Enrichment of the Murine Natural Killer (NK) and Mitogen Induced Cellular Cytotoxicity (MICC) Cells Using Preparative Free-Flow High Voltage Electrophoresis

A procedure using preparative free-flow high voltage electrophoresis is described for the fractionation of murine spleen and bone marrow cells so as to obtain cell subpopulations that are either enriched in or depleted of “natural killer” (NK) cells and “mitogen-induced cellular cytotoxicity” (MICC) effector cells. A nearly three fold enrichment in the NK and MICC activities of spleen cells was achieved. The enrichment in these cells could be further increased if the phagocytic cells were removed prior to electrophoresis. When bone marrow cells were fractionated a two and a half fold increase of NK activity, and a one and a half fold enrichment of MICC activity was achieved. In both cases, other fractions were nearly devoid of NK and MICC activity. The cell recovery after electrophoresis averages 70% of the cells applied, and at least 90% of these cells were viable. MICC and NK effector cells could not be separated to a useful extent electro-phoretically but were found to be separable using Sephadex G-10 gel filtration columns. The MICC but not the NK cells were retained on these columns.     

Enrichment of the murine natural killer (NK) and mitogen induced cellular cytotoxicity (MICC) cells using preparative free-flow high voltage electrophoresis.

A procedure using preparative free-flow high voltage electrophoresis is described for the fractionation of murine spleen and bone marrow cells so as to obtain cell subpopulations that are either enriched in or depleted of "natural killer" (NK) cells and "mitogen-induced cellular cytotoxicity" (MICC) effector cells. A nearly three fold enrichment in the NK and MICC activities of spleen cells was achieved. The enrichment in these cells could be further increased if the phagocytic cells were removed prior to electrophoresis. When bone marrow cells were fractionated a two and a half fold increase of NK activity, and a one and a half fold enrichment of MICC activity was achieved. In both cases, other fractions were nearly devoid of NK and MICC activity. The cell recovery after electrophoresis averages 70% of the cells applied, and at least 90% of these cells were viable. MICC and NK effector cells could not be separated to a useful extent electrophoretically but were found to be separable using Sephadex C-10 gel filtration columns. The MICC but not the NK cells were retained on these columns.     

Enantioseparation of tetrahydropalmatine and Tröger's base by molecularly imprinted monolith in capillary electrochromatography

Two chiral compounds, Tr?ger's base and tetrahydropalmatine, were enantioseparated on the (5S, 11S)-(-)-Tr?ger's base and l-tetrahydropalmatine imprinted monolithic capillary columns with CEC, respectively. The monoliths were prepared by in situ thermal-initiated copolymerization of methacrylic acid (MAA) and ethylene dimethacrylate (EDMA). After optimizing the ratio of porogens (toluene and dodecanol), the obtained monolithic capillary columns show good flow-through property and enantioselectivity. The influences of CEC parameters such as pH of the buffer, organic solvent and salt concentration on the electroosmotic flow (EOF) and recognition selectivity were systematically investigated. Under the optimal conditions, baseline resolutions of two chiral compounds were achieved. In addition, the fast separation was obtained within 4 min by applying higher voltage and assisting pressure of 6 bar.     

Monocyte involvement in PHA induced cytotoxicity of chicken erythrocytes in man.

Phytohemagglutinin (PHA) induced cell cytotoxicity was studied in man using chromated chicken red blood cells (CRBC) as target cells. A phagocytic, adherent monocyte was found to be necessary for lysis of target cells. Results using E rosette depletion showed that this procedure markedly increased mitogen-induced cellular cytotoxicity (MICC). Carbonyl iron treatment of peripheral blood cell suspensions to remove phagocytic cells abolished MICC, as did removal of adherent cells by glass wool columns. Complement mediated lysis of B cells did not substantially reduce MICC. However, pretreatment of cells with silica or hydrocortisone did reduce MICC. The mechanism of mitogen-induced lysis appears to require direct cell contact between effector cells and target cells.     

Mitogen induced cellular cytotoxicity (MICC) in multiple myeloma.

Mitogen induced cellular cytotoxicity (MICC) was noted to be markedly increased in patients with multiple myeloma as compared to normal controls and to patients with chronic lymphocytic leukemia (CLL). Enhanced MICC was present at various effector-to-target cell ratios and at several mitogen concentrations. Removal of adherent, phagocytic cells by carbonyl iron, glass wool, or rayon columns abolished the MICC response from the peripheral blood of both multiple myeloma patients and normal controls. Thus, the effector cell mediating MICC may be monocytic in origin and closely resembles the suppressor cell for immunoglobulin synthesis described in patients with multiple myeloma. Our data suggest that the MICC assay with chicken red blood cells as targets may provide a convenient method for identifying pathologic conditions where this cytotoxic effector cell population plays an active role.     

Synthesis and evaluation of polymeric continuous bed (monolithic) reversed-phase gradient stationary phases for capillary liquid chromatography and capillary electrochromatography

There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversed-phase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.     

Molecularly imprinted composite cryogels for hemoglobin depletion from human blood

A molecularly imprinted composite cryogel (MICC) was prepared for depletion of hemoglobin from human blood prior to use in proteome applications. Poly(hydroxyethyl methacrylate) based MICC was prepared with high gel fraction yields up to 90%, and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, swelling studies, flow dynamics and surface area measurements. MICC exhibited a high binding capacity and selectivity for hemoglobin in the presence of immunoglobulin G, albumin and myoglobin. MICC column was successfully applied in fast protein liquid chromatography system for selective depletion of hemoglobin for human blood. The depletion ratio was highly increased by embedding microspheres into the cryogel (93.2%). Finally, MICC can be reused many times with no apparent decrease in hemoglobin adsorption capacity. Copyright © 2014 John Wiley & Sons, Ltd.     

Preparation and evaluation of bovine serum albumin immobilized chiral monolithic column for affinity capillary electrochromatography

A system of capillary silica monolith with bovine serum albumin (BSA) functionalized through two approaches for affinity monolithic capillary electrochromatography (AMCEC) was developed. Covalent immobilization conditions for two different Schiff base methods, which employed 3-glycidopropyl trimethoxysilane (GPTS) and 3-aminopropyl trimethoxysilane (APTS) as starting materials, respectively, were investigated to obtain good and stable chiral separation. The BSA immobilized silica monoliths were evaluated in terms of morphology, electroosmotic flow, retention time, column efficiency and resolution of model compound (±)-tryptophan. The columns exhibited satisfactory run-to-run, column-to-column repeatability and maintained their enantioselectivity for more than 3 months. Both developed methods can baseline separate tryptophan enantiomers, whereas shorter retention time, better column efficiency, and enantiomeric recognition between two pairs of drug enantiomers (pantoprazole and atenolol) were obtained by the GPTS method.     

Electrochromatographic characterization of methacrylate ester-based monolith and capillary electrochromatography separation of flavonoids

A porous polymethacrylate ester-based monolithic column for capillary electrochromatography (CEC) was designed by mean of in situ co-polymerizing lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent including cyclohexanol, 1,4-butanediol and water. After investigating the influence factors of the CEC monolithic columns, four flavonoids (i.e., Rutin, Quercetin, Kaempferol, and Quercitrin) were separated and assayed to evaluate this monolithic column with CEC method. Under optimum conditions, the CEC method exhibited high separation efficiency, with rapid separation time of 3–4 min, for the four flavonoid samples using 10 mM phosphate buffer containing 70% acetonitrile (pH 9.0). Importantly, the proposed method could provide a promising approach for rapid separation and detection in biomedicine.     

Fc receptors on human T lymphocytes. II. Cytotoxic capabilities of human T gamma, T mu, B, and L cells.

Subpopulations of human peripheral blood lymphocytes were prepared by rosetting techniques employing neuraminidase-treated sheep erythrocytes (SRBC_n), sheep erythrocytes coated with IgM and murine complement (EAC′), and bovine erythrocytes coated with IgG and IgM. The isolated subpopulations were tested in assays of natural cytotoxicity (NC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). B cells (SRBC_n?, EAC′+) did not mediate cytotoxicity. L cells (SRBC_n?, EAC′?) mediated NC and ADCC but not MICC. T cells (SRBC_n+) mediated NC, ADCC, and MICC. Separation of T cells into Fc-IgG (Tγ) and Fc-IgM (Tμ) subsets revealed that Tγ cells mediated NC, ADCC, and MICC while Tμ cells mediated only MICC. Thus MICC but not NC or ADCC was solely T-cell mediated. Tγ and L cells were functionally distinguishable in that Tγ cells but not L cells mediated MICC. Tγ cells and Tμ cells differed with regard to NC and ADCC effector function while both subsets mediated MICC.     

Surface receptors and immune activity of purified human circulating mononuclear cells. III. The mechanisms of lysis, by lymphocytes and monocytes, of target cells in the lymphocyte-dependent and monocyte-dependent ADCC, NK, NOCC, and MICC cytotoxic reactions

The abilities of unfractionated mononuclear cells (MNC), monocytes (98-99% pure), and lymphocytes (98-99% pure) to carry out the lysis of target cells in the ADCC, NK, NOCC, and MICC assays were compared. Lymphocytes by themselves were able to lyse the CRBC (ADCC), K-562 (NK), and RRBC (MICC) target cells. The monocytes were very effective in the lysis of the CRBC (MICC) target cells. However, the lysis of two other target cells--RRBC (NOCC) and HRBC (ADCC)--required the simultaneous presence of both lymphocytes and monocytes in order to effect optimal lysis. Soluble factor(s) secreted by the cytotoxic cells capable of lysing the target cells were detected only in the NK assay. The activity of the soluble cytotoxic factor (NKCF) was only 25-40% of that exhibited by the cytotoxic NK cells and it was secreted by the cytotoxic cells after 48 hr of culture and not 24 hr of culture which is the usual assay condition. The NKCF was cytotoxic only to the NK target cells and not to the target cells used in the ADCC, NOCC, and MICC cytotoxic assays. Different classes of lymphocytes were cytotoxic in the monocyte-independent assays [ADCC (CRBC), NK (K-562), and MICC (RRBC)]. The null lymphocytes and the T lymphocytes were the primary cytotoxic cells in the ADCC and MICC assays, respectively, whereas the T, B, and null cells were almost equally cytotoxic in the NK assay. With respect to the monocyte-dependent assays [ADCC (HRBC), NOCC (RRBC), and MICC (CRBC)], the cytotoxic activity of any one class of lymphocytes failed to approach that of the unfractionated MNC. The T cells were the most cytotoxic; the B cells exhibited limited cytotoxic activity in only the ADCC assay and the null cells showed no cytotoxic activity. However, the combination of T and non-T cells and, to a lesser extent, T and B cells, exhibited much greater cytotoxic activity than the individual cells and together were as cytotoxic as the unfractionated MNC. It is concluded that, depending upon the selection of the target cells, lysis in the ADCC, NK, NOCC, and MICC assays may be effected by lymphocytes only, by monocytes only, by both monocytes and lymphocytes, or as a result of lymphocyte-monocyte collaboration. In the latter instance more than one class of lymphocytes must be present in order for maximum cytotoxic activity to be expressed.     

High-Throughput Amplicon Sequencing Reveals Distinct Communities within a Corroding Concrete Sewer System

Microbially induced concrete corrosion (MICC) is an important problem in sewers. Here, small-subunit (SSU) rRNA gene amplicon pyrosequencing was used to characterize MICC communities. Microbial community composition differed between wall- and ceiling-associated MICC layers. Acidithiobacillus spp. were present at low abundances, and the communities were dominated by other sulfur-oxidizing-associated lineages.     

荧光标记复合PCR扩增微卫星位点的构建与优化

本研究采用毛细管电泳技术,构建并优化了荧光标记复合PCR同时扩增多个微卫星位点。主要过程为：首先根据设计所扩增微卫星位点的期望长度,将9个微卫星位点分成两组,5个位点用FAM（蓝色）标记,4个位点用HEX（绿色）标记;两种荧光类型分组优化,用琼脂糖胶电泳检测。其次,荧光标记的复合PCR扩增8个中华绒螯蟹样品的9个微卫星位点,采用ABI3730xl毛细管电泳检测,以ROX500（红色）为长度标准物,结果经Genemapper3.5软件 分析,检测结果表明毛细管电泳检测荧光标记复合PCR产物不仅精确读取微卫星位点的长度（分辨率高达1bp）,还能区分微卫星位点复制时滑链所引起的“回声斑”;调整各微卫星位点引物比列使所有位点扩增强弱均匀。最后,逐一检测复合PCR基本参数（dNTP浓度、 PCR程序和模版DNA用量）对复合PCR产物的影响,优化PCR。结果表明通过毛细管电泳检测荧光标记复合PCR产物来读取微卫星位点的基因型具有精确性、高效性和稳定性。     

Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.     

Mitogen induced cellular cytotoxic activity as a monocyte function in human peripheral blood preparations

The effector cell population in man responsible for mitogen induced cellular cytotoxicity (MICC) of chicken erythrocytes was investigated using several separation techniques, including free flow electrophoresis. Electrophoresis produced substantial monocyte enrichment in some fractions with substantial depletion in others. MICC activity was seen to correlate with monocyte content in these fractions. Furthermore, removal of phagocytic cells with carbonyl iron and removal of adherent cells on plastic petri dishes depleted preparations of MICC activity. Thus it is suggested that under conditions described in this paper, the effector cell of the MICC assay in man appears to be a monocyte. This MICC effector cell was shown to be different from the effector cell in the natural killing (NK cells) of RPMI 6410 cells.     

The nature of the effector cells mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC).

The nature of the cell types capable of mediating mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) was investigated utilizing effector cells from athymic nude and euthymic heterozygous control littermate mice as well as Sephadex anti-Fab immunoabsorbent column purified spleen cell populations from normal (CS7BL/6) mice. Chicken erythrocytes (CRBC) and the mouse lymphoma, EL-4, were used as target cells in both cytotoxicity assays. MICC utilizing CRBC targets was mediated by several effector cell types whereas MICC utilizing EL-4 lymphoma targets was T-cell dependent. ADCC against both CRBC and EL-4 lymphoma targets occurred independently of the presence of T-cells. In addition, effector cell populations incapable of mediating MICC against EL-4 lymphoma targets were capable of mediating ADCC against the same EL-4 targets. Thus, utilizing the appropriate target cells, EL-4 but not CRBC, a sharp distinction can be made between the effectors for ADCC and MICC: ADCC is T-cell independent while MICC is dependent on the presence of mature thymus-derived cells. Furthermore these studies demonstrate that the nature of the target cell employed in MICC and ADCC reactions plays a critical role in defining the types of effector cells capable of mediating these cytotoxicity reactions.     

Polydopamine assisted fabrication of titanium oxide nanoparticles modified column for proteins separation by capillary electrochromatography

Development of a simple method for preparation of stable open tubular (OT) columns for proteins separation by capillary electrochromatography is still challenging. In this work, the titanium oxide (TiO₂) nanoparticles coated OT column was successfully prepared for separation of proteins by capillary electrochromatography. The polydopamine (PDA) film was first formed in the inner surface of a fused-silica capillary by the self-polymerization of dopamine under alkaline conditions. Then the TiO₂ coating was deposited onto the surface of pre-modified capillary with PDA by a liquid phase deposition process. The plentifully active hydroxyl groups in PDA coating can chelate with Ti⁴⁺ to boost the nucleation and growth of TiO₂ film. The as-prepared TiO₂ coated OT column was characterized by scanning electron microscopy and measurement of electroosmotic flow. Furthermore, the influence of liquid phase deposition time on the TiO₂ coating was investigated. The TiO₂ coated OT column was used for successful separation of two variants of β-lactoglobulin and eight glycoisoforms of ovalbumin. The column demonstrated good repeatability and stability. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.7%. Moreover, the application of the column was verified by successful separation of acidic proteins in egg white.     

Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

Evaluation of four capillary columns for the analysis of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers in human serum for epidemiologic studies

The separation of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether (PBDE) congeners was evaluated on four capillary columns: 60 m x 0.25 mm i.d., 0.25 microm film thickness RTX-5MS and DB-XLB capillary columns, and 60 m x 0.18 mm i.d., 0.25 microm film thickness DB-XLB and DB-5MS capillary columns. Based on performance, capacity, and cost, the RTX-5MS (60 m x 0.25 mm i.d., 0.25 microm thickness) and the DB-XLB (60 m x 0.25 mm i.d., 0.25 microm film thickness) were selected for the analysis of human serum extracts by using gas chromatography/electron-capture detection. In contrast to previous studies, the oven temperature program affords the separation of congeners that are not separated by using other combinations of capillary columns, most notably PBDE-47 and PCB 180. In addition, the method enables determination of OCPs, PCBs, and PBDEs prevalent in a single extract of serum, which can lead to considerable time savings in the analysis of large number of samples collected for epidemiologic studies.     

A gold nanoparticle-mediated enzyme bioreactor for inhibitor screening by capillary electrophoresis

A facile protocol to prepare highly effective and durable in-line enzyme bioreactors inside capillary electrophoresis (CE) columns was developed. To demonstrate the methodology, l-glutamic dehydrogenase (GLDH) was selected as the model enzyme. GLDH was first immobilized onto 38-nm-diameter gold nanoparticles (GNPs), and the functionalized GNPs were then assembled on the inner wall at the inlet end of the CE capillary treated with polyethyleneimine (PEI), producing an in-line GLDH bioreactor. Compared with a GLDH bioreactor prepared by immobilizing GLDH directly on PEI-treated capillary, the GNP-mediated bioreactor showed a higher enzymatic activity and a much better stability. The in-capillary enzyme bioreactor was proven to be very useful for screening of GLDH inhibitors deploying the GLDH-catalyzed α-ketoglutaric acid reaction. The screening assay was preliminarily validated by using a known GLDH inhibitor, namely perphenazine. A Z′ factor value of 0.95 (n = 10) was obtained, indicating that the screening results were highly reliable. Screening of GLDH inhibitors present in medicinal plant extracts by the proposed method was demonstrated. The inhibition percentages were found to be 53% for Radix scutellariae, 45% for Radix codonopsis, 37% for Radix paeoniae alba, and 0% for the other 22 extracts tested at a concentration of 0.6 mg extract/ml.