Murine Splenocytes Research Articles

Acute toxicity and genotoxicity assessment of PgTeL, a lectin from pomegranate sarcotesta, in mice

• PgTeL did not show hemolytic and cytotoxic effects in vitro . • It also did not cause toxicity when administered intraperitoneally in mice. • No damage to genetic material was observed. • PgTeL induced an increase in HDL levels and a decrease in TG and LDL levels, suggesting a hypolidemic effect. • The results open several possibilities for the application of PgTeL in vivo . PgTeL is a chitin-binding lectin from Punica granatum sarcotesta and shows antimicrobial activity against a wide range of microorganisms of medical interest. PgTeL did not show cytotoxicity in human peripheral blood mononuclear cells; however, there have been no studies to date on PgTeL toxicity in vivo . The present study evaluated PgTeL for cytotoxicity in murine splenocyte cells, in vitro hemolytic activity, and in vivo acute toxicity and genotoxicity in mice. In the acute toxicity assay with PgTeL at 100 mg/kg (i.p.), behavioral, biochemical, hematological, hemostatic, and histopathological parameters were evaluated. Comet and micronucleus tests were performed to assess the genotoxicity. After 24 h of incubation, there was only a slight decrease in splenocyte viability in treatments with PgTeL at 200 μg/mL (13.2%) and 100 μg/mL (9.9%) when compared to the control. No damage to the membranes of erythrocytes (0% hemolysis) was observed when they were incubated with all concentrations (3.9–1000 µg/mL) of PgTeL. There were no behavioral, hematological, hemostatic, or histopathological abnormalities in the in vivo acute toxicity assay. PgTeL caused an increase in HDL levels and decreased levels of triglycerides, LDL, and VLDL. The results provide several possibilities for the application of PgTeL in vivo , such as in formulations with antimicrobial potential.

A Defucosylated Anti-EpCAM Monoclonal Antibody (EpMab-37-mG2a-f) Exerts Antitumor Activity in Xenograft Model.

The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor prognosis. Therefore, EpCAM has been considered as a promising target for tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method, we previously established an anti-EpCAM monoclonal antibody (EpMab-37; mouse IgG1, kappa). In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG2a-type of EpMab-37 (EpMab-37-mG2a-f) against a breast cancer cell line (BT-474) and a pancreatic cancer cell line (Capan-2), both of which express EpCAM. EpMab-37-mG2a-f recognized BT-474 and Capan-2 cells with a moderate binding-affinity [apparent dissociation constant (KD): 2.9 × 10-8 M and 1.8 × 10-8 M, respectively] by flow cytometry. EpMab-37-mG2a-f exhibited ADCC and CDC for both cells by murine splenocytes and complements, respectively. Furthermore, administration of EpMab-37-mG2a-f significantly suppressed the xenograft tumor development compared with the control mouse IgG. These results indicated that EpMab-37-mG2a-f exerts antitumor activities and could provide valuable therapeutic regimen for breast and pancreatic cancers.

Key Subsets of Immune Cells in Bone Marrow Myeloid Compartment Indicate Degree of Tumor Infiltration and Avenues for Successful Immunotherapy in Multiple Myeloma

With the onset of single-cell omics, the complete gene expression landscape of immune cells can be leveraged to differentiate distinct subsets with inherently diverse functions. By analyzing the sample-specific composition of these distinct cellular groups, we can decipher the hallmarks of healthy and fully tumor-infiltrated bone marrow microenvironments as well as compositional modulations during effective immunotherapy. Unique to our approach, our analyses leverage over 500,000 existing RNA-Seq experiments packaged in the ARCHS4 repository to strengthen the biological signal and quality of our tertiary analyses by informing us in an unbiased way about lesser known immune-typologies. We applied this technique to our well-established immunocompetent mouse model of multiple myeloma (MM) which we rendered sensitive to IMiDs by introducing a human CRBN transgene: Vk*MYChCRBN. In multiple single cell experiments measuring effectiveness of Iberdomide in combination with Dexamethasone and an additional experiment comparing aged and untreated Vk*MYC mice with multiple myeloma (MM), key compositional patterns emerge in the murine bone marrow niche. Two distinct polarities of C1q expressing macrophages are present within murine bone marrow and spleen-one expressing Fcgr4 and present in untreated, tumor-bearing samples and a separate population elevated in Apoe, Vcam1, and MHC-II markers that can be found in healthy and tumor-bearing mice. Among splenocytes, we observed that the proportion of C1q macrophages correlates strongly with tumor burden and that BM and spleen samples in the process of tumor debulking via effective immunotherapy dramatically reduce relative frequencies of C1q macrophages. We hypothesize that the C1q macrophages without MHC-II features represent a tumor-associated macrophage (TAM) phenotype that is MM dependent and promotes tumor progression within the BM niche.1 Additional analyses of murine splenocytes have revealed that effective immunotherapy via an IAP antagonist induces the presence of immature or migratory dendritic cells (high expressers of Ccl5 and likely key immune recruiters) and that tumor-bearing splenocytes typically maintain large segments of immature neutrophils (high expressers of Ngp and Camp) without other neutrophils in more advanced stages of development2-while both immature and mature neutrophils are found in healthy controls. When comparing the BM composition of an aged, tumor-bearing mouse with that of a young, healthy mouse, key differences emerge. While multiple subsets of progenitors in murine BM niche can be identified via single cell, a unique population of cells over-expressing Cxcl12 that resemble Lepr+ mesenchymal stem cells from existing experiments (GSM3676085) are nearly absent in the young mouse, highly represented in the aged mouse, and may be a critical in creating a BM niche that supports tumor expansion.3 Curiously, a sizeable population of basophils is evident in the young, non-tumor bearing mouse but not found in the adult, tumor-bearing mouse. In addition, both types of C1q macrophages have expanded frequencies in the aged mouse. In total, single-cell analyses provide a snapshot of the compositional landscape of immune cell types thus illustrating hallmarks of tumor-infiltrated, healthy, and therapy modulated BM niches, providing potential new therapeutic targets.

Isolating and targeting a highly active, stochastic dendritic cell subpopulation for improved immune responses.

Dendritic cell (DC) activation via pathogen-associated molecular patterns (PAMPs) is critical for antigen presentation and development of adaptive immune responses, but the stochastic distribution of DC responses to PAMP signaling, especially during the initial stages of immune activation, is poorly understood. In this study, we isolate a unique DC subpopulation via preferential phagocytosis of microparticles (MPs) and characterize this subpopulation of "first responders" (FRs). We present results that show these cells (1) can be isolated and studied via both increased accumulation of the micron-sized particles and combinations of cell surface markers, (2) show increased responses to PAMPs, (3) facilitate adaptive immune responses by providing the initial paracrine signaling, and (4) can be selectively targeted by vaccines to modulate both antibody and Tcell responses invivo. This study presents insights into a temporally controlled, distinctive cell population that influences downstream immune responses. Furthermore, it demonstrates potential for improving vaccine designs via FR targeting.

Effects of Oleacein, a New Epinutraceutical Bioproduct from Extra Virgin Olive Oil, in LPS-Activated Murine Immune Cells.

The present study was designed to evaluate the immunomodulatory effects of the secoiridoid from extra virgin olive oil, oleacein (OLA), deepening into the possible signaling pathways involved in LPS-activated murine peritoneal macrophages. Moreover, we have explored OLA-induced epigenetic changes in histone markers and related cytokine production in murine LPS-stimulated murine splenocytes. Murine cells were treated with OLA in the presence or absence of LPS (5 μg/mL) for 18 or 24 h. OLA modulated the oxidative stress and the inflammatory response produced by LPS stimulation in murine peritoneal macrophages, by the inhibition of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-γ, IL-17 and IL-18) and ROS production and the expression of pro-inflammatory enzymes such as iNOS, COX-2 and m-PGES1. These protective effects could be due to the activation of the Nrf-2/HO-1 axis and the inhibition of JAK/STAT, ERK and P38 MAPKs and inflammasome canonical and non-canonical signaling pathways. Moreover, OLA modulated epigenetic modifications throughout histone methylation deacetylation (H3K18ac) and (H3K9me3 and H3K27me) in LPS-activated spleen cells. In conclusion, our data present OLA as an interesting anti-inflammatory and antioxidant natural compound that is able to regulate histone epigenetic markers. Nevertheless, additional in vivo studies are required to further investigate the beneficial effects of this EVOO secoiridoid, which might be a promising epinutraceutical bioproduct for the management of immune-related inflammatory diseases.

Differential Affinity Chromatography Coupled to Mass Spectrometry: A Suitable Tool to Identify Common Binding Proteins of a Broad-Range Antimicrobial Peptide Derived from Leucinostatin.

Leucinostatins are antimicrobial peptides with a broad range of activities against infectious agents as well as mammalian cells. The leucinostatin-derivative peptide ZHAWOC_6027 (peptide 6027) was tested in vitro and in vivo for activity against the intracellular apicomplexan parasite Toxoplasma gondii. While highly efficacious in vitro (EC50 = 2 nM), subcutaneous application of peptide 6027 (3 mg/kg/day for 5 days) in mice experimentally infected with T. gondii oocysts exacerbated the infection, caused mild clinical signs and elevated cerebral parasite load. Peptide 6027 also impaired the proliferation and viability of mouse splenocytes, most notably LPS-stimulated B cells, in vitro. To identify common potential targets in Toxoplasma and murine splenocytes, we performed differential affinity chromatography (DAC) with cell-free extracts from T. gondii tachyzoites and mouse spleens using peptide 6027 or an ineffective analogue (peptide 21,358) coupled to N-hydroxy-succinimide sepharose, followed by mass spectrometry. Proteins specifically binding to peptide 6027 were identified in eluates from the peptide 6027 column but not in peptide 21,358 nor the mock column eluates. In T. gondii eluates, 269 proteins binding specifically to peptide 6027 were identified, while in eluates from mouse spleen extracts 645 proteins specifically binding to this peptide were detected. Both datasets contained proteins involved in mitochondrial energy metabolism and in protein processing and secretion. These results suggest that peptide 6027 interacts with common targets in eukaryotes involved in essential pathways. Since this methodology can be applied to various compounds as well as target cell lines or organs, DAC combined with mass spectrometry and proteomic analysis should be considered a smart and 3R-relevant way to identify drug targets in pathogens and hosts, thereby eliminating compounds with potential side effects before performing tedious and costly safety and efficacy assessments in animals or humans.

Immunomodulatory Effects of (R)-Sulforaphane on LPS-Activated Murine Immune Cells: Molecular Signaling Pathways and Epigenetic Changes in Histone Markers.

The aim of this study was to explore the immunomodulatory effects of the natural enantiomer (R)-Sulforaphane (SFN) and the possible signaling pathways involved in an ex vivo model of LPS-stimulated murine peritoneal macrophages. Furthermore, we studied the epigenetic changes induced by (R)-SFN as well as the post-translational modifications of histone H3 (H3K9me3 and H3K18ac) in relation to the production of cytokines in murine splenocytes after LPS stimulation. (R)-SFN was able to modulate the inflammatory response and oxidative stress induced by LPS stimulation in murine peritoneal macrophages through the inhibition of reactive oxygen species (ROS), nitric oxide (NO) and cytokine (IL-1β, IL-6, IL-17, IL-18 and TNF-α) production by down-regulating the expression of pro-inflammatory enzymes (iNOS, COX-2 and mPGES-1). We also found that activation of the Nrf-2/HO-1 axis and inhibition of the JAK2/STAT-3, MAPK, canonical and non-canonical inflammasome signaling pathways could have been responsible for the immunomodulatory effects of (R)-SFN. Furthermore, (R)-SFN modulated epigenetic modifications through histone methylation (H3K9me3) and deacetylation (H3K18ac) in LPS-activated spleen cells. Collectively, our results suggest that (R)-SFN could be a promising epinutraceutical compound for the management of immunoinflammatory diseases.

Modulation of local and systemic immune responses by fermented garlic extract

Fermented garlic (FG), prepared by fermenting garlic with <em>Bacillus subtilis</em> for 3‑15 days at 20‑40˚C has been linked to immune‑boosting properties. In the present study, FG was utilized to investigate its immunomodulatory effects using RAW264.7 cells, as well as BALB/c and C3H/HeN mice. Nitric oxide (NO) and pro‑inflammatory cytokine levels were quantified in RAW264.7 cells treated with or without FG (0.016, 0.08, 0.4, 2 and 10 mg/ml), garlic extract (GE) (0.016, 0.08, 0.4, 2 and 10 mg/ml) and lipopolysaccharide (1 µg/ml). Peritoneal macrophages and splenocytes were obtained from BALB/c mice for <em>ex vivo</em> analyses. MTT and enzyme‑linked immunosorbent assays were used to determine splenocyte proliferation, natural killer (NK) cell activity and cytokine production. The intestinal immunostimulatory <em>in vitro</em> effects of FG were also investigated. In a concentration‑dependent manner, FG treatment markedly accelerated the proliferation of RAW264.7 cells, murine peritoneal macrophages and splenocytes. Moreover, FG initiated macrophage activation by significantly increasing the NO, tumor necrosis factor‑α, interleukin (IL)‑12 and IL‑6 levels in RAW264.7 cells and peritoneal macrophages. In contrast to FG, GE promoted RAW264.7 cell proliferation at lower concentrations and toxicity at higher concentrations, while no detectable NO synthesis was observed at the concentrations tested. Likewise, the FG extract at larger concentrations fortified the intestinal immune system and improved bone marrow cell proliferation by significantly elevating IL‑6 and granulocyte‑macrophage colony‑stimulating factor production in splenocytes and Peyer's patch cells of mice. In addition, the NK cell activities of splenocytes from FG‑treated mice at lower doses against YAC‑1 cells were significantly increased, which must have been fostered and activated by interferon‑γ generated by splenocytes. On the whole, these findings provide relevant <em>in vitro</em> and <em>ex vivo</em> information that describes the mechanisms through which FG can strengthen a compromised immune system by influencing local and systemic gut immune responses directly or indirectly.

TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1.

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory periodic fever syndrome associated with heterozygous mutations in TNFRSF1A, which encodes TNF receptor type I (TNFR1). Although possible proinflammatory mechanisms have been proposed, most previous studies were performed using in vitro overexpression models, which could lead to undesirable inflammatory responses due to artificial overexpression. It is crucial to reproduce heterozygous mutations at physiological expression levels; however, such studies remain limited. In this study, we generated TRAPS mutant mice and analyzed their phenotypes. Three Tnfrsf1a mutant strains were generated by introducing T79M, G87V, or T90I mutation. T79M is a known mutation responsible for TRAPS, whereas G87V is a TRAPS mutation that we have reported, and T90I is a variant of unknown significance. Using these murine models, we investigated whether TRAPS mutations could affect the inflammatory responses in vivo and in vitro. We found that none of the mutant mice exhibited detectable inflammatory phenotypes under standard housing conditions for 1 year. Interestingly, TRAPS mutant (T79M and G87V) mice had reduced mortality rates after the administration of lipopolysaccharide (LPS) and D-galactosamine, which induce TNFα-dependent lethal hepatitis. Moreover, TRAPS mutations strongly suppressed the development of TNFα-mediated arthritis when crossed with human TNFα transgenic mice. In in vitro primary bone marrow-derived macrophage cultures, the T79M and G87V mutations attenuated the inflammatory responses to TNFα compared with the wild-type, whereas these mutations did not alter the responsiveness of these cells to LPS. The T90I mutant macrophages behaved similarly to wild type in response to LPS and TNFα. The TNFR1 levels were increased in whole-cell lysates of TRAPS mutant macrophages, whereas the cell surface expression of TNFR1 was significantly decreased in TRAPS mutant macrophages. Taken together, TRAPS mutations did not augment the inflammatory responses to TNFα and LPS; instead, they suppressed the response to TNFα via decreased cell surface expression of TNFR1. The stimulation of lymphotoxin-α, adenosine triphosphate, and norepinephrine in primary macrophages or various stimuli in murine splenocytes did not induce detectable inflammatory responses. In conclusion, TRAPS mutations suppressed responsiveness to TNFα, and TRAPS-associated inflammation is likely induced by unconfirmed disease-specific proinflammatory factors.

Hypertensive Stimuli Indirectly Stimulate Lymphangiogenesis through Immune Cell Secreted Factors.

(1) Background: Renal immune cells and lymphatic vessel (LV) density have been reported previously to be increased in multiple mouse models of hypertension (HTN). However, whether interstitial levels of HTN stimuli such as angiotensin II, salt, or asymmetric dimethylarginine have a direct or indirect effect on lymphangiogenesis is unknown. We hypothesized that these 3 HTN stimuli directly increase lymphatic endothelial cell (LEC) proliferation, LEC 3-D matrix invasion and vessel formation, and sprouting of mouse mesometrial LVs. (2) Methods: Human LECs (hLECs) and mouse LECs (mLECs) were treated with HTN stimuli while explanted mouse mesometrial LVs were treated with either the same HTN stimuli or with HTN stimuli-conditioned media. Conditioned media was prepared by treating murine splenocytes with HTN stimuli. (3) Results: HTN stimuli had no direct effect on hLEC or mLEC proliferation. Treatment of hLECs with HTN stimuli increased the number of lumen-forming structures and invasion distance (both p < 0.05) in the 3-D matrix but decreased the average lumen diameter and the number of cells per invading structure (both p < 0.05). Conditioned media from HTN-stimuli-treated splenocytes significantly attenuated the decrease in sprout number (aside from salt) and sprout length of mouse mesometrial LVs that is found in the HTN stimuli alone. (4) Conclusions: These data indicate that HTN stimuli indirectly prevent a decrease in lymphangiogenesis through secreted factors from HTN-stimuli-treated immune cells.

Assessing the immunosuppressive activity of alginate-encapsulated mesenchymal stromal cells on splenocytes

Mesenchymal stromal cells (MSCs) show immunosuppressive effects both via cell-to-cell contact (direct) with immune cells and by producing paracrine factors and extracellular vesicles (indirect). A key challenge in delivering this therapeutic effect in vivo is retaining the MSCs at the site of injection. One way to address this is by encapsulating the MSCs within suitable biomaterial scaffolds. Here, we assess the immunosuppressive effect of alginate-encapsulated murine MSCs on proliferating murine splenocytes. Our results show that MSCs are able to significantly suppress splenocyte proliferation by ∼50% via the indirect mechanism and almost completely (∼98%) via the direct mechanism. We also show for the first time that MSCs as monolayers on tissue culture plastic or encapsulated within alginate, when physically isolated from the splenocytes via transwells, are able to sustain immunosuppressive activity with repeated exposure to fresh splenocytes, for as long as 9 days. These results indicate the need to identify design strategies to simultaneously deliver both modes of MSC immunosuppression. By designing cell-biomaterial constructs with tailored degradation profiles, we can achieve a more sustained (avoiding MSCs migration and apoptosis) and controlled release of both the paracrine signals and eventually the cells themselves enabling efficient MSC-based immunosuppressive therapies for wound healing.

Immune Memory After Respiratory Infection With Streptococcus pneumoniae Is Revealed by in vitro Stimulation of Murine Splenocytes With Inactivated Pneumococcal Whole Cells: Evidence of Early Recall Responses by Transcriptomic Analysis.

The in vitro stimulation of immune system cells with live or killed bacteria is essential for understanding the host response to pathogens. In the present study, we propose a model combining transcriptomic and cytokine assays on murine splenocytes to describe the immune recall in the days following pneumococcal lung infection. Mice were sacrificed at days 1, 2, 4, and 7 after Streptococcus pneumoniae (TIGR4 serotype 4) intranasal infection and splenocytes were cultured in the presence or absence of the same inactivated bacterial strain to access the transcriptomic and cytokine profiles. The stimulation of splenocytes from infected mice led to a higher number of differentially expressed genes than the infection or stimulation alone, resulting in the enrichment of 40 unique blood transcription modules, including many pathways related to adaptive immunity and cytokines. Together with transcriptomic data, cytokines levels suggested the presence of a recall immune response promoting both innate and adaptive immunity, stronger from the fourth day after infection. Dimensionality reduction and feature selection identified key variables of this recall response and the genes associated with the increase in cytokine concentrations. This model could study the immune responses involved in pneumococcal infection and possibly monitor vaccine immune response and experimental therapies efficacy in future studies.

Electrical stimulation of the splenic nerve bundle ameliorates dextran sulfate sodium-induced colitis in mice

BackgroundVagus nerve stimulation has been suggested to affect immune responses, partly through a neuronal circuit requiring sympathetic innervation of the splenic nerve bundle and norepinephrine (NE) release. Molecular and cellular mechanisms of action remain elusive. Here, we investigated the therapeutic value of this neuromodulation in inflammatory bowel disease (IBD) by applying electrical splenic nerve bundle stimulation (SpNS) in mice with dextran sulfate sodium (DSS)-induced colitis.MethodsCuff electrodes were implanted around the splenic nerve bundle in mice, whereupon mice received SpNS or sham stimulation. Stimulation was applied 6 times daily for 12 days during DSS-induced colitis. Colonic and splenic tissues were collected for transcriptional analyses by qPCR and RNA-sequencing (RNA-seq). In addition, murine and human splenocytes were stimulated with lipopolysaccharide (LPS) in the absence or presence of NE. Single-cell RNA-seq data from publicly available data sets were analyzed for expression of β-adrenergic receptors (β-ARs).ResultsColitic mice undergoing SpNS displayed reduced colon weight/length ratios and showed improved Disease Activity Index scores with reduced Tumor Necrosis Factor α mRNA expression in the colon compared with sham stimulated mice. Analyses of splenocytes from SpNS mice using RNA-seq demonstrated specific immune metabolism transcriptome profile changes in myeloid cells. Splenocytes showed expression of β-ARs in myeloid and T cells. Cytokine production was reduced by NE in mouse and human LPS-stimulated splenocytes.ConclusionsTogether, our results demonstrate that SpNS reduces clinical features of colonic inflammation in mice with DSS-induced colitis possibly by inhibiting splenic myeloid cell activation. Our data further support exploration of the clinical use of SpNS for patients with IBD.

Abstract 2122: CRD1600 is a potent HPK-I inhibitor with robust immune modulatory properties

Abstract Background: Haematopoietic Progenitor Kinase (HPK-1, MAP4K1) is a member of the STE20 family of serine/threonine kinases, expressed predominantly in hematopoietic cells. The kinase activity of HPK-1 provides an inhibitory signal that dampens immune responses1. Upon activation of T-cell and/or B-cell receptors, activated HPK-1 phosphorylates the adaptor protein SLP76 at Ser376 in T cells or BLNK at Thr152 in B-cells to trigger proteasomal degradation of the adaptors and disrupt downstream T-cell or B-cell activating signalosome complexes. Blocking HPK-1 extends immune action and since HPK-1 expression is primarily restricted to immune cells, it is a compelling drug target for immune modulation. Anti-tumor efficacy has been demonstrated in HPK-1 kinase dead knock-in mice, HPK-1 knockout mice or using small molecule pharmacological inhibitors of HPK-1 either as monotherapy or in combination regimens2. Methods: Curadev used a pharmacophore based approach to discover novel small molecule HPK-1 inhibitors. SAR studies were carried out using a screening funnel that included efficacy assessment by monitoring in vitro enzymatic and cellular activity, anti-target cross-kinase specificity by assessing cytotoxicity in fresh murine splenocytes and effector mobilization by monitoring IL2 production and human T-cell proliferation under anti-CD3/CD28 stimulation. Results: Curadev’s medicinal chemistry campaign yielded several highly potent HPK-1 inhibitors with sub-nanomolar to nanomolar potency in in vitro human HPK-1 enzyme assays which were active in the cellular SLP76 assay. A single dose of the lead compound CRD1600 to mice treated with an anti-CD3 antibody caused systemic enhancement of pro-inflammatory cytokines such as IL2 and reduced pSLP76 levels in the spleen. These pharmacodynamic effects correlated with plasma levels of CRD1600. Conclusions: Inhibition of HPK-1 is a promising therapeutic modality that could augment the effects of existing anti-cancer treatments including immunotherapy. CRD1600 is a potent, selective HPK-1 inhibitor that has been selected from a rich portfolio of active compounds and shows in vivo activity against the target. Further development of the compound is ongoing.

Protective Effect of Casticin on Experimental Skin Wound Healing of Rats.

The wound is known as damage to the skin structure by external stimuli, such as cut, bruises, and burn, which typically leaves the internal tissue exposed. The wound repair process when hampered leads to an excessive burden on the healthcare setting. Therefore, there is an urgent need to develop effective agents that can promote the wound healing process. In the present study, we intended to investigate the pharmacological benefit of Casticin (CST), a polymethylflavone against wound. The wound in Wistar rats was induced by a surgical procedure. After surgery, the wound was examined for wound size over a period and for the expression of cyclooxygenase-2 and induced collagen III expressions. The effect of CST was examined on tumor necrosis factor α, interferon gamma, interleukin (IL)-10, transforming growth factor beta, and IL-7 by real-time polymerase chain reaction. The expression of matrix metalloproteinases (MMP)-2 and MMP-9 and its natural inhibitor (tissue inhibitors of metalloproteinases, TIMP1), level of macrophages, and lymphocytes were also quantified. The effect of CST was determined also on apoptosis of rats' splenocytes. CST significantly enhances wound healing of rat postsurgery, with maximum activity achieved in the case of a 60μM treated group. The expression of cyclooxygenase-2 was found reduced together with an increase in collagen III, tumor necrosis factor α, interferon gamma, IL-10, transforming growth factor beta, and IL-7 in the CST group. The levels of MMP-2 and MMP-9 were also found reduced together with an increase in TIMP1 level in CST-treated group. The levels of CD4+, CD8+, and CD11b+ cells at the wound site 24 and 120h postsurgery was also found reduced in CST-treated group. However, it showed no apoptosis in murine splenocytes. Collectively, CST can promote the wound healing process by modulation of inflammation and immune response, which induces tissue remodeling.

Oxonitrogenated Derivatives of Eremophilans and Eudesmans: Antiproliferative and Anti-Trypanosoma cruzi Activity.

Cancer is one of the most important causes of death worldwide. Solid tumors represent the vast majority of cancers (>90%), and the chemotherapeutic agents used for their treatment are still characterized by variable efficacy and toxicity. Sesquiterpenes are a group of natural compounds that have shown a wide range of biological activities, including cytotoxic and antiparasitic activity, among others. The antiproliferative activity of natural sesquiterpenes, tessaric acid, ilicic acid, and ilicic alcohol and their semisynthetic derivatives against HeLa, T-47D, WiDr, A549, HBL-100, and SW1573 cell lines were evaluated. The effect of the compounds on Trypanosoma cruzi epimastigotes was also assessed. The selectivity index was calculated using murine splenocytes. Derivatives 13 and 15 were the most antiproliferative compounds, with GI50 values ranging between 5.3 (±0.32) and 14 (±0.90) μM, in all cell lines tested. The presence of 1,2,3-triazole groups in derivatives 15–19 led to improvements in activity compared to those corresponding to the starting natural product (3), with GI50 values ranging between 12 (±1.5) and 17 (±1.1) μM and 16 being the most active compound. In relation to the anti-T. cruzi activity, derivatives 7 and 16 obtained from tessaric acid and ilicic acid were among the most active and selective compounds with IC50 values of 9.3 and 8.8 µM (SI = 8.0 and 9.4), respectively.

Discovery of Undescribed Monoterpenoid Polyprenylated Acylphloroglucinols with Immunosuppressive Activities from Hypericum longistylum

Acylphlorostylums A–G (1–7), seven undescribed monoterpenoid polyprenylated acylphloroglucinols, were isolated and identified from Hypericum longistylum. Significantly, acylphlorostylums A and B were the first monoterpenoid polyprenylated acylphloroglucinols possessing a dodecahydro-1H-benzo [b]cyclopenta [e]oxepine moiety bearing a 6/7/5 fused tricyclic ring system that assembled by the attack from 4-OH to C-13. In addition, acylphlorostylums A–G exhibited moderate in vitro immunosuppressive activity in anti-CD3/anti-CD28 monoclonal antibodies, lipopolysaccharide and concanavalin A-induced murine splenocyte proliferation, with IC50 values ranging from 1.51 ± 0.12 to 18.49 ± 1.67 μM, underscoring those isolates as novel chemical templates in the development of novel immunosuppressors.

A selective luminescent probe to monitor cellular ATP: Potential application for in vivo imaging in zebrafish embryo

Adenosine 5′-triphosphate (ATP) is the major energy currency of living cells and its efficient real-time monitoring in vivo has increased the demand for the development of fluorescent chemosensors. For the prompt detection of ATP in submolar range, we have developed an easily accessible colorimetric and fluorescence probe 4-(Di-p-tolylphosphoryl)-7-hydroxychroman-2-one (DPC). The selective nature of DPC towards ATP has been confirmed through UV–vis, fluorescence titration, NMR titration as well as computational studies. Coupled with high mitochondrial membrane potential and ATP levels, the chemosensor DPC shows intense blue fluorescence signal at intracellular compartments in murine splenocytes and A549 human lung cancer cells. Moreover, using live zebrafish (Danio rerio)embryos with high energetic demands, we have successfully achieved fluorescence imaging of ATP at distinct sites in vivo(neural tube, myotome or embryonic muscle tissue, somite boundaries and tail regions) followed by estimation of endogenous ATP level from embryo extract. Collectively, our designed probe demonstrates excellent cellular permeability, sensitivity and specificity for intracellular ATP, and may provide valuable insights in the research of ATP-regulated biological processes in living cells or organisms.

Betulin Attenuates TGF-β1- and PGE&lt;sub&gt;2&lt;/sub&gt;-Mediated Inhibition of NK Cell Activity to Suppress Tumor Progression and Metastasis in Mice

Transforming growth factor (TGF)-β1 and prostaglandin E2 (PGE2) are humoral factors critically involved in the induction of immunosuppression in the microenvironment of various types of tumors, including melanoma. In this study, we identified a natural compound that attenuated TGF-β1- and PGE2-induced immunosuppression and examined its effect on B16 melanoma growth in mice. By screening 502 natural compounds for attenuating activity against TGF-β1- or PGE2-induced suppression of cytolysis in poly(I:C)-stimulated murine splenocytes, we found that betulin was the most potent compound. Betulin also reduced TGF-β1- and PGE2-induced downregulation of perforin and granzyme B mRNA expression and cell surface expression of NKG2D and CD69 in natural killer (NK) cells. Cell depletion and coculture experiments showed that NK cells, dendritic cells, B cells, and T cells were necessary for the attenuating effects of betulin. Structure-activity relationship analysis revealed that two hydroxyl groups at positions C3 and C28 of betulin, their cis-configuration, and methyl group at C30 played crucial roles in its attenuating activity. In a subcutaneous implantation model of B16 melanoma in mice, intratumor administration of betulin and LY2157299, a TGF-β1 type I receptor kinase inhibitor, significantly retarded the growth of B16 melanoma. Notably, betulin increased significantly the number of CD69 positive NK cells in tumor sites at early stages of post-tumor cell injection. Our data suggest that betulin inhibits the growth of B16 melanoma by enhancing NK cell activity through attenuating the immunosuppressive tumor microenvironment.

Gold Nanorods are Selective Cytotoxic Agents.

Gold nanorods (GNRs) are very promising agents with multiple applications in medicine and biology. However, the cytotoxic effects of GNRs have not been fully explored. Therefore, the main objective of this study was to determine the selective cytotoxic effect of GNRs towards several human tumor cell lines. To address this issue, three sizes of GNRs (10-nm, 25-nm, and 50-nm) were tested against two human tumor cell lines, namely, human hepatoma HepG2 and human prostate PC3 cancer cells. As GNRs are usually stored in soft tissues inside living bodies, we also tested the effect of GNRs on murine splenocyte viability. To determine if the GNRs displayed selective cytotoxicity towards cancer cells, active GNRs with the size showing the least cytotoxicity to splenocytes were then tested against a panel of 11 human tumor cell lines and two human non-tumor cell lines. Our results showed that the most cytotoxic size of GNRs is 10-nm, followed by the 25-nm GNRs, while the 50-nm GNRs did not show a significant effect. In addition, the 25-nm GNRs were the least cytotoxic to splenocytes when tested for 24 and 48 h. These GNRs showed a selective cytotoxic effect to prostate cancer PC3 cells with median inhibitory concentration (IC50) = 8.3 ± 0.37 μM, myeloblastic leukemia HL60 cells (IC50 = 19.7 ± 0.89 μM), cervical cancer HeLa cells (IC50 = 24.6 ± 0.37 μM), renal adenocarcinoma 786.0 cells (IC50 = 27.34 ± 0.6 μM), and hepatoma HepG2 cells (IC50 = 27.79 ± 0.03 μM) when compared to the effect on the non-tumor human cells; skin fibroblast BJ cell line (IC50 = 40.13 ± 0.7 μM) or epithelial breast MCF10A cells (IC50 = 33.2 ± 0.89 μM). High selectivity indices (SIs) were observed in GNRs-treated PC3 and HL60 cells with values ranging from 1.69 to 4.83, whereas moderate SIs were observed in GNRs-treated HeLa, 786.0, and HepG2 cells with values ranging from 1.19 to 1.63. Other cells did not show a similar selective effect, including human laryngeal HEp2 cells, colon HCT116, metastatic renal adenocarcinoma ACHN cells, and human breast cancer cells (MCF7, MDA-MB-231, and MDA-MB-468 cells). The effect of GNRs was confirmed using the colony formation assay and the effect was found to be cell cycle-specific. Finally, it was shown that laser treatment could potentiate the cytotoxic effect of the 25-nm GNRs. GNRs are selective cytotoxic agents and they have the potential to act as candidate anticancer agents.