Evaluation of tissues is a common and important aspect of translational research studies. Labeling techniques such as immunohistochemistry can stain cells/tissues to enhance identification of specific cell types, cellular activation states, and protein expression. While qualitative evaluation of labeled tissues has merit, use of semiquantitative and quantitative scoring approaches can greatly enhance the rigor of the tissue data. Adhering to key principles for reproducible scoring can enhance the quality and reproducibility of the tissue data so as to maximize its biological relevance and scientific impact.

CD4 T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4 T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1 hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1 mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Inflammation is a common feature in several types of lung disease and is a frequent end point to validate lung disease models, evaluate genetic or environmental impact on disease severity, or test the efficacy of new therapies. Questions relevant to a study should be defined during experimental design and techniques selected to specifically address these scientific queries. In this review, the authors focus primarily on the breadth of techniques to evaluate lung inflammation that have both clinical and preclinical applications. Stratification of approaches to assess lung inflammation can diminish weaknesses inherent to each technique, provide data validation, and increase the reproducibility of a study. Specialized techniques (eg, imaging, pathology) often require experienced personnel to collect, evaluate, and interpret the data; these experts should be active contributors to the research team through reporting of the data. Scoring of tissue lesions is a useful method to transform observational pathologic data into semiquantitative or quantitative data for statistical analysis and enhanced rigor. Each technique to evaluate lung inflammation has advantages and limitations; understanding these parameters can help identify approaches that best complement one another to increase the rigor and translational significance of data.

Our laboratory has developed a novel delivery platform using an attenuated non-toxic and non-pathogenic bacterium Listeria monocytogenes that infects tumor cells and selectively survives and multiplies in metastases and primary tumors with help of myeloid-derived suppressor cells (MDSC) and immune suppression in the tumor microenvironment (TME). 32P was efficiently incorporated into the Listeria bacteria by starvation of the bacteria in saline, and then cultured in phosphorus-free medium complemented with 32P as a nutrient. Listeria-32P kills tumor cells through both 32P-induced ionizing radiation and Listeria-induced reactive oxygen species (ROS). The levels of 32P and Listeria were studied in various normal and tumor tissues, at sequential time points after injection of mice with pancreatic cancer (syngeneic model Panc-02). We found that 32P and Listeria predominantly accumulated in tumors and metastases, with their highest accumulation 4 hrs (32P) and 3 days (Listeria) after injection. Listeria also penetrated the transgenic KPC (conditionally express endogenous Kras-G12D and p53-R172H mutant alleles) pancreatic tumors and metastases. This is remarkable since KPC tumors, like human tumors, exhibit a stromal barrier, which prevents most drugs from penetrating the pancreatic tumors. Therapeutic treatment with Listeria -32P resulted in a strong reduction of the growth of pancreatic cancer at early and late stages in Panc-02 and KPC mice. These results highlight the power of Listeria as new delivery platform of anticancer agents to the TME. Not only were therapeutic levels of radioactive Listeria reached in tumors and metastases but the selective delivery also led to minimal side effects.

Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments; however, therapies tested in such models often fail to translate into clinical settings. Therefore, a better preclinical model for cancer treatment testing is needed. Here we demonstrate that an immunodeficient line of pigs can host and support the growth of xenografted human tumors and has the potential to be an effective animal model for cancer therapy. Wild-type and immunodeficient pigs were injected subcutaneously in the left ear with human melanoma cells (A375SM cells) and in the right ear with human pancreatic carcinoma cells (PANC-1). All immunodeficient pigs developed tumors that were verified by histology and immunohistochemistry. Nonaffected littermates did not develop tumors. Immunodeficient pigs, which do not reject xenografted human tumors, have the potential to become an extremely useful animal model for cancer therapy because of their similarity in size, anatomy, and physiology to humans.

Translation of disease-modifying therapies in neurodegenerative disease has been disappointing. Parkinson’s disease (PD) was used to compare patterns of preclinical study design for symptomatic and potentially disease-modifying interventions. We examined the relationship of model, intervention type and timing, outcomes and outcome measures in 543 animal and human studies (1973–2015) across a contemporary cohort of animal and human interventional studies (n = 445), animal studies for approved interventions (n = 28), animal and human studies for those that failed to translate (n = 70). Detailed study design data were collected for 216 studies in non-human primate (NHP) and rodent toxin-induced models. Species-specific patterns of study design prevailed regardless of whether interventions were symptomatic or potentially disease-modifying. In humans and NHPs, interventions were typically given to both sexes well after the PD phenotype was established, and clinical outcome measures were collected at single (symptomatic) or multiple (disease-modifying) time-points. In rodents, interventions often preceded induction of the model, acute toxic protocols were common, usually given to young males, clinical outcome measures were used less commonly, and outcomes were less commonly assessed at multiple time points. These patterns were more prevalent in mice than rats. In contrast, study design factors such as randomization and blinding did not differ appreciably across symptomatic and disease-modifying intervention categories. The translational gap for potentially disease-modifying interventions in PD in part results from study designs, particularly in mice, that fail to model the progressive nature and relatively late intervention characteristic of PD, or that anchor mechanistic and neuropathologic data to longitudinal clinical outcomes. Even if measures to improve reproducibility are broadly adopted, perpetuation of these norms will continue to impede effective translation.

Failure to reproduce results from some scientific studies has raised awareness of the critical need for reproducibility in translational studies. Macroscopic and microscopic examination is a common approach to determine changes in tissues, but text descriptions and visual images have limitations for group comparisons. Semiquantitative scoring is a way of transforming qualitative tissue data into numerical data that allow more robust group comparisons. Semiquantitative scoring has broad uses in preclinical and clinical studies for evaluation of tissue lesions. Reproducibility can be improved by constraining bias through appropriate experimental design, randomization of tissues, effective use of multidisciplinary collaborations, and valid masking procedures. Scoring can be applied to tissue lesions (eg, size, distribution, characteristics) and also to tissues through evaluation of staining distribution and intensity. Semiquantitative scores should be validated to demonstrate relevance to biological data and to demonstrate observer reproducibility. Statistical analysis should make use of appropriate tests to give robust confidence in the results and interpretations. Following key principles of semiquantitative scoring will not only enhance descriptive tissue evaluation but also improve quality, reproducibility, and rigor of tissue studies.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease. Tumors are poorly immunogenic and immunosuppressive, preventing T cell activation in the tumor microenvironment. Here, we present a microbial-based immunotherapeutic treatment for selective delivery of an immunogenic tetanus toxoid protein (TT 856-1313 ) into PDAC tumor cells by attenuated Listeria monocytogenes . This treatment reactivated preexisting TT-specific memory T cells to kill infected tumor cells in mice. Treatment of KrasG12D,p53R172H, Pdx1-Cre (KPC) mice with Listeria -TT resulted in TT accumulation inside tumor cells, attraction of TT-specific memory CD4 T cells to the tumor microenvironment, and production of perforin and granzyme B in tumors. Low doses of gemcitabine (GEM) increased immune effects of Listeria -TT, turning immunologically cold into hot tumors in mice. In vivo depletion of T cells from Listeria -TT + GEM–treated mice demonstrated a CD4 T cell–mediated reduction in tumor burden. CD4 T cells from TT-vaccinated mice were able to kill TT-expressing Panc-02 tumor cells in vitro. In addition, peritumoral lymph node–like structures were observed in close contact with pancreatic tumors in KPC mice treated with Listeria -TT or Listeria -TT + GEM. These structures displayed CD4 and CD8 T cells producing perforin and granzyme B. Whereas CD4 T cells efficiently infiltrated the KPC tumors, CD8 T cells did not. Listeria -TT + GEM treatment of KPC mice with advanced PDAC reduced tumor burden by 80% and metastases by 87% after treatment and increased survival by 40% compared to nontreated mice. These results suggest that Listeria -delivered recall antigens could be an alternative to neoantigen-mediated cancer immunotherapy.