Antigen-specific electrophoretic cell separation for immunological investigations

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non-capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen-positive and antigen-negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence-labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen-positive cells in the fractions of electrophoresis. Carrier-free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti-IgM-antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate-labelled anti-IgG resulted in the large-scale separation of high pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen-specific induction and regulation of antibody synthesis in vitro. These separate lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell subpopulations. We studied the influence of the anesthetic thiopental on separated human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B-Cells, T-helper and T-suppressor cells were equally affected and showed the same dose response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luciferase-transfected colon adenocarcinoma cell line (DLD-1) for use in Orthotopic Xenotransplantation studies

ABSTRACT: BACKGROUND: Renilla Luciferase reporter gene (rLuc) GL4.82 and GL4.13 promoter are key player in transfection, but precise knowledge of its targets in colon cancer remains limited. The aim of this study was to characterize the best transfection technique to produce a stable transfected colon DLD1 (colorectal adenocarcinoma cell line), therefore imaging based approaches were employed. RESULTS: DLD1 cells were transfected with a Plasmid (SV40-RLuc) carrying Renilla luciferase under the control of the SV-40 promoter, by using two different transfection techniques. Cells expressing the required DNA were isolated after antibiotic (Puramycin) selection. Clones of DLD-1/SV40-RLuc were produced using two different techniques (96 well plates and Petri dish) and their florescence intensity was recorded using IVIS machine (Calliper Life Sciences, Hopkinton, USA). Both techniques were characterized with the help of serial dilution technique. Results from this study substantiated that electroporation is the best. As expected, clones varied in their specific luciferase activity along with the dilutions. With the increase in cell concentration increase in intensity of florescence was recorded. CONCLUSIONS: Based on the results we are confident that this transfected cell line DLD-1/SV40-RLuc (colorectal adenocarcinoma cell line) is the best for further Orthotopic Xenotransplantation Studies and in-vivo experiments as well. Investigation shows that DLD1/SV-rLuc cells have gained little bit resistance against both drugs therefore further study is suggested to know the reasons.     

A rapid and simple method for the isolation of mutant variants regulating tissue-specific expression of the tni gene through drug selection

TnINEO fusion gene was constructed by fusing 3.4-kbp of quailTnI genomic DNA sequences spanning the promoter to exon 5 and aneo gene in frame. A myoblast cell line was established after transfection of pTnINEO. Since this cell line was passaged several times, a high frequency of neomycin (G418) sensitivity conversion was detected. Two drug-resistant variants were analyzed through genomic Southern blot and S1 nuclease protection assay. One variant has a mutation(s) in the regulatory element that activated the dormantTnI promoter-enhancer in myoblast, and the other has shown the genomic rearrangement. This result presented the possibility of isolating factor(s) that activate the muscle-specificTnI promoter simply by screening drug-resistant cells having appropriate mutations.     

Multiplex, construct-specific, and real-time PCR-based analytical methods for Bt rice with cry1Ac gene

Qualitative and quantitative analytical methods based on PCR for Bacillus thuringiensis (Bt) rice hybrid, namely, MRP 5401 Bt expressing a modified version of the Bt cry1Ac gene, are reported here. Multiplex PCR assays were developed to target the cry1Ac transgene, Cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptLL) marker gene, and an endogenous a-tubulin (TubA) gene in Bt rice. The 3.178 kb region of inserted gene construct comprising the region of the CaMV 35S promoter and cry1Ac gene was amplified, and the construct integrity was confirmed by the nested PCR. The LOD for cry1Ac gene-specific simplex PCR was 0.01%, as established using Bt rice DNA dilutions with 100, 10, 1.0, 0.1, 0.05, 0.01, and 0.001% genetically modified trait. A real-time PCR assay was also developed to quantify the cry1Ac gene. The method performance of the reported real-time PCR assay was in line with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with LOD and LOQ values of 0.05%. The reliable PCR assays prior to commercial release of Bt rice would facilitate efficient regulatory compliance for identification of genetic trait, labeling requirements, and effective risk assessment and management. They could also address consumers' concerns and legal disputes that may arise.     

Human lymphocyte sorting by gravitational field-flow fractionation

Interest in biological studies on various cell types for many biomedical applications, from research to patient treatments, is constantly increasing. The ability to discriminate (sort) and/or quantify distinct subpopulations of cells has become increasingly important. For instance, not only detection but also the highest depletion of neoplastic cells from normal cells is an important requisite in the autologous transplantation of lymphocytes for blood cancer treatments. In this work, gravitational field-flow fractionation (GrFFF) is shown to be effective for sorting a heterogeneous mixture of human, living lymphocytes constituted of neoplastic B cells from a Burkitt lymphoma cell line and healthy T and B lymphocytes from blood samples. GrFFF does not require the use of fluorescent immunotags for sorting cells, and the sorted cells can be collected for their further characterization. Flow cytometry was used to assess the viability of the cells collected, and to evaluate the cell fractionation achieved. A low amount of neoplastic B lymphocytes (less than 2%) was found in a specific fraction obtained by GrFFF. The high depletion from neoplastic cells (more than 98%) was confirmed by a clonogenicity test.     

Centrifugal countercurrent chromatography to elucidate surface differences of adipose tissue-derived stem cells

The current methods of isolation of adipose tissue-derived stem cells result in a heterogeneous population that might interfere with their differentiation potential and makes it difficult to compare the results between different groups. Partition in aqueous two-phase systems is one of the few techniques that separate cells on the basis of surface properties, gentle enough to isolate fragile cell types in isotonic conditions without altering their structure, and can be easily scaled. In this study, stem cells isolated from human adipose tissue seeded and expanded in vitro were fractionated by using centrifugal countercurrent distribution in an aqueous two-phase system. The separated subpopulations revealed the high heterogeneity of adipose tissue-derived stem cell samples. Comparative partition analyses showed that aging induces a loss of heterogeneity, which is not due to a loss of cell viability associated to age. The phosphatidylserine externalization, an apoptotic feature, is the main factor in cell partition that results in a decreased hydrophobicity of the cell surface. This procedure may be suitable for separating adipose tissue-derived stem cell populations enriched in some functional and/or structural surface characteristics. The possibility of a very effective separation of different subpopulations in opposite phases would be an interesting development of the method.     

Facile assay of telomerase activity utilizing a DNA-detectable chemiluminogenic reagent.

Telomerase shows increased activity in most human cancers and germ line cells, but not in normal human somatic cells. We describe a novel chemiluminescence method for the facile assay of telomerase activity in human cells. The telomerase substrate was incubated with the cell lysate containing various amounts of telomerase, and then the telomerase product was amplified by the polymerase-chained reaction (PCR). The PCR products were separated from the excess substrate, primer and deoxyribonucleotide triphosphates by a centrifugal filter, which distinguished different molecular sizes. The isolated products were reacted with a DNA-detectable chemiluminogenic reagent, 3,4,5-trimethoxyphenylglyoxal. The proposed assay method gave linearity for the telomerase activity in 100 to 10000 cells (r2=0.997), and allowed the assay not only of lower activity, but also of higher activity of telomerase without the requirement of any special labeled-PCR primers in the assay system.     

Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation

We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations.     

Construction of HIV-1 Virus-like Particle Vaccine

The virus-like particle（VLPs） vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have been used, one can express the HIV-1 main structure proteins, Gagpol and Env, and the other contains an antibiotic gene. The two kinds of plasmids have been cotransfected into 293 cells. A stable cell line that can express Gagpol and Env proteins efficiently and lastingly has been screened. It has been confirmed that Gagpol and Env proteins in the cell culture supernatant can be self-assembled into virus-like particles. The authors have detected the secretion of VLPs in the cell medium, defined the peak of the secretion, and followed and monitored the stability of expression.     

Synergistic Roles of Curcumin in Sensitising the Cisplatin Effect on a Cancer Stem Cell-Like Population Derived from Non-Small Cell Lung Cancer Cell Lines

Cancer stem cells (CSCs) represent a small subpopulation within a tumour. These cells possess stem cell-like properties but also initiate resistance to cytotoxic agents, which contributes to cancer relapse. Natural compounds such as curcumin that contain high amounts of polyphenols can have a chemosensitivity effect that sensitises CSCs to cytotoxic agents such as cisplatin. This study was designed to investigate the efficacy of curcumin as a chemo-sensitiser in CSCs subpopulation of non-small cell lung cancer (NSCLC) using the lung cancer adenocarcinoma human alveolar basal epithelial cells A549 and H2170. The ability of curcumin to sensitise lung CSCs to cisplatin was determined by evaluating stemness characteristics, including proliferation activity, colony formation, and spheroid formation of cells treated with curcumin alone, cisplatin alone, or the combination of both at 24, 48, and 72 h. The mRNA level of genes involved in stemness was analysed using quantitative real-time polymerase chain reaction. Liquid chromatography-mass spectrometry was used to evaluate the effect of curcumin on the CSC niche. A combined treatment of A549 subpopulations with curcumin reduced cellular proliferation activity at all time points. Curcumin significantly (p < 0.001) suppressed colonies formation by 50% and shrank the spheroids in CSC subpopulations, indicating inhibition of their self-renewal capability. This effect also was manifested by the down-regulation of SOX2, NANOG, and KLF4. Curcumin also regulated the niche of CSCs by inhibiting chemoresistance proteins, aldehyde dehydrogenase, metastasis, angiogenesis, and proliferation of cancer-related proteins. These results show the potential of using curcumin as a therapeutic approach for targeting CSC subpopulations in non-small cell lung cancer.     

Analysis of U87 glioma cells by dielectrophoresis

Glioblastoma multiforme is the most aggressive and invasive brain cancer consisting of genetically and phenotypically altering glial cells. It has massive heterogeneity due to its highly complex and dynamic microenvironment. Here, electrophysiological properties of U87 human glioma cell line were measured based on a dielectrophoresis phenomenon to quantify the population heterogeneity of glioma cells. Dielectrophoretic forces were generated using a gold-microelectrode array within a microfluidic channel when 3 V_pp and 100, 200, 300, 400, 500 kHz, 1, 2, 5, and 10 MHz frequencies were applied. We analyzed the dielectrophoretic behavior of 500 glioma cells, and revealed that the crossover frequency of glioma cells was around 140 kHz. A quantifying dielectrophoretic movement of the glioma cells exhibited three distinct glioma subpopulations: 50% of the glioma cells experienced strong, 30% of the cells were spread in the microchannel by moderate, and the rest of the cells experienced very weak positive dielectrophoretic forces. Our results demonstrated the dielectrophoretic spectra of U87 glioma cell line. Dielectrophoretic responses of glioma cells linked population heterogeneity to membrane properties of glioma cells rather than their size distribution in the population.     

Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers

Phenotypically pure subpopulations of lymphocytes can provide valuable insights into the immune response to injury and disease. The isolation of these subpopulations presents unique challenges, particularly when preprocessing incubation to attach fluorescent or antibody tags is to be minimized. This paper examines the separation of T and B lymphocytes from mixtures using microfluidic chambers coated with antibodies, focusing on flow conditions and surface chemistry. The adhesion of both cell types decreases as shear stress increases irrespective of the surface chemistry. The incorporation of poly(ethylene glycol) chains along with the antibodies on the chamber surface is shown to significantly improve the reproducibility of cell adhesion and is thus an important part of the overall system design. Furthermore, this technique is shown to be an effective way of isolating highly pure subpopulations of lymphocytes from model mixtures, even when the target cell concentration is low.     

A new class of branched aminoglycosides: pseudo-pentasaccharide derivatives of neomycin B

[reaction: see text] The clinically important antibiotic neomycin B was modified at position C5' ' by adding one extra sugar ring in the beta-configuration, and the observed pseudo-pentasaccharides were tested against various bacterial strains, including pathogenic and resistant strains. The designed antibiotics show antibacterial activity superior to that of neomycin B against pathogenic and resistant bacterial strains.     

Restoration of full-length APC protein in SW480 colon cancer cells induces exosome-mediated secretion of DKK-4

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are common in both inherited and sporadic forms of colorectal cancer (CRC), and are associated with dysregulated Wnt signaling. Colon carcinoma SW480 cells restored with stable expression of wild-type APC (SW480APC cells) exhibit attenuated Wnt signaling, and reduced tumorigenicity, including increased cell adhesion. We performed a comparative proteomic analysis of exosomes isolated from SW480 and SW480APC cells to examine the effects of restored APC on exosome protein expression. A salient finding of our study was the unique expression of the Wnt antagonist Dickkopf-related protein 4 (DKK4) in SW480APC, but not parental SW480 cell-derived exosomes. Upregulation of DKK4 in SW480APC cells was confirmed by semiquantitative RT-PCR, immunoblotting, and immunogold electron microscopy. Analysis of the DKK4 gene promoter by methylation-specific PCR revealed reduced methylation in SW480APC cells, while RT-PCR demonstrated the downregulation of DNMT-3a, compared to the parental cell line. Our discovery of exosome-mediated secretion of DKK4 opens up the possibility that exosomal DKK4 may be a mechanism used by epithelial colon cells to regulate Wnt signaling which is lost during CRC progression.     

Microsample preparation by dielectrophoresis: isolation of malaria

An important enabling factor for realising integrated micro fluidic analysis instruments for medical diagnostics purposes is front-end sample preparation. Dielectrophoresis is a method that offers great potential for cell discrimination and isolation for sample processing, and here we have applied it to the problem of isolating malaria-infected cells from blood. During development of the malarial pathogen, Plasmodium falciparum, increases occur in the ionic permeability of the plasma membrane of infected erythrocytes. When challenged by suspension in a low conductivity medium, infected cells lose internal ions while uninfected cells retain them. The resultant dielectric differences between infected and uninfected cells were exploited by dielectrophoretic manipulation in spatially inhomogeneous, travelling electrical fields produced by two types of microelectrode arrays. Parasitised cells of ring form or later stage from cultures and clinical specimens were isolated by steric dielectric field-flow-fractionation, focused at the centre of a spiral electrode array, and identified and counted. The dielectrophoretic methods require only a few micro litres of blood, and should be applicable to the production of small, low-cost automated devices for assessing parasite concentrations with potential applicability to drug sensitivity studies and the diagnosis of malaria. By simple adjustment of the electrical field parameters, other cell subpopulations that characterise disease, such as residual cancer cells in blood, can be similarly isolated and analysed.     

A near-infrared spectroscopy method to determine aminoglycosides in pharmaceutical formulations

The analytical determination of aminoglycosides in pharmaceutical formulations is very difficult due to the lack of chromophores or fluorophores. Several analytical methods have been developed along the years mainly based on derivatization reactions. The European Pharmacopeia (EP) and the United States Pharmacopeia (USP) describe a microbiological assay to the quantification of aminoglycosides. Near infrared spectroscopy (NIRS) can be used alternatively to analyse aminoglycosides without the need of derivatization reactions or other type of sample processing. A new NIRS based method was developed for the analysis of the aminoglycoside antibiotic neomycin. The method was developed with samples based on a commercial formulation containing neomycin sulphate and three excipients: lactose, talc and magnesium stearate. Synthetic and doped samples were manufactured for this purpose. Three lots of a commercial solid formulation were also used to assess the validity of the method to quantify neomycin sulphate in the industrial pharmaceutical product. The method proposes measurements in reflectance mode using a Fourier-transform near infrared (FT-NIR) spectrometer. Partial least squares regression was the multivariate method adopted to calibrate the NIR spectra with the neomycin sulphate mass fraction. The concentration of neomycin sulphate present in the commercial samples was confirmed by HPLC with pre-column derivatization with phenylisocyanate. Results show that neomycin sulphate was determined successfully in the commercial samples using the method calibrated with the doped samples (mass fraction error of 6.6%). Moreover, the synthetic samples were found to be unqualified to develop the method, producing a biased calibration.     

Dielectrophoretic characterization and separation of monocytes and macrophages using 3D carbon‐electrodes

Monocyte heterogeneity and its prevalence are revealed as indicator of several human diseases ranking from cardiovascular diseases to rheumatoid arthritis, chronic kidney diseases, autoimmune multiple sclerosis, and stroke injuries. When monocytes and macrophages are characterized and isolated with preserved genetic, phenotypic and functional properties, they can be used as label‐free biomarkers for precise diagnostics and treatment of various diseases. Here, the dielectrophoretic responses of the monocytes and macrophages were examined. We present 3D carbon‐electrode dielectrophoresis (carbon‐DEP) as a separation tool for U937 monocytes and U937 monocyte‐differentiated macrophages. The carbon‐electrodes advanced the usability and throughput of DEP separation, presented wider electrochemical stability. Using the 3D carbon‐DEP chip, we first identified the selective positive and negative DEP responses and specific crossover frequencies of monocytes and macrophages as their signatures for separation. The crossover frequency of monocytes and macrophages was 17 and 30 kHz, respectively. Next, we separated monocyte and macrophage subpopulations using their specific dielectrophoretic responses. Afterward, we used a fluorescence‐activated cell sorter to confirm our results. Finally, we enriched 70% of monocyte cells from the mixed cell population, in other words, concentration of monocyte cells to macrophage cells was five times increased, using the 30‐kHz, 10‐Vpp electric field and 1 μL/min flow rate.     

Multisite modification of neomycin B: combined Mitsunobu and click chemistry approach

The aminoglycoside antibiotic neomycin B has been converted into several novel building blocks that can be used for the specific modification of three of the four ring systems. Under carefully controlled conditions, the Mitsunobu reaction can be used to selectively dehydrate the ido ring to give the talo epoxide. Subsequently however, under more forcing conditions, the 2-deoxy streptamine ring undergoes Mitsunobu dehydration to give an aziridine. An unusual remote neighboring group effect was observed. When the primary hydroxyl of the ribose ring was blocked, aziridine formation on the deoxystreptamine ring did not occur. Both the epoxide and epoxide-aziridine neomycin building blocks can be ring-opened with azide and subjected to "click" type chemistry with terminal alkynes to generate a series of new neomycin analogues. These reactions can all be carried out without recourse to O-protecting groups. A detailed conformational analysis by NMR revealed some unexpected conformer preferences in these systems.     

Screening of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities

Recently we constructed yeast cells that either express the human estrogen receptor α or the human androgen receptor in combination with a consensus ERE or ARE repeat in the promoter region of a green fluorescent protein (yEGFP) read-out system. These bioassays were proven to be highly specific for their cognate agonistic compounds. In this study the value of these yeast bioassays was assessed for analysis of compounds with antagonistic properties. Several pure antagonists, selective estrogen receptor modulators (SERMs) and plant-derived compounds were tested. The pure antiestrogens ICI 182,780 and RU 58668 were also classified as pure ER antagonists in the yeast estrogen bioassay and the pure antiandrogen flutamide was also a pure AR antagonist in the yeast androgen bioassay. The plant-derived compounds flavone and guggulsterone displayed both antiestrogenic and antiandrogenic activities, while 3,3′-diindolylmethane (DIM) and equol combined an estrogenic mode of action with an antiandrogenic activity. Indol-3-carbinol (I3C) only showed an antiandrogenic activity. Coumestrol, genistein, naringenin and 8-prenylnaringenin were estrogenic and acted additively, while the plant sterols failed to show any effect. Although hormonally inactive, in vitro and in vivo metabolism of the aforementioned plant sterols may still lead to the formation of active metabolites in other test systems.