IL10 deficiency promotes alveolar enlargement and lymphoid dysmorphogenesis in the aged murine lung

The connection between aging‐related immune dysfunction and the lung manifestations of aging is poorly understood. A detailed characterization of the aging IL10‐deficient murine lung, a model of accelerated aging and frailty, reconciles features of both immunosenescence and lung aging in a coherent model. Airspace enlargement developed in the middle‐aged (12 months old) and aged (20–22 months old) IL10‐deficient lung punctuated by an expansion of macrophages and alveolar cell apoptosis. Compared to wild‐type (WT) controls, the IL10‐deficient lungs from young (4‐month‐old) mice showed increased oxidative stress which was enhanced in both genotypes by aging. Active caspase 3 staining was increased in the alveolar epithelial cells of aged WT and mutant lungs but was greater in the IL10‐deficient milieu. Lung macrophages were increased in the aged IL10‐deficient lungs with exuberant expression of MMP12. IL10 treatment of naïve and M2‐polarized bone marrow‐derived WT macrophages reduced MMP12 expression. Conditioned media studies demonstrated the secretome of aged mutant macrophages harbors reduced AECII prosurvival factors, specifically keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF), promotes cell death, and reduces survival of primary alveolar epithelial cells. Compared to WT controls, aged IL10‐deficient mice have increased parenchymal lymphoid collections comprised of a reduced number of apoptotic cells and B cells. We establish that IL10 is a key modulator of airspace homeostasis and lymphoid morphogenesis in the aging lung enabling macrophage‐mediated alveolar epithelial cell survival and B‐cell survival within tertiary lymphoid structures.     

Clinical,Molecular and Genetic Validation of a Murine Orthotopic Xenograft Model of Pancreatic Adenocarcinoma Using Fresh Human Specimens

Background

Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC.

Methods

Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression.

Results

Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines.

Conclusions

The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy.     

Eubacterial SpoVG Homologs Constitute a New Family of Site-Specific DNA-Binding Proteins

A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins.     

Visualizing the Prostate Gland by MR Imaging in Young and Old Mice

Purpose

Prostate imaging requires optimization in young and old mouse models. We tested which MR sequences and field strengths best depict the prostate gland in young and old mice; and, whether prostate MR signal, size, and architecture change with age.

Technique

Magnetic resonance imaging (MRI) of the prostate of young (2 months) and old (18 months) male nude mice (n = 6) was performed at 4.7 and 7 T and SCID mice (n = 6) at 7 T field strengths, using T1, fat suppressed T1, DWI, T2, fat suppressed T2, as well as T2-based- and proton density-based Dixon “water only” sequences. Images were ranked for best overall sequence for prostate visualization, prostate delineation, and quality of fat suppression. Prostate volume and signal characteristics were compared and histology was performed.

Results

T2-based-Dixon “water only” images ranked best overall for prostate visualization and delineation as well as fat suppression (n = 6, P<0.001) at both 4.7 T and 7 T in nude and 7T in SCID mice. Evaluated in nude mice, T2-based Dixon “water only” had greater prostate CNR and lower fat SNR at 7 T than 4.7 T (P<0.001). Prostate volume was less in older than younger mice (n = 6, P<0.02 nude mice; n = 6, P<0.002 SCID mice). Prostate T2 FSE as well as proton density-based and T2-based-Dixon “water only” signal intensity was higher in younger than older mice (P<0.001 nude mice; P<0.01 SCID mice) both at 4.7 and 7 T. This corresponded to an increase in glandular hyperplasia in older mice by histology (P<0.01, n = 6).

Conclusion

T2-based Dixon “water only” images best depict the mouse prostate in young and old nude mice at 4.7 and 7 T. The mouse prostate decreases in size with age. The decrease in T2 and T2-based Dixon “water only” signal with age corresponds with glandular hyperplasia. Findings suggest age should be an important determinant when choosing models of prostate biology and disease.     

Population structure of the Chagas disease vector,Triatoma infestans,at the urban–rural interface

The increasing rate of biological invasions resulting from human transport or human‐mediated changes to the environment has had devastating ecological and public health consequences. The kissing bug, Triatoma infestans, has dispersed through the Peruvian city of Arequipa. The biological invasion of this insect has resulted in a public health crisis, putting thousands of residents of this city at risk of infection by Trypanosoma cruzi and subsequent development of Chagas disease. Here, we show that populations of Tria. infestans in geographically distinct districts within and around this urban centre share a common recent evolutionary history although current gene flow is restricted even between proximal sites. The population structure among the Tria. infestans in different districts is not correlated with the geographical distance between districts. These data suggest that migration among the districts is mediated by factors beyond the short‐range migratory capabilities of Tria. infestans and that human movement has played a significant role in the structuring of the Tria. infestans population in the region. Rapid urbanization across southern South America will continue to create suitable environments for Tria. infestans, and knowledge of its urban dispersal patterns may play a fundamental role in mitigating human disease risk.     

Metabolomic response to exercise training in lean and diet-induced obese mice

Exercise training is a common therapeutic approach known to antagonize the metabolic consequences of obesity. The aims of the present study were to examine 1) whether short-term, moderate-intensity exercise training alters the basal metabolite profile and 2) if 10 days of mild exercise training can correct obesity-induced shifts in metabolic spectra. After being weaned, male C57BL/6J littermates were randomly divided into two diet groups: low fat (LF) or high fat (HF). After 12 wk of dietary manipulation, HF animals were obese and hyperglycemic compared with LF animals. Mice from each group were further divided into sedentary or exercise treatments. Exercise training consisted of wheel running exercise (2 h/day, 10 days, 5.64 m/min). After exercise training, animals were rested (36 h) and fasted (6 h) before serum collection. Samples were analyzed by high-resolution one-dimensional proton NMR. Fifty high- and medium-concentration metabolites were identified. Pattern recognition algorithms and multivariate modeling were used to identify and isolate significant metabolites changing in response to HF and exercise training. The results showed that while exercise can mitigate some of the abnormal patterns in metabolic spectra induced by HF diet feeding, they cannot negate it. In fact, when the effects of diet and exercise were compared, diet was a stronger predictor and had the larger influence on the metabolic profile. External validation of models showed that diet could be correctly classified with an accuracy of 89%, whereas exercise training could be classified 73% of the time. The results demonstrate metabolomics to effectively characterize obesity-induced perturbations in metabolism and support the concept that exercise is beneficial for this condition.     

A Modified CDC Biofilm Reactor to Produce Mature Biofilms on the Surface of PEEK Membranes for an In Vivo Animal Model Application

Biofilm-related infections have become a major clinical concern. Typically, animal models that involve inoculation with planktonic bacteria have been used to create positive infection signals and examine antimicrobial strategies for eradicating or preventing biofilm-related infection. However, it is estimated that 99.9% of bacteria in nature dwell in established biofilms. As such, open wounds have significant potential to become contaminated with bacteria that reside in a well-established biofilm. In this study, a modified CDC biofilm reactor was developed to repeatably grow mature biofilms of Staphylococcus aureus on the surface of polyetheretherketone (PEEK) membranes for inoculation in a future animal model of orthopaedic implant biofilm-related infection. Results indicated that uniform, mature biofilms repeatably grew on the surface of the PEEK membranes.     

The phylogenetic distribution of the ampulla ureter and ampulla urogenital/uriniferous papilla in the Serpentes

The ampulla ureter and ampulla urogenital/uriniferous papilla represent differing morphologies of the caudal urogenital ducts in snakes. The ampulla ureter is an enlarged portion of the caudal extremity of the ureter that communicates the cranial regions of the ureter and the ductus deferens/Wolffian duct to the urodaeum. The ampulla urogenital/uriniferous papilla is an enlarged pouch, distinct from the ureter, which communicates the ureter and ductus deferens/Wolffian duct to the urodaeum. Although functional differences of these two structures are unknown, the ampulla urogenital/uriniferous papilla may have evolved for urine storage in males and females, and secondarily evolved a reproductive function in males. The most parsimonious optimization of the ampulla ureter and ampulla urogenital/uriniferous papilla indicates that the ampulla ureter is the ancestral state in snakes. Examining the presence or absence of the ampulla ureter and ampulla urogenital/uriniferous papilla in snakes on conflicting caenophidian phylogenies results in two hypotheses for the evolution of these variant morphologies: (1) The ampulla urogenital/uriniferous papilla evolved from the ampulla ureter independently in the Colubroidea and Elapoidea with subsequent losses of the ampulla urogenital/uriniferous papilla in the Elapoidea and (2) a single transition from the ampulla ureter to the ampulla urogenital/uriniferous papilla on the branch leading to the Colubroidea + Elapoidea with subsequent losses of the ampulla urogenital/uriniferous papilla in the Elapoidea and Colubroidea. The presence of the ampullae urogenital/uriniferous papilla in only the Colubroidea and Elapoidea highlights the affinity of these two taxonomic groups, a relationship that is strongly supported in published cladograms produced with molecular datasets.     

Synthesis and optimization of novel 4,4-disubstituted cyclohexylbenzamide derivatives as potent 11β-HSD1 inhibitors

The synthesis and SAR of a series of 4,4-disubstituted cyclohexylbenzamide inhibitors of 11β-HSD1 are described. Optimization rapidly led to potent, highly selective, and orally bioavailable inhibitors demonstrating efficacy in both rat and non-human primate ex vivo pharmacodynamic models.