Scientific and Regulatory Policy Committee Points to Consider: Biological Sample Retention From Nonclinical Toxicity Studies.

Samples of biologic specimens and their derivatives (eg, wet tissues, paraffin-embedded tissue blocks, histology slides, frozen tissues, whole blood, serum/plasma, and urine) are routinely collected during the course of nonclinical toxicity studies. Good Laboratory Practice regulations and/or guidance specify minimum requirements for specimen retention duration, with the caveat that retention of biologic specimens need not extend beyond the duration of sample stability. However, limited availability of published data regarding stability for various purposes following storage of each specimen type has resulted in confusion, uncertainty, and inconsistency as to the appropriate duration for storage of these specimens. To address these issues, a working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee was formed to review published information, regulations, and guidance pertinent to this topic and to summarize the current practices and rationales for retention duration through a survey-based approach. Information regarding experiences reaccessing biologic specimens and performing sample stability investigations was also collected. Based on this combined information, the working group developed several points to consider that may be referenced when developing or revising sample retention practices. [Box: see text].

UVB mutagenesis differs in Nras- and Braf-mutant mouse models of melanoma.

BRAF-mutant melanomas are more likely than NRAS-mutant melanomas to arise in anatomical locations protected from chronic sun damage. We hypothesized that this discrepancy in tumor location is a consequence of the differential sensitivity of BRAF and NRAS-mutant melanocytes to ultraviolet light (UV)-mediated carcinogenesis. We tested this hypothesis by comparing the mutagenic consequences of a single neonatal, ultraviolet-AI (UVA; 340-400 nm) or ultraviolet-B (UVB; 280-390 nm) exposure in mouse models heterozygous for mutant Braf or homozygous for mutant Nras Tumor onset was accelerated by UVB, but not UVA, and the resulting melanomas contained recurrent mutations affecting the RING domain of MAP3K1 and Actin-binding domain of Filamin A. Melanomas from UVB-irradiated, Braf-mutant mice averaged twice as many single-nucleotide variants and five times as many dipyrimidine variants than tumors from similarly irradiated Nras-mutant mice. A mutational signature discovered in UVB-accelerated tumors mirrored COSMIC signatures associated with human skin cancer and was more prominent in Braf- than Nras-mutant murine melanomas. These data show that a single UVB exposure yields a greater burden of mutations in murine tumors driven by oncogenic Braf.

Stable Attenuation of Human Respiratory Syncytial Virus for Live Vaccines by Deletion and Insertion of Amino Acids in the Hinge Region between the mRNA Capping and Methyltransferase Domains of the Large Polymerase Protein.

Human respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory tract disease in infants and children worldwide. Currently, there are no FDA-approved vaccines to combat this virus. The large (L) polymerase protein of RSV replicates the viral genome and transcribes viral mRNAs. The L protein is organized as a core ring-like domain containing the RNA-dependent RNA polymerase and an appendage of globular domains containing an mRNA capping region and a cap methyltransferase region, which are linked by a flexible hinge region. Here, we found that the flexible hinge region of RSV L protein is tolerant to amino acid deletion or insertion. Recombinant RSVs carrying a single or double deletion or a single alanine insertion were genetically stable, highly attenuated in immortalized cells, had defects in replication and spread, and had a delay in innate immune cytokine responses in primary, well-differentiated, human bronchial epithelial (HBE) cultures. The replication of these recombinant viruses was highly attenuated in the upper and lower respiratory tracts of cotton rats. Importantly, these recombinant viruses elicited high levels of neutralizing antibody and provided complete protection against RSV replication. Taken together, amino acid deletions or insertions in the hinge region of the L protein can serve as a novel approach to rationally design genetically stable, highly attenuated, and immunogenic live virus vaccine candidates for RSV.IMPORTANCE Despite tremendous efforts, there are no FDA-approved vaccines for human respiratory syncytial virus (RSV). A live attenuated RSV vaccine is one of the most promising vaccine strategies for RSV. However, it has been a challenge to identify an RSV vaccine strain that has an optimal balance between attenuation and immunogenicity. In this study, we generated a panel of recombinant RSVs carrying a single and double deletion or a single alanine insertion in the large (L) polymerase protein that are genetically stable, sufficiently attenuated, and grow to high titer in cultured cells, while retaining high immunogenicity. Thus, these recombinant viruses may be promising vaccine candidates for RSV.

Cotton Rat Placenta Anatomy and Fc Receptor Expression and Their Roles in Maternal Antibody Transfer.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral pneumonia in infants and young children worldwide. Currently no vaccine is available to prevent RSV infection, but virus-neutralizing monoclonal antibodies can be given prophylactically, emphasizing the protective potential of antibodies. One concept of RSV vaccinology is mothers' immunization to induce high antibody titers, leading to passive transfer of high levels of maternal antibody to the fetus through the placenta and to the neonate through colostrum. Cotton rats are an excellent small animal model for RSV infection and have been used to test maternal immunization. To mechanistically understand antibody transfer in the cotton rat model, we characterized the cotton rat placenta and Fc receptor localization. Placentas from cotton rats at midgestation (approximately day 14) and at late gestation (approximately day 25) and neonatal (younger than 1 wk) gastrointestinal tracts were collected for light microscopy, immunohistochemistry, and transmission electron microscopy. The cotton rat placenta is hemotrichorial and has 5 distinct layers: decidua, junctional zone, labyrinth, chorionic plate, and yolk sac. Consistent with the transfer of maternal antibodies, the majority of the Fc receptors are present in the yolk sac endoderm and fetal capillary endothelium of the chorionic plate, involving 10% of the cells within the labyrinth. In addition, Fc receptors are present on duodenal and jejunal enterocytes in cotton rats, similar to humans, mice, and rats. These findings provide the structural basis for the pre- and postnatal transfer of maternal antibodies described in cotton rats.

Akt isoform-specific effects on thyroid cancer development and progression in a murine thyroid cancer model.

The Akt family is comprised of three unique homologous proteins with isoform-specific effects, but isoform-specific in vivo data are limited in follicular thyroid cancer (FTC), a PI3 kinase-driven tumor. Prior studies demonstrated that PI3K/Akt signaling is important in thyroid hormone receptor ßPV/PV knock-in (PV) mice that develop metastatic thyroid cancer that most closely resembles FTC. To determine the roles of Akt isoforms in this model we crossed Akt1-/-, Akt2-/-, and Akt3-/- mice with PV mice. Over 12 months, thyroid size was reduced for the Akt null crosses (p < 0.001). Thyroid cancer development and local invasion were delayed in only the PVPV-Akt1 knock out (KO) mice in association with increased apoptosis with no change in proliferation. Primary-cultured PVPV-Akt1KO thyrocytes uniquely displayed a reduced cell motility. In contrast, loss of any Akt isoform reduced lung metastasis while vascular invasion was reduced with Akt1 or 3 loss. Microarray of thyroid RNA displayed incomplete overlap between the Akt KO models. The most upregulated gene was the dendritic cell (DC) marker CD209a only in PVPV-Akt1KO thyroids. Immunohistochemistry demonstrated an increase in CD209a-expressing cells in the PVPV-Akt1KO thyroids. In summary, Akt isoforms exhibit common and differential functions that regulate local and metastatic progression in this model of thyroid cancer.

Cause and Treatment of Exophthalmos in Aged Cotton Rats (Sigmodon hispidus).

Aged cotton rats (Sigmodon hispidus) from an established breeding colony displayed signs of spontaneous exophthalmos. Of a total of 118 colony animals that were older than 6 mo of age, 37 (31%) displayed signs of exophthalmos. These rats were clinically healthy and had no other signs of disease. Ophthalmic exams, molecular and microbiologic testing, and histopa- thology were performed to determine the cause of the exophthalmos and to provide appropriate treatment. Environmental monitoring records were also reviewed for vivarium rooms in which the cotton rats were housed. Histopathology findings supported that the exophthalmos in these cotton rats was secondary to retro-orbital thrombosis associated with cardiomyopathy. The exophthalmic eyes were treated by either removal of the affected eye (enucleation) or surgical closure of the eyelids (temporary tarsorraphy). Enucleation of the exophthalmic eye was the best intervention for these aged cotton rats. These findings demonstrate the potential for a high incidence of ocular problems occurring secondary to cardiomyopathy in aged cotton rats. Enucleation as a therapeutic intervention for exophthalmic eyes in aged cotton rats prolongs the morbidity-free time span during which these aged animals can be used experimentally.

Tagging enhances histochemical and biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin.

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.

Establishment of Chronic Typhoid Infection in a Mouse Carriage Model Involves a Type 2 Immune Shift and T and B Cell Recruitment to the Gallbladder.

Typhoid fever, caused primarily by Salmonella enterica serovar Typhi (S Typhi), is a life-threatening systemic disease responsible for significant morbidity and mortality worldwide. Three to 5% of individuals infected with S Typhi become chronic carriers due to bacterial persistence in the gallbladder. We have demonstrated that Salmonella forms biofilms on gallstones to establish gallbladder carriage. However, an in-depth molecular understanding of chronic carriage in the gallbladder, from the perspective of both the pathogen and host, is poorly defined. To examine the dynamics of the gallbladder in response to Salmonella infection, we performed transcriptional profiling in the mouse gallbladder at early (7 days) and chronic (21 days) time points. Transcriptome sequencing (RNA-Seq) revealed a shift from a Th1 proinflammatory response at 7 days postinfection (dpi) toward an anti-inflammatory Th2 response by 21 dpi, characterized by increased levels of immunoglobulins and the Th2 master transcriptional regulator, GATA3. Additionally, bioinformatic analysis predicted the upstream regulation of characteristic Th2 markers, including interleukin-4 (IL-4) and Stat6. Immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis confirmed a significant increase in lymphocytes, including T and B cells, at 21 dpi in mice with gallstones. Interestingly, the levels of Salmonella-specific CD4 T cells were 10-fold higher in the gallbladder of mice with gallstones at 21 dpi. We speculate that the biofilm state allows Salmonella to resist the initial onslaught of the Th1 inflammatory response, while yet undefined events influence a switch in the host immunity toward a more permissive type 2 response, enabling the establishment of chronic infection.IMPORTANCE The existence of chronic typhoid carriers has been in the public eye for over 100 years in part because of the publicity around Typhoid Mary. Additionally, it has been known for decades that the gallbladder is the main site of persistence and recently that gallstones play a key role. Despite this, very little is known about the physiological conditions that allow Salmonella enterica serovar Typhi to persist in the gallbladder. In this study, we analyze the transcriptional profile of the gallbladder in a mouse model of chronic carriage. We found a shift from an early proinflammatory immune response toward a later anti-inflammatory response, which could explain the stalemate that allows Salmonella persistence. Interestingly, we found a 10-fold increase in the number of Salmonella-specific T cells in mice with gallstones. This work moves us closer to understanding the mechanistic basis of chronic carriage, with a goal toward eradication of the disease.

Identification of Recurrent Activating HER2 Mutations in Primary Canine Pulmonary Adenocarcinoma.

PURPOSE: Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC). EXPERIMENTAL DESIGN: We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response. RESULTS: We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2 V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2 V659E tumors. HER2 V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines (IC50 < 200 nmol/L in HER2 V659E vs. IC50 > 2,500 nmol/L in HER2 WT). CONCLUSIONS: This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition.

Ribonuclease 7 Shields the Kidney and Bladder from Invasive Uropathogenic Escherichia coli Infection.

BACKGROUND: Evidence suggests that antimicrobial peptides, components of the innate immune response, protect the kidneys and bladder from bacterial challenge. We previously identified ribonuclease 7 (RNase 7) as a human antimicrobial peptide that has bactericidal activity against uropathogenic Escherichia coli (UPEC). Functional studies assessing RNase 7's contributions to urinary tract defense are limited. METHODS: To investigate RNase 7's role in preventing urinary tract infection (UTI), we quantified urinary RNase 7 concentrations in 29 girls and adolescents with a UTI history and 29 healthy female human controls. To assess RNase 7's antimicrobial activity in vitro in human urothelial cells, we used siRNA to silence urothelial RNase 7 production and retroviral constructs to stably overexpress RNase 7; we then evaluated UPEC's ability to bind and invade these cells. For RNase 7 in vivo studies, we developed humanized RNase 7 transgenic mice, subjected them to experimental UTI, and enumerated UPEC burden in the urine, bladder, and kidneys. RESULTS: Compared with controls, study participants with a UTI history had 1.5-fold lower urinary RNase 7 concentrations. When RNase 7 was silenced in vitro, the percentage of UPEC binding or invading human urothelial cells increased; when cells overexpressed RNase 7, UPEC attachment and invasion decreased. In the transgenic mice, we detected RNase 7 expression in the kidney's intercalated cells and bladder urothelium. RNase 7 humanized mice exhibited marked protection from UPEC. CONCLUSIONS: These findings provide evidence that RNase 7 has a role in kidney and bladder host defense against UPEC and establish a foundation for investigating RNase 7 as a UTI prognostic marker or nonantibiotic-based therapy.

MAPK- and AKT-activated thyroid cancers are sensitive to group I PAK inhibition.

The number of individuals who succumb to thyroid cancer has been increasing and those who are refractory to standard care have limited therapeutic options, highlighting the importance of developing new treatments for patients with aggressive forms of the disease. Mutational activation of MAPK signaling, through BRAF and RAS mutations and/or gene rearrangements, and activation of PI3K signaling, through mutational activation of PIK3CA or loss of PTEN, are well described in aggressive thyroid cancer. We previously reported overactivation and overexpression of p21-activated kinases (PAKs) in aggressive human thyroid cancer invasive fronts and determined that PAK1 functionally regulated thyroid cancer cell migration. We reported mechanistic crosstalk between the MAPK and PAK pathways that are BRAF-dependent but MEK independent, suggesting that PAK and MEK inhibition might be synergistic. In the present study, we tested this hypothesis. Pharmacologic inhibition of group I PAKs using two PAK kinase inhibitors, G-5555 or FRAX1036, reduced thyroid cancer cell viability, cell cycle progression and migration and invasion, with greater potency for G-5555. Combination of G-5555 with vemurafenib was synergistic in BRAFV600E-mutated thyroid cancer cell lines. Finally, G-5555 restrained thyroid size of BRAFV600E-driven murine papillary thyroid cancer by >50% (P < 0.0001) and reduced carcinoma formation (P = 0.0167), despite maintenance of MAPK activity. Taken together, these findings suggest both that group I PAKs may be a new therapeutic target for thyroid cancer and that PAK activation is functionally important for BRAFV600E-mediated thyroid cancer development.

Pathology of wild Norway rats in Vancouver, Canada.

To achieve a contemporary understanding of the common and rare lesions that affect wild, urban Norway rats ( Rattus norvegicus), we conducted a detailed pathology analysis of 672 rats from Vancouver, British Columbia, Canada. Grossly evident lesions, such as wounds, abscesses, and neoplasms, were present in 71 of 672 rats (11%) and tended to be severe. The most common and significant lesions were infectious and inflammatory, most often affecting the respiratory tract and associated with bite wounds. We assessed a subset of rats (up to n = 406 per tissue) for the presence of microscopic lesions in a variety of organ systems. The most frequent lesions that could impact individual rat health included cardiomyopathy (128 of 406; 32%), chronic respiratory tract infections as indicated by pulmonary inducible bronchus-associated lymphoid tissue (270 of 403; 67%), tracheitis (192 of 372; 52%), and thyroid follicular hyperplasia (142 of 279; 51%). We isolated 21 bacterial species from purulent lesions in rats with bacterial infections, the most frequent of which were Escherichia coli, Enterococcus sp., and Staphylococcus aureus. Parasitic diseases in rats resulted from infection with several invasive nematodes: Capillaria hepatica in the liver (242 of 672; 36%), Eucoleus sp. in the upper gastrointestinal tract (164 of 399; 41%), and Trichosomoides crassicauda in the urinary bladder (59 of 194; 30%). Neoplastic, congenital, and degenerative lesions were rare, which likely reflects their adverse effect on survival in the urban environment. Our results establish a baseline of expected lesions in wild urban rats, which may have implications for urban rat and zoonotic pathogen ecology, as well as rat control in cities worldwide.

Comparative Pathologists: Ultimate Control Freaks Seeking Validation!

Definable, reproducible, and meaningful are elemental features of grading/scoring systems, while thoroughness, accuracy, and consistency are quality indicators of pathology reports. The expertise of pathologists is significantly underutilized when it is limited to rendering diagnoses. The opportunity to provide guidance on animal model development, experimental design, optimal sample collection, and data interpretation not only contributes to job satisfaction but also, more importantly, promotes validation of the pathology data. Keys to validation include standard operating procedures, experimental controls, and standardized nomenclature applied throughout the experimental design and execution, tissue sampling, and slide preparation, as well as the creation or adaptation and application of semiquantitative grading/scoring systems. Diagnostic drift, thresholds, mental noise, and various diurnal fluctuations strongly influence the repeatability of grading/scoring systems used by the same or different pathologists. Quantitative image analyses are not plagued by the visual and cognitive traps that affect manual semiquantitative grading schemes but may still be affected by technical variables associated with necropsy, tissue sampling, and slide preparation. The validity of a grading scheme is ultimately assessed by its repeatability and biologic relevance, so it is important to correlate scores with comprehensive pathobiology data such as results of antemortem imaging, clinical pathology data, body and organ weights, and histopathologic evaluation of full tissue sets.

Genetic ablation of interacting with Spt6 (Iws1) causes early embryonic lethality.

IWS1 is an RNA-polymerase II (RNAPII)-associated transcription elongation factor whose biological functions are poorly characterized. To shed some light on the function of this protein at the organismal level, we performed a systematic tissue analysis of its expression and generated Iws1-deficient mice. A thorough immunohistochemical characterization shows that IWS1 protein is present in the nucleus of all cells in most of the examined tissues, with few notable exceptions. We also report that ablation of Iws1 consistently causes lethality at the pre-implantation stage with high expression of the gene in fertilized oocytes. In summary, we are providing evidence that Iws1 is expressed in all adult organs and it is an essential gene for mouse embryonic development.

RANBP9 affects cancer cells response to genotoxic stress and its overexpression is associated with worse response to platinum in NSCLC patients.

Although limited by severe side effects and development of resistance, platinum-based therapies still represent the most common first-line treatment for non-small cell lung cancer (NSCLC). However, a crucial need in the clinical management of NSCLC is represented by the identification of cases sensitive to DNA damage response (DDR)-targeting drugs, such as cisplatin or PARP inhibitors. Here, we provide a molecular rationale for the stratification of NSCLC patients potentially benefitting from platinum compounds based on the expression levels of RANBP9, a recently identified player of the cellular DDR. RANBP9 was found overexpressed by immunohistochemistry (IHC) in NSCLC compared to normal adjacent tissues (NATs) (n = 147). Moreover, a retrospective analysis of 132 platinum-treated patients from the multi-centric TAILOR trial showed that RANBP9 overexpression levels are associated with clinical response to platinum compounds [Progression Free Survival Hazard Ratio (RANBP9 high vs low) 1.73, 95% CI 1.15-2.59, p = 0.0084; Overall Survival HR (RANBP9 high vs low) 1.99, 95% CI 1.27-3.11, p = 0.003]. Accordingly, RANBP9 KO cells showed higher sensitivity to cisplatin in comparison with WT controls both in vitro and in vivo models. NSCLC RANBP9 KO cells were also more sensitive than control cells to the PARP inhibitor olaparib alone and in combination with cisplatin, due to defective ATM-dependent and hyper-activated PARP-dependent DDR. The current investigation paves the way to prospective studies to assess the clinical value of RANBP9 protein levels as prognostic and predictive biomarker of response to DDR-targeting drugs, leading to the development of new tools for the management of NSCLC patients.

Alterations in Sod2-Induced Oxidative Stress Affect Endocrine Cancer Progression.

Context: Although important advances have been made in understanding the genetics of endocrine tumors, cellular physiology is relatively understudied as a determinant of tumor behavior. Oxidative stress and reactive oxygen species are metabolic factors that may affect tumor behavior, and these are, in part, controlled by manganese-dependent superoxide dismutase (MnSod), the mitochondrial superoxide dismutase (encoded by SOD2). Objective: We sought to understand the role of MnSod in the prognosis of aggressive human endocrine cancers and directly assessed the effect of MnSod under- or overexpression on tumor behavior, using established mouse thyroid cancer models. Methods: We performed transcriptome analysis of human and mouse models of endocrine cancer. To address the role of Sod2 in endocrine tumors, we introduced a Sod2 null allele or a transgenic Sod2 overexpression allele into mouse models of benign thyroid follicular neoplasia or aggressive, metastatic follicular thyroid cancer (FTC) and monitored phenotypic changes in tumor initiation and progression. Results: In the thyroid, SOD2/Sod2 was downregulated in FTC but not papillary thyroid cancer. Reduced expression of SOD2 was correlated with poorer survival of patients with aggressive thyroid or adrenal cancers. In mice with benign thyroid tumors, Sod2 overexpression increased tumor burden. In contrast, in mice with aggressive FTC, overexpression of Sod2 reduced tumor proliferation and improved mortality rates, whereas its deficiency enhanced tumor growth. Conclusion: Overall, our results indicate that SOD2 has dichotomous roles in cancer progression and acts in a context-specific manner.

Deletion of PPARγ in lung macrophages provides an immunoprotective response against M. tuberculosis infection in mice.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear transcription factor belonging to the superfamily of ligand-activated nuclear receptors. It is activated by diverse endogenous lipid metabolites as well as by exogenous ligands such as the thiazolidinediones. It regulates cellular metabolism, proliferation, differentiation, and inflammation, the latter in part through trans-repression of pro-inflammatory cytokines. PPARγ is highly expressed in alternatively activated alveolar macrophages (AMs), a primary host cell for airborne Mycobacterium tuberculosis (M.tb). Our previous in vitro study identified the importance of PPARγ activation through the mannose receptor (CD206) on human macrophages in enabling M. tb growth. The aim of the current study was to investigate the role of PPARγ in vivo during M. tb infection using a macrophage-specific PPARγ knock out mouse model with special emphasis on the lung environment. Our data show that the absence of PPARγ in lung macrophages reduces the growth of virulent M. tb, enhances pro-inflammatory cytokines and reduces granulomatous infiltration. These findings demonstrate that PPARγ activation, which down-regulates macrophage pro-inflammatory responses, impacts the lung's response to M. tb infection, thereby supporting PPARγ's role in tuberculosis (TB) pathogenesis.

Deletion of Rap1b, but not Rap1a or Epac1, Reduces Protein Kinase A-Mediated Thyroid Cancer.

BACKGROUND: Thyroid cancer is an emerging health problem in the United States and worldwide. With incidence rates of thyroid cancer rapidly rising, the need to develop new treatment options is becoming a priority, and understanding the molecular mechanisms of this disease is crucial to furthering these efforts. Thyroid growth is driven by the TSH/cAMP/PKA signaling pathway, and it has previously been shown that activation of PKA through genetic ablation of the regulatory subunit Prkar1a (Prkar1a KO) is sufficient to cause follicular thyroid cancer in mouse models. cAMP also activates the Epac proteins and their downstream effectors, Rap1a and Rap1b. METHODS: Previously, the authors' laboratory generated a mouse model of follicular thyroid cancer by conferring thyroid-specific deletion of Prkar1a (R1a-TpoKO). To probe the roles of other components of the PKA signaling system in the development of thyroid cancer, this study deleted Rap1 and Epac1 in the setting of the Prkar1a knockout. RESULTS: Deletion of Rap1 significantly decreases thyroid size and cancer incidence in Prkar1a KO thyroids. Further, isoform-specific ablation of Rap1a and Rap1b implicates Rap1b as the downstream effector of PKA during thyroid carcinogenesis. In vivo modeling provides definitive evidence that Epac1 plays little role in thyroid proliferation and is dispensable for thyroid carcinogenesis arising from the deletion of Prkar1a. CONCLUSIONS: This study demonstrate that PKA signaling to Rap1b is a key signaling node for follicular thyroid carcinogenesis, while Epac1 activity is not required for tumor development. This work sheds new light on the pathways involved in FTC development and identifies a possible target for the development of new therapies in the treatment of FTC.

Role of Wild-type and Recombinant Human T-cell Leukemia Viruses in Lymphoproliferative Disease in Humanized NSG Mice.

Chronic infection with human T-cell leukemia virus type 1 (HTLV1) can lead to adult T-cell leukemia (ATL). In contrast, infection with HTLV2 does not lead to leukemia, potentially because of distinct virus-host interactions and an active immune response that controls virus replication and, therefore, leukemia development. We created a humanized mouse model by injecting human umbilical-cord stem cells into the livers of immunodeficient neonatal NSG mice, resulting in the development of human lymphocytes that cannot mount an adaptive immune response. We used these mice to compare the ability of molecular clones of HTLV1, HTLV2, and select recombinant viruses to induce leukemia-lymphoma in vivo. Infection with HTLV1 strongly stimulated the proliferation of CD4+ T cells, whereas HTLV2 preferentially stimulated the proliferation of CD8+ T cells; both HTLV1 and HTLV2 induced lymphoproliferative disease. Uninfected and HTLV-infected humanized mice both showed granulomatous inflammation as a background lesion. Similarly, recombinant viruses that expressed the HTLV1 envelope protein (Env) on an HTLV2 background (HTLV2-Env1) or Env2 on an HTLV1 background (HTLV1-Env2) induced lymphoproliferative disease. HTLV2-Env1 stimulated the proliferation of CD4+ T cells, whereas HTLV1-Env2 stimulated both CD4+ and CD8+ T-cell subsets. Our results show that T-cell transformation in vivo is guided by the Env protein of the virus. Furthermore, our humanized mouse model is useful for exploring the preferred T-cell tropisms of HTLV1 and HTLV2.