Mitotic Figures-Normal, Atypical, and Imposters: A Guide to Identification.

Counting mitotic figures (MF) in hematoxylin and eosin-stained histologic sections is an integral part of the diagnostic pathologist's tumor evaluation. The mitotic count (MC) is used alone or as part of a grading scheme for assessment of prognosis and clinical decisions. Determining MCs is subjective, somewhat laborious, and has interobserver variation. Proposals for standardizing this parameter in the veterinary field are limited to terminology (use of the term MC) and area (MC is counted in an area measuring 2.37 mm2). Digital imaging techniques are now commonplace and widely used among veterinary pathologists, and field of view area can be easily calculated with digital imaging software. In addition to standardizing the methods of counting MF, the morphologic characteristics of MF and distinguishing atypical mitotic figures (AMF) versus mitotic-like figures (MLF) need to be defined. This article provides morphologic criteria for MF identification and for distinguishing normal phases of MF from AMF and MLF. Pertinent features of digital microscopy and application of computational pathology (CPATH) methods are discussed. Correct identification of MF will improve MC consistency, reproducibility, and accuracy obtained from manual (glass slide or whole-slide imaging) and CPATH approaches.

International Guidelines for Veterinary Tumor Pathology: A Call to Action.

Standardization of tumor assessment lays the foundation for validation of grading systems, permits reproducibility of oncologic studies among investigators, and increases confidence in the significance of study results. Currently, there is minimal methodological standardization for assessing tumors in veterinary medicine, with few attempts to validate published protocols and grading schemes. The current article attempts to address these shortcomings by providing standard guidelines for tumor assessment parameters and protocols for evaluating specific tumor types. More detailed information is available in the Supplemental Files, the intention of which is 2-fold: publication as part of this commentary, but more importantly, these will be available as "living documents" on a website (www.vetcancerprotocols.org), which will be updated as new information is presented in the peer-reviewed literature. Our hope is that veterinary pathologists will agree that this initiative is needed, and will contribute to and utilize this information for routine diagnostic work and oncologic studies. Journal editors and reviewers can utilize checklists to ensure publications include sufficient detail and standardized methods of tumor assessment. To maintain the relevance of the guidelines and protocols, it is critical that the information is periodically updated and revised as new studies are published and validated with the intent of providing a repository of this information. Our hope is that this initiative (a continuation of efforts published in this journal in 2011) will facilitate collaboration and reproducibility between pathologists and institutions, increase case numbers, and strengthen clinical research findings, thus ensuring continued progress in veterinary oncologic pathology and improving patient care.

Third-party CD4+ invariant natural killer T cells protect from murine GVHD lethality.

Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third-party CD4(+) iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4(+) iNKT cells from third-party mice resulted in a significant survival benefit with retained graft-versus-tumor effects. In vivo expansion of alloreactive T cells was diminished while displaying a T helper cell 2-biased phenotype. Notably, CD4(+) iNKT cells from third-party mice were as protective as CD4(+) iNKT cells from donor mice although third-party CD4(+) iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third-party CD4(+) iNKT cells resulted in a robust expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third-party availability and feasibility of in vitro expansion provide the basis for clinical translation.

Crosstalking between androgen and PI3K/AKT signaling pathways in prostate cancer cells.

Both androgen action and PI3K medicated signaling pathways have been implicated in prostate tumorigenesis. Our androgen receptor (AR) conditional transgenic mice developed murine prostatic intraepithelial neoplasia (mPIN) and prostatic adenocarcinoma lesions recapitulating human prostate cancer development and progression. Role of transgenic AR contributing to malignancy was demonstrated by high degree of transgenic AR expression in atypical and tumor cells in mPIN as well as prostatic adenocarcinoma lesions of the transgenic mice, but not in adjacent normal tissue. Interestingly, reduced PI3K/Akt activation also appeared in these mouse atypical and tumor cells, suggesting an interaction between androgen and PI3K/AKT pathways. In this study, we further investigated this interaction. We showed that the androgen depletion or knockdown of AR expression results in elevated levels of active phosphorylated AKT in prostate cancer cells. Castration of conditional Pten knock-out mice showed increased Akt, phosphorylated Akt, and pS6 expression in the mouse prostate. Using a series of newly generated Ar reporter and Pten knock-out compound mice, we showed that Pten loss directly represses endogenous Ar expression in prostatic epithelial cells. Moreover, Pten loss and PI3K/Akt activation reduced Ar-mediated transcription in purified Pten-null cells. This study provides novel evidence demonstrating interplay between androgen and PI3K pathways, as well as introduces unique and relevant mouse models for further studies of PI3K and AR pathways in the context of prostate tumorigenesis.

CD4+ invariant natural killer T cells protect from murine GVHD lethality through expansion of donor CD4+CD25+FoxP3+ regulatory T cells.

Dysregulated donor T cells lead to destruction of host tissues resulting in graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). We investigated the impact of highly purified (>95%) donor CD4(+) invariant natural killer T (iNKT) cells on GVHD in a murine model of allogeneic HCT. We found that low doses of adoptively transferred donor CD4(+) iNKT cells protect from GVHD morbidity and mortality through an expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). These Tregs express high levels of the Ikaros transcription factor Helios and expand from the Treg pool of the donor graft. Furthermore, CD4(+) iNKT cells preserve T-cell-mediated graft-versus-tumor effects. Our studies reveal new aspects of the cellular interplay between iNKT cells and Tregs in the context of tolerance induction after allogeneic HCT and set the stage for clinical translation.

Wnt/ß-Catenin-Responsive Cells in Prostatic Development and Regeneration.

The precise role of Wnt/ß-catenin signaling during prostatic development and tumorigenesis is unclear. Axin2 is a direct transcriptional target of ß-catenin. Recent studies have shown that Axin2-expressing cells have stem/progenitor cell properties in a variety of mouse tissues. Here, we genetically labeled Axin2-expressing cells at various time points and tracked their cellular behavior at different developmental and mature stages. We found that prostatic Axin2-expressing cells mainly express luminal epithelial cell markers and are able to expand luminal cell lineages during prostatic development and maturation. They can also survive androgen withdrawal and regenerate prostatic luminal epithelial cells following androgen replacement. Deletion of ß-catenin or expression of stabilized ß-catenin in these Axin2-expressing cells results in abnormal development or oncogenic transformation, respectively. Our study uncovers a critical role of Wnt/ß-catenin-responsive cells in prostatic development and regeneration, and that dysregulation of Wnt/ß-catenin signaling in these cells contributes to prostatic developmental defects and tumorigenesis.

Transient CNS responses to repeated binge ethanol treatment.

The effects of ethanol (EtOH) on in vivo magnetic resonance (MR)-detectable brain measures across repeated exposures have not previously been reported. Of 28 rats weighing 340.66 ± 21.93 g at baseline, 15 were assigned to an EtOH group and 13 to a control group. Animals were exposed to five cycles of 4 days of intragastric (EtOH or dextrose) treatment and 10 days of recovery. Rats in both groups had structural MR imaging and whole-brain MR spectroscopy (MRS) scans at baseline, immediately following each binge period and after each recovery period (total = 11 scans per rat). Blood alcohol level at each of the five binge periods was ~300 mg/dl. Blood drawn at the end of the experiment did not show group differences for thiamine or its phosphate derivatives. Postmortem liver histopathology provided no evidence for hepatic steatosis, alcoholic hepatitis or alcoholic cirrhosis. Cerebrospinal fluid volumes of the lateral ventricles and cisterns showed enlargement with each binge EtOH exposure but recovery with each abstinence period. Similarly, changes in MRS metabolite levels were transient: levels of N-acetylaspartate and total creatine decreased, while those of choline-containing compounds and the combined resonance from glutamate and glutamine increased with each binge EtOH exposure cycle and then recovered during each abstinence period. Changes in response to EtOH were in expected directions based on previous single-binge EtOH exposure experiments, but the current MR findings do not provide support for accruing changes with repeated binge EtOH exposure.

Quantitative Assessment of Inflammation in a Porcine Acute Terminal Ileitis Model: US with a Molecularly Targeted Contrast Agent.

PURPOSE: To evaluate the feasibility and reproducibility of ultrasonography (US) performed with dual-selectin-targeted contrast agent microbubbles (MBs) for assessment of inflammation in a porcine acute terminal ileitis model, with histologic findings as a reference standard. MATERIALS AND METHODS: The study had institutional Animal Care and Use Committee approval. Acute terminal ileitis was established in 19 pigs; four pigs served as control pigs. The ileum was imaged with clinical-grade dual P- and E-selectin-targeted MBs (MBSelectin) at increasing doses (0.5, 1.0, 2.5, 5.0, 10, and 20 × 10(8) MB per kilogram of body weight) and with control nontargeted MBs (MBControl). For reproducibility testing, examinations were repeated twice after the MBSelectin and MBControl injections. After imaging, scanned ileal segments were analyzed ex vivo both for inflammation grade (by using hematoxylin-eosin staining) and for expression of selectins (by using quantitative immunofluorescence analysis). Statistical analysis was performed by using the t test, intraclass correlation coefficients (ICCs), and Spearman correlation analysis. RESULTS: Imaging signal increased linearly (P < .001) between a dose of 0.5 and a dose of 5.0 × 10(8) MB/kg and plateaued between a dose of 10 and a dose of 20 × 10(8) MB/kg. Imaging signals were reproducible (ICC = 0.70), and administration of MBSelectin in acute ileitis resulted in a significantly higher (P < .001) imaging signal compared with that in control ileum and MBControl. Ex vivo histologic grades of inflammation correlated well with in vivo US signal (ρ = 0.79), and expression levels of both P-selectin (37.4% ± 14.7 [standard deviation] of vessels positive; P < .001) and E-selectin (31.2% ± 25.7) in vessels in the bowel wall of segments with ileitis were higher than in control ileum (5.1% ± 3.7 for P-selectin and 4.8% ± 2.3 for E-selectin). CONCLUSION: Quantitative measurements of inflammation obtained by using dual-selectin-targeted US are reproducible and correlate well with the extent of inflammation at histologic examination in a porcine acute ileitis model as a next step toward clinical translation.

Assessing inflammatory liver injury in an acute CCl4 model using dynamic 3D metabolic imaging of hyperpolarized [1-(13)C]pyruvate.

To facilitate diagnosis and staging of liver disease, sensitive and non-invasive methods for the measurement of liver metabolism are needed. This study used hyperpolarized (13)C-pyruvate to assess metabolic parameters in a CCl4 model of liver damage in rats. Dynamic 3D (13)C chemical shift imaging data from a volume covering kidney and liver were acquired from 8 control and 10 CCl4-treated rats. At 12 time points at 5 s temporal resolution, we quantified the signal intensities and established time courses for pyruvate, alanine, and lactate. These measurements were compared with standard liver histology and an alanine transaminase (ALT) enzyme assay using liver tissue from the same animals. All CCl4-treated but none of the control animals showed histological liver damage and elevated ALT enzyme levels. In agreement with these results, metabolic imaging revealed an increased alanine/pyruvate ratio in liver of CCl4-treated rats, which is indicative of elevated ALT activity. Similarly, lactate/pyruvate ratios were higher in CCl4-treated compared with control animals, demonstrating the presence of inflammation. No significant differences in metabolite ratios were observed in kidney or vasculature. Thus this work shows that metabolic imaging using (13)C-pyruvate can be a successful tool to non-invasively assess liver damage in vivo.

Modulating the strength and threshold of NOTCH oncogenic signals by mir-181a-1/b-1.

Oncogenes, which are essential for tumor initiation, development, and maintenance, are valuable targets for cancer therapy. However, it remains a challenge to effectively inhibit oncogene activity by targeting their downstream pathways without causing significant toxicity to normal tissues. Here we show that deletion of mir-181a-1/b-1 expression inhibits the development of Notch1 oncogene-induced T cell acute lymphoblastic leukemia (T-ALL). mir-181a-1/b-1 controls the strength and threshold of Notch activity in tumorigenesis in part by dampening multiple negative feedback regulators downstream of NOTCH and pre-T cell receptor (TCR) signaling pathways. Importantly, although Notch oncogenes utilize normal thymic progenitor cell genetic programs for tumor transformation, comparative analyses of mir-181a-1/b-1 function in normal thymocyte and tumor development demonstrate that mir-181a-1/b-1 can be specifically targeted to inhibit tumor development with little toxicity to normal development. Finally, we demonstrate that mir-181a-1/b-1, but not mir-181a-2b-2 and mir-181-c/d, controls the development of normal thymic T cells and leukemia cells. Together, these results illustrate that NOTCH oncogene activity in tumor development can be selectively inhibited by targeting the molecular networks controlled by mir-181a-1/b-1.

Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis.

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with Kras(G12D) to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

Deletion of leucine zipper tumor suppressor 2 (Lzts2) increases susceptibility to tumor development.

Using an Lzts2 knock-out mouse model, we characterized the biological role of Lzts2 in tumorigenesis. Both heterozygous and homozygous deletion of the Lzts2-targeted allele in mice shows an increased incidence in spontaneous tumor development, although Lzts2 homozygous knock-out mice show significantly higher incidences than heterozygous mice. Treatment of Lzts2-deficient mice with a carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine, increases the susceptibility to N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder carcinoma development. Examination of human prostate cancer tissue specimens shows a reduction of LZTS2 protein expression in prostate cancer cells. Further analyses of mouse embryonic fibroblasts isolated from Lzts2 knock-out embryos show that loss of Lzts2 enhances cell growth. These data provide the first line of evidence demonstrating that deletion of Lzts2 increases susceptibility to spontaneous and carcinogen-induced tumor development.

Molecular imaging of inflammation in inflammatory bowel disease with a clinically translatable dual-selectin-targeted US contrast agent: comparison with FDG PET/CT in a mouse model.

PURPOSE: To develop and test a molecular imaging approach that uses ultrasonography (US) and a clinically translatable dual-targeted (P- and E-selectin) contrast agent (MBSelectin) in the quantification of inflammation at the molecular level and to quantitatively correlate selectin-targeted US with fluorodeoxyglucose (FDG) combined positron emission tomography (PET) and computed tomography (CT) in terms of visualization and quantification of different levels of inflammation in a murine acute colitis model. MATERIALS AND METHODS: Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. MBSelectin was developed by covalently binding an analog of the naturally occurring binding ligand P-selectin glycoprotein ligand 1 fused to a human fragment crystallizable(or Fc) domain onto the lipid shell of perfluorobutane and nitrogen-containing MBs. Binding specificity of MBSelectin was assessed in vitro with a flow chamber assay and in vivo with a chemically induced acute colitis murine model. US signal was quantitatively correlated with FDG uptake at PET/CT and histologic grade. Statistical analysis was performed with the Student t test, analysis of variance, and Pearson correlation analysis. RESULTS: MBSelectin showed strong attachment to both human and mouse P- and E-selectin compared with MBControl in vitro (P ≤ .002). In vivo, US signal was significantly increased (P < .001) in mice with acute colitis (173.8 arbitrary units [au] ± 134.8 [standard deviation]) compared with control mice (5.0 au ± 4.5). US imaging signal strongly correlated with FDG uptake on PET/CT images (ρ = 0.89, P < .001). Ex vivo analysis enabled confirmation of inflammation in mice with acute colitis and high expression levels of P- and E-selectin in mucosal capillaries (P = .014). CONCLUSION: US with MBSelectin specifically enables detection and quantification of inflammation in a murine acute colitis model, leveraging the natural pathway of leukocyte recruitment in inflammatory tissue. US imaging with MBSelectin correlates well with FDG uptake at PET/CT imaging.

Low doses of natural killer T cells provide protection from acute graft-versus-host disease via an IL-4-dependent mechanism.

CD4(+) natural killer T (NKT) cells, along with CD4(+)CD25(+) regulatory T cells (Tregs), are capable of controlling aberrant immune reactions. We explored the adoptive transfer of highly purified (> 95%) CD4(+)NKT cells in a murine model of allogeneic hematopoietic cell transplantation (HCT). NKT cells follow a migration and proliferation pattern similar to that of conventional T cells (Tcons), migrating initially to secondary lymphoid organs followed by infiltration of graft-versus-host disease (GVHD) target tissues. NKT cells persist for more than 100 days and do not cause significant morbidity or mortality. Doses of NKT cells as low as 1.0 × 10(4) cells suppress GVHD caused by 5.0 × 10(5) Tcons in an interleukin-4 (IL-4)-dependent mechanism. Protective doses of NKT cells minimally affect Tcon proliferation, but cause significant reductions in interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production by donor Tcons and in skin, spleen, and gastrointestinal pathology. In addition, NKT cells do not impact the graft-versus-tumor (GVT) effect of Tcons against B-cell lymphoma-1 (BCL-1) tumors. These studies elucidate the biologic function of donor-type CD4(+)NKT cells in suppressing GVHD in an allogeneic transplantation setting, demonstrating clinical potential in reducing GVHD in HCT.

A novel clinically translatable fluorescent nanoparticle for targeted molecular imaging of tumors in living subjects.

The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QD as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance, and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding nontumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD(2) to integrin α(v)ß(3)-positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD-modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron-based nanoprobes in the clinical setting in the near future.

Conditional expression of the androgen receptor induces oncogenic transformation of the mouse prostate.

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.

The leucine zipper putative tumor suppressor 2 protein LZTS2 regulates kidney development.

Members of the leucine zipper putative tumor suppressor (LZTS) family play crucial roles in transcription modulation and cell cycle control. We previously demonstrated that LZTS2 functions as a novel ß-catenin-interacting protein and represses ß-catenin-mediated transcription on T-cell factor/lymphoid enhancing factor. Here, we investigate the biological role of LZTS2 using newly established Lzts2 KO mice. Homozygosity for loss-of-function of the Lzts2-targeted allele resulted in severe kidney and urinary tract developmental defects, including renal/ureteral duplication, hydroureter, and hydronephrosis, which were visible prenatally. Altered ureteric bud outgrowth was identified in Lzts2 null embryos. Further analysis indicated that ß-catenin subcellular localization was altered in fibroblasts isolated from Lzts2 null embryos. In addition, Wnt growth factor-induced ß-catenin-mediated transcriptional activity was increased in Lzts2 null fibroblasts, suggesting a direct role for Lzts2 in the Wnt signaling pathway. These data demonstrate a critical role of LZTS2 in renal development and implicate LZTS2 as a critical regulator of ß-catenin-mediated nephrogenesis.

Infection of pregnant mice with Listeria monocytogenes induces fetal bradycardia.

INTRODUCTION: Listeriosis is one of the most lethal bacterial diseases for fetuses and infants. However, pregnant women who get infected with Listeria may experience only mild symptoms, making the diagnosis difficult, even when the fetus is fatally infected. METHODS: To reveal features of this infection, we conducted a multimodality imaging study of Listeria-induced miscarriage, using a pregnant mouse model. In this model, fetal morbidity and mortality can be observed in utero, noninvasively, and the timing and extent of infection can be carefully controlled. By employing in vivo bioluminescence imaging (BLI), perinatal infections were localized over time such that a correlation of infection to outcome could be determined without the need to kill the animal subject. The morbidity and viability of fetuses were assessed with ultrasound, and fetal morphology was imaged using magnetic resonance imaging (MRI). RESULTS: The ultrasound revealed sustained fetal bradycardia, the slowing of the fetal heartbeat, in infected fetuses, with an association between slowed fetal heart rate and strong bioluminescent signal. DISCUSSION: Uninfected fetuses showing no bioluminescent signal in the same uterine horn exhibited normal heartbeats. Thus, fetal bradycardia during infection was localized to the infected fetus and was not systemic or disseminated.

Ineffective vaccination against solid tumors can be enhanced by hematopoietic cell transplantation.

Vaccination with tumor Ags has not been an effective treatment for solid tumors. The goal of the current study was to determine whether a combination of vaccination and hematopoietic cell transplantation (HCT) can effectively treat primary, disseminated, or metastatic CT26 and MC38 murine colon tumors. Vaccination of tumor-bearing mice with irradiated tumor cells and CpG adjuvant failed to alter progressive tumor growth. However, mice bearing primary, disseminated lung, or metastatic liver tumors were uniformly cured after administration of total body irradiation, followed by the transplantation of hematopoietic progenitor cells and T cells from syngeneic, but not allogeneic vaccinated donors. Requirements for effective treatment of tumors included irradiation of hosts, vaccination of donors with both tumor cells and CpG, transfer of both CD4(+) and CD8(+) T cells along with progenitor cells, and ability of donor cells to produce IFN-gamma. Irradiation markedly increased the infiltration of donor T cells into the tumors, and the combined irradiation and HCT altered the balance of tumor-infiltrating cells to favor CD8(+) effector memory T cells as compared with CD4(+)CD25(+)FoxP3(+) T regulatory cells. The combination of vaccination and autologous hematopoietic cell transplantation was also effective in treating tumors. In conclusion, these findings show that otherwise ineffective vaccination to solid nonhematologic tumors can be dramatically enhanced by HCT.

Measurement of serum, liver, and brain cytokine induction, thiamine levels, and hepatopathology in rats exposed to a 4-day alcohol binge protocol.

BACKGROUND: In rodent and human studies, ethanol (EtOH) exposure is associated with elevated brain levels of the magnetic resonance spectroscopy (MRS) signal representing choline-containing compounds (Cho). One interpretation of elevated brain Cho is that it is a marker of neuroinflammation, and some evidence suggests that EtOH exposure promotes neuroinflammation. This study aimed to determine whether binge EtOH exposure (intragastric 3 g/kg 25% EtOH every 8 hours for 4 days) would induce the expression of certain cytokines in blood, liver, or brain, thereby supporting the neuroinflammation hypothesis of elevated Cho. METHODS: Ten of 18 wild-type male Wistar rats (~322 g at baseline) were exposed to EtOH and attained average blood alcohol levels of ~315 mg/dl across 4 days. Blood for cytokine immunoassays was collected at baseline, after 5 doses of EtOH (binge), and immediately preceding euthanasia either 4 or 24 hours after the last dose of EtOH. Blood was additionally assayed for the levels of thiamine and liver enzymes; liver histopathology was performed postmortem; and tissue from liver and 6 brain regions was assayed for the potential induction of 7 cytokines. RESULTS: There were no group effects on the levels of thiamine or its phosphate derivatives, thiamine monophosphate or thiamine diphosphate. ANOVAs of liver enzyme levels indicated that only alkaline phosphatase (ALP) levels were higher in the EtOH group than in control group at binge; ALP elevations, however, are difficult to explain in the absence of changes in the levels of additional liver enzymes. Postmortem liver pathology provided evidence for minimal microvesicular lipidosis and portocentric fibrosis in the EtOH group. Group effects on the levels of the measured cytokines in the blood (TNF-α, IFN-γ, IL-1ß, IL-4, IL-5, IL-13, and GRO/CXCL1) were not significant. Similarly, postmortem evaluation of liver cytokines did not reveal group effects. Postmortem evaluation of the 7 cytokines in 6 brain regions (anterior cerebellar vermis, cingulate cortex, frontal cortex, hippocampus, hypothalamus, striatum) also failed to identify group effects. CONCLUSIONS: A single 4-day bout of binge EtOH exposure alone was insufficient to induce the expression of 7 cytokines in blood, liver, or 6 brain regions of wild-type Wistar rats. Alternative interpretations for elevations in brain Cho in response to a 4-day binge EtOH treatment are therefore necessary and may include induction of cytokines not measured herein or other noninflammatory mechanisms.