The MexTAg collaborative cross: host genetics affects asbestos related disease latency, but has little influence once tumours develop.

Objectives: This study combines two innovative mouse models in a major gene discovery project to assess the influence of host genetics on asbestos related disease (ARD). Conventional genetics studies provided evidence that some susceptibility to mesothelioma is genetic. However, the identification of host modifier genes, the roles they may play, and whether they contribute to disease susceptibility remain unknown. Here we report a study designed to rapidly identify genes associated with mesothelioma susceptibility by combining the Collaborative Cross (CC) resource with the well-characterised MexTAg mesothelioma mouse model. Methods: The CC is a powerful mouse resource that harnesses over 90% of common genetic variation in the mouse species, allowing rapid identification of genes mediating complex traits. MexTAg mice rapidly, uniformly, and predictably develop mesothelioma, but only after asbestos exposure. To assess the influence of host genetics on ARD, we crossed 72 genetically distinct CC mouse strains with MexTAg mice and exposed the resulting CC-MexTAg (CCMT) progeny to asbestos and monitored them for traits including overall survival, the time to ARD onset (latency), the time between ARD onset and euthanasia (disease progression) and ascites volume. We identified phenotype-specific modifier genes associated with these traits and we validated the role of human orthologues in asbestos-induced carcinogenesis using human mesothelioma datasets. Results: We generated 72 genetically distinct CCMT strains and exposed their progeny (2,562 in total) to asbestos. Reflecting the genetic diversity of the CC, there was considerable variation in overall survival and disease latency. Surprisingly, however, there was no variation in disease progression, demonstrating that host genetic factors do have a significant influence during disease latency but have a limited role once disease is established. Quantitative trait loci (QTL) affecting ARD survival/latency were identified on chromosomes 6, 12 and X. Of the 97-protein coding candidate modifier genes that spanned these QTL, eight genes (CPED1, ORS1, NDUFA1, HS1BP3, IL13RA1, LSM8, TES and TSPAN12) were found to significantly affect outcome in both CCMT and human mesothelioma datasets. Conclusion: Host genetic factors affect susceptibility to development of asbestos associated disease. However, following mesothelioma establishment, genetic variation in molecular or immunological mechanisms did not affect disease progression. Identification of multiple candidate modifier genes and their human homologues with known associations in other advanced stage or metastatic cancers highlights the complexity of ARD and may provide a pathway to identify novel therapeutic targets.

Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment.

Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.

PD-L1+ dendritic cells in the tumor microenvironment correlate with good prognosis and CD8+ T cell infiltration in colon cancer.

BACKGROUND: The prognostic value of tumor-associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c+ DC co-expressing the immunoinhibitory molecule PD-L1 and their spatial relationship with CD8+ T-cells in patients treated for stage III colon cancer. METHODS: Tissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD-L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan-Meier estimates and Cox regression were used to assess survival. RESULTS: Intratumoral CD8+ cell density (HR = .52, 95% confidence interval [CI] .33-.83, P = .007), stromal CD11c+ cell density (HR = .52, 95% CI .33-.83, P = .006), intratumoral CD11c+ PD-L1+ cell density (HR = .57, 95% CI .35-.92, P = .021), and stromal CD11c+ PD-L1+ cell density (HR = .48, 95% CI .30-.77, P = .003) on leading-edge cores were all significantly associated with good survival. CD8+ cell density was positively correlated with both CD11c+ cell density and CD11c+ PD-L1+ cell density in tumor epithelium and stromal compartments. CONCLUSION: Here we showed that PD-L1-expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically "hot" tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor-associated DC may help to further elucidate their prognostic value.

Optimisation of multiplex immunofluorescence for a non-spectral fluorescence scanning system.

The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.

Tumour-infiltrating regulatory T cell density before neoadjuvant chemoradiotherapy for rectal cancer does not predict treatment response.

Neoadjuvant (preoperative) chemoradiotherapy (CRT) decreases the risk of rectal cancer recurrence and reduces tumour volume prior to surgery. However, response to CRT varies considerably between individuals and factors associated with response are poorly understood. Foxp3+ regulatory T cells (Tregs) inhibit anti-tumour immunity and may limit any response to chemotherapy and radiotherapy. We have previously reported that a low density of Tregs in the tumour stroma following neoadjuvant CRT for rectal cancer is associated with improved tumour regression. Here we have examined the association between Treg density in pre-treatment diagnostic biopsy specimens and treatment response, in this same patient cohort. We aimed to determine whether pre-treatment tumour-infiltrating Treg density predicts subsequent response to neoadjuvant CRT. Foxp3+, CD8+ and CD3+ cell densities in biopsy samples from 106 patients were assessed by standard immunohistochemistry (IHC) and evaluated for their association with tumour regression grade and survival. We found no association between the density of any T cell subset pre-treatment and clinical outcome, indicating that tumour-infiltrating Treg density does not predict response to neoadjuvant CRT in rectal cancer. Taken together with the findings of the previous study, these data suggest that in the context of neoadjuvant CRT for rectal cancer, the impact of chemotherapy and/or radiotherapy on anti-tumour immunity may be more important than the state of the pre-existing local immune response.

Reduction in Size and Number of Plantar Verrucae in Human Immunodeficiency Virus-Infected Individuals After the Implementation of Highly Active Antiretroviral Therapy.

BACKGROUND: Implementation of highly active antiretroviral therapy (HAART) significantly increased the life expectancy of those living with human immunodeficiency virus (HIV). Except for prevalence, scientific reports regarding clinical manifestations of plantar verrucae in the post-HAART era are lacking. The objective of this study was to compare clinical manifestations of plantar verrucae between HIV-infected and noninfected individuals and then to compare these findings with those observed before the implementation of HAART. METHODS: Nineteen patients with plantar verrucae (ten with HIV and nine without HIV) were examined to determine the size, number, and clinical type of verrucae present. The two groups were first compared with each other and then with previously collected data from a similar analysis conducted in 1995, before the implementation of HAART. Statistical significance was determined using the Fisher exact test or the Wilcoxon rank sum test. RESULTS: No significant differences were observed in the size, number, or clinical type of verrucae between HIV-negative and HIV-positive patients. Compared with the 1995 data, there was a significant decrease in the number of verrucae lesions per individual and a nonsignificant decrease in the average size of verrucae in HIV-positive patients. CONCLUSIONS: Study results indicate that the implementation of HAART has impacted the clinical manifestations of plantar verrucae in HIV-positive individuals. Further analyses with a larger number of patients are required to confirm and substantiate these findings.

Posterior Facet Settling and Changes in Bohler's Angle in Operatively and Nonoperatively Treated Calcaneus Fractures.

BACKGROUND: Patients with calcaneus fractures often exhibit settling of the posterior facet with a corresponding decrease in Bohler's angle (BA) following either operative or nonoperative treatment. Both injury BA and postoperative BA have been shown to be prognostic for outcomes; however, the demographic and surgeon-specific factors that may contribute to settling have not been critically examined in the literature. The purpose of this study was to identify these causative factors. METHODS: 234 patients with intra-articular calcaneus fractures were analyzed. All patients had preoperative plain radiographs, at least 5 months of orthopedic follow-up, and computed tomography scanning performed. BA was measured on the injury radiographs for all patients. For operatively treated patients, BA was measured on the immediate postoperative radiographs and compared with the last available radiograph. For nonoperatively treated patients, BA was measured on the last available radiograph. All patients were fully weightbearing at the time of final follow-up but not on initial radiographs due to their recent injury. Demographic data including age, gender, energy of injury mechanism, tobacco use, diabetes, osteoporosis, rheumatoid arthritis, and substance/alcohol abuse were retrospectively collected. Fractures were classified using the Essex-Lopresti and Sanders classifications. Time to full weightbearing was documented, as were any reports of noncompliance with weightbearing restrictions. For patients treated operatively, type of fixation (calcaneal-specific perimeter plate, nonperimeter plate, screw fixation), use of locking screws, use of bone graft or graft substitutes, and the number of screws supporting the posterior facet were documented. RESULTS: There was a statistically significant amount of settling within the operative and nonoperative groups, but there was no statistically significant difference in settling of BA between the groups. The average settling of BA for the operative and nonoperative group was 8 degrees. Age greater than 50 years, diabetes, and alcohol abuse were all statistically significant and independent predictors of BA settling irrespective of treatment. CONCLUSION: The amount of BA settling between the operative and nonoperative group was not significant and showed an average decrease of 8 degrees in each group. However, the amount of settling that we found, irrespective of treatment, increased with patient age, alcohol abuse, and diabetes. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

A cadaver study revisiting the original methodology of Lauge-Hansen and a commentary on modern usage.

BACKGROUND: The study by Lauge-Hansen published in the Archives of Surgery in 1950 still stands as the seminal work for our understanding of the pathomechanics of ankle fractures. The purpose of the present study was to recreate Lauge-Hansen's experiments for the supination-external rotation (SER) fracture mechanism and to determine whether the predicted sequence of osseous and soft-tissue injury is reproducible on the basis of his originally described methodology. METHODS: Ten fresh-frozen cadaver specimens amputated above the knee were utilized. The foot was axially loaded in a position of neutral dorsiflexion and supination. External rotation was applied manually in accordance with Lauge-Hansen's description until osseous and/or soft-tissue injury occurred. Fluoroscopic images were made and anatomic dissection was performed. RESULTS: Although several specimens exhibited findings consistent with certain stages of the SER injury pattern, no specimen demonstrated the complete sequence of predicted osseous and soft-tissue injury. CONCLUSIONS: Loading cadaver specimens with an SER mechanism utilizing a methodology similar to that in the original experiments by Lauge-Hansen does not reliably produce the sequence of osseous and soft-tissue injury predicted by Lauge-Hansen.

FXYD5 modulates Na+ absorption and is increased in cystic fibrosis airway epithelia.

FXYD5, also known as dysadherin, belongs to a family of tissue-specific regulators of the Na(+)-K(+)-ATPase. We determined the kinetic effects of FXYD5 on Na(+)-K(+)-ATPase pump activity in stably transfected Madin-Darby canine kidney cells. FXYD5 significantly increased the apparent affinity for Na(+) twofold and decreased the apparent affinity for K(+) by 60% with a twofold increase in V(max) of K(+), a pattern that would increase activity and Na(+) removal from the cell. To test the effect of increased Na(+) uptake on FXYD5 expression, we analyzed Madin-Darby canine kidney cells stably transfected with an inducible vector expressing all three subunits of the epithelial Na(+) channel (ENaC). Na(+)-K(+)-ATPase activity increased sixfold after 48-h ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na(+) absorption by itself downregulates epithelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of Na(+) in the airways, accompanied by increased Na(+)-K(+)-ATPase activity. However, FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry, and immunoblot analysis (P < 0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared with controls (P < 0.02). Treatment of human tracheal epithelial cells with a CFTR inhibitor (I-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P < 0.001), which was abrogated by an inhibitor of NF-kappaB. Thus FXYD5 is upregulated in CF epithelia, and this change may exacerbate the Na(+) hyperabsorption and surface liquid dehydration observed in CF airway epithelia.

S163 is critical for FXYD5 modulation of wound healing in airway epithelial cells.

The FXYD family, which contains seven members, are tissue specific regulators of the Na,K-ATPase. Increased expression of FXYD5, a cancer-cell-associated membrane glycoprotein, has been associated with increased cell motility and metastatic potential. To better understand how FXYD5 may modulate cell motility, we analyzed S163, a conserved residue in all FXYD family members located in the C-terminus. Ectopic expression of human FXYD5 S163 mutants in HEK 293 cells showed that negative charge at S163 (S163D) decreased membrane localization, assessed by immunofluorescence. Coimmunoprecipitation studies revealed decreased FXYD5/Na,K-ATPase interaction for S163D compared with wild-type or S163A mutants. Interestingly, FXYD5 overexpression induced expression of vimentin, a marker of epithelial-mesenchymal transition, in murine airway epithelial cells. Because Na,K-ATPase expression is decreased in some forms of cancer and is critical for establishing cell polarity and suppressing cell motility, we analyzed S163 mutants in an epithelial cell scratch-wound model as a measure of cell migration. Wild-type FXYD5 overexpression increased reepithelialization (p<0.0001), which was further increased in S163D mutants (p<0.005). However, S163A mutants inhibited epithelial cell migration compared with wild-type FXYD5 overexpression (p<0.0001). We conclude that negative charge at S163 regulates FXYD5/Na,K-ATPase interaction and that this interaction modulates cell migration across a wound in airway epithelial cells.

Four-cut sinus computed tomographic scanning in screening for sinus disease.

OBJECTIVE: We sought to calculate the sensitivity, specificity, negative predictive value, and positive predictive value of a limited sinus computed tomographic (CT) scan for sinus disease. METHODS: We conducted a retrospective case series. Inclusion criteria included a sinus CT scan obtained between April 1999 and November 2000. From the complete sinus CT scan, the limited series were obtained by blocking from view all the other cuts and leaving the radiologist only four slices to read (midfrontal, anterior maxillary sinuses, posterior maxillary sinuses, and midsphenoidal). The complete CT scan was the "gold standard." RESULTS: Fifty-one patients were eligible. We observed 81.3% sensitivity, 89.5% specificity, a 73.9% negative predictive value, and a 92.9% positive predictive value for the limited CT scan for the detection of sinus disease. This sensitivity and specificity were higher than reported in the literature for plain films. CONCLUSION: The limited sinus CT scan is superior to plain radiographs but is not as good as the full CT scan in the evaluation of sinusitis.

Nanoparticles of compacted DNA transfect postmitotic cells.

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.