Bilirubin Nanoparticle as an Anti-Inflammatory Therapy for Graft versus Host Disease

Graft versus host disease (GvHD) caused by alloreactive donor lymphocytes is a fatal complication of hematopoietic stem cell transplant (HSCT). Myeloablative conditioning regimen, consisting of chemotherapy and/or radiation, given prior to HSCT can cause tissue damage. This non-specific tissue damage triggers cross-presentation of alloantigens to the donor immune cells, causing recruitment of leukocytes and production of inflammatory cytokines. Targeting this inflammation without affecting the anti-leukemia effects of HSCT, continues to be one of the biggest challenge in finding a therapy for GvHD. Bilirubin is a tetrapyrrole pigment, found in the blood, with natural anti-oxidative and anti-inflammatory properties. Using mouse models of various inflammatory diseases, studies by our collaborating investigators have shown that, water-soluble PEGylated bilirubin nanoparticles (BRNP) selectively accumulate at the site of inflammation and prevent further tissue damage through scavenging reactive oxygen species. Therefore, we hypothesized that BRNP treatment after myeloablative conditioning regimen can reduce clinical symptoms of GvHD by abating the initial tissue damage in HSCT. We investigated the therapeutic efficacy of BRNP using murine GvHD model. Sublethally irradiated recipient mice (Balb/cJ) were infused with 5x106 bone marrow cells and 5x106 splenic cells from MHC-mismatched donor mice (C57/B6J) on day 1, with or without BRNP (10 mg/kg) on days 0-4. Clinical GvHD symptoms were monitored for 60 days, and mice were scored for fur, skin, posture, activity, and weight change. Untreated recipient mice (n=10) developed significantly worse GvHD (median GvHD score=3.4) compared to BRNP treated recipient mice (n=10, median GvHD score=0.3) (p=0.0003, Mann Whitney U Test). This translated into significantly better survival of BRNP treated mice with day 60 survival of 100% as compared to the untreated recipient with day 60 survival of 20% (p=0.0001, Log-rank (Mantel-Cox) Test). Histological analyses on day 8 post-transplantation, showed significantly lowered GvHD associated damage in liver, lung, skin, and gut, in BRNP treated mice as compared with untreated mice. In summary, we show that prophylactic treatment with BRNP can reduce clinical and pathological GvHD symptoms and thereby improve survival in mice. In future, we plan to investigate a treatment model of BRNP in relieving the clinical and pathological symptoms of GvHD. We also plan to explore the potential of BRNP as a drug conjugate for GvHD treatment

Aerobic exercise impacts the tumor microenvironment by altering CAF abundance and composition in pancreatic cancer

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A Novel T-Cell Engaging Bi-specific Antibody Targeting the Leukemia Antigen PR1/HLA-A2

Despite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2+ primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2+ primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy

Exercise Promotes Pro-Apoptotic Ceramide Signaling in a Mouse Melanoma Model

Ceramides are essential sphingolipids that mediate cell death and survival. Low ceramide content in melanoma is one mechanism of drug resistance. Thus, increasing the ceramide content in tumor cells is likely to increase their sensitivity to cytotoxic therapy. Aerobic exercise has been shown to modulate ceramide metabolism in healthy tissue, but the relationship between exercise and ceramide in tumors has not been evaluated. Here, we demonstrate that aerobic exercise causes tumor cell apoptosis and accumulation of pro-apoptotic ceramides in B16F10 but not BP melanoma models using mice. B16F10 tumor-bearing mice were treated with two weeks of moderate treadmill exercise, or were control, unexercised mice. A reverse-phase protein array was used to identify canonical p53 apoptotic signaling as a key pathway upregulated by exercise, and we demonstrate increased apoptosis in tumors from exercised mice. Consistent with this finding, pro-apoptotic C16-ceramide, and the ceramide generating enzyme ceramide synthase 6 (CerS6), were higher in B16F10 tumors from exercised mice, while pro-survival sphingosine kinase 1 (Sphk1) was lower. These data suggest that exercise contributes to B16F10 tumor cell death, possibly by modulating ceramide metabolism toward a pro-apoptotic ceramide/sphingosine-1-phosphate balance. However, these results are not consistent in BP tumors, demonstrating that exercise can have different effects on tumors of different patient or mouse origin with the same diagnosis. This work indicates that exercise might be most effective as a therapeutic adjuvant with therapies that kill tumor cells in a ceramide-dependent manner

Hydrogen sulphide triggers VEGF-induced intracellular Ca²⁺ signals in human endothelial cells but not in their immature progenitors

intracellular Ca(2+) concentration ([Ca(2+)]i) is central to endothelial proliferation and may be triggered by both VEGF and H2S. Albeit VEGFR-2 might serve as H2S receptor, the mechanistic relationship between VEGF- and H2S-induced Ca(2+) signals in endothelial cells is unclear. The present study aimed at assessing whether and how NaHS, a widely employed H2S donor, stimulates pro-angiogenic Ca(2+) signals in Ea.hy926 cells, a suitable surrogate for mature endothelial cells, and human endothelial progenitor cells (EPCs). We found that NaHS induced a dose-dependent increase in [Ca(2+)]i in Ea.hy926 cells. NaHS-induced Ca(2+) signals in Ea.hy926 cells did not require extracellular Ca(2+) entry, while they were inhibited upon pharmacological blockade of the phospholipase C/inositol-1,4,5-trisphosphate (InsP3) signalling pathway. Moreover, the Ca(2+) response to NaHS was prevented by genistein, but not by SU5416, which selectively inhibits VEGFR-2. However, VEGF-induced Ca(2+) signals were suppressed by dl-propargylglycine (PAG), which blocks the H2S-producing enzyme, cystathionine γ-lyase. Consistent with these data, VEGF-induced proliferation and migration were inhibited by PAG in Ea.hy926 cells, albeit NaHS alone did not influence these processes. Conversely, NaHS elevated [Ca(2+)]i only in a modest fraction of circulating EPCs, whereas neither VEGF-induced Ca(2+) oscillations nor VEGF-dependent proliferation were affected by PAG. Therefore, H2S-evoked elevation in [Ca(2+)]i is essential to trigger the pro-angiogenic Ca(2+) response to VEGF in mature endothelial cells, but not in their immature progenitors

Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis

BACKGROUND: An increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1. METHODOLOGY/PRINCIPAL FINDINGS: We utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2-3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective. CONCLUSIONS: Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Unlike N- and RCC-ECFCs, the InsP3-dependent SOCE does not drive PMF-ECFC proliferation

Enhanced Expression of Stim, Orai, and TRPC Transcripts and Proteins in Endothelial Progenitor Cells Isolated from Patients with Primary Myelofibrosis

<div><p>Background</p><p>An increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca²⁺ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP₃)-sensitive Ca²⁺ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1.</p><p>Methodology/Principal Findings</p><p>We utilized Ca²⁺ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP₃ signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2–3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La³⁺ and Gd³⁺, while CPA-elicited SOCE was insensitive to Gd³⁺. Finally, BTP-2 and La³⁺ weakly blocked PMF-ECFC proliferation, while Gd³⁺ was ineffective.</p><p>Conclusions</p><p>Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd³⁺-resistant, while the other one is regulated by the InsP₃-sensitive Ca²⁺ pool and is inhibited by Gd³⁺. Unlike N- and RCC-ECFCs, the InsP₃-dependent SOCE does not drive PMF-ECFC proliferation.</p></div

Demographic and clinical data of patients and healthy controls enrolled in the study.

<p>Demographic and clinical data of patients and healthy controls enrolled in the study.</p

The amplitude of store-operated Ca²⁺ entry is not reduced by a high-K⁺ extracellular solution in endothelial colony forming cells isolated from patients affected by primary myelofibrosis.

<p>100 mM NaCl in the extracellular solution was replaced with an equimolar amount of K⁺ (HighK) to clamp the membrane potential at 0 mV and observe the consequences on the extent of SOCE activation in PMF-ECFCs. A, HighK did not affect either the amplitude or the kinetics of CPA (10 µM)-induced Ca²⁺ signals in PMF-ECFCs. Black and grey tracings illustrate CPA-dependent Ca²⁺ signals in the absence and presence of HighK, respectively. B, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE in the absence (black bar; n = 76) and in the presence of HighK (white bar; n = 77). C, the biphasic Ca²⁺ response to ATP (100 µM) was not impaired by HighK. Black and grey tracings illustrate ATP-dependent Ca²⁺ signals in the absence and presence of HighK, respectively. D, mean±SE of the amplitude of ATP-elicited Ca²⁺ release and ATP-elicited SOCE in the absence (black bar; n = 88) and in the presence of HighK (white bar; n = 95). In panels A and C, each trace is representative of at least three independent experiments conducted on cells isolated from three distinct healthy donors and three PMF patients.</p

Expression of TRPC1 and TRPC4 proteins in endothelial colony forming cells isolated from patients affected by primary myelofibrosis.

<p>Western blot and densitometry representative of four separate experiments were shown. Major bands of the expected molecular weights for TRPC1 (A) and TRPC4 (B) were observed. Each bar in the upper panel represents the mean±SE of the densitometric analysis of four different experiments. The asterisk indicates p<0.01 (Student’s <i>t</i>-test).</p