Effects of Encapsulation of Caesalpinia sappan L. with Cyclodextrins for Bovine Mastitis.

The main components of Caesalpinia sappan L. (CS) are brazilin and brazilein, which show high potential in pharmacologic applications. However, these have been drastically limited by the poor water solubility and stability. The present study investigates the formation of inclusion complexes F1, F2, and F3 between CS and ß-cyclodextrin (ßCD), hydroxypropyl-ß-cyclodextrin (HPßCD), and methyl-ß-cyclodextrin (MßCD), respectively. These complexes were characterized by Fourier transform infrared spectroscopy (FT-IR). The results showed that the highest encapsulation efficiency and loading capacity of CS extract were 44.24% and 9.67%, respectively. The solubility and stability of CS extract were significantly increased through complexation in phase solubility and stability studies. The complexes F1-F3 showed mainly significant antibacterial activities on gram-positive bacteria pathogens causing mastitis. Moreover, the expression levels of COX-2 and iNOS were significantly decreased in LPS-induced inflammatory cells at concentrations of 50 and 100 µg/mL. In addition, treatment of complex F3 (CS/MßCD) in bovine endothelial cells remarkably increased the chemokine gene expression of CXCL3 and CXCL8, which were responsible for immune cell recruitment (9.92 to 11.17 and 8.23 to 9.51-fold relative to that of the LPS-treated group, respectively). This study provides a complete characterization of inclusion complexes between CS extract and ßCD, HPßCD, and MßCD for the first time, highlighting the impact of complex formation on the pharmacologic activities of bovine mastitis.

Effect of Holoptelea integrifolia (Roxb.) Planch. n-Hexane Extract and Its Bioactive Compounds on Wound Healing and Anti-Inflammatory Activity.

The stem bark of Holoptelea integrifolia (Roxb.) Planch. has been applied for the treatment of human cutaneous diseases as well as canine demodicosis in several countries. However, no detailed mechanistic studies have been reported to support their use. In this study, thin-layer chromatography and gas chromatography were used to screen phytochemicals from the fresh stem bark extract of H. integrifolia. We found the two major bioactive compounds, friedelin and lupeol, and their activity on wound healing was further investigated in keratinocytes. Both bioactive compounds significantly reduced wound area and increased keratinocyte migration by increasing matrix metalloproteinases-9 production. Subsequently, we found that the mRNA gene expressions of cadherin 1 and desmoglobin 1 significantly decreased, whereas the gene expression involved in keratinocyte proliferation and homeostasis (keratin-17) increased in compound-treated human immortalized keratinocytes cells. The expression of inflammatory genes (cyclooxygenase-2 and inducible nitric oxide synthase) and pro-inflammatory cytokine genes (tumor necrosis factor-alpha and interleukin-6) was reduced by treatment with n-hexane extract of H. integrifolia and its bioactive compounds. Our results revealed that H. integrifolia extract and its bioactive compounds, friedelin and lupeol, exhibit wound-healing activity with anti-inflammatory properties, mediated by regulating the gene expression involved in skin re-epithelialization.

Curcumin protects rats against gentamicin-induced nephrotoxicity by amelioration of oxidative stress, endoplasmic reticulum stress and apoptosis.

CONTEXT: Gentamicin (GM) is an aminoglycoside antibiotic which is commonly used against Gram-negative bacterial infection; however, serious complications including nephrotoxicity could limit its clinical use. OBJECTIVE: The present study examined the protective effects of curcumin (CUR) on endoplasmic reticulum (ER) stress-mediated apoptosis through its antioxidative property in GM-induced nephrotoxicity in rats. MATERIALS AND METHODS: Male Sprague-Dawley rats (n = 3) were divided into six groups to receive normal saline (control), GM (100 mg/kg/day), co-treatment with GM and CUR (100, 200 and 300 mg/kg/day) and CUR (200 mg/kg/day) alone for 15 days by gavage feeding. Then, the renal function, kidney injury as well as oxidative stress, antioxidative markers and ER stress-mediated apoptosis were evaluated. RESULTS: Pre-treatment of CUR rescued the nephrotoxicity in GM-treated rats. Several nephrotoxicity hallmarks were reversed in the CUR-pre-treatment group. At the dose of 200 mg/kg/day, it could significantly lower serum creatinine (from 0.95 to 0.50 mg/dL), blood urea nitrogen (from 35.00 to 23.50 mg/dL) and augmented creatinine clearance (from 0.83 to 1.71 mL/min). The normalized expression of oxidative stress marker, malondialdehyde was decreased (from 13.00 to 5.98) in line with the increase of antioxidant molecules including superoxide dismutase (from 5.59 to 14.24) and glutathione (from 5.22 to 12.53). Furthermore, the renal ER stress and apoptotic protein biomarkers were lowered in CUR treatment. DISCUSSION AND CONCLUSIONS: Our findings pave the way for the application of CUR as a supplement in the prevention of nephrotoxicity and other kidney diseases in the future.

Alpha-mangostin inhibits dengue virus production and pro-inflammatory cytokine/chemokine expression in dendritic cells.

Dengue virus (DENV) is transmitted to humans via the bite of an Aedes mosquito, causing dengue fever, dengue hemorrhagic fever, or dengue shock syndrome. In the human skin, DENV first infects keratinocytes, dendritic cells, and macrophages. Monocytes that are recruited to the site of infection and differentiate into monocyte-derived dendritic cells (moDCs) are also infected by DENV. DENV-infected DCs secrete pro-inflammatory cytokines and chemokines to modulate the immune response. The viral load and massive pro-inflammatory cytokine/chemokine production, referred to as a 'cytokine storm', are associated with disease severity. We propose that an ideal drug for treatment of DENV infection should inhibit both virus production and the cytokine storm, and previously, we reported that alpha-mangostin (α-MG) inhibits both DENV replication and cytokine production in hepatocytes. However, the effect of α-MG on DENV-infected moDCs remains unknown. In this study, we investigated the effects of α-MG on DENV infection and pro-inflammatory cytokine/chemokine production in primary moDCs generated ex vivo from monocytes of healthy individuals. α-MG at the non-toxic concentrations of 20 and 25 µM reduced DENV production by more than 10-fold and 1,000-fold, respectively. Treatment with α-MG efficiently inhibited the infection of immature moDCs by all four serotypes of DENV. Time-of-addition studies suggested that α-MG (25 µM) inhibits DENV at the early stage of replication. In addition, α-MG markedly reduced cytokine/chemokine (TNF-α, CCL4, CCL5, CXCL10, IL6, IL1ß, IL10, and IFN-α) transcription in DENV-infected immature moDCs. These findings suggest the potential of α-MG to be developed as a novel anti-DENV drug.

Cordycepin Sensitizes Cholangiocarcinoma Cells to Be Killed by Natural Killer-92 (NK-92) Cells.

Immunotherapy harnessing immune functions is a promising strategy for cancer treatment. Tumor sensitization is one approach to enhance tumor cell susceptibility to immune cell cytotoxicity that can be used in combination with immunotherapy to achieve therapeutic efficiency. Cordycepin, a bioactive compound that can be extracted from some Cordyceps spp. has been reported to effectively inhibit tumor growth, however, the mechanism of its tumor sensitization activity that enhances immune cell cytotoxicity is unknown. In the present study, we investigated the potency of cordycepin to sensitize a lethal cancer, cholangiocarcinoma (CCA), to natural killer (NK) cells. Treatment with cordycepin prior to and during co-culturing with NK-92 cells significantly increased cell death of KKU-213A as compared to solitary cordycepin or NK treatment. Moreover, sensitization activity was also observed in the combination of NK-92 cells and Cordyceps militaris extract that contained cordycepin as a major component. The cordycepin treatment remarkably caused an increase in TRAIL receptor (DR4 and DR5) expression in KKU-213A, suggesting the possible involvement of TRAIL signaling in KKU-213A sensitization to NK-92 cells. In conclusion, this is the first report on the sensitization activity of cordycepin on CCA cells to NK cytotoxicity, which supports that cordycepin can be further developed as an alternate immunomodulating agent.

Cordycepin Inhibits Virus Replication in Dengue Virus-Infected Vero Cells.

Dengue virus (DENV) infection causes mild to severe illness in humans that can lead to fatality in severe cases. Currently, no specific drug is available for the treatment of DENV infection. Thus, the development of an anti-DENV drug is urgently required. Cordycepin (3'-deoxyadenosine), which is a major bioactive compound in Cordyceps (ascomycete) fungus that has been used for centuries in Chinese traditional medicine, was reported to exhibit antiviral activity. However, the anti-DENV activity of cordycepin is unknown. We hypothesized that cordycepin exerts anti-DENV activity and that, as an adenosine derivative, it inhibits DENV replication. To test this hypothesis, we investigated the anti-DENV activity of cordycepin in DENV-infected Vero cells. Cordycepin treatment significantly decreased DENV protein at a half-maximal effective concentration (EC50) of 26.94 µM. Moreover, DENV RNA was dramatically decreased in cordycepin-treated Vero cells, indicating its effectiveness in inhibiting viral RNA replication. Via in silico molecular docking, the binding of cordycepin to DENV non-structural protein 5 (NS5), which is an important enzyme for RNA synthesis, at both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, was predicted. The results of this study demonstrate that cordycepin is able to inhibit DENV replication, which portends its potential as an anti-dengue therapy.

Cytotoxic activity of effector T cells against cholangiocarcinoma is enhanced by self-differentiated monocyte-derived dendritic cells.

Cholangiocarcinoma (CCA) is a cancer of the bile ducts that is associated with poor prognosis and poor treatment outcome. Approximately one-third of CCA patients can undergo surgery, but the recurrence rate is high and chemotherapy often cannot satisfactorily prolong survival. Cellular immunotherapy based on adoptive T-cell transfer is a potential treatment for CCA; however, the development of this technology and the search for an appropriate tumor-associated antigen are still ongoing. To enhance the cytotoxic activity of effector T cells against CCA, we developed self-differentiated monocyte-derived dendritic cells (SD-DC) presenting cAMP-dependent protein kinase type I-alpha regulatory subunit (PRKAR1A), which is an overexpressed protein that plays a role in the regulation of tumor growth to activate T cells for CCA cell killing. Dendritic cells (DCs) transduced with lentivirus harboring tri-cistronic cDNA sequences (SD-DC-PR) could produce granulocyte-macrophage colony-stimulating factor, interleukin-4, and PRKAR1A. SD-DC showed similar phenotypes to those of DCs derived by conventional method. Autologous effector T cells (CD3+, CD8+) activated by SD-DC-PR exhibited greater cytotoxic activity against CCA than those activated by conventionally-derived DCs. Effector T cells activated by SD-DC-PR killed 60% of CCA cells at an effector-to-target ratio of 15:1, which is approximately twofold greater than the cell killing performance of those stimulated with control DC. The cytotoxic activities of effector T cells activated by SD-DC-PR against CCA cells were significantly associated with the expression levels of PRKR1A in CCA cells. This finding that SD-DC-PR effectively stimulated autologous effector T cells to kill CCA cells may help to accelerate the development of novel therapies for treating CCA.

Vivo-morpholino oligomers strongly inhibit dengue virus replication and production.

Dengue virus (DENV) infection is a worldwide public health problem, which can cause severe dengue hemorrhagic fever (DHF) and life-threatening dengue shock syndrome (DSS). There are currently no anti-DENV drugs available, and there has been an intensive search for effective anti-DENV agents that can inhibit all four DENV serotypes. In this study, we tested whether vivo-morpholino oligomers (vivo-MOs), whose effect on DENV infection has not previously been studied, can inhibit DENV infection. Vivo-MOs were designed to target the top of 3' stem-loop (3' SL) in the 3' UTR of the DENV genome and tested for inhibition of DENV infection in monkey kidney epithelial (Vero) cells and human lung epithelial carcinoma (A549) cells. The results showed that vivo-MOs could bind to a DENV RNA sequence and markedly reduce DENV-RNA, protein, and virus production in infected Vero and A549 cells. Vivo-MOs at a concentration of 4 µM could inhibit DENV production by more than 104-fold when compared to that of an untreated control. In addition, vivo-MOs also inhibited DENV production in U937 cells and primary human monocytes. Therefore, vivo-MOs targeting to the 3' SL in the 3' UTR of DENV genomes are effective and have the potential to be developed as anti-DENV agents.

A novel method for dengue virus detection and antibody screening using a graphene-polymer based electrochemical biosensor.

Dengue fever is a major disease that kills many people in the developing world every year. During early infection, a patient displays a high temperature without other signs. After this stage, and without proper treatment, serious damage to internal organs can happen, which occasionally leads to death. A rapid technique for the early detection of dengue virus (DENV) could reduce the number of fatalities. This study presents a new technique for the detection, classification and antibody screening of DENV based on electrochemical impedance spectroscopy (EIS). We found that the charge transfer resistance (Rct) of a gold electrode coated with graphene oxide reinforced polymer was influenced by virus type and quantity exposed on the surface. Molecular recognition capability established during the GO-polymer composite preparation was used to explain this observation. The linear dependence of Rct versus virus concentrations ranged from 1 to 2×103pfu/mL DENV with a 0.12 pfu/mL detection limit.

Tri-specific killer engager: unleashing multi-synergic power against cancer.

Cancer continues to be a global health concern, necessitating innovative solutions for treatment. Tri-specific killer engagers (TriKEs) have emerged as a promising class of immunotherapeutic agents, offering a multifaceted approach to cancer treatment. TriKEs simultaneously engage and activate natural killer (NK) cells while specifically targeting cancer cells, representing an outstanding advancement in immunotherapy. This review explores the generation and mechanisms of TriKEs, highlighting their advantages over other immunotherapies and discussing their potential impact on clinical trials and cancer treatment. TriKEs are composed of three distinct domains, primarily antibody-derived building blocks, linked together by short amino acid sequences. They incorporate critical elements, anti-cluster of differentiation 16 (CD16) and interleukin-15 (IL-15), which activate and enhance NK cell function, together with specific antibody to target each cancer. TriKEs exhibit remarkable potential in preclinical and early clinical studies across various cancer types, making them a versatile tool in cancer immunotherapy. Comparative analyses with other immunotherapies, such as chimeric antigen receptor-T (CAR-T) cell therapy, immune checkpoint inhibitors (ICIs), cytokine therapies, and monoclonal antibodies (mAbs), reveal the unique advantages of TriKEs. They offer a safer pathway for immunotherapy by targeting cancer cells without hyperactivating T cells, reducing off-target effects and complications. The future of TriKEs involves addressing challenges related to dosing, tumor-associated antigen (TAA) expression, and NK cell suppression. Researchers are exploring innovative dosing strategies, enhancing specificity through tumor-specific antigens (TSAs), and combining TriKEs with other therapies for increased efficacy.

Anti-Herpes Simplex Virus and Anti-Inflammatory Activities of the Melittin Peptides Derived from Apis mellifera and Apis florea Venom.

Herpes simplex virus (HSV) is known to cause cold sores and various diseases in humans. Importantly, HSV infection can develop latent and recurrent infections, and it is also known to cause inflammation. These infections are difficult to control, and effective treatment of the disease remains a challenge. Thus, the search for new antiviral and anti-inflammatory agents is a necessity. Melittin is a major peptide that is present in the venom of the honeybee. It possesses a number of pharmacological properties. In this study, the effects of the melittin peptides from A. mellifera (MEL-AM) and A. florea (MEL-AF) against HSV-1 and HSV-2 were evaluated at different stages during the viral multiplication cycle in an attempt to define the mode of antiviral action using plaque reduction and virucidal assays. The results revealed a new finding that melittin at 5 µg/mL demonstrated the highest inhibitory effect on HSV through the direct inactivation of viral particles, and MEL-AF displayed a greater virucidal activity. Moreover, melittin was also observed to interfere with the process of HSV attachment to the host cells. MEL-AM exhibited anti-HSV-1 and anti-HSV-2 effects with EC50 values of 4.90 ± 0.15 and 4.39 ± 0.20 µg/mL, while MEL-AF demonstrated EC50 values of 4.47 ± 0.21 and 3.95 ± 0.61 µg/mL against HSV-1 and HSV-2, respectively. However, non-cytotoxic concentrations of both types of melittin produced only slight degrees of HSV-1 and HSV-2 inhibition after viral attachment, but melittin at 5 µg/mL was able to reduce the plaque size of HSV-2 when compared to the untreated group. In addition, MEL-AM and MEL-AF also exhibited anti-inflammatory activity via the inhibition of nitric oxide production in LPS-stimulated RAW 264.7 macrophage cells, and they were also found to down-regulate the expressions of the iNOS, COX-2 and IL-6 genes. The highest inhibition of IL-6 mRNA expression was found after treatment with 10 µg/mL of MEL-AM and MEL-AF. Therefore, melittin peptides have displayed strong potential to be used as an alternative treatment for HSV infection and inflammatory diseases in the future.

Inhibitory effects of algal polysaccharide extract from Cladophora spp. against herpes simplex virus infection.

Herpes simplex virus (HSV) is a causative agent of fever blister, genital herpes, and neonatal herpes. Nowadays, edible algae are recognized as health food due to high nutrition content and their many active compounds that are beneficial to health. The purpose of this study is to investigate the inhibitory effects of algal polysaccharide extract from Cladophora spp. against herpes simplex virus type 1 and type 2 on Vero cells. In this study, the structure of polysaccharide extract is presented as S=O and C-O-S of the sulfate group, as identified by the FT-IR technique. The toxicity of algal polysaccharide extract on Vero cells was determined by MTT assay. The algal extract showed low toxicity on the cells, with 50% cytotoxic concentration (CC50) value greater than 5000 µg mL-1. The inhibition of HSV infection by the algal extract was then evaluated on Vero cells using plaque reduction assay. The 50% effective concentration (EC50) values of algal extract exhibited antiviral activity against HSV-1 upon treatment before, during, and after viral adsorption with and without removal of the extract were 70.31, 15.17, > 5000 and 9.78 µg mL-1, respectively. Additionally, the EC50 values of algal extract against HSV-2 upon treatment before, during and after viral adsorption with, and without removal of the extract were 5.85, 2.57, > 5000 and 26.96 µg mL-1, respectively. Moreover, the algal extract demonstrated direct inactivation of HSV-1 and HSV-2 virions as well as inhibitory effect against HSV replication. Accordingly, algal polysaccharide extract containing sulfated polysaccharides showed strong activity against HSV. Therefore, it is proved to be useful to apply Cladophora spp. polysaccharide extract as an anti-HSV agent.

The Evaluation of Anti-Osteoclastic Activity of the Novel Calcium Hydroxide Biodegradable Nanoparticles as an Intracanal Medicament.

INTRODUCTION: The aim of this study was to evaluate the anti-osteoclastic activity of calcium hydroxide-loaded poly(lactic-co-glycolic acid) nanoparticles [Ca(OH)2-loaded PLGA NPs] in comparison to calcium hydroxide nanoparticles [Ca(OH)2 NPs]. METHODS: RAW 264.7 cell lines (third-fifth passage) were cultured and incubated with soluble receptor activator of nuclear factor kappa B ligand in triplicate. Subsequently, Ca(OH)2-loaded PLGA NPs and Ca(OH)2 NPs were added for 7 days to evaluate their effects on receptor activator of nuclear factor kappa B ligand-induced osteoclast differentiation of RAW 264.7 cells by tartrate-resistant acid phosphatase activity. Additionally, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to confirm the cytotoxicity of treatments to cells. RESULTS: Tartrate-resistant acid phosphatase staining showed a significant reduction in the osteoclast number when treated with Ca(OH)2-loaded PLGA NPs compared with Ca(OH)2 NPs (P < .01). In comparison to the control, the number of osteoclasts significantly reduced upon treatment with Ca(OH)2-loaded PLGA NPs (P < .05), but there was no significant difference in Ca(OH)2 NPs. Furthermore, osteoclast morphology in both treatment groups exhibited smaller sizes than the control group. Neither Ca(OH)2-loaded PLGA NPs nor Ca(OH)2 NPs demonstrated cytotoxic effects on RAW264.7 cells. CONCLUSIONS: Both Ca(OH)2 NPs with and without poly(lactic-co-glycolic acid) have the ability to inhibit osteoclast differentiation. However, Ca(OH)2-loaded PLGA NPs exhibit greater potential than Ca(OH)2 NPs, making them a promising intracanal medicament for cases of root resorption.

Dual action effects of ethyl-p-methoxycinnamate against dengue virus infection and inflammation via NF-κB pathway suppression.

Dengue virus (DENV) infection can lead to severe outcomes through a virus-induced cytokine storm, resulting in vascular leakage and inflammation. An effective treatment strategy should target both virus replication and cytokine storm. This study identified Kaempferia galanga L. (KG) extract as exhibiting anti-DENV activity. The major bioactive compound, ethyl-p-methoxycinnamate (EPMC), significantly reduced DENV-2 infection, virion production, and viral protein synthesis in HepG2 and A549 cells, with half-maximal effective concentration (EC50) values of 22.58 µM and 6.17 µM, and impressive selectivity indexes (SIs) of 32.40 and 173.44, respectively. EPMC demonstrated efficacy against all four DENV serotypes, targeting the replication phase of the virus life cycle. Importantly, EPMC reduced DENV-2-induced cytokines (IL-6 and TNF-α) and chemokines (RANTES and IP-10), as confirmed by immunofluorescence and immunoblot analyses, indicating inhibition of NF-κB activation. EPMC's role in preventing excessive inflammatory responses suggests it as a potential candidate for dengue treatment. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) and drug-likeness for EPMC were predicted using SwissADME and ProTox II servers, showing good drug-like properties without toxicity. These findings highlight KG extract and EPMC as promising candidates for future anti-dengue therapeutics, offering a dual-action approach by inhibiting virus replication and mitigating inflammatory reactions.

Inhibition of dengue virus infection in vitro by fucoidan and polysaccharide extract from marine alga Sargassum spp.

Dengue virus (DENV) infection poses a global health threat, leading to severe conditions with the potential for critical outcomes. Currently, there are no specific drugs available whereas the vaccine does not offer comprehensive protection across all DENV serotypes. Therefore, the development of potential anti-viral agents is necessary to reduce the severity risk and interrupt the transmission circuit. The search for effective antiviral agents against DENV has predominantly focused on natural resources, particularly those demonstrating diverse biological activities and high safety profiles. Cyanobacteria and algae including Leptolyngbya sp., Spirulina sp., Chlorella sp., and Sargassum spp., which are prevalent species in Thailand, have been reported for their diverse biological activities and high safety profile but not specifically for anti-DENV activity. In this study, the screening assay was performed to compare the anti-viral activity against DENV of crude polysaccharide and ethanolic extracts derived from 4 species of cyanobacteria and algae in Vero cells. Polysaccharide extracts from Sargassum spp. exhibited the most effective in inhibiting DENV-2 infection at co-infection conditions where the virus was exposed to the extract at the time of infection. Treatment of the extract significantly reduced the ability of DENV to bind to the host cells to 47.87 ±â€¯3.88 % while treatment upon virus binding step had no anti-viral effect suggesting the underlaying mechanism of the extract on interfering virus binding step. Fucoidan, a key bioactive substance in Sargassum polysaccharide, showed to reduce DENV-2 infection to 26.59 ±â€¯5.01 %, 20.46 ±â€¯6.58 % in co-infection condition in Vero cells and A549 cell line, respectively. In accompanied with Sargassum polysaccharide, fucoidan disturbed the virus binding to the host cells. These findings warrant further development and exploration of the Sargassum-derived polysaccharide, fucoidan, as a promising candidate for combating DENV infections.

Precision immunotherapy for cholangiocarcinoma: Pioneering the use of human-derived anti-cMET single chain variable fragment in anti-cMET chimeric antigen receptor (CAR) NK cells.

Cholangiocarcinoma (CCA) presents a significant clinical challenge which is often identified in advanced stages, therby restricting the effectiveness of surgical interventions for most patients. The high incidence of cancer recurrence and resistance to chemotherapy further contribute to a bleak prognosis and low survival rates. To address this pressing need for effective therapeutic strategies, our study focuses on the development of an innovative cellular immunotherapy, specifically utilizing chimeric antigen receptor (CAR)-engineered natural killer (NK) cells designed to target the cMET receptor tyrosine kinase. In this investigation, we initiated the screening of a phage library displaying human single-chain variable fragment (ScFv) to identify novel ScFv molecules with specificity for cMET. Remarkably, ScFv11, ScFv72, and ScFv114 demonstrated exceptional binding affinity, confirmed by molecular docking analysis. These selected ScFvs, in addition to the well-established anti-cMET ScFvA, were integrated into a CAR cassette harboring CD28 transmembrane region-41BB-CD3ζ domains. The resulting anti-cMET CAR constructs were transduced into NK-92 cells, generating potent anti-cMET CAR-NK-92 cells. To assess the specificity and efficacy of these engineered cells, we employed KKU213A cells with high cMET expression and KKU055 cells with low cMET levels. Notably, co-culture of anti-cMET CAR-NK-92 cells with KKU213A cells resulted in significantly increased cell death, whereas no such effect was observed with KKU055 cells. In summary, our study identified cMET as a promising therapeutic target for CCA. The NK-92 cells, armed with the anti-cMET CAR molecule, have shown strong ability to kill cancer cells specifically, indicating their potential as a promising treatment for CCA in the future.

Anti-Inflammatory Activity of Glyceryl 1,3-Distearate Identified from Clinacanthus nutans Extract against Bovine Mastitis Pathogens.

Clinacanthus nutans is widely used as a traditional medicine in Thailand and other countries in Southeast Asia. Although its effectiveness is well documented, its therapeutic use is limited to the treatment of only a few diseases; mostly it is used as an anti-viral agent against varicella-zoster and herpes simplex virus infections. Herein, we demonstrate the therapeutic activity of C. nutans extracts in lowering inflammation in a model of bovine mastitis caused by bacterial infection. Lipopolysaccharide (LPS), a gram-negative bacterial component, caused inflammation activation in bovine endothelial cells (CPAE) through the upregulation of proinflammatory cytokines (IL6 and IL1ß) and chemokines (CXCL3 and CXCL8) gene expression, partially leading to cell death. Treatment with C. nutans crude extract significantly diminished these responses in a dose-dependent manner. The solvent fractionation of C. nutans extract revealed that the ethyl acetate (C4H8O2) fractions had a high potential to protect against cell death and diminished IL1ß, IL6, CXCL3, and CXCL8 levels to less than 0.45 folds relative to the LPS-treated control. Glyceryl 1,3-distearate (C39H76O5) was identified as a bioactive compound responsible for the anti-inflammation activity but not the anti-cell death activity of C. nutans extract. This study highlighted the efficiency of C. nutans extracts as an alternative therapeutic option for the natural-product sustainable development of bovine mastitis treatment.

Enhancing cholangiocarcinoma immunotherapy with adoptive T cells targeting HLA-restricted neoantigen peptides derived from driver gene mutations.

Precision immunotherapy, driven by genomic and bioinformatic advancements, has emerged as a promising and viable approach to combat cancer. Targeting neoantigens offers the advantage of specific immune responses with minimal off-tumor toxicity. In this study, we investigated the potential of adoptive T cells activated by HLA-restricted neoantigen peptides from driver gene mutations for treating cholangiocarcinoma (CCA), a highly aggressive cancer with poor prognosis and high mortality rates. Through whole exome sequencing of CCA cell lines, KKU-213A and KKU-100, we identified mutations in common driver genes and predicted corresponding HLA-restricted peptides. Peptides from KRAS, RNF43, and TP53 mutations exhibited strong binding affinity to HLA-A11, as validated through molecular docking and T2-cell binding assays. Dendritic cells (DCs) from healthy donors expressing HLA-A* 11:01, pulsed with individual or pooled peptides, showed comparable levels of costimulatory molecules (CD11c, CD40, CD86, and HLA-DR) to conventional DCs but higher expression of maturation markers, CD80 and CD86. Autologous HLA-A* 11:01-restricted T cells, activated by peptide-pulsed DCs, effectively lysed KKU-213A (HLA-A*11:01) cells, outperforming conventional tumor lysate-pulsed DCs. This effect was specific to HLA-A* 11:01-restricted T cells and not observed in KKU-100 (HLA-A*33:03) cells. Moreover, HLA-A* 11:01-restricted T cells exhibited elevated levels of IFN-gamma, granulysin, and granzyme B, indicating their potent anti-tumor capabilities. These findings underscore the specificity and efficiency of HLA-A* 11:01-restricted T cells targeting KRAS, RNF43, TP53 mutated CCA cells, and offer valuable insights for developing immunotherapeutic strategies and therapeutic peptide-vaccines for CCA treatment.

Microalga Chlorella sp. extract induced apoptotic cell death of cholangiocarcinoma via AKT/mTOR signaling pathway.

Cancer is the leading cause of death worldwide. Drug resistance and relapse after current standard treatments frequently occur; thus, alternative and effective treatments are required. Algae and cyanobacteria are abundant organisms that serve as bioresources of nutrients/metabolites, which are attractive sources of numerous bioactive compounds for drug discovery. In the present study, we, therefore, investigated anti-cancer activities of crude polysaccharide and ethanolic extracts from Chlorella sp., Sargassum spp., and Spirulina sp. against cell lines of five top-leading cancers including lung cancer (A549), cervical cancer (Hela), breast cancer (MCF7), hepatocellular carcinoma (Huh7), and cholangiocarcinoma (CCA; KKU213A). Only ethanolic extracts of Chlorella sp. showed consistent inhibition of growth of all cancer cell types. CCA was the most sensitive to Chlorella sp. ethanolic extract with CC50 of 277.4, 400.5, and 313.4 µg/mL for KKU055, KKU100, and KKU213A cells, respectively. Flow cytometric analysis demonstrated that CCA cell death was triggered via apoptosis pathway in accompany with lowering procaspase-3, -8, and -9 and increasing caspase enzymatic activity in addition to reducing anti-apoptosis Bcl-2 protein. Interestingly, the treatment of the extract at 400 µg/mL greatly inhibited the AKT/mTOR survival signaling as evidenced by significant reduction of phosphorylated-AKT and phosphorylated-mTOR proteins. The presence of reported bioactive compounds, gallic acid, and lutein, were confirmed in Chlorella sp. extract by high-performance liquid chromatography. Gallic acid and lutein treatment caused a significant reduction of KKU055, KKU100, and KKU213A cell viability. This study demonstrated the anti-cancer effect of Chlorella sp. ethanolic extract to promote cancer cell death via inhibition of AKT/mTOR pathway.

Intraspecific Diversity and Pathogenicity of Bacillus thuringiensis Isolates from an Emetic Illness.

This study describes an emetic food-borne intoxication associated with a Bacillus cereus group species and the characterization of the bacterial isolates from the incident in aspects of molecular tying, genetic factors, cytotoxicity, and pathogenic mechanisms relating to emetic illness. Through the polyphasic identification approach, all seven isolates obtained from food and clinical samples were identified as Bacillus thuringiensis. According to multilocus sequence typing (MLST) analysis, intraspecific diversity was found within the B. thuringiensis isolates. Four allelic profiles were found, including two previously known STs (ST8 and ST15) and two new STs (ST2804 and ST2805). All isolates harbored gene fragments located in the cereulide synthetase (ces) gene cluster. The heat-treated culture supernatants of three emetic B. thuringiensis isolates, FC2, FC7, and FC8, caused vacuolation and exhibited toxicity to Caco-2 cells, with CC50 values of 56.57, 72.17, and 79.94 µg/mL, respectively. The flow cytometry with the Annexin V/PI assay revealed both apoptosis and necrosis mechanisms, but necrosis was the prominent mechanism that caused Caco-2 cell destruction by FC2, the most toxic isolate.