Trauma-hemorrhagic shock induces a CD36-dependent RBC endothelial-adhesive phenotype.

OBJECTIVE: Microvascular dysfunction is a key element in the development of the multiple organ dysfunction syndrome. Although the mechanisms for this response are unclear, RBC adhesion to endothelium may initiate intravascular occlusion leading to ischemic tissue injury. Thus, we tested the hypothesis that trauma-hemorrhage induces RBC-endothelial cell adhesion. DESIGN: Prospective in vivo and in vitro animal study and analysis of patient blood samples. SETTING: University research laboratory and hospital emergency and trauma units. INTERVENTION: We initially assayed RBC adhesion to endothelial cells in vitro using RBCs obtained from rats subjected to trauma-hemorrhagic shock or sham shock as well as from severely injured trauma patients. Subsequently, we measured the role of putative RBCs and endothelial cell receptors in the increased RBC-endothelial cell adhesive response. MAIN RESULTS: In both rats and humans, trauma-hemorrhagic shock increased RBC adhesion to endothelium as well as increasing several putative RBC surface adhesion molecules including CD36. The critical factor leading to RBC-endothelial cell adhesion was increased surface RBC CD36 expression. Adhesion of trauma-hemorrhagic shock RBCs was mediated, at least in part, by the binding of RBC CD36 to its cognate endothelial receptors (αVß3 and VCAM-1). Gut-derived factors carried in the intestinal lymphatics triggered these trauma-hemorrhagic shock-induced RBC changes because 1) preventing trauma-hemorrhagic shock intestinal lymph from reaching the systemic circulation abrogated the RBC effects, 2) in vitro incubation of naïve whole blood with trauma-hemorrhagic shock lymph replicated the in vivo trauma-hemorrhagic shock-induced RBC changes while 3) injection of trauma-hemorrhagic shock lymph into naïve animals recreated the RBC changes observed after actual trauma-hemorrhagic shock. CONCLUSIONS: 1) Trauma-hemorrhagic shock induces rapid RBC adhesion to endothelial cells in patients and animals. 2) Increased RBC CD36 expression characterizes the RBC-adhesive phenotype. 3) The RBC phenotypic and functional changes were induced by gut-derived humoral factors. These novel findings may explain the microvascular dysfunction occurring after trauma-hemorrhagic shock, sepsis, and other stress states.

Diverse roles and interactions of the SWI/SNF chromatin remodeling complex revealed using global approaches.

A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate, and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function, and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate, we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5' ends, RNA Polymerases II and III, and enhancers) as well as regions critical for chromosome organization (e.g. CTCF, lamins, and DNA replication origins). Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression, whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression. To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins, we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members, as well as with cellular constituents such as nuclear matrix proteins, key transcription factors, and centromere components, implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions than previously appreciated.

Comprehensive annotation of the transcriptome of the human fungal pathogen Candida albicans using RNA-seq.

Candida albicans is the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections that are often lifethreatening. We have used massively parallel high-throughput sequencing of cDNA (RNA-seq) to generate a high-resolution map of the C. albicans transcriptome under several different environmental conditions. We have quantitatively determined all of the regions that are transcribed under these different conditions, and have identified 602 novel transcriptionally active regions (TARs) and numerous novel introns that are not represented in the current genome annotation. Interestingly, the expression of many of these TARs is regulated in a condition-specific manner. This comprehensive transcriptome analysis significantly enhances the current genome annotation of C. albicans, a necessary framework for a complete understanding of the molecular mechanisms of pathogenesis for this important eukaryotic pathogen.

X chromosome-wide analyses of genomic DNA methylation states and gene expression in male and female neutrophils.

The DNA methylation status of human X chromosomes from male and female neutrophils was identified by high-throughput sequencing of HpaII and MspI digested fragments. In the intergenic and intragenic regions on the X chromosome, the sites outside CpG islands were heavily hypermethylated to the same degree in both genders. Nearly half of X chromosome promoters were either hypomethylated or hypermethylated in both females and males. Nearly one third of X chromosome promoters were a mixture of hypomethylated and heterogeneously methylated sites in females and were hypomethylated in males. Thus, a large fraction of genes that are silenced on the inactive X chromosome are hypomethylated in their promoter regions. These genes frequently belong to the evolutionarily younger strata of the X chromosome. The promoters that were hypomethylated at more than two sites contained most of the genes that escaped silencing on the inactive X chromosome. The overall levels of expression of X-linked genes were indistinguishable in females and males, regardless of the methylation state of the inactive X chromosome. Thus, in addition to DNA methylation, other factors are involved in the fine tuning of gene dosage compensation in neutrophils.

Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.

BACKGROUND: Short-read high-throughput DNA sequencing technologies provide new tools to answer biological questions. However, high cost and low throughput limit their widespread use, particularly in organisms with smaller genomes such as S. cerevisiae. Although ChIP-Seq in mammalian cell lines is replacing array-based ChIP-chip as the standard for transcription factor binding studies, ChIP-Seq in yeast is still underutilized compared to ChIP-chip. We developed a multiplex barcoding system that allows simultaneous sequencing and analysis of multiple samples using Illumina's platform. We applied this method to analyze the chromosomal distributions of three yeast DNA binding proteins (Ste12, Cse4 and RNA PolII) and a reference sample (input DNA) in a single experiment and demonstrate its utility for rapid and accurate results at reduced costs. RESULTS: We developed a barcoding ChIP-Seq method for the concurrent analysis of transcription factor binding sites in yeast. Our multiplex strategy generated high quality data that was indistinguishable from data obtained with non-barcoded libraries. None of the barcoded adapters induced differences relative to a non-barcoded adapter when applied to the same DNA sample. We used this method to map the binding sites for Cse4, Ste12 and Pol II throughout the yeast genome and we found 148 binding targets for Cse4, 823 targets for Ste12 and 2508 targets for PolII. Cse4 was strongly bound to all yeast centromeres as expected and the remaining non-centromeric targets correspond to highly expressed genes in rich media. The presence of Cse4 non-centromeric binding sites was not reported previously. CONCLUSION: We designed a multiplex short-read DNA sequencing method to perform efficient ChIP-Seq in yeast and other small genome model organisms. This method produces accurate results with higher throughput and reduced cost. Given constant improvements in high-throughput sequencing technologies, increasing multiplexing will be possible to further decrease costs per sample and to accelerate the completion of large consortium projects such as modENCODE.

Individual heritable differences result in unique cell lymphocyte receptor repertoires of naïve and antigen-experienced cells.

The adaptive immune system's capability to protect the body requires a highly diverse lymphocyte antigen receptor repertoire. However, the influence of individual genetic and epigenetic differences on these repertoires is not typically measured. By leveraging the unique characteristics of B, CD4(+) T and CD8(+) T-lymphocyte subsets from monozygotic twins, we quantify the impact of heritable factors on both the V(D)J recombination process and on thymic selection. We show that the resulting biases in both V(D)J usage and N/P addition lengths, which are found in naïve and antigen experienced cells, contribute to significant variation in the CDR3 region. Moreover, we show that the relative usage of V and J gene segments is chromosomally biased, with ∼1.5 times as many rearrangements originating from a single chromosome. These data refine our understanding of the heritable mechanisms affecting the repertoire, and show that biases are evident on a chromosome-wide level.

Assay of stanozolol for tumor initiating and promoting activity in two rat liver foci bioassays.

In this study stanozolol, one of the most abused anabolic steroids, was investigated for tumor initiating and promoting activity in two rat liver foci bioassays, using gamma-glutamyltranspeptidase (GGT) as marker for detection of putative preneoplastic foci. Stanozolol, orally administered for 2 weeks, at a dose level approximately 400-times larger than the human therapeutic dose, in rats initiated with N-nitroso-diethylamine according to the Solt-Farber system assay, did not produce any increase in the number and volume of GGT-positive liver foci. A 6-week oral treatment with stanozolol (430 ppm in the diet) followed by 2 weeks of 2-acetylaminofluorene (AAF) diet (200 ppm), carried out according to the Tatematsu assay system to evaluate the initiating activity, did not provoke any significant modification of the number and volume of GGT-positive foci as compared to the controls. In the rats receiving AAF (200 ppm in the diet for 2 weeks) followed by 6 weeks of stanozolol, to evaluate the promoting activity, an increase in number and volume of the GGT-positive foci was observed at the highest oral dose, but the differences from the corresponding control values which resulted were not statistically significant. Taken as a whole the results of this study do not provide any substantial evidence of carcinogenic activity of stanozolol in rat liver, even when orally administered at high doses.

Increased frequency of N-methyl-N'-nitro-N-nitrosoguanidine-induced nuclear anomalies in the forestomach of rats pretreated with sodium chloride.

The frequency of nuclear anomalies (micronuclei, pyknosis, and karyorrhexis) in the forestomach mucosa was examined in Sprague-Dawley male rats given a single oral dose of 50 or 150 mg/kg of the gastric carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 17 h after the administration of 2 ml of a 3 M NaCl solution. Rats pretreated with NaCl displayed an incidence of nuclear anomalies approximately 3-fold greater than the one observed in rats given MNNG alone, and micronucleated cells accounted to a significant extent for this increase. These findings confirm that NaCl presumably acts as co-carcinogen in the initial phase of gastric carcinogenesis, and suggest that its administration before the carcinogen might increase the sensitivity of short-term tests for the preliminary screening of potential gastric carcinogens.

Induction and promotion of gamma-glutamyltranspeptidase-positive foci in the liver of female rats treated with ethinyl estradiol, clomiphene, tamoxifen and their associations.

The objective of the present study was to determine whether a short exposure (6 weeks) to high doses of ethinyl estradiol (EE) could not only promote but also initiate hepatocarcinogenesis, and whether two antiestrogens, clomiphene (C) and tamoxifen (T), could influence EE activity. 2-Acetylaminofluorene (AAF), which has been shown to produce rat liver hyperplastic lesions characterized by the presence of estrogen receptors, was used either as a promoter to test for initiating activity, or as an initiator to test for promoting activity. Putative preneoplastic lesions were identified by means of a positive gamma-glutamyltranspeptidase (GGT) reaction. The results revealed that when administered alone in female Sprague-Dawley rats, not only E, but also C and T were clearly active in both initiating and promoting the development of GGT-positive foci. Moreover, in rats of the same strain treated with EE + C or EE + T a significant increase in the incidence of GGT foci demonstrated the occurrence of an additive effect in terms of both initiating and promoting activity. Fischer 344 rats were more susceptible than Sprague-Dawley rats to promotion by EE, C and T, but any substantial evidence of an additive effect was absent when the two anti-estrogens were administered in association with the estrogen.

Evaluation of the potential carcinogenic activity of Senna and Cascara glycosides for the rat colon.

Anthraquinone glycosides of Senna and Cascara were investigated for their ability to induce aberrant crypt foci (ACF) in the rat colon mucosa, which are considered putative preneoplastic lesions. Dietary exposure to high doses of these glycosides for 56 successive days did not cause the appearance of ACF or increase in incidence of ACF induced by 1,2-dimethyl-hydrazine (DMH). However, in rats treated with both DMH and the highest dose of glycosides, the average number of aberrant crypts per focus, considered a consistent predictor of tumor outcome, was higher than in rats given DMH alone. These findings suggest that Senna and Cascara glycoside might behave as weak promoters in rat colon carcinogenesis.

A possible medium-term assay for detecting the effects of liver and colon carcinogens in rats.

The aim of the present study was to verify whether the tumorigenic effect of a rat liver carcinogen, 2-acetylaminofluorene (AAF), and of a promoter of rat colon carcinogenesis, chenodeoxycholic acid (CDCA), could be detected with a single medium-term assay using as markers gamma-glutamyltranspeptidase (GGT)-positive foci in the liver and aberrant crypt foci (ACF) in colon mucosa. In rats given in the first 2 weeks of treatment both N-nitrosodiethylamine (NDEA), as initiator of liver carcinogenesis, and azoxymethane (AOM), as initiator of colon carcinogenesis, the subsequent 6-week feeding on a diet containing AAF (0.01%) produced a significant marked increase of the number and area of GGT-positive foci which is consistent with the results of long term assays. When rats initiated with both NDEA and AOM were fed for 6 weeks on a diet containing CDCA (0.1%) a significant increase of large ACF as well as of crypt multiplicity was observed, consistently with the promoting effect of CDCA in colon carcinogenesis. The results obtained in this preliminary study suggest that this medium-term assay might be able to screen both liver and colon carcinogens in rats.

Effect of aspirin on incidence and growth of aberrant crypt foci induced in the rat colon by 1,2-dimethylhydrazine.

The aim of this work was to investigate if the possible chemopreventive effect of aspirin (ASA) on rat colon carcinogenesis could be detected with a medium-term assay. The end-point chosen was the inhibition of incidence and growth of putative preneoplastic lesions, the aberrant crypt foci (ACF) induced in the rat colon by two administrations of 1,2-dimethylhydrazine (DMH, 25 mg/kg p.o.). At both 4 and 8 weeks after the starting of the carcinogenic treatment the incidence of total ACF was reduced of 60% in rats receiving ASA (10 mg/kg/day p.o.) for 12 consecutive days during the initiation treatment with DMH. Also the number of the larger foci (with 3 or more crypts) was significantly lower in ASA-treated rats at both time-points (about 70% reduction). Moreover, concomitant ASA treatment determined a significant decrease of the mean number of crypts per focus at week 8. These results indicate that the chemopreventive effect of ASA on chemically-induced rat colon carcinogenesis observed in long-term studies may be detected in this relatively short assay.

Oral administration of chlordiazepoxide plus sodium nitrite investigated for tumor initiating activity in the rat liver.

Chlordiazepoxide (CDE) reacts with sodium nitrite at acid pH yielding the genotoxic derivative N-nitrosochlordiazepoxide (NO-CDE). In the present study oral administration of CDE plus NaNO2, previously found to produce DNA fragmentation in the rat liver, was examined for its ability to initiate hepatocarcinogenesis. The oral treatment for 6 successive weeks with CDE + NaNO2, added to the diet at the levels of 290 + 270 and 870 + 800 ppm, did not significantly increase the number or volume of gamma-glutamyltranspeptidase-positive foci (putative preneoplastic lesions). These findings are in agreement with the negative results previously obtained in rodent carcinogenesis assays and indicate that NO-CDE belongs to the progressively expanding list of genotoxic non-carcinogens.

Induction of DNA fragmentation and DNA repair synthesis in human and rat hepatocytes by diethylstilbestrol.

The synthetic estrogen diethylstilbestrol (DES), a known human carcinogen, was examined for cytotoxicity, and the induction of DNA damage and repair in primary cultures of human and rat hepatocytes. In both species concentrations of DES ranging from 5.6 to 18 micrograms/ml constantly produced reduction of cell viability and DNA fragmentation in dose-related amounts. However, large individual quantitative differences in the sensitivity to the cytotoxic and DNA-damaging activities of DES were observed among cultures derived from the 5 human donors. DES capability of eliciting DNA-excision repair was weak but statistically significant in both human and rat hepatocytes. Taken as a whole these results contribute to support the hypothesis of a genotoxic mechanism in DES-induced carcinogenesis.

A study of the potential genotoxicity of cimetidine using human hepatocyte primary cultures: discrepancy from results obtained in rat hepatocytes.

The genotoxicity of cimetidine, a drug widely used in the treatment of peptic ulcer, was examined in human hepatocyte primary cultures. No induction of unscheduled DNA synthesis, as detected by autoradiography, or of DNA fragmentation, as measured by alkaline elution, was seen in metabolically competent human hepatocytes exposed for 20 h to cimetidine concentrations ranging from 0.33 to 9 mM. These findings, which are in contrast with the previously observed capability of cimetidine to induce DNA damage and repair in rat hepatocyte primary cultures, suggest that for some chemicals the rat hepatocyte model might be an inappropriate predictor of potential genotoxic effects in the analogous human cells.

Effects of ethinyl estradiol and epidermal growth-factor on DNA-synthesis in human cultured-hepatocytes.

It has been demonstrated that the liver tumor promoter ethinyl estradiol (EE) potentiates the DNA synthetic response of cultured rat hepatocytes to epidermal growth factor (EGF), probably acting as a comitogen with a growth factor of the EGF/TGF family. The present study investigated the effects of EE and EGF on DNA synthesis in cultures of rat and human hepatocytes obtained from 5 female donors. The cultures were given the following treatments: a. 15 muM EE for 30 h; b. 25 ng/ml of EGF for 12 h; c. combination of treatments a and b. The results obtained in rat hepatocytes confirm that EE potentiates the.DNA synthetic response to EGF, as evaluated by the labelling index. As to human hepatocytes, in only one case the response to EGF produced a significant increase of DNA synthesis and the EE pretreatment did not potentiate this effect. The results of this study indicate that, in contrast to the response observed in rat hepatocytes, EE does not seem to affect the DNA synthesis in primary cultures of human hepatocytes, and the association EE+EGF does not produce any synergic effect.

Effects of reduced glutathione and n-acetylcysteine on lidocaine metabolism in cimetidine treated rats.

The aim of this work was to evaluate the effects of exogenous glutathione (GSH) and N-acetylcysteine (NAC) on the formation of monoethylglycinexylidide (MEGX) from lidocaine in rats with and without the administration of cimetidine. GSH and NAC were administered intraperitoneally (i.p.) (1 mmol/kg) 1 hour before treatment with cimetidine (0.5 mmol/kg) or saline, and 1 hr later all rats were injected i.p. with lidocaine (1 mg/kg). Blood samples were drawn 30 min after the lidocaine injection. MEGX and lidocaine serum concentrations were determined by means of fluorescence polarization immuno-assay using the TDX system. Cimetidine produced a decrease in MEGX levels (from 210 +/- 18 to 164 +/- 13 ng/mL) and a parallel increase in lidocaine levels (from 73 +/- 22 to 172 +/- 47 ng/mL), consistent with cytochrome P-450 3A inhibition. Both GSH and NAC produce a significant decrease in MEGX levels (151 +/- 16 and 139 +/- 14 ng/mL, respectively), but no significant increase in lidocaine levels were found. As compared to the cimetidine group, pre-treatment using either GSH or NAC with cimetidine produced a marked decrease in lidocaine levels (37 +/- 27 and 63 +/- 28 ng/mL, respectively) and no modification of MEGX levels (155 +/- 12 and 165 +/- 22 ng/mL, respectively). These results suggest that GSH and NAC might accelerate the lidocaine metabolism while counteracting the inhibitory effect of cimetidine.

Inhibition of the development of rat liver preneoplastic lesions by verapamil and dexverapamil.

The effect of verapamil and dexverapamil on the development of liver preneoplastic foci was investigated in male Sprague-Dawley rats initiated with N-nitrosodiethylamine (200 mg/kg i.p.), fed on a diet containing 0.01% 2-acetylaminofluorene and subjected to partial hepatectomy, according to the hepatocarcinogenesis model developed by Solt and Farber. Administration of drinking water containing 0.03% verapamil or dexverapamil resulted in a decrease in the incidence and size of gamma-glutamyl transpeptidase-positive foci. The chemopreventive effect of dexverapamil was more marked than that of verapamil. These findings support the hypothesis that these two calcium channel blockers act by reducing the resistance of initiated hepatocytes to the mitoinhibitory and cytotoxic effects of 2-acetylaminofluorene.

Scintillometric determination of unscheduled DNA synthesis in primary cultures of rat-liver cells. A hydroxylapatite batch assay.

A new procedure has been examined for measuring unscheduled DNA synthesis (UDS) in hepatocyte primary cultures by liquid-scintillation counting. DNA of the hepatocyte lysates was eluted with K-phosphate buffers after absorption on hydroxylapatite in order to reduce the background produced by cytoplasmic radioactivity. To inhibit hepatocyte replicative synthesis, hydroxyurea (10 mM) and cytosine arabinoside (80 microM) were added to the cultures. This procedure was found capable of detecting UDS elicited by 0.3 - 10 mM N-nitrosodimethylamine.

Cinnamaldehyde-induced micronuclei in rodent liver.

Cinnamaldehyde, a widely used flavoring agent, has so far been subjected to a limited range of genotoxicity tests, mainly carried out in vitro, which produced contradictory results. Therefore we have examined cinnamaldehyde using additional in vivo genotoxicity end-points. In Sprague-Dawley rats, a single oral dose equal to 1/2 LD50 did not induce DNA fragmentation in liver and gastric mucosa as evaluated by the alkaline elution technique, increased the frequency of micronucleated hepatocytes but not of bone marrow micronucleated polychromatic erythrocytes, and gave rise to a significantly higher incidence of total nuclear anomalies but not of micronucleated cells in forestomach mucosa. In Swiss mice, the equitoxic dose of cinnamaldehyde caused the same clastogenic effect in the liver, whilst a negative response was observed in both bone marrow and forestomach mucosa. Finally, in rats initiated with N-nitrosodiethylamine the administration of 500 mg/kg/day cinnamaldehyde for 14 successive days produced a modest but statistically significant increase of the average diameter and area of gamma-glutamyltranspeptidase-positive foci that, together with changes observed in other parameters, might be considered indicative of a potential promoting activity. Taken as a whole, these findings confirm that high doses of cinnamaldehyde may induce genetic alterations at the chromosomal level, and suggest that the liver is the preferential target of its undesirable effects.