Mismatch epitope load predicts de novo-DSA-free survival in pediatric liver transplantation.

BACKGROUND: Our knowledge of de novo anti-HLA donor-specific antibodies (dnDSA) in liver transplantation continues to be defined. We hypothesized that differences of HLA-DR/DQ mismatches can improve precision in alloimmune risk categorization and be applied to tailor immunosuppression. METHODS: A retrospective chart review of 244 pediatric patients consecutively transplanted at our center between 2003 and 2019 was performed to identify patients tested for dnDSA. Records were queried for: demographics, pre-transplant diagnosis, biopsy-proven T-cell-mediated rejection (TCMR), radiology proven biliary complications, tacrolimus trough levels, dnDSA characteristics, and HLA typing. The eplet mismatch analyses were performed using HLAMatchmaker™ 3.1. All statistical analyses were conducted using R software version 3.40. RESULTS: There were 99 dnDSA-negative patients and 73 dnDSA-positive patients (n = 70 against class II and n = 3 against class I and II). ROC analysis identified optimal cutoff of eplet mismatch load for dnDSA and defined risk groups for an alloimmune outcome. Kaplan-Meier curves and log-rank tests showed high eplet mismatch load was associated with shorter dnDSA-free survival (log-rank p = .001). Multivariable Cox regression models showed that tacrolimus coefficient of variation and tacrolimus mean levels were significantly associated with dnDSA-free survival (p < .001 and p = .036). Fisher's exact test showed that dnDSA was associated with an increased likelihood of TCMR (OR 14.94; 95% CI 3.65 - 61.19; p < .001). Patients without TCMR were more likely to have dnDSA to HLA-DQ7 and less likely to have dnDSA to HLA-DQ2 (p = .03, p = .080). CONCLUSIONS: Mismatched epitope load predicts dnDSA-free survival in pediatric liver transplant, while dnDSA specificity may determine alloimmune outcome.

Operational tolerance in intestinal transplantation.

By presenting the first case report of true operational tolerance in an intestinal transplant patient, we aim to demonstrate that tolerance is possible in a field that has been hampered by suboptimal outcomes. Although operational tolerance has been achieved in liver and kidney transplantation, and some intestinal transplant patients have been able to decrease immunosuppression, this is the first instance of true operational tolerance after complete cessation of immunosuppression. A patient received a deceased-donor small intestinal and colon allograft with standard immunosuppressive treatment, achieving excellent graft function after overcoming a graft-versus-host-disease episode 5 months posttransplant. Four years later, against medical advice, the patient discontinued all immunosuppression. During follow-up visits 2 and 3 years after cessation of immunosuppression, the patient exhibited normal graft function with full enteral autonomy and without histological or endoscopic signs of rejection. Mechanistic analysis demonstrated immune competence against third party antigen, with in vitro evidence of donor-specific hyporesponsiveness in the absence of donor macrochimerism. This proof of principle case can stimulate future mechanistic studies on diagnostic and therapeutic strategies, for example, cellular therapy trials, that can lead to minimization or elimination of immunosuppression and, it is hoped, help revitalize the field of intestinal transplantation.

Guiding therapeutic plasma exchange for antibody-mediated rejection treatment in lung transplant recipients - a retrospective study.

Antibody-Mediated Rejection (AMR) due to donor-specific antibodies (DSA) is associated with poor outcomes after lung transplantation. Currently, there are no guidelines regarding the selection of treatment protocols. We studied how DSA characteristics including titers, C1q, and mean fluorescence intensity (MFI) values in undiluted and diluted sera may predict a response to therapeutic plasma exchange (TPE) and inform patient prognosis after treatment. Among 357 patients consecutively transplanted without detectable pre-existing DSAs between 01/01/16 and 12/31/18, 10 patients were treated with a standardized protocol of five TPE sessions with IVIG. Based on DSA characteristics after treatment, all patients were divided into three groups as responders, partial responders, and nonresponders. Kaplan-Meier Survival analyses showed a statistically significant difference in patient survival between those groups (P = 0.0104). Statistical analyses showed that MFI in pre-TPE 1:16 diluted sera was predictive of a response to standardized protocol (R2  = 0.9182) and patient survival (P = 0.0098). Patients predicted to be nonresponders who underwent treatment with a more aggressive protocol of eight TPE sessions with IVIG and bortezomib showed improvements in treatment response (P = 0.0074) and patient survival (P = 0.0253). Dilutions may guide clinicians as to which patients would be expected to respond to a standards protocol or require more aggressive treatment.

Assessing the utilization of high-resolution 2-field HLA typing in solid organ transplantation.

HLA typing in solid organ transplantation (SOT) is necessary for determining HLA-matching status between donor-recipient pairs and assessing patients' anti-HLA antibody profiles. Histocompatibility has traditionally been evaluated based on serologically defined HLA antigens. The evolution of HLA typing and antibody identification technologies, however, has revealed many limitations with using serologic equivalents for assessing compatibility in SOT. The significant improvements to HLA typing introduced by next-generation sequencing (NGS) require an assessment of the impact of this technology on SOT. We have assessed the role of high-resolution 2-field HLA typing (HR-2F) in SOT by retrospectively evaluating NGS-typed pre- and post-SOT cases. HR-2F typing was highly instructive or necessary in 41% (156/385) of the cases. Several pre- and posttransplant scenarios were identified as being better served by HR-2F typing. Five different categories are presented with specific case examples. The experience of another center (Temple University Hospital) is also included, whereby 21% of the cases required HR-2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti-HLA antibody issues.

Ytterbium increases transmembrane water transport in Zea mays roots via aquaporin modulation.

In our study, the rare earth element ytterbium (Yb3+) was demonstrated to affect water exchange in roots of Zea mays seedlings. Herewith, the overall membrane permeability (Pd) increased. The Pd increase was determined by aquaporin activity but not the membrane lipid component since the closure of aquaporin channels due to low intracellular pH abolished the positive effect of Yb3+ on Pd. Additionally, the expression level of aquaporin genes ZmPIP2;2, ZmPIP2;6 and ZmTIP2;2 was increased when plants were grown in the presence of Yb3+. Our results indicate that previously described positive influence of rare earth metals on plant growth and productivity may be mediated (at least partially) by the modification of the plant hydraulic system.

STAT3 suppresses transcription of proapoptotic genes in cancer cells with the involvement of its N-terminal domain.

Activation of STAT3 in cancers leads to gene expression promoting cell proliferation and resistance to apoptosis, as well as tumor angiogenesis, invasion, and migration. In the characterization of effects of ST3-H2A2, a selective inhibitor of the STAT3 N-terminal domain (ND), we observed that the compound induced apoptotic death in cancer cells associated with robust activation of proapoptotic genes. Using ChIP and tiling human promoter arrays, we found that activation of gene expression in response to ST3-H2A2 is accompanied by altered STAT3 chromatin binding. Using inhibitors of STAT3 phosphorylation and a dominant-negative STAT3 mutant, we found that the unphosphorylated form of STAT3 binds to regulatory regions of proapoptotic genes and prevents their expression in tumor cells but not normal cells. siRNA knockdown confirmed the effects of ST3-HA2A on gene expression and chromatin binding to be STAT3 dependent. The STAT3-binding region of the C/EBP-homologous protein (CHOP) promoter was found to be localized in DNaseI hypersensitive site of chromatin in cancer cells but not in nontransformed cells, suggesting that STAT3 binding and suppressive action can be chromatin structure dependent. These data demonstrate a suppressive role for the STAT3 ND in the regulation of proapoptotic gene expression in cancer cells, providing further support for targeting STAT3 ND for cancer therapy.

Mechanisms of unphosphorylated STAT3 transcription factor binding to DNA.

Phosphorylation of signal transducer and activator of transcription 3 (STAT3) on a single tyrosine residue in response to growth factors, cytokines, interferons, and oncogenes activates its dimerization, translocation to the nucleus, binding to the interferon γ (gamma)-activated sequence (GAS) DNA-binding site and activation of transcription of target genes. STAT3 is constitutively phosphorylated in various cancers and drives gene expression from GAS-containing promoters to promote tumorigenesis. Recently, roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in maintenance of heterochromatin stability. However, the mechanisms underlying U-STAT3 binding to DNA has not been fully investigated. Here, we explore STAT3-DNA interactions by atomic force microscopy (AFM) imaging. We observed that U-STAT3 molecules bind to the GAS DNA-binding site as dimers and monomers. In addition, we observed that U-STAT3 binds to AT-rich DNA sequence sites and recognizes specific DNA structures, such as 4-way junctions and DNA nodes, within negatively supercoiled plasmid DNA. These structures are important for chromatin organization and our data suggest a role for U-STAT3 as a chromatin/genome organizer. Unexpectedly, we found that a C-terminal truncated 67.5-kDa STAT3 isoform recognizes single-stranded spacers within cruciform structures that also have a role in chromatin organization and gene expression. This isoform appears to be abundant in the nuclei of cancer cells and, therefore, may have a role in regulation of gene expression. Taken together, our data highlight novel mechanisms by which U-STAT3 binds to DNA and supports U-STAT3 function as a transcriptional activator and a chromatin/genomic organizer.

ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.

We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

COVID-19 infection and vaccination rarely impact HLA antibody profile in waitlisted renal transplant candidates- a multicenter cohort.

Although rare, infection and vaccination can result in antibodies to human leukocyte antigens (HLA). We analyzed the effect of SARS-CoV-2 infection or vaccination on HLA antibodies in waitlisted renal transplant candidates. Specificities were collected and adjudicated if the calculated panel reactive antibodies (cPRA) changed after exposure. Of 409 patients, 285 (69.7 %) had an initial cPRA of 0 %, and 56 (13.7 %) had an initial cPRA > 80 %. The cPRA changed in 26 patients (6.4 %), 16 (3.9 %) increased, and 10 (2.4 %) decreased. Based on cPRA adjudication, cPRA differences generally resulted from a small number of specificities with subtle fluctuations around the borderline of the participating centers' cutoff for unacceptable antigen listing. All five COVID recovered patients with an increased cPRA were female (p = 0.02). In summary, exposure to this virus or vaccine does not increase HLA antibody specificities and their MFI in approximately 99 % of cases and 97 % of sensitized patients. These results have implications for virtual crossmatching at the time of organ offer after SARS-CoV-2 infection or vaccination, and these events of unclear clinical significance should not influence vaccination programs.

Current donor selection strategies for allogeneic hematopoietic cell transplantation.

Since the first allogeneic hematopoietic stem cell transplantation (HCT) was performed by Dr. E. Donnall Thomas in 1957, the field has advanced with new stem cell sources, immune suppressive regimens, and transplant protocols. Stem cells may be collected from bone marrow, peripheral or cord blood from an identical twin, a sibling, or a related or unrelated donor, which can be human leukocyte antigen (HLA) matched, mismatched, or haploidentical. Although HLA matching is one of the most important criteria for successful allogeneic HCT (allo-HCT) to minimize graft vs host disease (GVHD), prevent relapse, and improve overall survival, the novel immunosuppressive protocols for GVHD prophylaxis offered improved outcomes in haploidentical HCT (haplo-HCT), expanding donor availability for the majority of HCT candidates. These immunosuppressive protocols are currently being tested with the HLA-matched and mismatched donors to improve HCT outcomes further. In addition, fine-tuning the DPB1 mismatching and discovering the B leader genotype and mismatching may offer further optimization of donor selection and transplant outcomes. While the decision about a donor type largely depends on the patient's characteristics, disease status, and the transplant protocols utilized by an individual transplant center, there are general approaches to donor selection dictated by donor-recipient histocompatibility and the urgency for HCT. This review highlights recent advances in understanding critical factors in donor selection strategies for allo-HCT. It uses clinical vignettes to demonstrate the importance of making timely decisions for HCT candidates.

EGF-induced ERK-activation downstream of FAK requires rac1-NADPH oxidase.

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox-sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1-NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1-NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAK's ability to act as a scaffold for EGF-mediated signaling, including activation of ERK. Accordingly, we found that EGF-induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK-regulated S910-site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading.

Serum dilutions as a predictive biomarker for peri-operative desensitization: An exploratory approach to transplanting sensitized heart candidates.

Antibody-mediated rejection (AMR) of cardiac allografts mediated by anti-HLA Donor Specific Antibodies (DSA) is one of the major barriers to successful transplantation for the treatment of end-stage heart failure. Therapeutic plasma exchange (TPE) is a first-line treatment for pre-transplant desensitization. However, indications for treatment regimens and treatment end-points have not been well established. In this study, we investigated how sera dilutions could guide TPE regimens for effective peri-operative desensitization and early AMR treatment. Our data show that 1:16 dilutions of EDTA-treated sera and 1.5 volume TPE reduce anti-HLA class I and class II antibody levels in the same manner and, therefore, allows to predict which antibodies would respond to peri-operative TPE. We successfully applied this approach to transplanting three highly sensitized cardiac recipients (CPRA 85-93%) with peri-operative desensitization based on a virtual crossmatch performed on 1:16 diluted serum. Furthermore, we have used sera dilutions to guide DSA treatment post-transplant. Although these findings have to be confirmed in a larger prospective study, our data suggest that serum dilutions can serve as a predictive biomarker to guide peri-operative desensitization and post-transplant immunologic management.

Donor-Specific HLA Antibodies as Biomarkers of Transplant Rejection.

This article reviews the current evidence to classify donor-specific antibodies (DSAs) using Food and Drug Administration-National Institutes of Health Biomarkers, EndpointS, and other Tools (BEST) resource terms as diagnostic, prognostic, predictive, monitoring, and risk biomarkers for graft rejection. The emphasis is on DSA characteristics, including the DSA levels determined by mean fluorescence intensity and/or titers, the ability to activate a complement cascade (C1q, C3d, and C4d binding), and specific IgG subclasses to define distinct roles of DSAs as biomarkers in clinical practice. In addition, technical limitation of DSA testing is discussed.

Developmental diazinon neurotoxicity in rats: later effects on emotional response.

Developmental exposure to the organophosphorus pesticides chlorpyrifos and diazinon (DZN) alters serotonergic synaptic function at doses below the threshold for cholinesterase inhibition, however there are some indications that the two agents may differ in several important attributes. Previously, we found that low-dose chlorpyrifos exposure in neonatal rats causes lasting changes in emotional response and in the current study we did a comparable evaluation for DZN. Male and female Sprague-Dawley rat pups (N=10-12 of each sex per treatment group) were given 0, 0.5 or 2 mg/(kg day) of DZN s.c. daily on postnatal days (PND) 1-4. These doses bracket the threshold for barely-detectable cholinesterase inhibition. Starting on PND 52, these rats began a battery of tests to assess emotional reactivity. In the elevated plus maze, there was a slight decrease in the time spent in the open arms for DZN-exposed males, while DZN-exposed females were not different from control females. In the novelty-suppressed feeding test, DZN-exposed males had significantly shorter latencies to begin eating than did control males, reducing the values to those normally seen in females. DZN-exposed rats of either sex showed reduced preference for chocolate milk in the anhedonia test that compared the consumption of chocolate milk to water. These findings show that neonatal exposures to DZN at a dose range below the threshold for cholinesterase inhibition nevertheless evokes specific, later alterations in emotional behaviors, particularly in males. The effects show not only some similarities to those of chlorpyrifos but also some differences, in keeping with neurochemical findings comparing the two agents.

Effects of o-aminoazotoluene on liver regeneration and p53 activation in mice susceptible and resistant to hepatocarcinogenesis.

The susceptibility to hepatocellular carcinoma (HCC) varies greatly within human populations in response to environmental risk agents. The mechanisms underlying differential susceptibility are still largely unknown and need to be clarified to improve HCC chemoprevention and therapeutic treatment. Inbred rodent strains with established predispositions for hepatocarcinogenesis offer the opportunity to identify intrinsic susceptibility and resistance factors. Previously, we have characterized mouse strains showing differential susceptibility to o-aminoazotoluene (OAT) and established that susceptibility does not result from OAT metabolism or genotoxicity in the livers of resistant and susceptible mice. In this study we have found that OAT differently affects hepatocyte proliferation in mice after partial hepatectomy (PH). OAT inhibited hepatocyte proliferation by 60-80% in the livers of susceptible mice, whereas resistant mice showed less than 15% inhibition. The inhibition resulted in significant delay of hepatic mass recovery in susceptible mice. OAT induced p53 stabilization and transcriptional activation in response to carcinogen treatment to the same degree in both, susceptible and resistant mice. Taken together, our data support inhibition of hepatocyte proliferation as a major cause for increased mouse susceptibility to hepatocarcinogenesis, and acceleration of functional liver recovery may offer a way to increase resistance to hepatic neoplasms. These results may have relevance to clinical observations of HCCs and implications for HCC chemoprevention and treatment.

Idazoxan blocks the nicotine-induced reversal of the memory impairment caused by the NMDA glutamate receptor antagonist dizocilpine.

RATIONALE: Alpha2-adrenoreceptor (alpha(2)-AR) antagonists have been shown to improve, while alpha(2)-AR agonists impair cognitive function in subjects with functioning NMDA receptors (NMDAR). In subjects with inhibited NMDAR (a model of schizophrenia) alpha(2)-AR agonists attenuate the cognitive impairments. The effect with alpha(2)-AR antagonists remains unclear. OBJECTIVES: We investigated the effects of the alpha(2)-AR antagonist idazoxan on memory function in rats treated/not treated with NMDAR antagonist dizocilpine or a combination of dizocilpine and nicotine to clarify noradrenergic/cholinergic regulation of memory function. METHODS: Female Sprague-Dawley rats (n=12) were trained for food reward on the radial maze. Working and reference memory errors and response latency were assessed after injections of idazoxan (0.5, 1.0 mg/kg), dizocilpine (0.05 mg/kg), nicotine (0.2, 0.4 mg/kg) or vehicle, alone or in combination. RESULTS: Dizocilpine potently impaired memory. Nicotine (0.4 mg/kg) reversed this impairment. Idazoxan at the doses tested did not affect performance when given alone or with dizocilpine, but it did block the nicotine reversal of the dizocilpine-induced memory impairment. Three rats after 10-12 drug treatments developed limbic seizures. Our findings suggest that combination of drugs which block alpha(2)-AR with nicotinic agonists in schizophrenia may prevent therapeutic effect of nicotinic agonists and increase risk for convulsive activity with repeated administration.

Persistent cognitive alterations in rats after early postnatal exposure to low doses of the organophosphate pesticide, diazinon.

BACKGROUND: Developmental neurotoxicity of organophosphorous insecticides (OPs) involves multiple mechanisms in addition to cholinesterase inhibition. We have found persisting effects of developmental chlorpyrifos (CPF) and diazinon (DZN) on cholinergic and serotonergic neurotransmitter systems and gene expression as well as behavioral function. Both molecular/neurochemical and behavioral effects of developmental OP exposure have been seen at doses below those which cause appreciable cholinesterase inhibition. OBJECTIVES: We sought to determine if developmental DZN exposure at doses which do not produce significant acetylcholinesterase inhibition cause persisting cognitive deficits. METHODS: Rats were exposed to DZN on postnatal days 1-4 at doses (0.5 and 2 mg/kg/d) that span the threshold for cholinesterase inhibition. They were later examined with a cognitive battery tests similar to that used with CPF. RESULTS: In the T-maze DZN caused significant hyperactivity in the initial trials of the session, but not later. In a longer assessment of locomotor activity no DZN-induced changes were seen over a 1-hour session. Prepulse inhibition was reduced by DZN exposure selectively in males vs. females; DZN eliminated the sex difference present in controls. In the radial maze, the lower but not higher DZN dose significantly impaired spatial learning. This type of nonmonotonic dose-effect function has previously been seen with CPF as well. The lower dose DZN group also showed significantly greater sensitivity to the memory-impairing effects of scopolamine a muscarinic acetylcholine antagonist. CONCLUSIONS: Neonatal DZN exposure below the threshold for appreciable cholinesterase inhibition caused persisting neurocognitive deficits in adulthood. The addition of some inhibition of AChE with a higher dose reversed the cognitive impairment. This non-monotonic dose-effect function has also been seen with neurochemical effects. Some of the DZN effects on cognition resemble those seen earlier for CPF, some differ. Our data suggest that DZN and CPF affect transmitter systems supporting memory function, differently, implying participation of mechanisms other than their common inhibition of cholinesterase.

The role of ascorbate in the modulation of HIF-1alpha protein and HIF-dependent transcription by chromium(VI) and nickel(II).

Molecular oxygen is involved in hydroxylation and subsequent degradation of HIF-1alpha, a subunit of HIF-1 transcription factor; therefore oxygen shortage (hypoxia) stabilizes this protein. However, HIF-1alpha can also be stabilized by transition metal ions in the presence of oxygen, suggesting that a different mechanism is involved in metal-induced hypoxic stress. Recently, we showed that the depletion of intracellular ascorbate by metals may lead to the inhibition of hydroxylases. Because nickel(II) has similarity to iron(II), an alternative hypothesis suggests that iron substitution for nickel in the enzyme inhibits hydroxylase activity. Here we investigated the induction of HIF-1 by another metal, chromium, which cannot replace iron in the enzyme. We show that chromium(VI), but not chromium(III), can oxidize ascorbate both in cells and in a cell-free system. In agreement with these data chromium(VI) stabilizes HIF-1alpha protein in cells only until it is reduced to chromium(III). In contrast, nickel(II) was found to be a catalyst, which facilitated continuous oxidation of ascorbate by ambient oxygen. These data correlate with extended stabilization of HIF-1alpha after acute exposure to nickel(II). The HIF-1-dependent reporter assays revealed that 20-24 h was required to fully develop the HIF-1 transcriptional response, and the acute exposure to nickel(II), but not chromium(VI), meets this requirement. However, repeated (chronic) exposure to chromium(VI) can also lead to extended stabilization of HIF-1alpha. Thus, the obtained data emphasize the important role of ascorbate in regulation of HIF-1 transcriptional activity in metal-exposed human lung cells.

Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer.

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer.

o-Aminoazotoluene does induce the enzymes of its own mutagenic activation in mouse liver.

The objective of this study was to investigate the CYP1A1 and CYP1A2 mRNAs and enzyme activities in mouse liver during induction with o-aminoazotoluene (OAT) as well as the capability of the hepatic S9-fraction from OAT-treated mice to induce its own activation to mutagens in the Ames test using S. typhymurium strain TA98. The data obtained indicate that when used at appropriate doses, OAT is a PAH-type inducer of mouse hepatic microsomal monooxygenases, which activity is not less than that of the known inducer 3,4-benzo[alpha]pyrene. In the absence of S9-fraction enzymes no OAT-mediated mutagenicity was observed in the Ames test. In the presence of the S9-fraction from OAT-pretreated mice, OAT induced as high revertant numbers, as it did in the presence of the S9 fraction from the liver of Aroclor 1254-treated mice. Thus, OAT does induce the enzymes of its own mutagenic activation in mouse liver.