Inverse-Vaccines for Rheumatoid Arthritis Re-establish Metabolic and Immunological Homeostasis in Joint Tissues.

Rheumatoid arthritis (RA) causes immunological and metabolic imbalances in tissue, exacerbating inflammation in affected joints. Changes in immunological and metabolic tissue homeostasis at different stages of RA are not well understood. Herein, the changes in the immunological and metabolic profiles in different stages in collagen induced arthritis (CIA), namely, early, intermediate, and late stage is examined. Moreover, the efficacy of the inverse-vaccine, paKG(PFK15+bc2) microparticle, to restore tissue homeostasis at different stages is also investigated. Immunological analyses of inverse-vaccine-treated group revealed a significant decrease in the activation of pro-inflammatory immune cells and remarkable increase in regulatory T-cell populations in the intermediate and late stages compared to no treatment. Also, glycolysis in the spleen is normalized in the late stages of CIA in inverse-vaccine-treated mice, which is similar to no-disease tissues. Metabolomics analyses revealed that metabolites UDP-glucuronic acid and L-Glutathione oxidized are significantly altered between treatment groups, and thus might provide new druggable targets for RA treatment. Flux metabolic modeling identified amino acid and carnitine pathways as the central pathways affected in arthritic tissue with CIA progression. Overall, this study shows that the inverse-vaccines initiate early re-establishment of homeostasis, which persists through the disease span.

Rescue of dendritic cells from glycolysis inhibition improves cancer immunotherapy in mice.

Inhibition of glycolysis in immune cells and cancer cells diminishes their activity, and thus combining immunotherapies with glycolytic inhibitors is challenging. Herein, a strategy is presented where glycolysis is inhibited in cancer cells using PFK15 (inhibitor of PFKFB3, rate-limiting step in glycolysis), while simultaneously glycolysis and function is rescued in DCs by delivery of fructose-1,6-biphosphate (F16BP, one-step downstream of PFKFB3). To demonstrate the feasibility of this strategy, vaccine formulations are generated using calcium-phosphate chemistry, that incorporate F16BP, poly(IC) as adjuvant, and phosphorylated-TRP2 peptide antigen and tested in challenging and established YUMM1.1 tumours in immunocompetent female mice. Furthermore, to test the versatility of this strategy, adoptive DC therapy is developed with formulations that incorporate F16BP, poly(IC) as adjuvant and mRNA derived from B16F10 cells as antigens in established B16F10 tumours in immunocompetent female mice. F16BP vaccine formulations rescue DCs in vitro and in vivo, significantly improve the survival of mice, and generate cytotoxic T cell (Tc) responses by elevating Tc1 and Tc17 cells within the tumour. Overall, these results demonstrate that rescuing glycolysis of DCs using metabolite-based formulations can be utilized to generate immunotherapy even in the presence of glycolytic inhibitor.

Biomaterial mediated simultaneous delivery of spermine and alpha ketoglutarate modulate metabolism and innate immune cell phenotype in sepsis mouse models.

Although different metabolic pathways have been associated with distinct macrophage phenotypes, the field of utilizing metabolites to modulate macrophage phenotype is in a nascent stage. In this report, we developed microparticles based on polymerization of alpha-ketoglutarate (a Krebs cycle metabolite), with or without encapsulation of spermine (a polyamine metabolite), to modulate cell phenotype that are critical for resolution of inflammation. Poly (alpha-ketoglutarate) microparticles encapsulated and released spermine (spermine (encap)paKG MPs) in vitro, which was accelerated in an acidic environment. When delivered to bone marrow-derived-macrophages, spermine (encap)paKG MPs induced a complex phenotypic profile outside of the typical M1/M2 paradigm, with distinct effects in the presence or absence of the pro-inflammatory stimulus lipopolysaccharide. Of particular interest was the increase in expression of CD163, which has been linked to anti-inflammatory responses in sepsis. Therefore, we systemically administered spermine (encap)paKG MPs to two different murine models of sepsis using acute or chronic injection of LPS. Macrophages and neutrophils in the liver and spleen of animals treated with spermine (encap)paKG MPs increased expression of CD163, concomitant with normalizing of glycolysis and oxidative phosphorylation, in both models. Overall, these results show that spermine (encap)paKG MPs modulate macrophage phenotype in vitro and in vivo, with potential applications in inflammation-associated diseases.

Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection.

High throughput automation is greatly enhanced using techniques that employ conveyor belt strategies with un-interrupted streams of flow. We have developed a 'conveyor belt' analog for high throughput real-time quantitative Polymerase Chain Reaction (qPCR) using droplet emulsion technology. We developed a low power, portable device that employs LED and fiber optic fluorescence excitation in conjunction with a continuous flow thermal cycler to achieve multi-channel fluorescence detection for real-time fluorescence measurements. Continuously streaming fluid plugs or droplets pass through tubing wrapped around a two-temperature zone thermal block with each wrap of tubing fluorescently coupled to a 64-channel multi-anode PMT. This work demonstrates real-time qPCR of 0.1-10 µL droplets or fluid plugs over a range of 7 orders of magnitude concentration from 1 × 10(1) to 1 × 10(7). The real-time qPCR analysis allows dynamic range quantification as high as 1 × 10(7) copies per 10 µL reaction, with PCR efficiencies within the range of 90-110% based on serial dilution assays and a limit of detection of 10 copies per rxn. The combined functionality of continuous flow, low power thermal cycling, high throughput sample processing, and real-time qPCR improves the rates at which biological or environmental samples can be continuously sampled and analyzed.

Isotropic 3D nuclear morphometry of normal, fibrocystic and malignant breast epithelial cells reveals new structural alterations.

BACKGROUND: Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria. METHODOLOGY: We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure. PRINCIPAL FINDINGS: We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations. CONCLUSIONS: Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.