搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Induction of fetal hemoglobin (HbF) has been shown to be a viable therapeutic approach to treating sickle cell disease and potentially other β-hemoglobinopathies. To identify targets and target-modulating small molecules that enhance HbF expression,we engineered a human umbilical-derived erythroid progenitor reporter cell line (HUDEP2_HBG1_HiBiT) by genetically tagging a HiBiT peptide to the carboxyl (C)-terminus of the endogenous HBG1 gene locus,which codes for γ-globin protein,a component of HbF. Employing this reporter cell line,we performed a chemogenomic screen of approximately 5000 compounds annotated with known targets or mechanisms that have achieved clinical stage or approval by the US Food and Drug Administration (FDA). Among them,10 compounds were confirmed for their ability to induce HbF in the HUDEP2 cell line. These include several known HbF inducers,such as pomalidomide,lenalidomide,decitabine,idoxuridine,and azacytidine,which validate the translational nature of this screening platform. We identified avadomide,autophinib,triciribine,and R574 as novel HbF inducers from these screens. We orthogonally confirmed HbF induction activities of the top hits in both parental HUDEP2 cells as well as in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs). Further,we demonstrated that pomalidomide and avadomide,but not idoxuridine,induced HbF expression through downregulation of several transcriptional repressors such as BCL11A,ZBTB7A,and IKZF1. These studies demonstrate a robust phenotypic screening workflow that can be applied to large-scale small molecule profiling campaigns for the discovery of targets and pathways,as well as novel therapeutics for sickle cell disease and other β-hemoglobinopathies. View Publication

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the Slc6a8 gene. The impaired creatine uptake in the brain leads to developmental delays with intellectual disability. We hypothesized that deficient creatine uptake in CTD cerebral cells impact methylation balance leading to alterations of genes and proteins expression by epigenetic mechanism. In this study,we determined the status of nucleic acid methylation in both Slc6a8 knockout mouse model and brain organoids derived from CTD patients’ cells. We also investigated the effect of dodecyl creatine ester (DCE),a promising prodrug that increases brain creatine content in the mouse model of CTD. The level of nucleic acid methylation was significantly reduced compared to healthy controls in both in vivo and in vitro CTD models. This hypo-methylation tended to be regulated by DCE treatment in vivo. These results suggest that increased brain creatine after DCE treatment restores normal levels of DNA methylation,unveiling the potential of using DNA methylation as a marker to monitor the drug efficacy. View Publication

The efficacy of immunotherapy is often hindered by the suppression of immune responses via the tumor microenvironment (TME). The presence of cancer cells forces other proximal non-cancerous cells to support tumor growth and persistence. A clear example of this cancerous-to-non-cancerous communication is represented by the accumulation of myeloid-derived suppressor cells (MDSCs) within the TME. Several studies have convergently shown that the overexpression of DNA-methyl-transferase-1 (DNMT1) in these cells results in protection from necroptosis and enhanced accumulation in vivo. Conversely,targeting DNMT1 through hypo-methylating agents has shown promising therapeutic potential by not only reducing the levels of MDSCs but also enhancing cancer immunogenicity and the efficacy of immune checkpoint inhibitors (ICI). Methods: Murine 4T1 (triple-negative breast cancer (TNBC)) and CT26 (colon carcinoma) cell lines were cultured under standard conditions and used to generate tumor models in BALB/c mice. An oncolytic adenovirus expressing a DNMT1-targeting short hairpin RNA (OAd.shDNMT1) was engineered and validated for DNMT1 knockdown and genome-wide methylation reduction. Small extracellular vesicles (sEVs) were isolated from virus-infected cancer cells and characterized for RNA content and uptake by MDSCs. MDSC differentiation and suppressive function were assessed in vitro using flow cytometry and co-culture assays with murine splenocytes. In vivo,tumor-bearing mice received intratumoral OAd.shDNMT1,systemic decitabine,or immune checkpoint inhibitors (anti-Programmed cell Death protein-1),and tumor growth,immune infiltration,and systemic MDSC levels were evaluated. Results: In this study,we report that,by using virally infected TNBC murine cells as a source for shDNMT1-loaded sEVs,OAd.shDNMT1 successfully reduced MDSC levels in vitro and in vivo. Furthermore,the co-administration with ICI resulted in a significant tumor growth reduction in mice bearing poorly immunogenic TNBC 4T1 cells. Also,our treatment promoted antitumor immunity,prolonged survival,and complete tumor eradication in modestly immunogenic colon CT26 cancer cells. Conclusions: This multifaceted strategy,based on OV-mediated immune stimulation and reduction of MDSC levels via sEVs,may improve clinical outcomes and the success of immuno-based regimens for patients facing MDSC-rich and highly aggressive cancer subtypes. View Publication

In dynamic light microscopy applications,imaging specimens from multiple angles while maintaining controlled temperature conditions is crucial for comprehensive and accurate analysis. To address these challenges,we present iCAT,an open-source multifunctional accessory designed to enhance light microscopy. It enables specimen rotation along the axial plane,incorporates built-in modules for precise temperature control,features an integrated LED,and includes a camera for real-time specimen monitoring. It can be easily 3D-printed and assembled using readily available electrical components. Combined with any up-right microscope,this versatile device allows researchers to capture detailed images and videos of organoid cultures and live or fixed specimens,such as C. elegans,zebrafish,drosophila,or mouse embryos. The potential applications of iCAT in investigating dynamic cellular processes and complex developmental phenomena are vast,inspiring researchers to explore its possibilities and push the boundaries of biological research. iCAT,an open-source,3D-printable microscopy accessory,enables axial specimen rotation,temperature control,and live monitoring for high-resolution imaging of organoids and diverse model organisms. View Publication

Adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) infection,occurs in 2% to 4% of the HTLV-1 carriers with a long latent period,suggesting that additional alterations participate in the development of ATL. To characterize and identify novel markers of ATL,we examined the expression profiles of more than 12 000 genes in 8 cases of acute-type ATL using microarray. One hundred ninety-two genes containing interleukin 2 (IL-2) receptor alpha were up-regulated more than 2-fold compared with CD4(+) and CD4(+)CD45RO(+) T cells,and tumor suppressor in lung cancer 1 (TSLC1),caveolin 1,and prostaglandin D2 synthase showed increased expression of more than 30-fold. TSLC1 is a cell adhesion molecule originally identified as a tumor suppressor in the lung but lacks its expression in normal or activated T cells. We confirmed ectopic expression of the TSLC1 in all acute-type ATL cells and in 7 of 10 ATL- or HTLV-1-infected T-cell lines. Introduction of TSLC1 into a human ATL cell line ED enhanced both self-aggregation and adhesion ability to vascular endothelial cells. These results suggested that the ectopic expression of TSLC1 could provide a novel marker for acute-type ATL and may participate in tissue invasion,a characteristic feature of the malignant ATL cells. View Publication

Many drug-resistant tumors and cancer stem cells (CSC) express elevated levels of CD44 receptor,a cellular glycoprotein binding hyaluronic acid (HA). Here,we report the synthesis of nanogel-drug conjugates based on membranotropic cholesteryl-HA (CHA) for efficient targeting and suppression of drug-resistant tumors. These conjugates significantly increased the bioavailability of poorly soluble drugs with previously reported activity against CSC,such as etoposide,salinomycin,and curcumin. The small nanogel particles (diameter 20-40 nm) with a hydrophobic core and high drug loads (up to 20%) formed after ultrasonication and demonstrated a sustained drug release following the hydrolysis of biodegradable ester linkage. Importantly,CHA-drug nanogels demonstrated 2-7 times higher cytotoxicity in CD44-expressing drug-resistant human breast and pancreatic adenocarcinoma cells compared to that of free drugs and nonmodified HA-drug conjugates. These nanogels were efficiently internalized via CD44 receptor-mediated endocytosis and simultaneous interaction with the cancer cell membrane. Anchoring by cholesterol moieties in the cellular membrane after nanogel unfolding evidently caused more efficient drug accumulation in cancer cells compared to that in nonmodified HA-drug conjugates. CHA-drug nanogels were able to penetrate multicellular cancer spheroids and displayed a higher cytotoxic effect in the system modeling tumor environment than both free drugs and HA-drug conjugates. In conclusion,the proposed design of nanogel-drug conjugates allowed us to significantly enhance drug bioavailability,cancer cell targeting,and the treatment efficacy against drug-resistant cancer cells and multicellular spheroids. View Publication

Chlamydia infection and disease are endemic in free-ranging koalas. Antibiotics remain the front line treatment for Chlamydia in koalas,despite their rates of treatment failure and adverse gut dysbiosis outcomes. A Chlamydia vaccine for koalas has shown promise for replacing antibiotic treatment in mild ocular Chlamydia disease. In more severe disease presentations that require antibiotic intervention,the effect of vaccinating during antibiotic use is not currently known. This study investigated whether a productive immune response could be induced by vaccinating koalas during antibiotic treatment for Chlamydia-induced cystitis. Plasma IgG antibody levels against the C. pecorum major outer membrane protein (MOMP) dropped during antibiotic treatment in both vaccinated and unvaccinated koalas. Post-treatment,IgG levels recovered. The IgG antibodies from naturally-infected,vaccinated koalas recognised a greater proportion of the MOMP protein compared to their naturally-infected,unvaccinated counterparts. Furthermore,peripheral blood mononuclear cell gene expression revealed an up-regulation in genes related to neutrophil degranulation in vaccinated koalas during the first month post-vaccination. These findings show that vaccination of koalas while they are being treated with antibiotics for cystitis can result in the generation of a productive immune response,in the form of increased and expanded IgG production and host response through neutrophil degranulation. View Publication

Hepatocyte death during acetaminophen (APAP) intoxication elicits a reactive inflammatory response,with hepatic recruitment of neutrophils and monocytes,which further aggravates liver injury. Neutrophil elastase (NE),secreted by activated neutrophils,carries degradative and cytotoxic functions and maintains a proinflammatory state. We investigated NE as a therapeutic target in acetaminophen-induced liver injury (AILI). C57BL/6 mice were administered a toxic dose of APAP,2 h prior to receiving the NE inhibitor sivelestat,N-acetylcysteine (NAC),or a combination therapy,and were euthanized after 24 and 48 h. Upon APAP overdose,neutrophils and monocytes infiltrate the injured liver,accompanied by increased levels of NE. Combination therapy of NAC and sivelestat significantly limits liver damage,as evidenced by lower serum transaminase levels and less hepatic necrosis compared to mice that received APAP only,and this to a greater extent than NAC monotherapy. Lower hepatic expression of proinflammatory markers was observed in the combination treatment group,and flow cytometry revealed significantly less monocyte influx in livers from mice treated with the combination therapy,compared to untreated mice and mice treated with NAC only. The potential of NE to induce leukocyte migration was confirmed in vitro. Importantly,sivelestat did not impair hepatic repair. In conclusion,combination of NE inhibition with sivelestat and NAC dampens the inflammatory response and reduces liver damage following APAP overdose. This strategy exceeds the standard of care and might represent a novel therapeutic option for AILI. View Publication

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However,little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional,or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite,thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria. View Publication

Mesenchymal stem cells (MSCs) are capable of down-regulating alloimmune responses and promoting the engraftment of hematopoietic stem cells. MSCs may therefore be suitable for improving donor-specific tolerance induction in solid-organ transplantation. Cells from cadaveric vertebral bone marrow (V-BM),aspirated iliac crest-BM,and peripheral blood progenitor cells were compared. Cells were characterized by flow cytometry and colony assays. MSCs generated from V-BM were assayed for differentiation capacity and immunomodulatory function. A median 5.7 x 10(8) nucleated cells (NCs) were recovered per vertebral body. The mesenchymal progenitor,colony-forming unit-fibroblast,frequency in V-BM (11.6/10(5) NC,range: 6.0-20.0) was considerably higher than in iliac crest-BM (1.4/10(5) NC,range: 0.4-2.6) and peripheral blood progenitor cells (not detectable). MSC generated from V-BM had the typical MSC phenotype (CD105(pos)CD73(pos)CD45(neg)CD34(neg)),displayed multilineage differentiation potential,and suppressed alloreactivity in mixed lymphocyte reactions. V-BM may be an excellent source for MSC cotransplantation approaches. View Publication

This review discusses the challenges of chemotherapy for human African trypanosomiasis (HAT). The few drugs registered for use against the disease are unsatisfactory for a number of reasons. HAT has two stages. In stage 1 the parasites proliferate in the haemolymphatic system. In stage 2 they invade the central nervous system and brain provoking progressive neurological dysfunction leading to symptoms that include the disrupted sleep wake patterns that give HAT its more common name of sleeping sickness. Targeting drugs to the central nervous system offers many challenges. However,it is the cost of drug development for diseases like HAT,that afflict exclusively people of the world's poorest populations,that has been the principal barrier to new drug development and has led to them becoming neglected. Here we review drugs currently registered for HAT,and also discuss the few compounds progressing through clinical trials. Finally we report on new initiatives that might allow progress to be made in developing new and satisfactory drugs for this terrible disease. View Publication

Mycoplasma pneumoniae is a human respiratory tract pathogen causing acute and chronic airway disease states that can include long-term carriage and extrapulmonary spread. The mechanisms of persistence and migration beyond the conducting airways,however,remain poorly understood. We previously described an acute exposure model using normal human bronchial epithelium (NHBE) in air-liquid interface culture,showing that M. pneumoniae gliding motility is essential for initial colonisation and subsequent spread,including localisation to epithelial cell junctions. We extended those observations here,characterizing M. pneumoniae infection of NHBE for up to 4 weeks. Colonisation of the apical surface was followed by pericellular invasion of the basolateral compartment and migration across the underlying transwell membrane. Despite fluctuations in transepithelial electrical resistance and increased NHBE cell desquamation,barrier function remained largely intact. Desquamation was accompanied by epithelial remodelling that included cytoskeletal reorganisation and development of deep furrows in the epithelium. Finally,M. pneumoniae strains S1 and M129 differed with respect to invasion and histopathology,consistent with contrasting virulence in experimentally infected mice. In summary,this study reports pericellular invasion,NHBE cytoskeletal reorganisation,and tissue remodelling with persistent infection in a human airway epithelium model,providing clear insight into the likely route for extrapulmonary spread. View Publication