Step-By-Step Procedure to Identify Previously Unknown Compounds by LC-QTOF-MS Exemplified by an Intoxication With the Methaqualone Analog SL-164.

In September 2019, a 22-year-old man with a history of drug abuse presented to the hospital with altered mental status. Due to a suspected drug overdose, a blood sample taken on admission and a urine sample collected 30 h thereafter were submitted to our laboratory to test for illegal drugs, pharmaceutical substances, and designer drugs. During the routine toxicological analysis of the serum sample, morphine and phenobarbital were identified by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Additionally, two compounds showing identical accurate masses and isotope ratios as the designer benzodiazepine diclazepam and the benzodiazepine lormetazepam were found. However, retention times differed significantly from the expected values, and the acquired MS/MS spectra did not match the library entries of the two compounds, indicating the presence of two previously unknown substances. After further investigation, SL-164 (5-chloro-3-(4-chloro-2-methylphenyl)-2-methyl-4(3H)-quinazolinone), a methaqualone analog, which has recently emerged on the research chemical market, and its hydroxy metabolite were tentatively identified by accurate mass, isotope matching, and plausible fragmentation. However, for unequivocal confirmation and quantification, a reference standard is required. As no reference material was available by the end of 2019, SL-164 was obtained from an online shop, and its identity and purity (97.8%) were confirmed by nuclear magnetic resonance spectroscopy. The subsequent quantitative analysis revealed a concentration of 390 ng/mL SL-164 in serum. In the urine sample, the parent compound was not detected, but three suspected monohydroxylated metabolites were found. This example shows that LC-QTOF-MS is a powerful approach for the (tentative) identification of unknown compounds in biological matrices.

Mortality Prediction by Kinetic Parameters of Lactate and S-Adenosylhomocysteine in a Cohort of Critically Ill Patients.

Tissue hypoxia is associated with the development of organ dysfunction and death in critically ill patients commonly captured using blood lactate. The kinetic parameters of serial lactate evaluations are superior at predicting mortality compared with single values. S-adenosylhomocysteine (SAH), which is also associated with hypoxia, was recently established as a useful predictor of septic organ dysfunction and death. We evaluated the performance of kinetic SAH parameters for mortality prediction compared with lactate parameters in a cohort of critically ill patients. For lactate and SAH, maxima and means as well as the normalized area scores were calculated for two periods: the first 24 h and the total study period of up to five days following ICU admission. Their performance in predicting in-hospital mortality were compared in 99 patients. All evaluated parameters of lactate and SAH were significantly higher in non-survivors compared with survivors. In univariate analysis, the predictive power for mortality of SAH was higher compared with lactate in all forms of application. Multivariable models containing SAH parameters demonstrated higher predictive values for mortality than models based on lactate parameters. The optimal models for mortality prediction incorporated both lactate and SAH parameters. Compared with lactate, SAH displayed stronger predictive power for mortality in static and dynamic application in critically ill patients.

Sepsis and delayed cerebral ischemia are associated and have a cumulative effect on poor functional outcome in aneurysmal subarachnoid hemorrhage.

Objective: Although sepsis and delayed cerebral ischemia (DCI) are severe complications in patients with aneurysmal subarachnoid hemorrhage (aSAH) and share pathophysiological features, their interrelation and additive effect on functional outcome is uncertain. We investigated the association between sepsis and DCI and their cumulative effect on functional outcome in patients with aSAH using current sepsis-3 definition. Methods: Patients admitted to our hospital between 11/2014 and 11/2018 for aSAH were retrospectively analyzed. The main explanatory variable was sepsis, diagnosed using sepsis-3 criteria. Endpoints were DCI and functional outcome at hospital discharge (modified Rankin Scale (mRS) 0-3 vs. 4-6). Propensity score matching (PSM) and multivariable logistic regressions were performed. Results: Of 238 patients with aSAH, 55 (23.1%) developed sepsis and 74 (31.1%) DCI. After PSM, aSAH patients with sepsis displayed significantly worse functional outcome (p < 0.01) and longer ICU stay (p = 0.046). Sepsis was independently associated with DCI (OR = 2.46, 95%CI: 1.28-4.72, p < 0.01). However, after exclusion of patients who developed sepsis before (OR = 1.59, 95%CI: 0.78-3.24, p = 0.21) or after DCI (OR = 0.85, 95%CI: 0.37-1.95, p = 0.70) this statistical association did not remain. Good functional outcome gradually decreased from 56.3% (76/135) in patients with neither sepsis nor DCI, to 43.8% (21/48) in those with no sepsis but DCI, to 34.5% (10/29) with sepsis but no DCI and to 7.7% (2/26) in patients with both sepsis and DCI. Conclusion: Our study demonstrates a strong association between sepsis, DCI and functional outcome in patients with aSAH and suggests a complex interplay resulting in a cumulative effect towards poor functional outcome, which warrants further studies.

S-Adenosylhomocysteine Is a Useful Metabolic Factor in the Early Prediction of Septic Disease Progression and Death in Critically Ill Patients: A Prospective Cohort Study.

A common final pathway of pathogenetic mechanisms in septic organ dysfunction and death is a lack or non-utilization of oxygen. Plasma concentrations of lactate serve as surrogates for the oxygen-deficiency-induced imbalance between energy supply and demand. As S-adenosylhomocysteine (SAH) was shown to reflect tissue hypoxia, we compared the ability of SAH versus lactate to predict the progression of inflammatory and septic disease to septic organ dysfunction and death. Using univariate and multiple logistic regression, we found that SAH but not lactate, taken upon patients' inclusion in the study close to ICU admission, significantly and independently contributed to the prediction of disease progression and death. Due to the stronger increase in SAH in relation to S-adenosylmethionine (SAM), the ratio of SAM to SAH, representing methylation potential, was significantly decreased in patients with septic organ dysfunction and non-survivors compared with SIRS/sepsis patients (2.8 (IQR 2.3-3.9) vs. 8.8 (4.9-13.8); p = 0.003) or survivors (4.9 (2.8-9.5) vs. 8.9 (5.1-14.3); p = 0.026), respectively. Thus, SAH appears to be a better contributor to the prediction of septic organ dysfunction and death than lactate in critically ill patients. As SAH is a potent inhibitor of SAM-dependent methyltransferases involved in numerous vital biochemical processes, the impairment of the SAM-to-SAH ratio in severely critically ill septic patients and non-survivors warrants further studies on the pathogenetic role of SAH in septic multiple organ failure.

Will tetrahydrocannabinol be formed from cannabidiol in gastric fluid? An in vivo experiment.

Cannabidiol (CBD) products have ascribed an uprising trend for their health-promoting effects worldwide. In contrast to Δ9-tetrahydrocannabinol (THC), CBD exhibits no state of euphoria. Since conversion of CBD into THC in an acidic environment has been reported, it has not been proved whether this degradation will also occur in human gastric fluid. A total of 9 subjects ingested 400 mg CBD as a water-soluble liquid together with lecithin as an emulsifier and ethanol as a solubilizer. Blood samples were taken up to 4 h, and urine samples were submitted up to 48 h. THC, 11-hydroxy-Δ9-THC (THC-OH), 11-nor-9-carboxy-Δ9-THC (THC-COOH), CBD, 7-hydroxy cannabidiol (7-OH-CBD), and 7-carboxy cannabidiol (7-CBD-COOH) were determined in blood and THC-COOH and 7-CBD-COOH in urine by LC-MS/MS. Neither THC, THC-OH, nor THC-COOH were detectable in any serum specimen. Only two urine samples revealed THC-COOH values slightly above the threshold of 10 ng/ml, which could also be caused by trace amounts of THC being present in the CBD liquid. It can be concluded that negative consequences for participants of a drug testing program due to a conversion of CBD into THC in human gastric fluid appear unlikely, especially considering a single intake of dosages of less than 400 mg. Nevertheless, there is a reasonable risk for consumers of CBD products being tested positive for THC or THC metabolites. However, this is probably not caused by CBD cyclization into THC in human gastric fluid but is most likely due to THC being present as an impurity of CBD products.

Pneumonia in the first week after polytrauma is associated with reduced blood levels of soluble herpes virus entry mediator.

Background: Pneumonia develops frequently after major surgery and polytrauma and thus in the presence of systemic inflammatory response syndrome (SIRS) and organ dysfunction. Immune checkpoints balance self-tolerance and immune activation. Altered checkpoint blood levels were reported for sepsis. We analyzed associations of pneumonia incidence in the presence of SIRS during the first week of critical illness and trends in checkpoint blood levels. Materials and methods: Patients were studied from day two to six after admission to a surgical intensive care unit (ICU). Blood was sampled and physician experts retrospectively adjudicated upon the presence of SIRS and Sepsis-1/2 every eight hours. We measured the daily levels of immune checkpoints and inflammatory markers by bead arrays for polytrauma patients developing pneumonia. Immune checkpoint time series were additionally determined for clinically highly similar polytrauma controls remaining infection-free during follow-up. We performed cluster analyses. Immune checkpoint time trends in cases and controls were compared with hierarchical linear models. For patients with surgical trauma and with and without sepsis, selected immune checkpoints were determined in study baseline samples. Results: In polytrauma patients with post-injury pneumonia, eleven immune checkpoints dominated subcluster 3 that separated subclusters 1 and 2 of myeloid markers from subcluster 4 of endothelial activation, tissue inflammation, and adaptive immunity markers. Immune checkpoint blood levels were more stable in polytrauma cases than controls, where they trended towards an increase in subcluster A and a decrease in subcluster B. Herpes virus entry mediator (HVEM) levels (subcluster A) were lower in cases throughout. In unselected surgical patients, sepsis was not associated with altered HVEM levels at the study baseline. Conclusion: Pneumonia development after polytrauma until ICU-day six was associated with decreased blood levels of HVEM. HVEM signaling may reduce pneumonia risk by strengthening myeloid antimicrobial defense and dampening lymphoid-mediated tissue damage. Future investigations into the role of HVEM in pneumonia and sepsis development and as a predictive biomarker should consider the etiology of critical illness and the site of infection.

Unexpected results found in larvae samples from two postmortem forensic cases.

PURPOSE: In forensics, entomological specimens can be used as additional/alternative matrices to detect xenobiotics when human specimens are limited in their application. Despite some advantages over implementing putrefied human remains, most medico-legal laboratories do not include entomotoxicological procedures as routine analytical methods. We thus applied two authentic cases to evaluate necrophagous larvae's potential as complementary matrices for toxicological analysis after extensive postmortem decomposition. METHODS: Larvae and postmortem human samples, including hair, stomach contents, pericardial fluid, liver, lung, and skeletal muscle, were collected at autopsy. Samples were analyzed by liquid chromatography-tandem mass spectrometry and liquid chromatography-quadrupole time-of-flight mass spectrometry for pharmaceutical substances, illicit drugs, and new psychoactive substances, including synthetic cannabinoids, benzodiazepines, new synthetic opioids, and stimulants. RESULTS: Nearly all substances detected in human specimens, including several benzodiazepines and synthetic cannabinoids, were also detected in larvae. Surprisingly, some drugs, including the new psychoactive substances EAM-2201 and U-47700, were found exclusively in larvae and hair. The benzodiazepine etizolam was detected only in liver, lungs, and stomach contents, possibly resulting from characteristic tissue distribution in humans and/or larvae. CONCLUSIONS: Antemortem external hair contamination with synthetic cannabinoids from side-stream smoke and postmortem hair contamination with substances in putrefaction fluids can be supposed in these cases. Our findings suggest that supplementary information can indeed be gained from analyzing larvae additional to those human specimens that are typically used for toxicological analysis after extensive postmortem decomposition. Nevertheless, these results represent merely two cases, requiring in-depth studies to determine whether such findings can identify acute intoxications as possible causes of death.

Comparative Analyses of the Impact of Different Criteria for Sepsis Diagnosis on Outcome in Patients with Spontaneous Subarachnoid Hemorrhage.

Data on sepsis in patients with a subarachnoid hemorrhage (SAH) are scarce. We assessed the impact of different sepsis criteria on the outcome in an SAH cohort. Adult patients admitted to our ICU with a spontaneous SAH between 11/2014 and 11/2018 were retrospectively included. In patients developing an infection, different criteria for sepsis diagnosis (Sepsis-1, Sepsis-3_original, Sepsis-3_modified accounting for SAH-specific therapy, alternative sepsis criteria compiled of consensus conferences) were applied and their impact on functional outcome using the modified Rankin Scale (mRS) on hospital discharge and in-hospital mortality was evaluated. Of 270 SAH patients, 129 (48%) developed an infection. Depending on the underlying criteria, the incidence of sepsis and septic shock ranged between 21-46% and 9-39%. In multivariate logistic regression, the Sepsis-1 criteria were not associated with the outcome. The Sepsis-3 criteria were not associated with the functional outcome, but in shock with mortality. Alternative sepsis criteria were associated with mortality for sepsis and in shock with mortality and the functional outcome. While Sepsis-1 criteria were irrelevant for the outcome in SAH patients, septic shock, according to the Sepsis-3 criteria, adversely impacted survival. This impact was higher for the modified Sepsis-3 criteria, accounting for SAH-specific treatment. Modified Sepsis-3 and alternative sepsis criteria diagnosed septic conditions of a higher relevance for outcomes in patients with an SAH.

Ground truth labels challenge the validity of sepsis consensus definitions in critical illness.

BACKGROUND: Sepsis is the leading cause of death in the intensive care unit (ICU). Expediting its diagnosis, largely determined by clinical assessment, improves survival. Predictive and explanatory modelling of sepsis in the critically ill commonly bases both outcome definition and predictions on clinical criteria for consensus definitions of sepsis, leading to circularity. As a remedy, we collected ground truth labels for sepsis. METHODS: In the Ground Truth for Sepsis Questionnaire (GTSQ), senior attending physicians in the ICU documented daily their opinion on each patient's condition regarding sepsis as a five-category working diagnosis and nine related items. Working diagnosis groups were described and compared and their SOFA-scores analyzed with a generalized linear mixed model. Agreement and discriminatory performance measures for clinical criteria of sepsis and GTSQ labels as reference class were derived. RESULTS: We analyzed 7291 questionnaires and 761 complete encounters from the first survey year. Editing rates for all items were > 90%, and responses were consistent with current understanding of critical illness pathophysiology, including sepsis pathogenesis. Interrater agreement for presence and absence of sepsis was almost perfect but only slight for suspected infection. ICU mortality was 19.5% in encounters with SIRS as the "worst" working diagnosis compared to 5.9% with sepsis and 5.9% with severe sepsis without differences in admission and maximum SOFA. Compared to sepsis, proportions of GTSQs with SIRS plus acute organ dysfunction were equal and macrocirculatory abnormalities higher (p < 0.0001). SIRS proportionally ranked above sepsis in daily assessment of illness severity (p < 0.0001). Separate analyses of neurosurgical referrals revealed similar differences. Discriminatory performance of Sepsis-1/2 and Sepsis-3 compared to GTSQ labels was similar with sensitivities around 70% and specificities 92%. Essentially no difference between the prevalence of SIRS and SOFA ≥ 2 yielded sensitivities and specificities for detecting sepsis onset close to 55% and 83%, respectively. CONCLUSIONS: GTSQ labels are a valid measure of sepsis in the ICU. They reveal suspicion of infection as an unclear clinical concept and refute an illness severity hierarchy in the SIRS-sepsis-severe sepsis spectrum. Ground truth challenges the accuracy of Sepsis-1/2 and Sepsis-3 in detecting sepsis onset. It is an indispensable intermediate step towards advancing diagnosis and therapy in the ICU and, potentially, other health care settings.

The effect of creatine ingestion on urinary creatinine concentration: Does supplementation mask a heavy dilution?

A high volume of fluid can strongly reduce a drug's concentration in urine. Therefore, to detect diluted samples, the concentration of creatinine in urine is determined during testing drugs of abuse. If the concentration is below 20 mg creatinine/dl urine, the urine sample is usually rejected for drug testing. It should be examined whether creatine or creatinine ingestion can mask urine dilution by increasing the creatinine concentration. A total of 18 subjects drank 1.3 L of water and 0.2 L of orange juice on each of the three testing days: (1) without creatine, (2) with 20 g of creatine, and (3) with 20 g of creatine following incubation for 4 days in orange juice at room temperature; an acidic environment should promote conversion of creatine to creatinine. The lowest creatinine concentrations in urine were observed on average 2 h after fluid intake. At that time, ingestion of fluid without creatine, with creatine, and with creatine(ine)-orange juice mixture resulted in mean values of 11.6, 22.5, and 28.3 mg creatinine/dl urine, respectively. It can be concluded that ingestion of creatine or creatinine can increase the concentration of creatinine in urine and thus mask dilution of a sample. The conversion of creatine in orange juice further increases availability of creatinine as it is obvious from urine creatinine concentration. Therefore, creatine ingestion during drug testing will give rise to negative results due to matrix adulteration. In a case of suspected creatine supplementation, the creatine content of the sample should be determined in addition to creatinine.

Maintaining oxygen delivery is crucial to prevent intestinal ischemia in critical ill patients.

BACKGROUND: Intestinal ischemia is a common complication with obscure pathophysiology in critically ill patients. Since insufficient delivery of oxygen is discussed, we investigated the influence of oxygen delivery, hemoglobin, arterial oxygen saturation, cardiac index and the systemic vascular resistance index on the development of intestinal ischemia. Furthermore, we evaluated the predictive power of elevated lactate levels for the diagnosis of intestinal ischemia. METHODS: In a retrospective case-control study data (mean oxygen delivery, minimum oxygen delivery, systemic vascular resistance index) of critical ill patients from 02/2009-07/2017 were analyzed using a proportional hazard model. General model fit and linearity were tested by likelihood ratio tests. The components of oxygen delivery (hemoglobin, arterial oxygen saturation and cardiac index) were individually tested in models. RESULTS: 59 out of 874 patients developed intestinal ischemia. A mean oxygen delivery less than 250ml/min/m2 (LRT vs. null model: p = 0.018; LRT for non-linearity: p = 0.012) as well as a minimum oxygen delivery less than 400ml/min/m2 (LRT vs null model: p = 0.016; LRT for linearity: p = 0.019) were associated with increased risk of the development of intestinal ischemia. We found no significant influence of hemoglobin, arterial oxygen saturation, cardiac index or systemic vascular resistance index. Receiver operating characteristics analysis for elevated lactate levels, pH, CO2 and central venous saturation was poor with an area under the receiver operating characteristic of 0.5324, 0.52, 0.6017 and 0.6786. CONCLUSION: There was a significant correlation for mean and minimum oxygen delivery with the incidence of intestinal ischemia for values below 250ml/min/m2 respectively 400ml/min/m2. Neither hemoglobin, arterial oxygen saturation, cardiac index, systemic vascular resistance index nor elevated lactate levels could be identified as individual risk factors.

Verifying the validity of creatinine measurement in low-concentrated urine spot samples-Photospectrometry versus liquid chromatography-tandem mass spectrometry.

One of the major challenges of testing drugs of abuse is the detection of highly diluted urine samples. The ingestion of a large amount of fluid can considerably reduce the concentration of substances, possibly resulting in inaccurate drug testing. For detection, determination of urinary creatinine is a widely established procedure. In this study, results from the most popular methods, including photospectrometry (Jaffe) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been compared regarding 327 urine abstinence control samples. Because samples with creatinine concentrations close to the cutoff of 20 mg/dL are of particular interest, only samples below 50 mg/dL were considered. Results revealed a close correlation of creatinine concentrations by both analytical methods with an R2 value of 0.9005. A mean concentration difference of 3.30 ± 3.45 mg/dL was observed, indicating a moderate underestimation by the Jaffe reaction. Graphical analyses showed high accordance between both methods with only a few outliers. Due to easy handling and for economic reasons, the spectrometric method is often preferred over LC-MS/MS. For urine samples with creatinine concentrations close to the cutoff, confirmation through a second method should be performed to avoid a possible disadvantage or even severe consequences for the respective individual. It is recommended that each laboratory establishes a reliable verification method.

A quantitative CT parameter for the assessment of pulmonary oedema in patients with acute respiratory distress syndrome.

OBJECTIVES: The aim of this study was to establish quantitative CT (qCT) parameters for pathophysiological understanding and clinical use in patients with acute respiratory distress syndrome (ARDS). The most promising parameter is introduced. MATERIALS AND METHODS: 28 intubated patients with ARDS obtained a conventional CT scan in end-expiratory breathhold within the first 48 hours after admission to intensive care unit (ICU). Following manual segmentation, 137 volume- and lung weight-associated qCT parameters were correlated with 71 clinical parameters such as blood gases, applied ventilation pressures, pulse contour cardiac output measurements and established status and prognosis scores (SOFA, SAPS II). RESULTS: Of all examined qCT parameters, excess lung weight (ELW), i.e. the difference between a patient's current lung weight and the virtual lung weight of a healthy person at the same height, displayed the most significant results. ELW correlated significantly with the amount of inflated lung tissue [%] (p<0.0001; r = -0.66) and was closely associated with the amount of extravascular lung water (EVLW) (p<0.0001; r = 0.72). More substantially than the oxygenation index (PaO2/FiO2) or any other clinical parameter it correlated with the patients' mean SOFA- (p<0.0001, r = 0.69) and SAPS II-Score (p = 0.0005, r = 0.62). Patients who did not survive intensive care treatment displayed higher values of ELW in the initial CT scans. CONCLUSIONS: ELW could serve as a non-invasive method to quantify the amount of pulmonary oedema. It might serve as an early radiological marker of severity in patients with ARDS.

Impact of different consensus definition criteria on sepsis diagnosis in a cohort of critically ill patients-Insights from a new mathematical probabilistic approach to mortality-based validation of sepsis criteria.

BACKGROUND: Sepsis-3 definition uses SOFA score to discriminate sepsis from uncomplicated infection, replacing SIRS criteria that were criticized for being inaccurate. Eligibility of sepsis-3 criteria for sepsis diagnosis and the applied validation methodology using mortality as endpoint are topic of ongoing debate. We assessed the impact of different criteria on sepsis diagnosis in our ICU and devised a mathematical approach for mortality-based validation of sepsis criteria. As infectious status is often unclear at clinical deterioration, we integrated non-infected patients into analysis. METHODS: Suspected infection, SOFA and SIRS were captured for an ICU cohort of a university center over one year. For raw scores (SIRS/SOFA) and sepsis criteria (SIRS≥2/SOFA≥2/SOFA_change≥2) frequencies and associations with in-hospital mortality were assessed. Using a mathematical approach, we estimated the correlation between sepsis and in-hospital mortality serving as reference for evaluation of observed mortality correlations of sepsis criteria. RESULTS: Of 791 patients, 369 (47%) were infected and 422 (53%) non-infected, with an in-hospital mortality of 39% and 15%. SIRS≥2 indicated sepsis in 90% of infected patients, SOFA≥2 in 99% and SOFA_change≥2 in 77%. In non-infected patients, SIRS, SOFA and SOFA_change were ≥2 in 78%, 88% and 58%. In AUROC analyses neither SOFA nor SIRS displayed superior mortality discrimination in infected compared to non-infected patients. The mathematically estimated correlation of sepsis and in-hospital mortality was 0.10 in infected and 0 in non-infected patients. Among sepsis criteria, solely SIRS≥2 agreed with expected correlations in both subgroups (infected: r = 0.19; non-infected: r = 0.02). CONCLUSIONS: SOFA≥2 yielded a more liberal sepsis diagnosis than SIRS≥2. None of the criteria showed an infection specific occurrence that would be essential for reliable sepsis detection. However, SIRS≥2 matched the mortality association pattern of a valid sepsis criterion, whereas SOFA-based criteria did not. With this study, we establish a mathematical approach to mortality-based evaluation of sepsis criteria.

AKIRIN1: A Potential New Reference Gene in Human Natural Killer Cells and Granulocytes in Sepsis.

Timely and reliable distinction of sepsis from non-infectious systemic inflammatory response syndrome (SIRS) supports adequate antimicrobial therapy and saves lives but is clinically challenging. Blood transcriptional profiling promises to deliver insights into the pathomechanisms of SIRS and sepsis and to accelerate the discovery of urgently sought sepsis biomarkers. However, suitable reference genes for normalizing gene expression in these disease conditions are lacking. In addition, variability in blood leukocyte subtype composition complicates gene profile interpretation. Here, we aimed to identify potential reference genes in natural killer (NK) cells and granulocytes from patients with SIRS and sepsis on intensive care unit (ICU) admission. Discovery by a two-step probabilistic selection from microarray data followed by validation through branched DNA assays in independent patients revealed several candidate reference genes in NK cells including AKIRIN1, PPP6R3, TAX1BP1, and ADRBK1. Initially, no candidate genes could be validated in patient granulocytes. However, we determined highly similar AKIRIN1 expression also in SIRS and sepsis granulocytes and no change by in vitro LPS challenge in granulocytes from healthy donors. Inspection of external neutrophil transcriptome datasets further support unchanged AKIRIN1 expression in human systemic inflammation. As a potential new reference gene in NK cells and granulocytes in infectious and inflammatory diseases, AKIRIN1 may improve our pathomechanistic understanding of SIRS and sepsis and help identifying new sepsis biomarkers.

Creatinine excretion in consecutive urine samples after controlled ingestion of water.

Highly diluted urine is among the most commonly observed factors affecting the validity of urine testing for drug abuse. A minimum creatinine concentration of 20 mg/dL urine has been proposed as a marker for dilution of a urine sample. This study investigates the effect of water consumption on creatinine concentration, as well as its effect on specific gravity and osmolality. In this study, 22 subjects (17 women and 5 men) were included to determine the influence of sex and weight on the impact of excessive water consumption on these markers of urine dilution. The subjects consumed 0.5 L, 1.0 L, and 1.5 L of water, respectively, within 15 minutes. The mean minimum creatinine concentrations (Jaffé reaction) for the void 2 hours after fluid intake were 60.4 mg/dL, 15.8 mg/dL, and 10.9 mg/dL for the respective ingested volumes of water. Mean creatinine concentrations excreted by men were significantly higher than those excreted by women. Participants with a weight below 60 kg tended to excrete lower urine creatinine concentrations. 50% of the volunteers with a BMI < 20 kg/m2 and 20% of the volunteers with a BMI > 20 kg/m2 exhibited creatinine concentrations below the threshold value of 20 mg/dL. A similar pattern was established for gravity and osmolality. Due to its simple determination, creatinine may be preferred over specific gravity or osmolality. In order to evaluate the internal dilution of a urine sample for legally defensible drug testing, it may be necessary to account for sex and body weight.

Tumor Budding, uPA, and PAI-1 in Colorectal Cancer: Update of a Prospective Study.

Aims. The prognostic role of the proteases uPA and PAI-1, as well as tumor budding, in colon cancer, has been investigated previously. Methods. We provide 6-year follow-up data and results of the validation set. The initial test set and validation set consisted of 55 colon cancers and 68 colorectal cancers, respectively. Tissue samples were analyzed for uPA and PAI-1 using a commercially available Enzyme-Linked Immunosorbent Assay (ELISA). Tumor budding was analyzed on cytokeratin-stained slides. Survival analyses were performed using cut-offs that were determined previously. Results. uPA was not prognostic for outcome. PAI-1 showed a trend towards reduced cancer specific survival in PAI-1 high-grade cases (68 versus 83 months; P = 0.091). The combination of high-grade PAI-1 and tumor budding was associated with significantly reduced cancer specific survival (60 versus 83 months; P = 0.021). After pooling the data from both sets, multivariate analyses revealed that the factors pN-stage, V-stage, and a combination of tumor budding and PAI-1 were independently prognostic for the association with distant metastases. Conclusions. A synergistic adverse effect of PAI-1 and tumor budding in uni- and multivariable analyses was found. PAI-1 could serve as a target for anticancer therapy.

Potential canonical wnt pathway activation in high-grade astrocytomas.

Aberrant wnt pathway activation through cytoplasmic stabilization of ß-catenin is crucial for the development of various human malignancies. In gliomagenesis, the role of canonical (i.e., ß-catenin-dependent) signalling is largely unknown. Here, we studied canonical wnt pathway activation in 15 short-term cultures from high-grade gliomas and potential pathomechanisms leading to cytoplasmic ß-catenin accumulation. Furthermore, we assessed the prognostic relevance of ß-catenin expression in a tissue microarray comprising 283 astrocytomas. Expression of ß-catenin, its transcriptional cofactors TCF-1 and TCF-4 as well as GSK-3ß and APC, constituents of the ß-catenin degradation complex was confirmed by RT-PCR in all cultures. A cytoplasmic ß-catenin pool was detectable in 13/15 cultures leading to some transcriptional activity assessed by luciferase reporter gene assay in 8/13. Unlike other malignancies, characteristic mutations of ß-catenin and APC leading to cytoplasmic stabilization of ß-catenin were excluded by direct sequencing or protein truncation test. In patient tissues, ß-catenin expression was directly and its degradation product's (ß-catenin-P654) expression was inversely correlated with WHO grade. Increased ß-catenin expression and low ß-catenin-P654 expression were associated with shorter survival. Altogether, we report on potential canonical wnt pathway activation in high-grade gliomas and demonstrate that ß-catenin expression in astrocytomas is associated with increased malignancy and adverse outcome.

Aberrant expression of retinoic acid signaling molecules influences patient survival in astrocytic gliomas.

Undifferentiated cell populations may influence tumor growth in malignant glioma. We investigated potential disruptions in the retinoic acid (RA) differentiation pathway that could lead to a loss of differentiation capacity, influencing patient prognosis. Expression of key molecules belonging to the RA differentiation pathway was analyzed in 283 astrocytic gliomas and was correlated with tumor proliferation, tumor differentiation, and patient survival. In addition, in situ concentrations of retinoids were measured in tumors, and RA signaling events were studied in vitro. Unlike other tumors, in gliomas expression of most RA signaling molecules increased with malignancy and was associated with augmented intratumoral retinoid levels in high-grade gliomas. Aberrantly expressed RA signaling molecules included i) the retinol-binding protein CRBP1, which facilitates cellular retinoid uptake; ii) ALDH1A1, capable of activating RA precursors; iii) the RA-degrading enzyme CYP26B1; and iv) the RA-binding protein FABP5, which can inhibit RA-induced differentiation. In contrast, expression of the RA-binding protein CRABP2, which fosters differentiation, was decreased in high-grade tumors. Moreover, expression of CRBP1 correlated with tumor proliferation, and FABP5 expression correlated with an undifferentiated tumor phenotype. CRBP1 and ALDH1A1 were independent prognostic markers for adverse patient survival. Our data indicate a complex and clinically relevant deregulation of RA signaling, which seems to be a central event in glioma pathogenesis.