Andexanet alfa after 4-factor PCC administration for intracranial hemorrhage: a case series

Journal of Thrombosis and Thrombolysis - The ongoing controversy regarding optimal reversal agent for factor Xa-inhibitors is mainly due to lack of comparative data of andexanet alfa (AA) to...     

Common denominator genes that distinguish colorectal carcinoma from normal mucosa

Purpose Microarray technology has been used by a growing number of investigators and several studies have been published that list hundreds of genes differentially expressed by colorectal carcinoma (CRC) and normal mucosa (MC). On the basis of our own and other investigators microarray data, our goal was to identify a common denominator gene cluster distinguishing CRC from MC.Methods Thirty GeneChips (HG-U133A, Affymetrix) were hybridized, 20 with RNA of CRC stages I–IV (UICC) and 10 with MC. Expression signals showing at least a 4-fold difference between CRC and MC (p<0.01) were identified as differentially expressed. In addition, in our integrative data analysis approach only those genes whose expression was altered simultaneously in at least 2 of 5 recently published studies were subjected to an unsupervised hierarchical cluster analysis.Results We detected 168 up- and 283 down-regulated genes in CRC relative to MC. Twenty-three genes were filtered from the five articles reviewed. An unsupervised hierarchical cluster analysis of these 23 genes confirmed the high specificity of these genes to differentiate between CRC and MC in our microarray data.Conclusions Colorectal cancer and mucosa could be clearly separated by 23 genes selected for being differentially expressed more than once in a recent literature review. These genes represent a common denominator gene cluster that can be used to distinguish colorectal MC from CRC.     

The effect of gestation and fetal mismatching on the development of autoimmune diabetes in non-obese diabetic mice

The impact of gestation and fetal-maternal interactions on pre-existent autoimmune beta cell destruction is widely unknown. The aim of this study was to investigate the influence of gestation per se and fetal mismatching on the onset of autoimmune diabetes in female non-obese diabetic (NOD) mice. We examined cumulative diabetes frequencies of NOD dams mated to syngeneic NOD, haploidentical CByB6F1/J and fully mismatched C57BL/6J male mice. Pregnancy from NOD males neither increased nor accelerated the diabetes onset of NOD dams (71% by age 28 weeks) compared to unmated female NOD mice (81% by age 28 weeks; P = 0·38). In contrast, delayed diabetes onset was observed when NOD dams were mated at 10 weeks of age with major histocompatibility complex (MHC) haploidentical CByB6F1/J male mice (38% at age 28 weeks; P = 0·01). Mating with fully MHC mismatched C57BL/6J male mice (72% diabetes by age 28 weeks; P = 0·22) or mating with the haploidentical males at the later time-point of age 13 weeks (64% versus 91% in unmated litter-matched controls; P = 0·13) did not delay diabetes significantly in NOD females. Because infusion of haploidentical male mouse splenocytes was found previously to prevent diabetes in NOD mice we looked for, but found no evidence of, persistent chimeric lymphocytes from haploidentical paternal origin within the dams' splenocytes. Gestation per se appears to have no aggravating or ameliorating effects on pre-existent autoimmune beta cell destruction, but pregnancy from MHC partially mismatched males delays diabetes onset in female NOD mice.     

The direct Peptide reactivity assay: selectivity of chemical respiratory allergens

It is well known that some chemicals are capable of causing allergic diseases of the skin and respiratory tract. Commonly, though not exclusively, chemical allergens are associated with the selective development of skin or respiratory sensitization. The reason for this divergence is unclear, although it is hypothesized that the nature of interactions between the chemical hapten and proteins is influential. The direct peptide reactivity assay (DPRA) has been developed as a screen for the identification of skin-sensitizing chemicals, and here we describe the use of this method to explore whether differences exist between skin and respiratory allergens with respect to their peptide-binding properties. Known skin and respiratory sensitizers were reacted with synthetic peptides containing either lysine (Lys) or cysteine (Cys) for 24h. The samples were analyzed by HPLC/UV, and the loss of peptide from the reaction mixture was expressed as the percent depletion compared with the control. The potential for preferential reactivity was evaluated by comparing the ratio of Lys to Cys depletion (Lys:Cys ratio). The results demonstrate that the majority of respiratory allergens are reactive in the DPRA, and that in contrast to most skin-sensitizing chemicals, preferentially react with the Lys peptide. These data suggest that skin and respiratory chemical allergens can result in different protein conjugates, which may in turn influence the quality of induced immune responses. Overall, these investigations reveal that the DPRA has considerable potential to be incorporated into tiered testing approaches for the identification and characterization of chemical respiratory allergens.     

Thermography – a valuable tool to test hydrocephalus shunt patency

Summary Introduction. Shunt-function in hydrocephalic patients is verified by clinical examination and repeated cranial computed tomography (CCT) in most cases. Because of the disadvantages of multiple radiation especially in children it was our aim to introduce video-thermography as a simple and non-invasive methodology to evaluate shunt function. Methods. 54 patients treated with shunts for hydrocephalus were tested. A ventriculo-peritoneal shunt had been implanted in 38 patients, a ventriculo-atrial shunt in 16 patients. Recent CCT-scans were available for all patients and served as control. None of the patients presented with clinical signs of shunt-dysfunction. The temperature of the skin covering the drainage catheter distal to the valve was recorded real-time by a calibrated infrared camera. After cooling the skin area downstream of the valve for exactly 1 min with an ice pack, changes of the skin temperature in the area downstream were registered by a thermocamera. The signals were transferred to a video screen and recorded on videotape. By off-line analysis of the obtained pseudo colour images variations of 0.1 °C in skin temperature could be measured. Results. Temperature distribution of the area under investigation revealed a significant reduction of the skin temperature according to the location of the downstream catheter segment in 48 patients after cooling. In 6 patients skin temperature remained constant, although clinical evaluation and CCT-scan showed no signs of shunt dysfunction. Shunt patency could be verified in more than 85% of the patients by thermal imaging. Conclusion. Infrared-thermography is a valuable and promising tool for replacing CCT-scanning as a screening method to test shunt function in hydrocephalic patients.     

Tissue preparation for gene expression profiling of colorectal carcinoma: three alternatives to laser microdissection with preamplification

Colorectal-carcinoma specimens are heterogeneous and include areas of nonmalignant mucosal and connective tissue. For those study designs in which laser microdissection and RNA preamplification are impracticable, the optimal yield of genuine cancer RNA is a key factor in gene-expression analysis. In this study we compared alternative methods of tissue purification. Three contiguous 0.5-cm(3) samples taken from an advanced primary adenocarcinoma of the sigmoid colon were processed immediately after surgery with the use of the following methods: (1) cryotomy after manual dissection (CMD), (2) microscopically assisted manual dissection (MAMD), and (3) tumor-cell isolation with the use of Ber-EP4 antibodies and Dynabeads (Dynal Biotech GmbH, Hamburg, Germany; technique abbreviated as DB). We generated gene-expression profiles with the use of GeneChip technology (Affymetrix, Santa Clara, Calif) and recorded preparation times, costs, and RNA quantity and quality. CMD took 60 minutes, MAMD 180 minutes, and DB 90 minutes to isolate 22, 8, and 23 microg of RNA, respectively. Expenses for materials amounted to 41, 23, and 91 US dollars for CMD, MAMD, and DB, respectively. The 3'/5' ratio, as determined with the GeneChips, for GAPDH/beta-actin was 1.01:1.03 for CMD, 1.13:1.28 for MAMD, 1.43:1.68 for DB, K-ras, APC, smad 2, transforming growth factor-beta, and p53 were marked as present in all cases, with the exception of APC, which was graded as marginal on DB. The correlation values of gene-expression profiles were 91% (CMD/DB), 93% (CMD/MAMD), and 97% (DB/MAMD). All 3 methods provided enough RNA, of sufficient quality, for gene-expression microarray analysis in colorectal carcinoma. Cross-methodologic analyses of array data should not be performed uncritically.     

High post surgical opioid requirements in Crohn's disease are not due to a general change in pain sensitivity

Crohn's disease (CD) is a painful inflammatory bowel disease with complex multigenic inheritance. Suggested on the basis of a few isolated reports CD patients require significantly higher post operative opioid doses than patients undergoing comparable severe abdominal surgery. Crohn's disease therefore may be a suitable model for the identification of novel pain susceptibility genes. In order to confirm this observation and to elucidate the underlying molecular mechanisms, we investigated if higher opioid needs of CD patients are due to a general change in pain sensitivity. Quantitative sensory testing (QST) was applied to a subgroup of patients and polymorphisms in the μ‐opioid receptor (OPRM1) and catechol‐O‐methyltransferase (COMT) were investigated. Significantly increased post operative opioid requirements in CD patients were confirmed and QST assessment demonstrates that CD patients do not display increased pain sensitivity in terms of lowered thresholds to thermal and mechanical stimuli. The data also suggest that common variants in OPRM1 and specific ‘high pain sensitivity’ COMT haplotypes may not be the cause of high opioid needs. The results indicate that a more complex pathway is involved in the greater post operative opioid demand in CD. Therefore the presence of other, as yet unknown, genes could modulate opioid requirements in CD patients.     

Identification of gene expression changes induced by chemical allergens in dendritic cells: opportunities for skin sensitization testing

Cellular changes within resident skin dendritic cells (DCs) after allergen uptake and processing are critical events in the acquisition of skin sensitization. Here we describe the development of a set of selection criteria to derive a list of potential target genes from previous microarray analyses of human peripheral blood-derived (peripheral blood mononuclear cells (PBMCs)-DCs) treated with dinitrobenzene sulfonic acid for predicting skin-sensitizing chemicals. Based on those criteria, a probing evaluation of the target genes has been conducted using an extended chemical data set, comprising five skin irritants and 11 contact allergens. PBMCs-DCs were treated for 24 hours with various concentrations of chemicals and in each instance the expression of up to 60 genes was examined by real-time PCR analysis. Consistent allergen-induced changes in the expression of many genes were observed and further prioritization of the targets was conducted by analysis of the same genes in DCs treated with non-sensitizing chemicals to determine their specificity for skin sensitization. Real-time PCR analyses of multiple chemical allergens, irritants, and non-sensitizers have identified 10 genes that demonstrate reproducibly high levels of selectivity, specificity, and dynamic range consistent with providing the basis for robust and sensitive alternative approaches for the identification of skin-sensitizing chemicals.     

A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation

Skin sensitization is a key endpoint for cosmetic ingredients, with a forthcoming ban for animal testing in Europe. Four alternative tests have so far been submitted to ECVAM prevalidation: (i) MUSST and (ii) h‐Clat assess surface markers on dendritic cell lines, (iii) the direct peptide reactivity assay (DPRA) measures reactivity with model peptides and (iv) the KeratinoSens^TM assay which is based on detection of Nrf2‐induced luciferase. It is anticipated that only an integrated testing strategy (ITS) based on a battery of tests might give a full replacement providing also a sensitization potency assessment, but this concept should be tested with a data‐driven analysis. Here we report a database on 145 chemicals reporting the quantitative endpoints measured in a U937‐ test, the DPRA and KeratinoSens^TM . It can serve to develop data‐driven ITS approaches as we show in a parallel paper and provides a view as to the current ability to predict with in vitro tests as we are entering 2013. It may also serve as reference database when benchmarking new molecules with in vitro based read‐across and find use as a reference database when evaluating new tests. The tests and combinations thereof were evaluated for predictivity, and overall a similar predictivity was found as before on three‐fold smaller datasets. Analysis of the dose–response parameters of the individual tests indicates a correlation to sensitization potency. Detailed analysis of chemicals false‐negative and false‐positive in two tests helped to define limitations in the tests but also in the database derived from animal studies. Copyright © 2013 John Wiley & Sons, Ltd.