Development of fatty liver disease model using high cholesterol and low choline diet in white leghorn chickens.

Nonalcoholic fatty liver disease (NAFLD), which shows similar symptoms as fatty liver hemorrhage syndrome (FLHS) in chickens, is the most common cause of chronic liver disease and cancer in humans. NAFLD patients and FLHS in chickens have demonstrated severe liver disorders when infected by emerging strains of human hepatitis E virus (HEV) and avian HEV, respectively. We sought to develop a fatty liver disease chicken model by altering the diet of 3-week-old white leghorn chickens. The high cholesterol, and low choline (HCLC) diet included 7.6% fat with additional 2% cholesterol and 800 mg/kg choline in comparison to 5.3% fat, and 1,300 mg/kg choline in the regular diet. Our diet induced fatty liver avian model successfully recapitulates the clinical features seen during NAFLD in humans and FLHS in chickens, including hyperlipidemia and hepatic steatosis, as indicated by significantly higher serum triglycerides, serum cholesterol, liver triglycerides, cholesterol, and fatty acids. By developing this chicken model, we expect to provide a platform to explore the role of lipids in the liver pathology linked with viral infections and contribute to the development of prophylactic interventions.

Gene expression landscape of cutaneous squamous cell carcinoma progression.

BACKGROUND: Cutaneous squamous cell carcinomas (SCC) are the second most common human cancer and have been characterized by RNA sequencing (RNA-Seq); however, the transferability of findings from individual studies may be limited by small sample sizes and diverse analysis protocols. OBJECTIVES: To define the transcriptome landscape at different stages in the progression of normal skin to SCC through a meta-analysis of publicly available RNA-Seq samples. METHODS: Whole-transcriptome data from 73 normal skin samples, 46 actinic keratoses (AK), 16 in situ SCC, 13 keratoacanthomas (KA), and 147 SCC (including 30 SCC from immunocompromised patients and 8 SCC from individuals with recessive dystrophic epidermolysis bullosa [RDEB]) was uniformly processed to harmonize gene expression. Differential expression, fusion detection, and cell-type deconvolution analyses were performed. RESULTS: Individual RNA-Seq studies of SCC demonstrated study-specific clustering and varied widely in their differential gene expression detection. Following batch correction, we defined a consensus set of differentially expressed genes (DEGs), including those altered in the preinvasive stages of SCC development, and used single-cell RNA-Seq data to demonstrate that DEGs are often, but not always, expressed by tumor-specific keratinocytes (TSKs). Analysis of the cellular composition of SCC, KA, and RDEB-SCC identified an increase in differentiated keratinocytes in KA, while RDEB-SCC contained the most TSKs. Compared to SCC arising in immunocompetent patients, SCC from immunosuppressed individuals demonstrated fewer memory B cells and CD8 T cells. A comprehensive and unbiased search for fusion transcripts in SCC and intermediate disease stages identified few candidates that recur in >1% of all specimens, suggesting most SCC are not driven by oncogenic gene fusions. Finally, using GTEx data, we distilled a novel 300-gene signature of chronic sun exposure that affirms greater cumulative ultraviolet (UV) exposure in later stages of SCC development. CONCLUSIONS: Our results define the gene expression landscape of SCC progression, characterize cell subpopulation heterogeneity in SCC subtypes that contribute to their distinct clinical phenotypes, demonstrate that gene fusions are not a common cause of SCC, and identify UV-responsive genes associated with SCC development.

Unravelling the landscape of skin cancer through single-cell transcriptomics.

The human skin is a complex organ that forms the first line of defense against pathogens and external injury. It is composed of a wide variety of cells that work together to maintain homeostasis and prevent disease, such as skin cancer. The exponentially rising incidence of skin malignancies poses a growing public health challenge, particularly when the disease course is complicated by metastasis and therapeutic resistance. Recent advances in single-cell transcriptomics have provided a high-resolution view of gene expression heterogeneity that can be applied to skin cancers to define cell types and states, understand disease evolution, and develop new therapeutic concepts. This approach has been particularly valuable in characterizing the contribution of immune cells in skin cancer, an area of great clinical importance given the increasing use of immunotherapy in this setting. In this review, we highlight recent skin cancer studies utilizing bulk RNA sequencing, introduce various single-cell transcriptomics approaches, and summarize key findings obtained by applying single-cell transcriptomics to skin cancer.

MAB21L4 Deficiency Drives Squamous Cell Carcinoma via Activation of RET.

Epithelial squamous cell carcinomas (SCC) most commonly originate in the skin, where they display disruptions in the normally tightly regulated homeostatic balance between keratinocyte proliferation and terminal differentiation. We performed a transcriptome-wide screen for genes of unknown function that possess inverse expression patterns in differentiating keratinocytes compared with cutaneous SCC (cSCC), leading to the identification of MAB21L4 (C2ORF54) as an enforcer of terminal differentiation that suppresses carcinogenesis. Loss of MAB21L4 in human cSCC organoids increased expression of RET to enable malignant progression. In addition to transcriptional upregulation of RET, deletion of MAB21L4 preempted recruitment of the CacyBP-Siah1 E3 ligase complex to RET and reduced its ubiquitylation. In SCC organoids and in vivo tumor models, genetic disruption of RET or selective inhibition of RET with BLU-667 (pralsetinib) suppressed SCC growth while inducing concomitant differentiation. Overall, loss of MAB21L4 early during SCC development blocks differentiation by increasing RET expression. These results suggest that targeting RET activation is a potential therapeutic strategy for treating SCC. SIGNIFICANCE: Downregulation of RET mediated by MAB21L4-CacyBP interaction is required to induce epidermal differentiation and suppress carcinogenesis, suggesting RET inhibition as a potential therapeutic approach in squamous cell carcinoma.

Mutant collagen COL11A1 enhances cancerous invasion.

Collagens are the most abundant proteins in the body and comprise the basement membranes and stroma through which cancerous invasion occurs; however, a pro-neoplastic function for mutant collagens is undefined. Here we identify COL11A1 mutations in 66 of 100 cutaneous squamous cell carcinomas (cSCCs), the second most common U.S. cancer, concentrated in a triple helical region known to produce trans-dominant collagens. Analysis of COL11A1 and other collagen genes found that they are mutated across common epithelial malignancies. Knockout of mutant COL11A1 impairs cSCC tumorigenesis in vivo. Compared to otherwise genetically identical COL11A1 wild-type tissue, gene-edited mutant COL11A1 skin is characterized by induction of ß1 integrin targets and accelerated neoplastic invasion. In mosaic tissue, mutant COL11A1 cells enhanced invasion by neighboring wild-type cells. These results suggest that specific collagens are commonly mutated in cancer and that mutant collagens may accelerate this process.

Outcomes of Percutaneous Coronary Intervention in Patients with Spontaneous Coronary Artery Dissection.

OBJECTIVES: To compare outcomes of percutaneous coronary intervention (PCI) in spontaneous coronary artery dissection (SCAD) patients versus conservative therapy. BACKGROUND: SCAD is an important cause of myocardial infarction (MI) in young-to-middle-aged women. Percutaneous coronary intervention (PCI) is often pursued, but outcomes compared to conservative therapy are unclear. METHODS: 403 nonatherosclerotic SCAD patients were enrolled between 2011 and 2017 and prospectively followed up in our Vancouver General Hospital registries. Detailed baseline, hospital, PCI, and outcomes were recorded. We explored the outcomes of SCAD patients who underwent PCI during their initial presentation. RESULTS: PCI was performed in 75 patients, the average age was 48.9 ± 10.1 yrs, and 94.7% were women. All presented with MI; 50.7% STEMI, 49.3% NSTEMI, and 13.3% had VT/VF. PCI was successful in 34.7%, partially successful in 37.3%, and unsuccessful in 28.0%. Stents were deployed in 73.3%, 16.0% had balloon angioplasty alone, 10.7% had wiring attempts only, and 5.3% required bailout surgery. Major adverse cardiovascular event rates (MACE) were significantly higher with the PCI group in hospital (29.3% versus 2.8%, p < 0.001), and at median follow-up of 3.7 yrs (58.7% versus 22.6% (p < 0.001) compared to the non-PCI group. CONCLUSION: PCI in SCAD patients was associated with high failure rate and MACE in hospital and at long-term follow-up. These findings support the recommendation of conservative therapy as first-line management unless high-risk features are present.

PD-L1 Expression and Tumor-Infiltrating Lymphocytes in High-Risk and Metastatic Cutaneous Squamous Cell Carcinoma.

OBJECTIVE: To characterize programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) positivity for locally aggressive or regionally metastatic cutaneous head and neck squamous cell carcinoma (cHNSCC). STUDY DESIGN: Retrospective chart review, followed by immunohistochemical staining of archived tumor specimens. SETTING: Tertiary academic medical center. SUBJECTS AND METHODS: After identification of 101 patients treated surgically for locally advanced or regionally metastatic cHNSCC, archived tissue was stained and graded for PD-L1 expression in addition to TIL presence. Cross-tabulation was performed to examine the association between either of these variables and clinicopathologic features and outcomes. RESULTS: A total of 101 patients met inclusion criteria, but archived tissue was available only for 83 (31 primaries, 52 metastases). The majority of primary tumors demonstrated grade 1 PD-L1 staining, while grade 2 staining was more likely for metastases. Neither high- nor low-grade PD-L1 expression correlated with any clinicopathologic variable for primary tumors. However, for metastases, high-grade staining was significantly associated with regional recurrence (15 of 19, P = .02). TILs were present for 65% of primary tumors and 90% of regional metastases but did not correlate with any clinicopathologic variables. CONCLUSION: Diffuse expression of PD-L1 in this study highlights the possibility of using immunotherapy in the form of programmed death 1/PD-L1 blockade to improve treatment for this devastating disease. However, further studies are needed to clarify the significance of PD-L1 expression and TIL positivity for locally advanced or regionally metastatic cHNSCC.

Case reports of coronary fibromuscular dysplasia and spontaneous coronary artery dissections.

Spontaneous coronary artery dissection (SCAD) is an important cause of acute coronary syndrome especially in women. The most common underlying predisposing cause of SCAD is fibromuscular dysplasia (FMD), a non-inflammatory arteriopathy that results in weakening of the affected arteries, and can cause dissection or aneurysm. Coronary FMD (CFMD) was described as rare, and was shown to cause SCAD in histopathological case reports. Unfortunately, CFMD is challenging to diagnose on coronary angiography, as the findings can be similar to other causes of coronary artery disease. Therefore, we illustrate two case examples of CFMD on coronary angiography, and highlight findings on optical coherence tomography to aid diagnosis.

Node-positive cutaneous squamous cell carcinoma of the head and neck: Survival, high-risk features, and adjuvant chemoradiotherapy outcomes.

BACKGROUND: Data lacks to guide treatment of regionally metastatic cutaneous head and neck squamous cell carcinoma (HNSCC). METHODS: We conducted a retrospective review of 80 patients treated for regionally metastatic cutaneous HNSCC. The effect of various clinicopathologic variables on overall survival (OS) was investigated, in addition to outcomes by treatment modality. RESULTS: On multivariate regression, cutaneous primary >2 cm (p = .03) and extracapsular spread (ECS; p = .01) were significantly associated with decreased OS. Location of regional metastasis (neck vs parotid vs both) had no effect on OS (p = .2), nor did the presence of a cutaneous primary at the time of presentation (p = .9). The 3-year survival was 43%, 52%, and 49% for surgery alone, adjuvant radiation, and adjuvant chemoradiation, respectively. Fifty-one percent of patients had a recurrence of their disease. CONCLUSION: Regionally metastatic cutaneous HNSCC is an aggressive disease associated with high recurrence rates. Patients with tumors >2 cm and ECS have poorer OS despite adjuvant therapy. © 2017 Wiley Periodicals, Inc. Head Neck 39: 881-885, 2017.

Body Image & Quality of Life: Changes With Gastric Bypass and Body Contouring.

INTRODUCTION: Bariatric surgery has emerged as an effective method of combating the morbid obesity epidemic. However, the massive weight loss that follows may result in contour changes that can affect body image and quality of life. Our study examines the effects and consequences of bariatric surgery and subsequent body contouring on body image and quality of life. METHODS: Patients were prospectively followed up through their experience with bariatric surgery and subsequent body contouring surgery. Using 2 validated survey instruments, the Multidimensional Body-Self Relations Questionnaire and the Short Form 36 (SF-36), patients completed questionnaires preoperatively and at 6, 12, and 24 months postoperatively. Mean scores were determined by repeated measures analyses of variance F tests. RESULTS: One hundred seventy-five patients were surveyed before bariatric surgery, with noted declines in survey completion at 6, 12, and 24 months. Appearance Evaluation scores improved significantly at all intervals (P = 0.0033), as did Body Area Satisfaction Scale and Appearance Orientation scores (P = 0.0079 and P = 0.044, respectively). While Overweight Preoccupation and Self-Classified Weight scores decreased over time, only the latter was significant (P < 0.0001). The composite SF-36 score for patients awaiting bariatric surgery (54.1%) with postoperative scores at 6 (67.6%,), 12 (at 74.0%), and 24 (76.7%) months being significantly higher (P < 0.0001). The body contouring group consisted of 41 patients who primarily had lower body procedures, with 31 patients surveyed at 6 months and 27 patients at 12 months. For this cohort, Appearance Evaluation and Body Area Satisfaction Scale scores both improved significantly (P = 0.0001 and P = 0.0005, respectively) whereas Appearance Orientation scores declined significantly (P = 0.0055). Both Overweight Preoccupation and Self-Classified Weight scores decreased with only the latter being statistically significant (P = 0.0286). Postoperative SF-36 scores at 6 (72.9%) and 12 (64.5%) months were no different than patients awaiting body contouring (71.3%). CONCLUSIONS: Using 2 validated survey instruments, we show that patients undergoing bariatric surgery have improvements in body image and quality of life. Subsequent postbariatric body contouring surgery results in further improvements in body image. Our findings provide measurable evidence for the value of body contouring after significant weight loss, which may favor greater insurance coverage for this patient population.

Intestinal Monocyte-Derived Macrophages Control Commensal-Specific Th17 Responses.

Generation of different CD4 T cell responses to commensal and pathogenic bacteria is crucial for maintaining a healthy gut environment, but the associated cellular mechanisms are poorly understood. Dendritic cells (DCs) and macrophages (Mfs) integrate microbial signals and direct adaptive immunity. Although the role of DCs in initiating T cell responses is well appreciated, how Mfs contribute to the generation of CD4 T cell responses to intestinal microbes is unclear. Th17 cells are critical for mucosal immune protection and at steady state are induced by commensal bacteria, such as segmented filamentous bacteria (SFB). Here, we examined the roles of mucosal DCs and Mfs in Th17 induction by SFB in vivo. We show that Mfs, and not conventional CD103(+) DCs, are essential for the generation of SFB-specific Th17 responses. Thus, Mfs drive mucosal T cell responses to certain commensal bacteria.

Genomic analysis of mycosis fungoides and Sézary syndrome identifies recurrent alterations in TNFR2.

Mycosis fungoides and Sézary syndrome comprise the majority of cutaneous T cell lymphomas (CTCLs), disorders notable for their clinical heterogeneity that can present in skin or peripheral blood. Effective treatment options for CTCL are limited, and the genetic basis of these T cell lymphomas remains incompletely characterized. Here we report recurrent point mutations and genomic gains of TNFRSF1B, encoding the tumor necrosis factor receptor TNFR2, in 18% of patients with mycosis fungoides and Sézary syndrome. Expression of the recurrent TNFR2 Thr377Ile mutant in T cells leads to enhanced non-canonical NF-κB signaling that is sensitive to the proteasome inhibitor bortezomib. Using an integrative genomic approach, we additionally discovered a recurrent CTLA4-CD28 fusion, as well as mutations in downstream signaling mediators of these receptors.

Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs.

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αß unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αß is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αß to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αß in the absence of Stat1. These studies reveal that IFN-αß is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.

Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma.

Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.

Transcriptome sequencing in Sezary syndrome identifies Sezary cell and mycosis fungoides-associated lncRNAs and novel transcripts.

Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma (CTCL) of unknown etiology in which malignant cells circulate in the peripheral blood. To identify viral elements, gene fusions, and gene expression patterns associated with this lymphoma, flow cytometry was used to obtain matched pure populations of malignant Sézary cells (SCs) versus nonmalignant CD4(+) T cells from 3 patients for whole transcriptome, paired-end sequencing with an average depth of 112 million reads per sample. Pathway analysis of differentially expressed genes identified mis-regulation of PI3K/Akt, TGFß, and NF-κB pathways as well as T-cell receptor signaling. Bioinformatic analysis did not detect either nonhuman transcripts to support a viral etiology of SS or recurrently expressed gene fusions, but it did identify 21 SC-associated annotated long noncoding RNAs (lncRNAs). Transcriptome assembly by multiple algorithms identified 13 differentially expressed unannotated transcripts termed Sézary cell-associated transcripts (SeCATs) that include 12 predicted lncRNAs and a novel transcript with coding potential. High-throughput sequencing targeting the 3' end of polyadenylated transcripts in archived tumors from 24 additional patients with tumor-stage CTCL confirmed the differential expression of SC-associated lncRNAs and SeCATs in CTCL. Our findings characterize the SS transcriptome and support recent reports that implicate lncRNA dysregulation in human malignancies.

The overlapping host responses to bacterial cyclic dinucleotides.

Macrophages respond to infection with Legionella pneumophila by the induction of inflammatory mediators, including type I Interferons (IFN-Is). To explore whether the bacterial second messenger cyclic 3'-5' diguanylate (c-diGMP) activates some of these mediators, macrophages were infected with L. pneumophila strains in which the levels of bacterial c-diGMP had been altered. Intriguingly, there was a positive correlation between c-diGMP levels and IFN-I expression. Subsequent studies with synthetic derivatives of c-diGMP, and newly described cyclic 3'-5' diadenylate (c-diAMP), determined that these molecules activate overlapping inflammatory responses in human and murine macrophages. Moreover, UV crosslinking studies determined that both dinucleotides physically associate with a shared set of host proteins. Fractionation of macrophage extracts on a biotin-c-diGMP affinity matrix led to the identification of a set of candidate host binding proteins. These studies suggest that mammalian macrophages can sense and mount a specific inflammatory response to bacterial dinucleotides.