Challenges with sirolimus experimental data to inform QSP model of post‐transplantation cyclophosphamide regimens

Dose optimization of sirolimus may further improve outcomes in allogeneic hematopoietic cell transplant (HCT) patients receiving post-transplantation cyclophosphamide (PTCy) to prevent graft-versus-host disease (GVHD). Sirolimus exposure-response association studies in HCT patients (i.e., the association of trough concentration with clinical outcomes) have been conflicting. Sirolimus has important effects on T-cells, including conventional (Tcons) and regulatory T-cells (Tregs), both of which have been implicated in the mechanisms by which PTCy prevents GVHD, but there is an absence of validated biomarkers of sirolimus effects on these cell subsets. Considering the paucity of existing biomarkers and the complexities of the immune system, we conducted a literature review to inform a quantitative systems pharmacology (QSP) model of GVHD. The published literature presented multiple challenges. The sirolimus pharmacokinetic models insufficiently describe sirolimus distribution to relevant physiological compartments. Despite multiple publications describing sirolimus effects on Tcons and Tregs in preclinical and human ex vivo models, consistent parameters relating sirolimus concentrations to circulating Tcons and Tregs could not be found. Each aspect presents a challenge in building a QSP model of sirolimus and its temporal effects on T-cell subsets and GVHD prevention. To optimize GVHD prevention regimens, phase I studies and systematic studies of immunosuppressant concentration-effect association are needed for QSP modeling

Akt-mediated signaling is induced by cytokines and cyclic adenosine monophosphate and suppresses hepatocyte inducible nitric oxide synthase expression independent of MAPK P44/42

AbstractCyclic AMP inhibits the expression of nitric oxide synthase (Harbrecht et al., 1995 [1]) in hepatocytes but the mechanism for this effect is incompletely understood. Cyclic AMP can activate several intracellular signaling pathways in hepatocytes including Protein Kinase A (PKA), cAMP regulated guanine nucleotide exchange factors (cAMP-GEFs), and calcium-mediated Protein Kinases. There is considerable overlap and cross-talk between many of these signaling pathways, however, and how these cascades regulate hepatocyte iNOS is not known. We hypothesized that Akt mediates the effect of cAMP on hepatocyte iNOS expression. Hepatocytes cultured with cytokines and dbcAMP increased Akt phosphorylation up to 2h of culture. Akt phosphorylation was inhibited by the PI3K inhibitor LY294002 (10μM), farnyltranferase inhibitor FTI-276, or transfection with a dominant negative Akt. The cyclic AMP-induced suppression of cytokine-stimulated iNOS was partially reversed by LY294002 and FTI-276. LY294002 also increased NFκB nucleus translocation by Western blot analysis in nuclear extracts. Cyclic AMP increased phosphorylation of Raf1 at serine 259 which was blocked by LY294002 and associated with decreased MAPK P44/42 phosphorylation. However, inhibition of MAPK P44/42 signaling with PD98059 failed to suppress cytokine-induced hepatocyte iNOS expression and did not enhance the inhibitory effect of dbcAMP on iNOS production. A constitutively active MAPK P44/42 plasmid had no effect on cytokine-stimulated NO production. These data demonstrate that dbcAMP regulates hepatocyte iNOS expression through an Akt-mediated signaling mechanism that is independent of MAPK P44/42

When Science Becomes Relevant

This chapter explores the use of indigenous knowledge for empowerment purposes. The White Angels Yoghurt and Dairy business in Molepolole, a wholly women owned business relied on indigenous knowledge of science with no formal scientific school background. The owners of this business have low education; they stay in a rural area and are poor. Their efforts are noteworthy as a symbol of women's empowerment that has relied on indigenous knowledge of milk pasteurisation and sweetening, a cultural tradition that Batswana have practiced for years. Their business has also demonstrated that combining indigenous knowledge with the current scientific and technological know-how can sustain and yield more gains for the business. Empowerment here can thus be defined as the ability to combine local/indigenous resources and current scientific knowledge and technologies to propel success and more gains from an empowerment project. </jats:p

Comparative Cytotoxicity and Intracellular Accumulation of Five Polybrominated Diphenyl Ether Congeners in Mouse Cerebellar Granule Neurons

Polybrominated diphenyl ethers (PBDEs), a group of flame retardants comprising 209 congeners, have become widespread environmental pollutants. High levels of PBDEs have been detected in human tissues, particularly in North America, and body burden is especially high in infants and toddlers because of exposure through breast milk and house dust. Increasing evidence, provided by animal studies, suggests that PBDEs are developmental neurotoxicants, although the underlying mechanisms are still unknown. Various PBDEs have been reported to cause oxidative stress and to induce apoptotic cell death in several cell types. In the present study, we investigated the comparative neurotoxicity in mouse cerebellar granule neurons of five brominated diphenyl ether (BDE) congeners, chosen among the most commonly found at the highest levels in human tissues. All BDE congener tested (BDE-47, BDE-99, BDE-100, BDE-153, and BDE-209) decreased cell viability and induced apoptotic cell death, which was prevented by antioxidants. They also caused oxidative stress, as indicated by an increase in reactive oxygen species and in lipid peroxidation. For all end points measured, the potency ranking of the congeners was BDE-100 > BDE-47 > BDE-99 > BDE-153 >> BDE-209. Measurement of BDE congener levels in neurons after exposure to different concentrations showed a significant accumulation in cells, which followed the same relative ranking. The findings suggest that all BDE congeners tested exhibit the same general mode of action (induction of oxidative stress–mediated apoptosis) and that the ability of each isomer to elicit such effects is dependent upon their accumulation in neurons, particularly in the microsomal fraction and the mitochondria

Enrichment of cancer stem cells via β-catenin contributing to the tumorigenesis of hepatocellular carcinoma

Abstract Background Hepatocellular carcinoma (HCC) is among the deadliest cancers due to its heterogeneity, contributing to chemoresistance and recurrence. Cancer stem-like cells (CSCs) are suggested to play an important role in HCC tumorigenesis. This study investigates the role of Wnt/β-catenin pathway in CSC enrichment and the capabilities of these CSCs in tumor initiation in orthotopic immunocompetent mouse model. Methods HCC-CSCs were enriched using established serum-free culture method. Wnt/β-catenin pathway activation and its components were analyzed by western blot and qRT-PCR. The role of β-catenin in enrichment of CSC spheroids was confirmed using siRNA interference. Tumorigenic capabilities were confirmed using orthotopic immunocompetent mouse model by injecting 2 × 106 Hepa1–6 CSC spheroids or control cells in upper left liver lobe. Results The serum-free cultured Hepa1–6 cells demonstrated self-renewal, spheroid formation, higher EpCAM expression, increased Hoechst-33342 efflux, and upregulated Wnt/β-catenin signaling. Wnt/β-catenin pathway upregulation was implicated with the downstream targets, i.e., c-MYC, Cyclin-D1, and LEF1. Also, we found that GSK-3β serine-9 phosphorylation increased in Hepa1–6 spheroids. Silencing β-catenin by siRNA reversed spheroid formation phenotype. Mice injected with Hepa1–6 CSC spheroids showed aggressive tumor initiation and growth compared with mice injected with control cells. Conclusions Successfully induced Hepa1–6 spheroids were identified with CSC-like properties. Aberrant β-catenin upregulation mediated by GSK-3β was observed in the Hepa1–6 spheroids. The β-catenin mediated CSC enrichment in the induced spheroids possesses the capability of tumor initiation in immunocompetent mice. Our study suggests plausible cell dedifferentiation mediated by β-catenin contributes to CSC-initiated HCC tumor growth in vivo

Co-Therapy of Pegylated G-CSF and Ghrelin for Enhancing Survival After Exposure to Lethal Radiation

Exposure to ionizing radiation (radiation injury, RI) in nuclear-related episode is evident to be life-threatening. RI occurs at levels of organs, tissues, cytosols, or nucleus. Their mechanisms are still not fully understood. FDA approves pegylated granulocyte colony-stimulating factor (Neulasta™, Peg-G-CSF) for acute hematopoietic syndrome and has been shown to save lives after lethal RI. We aimed to test whether Ghrelin enhanced Peg-G-CSF’s efficacy to save more lives after lethal RI. B6D2F1/J female mice were used for the study. They received 9.5 Gy (LD50/30 at 0.4 Gy/min) emitted from the60Co-γ-photon radiation facility. Peg-G-CSF was injected subcutaneously at 1 mg/kg once on days 1, 8, and 15 after irradiation. Ghrelin contains 28 amino acid and is a hunger peptide that has been shown to stimulate food intake, promote intestinal epithelial cell proliferation, elevates immunity, inhibits brain hemorrhage, and increases stress-coping. Ghrelin was injected subcutaneously at 113 μg/kg once on days 1, 2, and 3 after irradiation. Survival, body weight, water consumption, hematology, spleen weight, splenocytes, bone marrow cells, and histology of bone marrow and ileum were performed. We observed that radiation resulted in 30-days survival by 30%. RI decreased their body weights and water consumption volumes. On the 30th day post-RI, platelets and WBCs such as basophils, eosinophils, monocytes, lymphocytes, neutrophils and leukocytes were still significantly decreased in surviving mice. Likewise, their RBC, hemoglobin, hematocrit, and splenocytes remained low; splenomegaly was found in these mice. Bone marrow in surviving RI animals maintained low cellularity with high counts of fat cells and low counts of megakaryocytes. Meanwhile, ileum histology displayed injury. However, mice co-treated with both drugs 24 h after RI resulted in 30-days survival by 45% above the vehicle group. Additionally, the body-weight loss was mitigated, the acute radiation syndrome was reduced. This co-therapy significantly increased neutrophils, eosinophils, leukocytes, and platelets in circulation, inhibited splenomegaly, and increased bone marrow cells. Histopathological analysis showed significant improvement on bone marrow cellularity and ileum morphology. In conclusion, the results provide a proof of concept and suggest that the co-therapy of Peg-G-CSF and Ghrelin is efficacious to ameliorate RI.</jats:p