Phthalate-Induced Liver Protection against Deleterious Effects of the Th1 Response: A Potentially Serious Health Hazard

Infection with Mycobacterium tuberculosis (TB) induces pulmonary immunopathology mediated by classical Th1 type of acquired immunity with hepatic involvement in up to 80% of disseminated cases. Since PPAR agonists cause immune responses characterized by a decrease in the secretion of Th1 cytokines, we investigated the impact of activating these receptors on hepatic pathology associated with a well-characterized model of Th1-type pulmonary response. Male Fischer 344 rats were either maintained on a drug-free diet (groups I and II), or a diet containing diethylhexylphthalate (DEHP), a compound transformed in vivo to metabolites known to activate PPARs, for 21 days (groups III and IV). Subsequently, animals were primed with Mycobacterium bovis purified protein derivative (PPD) in a Complete Freund's Adjuvant. Fifteen days later, animals in groups II and IV were challenged with Sepharose 4B beads covalently coupled with PPD, while animals in groups I and III received blank Sepharose beads. Animals with Th1 response (group II) showed a marked structural disruption in the hepatic lobule. Remarkably, these alterations were conspicuously absent in animals which received DEHP (group IV), despite noticeable accumulation of T cells in the periportal triads. Immunostaining and confocal microscopy revealed hepatic accumulation of IFNγ+ Th1 and IL-4+ Th2 cells in animals from groups II and IV, respectively. Our data suggest a PPARα-mediated suppression of the development of a Th1 immune response in the liver, resulting in hepatoprotective effect. However, potentially negative consequences of PPAR activation, such as decreased ability of the immune system to fight infection and interference with the efficacy of vaccines designed to evoke Th1 immune responses, remain to be investigated

Genomic Instability Profiles at the Single Cell Level in Mouse Colorectal Cancers of Defined Genotypes

The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with Apclox/lox, LSL-KrasG12D, and Tp53lox/lox targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation

Graft-versus-host disease in recipients of male unrelated donor compared with parous female sibling donor transplants

Optimal donor selection is critical for successful allogeneic hematopoietic cell transplantation (HCT). Donor sex and parity are well-established risk factors for graft-versus-host disease (GVHD), with male donors typically associated with lower rates of GVHD. Well-matched unrelated donors (URDs) have also been associated with increased risks of GVHD as compared with matched sibling donors. These observations raise the question of whether male URDs would lead to more (or less) favorable transplant outcomes as compared with parous female sibling donors. We used the Center for International Blood and Marrow Transplant Research registry to complete a retrospective cohort study in adults with acute myeloid leukemia, acute lymphoblastic leukemia, or myelodysplastic syndrome, who underwent T-cell replete HCT from these 2 donor types (parous female sibling or male URD) between 2000 and 2012. Primary outcomes included grade 2 to 4 acute GVHD (aGVHD), chronic GVHD (cGVHD), and overall survival. Secondary outcomes included disease-free survival, transplant-related mortality, and relapse. In 2813 recipients, patients receiving male URD transplants (n = 1921) had 1.6 times higher risk of grade 2 to 4 aGVHD (P \u3c .0001). For cGVHD, recipient sex was a significant factor, so donor/recipient pairs were evaluated. Female recipients of male URD grafts had a higher risk of cGVHD than those receiving parous female sibling grafts (relative risk [RR] = 1.43, P \u3c .0001), whereas male recipients had similar rates of cGVHD regardless of donor type (RR = 1.09, P = .23). Donor type did not significantly affect any other end point. We conclude that when available, parous female siblings are preferred over male URDs

Hematopoietic Cell Transplantation Outcomes in Monosomal Karyotype Myeloid Malignancies

The presence of monosomal karyotype (MK+) in acute myeloid leukemia (AML) is associated with dismal outcomes. We evaluated the impact of MK+ in AML (MK+AML, N=240) and in myelodysplastic syndrome (MK+MDS, N=221) on hematopoietic cell transplantation (HCT) outcomes compared to other cytogenetically defined groups (AML, N=3,360; MDS, N=1,373) as reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) from 1998 to 2011. MK+AML was associated with higher disease relapse (hazard ratio [HR] 1.98, p<0.01), similar transplant related mortality (TRM, HR 1.01, p=0.9) and worse survival (HR 1.67, p<0.01) compared to other cytogenetically defined AML. Among patients with MDS, MK+MDS was associated with higher disease relapse (HR 2.39, p<0.01), higher TRM (HR 1.80, p<0.01) and worse survival (HR 2.02, p<0.01). Subset analyses comparing chromosome 7 abnormalities (del7/7q) with or without MK+ demonstrated higher mortality for MK+ disease in for both AML (HR 1.72, p<0.01) and MDS (HR1.79, p<0.01). The strong negative impact of MK+ in myeloid malignancies was observed in all age groups and using either myeloablative or reduced intensity conditioning regimens. Alternative approaches to mitigate disease relapse in this population are needed

Significant Improvements in the Practice Patterns of Adult Related Donor Care in US Transplantation Centers

Recent investigations have found a higher incidence of adverse events associated with hematopoietic cell donation in related donors (RDs) who have morbidities that if present in an unrelated donor (UD) would preclude donation. In the UD setting, regulatory standards ensure independent assessment of donors, one of several crucial measures to safeguard donor health and safety. A survey conducted by the Center for International Blood and Marrow Transplant Research (CIBMTR) Donor Health and Safety Working Committee in 2007 reported a potential conflict of interest in >70% US centers, where physicians had simultaneous responsibility for RDs and their recipients. Consequently, several international organizations have endeavored to improve practice through regulations and consensus recommendations. We hypothesized that the changes in the 2012 FACT-JACIE Standards, resulting from the CIBMTR study, will have significantly impacted practice. Accordingly, a follow-up survey of US transplant centers was conducted to assess practice changes since 2007, and investigate additional areas where RD care was predicted to differ from UD care. 73 centers (53%), performing 79% of US RD transplants responded. Significant improvements were observed since the earlier survey; 62% centers now ensure separation of RD and recipient care (P<0.0001). However, this study identifies several areas where RD management does not meet international donor care standards. Particular concerns include counseling and assessment of donors before HLA typing, with 61% centers first disclosing donor HLA results to an individual other than the donor, the use of unlicensed mobilization agents, and the absence of long-term donor follow-up. Recommendations for improvement are described

Intestinal stem cells differentiation and its application in cancer therapy

Colorectal cancer (CRC) is one of the deadliest cancers worldwide. Despite extensive research in the field, new therapies targeting CRC are in urgent need. CRC is a highly heterogeneous disease with high levels of intra- and inter-patient heterogeneity. Cancer stem cells are the driving force of tumor development and metastasis. One of the approaches to treat CRC is differentiation therapy thereby induction of cancer stem cell differentiation may lead to tumor regression. Intestinal normal and cancer organoids represent an ideal model for studying the differentiation process ex vivo. Gene expression profiling of drug-treated organoids can be used for quantification of the differentiation process with high precision. RNA-seq based drug-screening platforms in organoids are not yet established. Here we developed a high-throughput high-content targeted RNA-seq-based platform (TORNADO-seq) for monitoring gene expression of large gene signatures in intestinal organoids which allowed us to quantify and evaluate complex cellular phenotypes and cellular alterations upon drug treatment. We applied TORNADO-seq in drug screening in wt and cancer intestinal organoids. We found many differentiation-inducing drugs in wt organoids, including some drugs specifically enriching for certain cell phenotypes, e.g. enteroendocrine cells. Further, we applied identified differentiation-inducing drugs to cancer organoids (APClof;KRASG12D;TP53lof) and discovered that the majority of those differentiation-inducing drugs also targeted cancer organoids. Differentiation induction seems to be a common mode of action of cancer-targeting drugs. Based on gene expression profiles of treated cancer organoids, we were able to propose and confirm the mechanism of action of several drugs indicating that cancer cells may to be sensitive to alterations in the cholesterol biosynthesis pathway. In general, utilization of TORNADO-seq allows predicting connections between cellular phenotypes and signaling pathways in organoids which can be used for hypothesis generation in a variety of biological systems. The second part of the thesis is devoted to the investigation of mechanisms underlying the accumulation of genetic alterations in CRC tumors. It is not known whether cancer driver genes affect the rate of mutation acquisition. Here we performed exome sequencing of single cell-derived organoids obtained from tumors with either APClof, APClof;KRASG12D or APClof;KRASG12D;TP53lof mutation phenotypes that reflect early stages of CRC development. We discovered that presence of KRAS and P53 mutations didn't affect the number of single nucleotide substitutions (SNSs) in tumors, while the latter was increased in APC mutated tumors compared to wt cells and was highly correlated with the tumor age. P53 inactivation was highly correlated with the presence of large-scale (>10 Mb) copy number alterations (CNAs)

High-content, targeted RNA-seq screening in organoids for drug discovery in colorectal cancer

Organoids allow the recapitulation of intestinal homeostasis and cancerogenesis in vitro; however, RNA sequencing (RNA-seq)-based methods for drug screens are missing. We develop targeted organoid sequencing (TORNADO-seq), a high-throughput, high-content drug discovery platform that uses targeted RNA-seq to monitor the expression of large gene signatures for the detailed evaluation of cellular phenotypes in organoids. TORNADO-seq is a fast, highly reproducible time- and cost-effective ($5 per sample) method that can probe cell mixtures and their differentiation state in the intestinal system. We apply this method to isolate drugs that enrich for differentiated cell phenotypes and show that these drugs are highly efficacious against cancer compared to wild-type organoids. Furthermore, TORNADO-seq facilitates in-depth insight into the mode of action of these drugs. Our technology can easily be adapted to many other systems and will allow for more systematic, large-scale, and quantitative approaches to study the biology of complex cellular systems