentrations for metabolites may be used as normal reference values. The homogeneity of the obtained results and the low biologic variability show that the 1H NMR analysis of the dog’s cerebrospinal fluid is reliable in designing and interpreting clinical and therapeutic trials in dogs with central nervous system pathologies. The availability of data for reference values in cerebrospinal fluid for healthy humans is limited due to obvious practical and ethical issues. The variability of reported values for metabolites in human cerebrospinal fluid is quite large. Dogs present great similarities with humans, including in cases of central nervous system pathologies. The paper presents the first study on healthy dog cerebrospinal fluid metabolomic profile using  Metabolomics, one of the young “omics” sciences studies small molecules (metabolites) simultaneously from various natural occurring samples . The term metabolomics was first introduced by Oliver , and latThe simultaneous determination of concentrations for several metabolites investigated in metabolomics and metabonomics relies on complex analytical techniques , including hyphenated ones . There are many reviews describing applications in various fields, definitions and history of the field ,5.1H NMR spectroscopy offers several advantages as compared with classical methods. Thus, the 1H NMR spectrum represents the global profile of the analyzed sample, making possible the identification and quantification of more compounds, without the need for individual isolation. A small volume of sample is required for the NMR analysis, from 1 mL down to few microliters with the existing state-of-the-art accessories (e.g. cryoprobes). There is very simple or no sample preparation required for NMR analysis of biological fluids, thus only addition of a small volume of deuterated water , with or without centrifugation being needed. If desired, the pH can be adjusted with buffer solutions or acids / bases solutions. For longer time storage, a preservative (e.g. sodium azide) was added to the sample, to reduce chemical deterioration until NMR analysis. 1H NMR spectroscopy analysis can be used for transforming it in individual concentrations of some metabolites -1-propionate  and sodium azide (used as preservative). The final sample pH was 7.0. The entire volume of sample (600 μL) were transferred into a 5 mm NMR tube and subjected to the analysis. Before NMR analysis the CSF samples were allowed to reach room temperature  and were centrifuged at 7,000 rotations per minute for 10 minutes. The samples were prepared by transferring a 540 μL aliquot of CSF to a 1.5 mL Eppendorf tube followed by the addition of 60 μL phosphate buffer solution (pH 7.5) in deuterated water  referred to TSP (0.0 ppm) as internal standard. The spectra were acquired and processed with the Bruker TopSpin 2.1pl6 software. The NMR protocol allows full relaxation of signals over a 37 s overall relaxation delay, as previously employed by us for other biological samples . Previous reports indicated higher concentrations of acetone in CSF in patients with amyotrophic lateral sclerosis [24] and also as much as 100% variation in humanThe quantitative analysis of the 13 metabolites in the present study showed closely resembling values with the ones registered in humans ,47, the s. The most important factors influencing the concentration of CSF metabolites (and implicitly their RSD) were supposed to be genetics, breeding, diet, environment and age . The ageAn important aspect that could be assessed in this study is the individual’s health status. The difficulty to obtain CSF samples from healthy humans, limited the reference values to patients with meningitis suspicion , unconfir = 0.623, P = 0.462). In addition to that, the glutamine was significantly correlated with the lactate in human multiple sclerosis [r = 0.437, P = 0.206).The correlations between the determined metabolites in dogs were similar with those reported by the studies performed on human CSF in physiologic conditions. Specific differences were observed in cases of data obtained in studies on human pathologic CSF. Levine et al.  comparinclerosis  as an efr = - 0.73, P < 0.000001) unlike the one noticed by us where the correlation was positive with no statistical significance .The ratio lactate/pyruvate (L/P) is considered to be an indicator of the redox cerebral status. However, this ratio was considered without relevance in dogs in some circumstances . This st1H NMR contributed to the simultaneous identification of a great number of compounds, thus helping in the integrated analysis of the physiological relations among various systems and metabolic ways of the CSF. Although the study was made on a limited number of dogs, the 1H NMR derived concentrations allowed correlations between various metabolites. This validates the results by highlighting interconnections of metabolic pathways previously described in humans as well as those specific in dogs. Sometimes, in physiological conditions, it is difficult to assign metabolites to a particular metabolism sequence because of the existence of multiple interferences among the metabolic ways and of the involvement of a large number of regulating enzymes. The analysis of CSF through 1H NMR analysis has an important potential for further research of the CSF of canine patients with neurological, or even other types of pathologies. To our knowledge there is no previous study undertaken on dogs CSF considering simultaneously 13 metabolites for quantification and correlations among them. The obtained data offered the possibility for higher fidelity analysis of the cerebral metabolic profile and for the assessment of CSF homeostasis in healthy dogs. The reduced degree of metabolites variability and the great number of simultaneously identified metabolites demonstrate the fact that the Data reported in this study may be used as reference values for further studies of CSF metabolomics in dogs and contribute to the understanding of dog metabolism. Moreover, due to the closely resembling interspecies metabolites concentrations, shorter lifespan and great similarities with the pathogenesis of human CNS disease, canine species is a valuable alternative in clinical research of the human CNS. Further studies with other control dogs and possibly larger cohorts will have to validate the concentration ranges of metabolites reported here. Until then, this study is the most extensive set of metabolite concentrations for CSF healthy dogs reported to date. 1H NMR spectroscopy. In addition to that, no other study has been simultaneously quantifying and analysing correlations for as many as 13 metabolites in the same sample of dog CSF. The homogeneity of the obtained results and the low biologic variability allow for the reported data to be used as reference values in further studies and indicate that the 1H NMR spectroscopy analysis of dog’s CSF could help in designing and interpreting clinical and therapeutic trials in dogs with CNS pathology.To our knowledge, this is the first report of dogs CSF analysis through


Different donor/acceptor conformations can lead to κ2 values in the 0 ≤ κ2 ≤ 4 range. Because the characteristic distance for FRET, R0, is proportional to (κ2)1/6, uncertainties in the orientation factor are reflected in the quality of information that can be retrieved from a FRET experiment. In most cases, the average value of κ2 corresponding to the dynamic isotropic limit (<κ2> = 2/3) is used for computation of R0 and hence donor-acceptor distances and acceptor concentrations. However, this can lead to significant error in unfavorable cases. This issue is more critical in membrane systems, because of their intrinsically anisotropic nature and their reduced fluidity in comparison to most common solvents. Here, a simple numerical simulation method for estimation of the probability density function of κ2 for membrane-embedded donor and acceptor fluorophores in the dynamic regime is presented. In the simplest form, the proposed procedure uses as input the most probable orientations of the donor and acceptor transition dipoles, obtained by experimental (including linear dichroism) or theoretical (such as molecular dynamics simulation) techniques. Optionally, information about the widths of the donor and/or acceptor angular distributions may be incorporated. The methodology is illustrated for special limiting cases and common membrane FRET pairs.Because of its acute sensitivity to distance in the nanometer scale, Förster resonance energy transfer (FRET) has found a large variety of applications in many fields of chemistry, physics, and biology. One important issue regarding the correct usage of FRET is its dependence on the donor-acceptor relative orientation, expressed as the orientation factor κ Fluorescence spectroscopy has been utilized as a major tool in biophysics in general, and membrane studies in particular, for decades. Several parameters can be measured in steady-state  or time-resolved (intensity and anisotropy decays) conditions, and it allows monitoring of photophysical processes that are sensitive to distance, concentration , aggregation  or diffusion . Fluorescence’s versatility and sensitivity have earned it a place among the most useful biophysical techniques . In part2. Because the characteristic distance for FRET, R0, is proportional to (κ2)1/6, uncertainties in the orientation factor are reflected on the quality of information that can be retrieved from a FRET experiment. Most often, the theoretical value for the so-called dynamic isotropic limit (<κ2> = 2/3) is used, but <κ2> uncertainty is still widely regarded as an inconvenience  interval for which S = 0 ↔ cosθD = ± (1/3)1/2  and S = 2/3 ↔ cosθD = ± (7/9)1/2 . However, despite <κ2> being identical to the isotropic value, the κ2 distributions are quite different. As shown in 2 distribution for θD = 54.74° shows peaks at κ2 ≈ 0.12 and κ2 ≈ 1 (absent in the isotropic distribution), while it vanishes abruptly for κ2 > 25/9 = 2.778. This latter higher limiting value corresponds to the situation of coplanar donor dipole, acceptor dipole and separation vector, with the two dipoles facing each other. On the contrary, in the isotropic limit, values of κ2 near 4 (corresponding to collinear dipoles) are allowed and no cutoff is observed.2>, we consider the recent MD study of 1-palmitoyl,2-[12-amino]dodecanoyl-sn-glycero-3-phosphocholine (C12-NBD-PC) in a fluid bilayer of 1,2-dipalmitoyl-sn-3-glycerophoshocholine . The angular distribution of the transition dipole has a maximum around ~120–125° relative to the bilayer normal  interval by inversion.2.3. Set donor and acceptor dipole orientation coordinates,  and .2.4. Calculate Cartesian coordinates of unit dipole vectors 2 using 2.5. Calculate κ2 distribution , and partial sum of κ2 values /N at the end of the cycle).2.6. Update histogram of κN.2.7 Increment i and repeat until i > 2 average value and distribution, θD and θA distributions.Write outputs to the active worksheet: κAll calculations and distributions shown in 2 for the ith donor-acceptor pair. However, it computes an average κ2 value using Ri−6 as weight for each pair. It may be used for both one and two acceptor planes. The program reads θD,max, θA,max, σD, σA, l, N, and, for both planes, the respective donor-acceptor interplanar (h1 and h2) and exclusion (Re1 and Re2) distances. Two main cycles are carried out, one for each acceptor plane, with calculation of κ2 distribution in each case. For both cycles, calculation of κ2 for the ith donor-acceptor pair is identical to that in the first program, with an additional check: if Ri < Re1 (first cycle) or Ri < Re2 (second cycle), then new donor and acceptor distance coordinates are allotted and a corresponding new Ri is calculated. Partial sums of κ2/R6 values are now computed, to obtain <κ2> = sum(κ2i/Ri6)/sum(1/Ri6). As described in Section 3, this weighting scheme reflects the dependence of the FRET interaction with distance, and was used to compute the values of the right column of Program 2 follows a similar algorithm for calculation of κ2, for membrane-located donor and acceptor fluorophores, is presented. The method is implemented in two programs that are available with the present article. The programs use as inputs the donor and acceptor transverse locations, orientation and orientational heterogeneity. Analytical results previously obtained in limiting cases were verified, validating our approach. A number of donor/acceptor orientation distributions tested in this work produce κ2 distributions and average values which are significantly different to that corresponding to the isotropic limit, characterized by <κ2> = 2/3. Considerable dependence of <κ2> on system size l when the latter is <<100 nm was verified, which presents a problem regarding the most appropriate <κ2> to consider for the purpose of quantitative analysis of FRET data. It is proposed that calculating <κ2> using the sixth inverse power of the donor-acceptor distance is the most natural way to average κ2 in this situation, and estimates of <κ2> were obtained for common membrane FRET pairs using available fluorophore location and orientation data. It was shown that for most of these systems, the calculated <κ2> lies within ±15% of the 2/3 value, possibly because probes in fluid bilayers have very broad orientation distributions. In these cases, substantial effects on FRET efficiency are not expected, provided that the Förster radius is significantly larger than the donor-acceptor exclusion distance. For more ordered bilayers, narrow orientation distributions may produce <κ2> further form the 2/3 value. In these ordered systems, an additional problem is that fluorophore dipole orientation randomization may occur in a time scale of the order or slower than the donor excited state lifetime. In case reorientation proceeds much slower than transfer, the opposite regime (static limit) is operative. As discussed in detail elsewhere [2 averages to calculate donor-acceptor distances in intramolecular FRET . In the intermediate case of comparable rates of reorientation and transfer, FRET kinetics becomes very complex [2 distributions and averages corresponding to the dynamic limit, care must be taken when addressing these systems. However, even in such cases, the ease of application of this method—as long as fluorophore location and orientation data  are available (in the literature or upon measurement) for the pair at hand—allows calculation of <κ2> which will constitute an improvement over the widely used but very seldom justified <κ2> = 2/3 assumption.A simple method for calculation of the distribution and average value of the FRET orientation factor κlsewhere ,5, in th complex . Because


Central nervous system (CNS) infection is a nearly universal feature of untreated systemic HIV infection with a clinical spectrum that ranges from chronic asymptomatic infection to severe cognitive and motor dysfunction. Analysis of cerebrospinal fluid (CSF) has played an important part in defining the character of this evolving infection and response to treatment. To further characterize CNS HIV infection and its effects, we applied advanced high-throughput proteomic methods to CSF to identify novel proteins and their changes with disease progression and treatment.accurate mass and time (AMT) tag database containing 23,141 AMT tags for CSF peptides, we analyzed 91 CSF samples by LC-MS from 12 HIV-uninfected and 14 HIV-infected subjects studied in the context of initiation of antiretroviral therapy and correlated abundances of identified proteins a) within and between subjects, b) with all other proteins across the entire sample set, and c) with "external" CSF biomarkers of infection (HIV RNA), immune activation (neopterin) and neural injury . We identified a mean of 2,333 +/- 328 (SD) peptides covering 307 +/-16 proteins in the 91 CSF sample set. Protein abundances differed both between and within subjects sampled at different time points and readily separated those with and without HIV infection. Proteins also showed inter-correlations across the sample set that were associated with biologically relevant dynamic processes. One-hundred and fifty proteins showed correlations with the external biomarkers. For example, using a threshold of cross correlation coefficient (Pearson's) ≤ -0.3 and ≥0.3 for potentially meaningful relationships, a total of 99 proteins correlated with CSF neopterin (43 negative and 56 positive correlations) and related principally to neuronal plasticity and survival and to innate immunity. Pathway analysis defined several networks connecting the identified proteins, including one with amyloid precursor protein as a central node.After establishing an Advanced CSF proteomic analysis enabled the identification of an array of novel protein changes across the spectrum of CNS HIV infection and disease. This initial analysis clearly demonstrated the value of contemporary state-of-the-art proteomic CSF analysis as a discovery tool in HIV infection with likely similar application to other neurological inflammatory and degenerative diseases. While central nervous system (CNS) HIV infection is a nearly universal facet of systemic infection, its course and clinical manifestations are highly variable and, indeed, evolve over time within individual patients. CNS infection can be detected soon after primary exposure and initial viremia ,2, and cBecause of its close proximity to the brain parenchyma and interchange with brain extracellular fluid, the composition of CSF can provide insights into the brain chemistry and cellular processes of infection and brain perturbation, and because it is also separated from the blood by barriers that restrict molecular and cellular passage, it can also serve as a "model" of an isolated and protected tissue reacting to infection that shares properties with brain ,19. ThesAccurate Mass and Time (AMT) tag strategy for high-throughput label-free quantitative analytical measurements of protein abundance [In this study, we applied recent advances in high-throughput, mass spectrometry-based proteomic methods to CSF across the spectrum of HIV infection and resultant CNS disease. Proteomic technologies have significantly matured in the last few years, particularly for application in the clinical realm, where robust measurements can now be made with sufficient sensitivity and throughput to allow both the depth and breadth of analysis required for confident global comparisons of larger sample sets ,34. Pastbundance ,43, haveWe applied proteomic analysis to a group of specimens from well-characterized subjects. Because of anticipated individual between-subject differences unrelated to HIV infection, these initial efforts focused on analysis of longitudinal sample sets, and particularly sample sets for subjects who changed during the period of observation related to either the development of neurological disease or by responses to cART. In addition to analyzing correlations within the proteomic data, we analyzed correlations of identified protein with three orthogonal biomarkers related to main pathobiological disease components. These markers included CSF HIV RNA to assess relations to the local viral replication; CSF neopterin to compare to the state of intrathecal immune activation and particularly macrophage activation ,44, and Proteomic analysis of CSF involved two stages. The first established an AMT tag database by detailed analysis of four CSF samples subjected to extensive fractionation, allowing for a deep view into the CSF proteome and defining a large peptide-protein library of AMT tags for subsequent application to the larger sample set. Two of the samples were from HIV-uninfected, neurologically normal control volunteers and two from HIV-infected subjects with more advanced infection. The two control subjects, both male ages 45 and 43, had normal CSF WBC counts, total proteins, and albumin ratios along with normal bedside neurological examinations and QNPZ-4 test scores. The two HIV-infected subjects, ages 38 and 45, had advanced systemic infection with blood CD4+ counts of 98 and 84 cells/μL, blood HIV RNA concentrations of 522,000 and 109,000 copies/mL and CSF HIV RNA concentrations of 1,202,000 and 22,600 copies/mL. Their CSF total proteins were elevated at 93 and 60 mg/dL with albumin ratios of 12.79 and 7.63 and CSF cell counts of 35 and 44 WBCs/μL. They also tested abnormally with QNPZ-4 scores of -1.8 and -1.0.The four samples were subjected to protein isolation, tryptic digestion and pre-MS SCX fractionation. Each SCX fraction was analyzed by high resolution capillary LC-MS/MS for identification of peptide sequences and inclusion into a CSF exclusive AMT tag database. A total 23,141 confident peptide mass and time tag identifications were then included in the CSF database that served as the basis for protein identification in the subsequent main clinical study.Analysis of the CSF proteome across the spectrum of HIV infection included 79 samples from 14 HIV-infected subjects with varying systemic and CNS disease severity. The longitudinal samples included multiple time points for 11 of the subjects  Table  the mediThree approaches were then taken in the analysis of the proteomic results across the sample set: characterization of peptide and protein abundances across all subjects, correlation among proteins across sample sets, and correlation of protein abundances with three external CSF biomarkers.Analysis of individual CSF samples identified 9,074 peptides  were selected for further analysis , a highly connected node in the network shown in Figure Proteins correlating with NFL levels in the CSF were used to create the network shown in Figure validation studies where targeted approaches are used do require knowledge of target proteins. In searching for disease biomarkers or disease associated protein profiles, the discovery phase should provide a list of candidate proteins and serves as a precursor phase for targeted quantitative approaches. These subsequent targeted approaches, whether they use MS techniques or are immunobased, are designed to validate the use of the biomarker proteins [This exploratory study demonstrates the power of discovery methods applied to CSF in HIV infection and establishes a strong foundation for further extension of these techniques in this condition and to other neurological diseases. In this discovery-phase investigation, MS based label-free quantification was advantageous given no required a priori knowledge of CSF proteome targets. Follow up proteins . The resClinical and research studies of CSF currently exploit only a narrow range of its content. The improved definition of the CSF proteome presented here indicates considerable potential to expand the range of useful measurements for diagnosis, assessing treatment responses and understanding underlying disease processes in HIV infection. Thus, the study identified a mean of 2,333 +/-328 (SD) peptides per patient sample corresponding to 307 +/-16 proteins and a total of 344 proteins in all CSF samples analyzed by LC-MS  protein "signatures" on which samples can be segregated. This was anticipated in the design of the study, and was the rationale for including longitudinal samples from a relatively small group of subjects with changing clinical state related to cART treatment. While not eliminating the contribution of individual protein profiles, it reduced their impact on the analysis related to changing disease state and treatment effects. It also showed a clear segregation of HIV-infected and -uninfected subject CSF proteome "signatures".Analysis of protein abundances showed that the majority of samples from the longitudinally-studied individual subjects clustered together in the analysis of overall protein abundances Figure . This waThe correlations among proteins across the larger sample set allowed grouping of proteins as they varied in abundance across the disease spectrum, suggesting common or system-level group responses to the pathological state of infection, immune reaction and/or brain injury. These grouped changes are now being explored in more depth within this dataset, through additional proteomic studies of HIV infection, and using alterative assays with greater precision, including dedicated immunoassays and "targeted" LC-MS assays using multiple reaction monitoring MS -56.While the relatedness of some of the identified proteins was obvious, reflecting the known linkages as part of the systemic host response to HIV infection and CNS injury, many were unknown or unexplored in this context. Thus, the protein abundance correlations provide new insight into the content and breadth of the changes in the CSF proteome over the course of HIV infected subjects. These correlations now need to be examined and dissected in more detail to define the underlying mechanistic pathways.Analysis of this varied patient cohort allowed for capturing a wide range of CNS states related to disease severity Figure . FollowiHIV RNA in CSF serves as an index of local infection, and directly traces the responses to antiretroviral therapy. However, the character and origins of CSF HIV are heterogeneous and can vary in genotype, receptor utilization, tropism, and resistance mutations ,29,57-62Neopterin, is a low-molecular mass pteridine metabolite of GTP that is produced largely by cells of the monocyte-macrophage lineage after activation, primarily by IFN-gamma . Its conCSF NFL can serve as an indicator of axonal injury in a wide variety of neurodegenerative conditions -72, inclHIV encephalitis is sustained primarily in brain macrophages and is associated with widespread macrophage and microglial cells activation ,74. A siThe correlations within the proteome and with the three independent CSF biomarkers identified a number of proteins suitable for future exploration. For example procollagen C-proteinase enhancer-1  was negatively correlated (R = -0.29) with NFL and present at reduced levels (1.5 fold) in pretreatment HIV infected relative to post treatment samples. This finding is consistent with a previous report . ProteinHigh levels of oxidative damage and oxidative stress have been linked to several neurodegenerative diseases including multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis . SuperoxApplication of Ingenuity Pathway Analysis database suggests previously reported connections that may be involved in CNS HIV infection. Supporting the utility of this approach was the identification of APP as a "linking" node in the network identified among the neopterin correlations. A previous independent study showed that CSF concentrations of soluble APPs  were reduced in ADC patients, and that this finding helped distinguish the CSF profile of ADC from that of Alzheimer's disease . While tpanomics - in CSF is likely to provide a rich and fruitful view of HIV-related CNS disease processes.Autotaxin  has lysophospholipase D activity, converting lysophosphatidylcholine into lysophosphatidic acid (LPA) (reviewed in ) and sigAnalysis of the CSF proteome of HIV infected individuals prior to and coincident with cART allowed the analysis of a wide range of disease severity in a relatively small number of samples. Proteome analysis has revealed many proteins exhibiting strong positive and negative correlation with CSF neopterin which serves as a broad marker of disease severity. This highlights the potential that these proteins may be participating in the ongoing CNS pathology and as such constitute targets of interest for development of better diagnostic, prognostic, and therapeutic tools to identify and manage ongoing disease related processes. Surprisingly, we found that different groups of proteins correlated with neopterin and NFL levels in the CSF, suggesting a decoupling of the processes associated with those once linked to pathological markers of poor patient prognosis in the absence of therapeutic intervention.This exploratory study clearly shows the power and potential utility of CSF proteome analysis in providing a new and rich view of the effects of HIV infection on the CNS. Mass spectrometry-based proteome identification and label-free quantification of the CSF provides a wealth of data contributing to the characterization of CSF changes across the spectrum of disease and therapy. The findings now need to be explored in more depth as well as verified, validated, and extended with further studies. We are now pursuing these issues, and the lists of identified proteins in the supplementary tables should facilitate similar studies by other groups. The present studies of HIV also support a similar discovery approach to the study of other inflammatory and neurodegenerative conditions.CSF samples were selected from archived specimens obtained during two observational studies conducted at the University of California San Francisco (UCSF), one following the course of a cohort of HIV-infected subjects with periodic evaluations that included lumbar punctures (LPs), and the other study more directly evaluated CSF responses to initiation of cART ,6. The sCSF total white blood cell (WBC) counts and differential, protein and albumin, along with blood albumin and CD4+ and CD8+ T lymphocyte counts by flow cytometry were performed in the San Francisco General Hospital (SFGH) Clinical Laboratories using routine methods in real time. For other CSF assays, including proteomics, CSF was first subjected to low-speed centrifugation to remove cells before aliquoting for storage at -80°C and later use.10 values before analysis.HIV RNA was measured in cell-free CSF and plasma using the ultrasensitive Amplicor HIV Monitor assay  performed in the UCSF Virology Research Core Laboratory. We used a detection limit of 40 copies/mL and assigned a "floor" value of 39 copies/mL to lower values. HIV RNA levels were transformed to loghttp://www.umandiagnostics.com/ "converted" to values comparable to the older method using linear regression to create a conversion formula. The aggregate results used the lower limit cutoff of 125 ng/mL from the older assay, and results below this concentration were expressed as 124 ng/mL for analysis [CSF neopterin concentrations were determined by enzyme-linked immunoassay in Innsbruck, Austria  . CSF neuanalysis ,46.2; the digestion reaction was carried out for 4 h at 37°C by addition of sequencing grade, modified porcine trypsin  at a trypsin:protein ratio of 1:50, and then a second digestion reaction was performed after 2-fold dilution with the same buffer with the trypsin at a 1:50 trypsin/protein ratio at 37°C overnight. Tryptic peptide mixtures were cleaned by solid phase extraction using a 1-mL SPE C18 column  as described previously [Proteins in CSF samples were first denatured using 8 M urea, and disulfides were reduced following the addition of 5 mM dithiothreitol and incubating at 37°C for 60 min. Trypsin digestion was performed following diluting samples 4-fold with 50 mM ammonium bicarbonate (pH 8) and adding 1 mM CaCleviously . Final pAmmonium bicarbonate and acetonitrile were purchased from Fisher Scientific . Sequencing grade modified trypsin was purchased from Promega . Bicinchoninic acid (BCA) assay reagents and standards were from Pierce . All other reagents were purchased from Sigma-Aldrich . Water was purified using a Barnstead Nanopure Infinity water purification system .To develop the AMT tag database, tryptic peptide mixtures from CSF samples from two HIV- and two HIV + patients (see below for subject characteristics) were fractionated by strong cation exchange chromatography to reduce sample complexity. 150 μg of tryptic peptides per sample was resuspended in 900 μL 10 mM ammonium formate, pH 3.0; 25% acetonitrile and fractionated by strong cation exchange chromatography as described previously . 15 fracFor analysis of the main sample group, peptide samples were separated by high resolution, reversed phase liquid chromatography (LC) as previously described using a four-column capillary LC system  in-line LC-LTQ MS/MS raw data was extracted using Extract_MSn (version 3.0) and analyzed with the SEQUEST algorithm (V27 revision 12) searching the MS/MS data against the human IPI database which contained 69,731 total protein entries . Precursor mass tolerance of 3 Daltons and 1 Dalton for MS/MS ion masses without an enzyme defined were used for search parameters. The LTQ MS/MS data were processed by in-house software DeconMSn  accurateThe accurate mass and time (AMT) tag strategy  was usedhttp://www.R-project.org.To determine quantitative changes in protein abundances between control HIV-uninfected and HIV-infected subjects' CSF samples, we used in-house developed software DAnTE . Briefly® Systems, http://www.ingenuity.com). The functional analysis identified the biological functions and/or diseases that were most significant to the dataset. Molecules from the dataset that met the cross correlation filter cutoff of R ≤ -0.3 or ≥ 0.3 and a P value of ≤ 0.05 by t-test (Pearson) and were associated with biological functions and/or diseases in Ingenuity's Knowledge Base were considered for the analysis. Right-tailed Fisher's exact test was used to calculate P-values determining the probability that each biological function and/or disease assigned to that dataset was due to chance alone. Correlations of the CSF marker findings across the sample set used Spearman's non-parametric correlation.Determination of functional enrichment of proteins clustered together by cross correlation was performed employing Gominer . NetworkThe authors declare that they have no competing interests.TEA performed data analysis, interpretation and manuscript preparation. JMJ participated in study design, data analysis, interpretation, and manuscript preparation. SSS participated in study design, sample acquisition, data analysis, interpretation and manuscript preparation. MAG participated in sample preparation and data acquisition and manuscript preparation. DF participated in data acquisition, analysis, and manuscript preparation. TL participated in sample acquisition and preparation. HZ participated in data acquisition, data analysis, and manuscript preparation. DGC participated study design, data interpretation, and manuscript preparation. RWP participated in study design, sample acquisition, data analysis, interpretation, and manuscript preparation. RDS participated in study design, data analysis, interpretation, and manuscript preparation. All authors read and approved the final manuscript.Figure S1. Comparison of concentrations of three orthogonal CSF biomarkers across the sample set. In order to visually compare the range of concentrations of the three CSF biomarkers across the entire sample sets, they were independently ordered from highest to lowest and this ranking was then applied to the other two biomarkers and displayed on the same row. The top row shows results ranked by CSF HIV RNA concentration for these values (left), CSF neopterin (middle) and CSF NFL (right). Both visually and by Spearman's rank  it can be seen that CSF neopterin values correlate more closely with HIV RNA (r = 0.612) than do CSF NFL values (r = 0.289). Also note that the low CSF HIV RNA values are at the limit of detection (dotted line) and that this was not measured in the HIV- subjects. The second row ranks results by CSF neopterin. In addition to the correlation with CSF HIV RNA (right), it also shows that the CSF NFL values are less well correlated (r = 0.235) and that many high NFL values were noted in patients with lower CSF neopterin. The bottom row shows ranking by CSF NFL and again emphasizes that elevated CSF HIV RNA and neopterin was noted even in the subjects with CSF NFL below the level of detection (dotted line in lower right graph).Click here for fileTables S1. Peptides identified in the cerebrospinal fluid.Click here for fileTables S2. Proteins identified in the cerebrospinal fluid of subject samples and correlation analysis of protein abundance with orthogonal markers of HIV CNS infection and pathology.Click here for file


Use of midazolam infusion in mechanically ventilated patient is an established practice in critical care. In our case, the use of erythromycin as a prokinetic agent for better tolerance of enteral feeding and paralytic ileus led to an interaction between midazolam and erythromycin, which resulted in prolonged and deeply sedated patient. In a critically ill patient, there is always a possibility of multiple drug interactions. It is important to understand them and they should be considered before starting new medication. Drug interactions in critical care setting are of utmost importance. We report an interesting case of prolonged sedation due to erythromycin and midazolam interaction in a post cardiac surgery critically ill patient.2–V6. His coronary angiogram revealed thrombus in left main coronary artery, left anterior descending artery (LAD) showing 99% occlusion, and right coronary artery showing 90% occlusion. A preoperative transthoracic echocardiography showed normal left ventricular systolic function (ejection fraction 60%). An urgent coronary bypass graft surgery was planned. Other comorbidities included benign prostatic hypertrophy and chronic constipation (able to pass motions only one to two times a week).A 66-year-old male patient, who was a known case of hypertension, old cerebrovascular episode, post lumbar laminectomy with restricted mobility due to bilateral knee osteoarthritis, presented with history of unstable angina, 1 day prior to admission. His electrocardiogram confirmed anterolateral ischemia showing ST-T segment depression in anterolateral precordial leads VThe patient underwent off-pump coronary artery bypass surgery with grafts to LAD, obtuse marginal branch of left circumflex coronary artery and posterior descending branch of right coronary artery. Intraoperative anesthesia management included midazolam 5 mg, fentanyl 500 μg, and vecuronium and sevoflurane 2–2.5%. Total urine output was 400 ml. The total duration in the operating theater was 5 hours. The patient was extubated at 7 hours after surgery in the recovery unit. The patient was stable on postoperative day 1, tolerating oral diet, and was ambulated out of bed.nd postoperative day, the patient was noted to have abdominal distension with weak peristaltic activity and tachycardia 120/minute. He was off inotropic support with mean arterial pressure of 70–80 mmHg and central venous pressure of 5–7 mmHg. Nasogastric tube aspiration drained 1.4 l/24 hour of bilious fluid, suggesting decreased gastrointestinal (GI) motility.On the 2For improving the GI motility and tolerance of enteral feeding, serum potassium was optimized between 4.0 and 4.5 mEq/l; IV metoclopramide 10 mg 12 hourly, Tab. itopride 50 mg once daily and Tab. erythromycin 250 mg 6 hourly were started. Flatus tube was inserted for relieving abdominal distention.Despite prokinetic therapy, ileus persisted and significant abdominal distention led to respiratory distress, tachycardia and appearance of hypoxemia. Serum creatinine increased to 2.01 mg/dl.Patient had to be intubated on the 4th postoperative day and ventilated for worsening respiratory distress and hemodynamic instability. Circulatory support  was started and midazolam infusion commenced at 2–3 mg/hour for sedation. Midazolam infusion was stopped the next morning in view of weaning from ventilation. The patient received midazolam at a cumulative dose of 46 mg (15 hours). The patient remained sedated but arousable with no focal neurologic signs. Supportive therapy was continued .th day postoperatively.GI ileus steadily improved by the 6th postoperative day in the form of decreased nasogastric aspirate and passage of feces. On postoperative day 6, he remained unresponsive with intact brainstem reflexes and normal electrolytes. Differential diagnosis included were metabolic encephalopathy, cerebrovascular event or prolonged midazolam effect. Intravenous flumazenil (antidote for midazolam) 200 mcg bolus was diagnostic of prolonged midazolam effect. Hence, computed tomography study of brain could be avoided. A possibility of prolonged midazolam effect due to erythromycin was entertained and Tab. erythromycin was stopped. Enteral feeding was established on 7Following satisfactory arousal from sedation, the patient was extubated on day 8 postoperatively. Further recovery remained uneventful and the patient was discharged home on day 15 postoperatively.It is not very uncommon to notice paralytic ileus in an elderly patient, especially after cardiac surgery. These paUnexpected delay in waking up after stopping midazolam infusion evokes suspicion of electrolyte imbalance, cerebrovascular episode or prolonged sedation due to midazolam. Probability of prolonged sedation due to midazolam was confirmed by awakening after flumazenil injection.Erythromycin 250 mg 6–8 hourly has been shown to improve gastrointestinal motility and increase tolerance to enteral feeding in intensive care.Erythromycin and midazolam both are substrates for the same enzyme, cytochrome P450 IIIA4. So, there is competition for enzyme sites and metabolism of midazolam is inhibited leading to elevated levels and delayed elimination.[This has been demonstrated clinically in cross-over studies, in which in patients receiving erythromycin, the effect of single dose midazolam was prolonged for 6 hours.6Loss of consciousness following intravenous erythromycin administration was reported in an 8-year-child who underwent adenoidectomy due to interaction with oral midazolam administered as premedication.We believe that in our patient, the presence of erythromycin therapy was responsible for delayed arousal from midazolam and prolonged mechanical ventilation.Drug interactions in critically ill patients, such as midazolam–erythromycin, can increase morbidity by prolonged sedation and delayed weaning from the ventilator. So, we suggest that concomitant use of macrolide antibiotics and midazolam should be avoided.


Considering that handwashing responsabilityis mainly on health area professionals, the survey of employees' perception helps to orientate the implementation of educational measures in the promotion of improvements and and can also offer subsidies to stimulate patients' active participation in the search for health.Evaluate the health care employees perception regarding the best practices of handwashing, including the patient involvement in the shared responsability of this handwashing practiceIt is about a descriptive, quantitative study performed at a midsize, private hospital located in São Paulo, Brazil.Among the actions performed in the Annual Handwashing Campaign in 2012, pamphlets with closed questions were distributed, allowing the evaluation of 363 healthcare employees.The variables for evaluation were considered: the importance of the 5 moments practice, the performance of handwashing opportunities, the importance given to the patient involvement, handwashing as part of care and the quality of alcohol gel.The analysis of collected data has showed that 99% of employees consider of “much importance” the 5 Moments practice, 58% consider fulfill 100% of opportunities. When evaluated the importance given to the patient involvement in the improvement process, 93% of the employees consider it very important, 3% indifferent and 4% refer moderate importance. 100% of the employees consider that handwashing is part of the health process. 81% of these consider that the family and patient proactivity help increase the implementation of handwashing measures, while 19% of the employees do not agree. The employees evaluated the quality of alcohol gel as: 61% very good and 38% good.The results found show a homogeneous awareness on the practice of handwashing and also that the perception of the importance of the patient participation in the shared responsability already is a fact, which allows the implementation of new multiprofessional strategies, stimulating the patient to take an active part in his own care.None declared