Practical Strategies for Navigating Toxicologic Pathology in One's Early Career and Beyond!

The toxicologic pathologist plays a vital role in the scientific community, using their unique blend of diagnostic and investigative skills to advance biomedical research, public health, drug discovery, or regulatory practices. But what exactly do toxicologic pathologists contribute? Where do these specialized professionals work? How can toxicologic pathologists maximize their efficiency and potential? To enlighten students and trainees, as well as early- or mid-career toxicologic pathologists, or even those approaching retirement, the Career Development and Outreach Committee of the Society of Toxicologic Pathology (STP) sponsored a career development workshop entitled "Practical Strategies for Navigating Toxicologic Pathology in One's Early Career…and Beyond!" in conjunction with the STP 37th annual symposium. The workshop featured toxicologic pathologists from contract research organizations and the pharmaceutical industry, who provided their perspectives on career preparation, evolving veterinary pathologist roles within various sectors of toxicologic pathology, the fundamentals of safety assessment, logistics of projects involving good laboratory practices, tools for effective interpretation and communication of anatomic and clinical pathology results, and a recap of scientific resources available to support the toxicologic pathologist in his or her journey. This article provides brief summaries of the talks presented during this career development workshop.

Scientific and Regulatory Policy Committee Points to Consider: Histopathologic Evaluation in Safety Assessment Studies for PEGylated Pharmaceutical Products.

Colorless, intracytoplasmic vacuoles occur in multiple tissues in animals following repeated administration of polyethylene glycol (PEG)-conjugated molecules. The extent of vacuolation depends on physical characteristics and molecular backbone of the PEG and the dose, product, drug target/pharmacology, and duration of exposure. The collective experience gathered from multiple nonclinical toxicology studies of PEGylated biopharmaceuticals indicates that in general, PEG-related vacuolation is not associated with demonstrable cell and tissue damage or dysfunction and is reversible with sufficient duration of drug-free periods. Existing data are insufficient to predict whether nonclinical animal species differ in their sensitivity to develop PEG-associated vacuoles; however, recent data suggest that there may be species differences. Recent comprehensive reviews have addressed the basic challenges in developing PEGylated pharmaceutical products, including general reference to and description of PEG-associated tissue findings. These manuscripts have identified gaps in our current understanding of PEG-associated vacuolation, including the lack of a widely accepted standardized histological terminology and criteria to record and grade the severity of vacuolation as well as insufficient knowledge regarding the nature of the contents of these vacuoles. The goal of this article is to help address some of the gaps identified above by providing points to consider, including a pictorial review of PEG-associated microscopic findings, when evaluating and reporting the extent, severity, and significance (adversity or lack of adversity) of PEG-associated cytoplasmic vacuolation in safety assessment studies. [Box: see text].

Occurrence of spontaneous amphophilic-vacuolar renal tubule tumors in Sprague-Dawley rats from subchronic toxicity studies.

The low background incidence of tumors in rodents from subchronic toxicity studies makes it difficult to assess their relevance, especially when present only in treated animals. This report investigates the occurrence of renal tubule tumors (RTTs), specifically the amphophilic-vacuolar (AV) phenotypic variant, in young Sprague-Dawley (SD) rats from a survey of laboratories conducting subchronic toxicity studies spanning a period of 10 years (2002-2012). This survey establishes a general profile of tumor occurrence; it does not estimate overall incidence or prevalence. AV tumors are spontaneous, nontreatment-related tumors of familial origin, and morphologically distinct from conventional RTTs induced by exposure to renal carcinogens. They are composed of distinct lobules of large, round to polyhedral cells with vacuolated amphophilic to eosinophilic cytoplasm and prominent nucleoli. Data from five collaborating laboratories, representing 37 qualifying studies, are presented. In total, 58 renal tubule neoplasms were recorded in this data set. The AV tumor variant was reported more commonly than the conventional RTT (n = 45 and 13, respectively), and it was recorded in both experimental (n = 32) and control (n = 13) groups. AV tumors occurred more often in females (n = 34) than in males (n = 11); conventional RTTs were recorded more often in males (n = 9) than in females (n = 4). AV tumors often occurred in more than one rat within the same study (up to 7) and were documented to occur in rats as young as 7 to 10 weeks of age. Results from this survey indicate that AV tumors are being reported more commonly in recent years; the majority (n = 33) were reported in studies commencing since 2009. In conclusion, this study reaffirms that AV tumors are spontaneous, nontreatment-related lesions, and suggests that they may be more common than conventional RTTs in young SD rats. The authors propose that AV tumors be recorded separately from conventional RTTs in order to clearly distinguish these two renal tubule neoplasms from one another and allow for appropriate interpretation of a compound's potential carcinogenic effect in the kidney.

Cyclosporine-induced immune suppression alters establishment of HTLV-1 infection in a rabbit model.

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia and several lymphocyte-mediated inflammatory diseases. Persistent HTLV-1 infection is determined by a balance between host immune responses and virus spread. Immunomodulatory therapy involving HTLV-1-infected patients occurs in a variety of clinical settings. Knowledge of how these treatments influence host-virus relationships is not understood. In this study, we examined the effects of cyclosporine A (CsA)-induced immune suppression during early infection of HTLV-1. Twenty-four New Zealand white rabbits were split into 4 groups. Three groups were treated with either 10 or 20 mg/kg CsA or saline before infection. The fourth group was treated with 20 mg/kg CsA 1 week after infection. Immune suppression, plasma CsA concentration, ex vivo lymphocyte HTLV-1 p19 production, anti-HTLV-1 serologic responses, and proviral load levels were measured during infection. Our data indicated that CsA treatment before HTLV-1 infection enhanced early viral expression compared with untreated HTLV-1-infected rabbits, and altered long-term viral expression parameters. However, CsA treatment 1 week after infection diminished HTLV-1 expression throughout the 10-week study course. Collectively, these data indicate immunologic control is a key determinant of early HTLV-1 spread and have important implications for therapeutic intervention during HTLV-1-associated diseases.

Measles virotherapy in a mouse model of adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATL) is a highly aggressive CD4(+) T-cell malignancy caused by human T-cell leukaemia virus type 1. Measles virus (MV) oncolytic therapy has been reported to be efficient in reducing tumour burden in subcutaneous xenograft models of lymphoproliferative disorders such as myeloma, B-cell lymphoma and cutaneous T-cell lymphoma, but its potential to reduce tumour burden in disseminated lymphoproliferative disorders such as ATL remains to be determined. In this study, MV oncolytic therapy was evaluated in the MET-1/NOD/SCID xenograft mouse model of ATL. Treatment with the vaccine-related strain MV-NSE led to a significant reduction in tumour burden. In mice with a high tumour burden, therapy with MV-NSE significantly increased survival beyond any other single treatment tested previously using this model. Interestingly, signs of morbidity (cachexia) in mice treated with MV were not directly associated with tumour burden, but were correlated with the secretion of interleukin-6 by MET-1 cells and host cells. The results suggest that MV therapy could be a promising therapy for generalized lymphoproliferative disease.

Early spatial and temporal events of human T-lymphotropic virus type 1 spread following blood-borne transmission in a rabbit model of infection.

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia/lymphoma (ATL) and is associated with a variety of lymphocyte-mediated disorders. HTLV-1 transmission occurs by transmission of infected cells via breast-feeding by infected mothers, sexual intercourse, and contaminated blood products. The route of exposure and early virus replication events are believed to be key determinants of virus-associated spread, antiviral immune responses, and ultimately disease outcomes. The lack of knowledge of early events of HTLV-1 spread following blood-borne transmission of the virus in vivo hinders a more complete understanding of the immunopathogenesis of HTLV-1 infections. Herein, we have used an established animal model of HTLV-1 infection to study early spatial and temporal events of the viral infection. Twelve-week-old rabbits were injected intravenously with cell-associated HTLV-1 (ACH-transformed R49). Blood and tissues were collected at defined intervals throughout the study to test the early spread of the infection. Antibody and hematologic responses were monitored throughout the infection. HTLV-1 intracellular Tax and soluble p19 matrix were tested from ex vivo cultured lymphocytes. Proviral copy numbers were measured by real-time PCR from blood and tissue mononuclear leukocytes. Our data indicate that intravenous infection with cell-associated HTLV-1 targets lymphocytes located in both primary lymphoid and gut-associated lymphoid compartments. A transient lymphocytosis that correlated with peak virus detection parameters was observed by 1 week postinfection before returning to baseline levels. Our data support emerging evidence that HTLV-1 promotes lymphocyte proliferation preceding early viral spread in lymphoid compartments to establish and maintain persistent infection.

Human T-cell leukemia virus type-1 antisense-encoded gene, Hbz, promotes T-lymphocyte proliferation.

Human T-cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ) is dispensable for HTLV-1-mediated cellular transformation in cell culture, but is required for efficient viral infectivity and persistence in rabbits. In most adult T-cell leukemia (ATL) cells, Tax oncoprotein expression is typically low or undetectable, whereas Hbz gene expression is maintained, suggesting that Hbz expression may support infected cell survival and, ultimately, leukemogenesis. Emerging data indicate that HBZ protein can interact with cAMP response element binding protein (CREB) and Jun family members, altering transcription factor binding and transactivation of both viral and cellular promoters. Herein, lentiviral vectors that express Hbz-specific short hairpin (sh)-RNA effectively decreased both Hbz mRNA and HBZ protein expression in transduced HTLV-1-transformed SLB-1 T cells. Hbz knockdown correlated with a significant decrease in T-cell proliferation in culture. Both SLB-1 and SLB-1-Hbz knockdown cells engrafted into inoculated NOD/SCID(gammachain-/-) mice to form solid tumors that also infiltrated multiple tissues. However, tumor formation and organ infiltration were significantly decreased in animals challenged with SLB-1-Hbz knockdown cells. Our data indicate that Hbz expression enhances the proliferative capacity of HTLV-1-infected T cells, playing a critical role in cell survival and ultimately HTLV-1 tumorigenesis in the infected host.

Creation and characterization of a doxycycline-inducible mouse model of thyroid-targeted RET/PTC1 oncogene and luciferase reporter gene coexpression.

BACKGROUND: RET/PTC1 chromosomal rearrangement is associated with papillary thyroid carcinoma formation in children exposed to ionizing radiation. We previously created a transgenic mouse model with thyroid-targeted constitutive RET/PTC1 expression and demonstrated papillary thyroid carcinoma formation. OBJECTIVE: In this study, we aimed to create a doxycycline-inducible mouse model of thyroid RET/PTC1 and luciferase reporter gene coexpression to allow for noninvasive monitoring of transgene expression in mice of various ages and timepoints after induction. DESIGN: Transgenic mice carrying the rtTA gene driven by the thyroglobulin promoter were generated, and crossed with responder mice carrying RET/PTC1 and firefly luciferase genes under control of a bidirectional tetracycline response element. MAIN OUTCOMES: Most bitransgenic mice had thyroid-targeted, doxycycline-independent transgene expression. Only one line had thyroid-targeted, doxycycline-regulated RET/PTC1 and luciferase coexpression, in which doxycycline induction of RET/PTC1 led to Erk phosphorylation and reduced expression of the sodium/iodide symporter (NIS). However, thyroid lesions were not found in any bitransgenic mice examined. CONCLUSIONS: We found that acute RET/PTC1 expression can activate the MEK/Erk pathway and downregulate NIS expression in the mouse thyroid gland. However, a higher level of RET/PTC1 is likely necessary for tumor formation. Thyroid luciferase induction was detectable noninvasively using IVIS in vivo imaging.

Efficacy of novel histone deacetylase inhibitor, AR42, in a mouse model of, human T-lymphotropic virus type 1 adult T cell lymphoma.

Human T-lymphotropic virus type 1 (HTLV-1) causes a variety of forms of adult T-cell leukemia/lymphoma (ATL), a refractory CD4+/CD25+ T-cell malignancy. Novel approaches to treat ATL patients are required due to the resistance of ATL to conventional chemotherapies. Histone deacetylase inhibitors (HDACi), which induce histone hyperacetylation leading to chromatin remodeling and reactivation of transcriptionally repressed genes have shown efficacy against a variety of cancers. Herein, we tested if valproic acid and the novel orally bioavailable HDACi, AR-42 reduced the proliferation of ATL cell lines by promoting apoptosis and histone hyperacetylation. Both compounds were cytotoxic and elicited a dose dependent increase in cytochrome C and cleaved Poly (ADP-ribose) polymerase (PARP) indicating the induction of cell death by apoptosis and promoted acetylation of histone H3 in both MT-2 and C8166 cell lines. We then evaluated the effects of AR-42, for survival in an ATL NOD/SCID mouse model. A dietary formulation of AR-42 prolonged survival of ATL engrafted mice compared to controls. Our data provide new directions for the treatment of ATL and support the further development of AR-42 against HTLV-1-associated lymphoid malignancies.

Effect of treatment of Strongyloides infection on HTLV-1 expression in a patient with adult T-cell leukemia.

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia-lymphoma (ATLL) in about 5% of infected individuals. Coinfection by Strongyloides stercoralis has been suggested to be a cofactor for development of ATLL. We describe a patient who presented with HTLV-1-associated chronic ATLL and Strongyloides infection. Studies of this patient's viral RNA levels demonstrated stimulation of HTLV-1 replication by Strongyloides, which resolved with anti-helminthic therapy. This case provides support for the hypothesis that Strongyloides is a cofactor for ATLL via T-cell stimulation.