Culture Model Research Articles

Hypersensitive blood vessels in Clarkson disease.

Idiopathic systemic capillary leak syndrome (ISCLS) is a rare, recurrent condition with dramatically increased blood vessel permeability and, therefore, induction of systemic edema, which may lead to organ damage and death. In this issue of the JCI, Ablooglu et al. showed that ISCLS vessels were hypersensitive to agents known to increase vascular permeability, using human biopsies, cell culture, and mouse models. Several endothelium-specific proteins that regulate endothelial junctions were dysregulated and thereby compromised the vascular barrier. These findings suggest that endothelium-intrinsic dysregulation underlies hyperpermeability and implicate the cytoplasmic serine/threonine protein phosphatase 2A (PP2A) as a potential drug target for the treatment of ISCLS.

ApTOLL: A new therapeutic aptamer for cytoprotection and (re)myelination after multiple sclerosis.

ApTOLL is an aptamer selected to antagonize toll-like receptor 4 (TLR4), a relevant actor for innate immunity involved in inflammatory responses in multiple sclerosis (MS) and other diseases. The currently available therapeutic arsenal to treat MS is composed of immunomodulators but, to date, there are no (re)myelinating drugs available in clinics. In our present study, we studied the effect of ApTOLL on different animal models of MS. The experimental autoimmune encephalomyelitis (EAE) model was used to evaluate the effect of ApTOLL on reducing the inflammatory component. A more direct effect on oligodendroglia was studied with the cuprizone model and purified primary cultures of murine and human oligodendrocyte precursor cells (OPCs) isolated through magnetic-activated cell sorting (MACS) from samples of brain cortex. Also, we tested these effects in an ex vivo model of organotypic cultures demyelinated with lysolecithin (LPC). ApTOLL treatment positively impacted the clinical symptomatology of mice in the EAE and cuprizone models, which was associated with better preservation plus restoration of myelin and oligodendrocytes in the demyelinated lesions of animals. Restoration was corroborated on purified cultures of rodent and human OPCs. Our findings reveal a new therapeutic approach for the treatment of inflammatory and demyelinating diseases such as MS. The molecular nature of the aptamer exerts not only an anti-inflammatory effect but also neuroprotective and remyelinating effects. The excellent safety profile demonstrated by ApTOLL in animals and humans opens the door to future clinical trials in MS patients.

The Transformative Role of 3D Culture Models in Triple-Negative Breast Cancer Research

Advancements in cell culturing techniques have allowed the development of three-dimensional (3D) cell culture models sourced directly from patients’ tissues and tumors, faithfully replicating the native tissue environment. These models provide a more clinically relevant platform for studying disease progression and treatment responses compared to traditional two-dimensional (2D) models. Patient-derived organoids (PDOs) and patient-derived xenograft organoids (PDXOs) emerge as innovative 3D cancer models capable of accurately mimicking the tumor’s unique features, enhancing our understanding of tumor complexities, and predicting clinical outcomes. Triple-negative breast cancer (TNBC) poses significant clinical challenges due to its aggressive nature, propensity for early metastasis, and limited treatment options. TNBC PDOs and PDXOs have significantly contributed to the comprehension of TNBC, providing novel insights into its underlying mechanism and identifying potential therapeutic targets. This review explores the transformative role of various 3D cancer models in elucidating TNBC pathogenesis and guiding novel therapeutic strategies. It also provides an overview of diverse 3D cell culture models, derived from cell lines and tumors, highlighting their advantages and culturing challenges. Finally, it delves into live-cell imaging techniques, endpoint assays, and alternative cell culture media and methodologies, such as scaffold-free and scaffold-based systems, essential for advancing 3D cancer model research and development.

Immortalized hepatocyte-like cells: A competent hepatocyte model for studying clinical HCV isolate infection.

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.

The role of claudins in renal transepithelial transport and kidney disease.

25 years after the discovery of claudins as the central constituents of tight junctions, the "hunter-gatherer phase" of claudin research is coming to an end. Deficiency in individual claudins as a cause of rare hereditary diseases is well documented. However, knowledge about the involvement of renal claudins in common kidney diseases and strategies to utilize claudins or their regulators for intervention are still scarce. The present review summarizes novel approaches to address these questions. Publicly accessible omics data provide new insights not only into general claudin expression patterns along the nephron, but also into sex-specific differences in claudin expression and into claudin dysregulation in renal injury. Computational association studies identify claudin variants as risk factors for kidney disease such as nephrolithiasis or loss of filtration capacity. The establishment of innovative cell culture and organoid models contributes to a better understanding of junctional and extra-junctional functions of individual claudins. The current studies lay the foundation for the identification of upstream regulators of renal claudin expression and thus for the development of new concepts for the treatment of kidney disease.

An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro

An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

Targeting cellular cathepsins inhibits hepatitis E virus entry.

The hepatitis E virus (HEV) is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potentials of cellular proteases during HEV infection. Using our established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors, impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 suppressed HEV infections with an EC50 of ~ 0.01 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells, was also observed in HepaRG and primary human hepatocytes. Furthermore, through time-of-addition and RNAscope experiments, we confirmed that HEV entry is blocked by inhibition of cathepsins. Cathepsin L (CTSL) knockout cells were less permissive to HEV, suggesting that CTSL is critical for HEV infection. Finally, we observed cleavage of the glycosylated ORF2 protein and virus particles by recombinant CTSL. In summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor, K11777, especially with its notable safety profile in primary cells, further underscores its potential as a therapeutic candidate.

Methods for assessing and removing non-specific photoimmunotherapy damage in patient-derived tumor cell culture models.

Tumor-targeted, activatable photoimmunotherapy (taPIT) has been shown to selectively destroy tumor in a metastatic mouse model. However, the photoimmunoconjugate (PIC) used for taPIT includes a small fraction of non-covalently associated (free) benzoporphyrin derivative (BPD), which leads to non-specific killing invitro. Here, we report a new treatment protocol for patient-derived primary tumor cell cultures ultrasensitive to BPD photodynamic therapy (BPD-PDT). Based on free BPD efflux dynamics, the updated invitro taPIT protocol precludes non-specific BPD-PDT by silencing the effect of free BPD. Following incubation with PIC, incubating cells with PIC-free medium allows time for expulsion of free BPD whereas BPD covalently bound to PIC fragments is retained. Administration of the light dose after the intracellular free BPD drops below the threshold for inducing cell death helps to mitigate non-specific damage. In this study, we tested two primary ovarian tumor cell lines that are intrinsically chemoresistant, yet ultrasensitive to BPD-PDT such that small amounts of free BPD (afew percent of the total BPD dose) lead to potent induction of cell death upon irradiation. The modifications in the protocol suggested here improve invitro taPIT experiments that lack invivo mechanisms of free BPD clearance (i.e., lymph and blood flow).

Targeting resistant breast cancer stem cells in a three-dimensional culture model with oleuropein encapsulated in methacrylated alginate microparticles.

Cancer stem cells (CSCs) are a subpopulation of cancer cells that are believed to be responsible for tumor initiation, progression, metastasis, and resistance to conventional therapies. Oleuropein as a natural compound found in olive leaves and olive oil, has potential therapeutic effects in cancer treatment, particularly in targeting CSCs. It induces apoptosis in CSCs while sparing normal cells, inhibit proliferation, migration, and invasion, and suppress the self-renewal ability of CSCs. Additionally, oleuropein has shown synergistic effects with conventional chemotherapy drugs, enhancing their efficacy against CSCs. This study aims to selectively target therapeutically resistant cancer stem cells (CSCs) within a heterogeneous tumor population by utilizing oleuropein (OLE) encapsulated in methacrylated alginate (OLE-mALG) within an in vivo-like microenvironment. This study aims to target therapeutically resistant cancer stem cells (CSCs) with oleuropein (OLE) encapsulated in the methacrylated alginate (OLE-mALG) in a heterogeneous tumor population with an in vivo-like microenvironment. Co-culture of CSCs with non-tumorogenic MCF-12A cells was performed, the 3D breast cancer model was supported with methocel/matrigel/collagen-I, and vascularization was ensured with human umbilical vein endothelial cells (HUVEC). Then, OLE-loaded methacrylated alginate microparticles (mALG) were formed by dual crosslinking in the presence of both ionic and visible light obtained with a droplet based microfluidic system. The characterization and effectiveness of the produced OLE-mALG were evaluated by the FTIR, swelling/degradation/release analysis. Before producing OLE loaded mALG microparticles, a preliminary study was carried out to determine the effective dose of OLE for cells and the duration of OLE action on MCF-7, CSCs and MCF-12A. Subsequently, CSC viability (WST-1), apoptosis (Bcl-2, Bax, caspase-3, caspase-9), stemness (OCT3/4, NANOG, SOX2), EMT profile (E-cadherin, Vimentin, Slug) and proliferation (SURVIVIN, p21, CYCLIN D1) after OLE-mALG treatment were all evaluated in the 3D model. OLE was encapsulated in mALG with an efficiency of 90.49% and released 73% within 7h. OLE-mALG induced apoptosis through the decrease in anti-apoptotic Bcl-2 and an increase in pro-apoptotic Bax, caspase-3, and caspase-9 protein levels. While Vimentin and Slug protein levels decreased after 200µg/mL OLE-mALG treatment to 3D breast cancer culture, E-cadherin levels increased. OLE-mALG treatment to CSC co-culture led to a decrease in proliferation by triggering p21/SURVIVIN expressions, and also resulted in an increase in stemness genes (OCT3/4/NANOG/SOX2). 200µg/mL OLE-loaded mALG microparticles suppressed epithelial-to-mesenchymal transition by suppressing Vimentin and Slug protein levels, and increased E-cadherin levels in the 3D breast cancer model we created with CSCs, MCF-12A and HUVECs. This complex system may allow the use of personalized cells for rapid drug screening in preclinical studies compared to animal experiments. OLE-mALG showed apoptotic and metastasis suppressive properties in cancer cells and it was concluded that it can be used in combination with or alternatively with chemotherapeutic agents to target breast cancer stem cells.

Pentoxifylline and Norcantharidin Modify p62 Expression in 2D and 3D Cultures of B16F1 Cells

Three-dimensional cell cultures have improved the evaluation of drugs for cancer therapy, due to their high similarity to solid tumors. In melanoma, autophagy appears to show a dual role depending on the progression of the disease. p62 protein has been proposed for the evaluation of autophagic flux since its expression is an indicator of the state of autophagy. Pentoxifylline (PTX) and Norcantharidin (NCTD) are drugs that have been shown to possess anticancer effects. In this work, we used B16F1 mouse melanoma cells in two-dimensional (2D) monolayer cultures and three-dimensional (3D) spheroids to test the effect of PTX and NCTD over the p62 expression. We analyzed the effect on p62 expression through Western blot and immunofluorescence assays. Our results indicate that PTX decreases p62 expression in both cell culture models, while Norcantharidin increases its expression in 3D cultures at 24 h. Therefore, these drugs could have a potential therapeutic use for the regulation of autophagy in melanoma, depending on the state of evolution of the disease.

Left ventricular global longitudinal strain is worse in BRCA mutation positive breast cancer patients prior to cancer treatment and premature menopause.

Breast cancer patients with mutations in human tumor suppressor genes BRCA1 and BRCA2 are at higher risk of cardiovascular disease (CVD) than the general population, as they are frequently exposed to cardiotoxic chemotherapy, anti-estrogen therapy, radiation, and/or oophorectomy for cancer-related treatment and prophylaxis. Animal and cell culture models suggest that BRCA mutations may play an independent role in heart failure. We sought to evaluate cardiac structure and function in female BRCA1 and BRCA2 mutation carriers with breast cancer compared to BRCA wildtype women with breast cancer. We performed a 1:2 age- and hypertension-matched retrospective cohort study comparing BRCA1 and BRCA2 mutation carriers (n = 38) versus BRCA wildtype controls (n = 76) with a new diagnosis of breast cancer. Echocardiographic data were obtained within 6months of breast cancer diagnosis and prior to chemotherapy, anti-estrogen therapy, radiation, or oophorectomy. Left ventricular global longitudinal strain (LV-GLS), a highly sensitive marker of LV function, was measured using QLab 15 (Philips Healthcare). In the total cohort of 114 patients with a new diagnosis of breast cancer, the median age was 45 ± 11years and the prevalence of hypertension was 8%. There were no differences in traditional cardiovascular disease risk factors between cases and controls. BRCA carriers had lower LV-GLS (-18.1% ± 4.7% vs. -20.1% ± 3.8%, p = 0.02) and greater right atrial area (12.9cm2 ± 2.7cm2 vs. 11.8cm2 ± 2.0cm2, p = 0.04) compared to controls; however, both LV-GLS and right atrial area were within the normal range. Compared to controls, BRCA carriers had a trend toward worse LV posterior wall thickness (0.89cm ± 0.15cm vs. 0.83cm ± 0.16cm, p = 0.06) although not statistically significant. In women with newly diagnosed breast cancer and prior to treatment, LV-GLS was worse in BRCA1 and BRCA2 mutation carriers compared to those with BRCA wildtype. These findings suggest that BRCA mutations may be associated with subtle changes in cardiac function. Whether differences in GLS translate to increased cardiovascular risk in women with BRCA mutations needs to be further characterized.

Hyperglycaemia perturbs blood-brain barrier integrity through its effects on endothelial cell characteristics and function

ABSTRACT Breakdown of blood-brain barrier (BBB) represents a key pathology in hyperglycemia-mediated cerebrovascular damage after an ischemic stroke. As changes in the level and nature of vasoactive agents released by endothelial cells (ECs) may contribute to BBB dysfunction, this study first explored the specific impact of hyperglycemia on EC characteristics and secretome. It then assessed whether secretome obtained from ECs subjected to normoglycaemia or hyperglycemia might regulate pericytic cytokine profile differently. Using a triple cell culture model of human BBB, composed of brain microvascular EC (BMEC), astrocytes and pericytes, this study showed that exposure to hyperglycemia (25 mM D-glucose) for 72 h impaired the BBB integrity and function as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux of sodium fluorescein. Dissolution of zonula occludens-1, a tight junction protein, and appearance of stress fibers appeared to play a key role in this pathology. Despite elevations in angiogenin, endothelin-1, interleukin-8 and basic fibroblast growth factor levels and a decrease in placental growth factor levels in BMEC subjected to hyperglycemia vs normoglycaemia (5.5 mM D-glucose), tubulogenic capacity of BMECs remained similar in both settings. Similarly, pericytes subjected to secretome obtained from hyperglycemic BMEC released higher quantities of macrophage migration inhibitory factor and serpin and lower quantities of monocyte chemoattractant protein-1, intercellular adhesion molecule, interleukin-6 and interleukin-8. Taken together these findings indicate the complexity of the mechanisms leading to BBB disruption in hyperglycemic settings and emphasize the importance of endothelial cell-pericyte axis in the development of novel therapeutic strategies.

To internalize or not? Addressing key differences between Turkish migrant and native workers in construction employment

PurposeMigration is on the rise due to globalization and human mobility. This has led to increased impacts that have affected many industries, including the construction industry. A large number of migrants are employed in the construction sector, and employers are challenged to make sure all employees are properly integrated to meet the demands needed for construction projects. This article addresses key differences between migrant and native workers to help hiring departments in the construction industry analyse workers' attitudes based on cultural and motivational factors to have the workforce they need to succeed.Design/methodology/approachThe research used both quantitative and qualitative surveys. A two-part questionnaire, designed through a comprehensive literature review, was carried out to identify key differences between native and migrant workers. The data were obtained and then analysed using different statistical approaches, including factor analysis protocol, factor structure model, reliability analysis, relative importance index and nonparametric test analysis. A semi-structured interview was then conducted to discuss all the findings.FindingsThe study indicated that migrant workers, compared to natives, tend to give more importance to their working environment, particularly accommodation, work safety and relations with teammates. Also, migrants typically take a socialistic approach instead of an individual approach while at work and reveal an extensive range of behaviours based on a sense of belonging. It might be more important for migrants to have a place in society, to have a settled life and to be integrated into an established order than to improve their rights and benefits. On the other hand, the study argued that native workers tend to prioritize their benefits at work, such as regular payments for overtime and insurance premiums. Their behaviours might carry a more neutral and individual attitude as well as specific cultural traces.Research limitations/implicationsThe study is limited to a sample of participants in the Turkish construction sector. Further research based on more cultural models and motivational factors with a larger group of respondents from different countries could offer better results. The results of the study might not apply to a broad context due to many other factors that affect worker behaviours, such as geography, cultural structures and working conditions. Despite these drawbacks, the present paper may help employers and other stakeholders understand the best way to incorporate migrants into the construction industry.Originality/valueThis research is very important for the construction industry in various countries that are currently employing thousands of migrants. Being able to address the key differences between migrants and native workers based on cultural and motivational factors might help with engagement and create a level of harmony in the field for greater productivity.

A Latent Profile Analysis of COVID-19 and Influenza Vaccine Hesitancy among Economically Marginalized Hispanic Mothers of Children under Five Years of Age in the US.

Rates of COVID-19 and influenza vaccine coverage among Hispanic young children continue to be low in comparison to other racial and ethnic groups in the United States. This study utilized a person-centered approach to understand COVID-19 and influenza vaccination hesitancy for young children under the age of five among 309 economically marginalized Hispanic mothers. Drawing on the cultural health belief model, in 2022, following FDA approval of the COVID-19 vaccine for young children, a latent profile analysis was conducted from which three profiles emerged. The Low Acculturation group (Profile 1), was notable for lower acculturation, moderate cultural medical mistrust, lower access to vaccines, and higher financial security. Compared to Profile 1, the two remaining profiles had higher acculturation and lower levels of financial security, but differed in that the High Acculturation group (Profile 2) had higher vaccine accessibility and the Moderate Acculturation group (Profile 3) had higher cultural medical mistrust. Relative to other profiles, Low Acculturation mothers were more likely to plan to vaccinate their child against current and seasonal COVID-19 and seasonal influenza, report that their child's health provider recommended the COVID-19 shot and reported lower COVID-19 and influenza vaccine mistrust. However, they also reported lower vaccine accessibility and moderate levels of cultural medical mistrust. The study highlights the importance of developing person-centered public health strategies that draw on Hispanic cultural values and consider diversity within lower income Hispanic populations to increase future pediatric COVID-19 and flu vaccination coverage among young Hispanic children.

NRN1 interacts with Notch to increase oncogenic STAT3 signaling in melanoma

BackgroundMelanoma is a highly heterogeneous cancer, in which frequent changes in activation of signaling pathways lead to a high adaptability to ever changing tumor microenvironments. The elucidation of cancer specific signaling pathways is of great importance, as demonstrated by the inhibitor of the common BrafV600E mutation PLX4032 in melanoma treatment. We therefore investigated signaling pathways that were influenced by neurotrophin NRN1, which has been shown to be upregulated in melanoma.MethodsUsing a cell culture model system with an NRN1 overexpression, we investigated the influence of NRN1 on melanoma cells’ functionality and signaling. We employed real time cell analysis and spheroid formation assays, while for investigation of molecular mechanisms we used a kinase phosphorylation kit as well as promotor activity analysis followed by mRNA and protein analysis.ResultsWe revealed that NRN1 interacts directly with the cleaved intracellular domain (NICD) of Notch1 and Notch3, causing a potential retention of NICD in the cytoplasm and thereby reducing the expression of its direct downstream target Hes1. This leads to decreased sequestration of JAK and STAT3 in a Hes1-driven phosphorylation complex. Consequently, our data shows less phosphorylation of STAT3 while presenting an accumulation of total protein levels of STAT3 in association with NRN1 overexpression. The potential of the STAT3 signaling pathway to act in both a tumor suppressive and oncogenic manner led us to investigate specific downstream targets – namely Vegf A, Mdr1, cMet - which were found to be upregulated under oncogenic levels of NRN1.ConclusionsIn summary, we were able to show that NRN1 links oncogenic signaling events between Notch and STAT3 in melanoma. We also suggest that in future research more attention should be payed to cellular regulation of signaling molecules outside of the classically known phosphorylation events.Graphical

Opaganib Downregulates N-Myc Expression and Suppresses In Vitro and In Vivo Growth of Neuroblastoma Cells.

Neuroblastoma (NB), the most common cancer in infants and the most common solid tumor outside the brain in children, grows aggressively and responds poorly to current therapies. We have identified a new drug (opaganib, also known as ABC294640) that modulates sphingolipid metabolism by inhibiting the synthesis of sphingosine 1-phosphate (S1P) by sphingosine kinase-2 and elevating dihydroceramides by inhibition of dihydroceramide desaturase. The present studies sought to determine the potential therapeutic activity of opaganib in cell culture and xenograft models of NB. Cytotoxicity assays demonstrated that NB cells, including cells with amplified MYCN, are effectively killed by opaganib concentrations well below those that accumulate in tumors in vivo. Opaganib was shown to cause dose-dependent decreases in S1P and hexosylceramide levels in Neuro-2a cells, while concurrently elevating levels of dihydroceramides. As with other tumor cells, opaganib reduced c-Myc and Mcl-1 protein levels in Neuro-2a cells, and also reduced the expression of the N-Myc protein. The in vivo growth of xenografts of human SK-N-(BE)2 cells with amplified MYCN was suppressed by oral administration of opaganib at doses that are well tolerated in mice. Combining opaganib with temozolomide plus irinotecan, considered the backbone for therapy of relapsed or refractory NB, resulted in increased antitumor activity in vivo compared with temozolomide plus irinotecan or opaganib alone. Mice did not lose additional weight when opaganib was combined with temozolomide plus irinotecan, indicating that the combination is well tolerated. Opaganib has additive antitumor activity toward Neuro-2a tumors when combined with the checkpoint inhibitor anti-CTLA-4 antibody; however, the combination of opaganib with anti-PD-1 or anti-PD-L1 antibodies did not provide increased antitumor activity over that seen with opaganib alone. Overall, the data demonstrate that opaganib modulates sphingolipid metabolism and intracellular signaling in NB cells and inhibits NB tumor growth alone and in combination with other anticancer drugs. Amplified MYCN does not confer resistance to opaganib, and, in fact, the drug attenuates the expression of both c-Myc and N-Myc. The safety of opaganib has been established in clinical trials with adults with advanced cancer or severe COVID-19, and so opaganib has excellent potential for treating patients with NB, particularly in combination with temozolomide and irinotecan or anti-CTLA-4 antibody.

PLGA nanocapsules as a delivery system for a recombinant LRP-based therapeutic.

Telomerase activity is directly affected by the laminin receptor precursor (LRP) protein, a highly conserved nonintegrin transmembrane receptor, which has been shown to have therapeutic effects in ageing, and age-related diseases. Recently, it has been found that overexpression of LRP-FLAG, by plasmid transfection, leads to a significant increase in telomerase activity in cell culture models. This may indicate that upregulation of LRP can be used to treat various age-related diseases. However, transfection is not a viable treatment strategy for patients. Therefore, we present a nanoencapsulated protein-based drug synthesised using poly(lactic-co-glycolic acid) (PLGA) nanocapsules for delivery of the 37 kDa LRP protein therapeutic. PLGA nanocapsules were synthesised using the double emulsification-solvent evaporation technique. Different purification methods, including filtration and centrifugation, were tested to ensure that the nanocapsules were within the optimal size range, and the BCA assay was used to determine encapsulation efficiency. The completed drug was tested in a HEK-293 cell culture model, to investigate the effect on cell viability, LRP protein levels and telomerase activity. A significant increase in total LRP protein levels with a concomitant increase in cell viability and telomerase activity was observed. Due to the observed increase in telomerase activity, this approach could represent a safer alternative to plasmid transfection for the treatment of age-related diseases.

Three-Dimensional Culture Modelling Reveals Divergent Mycobacterium tuberculosis Virulence and Antimicrobial Treatment Response

Tuberculosis (TB) remains a persistent epidemic, and the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) presents a global healthcare threat. While some new agents have been successfully introduced, innovative technologies to evaluate emerging anti-TB compounds are required to inform transformative approaches. Mtb is an obligate human pathogen, and consequently utilizing models that are consistent with human disease is likely to be critical. We have developed a human 3-dimensional (3-D) cell culture model that reflects human disease gene expression patterns and causes Mtb to become pyrazinamide sensitive in vitro. Here, we identify key differences in virulence between the standard laboratory strain, Mtb H37Rv, and clinical isolates. We demonstrate that Mtb H37Rv is attenuated in the 3-D system, more susceptible to antibiotics and hyperinflammatory compared to clinical isolates. Prolonged in vitro culture of a clinical strain leads to attenuation. We then characterise antibiotic sensitivity of multi-drug-resistant Mtb within the 3-D model and define relative bactericidal activity. Finally, we demonstrate that verapamil increases efficacy of bedaquiline and delamanid antibiotic therapy. Taken together, our findings suggest that studying virulent clinical strains in an advanced cell culture system is a powerful adjunct to established methodologies to evaluate new interventions for TB.

Apoptotic effect of 5-fluorouracil-doxorubicin combination on colorectal cancer cell monolayers and spheroids.

Drug combination studies help to improve new treatment approaches for colon cancer. Tumor spheroids (3D) are better models than traditional 2-dimensional cultures (2D) to evaluate cellular responses to chemotherapy drugs. The cultivation of cancer cells in 2D and 3D cultures affects the apoptotic process, which is a major factor influencing the response of cancer cells to chemotherapeutic drugs. In this study, the antiproliferative effects of 5-fluorouracil (5-FU) and doxorubicin (DOX) were investigated separately and in combination using 2D and 3D cell culture models on two different colon cancer cell lines, HT-29 (apoptosis-resistant cells) and Caco-2 2 (apoptosis-susceptible cells). The effect of the drugs on the proliferation of both colon cancer cells was determined by performing an MTT assay in 2D culture. The apoptotic effect of 5-FU and DOX, both as single agents and in combination, was assessed in 2D and 3D cultures through quantitative real-time polymerase chain reaction analysis. The expression of apoptotic genes, such as caspases, p53, Bax, and Bcl-2, was quantified. It was found that the mRNA expression of proapoptotic genes was significantly upregulated, whereas the mRNA expression of the antiapoptotic Bcl-2 gene was significantly downregulated in both colon cancer models treated with 5-FU, DOX, and 5-FU + DOX. The results indicated that the 5-FU + DOX combination therapy induces apoptosis and renders 5-FU and DOX more effective at lower concentrations compared to their alone use. This study reveals promising results in reducing the potential side effects of treatment by enabling the use of lower drug doses.