Phase I dose-escalation study of procaspase-activating compound-1 in combination with temozolomide in patients with recurrent high-grade astrocytomas.

Background: Procaspase-3 (PC-3) is overexpressed in various tumor types, including gliomas. Targeted PC-3 activation combined with chemotherapy is a novel strategy for treating patients with high-grade gliomas, with promising preclinical activity. This study aimed to define safety and tolerability of procaspase-activating compound-1 (PAC-1) in combination with temozolomide (TMZ) for patients with recurrent high-grade astrocytomas. Methods: A modified-Fibonacci dose-escalation 3 + 3 design was used. PAC-1 was administered at increasing dose levels (DL; DL1 = 375 mg) on days 1-21, in combination with TMZ 150 mg/m2/5 days, per 28-day cycle. Dose-limiting toxicity was assessed during the first 2 cycles. Neurocognitive function (NCF) testing was conducted throughout the study. Results: Eighteen patients were enrolled (13 GBM, IDH-wild type; 2 astrocytoma, IDH-mutant, grade 3; 3 astrocytoma, IDH-mutant, grade 4). Dose escalation was discontinued after DL3 (ie, PAC-1, 625 mg) due to lack of additional funding. Grade 3 toxicity was observed in 1 patient at DL1 (elevated liver transaminases) and 1 at DL 2 (headache). Two partial responses were observed at DL1 in patients with GBM, O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated. Two patients had stable disease, and 11 experienced progression. NCF testing did not show a clear relationship between PAC-1 dose, treatment duration, and declines in NCF. Conclusions: Combination of PAC-1 and TMZ was well tolerated up to 625 mg orally daily and TMZ orally 150 mg/m2/5 days per 28-day cycle. The maximum tolerated dose was not reached. Further dose escalation of PAC-1 in combination with TMZ is advised before conducting a formal prospective efficacy study in this patient population.

Depatuxizumab mafodotin in EGFR-amplified newly diagnosed glioblastoma: A phase III randomized clinical trial.

BACKGROUND: Approximately 50% of newly diagnosed glioblastomas (GBMs) harbor epidermal growth factor receptor gene amplification (EGFR-amp). Preclinical and early-phase clinical data suggested efficacy of depatuxizumab mafodotin (depatux-m), an antibody-drug conjugate comprised of a monoclonal antibody that binds activated EGFR (overexpressed wild-type and EGFRvIII-mutant) linked to a microtubule-inhibitor toxin in EGFR-amp GBMs. METHODS: In this phase III trial, adults with centrally confirmed, EGFR-amp newly diagnosed GBM were randomized 1:1 to radiotherapy, temozolomide, and depatux-m/placebo. Corneal epitheliopathy was treated with a combination of protocol-specified prophylactic and supportive measures. There was 85% power to detect a hazard ratio (HR) ≤0.75 for overall survival (OS) at a 2.5% 1-sided significance level (ie traditional two-sided p ≤ 0.05) by log-rank testing. RESULTS: There were 639 randomized patients (median age 60, range 22-84; 62% men). Prespecified interim analysis found no improvement in OS for depatux-m over placebo (median 18.9 vs. 18.7 months, HR 1.02, 95% CI 0.82-1.26, 1-sided p = 0.63). Progression-free survival was longer for depatux-m than placebo (median 8.0 vs. 6.3 months; HR 0.84, 95% confidence interval [CI] 0.70-1.01, p = 0.029), particularly among those with EGFRvIII-mutant (median 8.3 vs. 5.9 months, HR 0.72, 95% CI 0.56-0.93, 1-sided p = 0.002) or MGMT unmethylated (HR 0.77, 95% CI 0.61-0.97; 1-sided p = 0.012) tumors but without an OS improvement. Corneal epitheliopathy occurred in 94% of depatux-m-treated patients (61% grade 3-4), causing 12% to discontinue. CONCLUSIONS: Interim analysis demonstrated no OS benefit for depatux-m in treating EGFR-amp newly diagnosed GBM. No new important safety risks were identified.

Phase I study of procaspase-activating compound-1 (PAC-1) in the treatment of advanced malignancies.

BACKGROUND: Procaspase-3 (PC-3) is overexpressed in multiple tumour types and procaspase-activating compound 1 (PAC-1) directly activates PC-3 and induces apoptosis in cancer cells. This report describes the first-in-human, phase I study of PAC-1 assessing maximum tolerated dose, safety, and pharmacokinetics. METHODS: Modified-Fibonacci dose-escalation 3 + 3 design was used. PAC-1 was administered orally at 7 dose levels (DL) on days 1-21 of a 28-day cycle. Dose-limiting toxicity (DLT) was assessed during the first two cycles of therapy, and pharmacokinetics analysis was conducted on days 1 and 21 of the first cycle. Neurologic and neurocognitive function (NNCF) tests were performed throughout the study. RESULTS: Forty-eight patients were enrolled with 33 completing ≥2 cycles of therapy and evaluable for DLT. DL 7 (750 mg/day) was established as the recommended phase 2 dose, with grade 1 and 2 neurological adverse events noted, while NNCF testing showed stable neurologic and cognitive evaluations. PAC-1's t1/2 was 28.5 h after multi-dosing, and systemic drug exposures achieved predicted therapeutic concentrations. PAC-1 clinical activity was observed in patients with neuroendocrine tumour (NET) with 2/5 patients achieving durable partial response. CONCLUSIONS: PAC-1 dose at 750 mg/day was recommended for phase 2 studies. Activity of PAC-1 in treatment-refractory NET warrants further investigation. CLINICAL TRIAL REGISTRATION: Clinical Trials.gov: NCT02355535.

Optimization of decalcification techniques for histologic examination of the rat maxillary and mandibular incisors for toxicity studies.

The objective of this study was to provide optimized processing for examination of rat incisors in nonclinical toxicity studies that enables analysis using immunohistochemistry (IHC). Rat maxillas and mandibles were decalcified in Immunocal, a formic acid decalcifier, and Decal Stat, a hydrochloric acid decalcifier, to evaluate tissue quality when with hematoxylin and eosin (H&E) stain and an IHC. Following necropsy of 10 to 13-week-old male Sprague Dawley rats, tissues were collected, trimmed, fixed in neutral buffered formalin (NBF), and placed into the corresponding decalcifying solution. After a pilot study with multiple timepoints for both decalcifying solutions, times were selected for the definitive study. Incisors in the definitive study were decalcified for 72, 96 or 120 hours in Immunocal and 24 hours in Decal Stat, trimmed, processed, embedded in paraffin, and sectioned. The microtomy process and sections were evaluated by histotechnologists. Sections were stained withH&E or an IHC to detect vimentin. Veterinary pathologists used blinded assessment to evaluate staining and tissue quality. The H&E sections from Immunocal timepoints scored higher based on criteria such as cellular morphology. However, tissue quality decreased at 120 hours with Immunocal but was adequate after 24 hours with Decal Stat. For IHC, moderate to excellent expression of vimentin was observed at timepoints for both decalcifiers. Optimal tissue sectioning and histological quality were achieved on incisor sections decalcified for 96 hours with Immunocal and 24 hours with Decal Stat.

Marital Status and Overall Survival in Patients with Resectable Pancreatic Cancer: Results of an Ancillary Analysis of NRG Oncology/RTOG 9704.

BACKGROUND: Several registry-based analyses suggested a survival advantage for married versus single patients with pancreatic cancer. The mechanisms underlying the association of marital status and survival are likely multiple and complex and, therefore, may be obscured in analyses generated from large population-based databases. The goal of this research was to characterize this potential association of marital status with outcomes in patients with resected pancreatic cancer who underwent combined modality adjuvant therapy on a prospective clinical trial. MATERIALS AND METHODS: This is an ancillary analysis of 367 patients with known marital status treated on NRG Oncology/RTOG 97-04. Survival analysis was performed using the Kaplan-Meier method and compared using the log-rank test. Multivariate analysis was performed using the Cox proportional hazards regression model. RESULTS: Of 367 patients, 271 (74%) were married or partnered and 96 (26%) were single. Married or partnered patients were more likely to be male. There was no association between marital status and overall survival (OS) or disease-free survival (DFS) on univariate (hazard ratio [HR], 1.09 and 1.01, respectively) or multivariate analyses (HR, 1.05 and 0.98, respectively). Married or partnered male patients did not have improved survival compared with female or single patients. CONCLUSION: Ancillary analysis of data from NRG Oncology/RTOG 97-04 demonstrated no association between marital and/or partner status and OS or DFS in patients with resected pancreatic cancer who received adjuvant postoperative chemotherapy followed by concurrent external beam radiation therapy and chemotherapy. Clinical trial identification number. NCT00003216. IMPLICATIONS FOR PRACTICE: Several population-based studies have shown an epidemiological link between marital status and survival in patients with pancreatic cancer. A better understanding of this association could offer an opportunity to improve outcomes through psychosocial interventions designed to mitigate the negative effects of not being married. Based on the results of this analysis, patients who have undergone a resection and are receiving adjuvant therapy on a clinical trial are unlikely to benefit from such interventions. Further efforts to study the association between marital status and survival should be focused on less selected subgroups of patients with pancreatic cancer.

Unique Case Report of a Meningeal Sarcoma Arising during Ongoing Treatment for Progressing Intraparenchymal Glioma.

Radiation-induced sarcomas in the brain are extremely rare, usually occur with an average latency of 9 years, and are associated with poor outcomes. Latency periods shorter than 1 year may indicate a genetic predisposition such as Li-Fraumeni syndrome. A 34-year-old man underwent initial tumor resection and radiation therapy for a World Health Organization (WHO) Grade II Astrocytoma. Within 6 months, the tumor recurred as WHO Grade III and was treated with temozolomide and then bevacizumab. Despite the patient's apparent improving condition, MRI revealed new dural-based lesions 10 months after radiation therapy and identified as high-grade sarcoma. The patient resumed bevacizumab, began NovoTTF treatment for progressing glioma, and ifosfamide/doxorubicin for the sarcoma. Genetic testing revealed no pathogenic mutation in the TP53 gene. Ultimately, treatment was unsuccessful and the patient succumbed to glioma and sarcoma within 2 years of initial diagnosis. This case was unique due to the rapidly progressing glioma and sudden appearance of a high-grade sarcoma. It is unusual to have two separate intracranial primary cancers with each requiring a different chemotherapy regimen. We discuss the difficulty of simultaneously treating with separate chemotherapy regimens. It remains unclear whether the sarcoma was induced by the radiation treatment or a genetic predisposition.

Scientific and Regulatory Policy Committee Points to Consider*: Review of Scientific and Regulatory Policy Committee Points to Consider: Review of Current Practices for Ultrastructural Pathology Evaluations in Support of Nonclinical Toxicology Studies.

Anatomic pathology and clinical pathology end points are standard components of almost every nonclinical general toxicity study conducted during the risk assessment of novel pharmaceuticals and chemicals. On occasion, an ultrastructural pathology evaluation using transmission electron microscopy (TEM) may be included in nonclinical toxicity studies. Transmission electron microscopy is most commonly used when a light microscopic finding may require further characterization that could inform on the pathogenesis and/or mechanism of action. Regulatory guidance do not address the use of TEM in general study designs nor whether these assessments should be performed in laboratories conducted in compliance with Good Laboratory Practices. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) formed a Working Group to assess the current practices on the use of TEM in nonclinical toxicity studies. The Working Group constructed a survey sent to members of societies of toxicologic pathology in the United States, Europe, Britain, and Japan, and responses were collected through the STP for evaluation by the Working Group. The survey results and regulatory context are discussed, as are "points to consider" from the collective experience of the Working Group. This survey indicates that TEM remains an essential diagnostic option for complementing toxicologic pathology evaluations. *This Points to Consider article is a product of a Society of Toxicologic Pathology (STP) Working Group commissioned by the Scientific and Regulatory Policy Committee (SRPC) of the STP. It has been reviewed and approved by the SRPC and Executive Committee of the STP but it does not represent a formal Best Practice recommendation of the Society; rather, it is intended to provide key "points to consider" in designing nonclinical studies or interpreting data from toxicity and safety studies intended to support regulatory submissions. The points expressed in this document are those of the authors and do not reflect views or policies of the employing institutions. Readers of Toxicologic Pathology are encouraged to send their thoughts on these articles or ideas for new topics to the Editor.

Kidney Pathology and Investigative Nephrotoxicology Strategies Across Species.

Drug-induced kidney toxicity is a significant contributor to acute kidney injury. Nephrotoxic drugs need to be identified during nonclinical testing to highlight potential risk translatable to the intended patient population. When nonclinical kidney toxicity signals arise, scientists and physicians affiliated with clinical trials need to be familiar with commonly encountered drug-induced perturbations in the kidney, terminology, and how these changes relate to clinical risk. Mechanistic and translational toxicologic studies beyond routine histopathology and clinical pathology approaches may be needed to elucidate the pathogenesis and human relevance to inform clinical risk assessment. Investigational studies may help elucidate specific sites of injury within the nephron, the presence of reactive metabolites, mechanisms of membrane transport or tissue distribution, potential drug-drug interactions, or the ability to recover function after drug withdrawal. Cutting-edge techniques such as in vitro alternative platforms, humanized animal models, translational imaging/microscopy or circulating/secretory biomarkers, omics platforms at the interface of genes, proteins, metabolites, or advanced molecular and biochemical approaches provide toxicologists and pathologists with a wide variety of potential experimental modalities to investigate mechanisms of kidney toxicity.

A multi-center preclinical study of gadoxetate DCE-MRI in rats as a biomarker of drug induced inhibition of liver transporter function.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and transplantation. DILI can be the result of impaired hepatobiliary transporters, with altered bile formation, flow, and subsequent cholestasis. We used gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), combined with pharmacokinetic modelling, to measure hepatobiliary transporter function in vivo in rats. The sensitivity and robustness of the method was tested by evaluating the effect of a clinical dose of the antibiotic rifampicin in four different preclinical imaging centers. The mean gadoxetate uptake rate constant for the vehicle groups at all centers was 39.3 +/- 3.4 s-1 (n = 23) and 11.7 +/- 1.3 s-1 (n = 20) for the rifampicin groups. The mean gadoxetate efflux rate constant for the vehicle groups was 1.53 +/- 0.08 s-1 (n = 23) and for the rifampicin treated groups was 0.94 +/- 0.08 s-1 (n = 20). Both the uptake and excretion transporters of gadoxetate were statistically significantly inhibited by the clinical dose of rifampicin at all centers and the size of this treatment group effect was consistent across the centers. Gadoxetate is a clinically approved MRI contrast agent, so this method is readily transferable to the clinic. CONCLUSION: Rate constants of gadoxetate uptake and excretion are sensitive and robust biomarkers to detect early changes in hepatobiliary transporter function in vivo in rats prior to established biomarkers of liver toxicity.

Successful treatment of pituitary carcinoma with concurrent radiation, temozolomide, and bevacizumab after resection.

The optimal treatment of pituitary carcinomas (PC) is unknown. Treatment includes surgical resection, radiation, and more recently, temozolomide (TMZ). Pituitary adenomas have relatively high expression of vascular endothelial growth factor; therefore, bevacizumab, an antiangiogenic agent, has been used in a small number of aggressive or malignant pituitary tumors after recurrence. However, it has not been administered concurrently with other chemotherapeutic agents or combined with radiation therapy in PC. We present a 63-year-old man with an adrenocorticotropic hormone (ACTH)-secreting PC, causing visual loss. It was resected transsphenoidally. There were several notable factors placing the patient at high risk for recurrence including distant metastasis in the form of a pulmonary nodule. Morphologically, his tumor was a pituitary neoplasm with malignant histopathologic features. It had abundant mitotic figures and zones of necrosis. Six weeks post-surgery, the patient started concurrent chemoradiation, using combination therapy with TMZ and bevacizumab. TMZ was continued for 12 cycles in the adjuvant setting. The ACTH was effective as a serum-based tumor marker and normalized during treatment. The patient is alive, five years after diagnosis, with no recurrence to date. This is the first case of pituitary carcinoma treated successfully with concurrent chemoradiation therapy that combined TMZ and bevacizumab with a long-term follow up.

Acute Alpha-Naphthylisothiocyanate-induced Liver Toxicity in Germfree and Conventional Male Rats.

Differences in the responses of conventional and germfree male Sprague-Dawley rats to acute injury induced by alpha-naphthylisothiocyanate (ANIT), a well-characterized biliary epithelial toxicant, were evaluated. Conventional and germfree rats were dosed once orally with 50 mg/kg of ANIT or corn oil alone and serially sacrificed daily for the next 3 days. Germfree rats treated with ANIT tended to have greater increases in virtually all liver and biliary-related analytes compared with conventional rats treated with ANIT; however, significant differences were found only in a few of these analytes including increased bile acids on day 3, total bilirubin on day 4, glutamate dehydrogenase (GLDH) on day 3, and reduced paraoxonase 1 (PON1) on days 2 and 3. Histologic differences between the conventional and germfree rats were modest, but most pronounced on day 2 (24-hr post dosing). Based on subjective scoring, biliary necrosis, neutrophilic cholangitis, and portal tract edema were more severe in germfree rats at 24 hr post dosing compared with conventional rats. Biliary epithelial replication did not differ between treated groups, however. Overall, germfree rats had a modestly greater level of biliary tract injury based on subjective histologic scoring and clinical chemistry measurements following an acute exposure to the well-characterized biliary toxin, ANIT; however, the difference between the ANIT-treated germfree and conventional groups was modest and most evident only within the first day following exposure. These findings suggest that the microbiome did not significantly affect ANIT-induced acute biliary tract injury in the conditions of this study.

Hepatic Phospholipidosis Is Associated with Altered Hepatobiliary Function as Assessed by Gadoxetate Dynamic Contrast-enhanced Magnetic Resonance Imaging.

To determine if amiodarone induces hepatic phospholipidosis (PLD) sufficient to detect changes in hepatobiliary transporter function as assessed by gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), rats were orally dosed with vehicle (1% methyl cellulose) or amiodarone (300 mg/kg/day) for 7 consecutive days. Gadoxetate DCE-MRI occurred at baseline, day 7, and following a 2-week washout of amiodarone. At day 7, the gadoxetate washout rate was significantly decreased compared to the vehicle group. Blood chemistry analysis revealed no significant changes in liver enzymes (alanine aminotransferase [ALT]/aspartate aminotransferase [AST]/alkaline phosphatase [ALP]), bilirubin, or bile acids between vehicle or amiodarone groups. Hepatic PLD was confirmed in all rats treated with amiodarone at day 7 by transmission electron microscopy. Following the 2-week washout, there was no ultrastructural evidence of hepatic PLD in rats and the gadoxetate washout rate returned to baseline levels. This is the first study to show the application of gadoxetate DCE-MRI to detect hepatobiliary functional changes associated with PLD and offer a potential new technique with clinical utility in patients suspected of having PLD. These results also suggest PLD itself has functional consequences on hepatobiliary function in the absence of biomarkers of toxicity, given the cause/effect relationship between PLD and function has not been fully established.

Effects of mid-respiratory chain inhibition on mitochondrial function in vitro and in vivo.

Relating the in vitro mitochondrial effects of drug candidates to likely in vivo outcomes remains challenging. Better understanding of this relationship, alongside improved methods to assess mitochondrial dysfunction in vivo, would both guide safer drug candidate selection and better support discovery programmes targeting mitochondria for pharmacological intervention. The aim of this study was to profile the in vivo effects of a compound with suspected complex III electron transport chain (ETC) inhibitory activity (GSK932121A) at doses associated with clinical signs, and relate findings back to in vitro data with the same compound. Control liver mitochondria or HepG2 cells were treated in vitro with GSK932121A to assess mitochondrial effects on both calcium retention capacity (CRC) and oxygen consumption rate (OCR) respectively. The same assessments were then performed on liver mitochondria isolated from Crl:CD(SD) rats, 5 hours following intraperitoneal (IP) administration of GSK932121A. Lactate/pyruvate assessment, hepatic microscopy, blood gas analysis, glutathione profiling and transcriptomics were used to characterise the acute toxicity. In vivo, GSK932121A caused hypothermia, increased levels of hepatocellular oxidative stress and a metabolic shift in energy production, resulting in an increased lactate/pyruvate ratio, liver steatosis and glycogen depletion, together with gene expression changes indicative of a fasted state. As would be expected of an ETC inhibitor, GSK932121A reduced the CRC of liver mitochondria isolated from naive control animals and the OCR of HepG2 cells when treated directly in vitro. In contrast, mitochondria isolated from animals treated with GSK932121A in vivo unexpectedly showed an increase in CRC and basal OCR. Whilst seemingly contradictory, these differences likely reflect an adapted state in vivo resulting from the initial insult in combination with compensatory changes made by the tissue to maintain energy production. Only the initial, unconfounded, response is observable in vitro. These findings improve current understanding of the toxicological and molecular consequences of ETC inhibition. Furthermore, this work highlights key differences in the way that mitochondrial perturbation is manifest in vivo versus in vitro in terms of functional endpoints and helps guide endpoint selection for future studies with potential mitochondrial toxicants or drugs designed to modulate mitochondrial function for therapeutic benefit.

Dermal toxicity studies: factors impacting study interpretation and outcome.

The field of dermal toxicity continues to evolve in order to accurately predict dermal (and systemic) responses in humans to topically applied chemicals. Although the testing methods have undergone extensive refinements, idiosyncrasies and unexpected issues during the conduct of these studies are not unusual due to the plethora of new vehicles available for formulating test substances, changing regulatory requirements, and introducting new strain and/or species of laboratory animals as no single species or method seems to suffice for evaluating skin toxicity. The objective of this article is to illustrate some pragmatic issues that should be considered during the conduct as well as interpretation of dermal toxicity studies. Routine procedure-related issues such as hair clipping, tape stripping, and wrapping the animal's torso to prevent oral ingestion can influence the interpretation. Excipients used in dermal toxicity studies may be nontoxic when used alone but complex dermal formulations can result in unexpected irritation and toxicity. In conclusion, interpretation and risk assessment of dermal toxicity studies should be done in a comprehensive manner, taking into account procedure-related impact on study results, unique species susceptibility, limitation of gross visual (naked eye) observation for evidence of toxicity, and normal anatomical variation.

Cardiolipin profiles as a potential biomarker of mitochondrial health in diet-induced obese mice subjected to exercise, diet-restriction and ephedrine treatment.

Cardiolipin (CL) is crucial for mitochondrial energy metabolism and structural integrity. Alterations in CL quantity or CL species have been associated with mitochondrial dysfunction in several pathological conditions and diseases, including mitochondrial dysfunction-related compound attrition and post-market withdrawal of promising drugs. Here we report alterations in the CL profiles in conjunction with morphology of soleus muscle (SM) and brown adipose tissue (BAT) in diet-induced obese (DIO) mice, subjected to ephedrine treatment (EPH: 200 mg kg(-1) day(-1) orally), treadmill exercise (EX: 10 meters per min, 1 h per day), or dietary restriction (DR: 25% less of mean food consumed by the EX group) for 7 days. Mice from the DR and EPH groups had a significant decrease in percent body weight and reduced fat mass compared with DIO controls. Morphologic alterations in the BAT included brown adipocytes with reduced cytoplasmic lipid droplets and increased cytoplasmic eosinophilia in the EX, DR and EPH groups. Increased cytoplasmic eosinophilia in the BAT was ultrastructurally manifested by increased mitochondrial cristae, fenestration of mitochondrial cristae, increased electron density of mitochondrial matrix, and increased complexity of shape and elongation of mitochondria. Mitochondrial ultrastructural alterations in the SM of the EX and DR groups included increased mitochondrial cristae, cup-shaped mitochondria and mitochondrial degeneration. All four CL species (tri-linoleoyl-mono-docosahexaenoyl, tetralinoleoyl, tri-linoleoyl-mono-oleoyl, and di-linoleoyl-di-oleoyl) were increased in the BAT of the DR and EPH groups and in the SM of the EPH and EX groups. In conclusion, cardiolipin profiling supported standard methods for assessing mitochondrial biogenesis and health, and may serve as a potential marker of mitochondrial dysfunction in preclinical toxicity studies.

Effects of Kupffer cell depletion on acute alpha-naphthylisothiocyanate-induced liver toxicity in male mice.

Depletion of Kupffer cells, known to modulate chemical-induced hepatocellular injury, has not been studied with regard to biliary epithelial injury. Here, the authors investigated the effect of Kupffer cell depletion by clodronate on the toxicity of alpha-naphthylisothiocyanate (ANIT), known to injure biliary epithelium as well as hepatocytes. Up to 99% depletion of Kupffer cells occurred in ANIT and liposome-encapsulated clodronate-treated mice. The effect of Kupffer cell depletion was most evident one day following ANIT treatment. Histologically, there was a modest increase in neutrophil infiltration of the bile ducts, hepatocytic necrosis, and microvesicular vacuolization in the ANIT and clodronate-treated mice, but differences between other groups did not persist. Clinical pathology analytes related to the biliary or hepatocellular injury were significantly elevated in ANIT and clodronate-treated mice compared to mice given clodronate only. This was also true for mice given ANIT and empty liposomes in the case of the biliary analytes. However, group means were typically higher for the ANIT and clodronate-treated group than others on the first 2 days following ANIT injection. These findings suggest that Kupffer cell reduction increases hepatobiliary damage due to ANIT treatment.

Regulatory T-cells: diverse phenotypes integral to immune homeostasis and suppression.

Regulatory T-cells (T(REG)) are diverse populations of lymphocytes that regulate the adaptive immune response in higher vertebrates. T(REG) delete autoreactive T-cells, induce tolerance, and dampen inflammation. T(REG) cell deficiency in humans (i.e., IPEX [Immunodysregulation, Polyendocrinopathy and Enteropathy, X-linked syndrome]) and animal models (e.g., "Scurfy" mouse) is associated with multisystemic autoimmune disease. T(REG) in humans and laboratory animal species are similar in type and regulatory function. A molecular marker of and the cell lineage specification factor for T(REG) is FOXP3, a forkhead box transcription factor. CD4(+) T(REG) are either natural (nT(REG)), which are thymus-derived CD4(+)CD25(+)FOXP3(+) T-cells, or inducible (i.e., Tr1 cells that secrete IL-10, Th3 cells that secrete TGF-ß and IL-10, and Foxp3(+) Treg). The proinflammatory Th17 subset has been a major focus of research. T(H)17 CD4(+) effector T-cells secrete IL-17, IL-21, and IL-22 in autoimmune and inflammatory disease, and are dynamically balanced with T(REG) cell development. Other lymphocyte subsets with regulatory function include: inducible CD8(+) T(REG), CD3(+)CD4(-)CD8(-) T(REG) (double-negative), CD4(+)Vα14(+) (NKT(REG)), and γδ T-cells. T(REG) have four regulatory modes of action: secretion of inhibitory cytokines (e.g., IL-10 and TGF-ß), granzyme-perforin-induced apoptosis of effector lymphocytes, depriving effector T-cells of cytokines leading to apoptosis, or inhibition of dendritic cell function. The role of T(REG) in mucosal sites, inflammation/infection, pregnancy, and cancer as well as a review of T(REG) as a modulatory target in drug development will be covered.

Preclinical applications of quantitative imaging cytometry to support drug discovery.

Preclinical drug development is actively involved in testing compounds to find cures or to manage the effects of disease, such as diabetes. Animal models, such as the Zucker diabetic fatty (ZDF) rat, are used to measure efficacy of candidate drugs. This animal model was selected because of its clinical and pathological similarities to diabetic human patients. A method using immunofluorescence and laser scanning cytometry (LSC) technology has been used to measure the development of diabetic phenotype in the ZDF rat during a 17-week time course. The expression levels of insulin, glucagon, voltage-dependent anion channel (VDAC), and Ki67 were quantified. Insulin and VDAC expression were reduced in the ZDF animals in comparison to the lean control rats, while no significant change was seen in glucagon and Ki67 expression at week 17. This information is useful in the design of studies to test experimental compounds in this model. Screening drug targets or biomarkers in tissue sections is another important activity in drug development. Tissue microarrays (TMAs) are composed of 60 or more tissue cores from humans or animal models and may contain healthy and/or diseased tissues. Antibodies against target proteins are applied to TMAs using routine immunohistochemical reagents and protocols. The protein expression across the cores, as labeled by immunohistochemistry, is measured using LSC technology. The process provides an efficient and cost-effective method for evaluating multiple targets in a large number of tissue samples. More recently, IHC and LSC have been taken to the next level to quantify biopharmaceutical drug and target co-localization in tissue sections.

Continuing education course #1: non-invasive imaging as a problem-solving tool and translational biomarker strategy in toxicologic pathology.

The continuing education course "Non-Invasive Imaging as a Problem-Solving Tool and Translational Biomarker Strategy in Toxicologic Pathology" provided a thorough overview of commonly used imaging modalities and the logistics required for integration of small animal imaging into toxicologic pathology. Non-invasive imaging (NIN) is gaining acceptance as an important modality in toxicologic pathology. This technology allows nonterminal, time-course evaluation of functional and morphologic endpoints and can be used to translate biomarkers between preclinical animal models and human patients. NIN can support drug development as well as basic research in academic or industrial environments. An initial overview of theoretical principles was followed by focused presentations on magnetic resonance imaging (MRI)/magnetic resonance microscopy (MRM), positron emission tomography (PET)/single proton emission computed tomography (SPECT), ultrasonography (US, primarily focused on echocardiography), optical (bioluminescent) imaging, and computed tomography (CT). The choice of imaging modality will depend on the research question and the needed resolution.

Acute ANIT toxicity in male IL-10 knockout and wild-type mice.

The innate immune response is known to modify hepatocellular injury induced by toxicants. To assess the role of IL-10, a component of the innate immune response, in toxicant-induced injury of biliary epithelium, wild-type (WT) and IL-10 knockout mice (KO) were given a single toxic dose (50 mg/kg) of alpha-napthylisothiocyanate (ANIT) and assessed at twenty-four-hour intervals for four days following treatment. Clinical signs of toxicity were greater in WT mice. Unexpectedly, over the course of the study, there was a consistent tendency for ANIT-treated IL-10 KO mice to have less hepatocellular injury than WT mice. However, changes in the biliary epithelium differed in that there was more histologic evidence of inflammation and necrosis on days 2 and 3, respectively, in ANIT-treated IL-10 KO mice compared with WT mice. Proliferation of biliary epithelium and hepatocytes was greater and/or occurred earlier in the ANIT-treated IL-10 KO mice compared with the ANIT-treated WT mice, suggesting a greater reparative response was needed for recovery after toxicant injury in the IL-10 KO mice. Overall, our data suggest that IL-10 KO mice have less hepatocellular injury than WT mice following a toxic dose of ANIT and that biliary epithelial injury is accentuated in the KO mice.