Macrophage Cytotoxicity Research Articles

A novel inhibitor of class IIa histone deacetylases attenuates collagen-induced arthritis.

Most inhibitors of histone deacetylases (HDACs) are not selective and are cytotoxic. Some have anti-inflammatory activity in disease models, but cytotoxicity prevents long-term uses in non-fatal diseases. Inhibitors selective for class IIa HDACs are much less cytotoxic and may have applications in management of chronic inflammatory diseases. LL87 is a novel HDAC inhibitor examined here for HDAC enzyme selectivity. It was also investigated in macrophages for cytotoxicity and for inhibition of lipopolysaccharide (LPS)-stimulated cytokine secretion. In a rat model of collagen-induced arthritis, LL87 was investigated for effects on joint inflammation in Dark Agouti rats. Histological, immunohistochemical, micro-computed tomography and molecular analyses characterise developing arthritis and anti-inflammatory efficacy. LL87 was significantly more inhibitory against class IIa than class I or IIb HDAC enzymes. In macrophages, LL87 was not cytotoxic and reduced both LPS-induced secretion of pro-inflammatory cytokines, and IL6-induced class IIa HDAC activity. In rats, LL87 attenuated paw swelling and clinical signs of arthritis, reducing collagen loss and histological damage in ankle joints. LL87 decreased immune cell infiltration, especially pro-inflammatory macrophages and osteoclasts, into synovial joints and significantly reduced expression of pro-inflammatory cytokines and tissue-degrading proteases. A novel inhibitor of class IIa HDACs has been shown to have an anti-inflammatory and anti-arthritic profile distinct from current therapies. It is efficacious in reducing macrophage infiltration and joint inflammation in a chronic model of rat arthritis.

Effect of a thiohydantoin salt derived from l-Arginine on Leishmania amazonensis and infected cells: Insights from biological effects to molecular docking interactions

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor.

TolC facilitates the intracellular survival and immunomodulation of Salmonella Typhi in human host cells

ABSTRACT Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a systemic infection that affects millions of people worldwide. S. Typhi can invade and survive within host cells, such as intestinal epithelial cells and macrophages, by modulating their immune responses. However, the immunomodulatory capability of S. Typhi in relation to TolC-facilitated efflux pump function remains unclear. The role of TolC, an outer membrane protein that facilitates efflux pump function, in the invasion and immunomodulation of S. Typhi, was studied in human intestinal epithelial cells and macrophages. The tolC deletion mutant of S. Typhi was compared with the wild-type and its complemented strain in terms of their ability to invade epithelial cells, survive and induce cytotoxicity in macrophages, and elicit proinflammatory cytokine production in macrophages. The tolC mutant, which has a defective outer membrane, was impaired in invading epithelial cells compared to the wild-type strain, but the intracellular presence of the tolC mutant exhibited greater cytotoxicity and induced higher levels of proinflammatory cytokines (IL-1β and IL-8) in macrophages compared to the wild-type strain. These effects were reversed by complementing the tolC mutant with a functional tolC gene. Our results suggest that TolC plays a role in S. Typhi to efficiently invade epithelial cells and suppress host immune responses during infection. TolC may be a potential target for the development of novel therapeutics against typhoid fever.

Exploring antiviral and antiparasitic activity of gold N-heterocyclic carbenes with thiolate ligands.

Gold(I) N-heterocyclic carbenes have been explored for their therapeutic potential against several diseases. Neglected tropical diseases, including leishmaniasis, Chagas disease, and viral infections, such as zika, mayaro, and chikungunya, urgently require new treatment options. The emergent SARS-CoV-2 also demands significant attention. Gold complexes have shown promise as alternative treatments for these conditions. Previously, gold(I)(1,3-bis(mesityl)imidazole-2-ylidene)Cl (AuIMesCl) demonstrated significant leishmanicidal and anti-Chikungunya virus activities. In this study, we synthesized and fully characterized a series of gold(I)(1,3-bis(mesityl)imidazole-2-ylidene)(SR) complexes, where SR includes thiolate donor species such as 1,3-thiazolidine-2-thione, 1,3-benzothiazole-2-thione, 2-mercaptopyrimidine, and 2-thiouracil. These compounds were stable in solution, and ligand exchange reactions with N-acetyl-L-cysteine indicated that complexes with SR ligands are more labile than those with chloride. Although the reactions are rapid, they reach equilibrium at varying molar ratios depending on the SR ligand. The increased lability of these compounds results in higher cytotoxicity to host cells, such as Vero E6 and bone marrow-differentiated macrophages, compared to AuIMesCl. Despite this, the compounds effectively inhibited viral replication, achieving 95.5% inhibition of Zika virus replication at 2 μM with 96% host cell viability. Although active at low concentrations (∼2 μM) against Leishmania (L.) amazonensis and Trypanosoma cruzi, their high cytotoxicity for macrophages confirmed AuIMesCl as a better candidate with a higher selectivity index. This work correlates the coordination chemistry of pyrimidines and thiazolidines with their in vitro biological activities against significant diseases.

Protein Corona-Directed Cellular Recognition and Uptake of Polyethylene Nanoplastics by Macrophages.

The widespread use of plastic products in daily life has raised concerns about the health hazards associated with nanoplastics (NPs). When exposed, NPs are likely to infiltrate the bloodstream, interact with plasma proteins, and trigger macrophage recognition and clearance. In this study, we focused on establishing a correlation between the unique protein coronal signatures of high-density (HDPE) and low-density (LDPE) polyethylene (PE) NPs with their ultimate impact on macrophage recognition and cytotoxicity. We observed that low-density and high-density lipoprotein receptors (LDLR and SR-B1), facilitated by apolipoproteins, played an essential role in PE-NP recognition. Consequently, PE-NPs activated the caspase-3/GSDME pathway and ultimately led to pyroptosis. Advanced imaging techniques, including label-free scattered light confocal imaging and cryo-soft X-ray transmission microscopy with 3D-tomographic reconstruction (nano-CT), provided powerful insights into visualizing NPs-cell interactions. These findings underscore the potential risks of NPs to macrophages and introduce analytical methods for studying the behavior of NPs in biological systems.

Nanostructured Hybrid Polymer‐Lipid Drug Delivery Platforms for Rapamycin Repositioning in Anticancer Therapy

AbstractHere, hybrid polymer‐lipid nanoparticles are designed as colloidal carriers for Rapamycin, to improve the aqueous drug stability and to support the drug repositioning for cancer treatment, that is, against rhabdomyosarcoma (RMS). With this aim, Rapamycin – loaded hybrid nanoparticles are produced by using as nanoparticle core a graft copolymer obtained from the functionalization of the α,β‐poly(N‐2‐hydroxyethyl)‐DL‐aspartamide (PHEA) with Rhodamine B (RhB), Polylactic acid (PLA), the PHEA‐g‐RhB‐g‐PLA, and different phospholipids, that is, 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) coated, pegylated and Mannose/PEG, for the surface coating. The drug loading of these samples allows for controlled release, and improves drug stability at pH 5.5 and 7.4 compared to the free drug. Chemical‐physical characterization confirms the nanostructure size below 200 nm, ideal for systemic administration, and easy re‐dispersibility in aqueous media. Moreover, biological characterization to test the potential use as antitumor agent shows induction of cytotoxicity in human rhabdomyosarcoma (RD) and macrophage (RAW) cell lines in a time‐ and concentration – dependent manner, and stimulated autophagy, comparable to the free drug. The uptake study following the fluorescence of the copolymer reveals that the hybrid nanoparticles are internalized by both tested cell lines, with a significantly higher amounts of internalized particles in the case of surface mannosylated and/or pegylated systems.

Immunomodulatory activity of polycaprolactone nanoparticles with calcium phosphate salts against Leishmania infantum infection

Objective: To prepare and characterize polycaprolactone (PCL) nanoparticles loaded with sonicator fragmented (SLA) and freeze- thaw Leishmania antigens (FTLA) and to investigate the in vitro immunogenicity of antigen-encapsulated nanoparticles with calcium phosphate adjuvant. Methods: The water/oil/water binary emulsion solvent evaporation method was used to synthesize antigen-loaded PCL nanoparticles. Particles were characterized by scanning electron microscopy and zeta potential measurements. Their cytotoxicity in J774 macrophages in vitro was determined by MTT analysis. In addition, the amount of nitric oxide and the level of cytokines produced by macrophages were determined by Griess reaction and ELISA method, respectively. The protective effect of the developed formulations was evaluated by determining the infection index percentage in macrophages infected with Leishmania infantum. Results: Compared to the control group, SLA PCL and FTLA PCL nanoparticles with calcium phosphate adjuvant induced a 6- and 7-fold increase in nitric oxide, respectively. Additionally, the vaccine formulations promoted the production of IFN-γ and IL-12. SLA PCL and FTLA PCL nanoparticles combined with calcium phosphate adjuvant caused an approximately 13- and 11-fold reduction in infection index, respectively, compared to the control group. Conclusions: The encapsulation of antigens obtained by both sonication and freeze-thawing into PCL nanoparticles and the formulations with calcium phosphate adjuvant show strong in vitro immune stimulating properties. Therefore, PCL-based antigen delivery systems and calcium phosphate adjuvant are recommended as a potential vaccine candidate against leishmaniasis.

Precursor B Cell Acute Lymphoblastic Leukemia Presenting with Repeated Episodes of Hemophagocytic Lymphohistiocytosis

AbstractHemophagocytic lymphohistiocytosis (HLH) is a rare disorder characterized by dysregulated activation of cytotoxic T-lymphocytes and macrophages, resulting in excessive cytokine release and tissue damage. Although hematological malignancies and the chemotherapies used to treat them are frequently identified as triggering factors for HLH, B lymphoid leukemias are rarely implicated. In this report, we present an interesting case of a patient who presented with symptoms of HLH and was subsequently diagnosed with B-cell acute lymphoblastic leukemia. The further course of chemotherapy was complicated by another episode of HLH. This case highlights the complexity and the diverse triggers of HLH in the context of B lymphoid leukemia. Recognizing this atypical presentation is important to institute timely management strategies.

A Second Wind for Inorganic APIs: Leishmanicidal and Antileukemic Activity of Hydrated Bismuth Oxide Nanoparticles.

American cutaneous leishmaniasis is a disease caused by protozoa of the genus Leishmania. Currently, meglumine antimoniate is the first-choice treatment for the disease. The limited efficacy and high toxicity of the drug results in the necessity to search for new active principles. Nanotechnology is gaining importance in the field, since it can provide better efficacy and lower toxicity of the drugs. The present study aimed to synthesize, characterize, and evaluate the in vitro leishmanicidal and antileukemic activity of bismuth nanoparticles (BiNPs). Promastigotes and amastigotes of L. (V.) guyanensis and L. (L.) amazonensis were exposed to BiNPs. The efficacy of the nanoparticles was determined by measurement of the parasite viability and the percentage of infected cells, while the cytotoxicity was characterized by the colorimetry. BiNPs did not induce cytotoxicity in murine peritoneal macrophages and showed better efficacy in inhibiting promastigotes (IC50 < 0.46 nM) and amastigotes of L. (L.) amazonensis. This is the first report on the leishmanicidal activity of Bi-based materials against L. (V.) guayanensis. BiNPs demonstrated significant cytotoxic activity against K562 and HL60 cells at all evaluated concentrations. While the nanoparticles also showed some cytotoxicity towards non-cancerous Vero cells, the effect was much lower compared to that on cancer cells. Treatment with BiNPs also had a significant effect on inhibiting and reducing colony formation in HL60 cells. These results indicate that bismuth nanoparticles have the potential for an inhibitory effect on the clonal expansion of cancer cells.

Bixin, a New Atheroprotective Carotenoid Candidate, Prevents oxLDL-Induced Cytotoxicity and Mitochondrial Dysfunction in Macrophages: Involvement of the Nrf2 and NF-κB Pathways.

The accumulation of oxidized low-density lipoprotein (oxLDL) and its toxicity in the arterial wall have been implicated in atherosclerosis. This study aimed to investigate the mechanisms underlying the atheroprotective effect of bixin, a carotenoid obtained from the seeds of the tropical plant Bixa orellana, on Cu2+-induced LDL oxidation and oxLDL-mediated effects in J774A.1 macrophage cells. Bixin's effects were compared to those of lycopene, a carotenoid widely studied for its cardiovascular protective effects. LDL was isolated from human plasma, incubated with bixin or lycopene (positive control), and subjected to oxidation with CuSO4. Afterward, bixin or lycopene was incubated with J774A.1 macrophage cells and exposed to oxLDL. The levels of ROS, RNS, GSH, nitrite, mitochondrial function, and foam cell formation, as well as the expression of proteins related to the antioxidant and inflammatory status, were evaluated. The effect of bixin in inhibiting in vitro human-isolated LDL oxidation was more potent (5-6-fold) than that of lycopene. Bixin pretreatment reduced the atherogenic signaling triggered by oxLDL in the macrophages, namely the generation of reactive species, disturbance of nitric oxide homeostasis, mitochondrial dysfunction, and foam cell formation. The cytoprotective effects of bixin were accompanied by the upregulation of Nrf2 and the downregulation of the NF-kB pathways. Lycopene showed the same protective effect as bixin, except that it did not prevent mitochondrial dysfunction. The efficient performance of bixin makes it an ideal candidate for further trials as a new nutraceutical compound for the prevention of atherosclerosis.

Spatial phenotyping of nodular lymphocyte predominant Hodgkin lymphoma and T-cell/histiocyte-rich large B-cell lymphoma

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma with sparse tumor B-cells and a favorable prognosis. Variant growth patterns of NLPHL, however, often show advanced stage, progression to T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) and a worse prognosis. We studied the tumor microenvironment (TME) of NLPHL and THRLBCL using highplex imaging and spatial profiling at the single cell level. Our findings show distinct differences in TME composition and spatial configuration that differ among typical and variant NLPHL and THRLBCL. Typical NLPHL show abundant helper T-cell subsets, while THRLBCL show abundant cytotoxic T-cells and macrophages. Tumor B-cell size and content is lowest in typical NLPHL, followed by variant NLPHL, and highest in THRLBCL, whereas an opposite trend characterized TME B-cells. CD4/CD8 double-positive T-cells are seen in all NLPHL but not in the majority of THRLBCL and are spatially distant from LP-cells and TFH-rosettes. The differences in macrophage/monocyte content in distinguishing NLPHL pattern E from THRLBCL is further corroborated in independent cohorts of cases. Our results validate the current approach to classification and in addition provide novel insights that could be leveraged to refine clinical management for patients with this spectrum of lymphomas.

Nigella sativa, active principle thymoquinone, alleviates palmitate-induced cytotoxicity, inflammation and bioenergetic disruptions in macrophages: An invitro study model

Thymoquinone (TQ) is one of the main phytochemical bioactive ingredients in Nigella sativa, with reported immunity-boosting properties. The current study evaluated the anti-inflammatory effect of TQ against inflammation brought on by free fatty acid Palmitate (PA) using macrophages raw 264.7 cell line. Data revealed that TQ significantly improved the viability of basal and PA stimulated Macrophages at concentrations of 50 and 100 μg/mL. Also, TQ significantly reduced nitric oxide and triglyceride levels in PA-stimulated macrophages at concentrations of 50 and 100 μg/mL. The pro-inflammatory cytokines studies revealed that PA significantly increased the release of the cytokines TNF-α, MHGB-1, IL-1β, and IL-6. TQ at concentrations 25, 50, and 100 μg/ml significantly decreases the release of the studied cytokines in PA-stimulated macrophages to variable extents with parallel inhibition to their corresponding gene expression. Bioenergetic assays showed that PA significantly decreased cellular ATP, mitochondrial complexes I and III activities and mitochondrial membrane potential with a subsequent significant increase in lactate production. At the same time, TQ can alleviate the effect of PA on macrophages' bioenergetics parameters to variable extent based on TQ concentration. To conclude, TQ could mitigate palmitate-induced inflammation and cytotoxicity in macrophages by improving macrophage viability and controlling cytokine release with improved PA-induced bioenergetics disruption.

Chlorpyrifos-induced suppression of the antioxidative defense system leads to cytotoxicity and genotoxicity in macrophages

Chlorpyrifos, widely used for pest control, is known to have various harmful effects, although its toxic effects in macrophages and the mechanisms underlying its toxicity remain unclear. The present study investigated the toxic effects of chlorypyrifos in a macrophage cell line. Here, we found that chlorpyrifos induced cytotoxicity and genotoxicity in RAW264.7 macrophages. Moreover, chlorpyrifos induced intracellular ROS production, subsequently leading to lipid peroxidation. Chlorpyrifos reduced the activation of antioxidative enzymes including superoxide dismutase, catalase, and glutathione peroxidase. Chlorpyrifos upregulated HO-1 expression and activated the Keap1-Nrf2 pathway, as indicated by enhanced Nrf2 phosphorylation and Keap1 degradation. Chlorpyrifos exerted effects on the following in a dose-dependent manner: cytotoxicity, genotoxicity, lipid peroxidation, intracellular ROS production, antioxidative enzyme activity reduction, HO-1 expression, Nrf2 phosphorylation, and Keap1 degradation. Notably, N-acetyl-L-cysteine successfully inhibited chlorpyrifos-induced intracellular ROS generation, cytotoxicity, and genotoxicity. Thus, chlorpyrifos may induce cytotoxicity and genotoxicity by promoting intracellular ROS production and suppressing the antioxidative defense system activation in macrophages.

Molecular incompatibility between pig CD200 and human CD200 receptor in in vitro xenogeneic immune responses.

Overexpression of human CD200 (hCD200) in porcine endothelial cells (PECs) has been reported to suppress xenogeneic immune responses of human macrophages againstporcine endothelial cells. The current study aimed to address whether the above-mentioned beneficial effect of hCD200 is mediated by overcoming the molecular incompatibility between porcine CD200 (pCD200) and hCD200 receptor or simply by increasing the expression levels of CD200 without any molecular incompatibility across the two species. We overexpressed hCD200 or pCD200 using lentiviral vectors with V5 marker in porcine endothelial cells and compared their suppressive activity against U937-derived human macrophage-like cells (hMCs) and primary macrophages. In xenogeneic coculture of porcine endothelial cellsand human macrophage-like cells or macrophages, hCD200-porcine endothelial cells suppressed phagocytosis and cytotoxicity of human macrophages to a greater extent than pCD200-porcine endothelial cells. Secretion of tumor necrosis factor-α, interleukin-1β, and monocyte chemoattractant protein-1 from human macrophages and expression of M1 phenotypes (inducible nitric oxide synthase, dectin-1, and CD86) were also suppressed by hCD200 to a greater extent than pCD200. Furthermore, in signal transduction downstream of CD200 receptor, hCD200 induced Dok2 phosphorylation and suppressed IκB phosphorylation to a greater extent than pCD200. The above data supported the possibility of a significant molecular incompatibility between pCD200 and human CD200 receptor, suggesting that the beneficial effects of hCD200 overexpression in porcine endothelial cellscould be mediated by overcoming the molecular incompatibility across the species barrier rather than by simple overexpression effects of CD200.

Nanostructured Lipid Carriers for oral treatment of leishmaniasis: Design and preclinical evaluation

Pentavalent antimonials are the primary treatment for leishmaniasis due to their proven efficacy and cost-effectiveness. However, they present poor oral absorption, parenteral administration, and serious adverse effects due to prolonged use. Nanostructured Lipid Carriers (NLC) offer a promising solution, providing prolonged release, enhanced intestinal permeability, and oral bioavailability. This study aimed to address these issues by developing Nanostructured Lipid Carrier loaded with meglumine antimoniate (NLC-MA) for oral leishmaniasis treatment. We used design of experiments (DoE) for optimization. The chosen NLC-MA was characterized assessing size, polydispersity index (PdI), zeta potential (ZP), entrapment efficiency (EE), pH, and by analytical techniques such as DSC, FTIR, XRD, and TEM. Stability under simulated gastrointestinal conditions, in vitro release kinetics, cytotoxicity in RAW 264.7 macrophages, and leishmanicidal activity were assessed. NLC-MA presented 41.13 nm, PdI 0.227, ZP −27.9 mV, EE of 62.43%, and pH 5.23. Characterization techniques confirmed drug incorporation and nanoparticles with reduced crystallinity. TEM images showed spherical morphology. NLC remained stable in gastrointestinal pH and under refrigeration storage, while lyophilized formulations retained their initial properties. MA release from NLC followed the Peppas-Sahlin model, an anomalous transport mechanism. Importantly, MA showed no cytotoxicity in macrophages, but the NLCs exhibited cytotoxicity beyond 50 µg/mL. NLC-MA displayed improved efficacy against Leishmania infantum. Overall, these results highlight the potential of utilizing a lipid nanocarrier incorporating meglumine antimoniate as an innovative drug delivery platform for oral treatment against leishmaniasis.

Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

Ruthenium-Cathepsin Inhibitor Conjugates for Green Light-Activated Photodynamic Therapy and Photochemotherapy.

Dysregulated cathepsin activity is linked to various human diseases including metabolic disorders, autoimmune conditions, and cancer. Given the overexpression of cathepsin in the tumor microenvironment, cathepsin inhibitors are promising pharmacological agents and drug delivery vehicles for cancer treatment. In this study, we describe the synthesis and photochemical and biological assessment of a dual-action agent based on ruthenium that is conjugated with a cathepsin inhibitor, designed for both photodynamic therapy (PDT) and photochemotherapy (PCT). The ruthenium-cathepsin inhibitor conjugate was synthesized through an oxime click reaction, combining a pan-cathepsin inhibitor based on E64d with the Ru(II) PCT/PDT fragment [Ru(dqpy)(dppn)], where dqpy = 2,6-di(quinoline-2-yl)pyridine and dppn = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine. Photochemical investigations validated the conjugate's ability to release a triazole-containing cathepsin inhibitor for PCT and to generate singlet oxygen for PDT upon exposure to green light. Inhibition studies demonstrated the conjugate's potent and irreversible inactivation of purified and intracellular cysteine cathepsins. Two Ru(II) PCT/PDT agents based on the [Ru(dqpy)(dppn)] moiety were evaluated for photoinduced cytotoxicity in 4T1 murine triple-negative breast cancer cells, L929 fibroblasts, and M0, M1, and M2 macrophages. The cathepsin inhibitor conjugate displayed notable selectivity for inducing cell death under irradiation compared to dark conditions, mitigating toxicity in the dark observed with the triazole control complex [Ru(dqpy)(dppn)(MeTz)]2+ (MeTz = 1-methyl-1H-1,2,4-triazole). Notably, our lead complex is among a limited number of dual PCT/PDT agents activated with green light.

Prognostic Significance of T-Cells and Macrophages in the Tumour Microenvironment of Nodal DLBCL.

Revised International Prognostic (R-IPI) score is used widely for risk stratification of DLBCL cases, yet some patients belonging to same risk category tend to exhibit different outcomes. The role of T-cells and macrophages in prognostication of lymphomas has been a point of interest of late. We aimed to study the association of FOXP3 positive T-regulatory cells, cytotoxic T-cells and macrophages with the immunophenotypic subtypes, clinicopathological characteristics, treatment response and survival in nodal diffuse large B-cell lymphoma (DLBCL) patients. The clinicopathological and treatment data of 83 DLBCL patients diagnosed and treated at our institute from January 2015 to December 2018 were collected and followed up till June 2020. CD8, FOXP3 and CD68 immunostains were performed to highlight the cytotoxic T-cells, T-regulatory cells and macrophages respectively on the lymph node biopsies and the distribution of these cells and their association with clinico-pathological factors, treatment response and survival was analyzed. DLBCL cases with higher percentage of CD3 positive T-cells and CD8 positive cytotoxic T-cells had significant association with attainment of complete response to treatment. In addition, CD8 positive T-cells of more than 6.5% proved to be an independent predictor of treatment outcome (p = 0.022). Multivariate Cox regression survival analysis revealed that cases with 'good' R-IPI prognostic score and 'high CD68 positive macrophages in tumor microenvironment' had a significantly longer overall survival. Increased number of cytotoxic T-cells was significantly associated with complete response to treatment and higher number of macrophages correlated significantly with better overall survival signifying their antitumor effects.

Evaluation of the immunomodulatory effect of Bougainvillea xbuttiana(Var. Orange) on the cytokine production induced by Botrhops jararacavenom in macrophages

To study the effect of 50% ethanol extract of Bougainvillea xbuttiana on the enzymatic activity, cell viability and cytokine production provoked by the venom of Bothrops jararacain macro-phages. Three assays were used to study the effects of B. xbuttiana extract on the damage produced by B. jararaca: Enzymatic activity was detected by measuring the proteolytic and phos-pholipase A2; macrophages cytotoxicity was determined by the MTT method; levels of cytokine were evaluated using ELISA and a biological assay. After treatment with 300 μg/mL B. xbuttiana extract for 30 min, the proteolytic and phospholipase A2 activities of the venom were reduced to 95 and 61%, respectively. In macrophages cultures treated with B. xbuttiana extract combined with venom, the production of TNF-α, IL-6 and IFN-γ was reduced, whereas IL-10 was potentiated. Our results support the potential effect of the B. xbuttiana extract as a complementary therapy against the toxicity caused by the venom of B. jararaca snakes.

Abstract 1341: SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status

Abstract Background: Monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) are frontline therapies for numerous EGFR-dependent epithelial tumors. However, their efficacy faces challenges due to on-target skin toxicity and intrinsic or acquired resistance. Within the tumor microenvironment, innate immune cells, including macrophages and natural killer (NK) cells, play a significant role but are hindered by the lack of specific cancer cell targeting and a suppressive microenvironment. To enhance innate immune cell cytotoxicity, we have developed SM2235, a humanized tetravalent bispecific VHH antibody. SM2235 partially blocks EGFR and EGF interaction while conditionally activating innate immune cells through CD16A upon engagement with cancer cells. Methods: SM2235 was engineered by combining a partially blocking EGFR single-domain antibody with a high-affinity CD16A-activating single-domain antibody on an inactivated human IgG1 Fc backbone. The binding affinity and specificity were confirmed using bio-layer interferometry and ELISA with recombinant proteins, followed by flow cytometry with EGFR+ human cancer cell lines and CD16A+ primary NK cells. SM2235's efficacy in blocking EGFR-EGF interaction and downstream signaling was assessed through competitive ELISA and proliferation assays in both EGFR-dependent and independent cancer cell lines. Its ability to induce target-dependent NK cell and macrophage activation was evaluated through activation markers as well as cytotoxicity and phagocytosis assays. In vivo efficacy of SM2235 against EGFR+ human tumors was measured using xenograft assays in NSG mice, covering cetuximab-sensitive and resistant cancer cell lines. Preliminary pharmacokinetics and safety studies were conducted in cynomolgus monkeys. Results: SM2235 exhibits sub-nanomolar affinity for EGFR+ human cancer cell lines and CD16A+ primary human NK cells. It partially inhibits EGFR-dependent cancer cell proliferation. Compared to cetuximab, SM2235 significantly enhances target-dependent CD16A-mediated NK cell and macrophage activation and cytotoxicity against EGFR+ cancer cells in ADCC and ADCP assays in vitro. SM2235 effectively inhibits EGFR+ xenograft tumors in vivo, irrespective of KRAS mutation status. In cynomolgus monkeys, SM2235 demonstrates excellent tolerance with a 20-hour serum half-life of approximately 20 hours and no dose-dependent skin toxicity. Conclusions: Preclinical characterization of SM2235 shows promising in vivo efficacy against EGFR+ human cancers, regardless of KRAS mutation, with a favorable safety profile marked by minimal skin toxicity. Citation Format: Huiqin Geng, Wenjing Song, Xiaodan Liu, Shihao Lu, Hong Zhou, Simin Yang, Siyao Xie, Yi Wei, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1341.