Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence

Anti-tumor CD8⁺ T cells are a key determinant for overall survival in patients following surgical resection for solid malignancies. Using a mouse model of cancer vaccination (adenovirus expressing melanoma tumor-associated antigen (TAA)—dopachrome tautomerase (AdDCT) and resection resulting in major surgical stress (abdominal nephrectomy), we demonstrate that surgical stress results in a reduction in the number of CD8⁺ T cell that produce cytokines (IFNγ, TNFα, Granzyme B) in response to TAA. This effect is secondary to both reduced proliferation and impaired T cell function following antigen binding. In a prophylactic model, surgical stress completely abrogates tumor protection conferred by vaccination in the immediate postoperative period. In a clinically relevant surgical resection model, vaccinated mice undergoing a positive margin resection with surgical stress had decreased survival compared to mice with positive margin resection alone. Preoperative immunotherapy with IFNα significantly extends survival in surgically stressed mice. Importantly, myeloid derived suppressor cell (MDSC) population numbers and functional impairment of TAA-specific CD8⁺ T cell were altered in surgically stressed mice. Our observations suggest that cancer progression may result from surgery-induced suppression of tumor-specific CD8⁺ T cells. Preoperative immunotherapies aimed at targeting the prometastatic effects of cancer surgery will reduce recurrence and improve survival in cancer surgery patients.     

Using comparative genomics to reorder the human genome sequence into a virtual sheep genome

Background

Is it possible to construct an accurate and detailed subgene-level map of a genome using bacterial artificial chromosome (BAC) end sequences, a sparse marker map, and the sequences of other genomes?

Results

A sheep BAC library, CHORI-243, was constructed and the BAC end sequences were determined and mapped with high sensitivity and low specificity onto the frameworks of the human, dog, and cow genomes. To maximize genome coverage, the coordinates of all BAC end sequence hits to the cow and dog genomes were also converted to the equivalent human genome coordinates. The 84,624 sheep BACs (about 5.4-fold genome coverage) with paired ends in the correct orientation (tail-to-tail) and spacing, combined with information from sheep BAC comparative genome contigs (CGCs) built separately on the dog and cow genomes, were used to construct 1,172 sheep BAC-CGCs, covering 91.2% of the human genome. Clustered non-tail-to-tail and outsize BACs located close to the ends of many BAC-CGCs linked BAC-CGCs covering about 70% of the genome to at least one other BAC-CGC on the same chromosome. Using the BAC-CGCs, the intrachromosomal and interchromosomal BAC-CGC linkage information, human/cow and vertebrate synteny, and the sheep marker map, a virtual sheep genome was constructed. To identify BACs potentially located in gaps between BAC-CGCs, an additional set of 55,668 sheep BACs were positioned on the sheep genome with lower confidence. A coordinate conversion process allowed us to transfer human genes and other genome features to the virtual sheep genome to display on a sheep genome browser.

Conclusion

We demonstrate that limited sequencing of BACs combined with positioning on a well assembled genome and integrating locations from other less well assembled genomes can yield extensive, detailed subgene-level maps of mammalian genomes, for which genomic resources are currently limited.     

Effect of chronic ethanol exposure and its withdrawal on the endocannabinoid system

The present study investigated the effect of ethanol (EtOH) exposure and its withdrawal on the central endocannabinoid system utilizing an EtOH vapor inhalation model, which is known to produce functional tolerance and dependence to EtOH. Swiss Webster mice (n=24) were exposed to EtOH vapors for 72h. Mice were sacrificed after 72h following EtOH exposure (n=12) and 24h after its withdrawal (n=12). Radioligand binding assays were performed to measure the density of CB(1) receptor and CB(1) receptor agonist-stimulated [(35)S]GTPgammaS binding in crude synaptic membranes isolated from the cortex, hippocampus, striatum and cerebellum. The density of CB(1) receptor was significantly decreased (31-39%) in all the brain regions when compared to the control group. The CB(1) receptor-stimulated G(i/o) protein activation was also found to be decreased (29-40%) in these brain regions of EtOH exposed mice. Recovery of the CB(1) receptor density, in addition to, the CB(1) receptor-mediated G-protein activation was observed after 24h withdrawal from EtOH. The levels of cortical anandamide, which was significantly increased (147%) by EtOH exposure, returned to basal levels after 24h of withdrawal from EtOH exposure. A significant reduction (21%) in the activity of fatty acid amide hydrolase was found in the cortex of EtOH administered mice. Taken together, the neuroadaptation in the EC system may have a potential role in development of tolerance and dependence to EtOH.     

Identification of genomic regions associated with phenotypic variation between dog breeds using selection mapping

The extraordinary phenotypic diversity of dog breeds has been sculpted by a unique population history accompanied by selection for novel and desirable traits. Here we perform a comprehensive analysis using multiple test statistics to identify regions under selection in 509 dogs from 46 diverse breeds using a newly developed high-density genotyping array consisting of >170,000 evenly spaced SNPs. We first identify 44 genomic regions exhibiting extreme differentiation across multiple breeds. Genetic variation in these regions correlates with variation in several phenotypic traits that vary between breeds, and we identify novel associations with both morphological and behavioral traits. We next scan the genome for signatures of selective sweeps in single breeds, characterized by long regions of reduced heterozygosity and fixation of extended haplotypes. These scans identify hundreds of regions, including 22 blocks of homozygosity longer than one megabase in certain breeds. Candidate selection loci are strongly enriched for developmental genes. We chose one highly differentiated region, associated with body size and ear morphology, and characterized it using high-throughput sequencing to provide a list of variants that may directly affect these traits. This study provides a catalogue of genomic regions showing extreme reduction in genetic variation or population differentiation in dogs, including many linked to phenotypic variation. The many blocks of reduced haplotype diversity observed across the genome in dog breeds are the result of both selection and genetic drift, but extended blocks of homozygosity on a megabase scale appear to be best explained by selection. Further elucidation of the variants under selection will help to uncover the genetic basis of complex traits and disease.     

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.     

Regulation of R7 and R8 differentiation by the spalt genes

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt and senseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression.     

RSC functions as an early double-strand-break sensor in the cell's response to DNA damage

The detection of a DNA double-strand break (DSB) is necessary to initiate DSB repair. Several proteins, including the MRX/N complex, Tel1/ATM (ataxia telangiectasia mutated), and Mec1/ATR (ATM and Rad3 related), have been proposed as sensors of DNA damage, yet how they recognize the breaks is poorly understood. DSBs occur in the context of chromatin, implicating factors capable of altering local and/or global chromatin structure in the cellular response to DNA damage, including DSB sensing. Emerging evidence indicates that ATP-dependent chromatin-remodeling complexes function in DNA repair. Here we describe an important and novel early role for the RSC ATP-dependent chromatin remodeler linked to DSB sensing in the cell's DNA-damage response. RSC is required for full levels of H2A phosphorylation because it facilitates the recruitment of Tel1/ATM and Mec1/ATR to the break site. Consistent with these results, we also show that Rsc2 is needed for efficient activation of the Rad53-dependent checkpoint, as well as for Cohesin's association with the break site. Finally, Rsc2 is needed for the DNA-damage-induced changes in nucleosome structure surrounding the DSB site. Together, these new findings functionally link RSC to DSB sensing, highlighting the importance of ATP-dependent chromatin-remodeling factors in the cell's early response to DNA damage.