Flow Cytometry Studies Research Articles

Magmas Inhibition in Prostate Cancer: A Novel Target for Treatment-Resistant Disease.

Simple SummaryMetastatic and treatment-resistant prostate cancer remains a life-threatening disease despite recent therapeutic advances. Literature suggests that treatment resistance and prostate cancer progression is associated with prostate cancer stem cells. In this study, we evaluated the role of the mitochondria-associated granulocyte–macrophage colony-stimulating factor signaling (Magmas) protein as a molecular target and applied a novel Magmas inhibitor, BT#9, on prostate cancer cells and normal control cells. We found that Magmas was overexpressed in human prostate cancers and its expression was linked to the aggressiveness of the disease. BT#9 downregulated Magmas expression, reduced viability and induced apoptotic cell death in prostate cancer cells. The mechanism of cell death by BT#9 is mainly caspase-independent and via a Reactive Oxygen Species (ROS)-mediated pathway. This is the first study that has evaluated targeting the Magmas protein in prostate cancer and, to our knowledge, the first to elucidate the potential molecular mechanism of BT#9 activity in prostate cancer, including the mode of cell death and the critical role of ROS accumulation. Our work may provide a potential clinical application for a novel prostate cancer treatment that can overcome cancer stem cell and therapeutic resistance.The purpose of our study was to evaluate Magmas as a potential target in prostate cancer. In addition, we evaluated our synthetic Magmas inhibitor (BT#9) effects on prostate cancer and examined the molecular mechanism of BT#9. A cell viability assay showed that treatment with BT#9 caused a significant decrease in the viability of DU145 and PC3 prostate cancer cells with little effect on the viability of WPMY-1 normal prostate cells. Western blot proved that BT#9 downregulated the Magmas protein and caspase-3 activation. Flow cytometry studies demonstrated increased apoptosis and disturbed mitochondrial membrane potential. However, the main mode of cell death was caspase-independent necrosis, which was correlated with the accumulation of mitochondrial and intra-cellular Reactive Oxygen Species (ROS). Taken together, our data suggest Magmas is a potential molecular target for the treatment of prostate cancer and that Magmas inhibition results in ROS-dependent and caspase-independent necrotic cell death.

CXCR7 Promotes Adipogenesis and Insulin Resistance in Human Visceral Adipocytes

Obesity is a multifactorial chronic condition that increases the risk of cardiovascular diseases, type II diabetes, and certain types of cancer. It has been known that some chemokines and their receptors facilitate the development of obesity, leading to other related comorbidities. However, the exact role of atypical chemokine receptor CXCR7 in obesity and obesity‐induced type II diabetes remains unknown. We aimed to study whether CXCR7 promotes adipogenesis and insulin resistance in human visceral adipocytes in vitro. Using RT‐PCR and flow cytometry studies, we found that both CXCR7 mRNA and its protein expressions were significantly upregulated during the differentiation of human visceral adipocytes. Stimulation of CXCR7 by SDF‐1 or LIH383 (a CXCR7‐selective peptide ligand) increased lipid droplets formation during adipocytes differentiation. In addition, we found that SDF‐1 or LIH383 treatment inhibited AKT phosphorylation in a dose‐dependent manner with no effect on ERK1/2 and JNK signaling pathways, which indicates a selective suppression of the AKT signaling axis by CXCR7. Furthermore, activation of CXCR7 by SDF‐1 inhibited insulin signaling to the AKT pathway and insulin‐induced glucose uptake. In contrast, ablation of CXCR7 by CRISPR/Cas9 system restored AKT signaling and enhanced insulin sensitivity in human visceral adipocytes. We concluded that CXCR7 plays a critical role in human visceral adipogenesis and insulin resistance by suppressing the AKT signaling pathway, highlighting CXCR7 as a potential new drug target against obesity and type II diabetes.

Neonate with Respiratory Distress, Bilateral Microtia, Hypocalcemia, and Lymphopenia.

Neonate with Respiratory Distress, Bilateral Microtia, Hypocalcemia, and Lymphopenia.

Mechanistic Investigation of Granzyme A inhibitory effects on intracellular Mycobacterium tuberculosis

Abstract One fourth of the world population is infected with Tuberculosis (TB). Our lab has identified γ9δ2 T cells that secrete Granzyme A (GzmA) with TB protective effects. In this study, we investigated the mechanism(s) by which human GzmA inhibits the intracellular replication of mycobacteria within infected human primary monocytes. GzmA was added to mycobacteria-infected monocytes for downstream analyses using 2D-DIGE and shotgun proteomics. We generated WT, enzymatically inactive (S195A), and monomeric only (C93S) recombinant GzmA and performed: flow cytometry studies using viable mycobacteria; intracellular inhibition assays neutralizing CD14, TLR4 and TLR2; and GzmA immunoprecipitation experiments. The 2D-DIGE proteomic analyses found the ER-stress response and ATP metabolism pathways as important for GzmA-mediated inhibition. Separately, shotgun proteomics uncovered the upregulation of Rab11FIP1 (important for phagocytosis). Both GzmA-WT and S195A proteins inhibited intracellular mycobacteria, but C93S did not. Neutralization of CD14 and TLR4, but not TLR2, reversed GzmA-inhibitory activity. GzmA-WT, S195A, and C93S all bound mycobacteria. However, GzmA-WT and S195A, but not GzmA-C93S, stably bound to TLR4 and CD14. Collectively, these studies demonstrate key structural, functional, and inter-/intra-molecular features required for GzmA-mediated inhibition of intracellular mycobacteria including interactions between GzmA, mycobacteria, TLR4 and CD14. These interactions result in the ER stress response, altered ATP metabolism, enhanced phagocytosis, and inhibition of mycobacteria. Thus, GzmA’s potential role as opsonin could lead to novel host-directed therapeutics for TB infections. Supported by grants from NIH (F30HL151136-01, R01AI048391-12)

Evaluation of dry DURAClone antibody panels for the characterization of human PBMCs, enriched T cell fractions and anti-BCMA CAR-T cells

Abstract CAR-T cells are becoming a more common therapeutic approach and are setting forth to targeting of hematological disorders other than CD19+ entities and also solid tumors. However, the immunological determinants of CAR-T efficacy still need comprehensive investigation from starting material to final product, covering different CAR constructs, transfection methods and targets. DURAClone dry antibody panels were designed to overcome standardization issues and operator-dependent impact on accuracy and precision in multicentric flow cytometry studies. The aim of this study was to evaluate the utility of DURAClone dry antibody panels for the characterization of PBMCs, derived T cell-enriched fractions as well as anti-BCMA CAR-T cells (ProMab Inc.), including MOCK-transfected controls. DURAClone dry antibody panels were used as pre-configured or extended with CAR-specific probes and further antibodies. Viable CD45+ leukocyte and CD3+ T cell count, leukocyte composition (CD3/CD19/CD56/CD14/CD16) and the T cell (CD3/CD4/CD8) maturation profile (CD45RA, CD197/CCR7, CD27, CD57) including expression of checkpoint markers (LAG-3, PD-1) were assessed. Regardless of sample type, absolute counts and cellular composition were assessed using streamlined procedures. Typical staining patterns for T cell maturation profile were observed, revealing unchanged proportions for the different stages between PBMCs and enriched T cell fractions and strong skewing toward effector cell phenotypes with elevated expression of a checkpoint markers in case of CAR-T cells. Our experiments demonstrate the flexible utility of DURAClone dry antibody panels for the phenotypic characterization from starting material to final CAR-T cell product.

TNFα signaling impairs endothelial calcium signaling and elevates blood pressure in obesity

Endothelial dysfunction in small arteries is a hallmark of obesity‐induced hypertension. Recent studies show that endothelial upregulation of inducible nitric oxide synthase (iNOS) and NADPH oxidase 1 (NOX1) plays a critical role in obesity‐induced endothelial dysfunction. Endothelial upregulation of iNOS and NOX1 results in excessive formation of peroxynitrite, a reactive nitrogen species, which impairs endothelium‐dependent vasodilation. It is well‐known that inflammatory mediators elevate cellular iNOS, NOX1, and peroxynitrite levels. In this regard, tumor necrosis factor alpha (TNFa) signaling has been proposed as a key contributor to endothelial dysfunction in obesity. Notably, vascular smooth muscle cells (SMCs) were shown to generate TNFa in heart failure and diabetes. Therefore, we hypothesized that vascular wall‐derived TNFa acts locally to impair endothelial function in obesity. Flow cytometry studies showed an increase in TNFa levels in freshly isolated SMCs from small mesenteric arteries (MAs) of high‐fat diet‐fed (14 weeks) obese mice compared to normal chow‐fed control mice. However, TNFa levels in endothelial cells (ECs) from obese mice were not different from those in ECs from control mice. The levels of two other cytokines, IL‐1b and IL‐6, were not different between in SMCs and ECs from normal and obese mice. Additionally, qPCR studies in endothelium‐denuded MAs provided further evidence that TNFa expression is increased in SMCs from obese mice compared to control mice. The pro‐inflammatory effects of TNFa are mediated mainly through TNFa receptor I activation. Treating the obese mice with a selective inhibitor of TNFa receptor I signaling (R7050, 10 mg/kg, i.p., once a day for 14 days) reduced the blood pressure to normal levels in obese mice, but had no effect on blood pressure in control mice. Treatment with R7050 also abolished obesity‐induced increase in iNOS and NOX1 expression in endothelial cells. Further, R7050 administration restored acetylcholine‐induced, endothelium‐dependent vasodilation of MAs in pressure myography experiments. Previous studies showed that a decrease in Ca2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4) channels contributes to blood pressure elevation in obese mice. Notably, R7050 administration restored the Ca2+ influx signals through endothelial TRPV4 channels in MAs, as studied using high‐speed confocal Ca2+ imaging. Collectively, our findings indicate that TNFa signaling contributes to the impairment of endothelial calcium signaling and blood pressure elevation in obesity, and highlight the role of SMCs as a source of TNFa in obesity.

Cu-Doped Hollow Bioactive Glass Nanoparticles for Bone Infection Treatment.

In search of new approaches to treat bone infection and prevent drug resistance development, a nanosystem based on hollow bioactive glass nanoparticles (HBGN) of composition 79.5SiO2-(18-x)CaO-2.5P2O5-xCuO (x = 0, 2.5 or 5 mol-% CuO) was developed. The objective of the study was to evaluate the capacity of the HBGN to be used as a nanocarrier of the broad-spectrum antibiotic danofloxacin and source of bactericidal Cu2+ ions. Core-shell nanoparticles with specific surface areas close to 800 m2/g and pore volumes around 1 cm3/g were obtained by using hexadecyltrimethylammonium bromide (CTAB) and poly(styrene)-block-poly(acrylic acid) (PS-b-PAA) as structure-directing agents. Flow cytometry studies showed the cytocompatibility of the nanoparticles in MC3T3-E1 pre-osteoblastic cell cultures. Ion release studies confirmed the release of non-cytotoxic concentrations of Cu2+ ions within the therapeutic range. Moreover, it was shown that the inclusion of copper in the system resulted in a more gradual release of danofloxacin that was extended over one week. The bactericidal activity of the nanosystem was evaluated with E. coli and S. aureus strains. Nanoparticles with copper were not able to reduce bacterial viability by themselves and Cu-free HBGN failed to reduce bacterial growth, despite releasing higher antibiotic concentrations. However, HBGN enriched with copper and danofloxacin drastically reduced bacterial growth in sessile, planktonic and biofilm states, which was attributed to a synergistic effect between the action of Cu2+ ions and danofloxacin. Therefore, the nanosystem here investigated is a promising candidate as an alternative for the local treatment of bone infections.

Spondias purpurea L. Bark Extract Protects against Oxidative Stress and Reduces Hypercholesterolemia in Mice Fed High-Fat Diet.

Oxidative stress plays a key role in the initiation and progression of metabolic diseases, including obesity. Preventing the accumulation of reactive oxygen species and oxidative damage to macromolecules is a beneficial strategy for reducing comorbidities associated with obesity. Fruits from the Spondias genus are known for their antioxidant activity, but they are not available year-round due to their seasonality. In this context, we investigated the antioxidant activity and identified the chemical constituents of the aqueous extract of the stem bark of Spondias purpurea L. (EBSp). Additionally, we evaluated the effect of EBSp consumption on metabolic parameters in mice with obesity induced by a high-fat diet. Chemical analyses revealed 19 annotated compounds from EBSp, including flavan-3-ols, proanthocyanidins, methoxylated coumarin, and gallic and ellagic acids, besides other phenolic compounds. In vitro, EBSp showed antioxidant activity through the scavenging of the free radicals and the protection of macromolecules against oxidative damage. Cellular assays revealed that EBSp reduced the levels of malondialdehyde produced by erythrocytes exposed to the oxidizing agent AAPH. Flow cytometry studies showed that EBSp reduced reactive oxygen species levels in human peripheral blood mononuclear cells treated with hydrogen peroxide. Obese mice treated with EBSp (400 mg.kg−1) for 60 days showed reduced levels of malondialdehyde in the heart, liver, kidneys, and nervous system. The total cholesterol levels in mice treated with EBSp reached levels similar to those after treatment with the drug simvastatin. Together, the results show that the combination of the different phenolic compounds in S. purpurea L. bark promotes antioxidant effects in vitro and in vivo, resulting in cytoprotection in the context of oxidative stress associated with obesity and a reduction in hypercholesterolemia. From a clinical perspective, the reduction in oxidative stress in obese individuals contributes to the reduction in the emergence of comorbidities associated with this metabolic syndrome.

Changes in Quality Characteristics of Crassostrea gigas Oysters during Hypothermic Storage

Now oysters are one of the most common representatives of aquaculture grown worldwide. Finding reliable ways to assess the quality of oysters is an important task for breeding professionals and users of these products. The Food and Agriculture Organization considers microbiological testing and organoleptic parameters as mandatory tests for evaluation of the mollusks’ safety. We believe these parameters in oysters are insufficient for unbiased evaluation of the condition of mollusks. In this research, we attempted to determine the most sensitive indices of oysters’ viability during hypothermic storage under anoxia conditions. We investigated the oysters during 6 days of their storage in a household refrigerator at a temperature of 5°C. According to microbiological studies, during the experiment, we expected a rise in the total number of microorganisms. However, there was a paradoxical situation when at the end of the observations the total number of microorganisms decreased significantly, which contradicted the negative assessment of the state of oysters by organoleptic parameters. Flow cytometry studies also showed no correlation between the percentages of necrotized and apoptotic cells to organoleptic parameters. Studies have shown that on day 3 of storage, the oysters demonstrated a partial deterioration in organoleptic characteristics, which is clearly reflected in the content of volatile basic nitrogen in the tissues of mollusks, which characterizes the protein breakdown and accumulation of toxic nitrogen compounds. Thus, the content of volatile basic nitrogen in the tissues of oysters is the most sensitive and significant parameter in terms of biosafety of mollusks.
 Keywords: hypothermic storage, oysters, microbial flora, food quality, nitrogen

Usnic acid induced changes in biomolecules and their association with apoptosis in squamous carcinoma (A-431) cells: A flow cytometry, FTIR and DLS spectroscopic study

Many natural products induce apoptotic cell death in cancer cells, though studies on their interactions with macromolecules are limited. For the first time, this study demonstrated the cytotoxic potential of usnic acid (UA) against squamous carcinoma (A-431) cells and the associated changes in cell surface proteins, lipids and DNA by attenuated total reflection- fourier transform infrared spectroscopy (ATR-FTIR) and dynamic light scattering (DLS) spectroscopic studies. The IC50 for UA was 98.9 µM after treatment of A-431 cells for 48 h, while the IC50 reduced to 39.2 µM after 72 h of incubation time. UA induced oxidative stress in treated cells as confirmed by DCFHDA flow cytometry assay, depletion in reduced glutathione and increase in lipid peroxidation. The oxidative stress resulted in conformation change in amide I, amide II protein bands and DNA as observed by ATR-FTIR in UA treated A-431 cells. Shift in secondary structures of proteins from α helix to β sheets and structural changes in DNA was observed in UA treated A-431 cells. An increase in the band intensity of phospholipids, increased distribution of lipid and change in membrane potential was noted in UA treated cells, which was confirmed by externalization of phosphatidylserine to the outer membrane by annexin V-FITC/PI assay. Increase in mitochondrial membrane potential, cell cycle arrest at G0/G1 phase by flow cytometry and activation of caspase-3/7 dependent proteins confirmed the UA induced apoptosis in treated A-431 cells. FTIR and DLS spectroscopy confirmed the changes in biomolecules after UA treatment, which were associated with apoptosis, as observed by flow cytometry.

Osteogenic Circulating Endothelial Progenitor Cells are Associated with Vascular Aging of the Large Arteries in Rheumatoid Arthritis

Background and AimRheumatoid arthritis is associated with both abnormal bone metabolism and accelerated vascular aging but a mechanistic link was lacking. This study aims to investigate the role of osteocalcin (OCN)-expressing circulating endothelial progenitor cells (EPCs) in vascular aging, as determined by arterial calcifications in rheumatoid arthritis.MethodsWe performed flow cytometry studies in 145 consecutive patients with rheumatoid arthritis to determine osteogenic circulating levels of OCN-positive (OCN+) CD34+KDR+ and OCN+CD34+ versus conventional early EPC CD34+CD133+KDR+. Total calcium load of the thoracic aorta (ascending plus descending) and the carotid arteries were assessed by non-contrast computed tomography (CT) and contrast CT angiography.ResultsOsteogenic EPCs OCN+CD34+KDR+ (P = 0.002) and OCN+CD34+ (P = 0.001), together with clinical parameters of age, history of hypertension, systolic blood pressure, serum levels of triglycerides, HbA1c and creatinine, use of leflunomide and brachial-ankle pulse-wave velocity (all P < 0.05), were associated with the clustered presence of aortic and carotid calcification. Multivariable analyses revealed that circulating OCN+CD34+KDR+ (B = 14.4 [95% CI 4.0 to 24.8], P = 0.007) and OCN+CD34+ (B = 9.6 [95% CI 4.9 to 14.3], P < 0.001) remained independently associated with increased aortic calcium load. OCN+CD34+ EPC (B = 0.8 [95% CI 0.1 to 1.5], P = 0.023), but not OCN+CD34+KDR+ EPC (B = 1.2 [95% CI −0.2 to 2.6], P = 0.09), was further independently associated with carotid calcium load. In comparison, conventional early EPC CD34+CD133+KDR+ had no significant association with aortic or carotid calcium load (P = 0.46 and 0.88, respectively).ConclusionCirculating level of osteogenic EPC is associated with increased vascular aging in terms of calcification of the large arteries in patients with rheumatoid arthritis. The findings may suggest a role of the bone-vascular axis underlying vascular aging in rheumatic diseases. Further research is needed to characterize the mechanistic links and basis of these observations.

Effect of Alpha and Beta Adrenergic Receptors on the Expression of Stk4, Caspase-3, and Sox2 Genes in Neural Stem Cells Derived From the Hippocampus and Treated With Norepinephrine in Rats

Background and Objectives Uncontrolled stress affects hippocampal-dependent memory through altering the morphology and the proliferation rate of hippocampal progenitor cells. Subjects and Methods In this experimental study, neural stem cells (NSCs) were isolated from the hippocampus of newborn rats and cultivated in a serum-free medium to generate neurosphere. To confirm the induced NSCs, immunocytochemistry and antibody nestin were used. The rate of cell proliferation and cytotoxicity caused by norepinephrine were measured by the MTT assay. NSCs were assigned in the following groups: Control (untreated NSCs), norepinephrine (NSCs treated with norepinephrine), propranolol (NSCs treated with beta receptor blocker propranolol plus norepinephrine), prazosin (NSCs treated with Alfa receptor blocker prazosin plus norepinephrine), and propranolol/ prazosin (NSCs treated with both propranolol and prazosin plus norepinephrine). Real-time PCR was conducted to measure the expression levels of Stk4, Caspase-3 and Sox2 genes. Results The flow cytometry study revealed that NSCs were nestin positive. Real-time PCR results showed that the expression level of Sox2 gene increased by norepinephrine. In addition, the expression level of Stk4 and Caspase-3 genes increased in the groups treated with prazosin. Conclusion The effect of norepinephrine on hippocampus-derived NSCs is receptor-dependent. The increase of norepinephrine under chronic stress can lead to either cell proliferation or apoptosis in NSCs; it acts as a double-edged sword.

Apoptosis Induction, Cell Cycle Arrest and Anti-Cancer Potential of Tamoxifen-Curcumin Loaded Niosomes Against MCF-7 Cancer Cells

Background & Objective: Breast cancer is the most common cancer among women. One of the most effective treatments for breast cancer is chemotherapy, in which specific drugs destroy the mass and its proliferation is inhibited. Chemotherapy is the most effective adjunctive therapy when multiple medications are used concurrently. Also, combining the drugs with nanocarrier has become an important strategy in targeted therapy. This study is designed to assess the apoptosis induction, cell cycle arrest, and anti-cancer potential of Tamoxifen-Curcumin-loaded niosomes against MCF-7 Cancer Cells.Methods: A novel niosomal formulation of tamoxifen-curcumin with Span 80 and lipid to drug ratio of 20 was employed. The MCF-7 cells were cultured and then treated with IC50 value of tamoxifen-curcumin-loaded niosomes, the combination of tamoxifen and curcumin, tamoxifen, and curcumin alone. Flow cytometry, Real-Time PCR, and cell cycle analysis tests were conducted to evaluate the induction of apoptosis.Results: Drug-loaded niosomes caused up-regulation of bax and p53 genes and down-regulation of bcl2 gene. Flow cytometry studies showed that niosomes containing tamoxifen-curcumin increased apoptosis rate in MCF-7 cells compared to the combination of tamoxifen and curcumin owing to the synergistic effect between the two drugs along with higher cell uptake by formulation niosomal. These results were also confirmed by cell cycle analysis.Conclusion: Co-delivery of curcumin and tamoxifen using optimized niosomal formulation revealed that at acidic pH of MCF-7 cancer cells, released drugs from niosomal carriers would be more effective than physiological pH. This feature of niosomal nanoparticles can reduce the side effects of drugs in normal cells. Niosomal nanoparticles might be used as a biological anti-cancer factor in treatment of breast cancer.

N-acetyl-d-glucosamine decorated nano-lipid-based carriers as theranostics module for targeted anti-cancer drug delivery

The present investigation was carried out to engineer N-acetyl-d-glucosamine (NADG) coupled quercetin-loaded (Q) nano-lipid-based carriers (NADG-Q-NLBCs) to target breast cancer. The constructed nano-bioconjugate was characterized by various techniques, and the in vitro drug release profiles were examined using zero and first-order kinetic models. The characterization data confirmed the morphology, size, charge distribution, crystallinity, and chemical interactions among the various moieties of the nano-bioconjugate. Further, the synthesized NADG-Q-NLBCs were applied to target the human breast cancer cells (MCF-7), which interestingly showed a more cytotoxic effect compared to the lone NLBCs and free Quercetin. The flow cytometry study confirmed NADG-Q-NLBCs induced apoptosis in MCF-7 cells in a targeted manner. The percentage of early apoptotic cells was found to be 25% in the case of NADG-Q-NLBCs, which is almost 2.5 times higher than the Q-NLBCs. However, the number of viable cells reached the maximum when treated with NLBCs. The present investigation suggests that the constructed nano-bioconjugate could be a capable carrier of drugs with sustained pharmacokinetics and improved physicochemical properties.

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy

CD38 Expression by Circulating and Skin-Infiltrating Lymphocytes from Sezary Syndrome Patients: A Flow Cytometry and Immunohistochemistry Study

Background Reports on the expression of CD38 in Sézary syndrome (SS), erythrodermic primary cutaneous T cell lymphoma with leukemic involvement, are limited. The aim of the present study is the analysis of the expression of CD38 by skin-infiltrating mononuclear cells and circulating T lymphocytes in a cohort of SS patients. Methods SS patients diagnosed since 1985 in our clinic were retrospectively analyzed for CD38 expression in biopsy and blood samples by immunohistochemistry and flow cytometry, respectively. Results SS patients show a predominant CD38-negative phenotype on both skin and blood. A subgroup of patients was found expressing CD38 (12 cases) in either the skin (>25% cell infiltrate) or blood (CD4+CD38+ >50%), among whom 4 in the blood, 7 in the skin, and 1 in both blood and skin. Conclusion The implications of these observations may be twofold: the relevance in basic science is related to a potential role in immune defense regulation, whilst in perspective CD38 may become a target for antibody therapy, considering the availability of different anti-CD38 monoclonal antibodies.

Amelioration of Lupus Serum-Induced Skin Inflammation in CD64-Deficient Mice.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder characterized by high autoantibodies levels and multiorgan tissue damage. The current study investigated the role of CD64 in SLE patients and animal models. According to a flow cytometry study, SLE patients showed an increase in CD64 expression in circulating monocytes. There was a correlation between CD64 and SLEDAI, blood urea nitrogen levels, and anti-Sm antibodies. In skin lesions of lupus MRL/lpr mice, there was high IgG deposition and CD64 expression. In vitro, cytokines IL-10 and IFN-γ upregulated CD64 expression in monocytes/macrophages that was inhibited by glucocorticoids. In CD64-deficient mice, skin inflammation induced by lupus serum was reduced. Furthermore, activation of spleen tyrosine kinase (Syk), Akt, and extracellular signal-regulated kinase (Erk) was inhibited in CD64-deficient monocytes. The results suggest that CD64 could be a biomarker for observing SLE progression, as well as a mechanistic checkpoint in lupus pathogenesis.

Effects of Each Domain in Recombinant Human Soluble Thrombomodulin on Prolongation of Murine Cardiac Allograft Survival

Thrombomodulin is used to manage disseminated intravascular coagulation. In our murine heart transplantation model, the administration of recombinant human soluble thrombomodulin (rTM) could induce the prolongation of cardiac allograft survival. However, there are limited data on the graft protective effects of each r domain (D1, D2, and D3). In this study, we investigated the effects of each domain of rTM on alloimmune responses in a murine model of cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA recipients using microsurgical techniques. CBA mice that underwent transplantation of C57BL/6 cardiac allografts were assigned to 4 groups: no treatment and each domain-exposed group. The dosage of each domain was determined based on our previous experiments. Flow cytometry and histologic studies were performed to determine whether Foxp3+ regulatory T cells were generated. Untreated and D2-exposed CBA recipients acutely rejected C57BL/6 cardiac allografts within 9 days. Administration of D3 resulted in modest prolongation of allograft survival, and administration of D1 significantly prolonged allograft survival. Histologic studies showed that myocardial damage of allografts from D1- and D3-exposed CBA recipients was controlled compared with that of untreated recipients. In particular, the CD4+CD25+Foxp3+ cell population in the splenocytes of D1-exposed CBA recipients was increased. In conclusion, D1 in rTM could help prolong cardiac allograft survival through regulatory T cell induction and graft protective effects.

Sodium butyrate reduces endoplasmic reticulum stress by modulating CHOP and empowers favorable anti-inflammatory adipose tissue immune-metabolism in HFD fed mice model of obesity

Over the past decade, the gut microbiome has been linked to several diseases including gastrointestinal diseases, cancer, immune disorder and metabolic syndrome. Shifts in the gut bacterial population affect the overall metabolic health status leading towards obesity and Type II diabetes mellitus. Secondary metabolites secreted by the gut microbiome interact with various host-sensing signalling pathways and are responsible for functional modulation of immune resident cells in metabolic tissues (Blüher, 2019). Of these, short- chain fatty acids (SCFAs) i.e., acetate, propionate and butyrate have been significantly correlated with the disposition of diabetes and metabolic disorder. The altered gut microbial population depletes the intestinal barrier causing entry of LPS into circulation and towards metabolic tissues triggering pro-inflammatory responses. As butyrate has been known to maintain intestinal integrity, we aimed to assess the apparent effect of externally given sodium butyrate [NaB] on immuno-metabolic profiling of adipose tissue, and its association with metabolic and inflammatory status of adipose tissue. To assess this, we put groups of C57BL/6 mice i.e., Control fed with a regular chow diet and another group that was fed on a high fat diet (HFD, 60%) for 8 weeks. Following this, the HFD group were further subdivided into two groups one fed with HFD and the other with HFD + NaB (5%w/w) for another 8 weeks. Body composition, weight gain, body adiposity and biochemical parameters were assessed. NaB fed group showed an improved metabolic profile compared to HFD fed group. Administration of NaB also improved glucose tolerance capacity and insulin sensitivity as determined by IPGTT and ITT profiles. Earlier reports have shown gut leakage and increased LPS in circulation is the primary cause of setting up inflammation at the tissue level. Our studies exhibited that, NaB increased the expression of tight junction proteins of intestinal linings and thereby enhanced intestinal barrier integrity. The FITC dextran permeability assay further confirmed this enhanced intestinal barrier integrity. We assessed the quantitative and relative population of different types of resident immune cells from a stromal vascular fraction of adipose tissue. Flow cytometry studies revealed significantly increased M2 (CD206+ ) macrophages and Tregs (CD25+ ) relative to the M1 macrophage population and CD4+ T cells respectively in NaB treated mice, suggesting its potential role in alleviating the inflammatory profile. In a nutshell, taken together better glucose tolerance, better gut health, reduced inflammatory adipose tissue immune cells, suggest potential beneficial role of sodium butyrate in alleviating overall inflammation and metabolic dysfunction associated with obesity.

Microemulsions and nanoemulsions modified with cationic surfactants for improving the solubility and therapeutic efficacy of loaded drug indomethacin

In this work, a noncovalent strategy was successfully used to modify colloidal stability and in vitro and in vivo efficacy of two amphiphilic formulations of the anti-inflammatory drug indomethacin. Namely, nanoemulsions and microemulsions based on oleic acid and nonionic surfactants have been produced and compared. The influence of cationic surfactants cetyltrimethylammonium bromide and its carbamate bearing analogue on the size characteristics, stability and ability to provide prolonged action of loaded drug indomethacin has been evaluated. Adding the positively charged molecules in the surface layer of nanoemulsions and microemulsions has shown the stability increase along with maintaining the size characteristics and homogeneity in time. Moreover, the carbamate modified analogue demonstrated beneficial behavior. Indomethacin loaded in microemulsions and nanoemulsions showed prolonged-release (10%–15% release for 5 h) compared to a free drug (complete release for 5 h). The rate of release of indomethacin from nanoemulsions was slightly higher than from microemulsions and insignificantly decreased with an increase in the concentration of the cationic surfactant. For carbamate surfactant nanocarrier loaded with fluorescence probe Nile Red, the ability to penetrate into the cell was supported by flow cytometry study and visualized by fluorescence microscopy. In vitro tests on anti-inflammatory activity of the systems demonstrated that the blood cell membrane stabilization increased in the case of modified microemulsion. The anti-inflammatory activity of the encapsulated drug was tested in rats using a carrageenan-induced edema model. Nanoemulsions without cationic surfactants appeared more efficient compared to microemulsions. Indomethacin emulsion formulations with carbamate surfactant added showed slower carrageenan-induced edema progression compared to unmodified compositions. Meanwhile, the edema completely disappeared upon treatment with emulsion loaded indomethacin after 4 h in the case of microemulsions versus 5 h in the case of nanoemulsions.