Ultrasound Neuromodulation of an Anti-Inflammatory Pathway at the Spleen Improves Experimental Pulmonary Hypertension.

BACKGROUND: Inflammation is pathogenically implicated in pulmonary arterial hypertension; however, it has not been adequately targeted therapeutically. We investigated whether neuromodulation of an anti-inflammatory neuroimmune pathway involving the splenic nerve using noninvasive, focused ultrasound stimulation of the spleen (sFUS) can improve experimental pulmonary hypertension. METHODS: Pulmonary hypertension was induced in rats either by Sugen 5416 (20 mg/kg SQ) injection, followed by 21 (or 35) days of hypoxia (sugen/hypoxia model), or by monocrotaline (60 mg/kg IP) injection (monocrotaline model). Animals were randomized to receive either 12-minute-long sessions of sFUS daily or sham stimulation for 14 days. Catheterizations, echocardiography, indices of autonomic function, lung and heart histology and immunohistochemistry, spleen flow cytometry, and lung single-cell RNA sequencing were performed after treatment to assess the effects of sFUS. RESULTS: Splenic denervation right before induction of pulmonary hypertension results in a more severe disease phenotype. In both sugen/hypoxia and monocrotaline models, sFUS treatment reduces right ventricular systolic pressure by 25% to 30% compared with sham treatment, without affecting systemic pressure, and improves right ventricular function and autonomic indices. sFUS reduces wall thickness, apoptosis, and proliferation in small pulmonary arterioles, suppresses CD3+ and CD68+ cell infiltration in lungs and right ventricular fibrosis and hypertrophy and lowers BNP (brain natriuretic peptide). Beneficial effects persist for weeks after sFUS discontinuation and are more robust with early and longer treatment. Splenic denervation abolishes sFUS therapeutic benefits. sFUS partially normalizes CD68+ and CD8+ T-cell counts in the spleen and downregulates several inflammatory genes and pathways in nonclassical and classical monocytes and macrophages in the lung. Differentially expressed genes in those cell types are significantly enriched for human pulmonary arterial hypertension-associated genes. CONCLUSIONS: sFUS causes dose-dependent, sustained improvement of hemodynamic, autonomic, laboratory, and pathological manifestations in 2 models of experimental pulmonary hypertension. Mechanistically, sFUS normalizes immune cell populations in the spleen and downregulates inflammatory genes and pathways in the lung, many of which are relevant in human disease.

AZGP1 deficiency promotes angiogenesis in prostate cancer.

BACKGROUND: Loss of AZGP1 expression is a biomarker associated with progression to castration resistance, development of metastasis, and poor disease-specific survival in prostate cancer. However, high expression of AZGP1 cells in prostate cancer has been reported to increase proliferation and invasion. The exact role of AZGP1 in prostate cancer progression remains elusive. METHOD: AZGP1 knockout and overexpressing prostate cancer cells were generated using a lentiviral system. The effects of AZGP1 under- or over-expression in prostate cancer cells were evaluated by in vitro cell proliferation, migration, and invasion assays. Heterozygous AZGP1± mice were obtained from European Mouse Mutant Archive (EMMA), and prostate tissues from homozygous knockout male mice were collected at 2, 6 and 10 months for histological analysis. In vivo xenografts generated from AZGP1 under- or over-expressing prostate cancer cells were used to determine the role of AZGP1 in prostate cancer tumor growth, and subsequent proteomics analysis was conducted to elucidate the mechanisms of AZGP1 action in prostate cancer progression. AZGP1 expression and microvessel density were measured in human prostate cancer samples on a tissue microarray of 215 independent patient samples. RESULT: Neither the knockout nor overexpression of AZGP1 exhibited significant effects on prostate cancer cell proliferation, clonal growth, migration, or invasion in vitro. The prostates of AZGP1-/- mice initially appeared to have grossly normal morphology; however, we observed fibrosis in the periglandular stroma and higher blood vessel density in the mouse prostate by 6 months. In PC3 and DU145 mouse xenografts, over-expression of AZGP1 did not affect tumor growth. Instead, these tumors displayed decreased microvessel density compared to xenografts derived from PC3 and DU145 control cells, suggesting that AZGP1 functions to inhibit angiogenesis in prostate cancer. Proteomics profiling further indicated that, compared to control xenografts, AZGP1 overexpressing PC3 xenografts are enriched with angiogenesis pathway proteins, including YWHAZ, EPHA2, SERPINE1, and PDCD6, MMP9, GPX1, HSPB1, COL18A1, RNH1, and ANXA1. In vitro functional studies show that AZGP1 inhibits human umbilical vein endothelial cell proliferation, migration, tubular formation and branching. Additionally, tumor microarray analysis shows that AZGP1 expression is negatively correlated with blood vessel density in human prostate cancer tissues. CONCLUSION: AZGP1 is a negative regulator of angiogenesis, such that loss of AZGP1 promotes angiogenesis in prostate cancer. AZGP1 likely exerts heterotypical effects on cells in the tumor microenvironment, such as stromal and endothelial cells. This study sheds light on the anti-angiogenic characteristics of AZGP1 in the prostate and provides a rationale to target AZGP1 to inhibit prostate cancer progression.

Multi-protein spatial signatures in ductal carcinoma in situ (DCIS) of breast.

BACKGROUND: There is limited knowledge about DCIS cellular composition and relationship with breast cancer events (BCE). METHODS: Immunofluorescence multiplexing (MxIF) was used to image and quantify 32 cellular biomarkers in FFPE DCIS tissue microarrays. Over 75,000 DCIS cells from 51 patients (median 9 years follow-up for non-BCE cases) were analysed for profiles predictive of BCE. K-means clustering was used to evaluate cellular co-expression of epithelial markers with ER and HER2. RESULTS: Only ER, PR and HER2 significantly correlated with BCE. Cluster analysis identified 6 distinct cell groups with different levels of ER, Her2, cMET and SLC7A5. Clusters 1 and 3 were not significant. Clusters 2 and 4 (high ER/low HER2 and SLC7A5/mixed cMET) significantly correlated with low BCE risk (P = 0.001 and P = 0.034), while cluster 6 (high HER2/low ER, cMET and SLC7A5) correlated with increased risk (P = 0.018). Cluster 5 (similar to cluster 6, except high SLC7A5) trended towards significance (P = 0.072). A continuous expression score (Escore) based on these 4 clusters predicted likelihood of BCE (AUC = 0.79, log-rank test P = 5E-05; LOOCV AUC = 0.74, log-rank test P = 0.006). CONCLUSION: Multiplexed spatial analysis of limited tissue is a novel method for biomarker analysis and predicting BCEs. Further validation of Escore is needed in a larger cohort.

Pathobiology of Candida auris infection analyzed by multiplexed imaging and single cell analysis.

Fungal organisms contribute to significant human morbidity and mortality and Candida auris (C. auris) infections are of utmost concern due to multi-drug resistant strains and persistence in critical care and hospital settings. Pathogenesis and pathology of C. auris is still poorly understood and in this study, we demonstrate how the use of multiplex immunofluorescent imaging (MxIF) and single-cell analysis can contribute to a deeper understanding of fungal infections within organs. We used two different neutrophil depletion murine models (treated with either 1A8-an anti-Ly6G antibody, or RB6-8C5-an anti-Ly6G/Ly6C antibody; both 1A8 and RB6-8C5 antibodies have been shown to deplete neutrophils) and compared to wildtype, non-neutropenic mice. Following pathologist assessment, fixed samples underwent MxIF imaging using a C. albicans antibody (shown to be cross-reactive to C. auris) and immune cell biomarkers-CD3 (T cells), CD68 (macrophages), B220 (B cells), CD45 (monocytes), and Ly6G (neutrophils) to quantify organ specific immune niches. MxIF analysis highlighted the heterogenous distribution of C. auris infection within heart, kidney, and brain 7 days post-infection. Size and number of fungal abscesses was greatest in the heart and lowest in brain. Infected mice had an increased count of CD3+, CD68+, B220+, and CD45+ immune cells, concentrated around C. auris abscesses. CD68+ cells were predominant in wildtype (non-neutropenic mice) and CD3+/CD45+ cells were predominant in neutropenic mice, with B cells being the least abundant. These findings suggest a Th2 driven immune response in neutropenic C. auris infection mice models. This study demonstrates the value of MxIF to broaden understanding of C. auris pathobiology, and mechanistic understanding of fungal infections.

3D reconstruction of skin and spatial mapping of immune cell density, vascular distance and effects of sun exposure and aging.

Mapping the human body at single cell resolution in three dimensions (3D) is important for understanding cellular interactions in context of tissue and organ organization. 2D spatial cell analysis in a single tissue section may be limited by cell numbers and histology. Here we show a workflow for 3D reconstruction of multiplexed sequential tissue sections: MATRICS-A (Multiplexed Image Three-D Reconstruction and Integrated Cell Spatial - Analysis). We demonstrate MATRICS-A in 26 serial sections of fixed skin (stained with 18 biomarkers) from 12 donors aged between 32-72 years. Comparing the 3D reconstructed cellular data with the 2D data, we show significantly shorter distances between immune cells and vascular endothelial cells (56 µm in 3D vs 108 µm in 2D). We also show 10-70% more T cells (total) within 30 µm of a neighboring T helper cell in 3D vs 2D. Distances of p53, DDB2 and Ki67 positive cells to the skin surface were consistent across all ages/sun exposure and largely localized to the lower stratum basale layer of the epidermis. MATRICS-A provides a framework for analysis of 3D spatial cell relationships in healthy and aging organs and could be further extended to diseased organs.

Deep learning-based automated pipeline for blood vessel detection and distribution analysis in multiplexed prostate cancer images.

Introduction: Prostate cancer is a highly heterogeneous disease, presenting varying levels of aggressiveness and response to treatment. Angiogenesis is one of the hallmarks of cancer, providing oxygen and nutrient supply to tumors. Micro vessel density has previously been correlated with higher Gleason score and poor prognosis. Manual segmentation of blood vessels (BVs) In microscopy images is challenging, time consuming and may be prone to inter-rater variabilities. In this study, an automated pipeline is presented for BV detection and distribution analysis in multiplexed prostate cancer images. Methods: A deep learning model was trained to segment BVs by combining CD31, CD34 and collagen IV images. In addition, the trained model was used to analyze the size and distribution patterns of BVs in relation to disease progression in a cohort of prostate cancer patients (N = 215). Results: The model was capable of accurately detecting and segmenting BVs, as compared to ground truth annotations provided by two reviewers. The precision (P), recall (R) and dice similarity coefficient (DSC) were equal to 0.93 (SD 0.04), 0.97 (SD 0.02) and 0.71 (SD 0.07) with respect to reviewer 1, and 0.95 (SD 0.05), 0.94 (SD 0.07) and 0.70 (SD 0.08) with respect to reviewer 2, respectively. BV count was significantly associated with 5-year recurrence (adjusted p = 0.0042), while both count and area of blood vessel were significantly associated with Gleason grade (adjusted p = 0.032 and 0.003 respectively). Discussion: The proposed methodology is anticipated to streamline and standardize BV analysis, offering additional insights into the biology of prostate cancer, with broad applicability to other cancers.

Keratinocytes are cell targets of West Nile virus in vivo.

West Nile virus (WNV) replicates in the skin; however, cell targets in the skin have not been identified. In the current studies, WNV infected the epidermis and adnexal glands of mouse skin, and the epidermal cells were identified as keratinocytes by double labeling for WNV antigen and keratin 10. Inoculation of mice with WNV replicon particles resulted in high levels of replication in the skin, suggesting that keratinocytes are an initial target of WNV. In addition, primary keratinocytes produced infectious virus in vitro. In conclusion, keratinocytes are cell targets of WNV in vivo and may play an important role in pathogenesis.

Novel DNA-based T-Cell Activator Promotes Rapid T-Cell Activation and Expansion.

Autologous chimeric antigen receptor engineered T-cell therapies are beginning to dramatically change the outlook for patients with several hematological malignancies. Yet methods to activate and expand these cells are limited, often pose challenges to automation, and have biological limitations impacting the output of the injectable dose. This study describes the development of a novel, highly flexible, soluble DNA-based T-cell activation and expansion platform which alleviates the limitations of current technologies and provides rapid T-cell activation and expansion.

Understanding heterogeneous tumor microenvironment in metastatic melanoma.

A systemic analysis of the tumor-immune interactions within the heterogeneous tumor microenvironment is of particular importance for understanding the antitumor immune response. We used multiplexed immunofluorescence to elucidate cellular spatial interactions and T-cell infiltrations in metastatic melanoma tumor microenvironment. We developed two novel computational approaches that enable infiltration clustering and single cell analysis-cell aggregate algorithm and cell neighborhood analysis algorithm-to reveal and to compare the spatial distribution of various immune cells relative to tumor cell in sub-anatomic tumor microenvironment areas. We showed that the heterogeneous tumor human leukocyte antigen-1 expressions differently affect the magnitude of cytotoxic T-cell infiltration and the distributions of CD20+ B cells and CD4+FOXP3+ regulatory T cells within and outside of T-cell infiltrated tumor areas. In a cohort of 166 stage III melanoma samples, high tumor human leukocyte antigen-1 expression is required but not sufficient for high T-cell infiltration, with significantly improved overall survival. Our results demonstrate that tumor cells with heterogeneous properties are associated with differential but predictable distributions of immune cells within heterogeneous tumor microenvironment with various biological features and impacts on clinical outcomes. It establishes tools necessary for systematic analysis of the tumor microenvironment, allowing the elucidation of the "homogeneous patterns" within the heterogeneous tumor microenvironment.

Stabilization of a recombinant ricin toxin A subunit vaccine through lyophilization.

Lyophilization was used to prepare dry, glassy solid vaccine formulations of recombinant ricin toxin A-chain containing suspensions of colloidal aluminum hydroxide adjuvant. Four lyophilized formulations were prepared by using combinations of rapid or slow cooling during lyophilization and one of two buffers, histidine or ammonium acetate. Trehalose was used as the stabilizing excipient. Aggregation of the colloidal aluminum hydroxide suspension was reduced in formulations processed with a rapid cooling rate. Aluminum hydroxide particle size distributions, glass transition temperatures, water contents, and immunogenicities of lyophilized vaccines were independent of incubation time at 40 °C for up to 15 weeks. Mice immunized with reconstituted ricin toxin subunit A (RTA) vaccines produced RTA-specific antibodies and toxin-neutralizing antibodies (TNAs) regardless of the length of high temperature vaccine storage or the degree of aluminum adjuvant aggregation that occurred during lyophilization. In murine studies, lyophilized formulations of vaccines conferred protection against exposure to lethal doses of ricin, even after the lyophilized formulations had been stored at 40 °C for 4 weeks. A corresponding liquid formulation of vaccine stored at 40 °C elicited RTA-specific antibody titers but failed to confer immunity during a ricin challenge.

LT-IIb(T13I), a non-toxic type II heat-labile enterotoxin, augments the capacity of a ricin toxin subunit vaccine to evoke neutralizing antibodies and protective immunity.

Currently, there is a shortage of adjuvants that can be employed with protein subunit vaccines to enhance protection against biological threats. LT-IIb(T13I) is an engineered nontoxic derivative of LT-IIb, a member of the type II subfamily of heat labile enterotoxins expressed by Escherichia coli, that possesses potent mucosal adjuvant properties. In this study we evaluated the capacity of LT-IIb(T13I) to augment the potency of RiVax, a recombinant ricin toxin A subunit vaccine, when co-administered to mice via the intradermal (i.d.) and intranasal (i.n.) routes. We report that co-administration of RiVax with LT-IIb(T13I) by the i.d. route enhanced the levels of RiVax-specific serum IgG antibodies (Ab) and elevated the ratio of ricin-neutralizing to non-neutralizing Ab, as compared to RiVax alone. Protection against a lethal ricin challenge was also augmented by LT-IIb(T13I). While local inflammatory responses elicited by LT-IIb(T13I) were comparable to those elicited by aluminum salts (Imject®), LT-IIb(T13I) was more effective than aluminum salts at augmenting production of RiVax-specific serum IgG. Finally, i.n. administration of RiVax with LT-IIb(T13I) also increased levels of RiVax-specific serum and mucosal Ab and enhanced protection against ricin challenge. Collectively, these data highlight the potential of LT-IIb(T13I) as an effective next-generation i.d., or possibly i.n. adjuvant for enhancing the immunogenicity of subunit vaccines for biodefense.

Biophysical characterization and immunization studies of dominant negative inhibitor (DNI), a candidate anthrax toxin subunit vaccine.

Dominant Negative Inhibitor (DNI) is a translocation-deficient homolog of recombinant protective antigen of Bacillus anthracis that is a candidate for a next generation anthrax vaccine. This study demonstrates that the biophysical characteristics of the DNI protein stored in lyophilized form at 4°C for 8 y were similar to recombinant Protective Antigen (rPA). To provide information on the accelerated stability of DNI, samples in the lyophilized form were subjected to thermal stress (40°C and 70°C for up to 4 weeks) and thoroughly evaluated using various biophysical and chemical characterization techniques. Results demonstrate preserved structural stability of the DNI protein under extreme conditions, suggesting long-term stability can be achieved for a vaccine that employs DNI, as desired for a biodefense countermeasure. Furthermore, the biological activity of the stressed DNI bound to the adjuvant Alhydrogel (®) was evaluated in mice and it was found that the immunogenicity DNI was not affected by thermal stress.