Ex vivo assays to detect complement activation in complementopathies.
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ABSTRACT:
SUBMITTER: Yuan X
PROVIDER: S-EPMC8609776 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundPlasmodium vivax is the second most prevalent cause of malaria yet remains challenging to study due to the lack of a continuous in vitro culture system, highlighting the need to establish a biobank of clinical isolates with multiple freezes per sample for use in functional assays. Different methods for cryopreserving parasite isolates were compared and subsequently the most promising one was validated. Enrichment of early- and late-stage parasites and parasite maturation were quantified to facilitate assay planning.MethodsIn order to compare cryopreservation protocols, nine clinical P. vivax isolates were frozen with four glycerolyte-based mixtures. Parasite recovery post thaw, post KCl-Percoll enrichment and in short-term in vitro culture was measured via slide microscopy. Enrichment of late-stage parasites by magnetic activated cell sorting (MACS) was measured. Short and long-term storage of parasites at either - 80 °C or liquid nitrogen were also compared.ResultsOf the four cryopreservation mixtures, one mixture (glycerolyte:serum:RBC at a 2.5:1.5:1 ratio) resulted in improved parasite recovery and statistically significant (P < 0.05) enhancement in parasite survival in short-term in vitro culture. A parasite biobank was subsequently generated using this protocol resulting in a collection of 106 clinical isolates, each with 8 vials. The quality of the biobank was validated by measuring several factors from 47 thaws: the average reduction in parasitaemia post-thaw (25.3%); the average fold enrichment post KCl-Percoll (6.65-fold); and the average percent recovery of parasites (22.0%, measured from 30 isolates). During short-term in vitro culture, robust maturation of ring stage parasites to later stages (> 20% trophozoites, schizonts and gametocytes) was observed in 60.0% of isolates by 48 h. Enrichment of mature parasite stages via MACS showed good reproducibility, with an average of 30.0% post-MACS parasitaemia and an average of 5.30 × 105 parasites/vial. Finally, the effect of storage temperature was tested, and no large impacts from short-term (7 days) or long-term (7-10 years) storage at - 80 °C on parasite recovery, enrichment or viability was observed.ConclusionsHere, an optimized freezing method for P. vivax clinical isolates is demonstrated as a template for the generation and validation of a parasite biobank for use in functional assays.
| S-EPMC10470152 | biostudies-literature
Project description:Complement is a major effector arm of the innate immune system that responds rapidly to pathogens or altered self. The central protein of the system, C3, participates in an amplification loop that can lead to rapid complement deposition on a target and, if excessive, can result in host tissue damage. Currently, complement activation is routinely monitored by assessing total C3 levels, which is an indirect and relatively insensitive method. An alternative approach would be to measure downstream C3 activation products such as C3a and iC3b. However, in vitro activation can produce falsely elevated levels of these biomarkers. To circumvent this issue, a lateral flow immunoassay system was developed that measures iC3b in whole blood, plasma, and serum and avoids in vitro activation by minimizing sample handling. This assay system returns results within 15 min and specifically measures iC3b while having minimal cross-reactivity to other C3 split products. While evaluating the potential of this assay, it was observed that circulating iC3b levels can distinguish healthy individuals from those with complement activation-associated diseases. This tool is engineered to provide an improved method to assess complement activation at point of care and could facilitate studies to monitor disease progression in a variety of inflammatory conditions.
| S-EPMC4404182 | biostudies-literature
Project description:The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.
| S-EPMC3606973 | biostudies-literature
Project description:Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods--in vitro, ex vivo and in vivo assays--to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA) and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages). However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.
| S-EPMC4133197 | biostudies-literature
Project description:Transplantation with low numbers of hematopoietic stem cells (HSCs), found in many of the publically accessible cryopreserved umbilical cord blood (UCB) units, leads to delayed time to engraftment, high graft failure rates, and early mortality in many patients. A chemical screen in zebrafish identified the prostaglandin compound, 16,16 dimethyl prostaglandin E2 (dmPGE2), to be a critical regulator of hematopoietic stem cell homeostasis. We hypothesized that an ex vivo modulation with dmPGE2 prior to transplantation would lead to enhanced engraftment by increasing the “effective” dose of hematopoietic stem cells (HSCs) in cord blood. A phase I trial of reduced-intensity double UCB transplantation was performed to evaluate safety, rates of engraftment and fractional chimerism of dmPGE2 enhanced UCB units. To explore potential causes of the lack of enhanced efficacy in the first cohort, we characterized HSCs to determine whether the prostaglandin pathway was being activated under the ex vivo incubation conditions (4°C, 10µM dmPGE2, 60 minutes). Incubation conditions were identified (37°C, 10µM dmPGE2, 120 minutes) that maximize the activation of the prostaglandin pathway by dmPGE2 in human CD34+ cells. Isolated human CD34+ from umbilical cord blood were incubated ex vivo in Stem Span media with 10uM 16,16-dmPGE2 or DMSO. Two treatment conditions were evaluated (4 deg C for 1 hour, 37 deg C for 2 hours) with either 3 or 7 biological replicates at each condition. Total RNA was isolated post incubation and analyzed on Affymetrix microarrays for pathway activation.
2012-04-15 | E-GEOD-36547 | biostudies-arrayexpress
Project description:We performed microRNA sequencing (miRNA-seq) according to Illumina sequencing protocols. Platelet-rich plasma (PRP) has been obtained by centrifuging whole blood at 160 x g for 15 min at room temperature. Platelets have been activated with 3 different physiologic agonists, namely ADP (20uM), collagen (60ug/mL), or Thrombin Receptor Activating Peptide (TRAP 25uM) under continuous stirring. An aliquot of platelets has been obtained at 120 minutes following ADP, collagen and TRAP and then processed to determine miRNA expression profiles. Time 0, indicating samples before any agonist treatment, was used as baseline. Total RNA was extracted by using mirVana Paris kit (life technologies) and then was subjected to size selection library preparation and retrotranscribed. Obtained cDNA was sequenced with the Illumina HiSeq 1000 sequencer .
2015-04-27 | E-MTAB-2078 | biostudies-arrayexpress
Project description:Impaired function of the retinal pigment epithelium (RPE) resembles a hallmark in many retinal diseases; thus, co-cultivation models with RPE and retinal explants are useful to investigate these. Here, we present an easy-to-handle direct co-cultivation protocol, containing a functional, primary RPE monolayer and retinal explants. We describe in detail steps for establishing the monolayer and the direct co-cultivation. Techniques, which allow users to investigate the integrity and functionality of the RPE monolayer, are presented and the co-cultivation was tested under stress conditions.
| S-EPMC10511932 | biostudies-literature
Project description:Data set presented in this article is related to the research paper entitled "Effect of amaranth proteins on the RAS system. In vitro, in vivo and ex vivo assays", available in Food Chemistry [1]. In this article, we evaluated the effect on systolic blood pressure of spontaneously hypertensive rats (SHR) of different samples with amaranth proteins/peptides. The effect of these samples on RAS system was evaluated using in vitro and ex vivo assays. The concentration of renin and angiotensin converting enzyme (ACE) was evaluated using two commercial ELISA kits. Renin concentration was estimated through a direct immunoassay and ACE concentration with an immunoassay based on a competitive inhibition. In addition, the ACE inhibitory activity in plasma was evaluated using a spectrophotometric assay according to [2]. Ex vivo experiments were done with thoracic aorta extracted during the surgical procedure employed to obtain blood samples according to [3]. Data presented in this article recollect a very extensive work on how can be affect the RAS system in SHR model using amaranth protein/peptides as potential antihypertensive samples. These data could be useful to design novel functional foods for hypertensive individuals.
| S-EPMC7011023 | biostudies-literature
Project description:INTRODUCTION:Bladder cancer (urothelial carcinoma) is a common malignancy characterized by high recurrence rates and intense clinical follow-up, indicating the necessity for more effective therapies. Current treatment regimens include intra-vesical administration of mitomycin C (MMC) for non-muscle invasive disease and systemic cisplatin for muscle-invasive or metastatic disease. Hyperthermia, heating a tumor to 40-44°C, enhances the efficacy of these chemotherapeutics by various modes of action, one of which is inhibition of DNA repair via homologous recombination. Here, we explore whether ex vivo assays on freshly obtained bladder tumors can be applied to predict the response towards hyperthermia. MATERIAL AND METHODS:The cytochrome C release assay (apoptosis) and the RAD51 focus formation assay (DNA repair) were first established in the bladder cancer cell lines RT112 and T24 as measurements for hyperthermia efficiency, and subsequently tested in freshly obtained bladder tumors (n = 59). RESULTS:Hyperthermia significantly increased the fraction of apoptotic cells after cisplatin or MMC treatment in both RT112 and T24 cells and in most of the bladder tumors (8/10). The RAD51 focus formation assay detected both morphological and numerical changes of RAD51 foci upon hyperthermia in the RT112 and T24 cell lines. In 64% of 37 analyzed primary bladder tumor samples, hyperthermia induced similar morphological changes in RAD51 foci. CONCLUSION:The cytochrome C assay and the RAD51 focus formation assay are both feasible on freshly obtained bladder tumors, and could serve to predict the efficacy of hyperthermia together with cytotoxic agents, such as MMC or cisplatin.
| S-EPMC6294360 | biostudies-literature
Project description:Immunostimulatory antibodies entering the clinic create challenge in terms of not only pharmacodynamics for monitoring anticipated mechanisms but also predetermining cytotoxicity. We show the use of ex vivo whole-blood samples to predict the activation requirements, cytokine signature, and adverse events of an anti-human-CD40 chimeric IgG1 antibody, ChiLob 7/4. Assessments were initially undertaken on human myeloid (mDC1) and plasmacytoid (pDC) dendritic cells, in which an absolute need for cross-linking was shown through the upregulation of activation markers CD83 and CCR7. Subsequent cytokine secretion evaluations of ex vivo whole blood showed the cross-linked antibody-induced increases in MIP1?, interleukin (IL)-8, IL-12, TNF?, and IL-6. This cytokine signature compared favorably with the Toll-like receptor (TLR) ligand lipopolysaccharide (LPS), in which levels of TNF? and IL-6 were significantly higher, suggesting a less intense proinflammatory response and possible modified cytokine release syndrome when used in human trials. Following first-in-human use of this agent within a dose escalation study, in vivo evaluations of dendritic cell activation and secreted cytokines closely matched the predetermined immunomonitoring endpoints. Patients showed a comparable pattern of MIP1?, IL-8, and IL-12 secretion, but no TNF? and IL-6 were identified. Mild symptoms relating to a cytokine release syndrome were seen at an equivalent dosage to that observed for dendritic cell activation and cytokine release. In summary, ChiLob 7/4 induces a distinctive pattern of dendritic cell activation and cytokine secretion in ex vivo assays that can be predictive of in vivo responses. Such preclinical approaches to monoclonal antibody evaluation may inform both the starting dosages and the anticipated cytokine release events that could occur, providing a valuable adjunct for future first-in-human assessments of immunostimulatory antibodies.
| S-EPMC4007630 | biostudies-literature