Whole transcriptome profiling of placental pathobiology in SARS-CoV-2 pregnancies identifies placental dysfunction signatures.

Objectives: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a 'preeclampsia-like syndrome'. However, the mechanisms underpinning SARS-CoV-2-induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS-CoV-2 infection. Methods: We utilised whole-transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS-CoV-2 in the third trimester of their pregnancy (n = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID-19) pandemic (n = 9). Results: Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS-CoV-2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH) and preeclampsia (FLT1, EGFR, KISS1, PAPPA2) were enriched with SARS-CoV-2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS-CoV-2 samples compared to uninfected controls. Conclusions: Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS-CoV-2 infection.

High risk clear cell renal cell carcinoma microenvironments contain protumour immunophenotypes lacking specific immune checkpoints.

Perioperative immune checkpoint inhibitor (ICI) trials for intermediate high-risk clear cell renal cell carcinoma (ccRCC) have failed to consistently demonstrate improved patient outcomes. These unsuccessful ICI trials suggest that the tumour infiltrating immunophenotypes, termed here as the immune cell types, states and their spatial location within the tumour microenvironment (TME), were unfavourable for ICI treatment. Defining the tumour infiltrating immune cells may assist with the identification of predictive immunophenotypes within the TME that are favourable for ICI treatment. To define the immunophenotypes within the ccRCC TME, fresh para-tumour (pTME, n = 2), low-grade (LG, n = 4, G1-G2) and high-grade (HG, n = 4, G3-G4) tissue samples from six patients with ccRCC presenting at a tertiary referral hospital underwent spatial transcriptomics sequencing (ST-seq). Within the generated ST-seq datasets, immune cell types and states, termed here as exhausted/pro-tumour state or non-exhausted/anti-tumour state, were identified using multiple publicly available single-cell RNA and T-cell receptor sequencing datasets as references. HG TMEs revealed abundant exhausted/pro-tumour immune cells with no consistent increase in expression of PD-1, PD-L1 and CTLA4 checkpoints and angiogenic genes. Additional HG TME immunophenotype characteristics included: pro-tumour tissue-resident monocytes with consistently increased expression of HAVCR2 and LAG3 checkpoints; an exhausted CD8+ T cells sub-population with stem-like progenitor gene expression; and pro-tumour tumour-associated macrophages and monocytes within the recurrent TME with the expression of TREM2. Whilst limited by a modest sample size, this study represents the largest ST-seq dataset on human ccRCC. Our study reveals that high-risk ccRCC TMEs are infiltrated by exhausted/pro-tumour immunophenotypes lacking specific checkpoint gene expression confirming that HG ccRCC TME are immunogenic but not ICI favourable.

Platelet-derived exerkine CXCL4/platelet factor 4 rejuvenates hippocampal neurogenesis and restores cognitive function in aged mice.

The beneficial effects of physical activity on brain ageing are well recognised, with exerkines, factors that are secreted into the circulation in response to exercise, emerging as likely mediators of this response. However, the source and identity of these exerkines remain unclear. Here we provide evidence that an anti-geronic exerkine is secreted by platelets. We show that platelets are activated by exercise and are required for the exercise-induced increase in hippocampal precursor cell proliferation in aged mice. We also demonstrate that increasing the systemic levels of the platelet-derived exerkine CXCL4/platelet factor 4 (PF4) ameliorates age-related regenerative and cognitive impairments in a hippocampal neurogenesis-dependent manner. Together these findings highlight the role of platelets in mediating the rejuvenating effects of exercise during physiological brain ageing.

Spatial Transcriptomics in Kidney Tissue.

Unlike bulk and single-cell/single-nuclei RNA sequencing methods, spatial transcriptome sequencing (ST-seq) resolves transcriptome expression within the spatial context of intact tissue. This is achieved by integrating histology with RNA sequencing. These methodologies are completed sequentially on the same tissue section placed on a glass slide with printed oligo-dT spots, termed ST-spots. Transcriptomes within the tissue section are captured by the underlying ST-spots and receive a spatial barcode in the process. The sequenced ST-spot transcriptomes are subsequently aligned with the hematoxylin and eosin (H&E) image, giving morphological context to the gene expression signatures within intact tissue. We have successfully employed ST-seq to characterize mouse and human kidney tissue. Here, we describe in detail the application of Visium Spatial Tissue Optimization (TO) and Visium Spatial Gene Expression (GEx) protocols for ST-seq in fresh frozen kidney tissue.

Induction of Reactive Bone Stromal Fibroblasts in 3D Models of Prostate Cancer Bone Metastases.

A dynamic interplay between prostate cancer (PCa) cells and reactive bone stroma modulates the growth of metastases within the bone microenvironment. Of the stromal cells, metastasis-associated fibroblasts (MAFs) are known to contribute but are the least studied cell type in PCa tumour progression. It is the aim of the current study to establish a biologically relevant 3D in vitro model that mimics the cellular and molecular profiles of MAFs found in vivo. Using 3D in vitro cell culture models, the bone-derived fibroblast cell line, HS-5, was treated with conditioned media from metastatic-derived PCa cell lines, PC3 and MDA-PCa 2b, or mouse-derived fibroblasts 3T3. Two corresponding reactive cell lines were propagated: HS5-PC3 and HS5-MDA, and evaluated for alterations in morphology, phenotype, cellular behaviour, plus protein and genomic profiles. HS5-PC3 and HS5-MDA displayed distinct alterations in expression levels of N-Cadherin, non-functional E-Cadherin, alpha-smooth muscle actin (α-SMA), Tenascin C, and vimentin, along with transforming growth factor receptor expression (TGF ß R1 and R2), consistent with subpopulations of MAFs reported in vivo. Transcriptomic analysis revealed a reversion of HS5-PC3 towards a metastatic phenotype with an upregulation in pathways known to regulate cancer invasion, proliferation, and angiogenesis. The exploitation of these engineered 3D models could help further unravel the novel biology regulating metastatic growth and the role fibroblasts play in the colonisation process.

Tolerogenic dendritic cells protect against acute kidney injury.

Ischemia reperfusion injury is a common precipitant of acute kidney injury that occurs following disrupted perfusion to the kidney. This includes blood loss and hemodynamic shock, as well as during retrieval for deceased donor kidney transplantation. Acute kidney injury is associated with adverse long-term clinical outcomes and requires effective interventions that can modify the disease process. Immunomodulatory cell therapies such as tolerogenic dendritic cells remain a promising tool, and here we tested the hypothesis that adoptively transferred tolerogenic dendritic cells can limit kidney injury. The phenotypic and genomic signatures of bone marrow-derived syngeneic or allogeneic, Vitamin-D3/IL-10-conditioned tolerogenic dendritic cells were assessed. These cells were characterized by high PD-L1:CD86, elevated IL-10, restricted IL-12p70 secretion and a suppressed transcriptomic inflammatory profile. When infused systemically, these cells successfully abrogated kidney injury without modifying infiltrating inflammatory cell populations. They also provided protection against ischemia reperfusion injury in mice pre-treated with liposomal clodronate, suggesting the process was regulated by live, rather than reprocessed cells. Co-culture experiments and spatial transcriptomic analysis confirmed reduced kidney tubular epithelial cell injury. Thus, our data provide strong evidence that peri-operatively administered tolerogenic dendritic cells have the ability to protect against acute kidney injury and warrants further exploration as a therapeutic option. This technology may provide a clinical advantage for bench-to-bedside translation to affect patient outcomes.

Multi-omic and spatial dissection of immunotherapy response groups in non-small cell lung cancer.

The composition and activation status of the cellular milieu contained within the tumour microenvironment (TME) is becoming increasingly recognized as a driving factor for immunotherapy response. Here, we employed multiplex immunohistochemistry (mIHC), and digital spatial profiling (DSP) to capture the targeted immune proteome and transcriptome of tumour and TME compartments from an immune checkpoint inhibitor (ICI)-treated (n = 41) non-small cell lung cancer (NSCLC) patient cohort. We demonstrate by mIHC that the interaction of CD68+ macrophages with PD1+ , FoxP3+ cells is enriched in ICI refractory tumours (p = 0.012). Patients responsive to ICI therapy expressed higher levels of IL2 receptor alpha (CD25, p = 0.028) within their tumour compartments, which corresponded with increased IL2 mRNA (p = 0.001) within their stroma. In addition, stromal IL2 mRNA levels positively correlated with the expression of pro-apoptotic markers cleaved caspase 9 (p = 2e-5 ) and BAD (p = 5.5e-4 ) and negatively with levels of memory marker, CD45RO (p = 7e-4 ). Immuno-inhibitory markers CTLA-4 (p = 0.021) and IDO-1 (p = 0.023) were suppressed in ICI-responsive patients. Tumour expression of CD44 was depleted in the responsive patients (p = 0.02), while higher stromal expression of one of its ligands, SPP1 (p = 0.008), was observed. Cox survival analysis also indicated tumour CD44 expression was associated with poorer prognosis (hazard ratio [HR] = 1.61, p = 0.01), consistent with its depletion in ICI-responsive patients. Through multi-modal approaches, we have dissected the characteristics of NSCLC immunotherapy treatment groups and provide evidence for the role of several markers including IL2, CD25, CD44 and SPP1 in the efficacy of current generations of ICI therapy.

The Utility of Spatial Transcriptomics for Solid Organ Transplantation.

Spatial transcriptomics (ST) measures and maps transcripts within intact tissue sections, allowing the visualization of gene activity within the spatial organization of complex biological systems. This review outlines advances in genomic sequencing technologies focusing on in situ sequencing-based ST, including applications in transplant and relevant nontransplant settings. We describe the experimental and analytical pipelines that underpin the current generation of spatial technologies. This context is important for understanding the potential role ST may play in expanding our knowledge, including in organ transplantation, and the important caveats/limitations when interpreting the vast data output generated by such methodological platforms.

Spatially Resolved Transcriptomes of Mammalian Kidneys Illustrate the Molecular Complexity and Interactions of Functional Nephron Segments.

Available transcriptomes of the mammalian kidney provide limited information on the spatial interplay between different functional nephron structures due to the required dissociation of tissue with traditional transcriptome-based methodologies. A deeper understanding of the complexity of functional nephron structures requires a non-dissociative transcriptomics approach, such as spatial transcriptomics sequencing (ST-seq). We hypothesize that the application of ST-seq in normal mammalian kidneys will give transcriptomic insights within and across species of physiology at the functional structure level and cellular communication at the cell level. Here, we applied ST-seq in six mice and four human kidneys that were histologically absent of any overt pathology. We defined the location of specific nephron structures in the captured ST-seq datasets using three lines of evidence: pathologist's annotation, marker gene expression, and integration with public single-cell and/or single-nucleus RNA-sequencing datasets. We compared the mouse and human cortical kidney regions. In the human ST-seq datasets, we further investigated the cellular communication within glomeruli and regions of proximal tubules-peritubular capillaries by screening for co-expression of ligand-receptor gene pairs. Gene expression signatures of distinct nephron structures and microvascular regions were spatially resolved within the mouse and human ST-seq datasets. We identified 7,370 differentially expressed genes (p adj < 0.05) distinguishing species, suggesting changes in energy production and metabolism in mouse cortical regions relative to human kidneys. Hundreds of potential ligand-receptor interactions were identified within glomeruli and regions of proximal tubules-peritubular capillaries, including known and novel interactions relevant to kidney physiology. Our application of ST-seq to normal human and murine kidneys confirms current knowledge and localization of transcripts within the kidney. Furthermore, the generated ST-seq datasets provide a valuable resource for the kidney community that can be used to inform future research into this complex organ.

Spatial Profiling Identifies Prognostic Features of Response to Adjuvant Therapy in Triple Negative Breast Cancer (TNBC).

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that has few effective treatment options due to its lack of targetable hormone receptors. Whilst the degree of tumour infiltrating lymphocytes (TILs) has been shown to associate with therapy response and prognosis, deeper characterization of the molecular diversity that may mediate chemotherapeutic response is lacking. Here we applied targeted proteomic analysis of both chemotherapy sensitive and resistant TNBC tissue samples by the Nanostring GeoMx Digital Spatial Platform (DSP). By quantifying 68 targets in the tumour and tumour microenvironment (TME) compartments and performing differential expression analysis between responsive and non-responsive tumours, we show that increased ER-alpha expression and decreased 4-1BB and MART1 within the stromal compartments is associated with adjuvant chemotherapy response. Similarly, higher expression of GZMA, STING and fibronectin and lower levels of CD80 were associated with response within tumour compartments. Univariate overall-survival (OS) analysis of stromal proteins supported these findings, with ER-alpha expression (HR=0.19, p=0.0012) associated with better OS while MART1 expression (HR=2.3, p=0.035) was indicative of poorer OS. Proteins within tumour compartments consistent with longer OS included PD-L1 (HR=0.53, p=0.023), FOXP3 (HR=0.5, p=0.026), GITR (HR=0.51, p=0.036), SMA (HR=0.59, p=0.043), while EPCAM (HR=1.7, p=0.045), and CD95 (HR=4.9, p=0.046) expression were associated with shorter OS. Our data provides early insights into the levels of these markers in the TNBC tumour microenvironment, and their association with chemotherapeutic response and patient survival.

Management of isoniazid-monoresistant tuberculosis (Hr-TB) in Queensland, Australia: a retrospective case series.

OBJECTIVES: Drug-resistance represents a major threat in the fight against tuberculosis. Globally, isoniazid-monoresistant tuberculosis (Hr-TB) is twice as common as multidrug/rifampicin-resistant (MDR/RR)-TB. Recently updated WHO guidelines now recommend treatment of Hr-TB with rifampicin, ethambutol, pyrazinamide and levofloxacin for at least six months. Our primary objective was to define the frequency, treatment and outcomes for Hr-TB in Queensland, Australia. We also sought to determine the frequency of fluoroquinolone use and whether its inclusion improved outcomes. METHODS: Retrospective case series of tuberculosis notifications in Queensland between 2000 and 2017 with at least low-level isoniazid resistance and preserved susceptibility to other first-line oral agents. RESULTS: Hr-TB was identified in 7.2% of all notifications. Where outcomes were assessable (163/198), 76.1% were treated with first-line agents only and 11.0% received at least six months of a fluoroquinolone-containing regimen (consistent with recent WHO guidelines). Favourable outcomes were achieved in 95.7%, comparable to fully susceptible disease (94.9%). Inclusion of a fluoroquinolone did not significantly improve outcomes compared with a regimen containing first-line agents only, although these cases were more likely to have high-level resistance. Previous treatment made an unfavourable outcome more likely. CONCLUSIONS: Hr-TB is prevalent in Queensland. Treatment outcomes in our cohort were comparable to fully susceptible disease. The current WHO-recommended regimen did not confer advantage over an appropriately constructed regimen containing first-line agents only. Our findings suggest that, in a well-resourced setting with good programmatic management, the addition of a fluoroquinolone may not substantially improve outcomes - potentially allowing these agents to be reserved for more extensively resistant disease.

Auranofin improves overall survival when combined with standard of care in a pilot study involving dogs with osteosarcoma.

Osteosarcoma is the most common paediatric primary bone malignancy. The major cause of death in osteosarcoma is drug-resistant pulmonary metastasis. Previous studies have shown that thioredoxin reductase 2 is a driver of metastasis in osteosarcoma and can be inhibited by auranofin (AF). Moreover, studies have shown that AF significantly reduces pulmonary metastases in xenotransplant models. Here, we describe a phase I/II study of AF in canine osteosarcoma, a well-recognized spontaneous model of human osteosarcoma. We performed a single-arm multicentre pilot study of AF in combination with standard of care (SOC) (amputation + carboplatin). We recruited 40 dogs to the trial and used a historical SOC-only control group (n = 26). Dogs >15 kg received 9 mg AF q3d PO and dogs <15 kg received 6 mg q3d. Follow-up occurred over at least a 3-year period. Auranofin plus SOC improved overall survival (OS) (P = .036) in all dogs treated. The improved outcome was attributable entirely to improved OS in male dogs (P = .009). At the time of writing, 10 dogs (25%) survive without measurable disease in the treatment group with survival times ranging between 806 and 1525 days. Our study shows that AF improves OS in male dogs when combined with SOC. Our findings have translational relevance for the management of canine and human osteosarcoma. Our data justify a larger multicentre phase 2 trial in dogs and a phase I/II trial in human patients with refractory disease at the time of initial surgery.

Multiple interaction nodes define the postreplication repair response to UV-induced DNA damage that is defective in melanomas and correlated with UV signature mutation load.

Ultraviolet radiation-induced DNA mutations are a primary environmental driver of melanoma. The reason for this very high level of unrepaired DNA lesions leading to these mutations is still poorly understood. The primary DNA repair mechanism for UV-induced lesions, that is, the nucleotide excision repair pathway, appears intact in most melanomas. We have previously reported a postreplication repair mechanism that is commonly defective in melanoma cell lines. Here we have used a genome-wide approach to identify the components of this postreplication repair mechanism. We have used differential transcript polysome loading to identify transcripts that are associated with UV response, and then functionally assessed these to identify novel components of this repair and cell cycle checkpoint network. We have identified multiple interaction nodes, including global genomic nucleotide excision repair and homologous recombination repair, and previously unexpected MASTL pathway, as components of the response. Finally, we have used bioinformatics to assess the contribution of dysregulated expression of these pathways to the UV signature mutation load of a large melanoma cohort. We show that dysregulation of the pathway, especially the DNA damage repair components, are significant contributors to UV mutation load, and that dysregulation of the MASTL pathway appears to be a significant contributor to high UV signature mutation load.

Outcomes of stage I/II follicular lymphoma in the PET era: an international study from the Australian Lymphoma Alliance.

Management practices in early-stage (I/II) follicular lymphoma (FL) are variable and include radiation (RT), systemic therapy, or combined modality therapy (CMT). There is a paucity of data regarding maintenance rituximab in this cohort. We conducted an international retrospective study of patients with newly diagnosed early-stage FL staged with positron emission tomography (PET)-computed tomography and bone marrow biopsy. Three hundred sixty-five patients (stage I, n = 221), median age 63 years, treated from 2005-2017 were included, with a median follow-up of 45 months. Management included watchful waiting (WW; n = 85) and active treatment (n = 280). The latter consisted of RT alone (n = 171) or systemic therapy (immunochemotherapy [n = 63] or CMT [n = 46]). Forty-nine systemically treated patients received maintenance rituximab; 72.7% of stage I patients received RT alone, compared to 42.6% with stage II (P < .001). Active therapies yielded comparable overall response rates (P = .87). RT alone and systemic therapy without maintenance rituximab yielded similar progression-free survival (PFS) (hazard ratio [HR], 1.32; 95% confidence interval [CI], 0.77-2.34; P = .96). Maintenance rituximab improved PFS (HR, 0.24; 95% CI, 0.095-0.64; P = .017). The incidence of transformation was lower with systemic therapy compared to RT or WW (HR, 0.20; 95% CI, 0.070-0.61; P = .034). Overall survival was similar among all practices, including WW (P = .40). In the largest comparative assessment of management practices in the modern era, variable practices each resulted in similar excellent outcomes. Randomized studies are required to determine the optimal treatment in early-stage FL.

Progression of Disease Within 24 Months in Follicular Lymphoma Is Associated With Reduced Intratumoral Immune Infiltration.

PURPOSE: Understanding the immunobiology of the 15% to 30% of patients with follicular lymphoma (FL) who experience progression of disease within 24 months (POD24) remains a priority. Solid tumors with low levels of intratumoral immune infiltration have inferior outcomes. It is unknown whether a similar relationship exists between POD24 in FL. PATIENTS AND METHODS: Digital gene expression using a custom code set-five immune effector, six immune checkpoint, one macrophage molecules-was applied to a discovery cohort of patients with early- and advanced-stage FL (n = 132). T-cell receptor repertoire analysis, flow cytometry, multispectral immunofluorescence, and next-generation sequencing were performed. The immune infiltration profile was validated in two independent cohorts of patients with advanced-stage FL requiring systemic treatment (n = 138, rituximab plus cyclophosphamide, vincristine, prednisone; n = 45, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone), with the latter selected to permit comparison of patients experiencing a POD24 event with those having no progression at 5 years or more. RESULTS: Immune molecules showed distinct clustering, characterized by either high or low expression regardless of categorization as an immune effector, immune checkpoint, or macrophage molecule. Low programmed death-ligand 2 (PD-L2) was the most sensitive/specific marker to segregate patients with adverse outcomes; therefore, PD-L2 expression was chosen to distinguish immune infiltrationHI (ie, high PD-L2) FL biopsies from immune infiltrationLO (ie, low PD-L2) tumors. Immune infiltrationHI tissues were highly infiltrated with macrophages and expanded populations of T-cell clones. Of note, the immune infiltrationLO subset of patients with FL was enriched for POD24 events (odds ratio [OR], 4.32; c-statistic, 0.81; P = .001), validated in the independent cohorts (rituximab plus cyclophosphamide, vincristine, prednisone: OR, 2.95; c-statistic, 0.75; P = .011; and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone: OR, 7.09; c-statistic, 0.88; P = .011). Mutations were equally proportioned across tissues, which indicated that degree of immune infiltration is capturing aspects of FL biology distinct from its mutational profile. CONCLUSION: Assessment of immune-infiltration by PD-L2 expression is a promising tool with which to help identify patients who are at risk for POD24.

Validation of the Boston University staging system in AL amyloidosis.

Objectives: We aimed to externally validate Lilleness' et al. Boston University (BU) prognostic score that replaced NT-proBNP with brain natriuretic peptide (BNP), which will allow centres without access to NT-proBNP to accurately stage and prognosticate AL amyloidosis. Patients/methods: Forty-four were identified that had BNP, NTpro-BNP and TnI taken simultaneously, with a mean follow up of 7.3 years. Median age of the 44 patients was 67 years and 27% were female, with 61% having cardiac involvement, and 61% having renal involvement. Results: Using the BU BNP-based staging system, we identified 12/44 (27%) of patients as stage I, 18/44 (41%) of patients as stage II and 14/44 (31%) of patients as stage III. This correlated closely with stratification via the Mayo score, with only one patient miscategorised (97.7% agreement, k = 0.98). Median overall survival for our BU stage I was not reached, stage II was 40 months and stage III was 5 months (long rank p = .0012). Mayo 2004 median overall survival was identical for stages I, II and III. Conclusion: We have provided external validation of the BU staging system, a novel prognostic scoring system incorporating BNP, instead of NT-proBNP, for AL amyloidosis.

IL-23 favours outgrowth of spondyloarthritis-associated pathobionts and suppresses host support for homeostatic microbiota.

OBJECTIVES: Certain gut bacterial families, including Bacteroidaceae, Porphyromonadaceae and Prevotellaceae, are increased in people suffering from spondyloarthropathy (SpA), a disease group associated with IL23R signalling variants. To understand the relationship between host interleukin (IL)-23 signalling and gut bacterial dysbiosis in SpA, we inhibited IL-23 in dysbiotic ZAP-70-mutant SKG mice that develop IL-23-dependent SpA-like arthritis, psoriasis-like skin inflammation and Crohn's-like ileitis in response to microbial beta 1,3-glucan (curdlan). METHODS: We treated SKG mice weekly with anti-IL-23 or isotype mAb for 3 weeks, rested them for 3 weeks, then administered curdlan or saline. We collected faecal samples longitudinally, assessed arthritis, spondylitis, psoriasis and ileitis histologically, and analysed the microbiota community profiles using next-generation sequencing. We used multivariate sparse partial least squares discriminant analysis to identify operational taxonomic unit (OTU) signatures best classifying treatment groups and linear regression to develop a predictive model of disease severity. RESULTS: IL-23p19 inhibition in naïve SKG mice decreased Bacteroidaceae, Porphyromonadaceae and Prevotellaceae. Abundance of Clostridiaceae and Lachnospiraceae families concomitantly increased, and curdlan-mediated SpA development decreased. Abundance of Enterobacteriaceae and Porphyromonadaceae family and reduction in Lachnospiraceae Dorea genus OTUs early in disease course were associated with disease severity in affected tissues. CONCLUSIONS: Dysbiosis in SKG mice reflects human SpA and is IL-23p19 dependent. In genetically susceptible hosts, IL-23p19 favours outgrowth of SpA-associated pathobionts and reduces support for homeostatic-inducing microbiota. The relative abundance of specific pathobionts is associated with disease severity.

Effect of Perioperative Opioids on Cancer-Relevant Circulating Parameters: Mu Opioid Receptor and Toll-Like Receptor 4 Activation Potential, and Proteolytic Profile.

Purpose: The purpose of this study is to investigate the potential interplay between opioid analgesia and tumor metastasis through modulation of µ-opioid receptor (MOR), Toll-like receptor 4 (TLR4) activation, and matrix degradation potential.Experimental Design: Plasma samples were collected from 60 patients undergoing elective lower limb joint replacement preoperatively and at 3, 6, and 24 hours after surgery; pain scores were documented at the same time points. Opioid administration was recorded and converted into morphine IV equivalents. Plasma samples were also collected from 10 healthy volunteers. Alphascreen cyclic AMP assay and MOR-overexpressing cells were employed to quantify MOR activation. HEK-Blue hTLR4 were utilized to measure TLR4 activation. Circulating matrix metalloprotease and tissue inhibitor of matrix protease activities were assessed by gelatin zymography and reverse zymography, respectively.Results: Postoperative plasma samples displayed the ability to activate MOR and to inhibit lipopolysaccharide (LPS)-induced TLR4 activation. Linear mixed model analysis revealed that MOR activation had a significant effect on inhibition of LPS-induced TLR4 activation. Furthermore, TLR4 had a significant effect to explain pain scores. Postoperative samples also displayed altered circulating matrix-degrading enzymes activity potential, but this was correlated neither to opioid administration nor to MOR activation potential.Conclusions: Our results show for the first time that (i) opioids administered to surgery patients result in modulation of ligand-induced TLR4 activation and (ii) postoperative pain is associated with increased circulating TLR4 activation potential. Our study further promotes the use of MOR activation potential rather than opioid intake in clinical studies measuring opioid exposure at a given time point. Clin Cancer Res; 24(10); 2319-27. ©2018 AACR.

Loss of Usp9x disrupts cell adhesion, and components of the Wnt and Notch signaling pathways in neural progenitors.

Development of neural progenitors depends upon the coordination of appropriate intrinsic responses to extrinsic signalling pathways. Here we show the deubiquitylating enzyme, Usp9x regulates components of both intrinsic and extrinsic fate determinants. Nestin-cre mediated ablation of Usp9x from embryonic neural progenitors in vivo resulted in a transient disruption of cell adhesion and apical-basal polarity and, an increased number and ectopic localisation of intermediate neural progenitors. In contrast to other adhesion and polarity proteins, levels of ß-catenin protein, especially S33/S37/T41 phospho-ß-catenin, were markedly increased in Usp9x -/Y embryonic cortices. Loss of Usp9x altered composition of the ß-catenin destruction complex possibly impeding degradation of S33/S37/T41 phospho-ß-catenin. Pathway analysis of transcriptomic data identified Wnt signalling as significantly affected in Usp9x -/Y embryonic brains. Depletion of Usp9x in cultured human neural progenitors resulted in Wnt-reporter activation. Usp9x also regulated components of the Notch signalling pathway. Usp9x co-localized and associated with both Itch and Numb in embryonic neocortices. Loss of Usp9x led to decreased Itch and Numb levels, and a concomitant increase in levels of the Notch intracellular domain as well as, increased expression of the Notch target gene Hes5. Therefore Usp9x modulates and potentially coordinates multiple fate determinants in neural progenitors.