Positive Selection Techniques Research Articles

Impact of positive selection technology on seed yam productivity

Positive Selection (PS) technique has been shown to reduce virus infection and increase yields, however there is insufficient empirical evidence on how this technology affects seed yam farm productivity. This study employed Propensity Score Matching (PSM) technique to evaluate the impact of PS on seed yam yields of 368 farmers randomly selected from Ghana and Nigeria.The findings showed that educational attainment, distance from the farm to the nearest market, cropping patterns, and other factors influenced farmers' adoption of PS. Furthermore, the adoption of PS technology resulted in a 16.98 % boost in farm productivity for PS seed yam farmers compared to their productivity without the technology. It is of the utmost importance that PS adoption be supported by developing tailored training materials for farmers to improve their use of the PS technology.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited or acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcomes of infected individuals. This article describes reproducible density-gradient-centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver, or the spleen. Mouse neutrophils isolated by these protocols can be used to examine several aspects of cellular function ex vivo, including pathogen binding, phagocytosis, and killing, neutrophil chemotaxis, oxidative burst, degranulation, and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow Using Positive Immunomagnetic Separation Alternate Protocol 1: Purification of Neutrophils from Bone Marrow Using Negative Immunomagnetic Separation Alternate Protocol 2: Purification of Neutrophils from Bone Marrow Using Histopaque-Based Density Gradient Centrifugation Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues Using Positive Immunomagnetic Separation Alternate Protocol 3: Isolation of Neutrophils from Mouse Tissues Using FACS.

Production and availability of good quality seed potatoes in the East African region: A review

Demand for potatoes has grown rapidly in eastern Africa due to rapid population growth, urbanisation and life style changes. This has made potato an important commodity crop in the region contributing to food security and enhanced livelihoods. However, potato productivity in the region is low; ranging from 6 to10 t/ha against potential yields of over 30 t/ha. One of the major causes of low yields is the chronic shortage of good quality seed potatoes for planting, which also limits adoption of new and improved varieties. Therefore, the objective of this paper is to highlight the opportunities and progresses in the production and availability of good quality seed potatoes in the East African region to bolster the productivity and commercial value of the crop. The first section of the paper discusses the status of good quality seed potatoes production and availability in the East African region. The second section looks at the farmer-based seed potato production technologies while the third section focuses on the efforts of both the private and public sector in production of good quality seed potatoes. Availability of good quality seed potatoes constitutes < 1% of the requirement in eastern Africa. These seeds are mainly produced by public research institutions as well as some private players. Consequently, most farmers opt to plant seed potatoes from informal sources. However, seeds from informal sources are mostly of poor quality and often accelerate the spread of tuber-borne diseases, especially viruses and bacterial wilt. To improve seeds from informal system, some technologies have been identified and promoted to enhance farmer-based seed production and distribution. These include seed plot technology (SPT), positive selection techniques (PST) and Quality Declared Seed (QDS) systems. In addition, there has been adoption and promotion of rapid multiplication techniques (RMT) such as stem/shoot cuttings, aeroponics, sandponics, and hydroponics among others. Other measures that have been taken include importing seeds of new potato varieties. The imports increase the quantity of seeds and the number of potato varieties available to farmers. Despite these efforts, the quantity of good quality seeds available is still far below the requirements. There is need for the governments, private sector players as well as farmers to enhance their efforts and save the situation. Governments need to invest seriously in local breeding activities and multiplication of early generation seeds. These and other measures may increase availability of good quality and affordable seed potatoes in East African region.

Profitability of positive selection technique for seed yam production in Ghana and Nigeria

Profitability of positive selection technique for seed yam production in Ghana and Nigeria

Use of positive selection technique to manage yam viruses in seed yam production by Ghanaian farmers

Positive selection technique (PST) involves physical examination of plants at vegetative stage for viral disease symptoms and using tubers from apparently symptomless plants as planting materials in seed yam production. This study determined PST use among farmers in Atebubu-Amantin and Ejura-Sekyedumase Municipalities of Ghana. Viral diseases were observed on 63 PST and 63 non-PST farms within these Municipalities. PST farms (two in third season and two starters) were selected to assess the impact of continuous PST practice on plant establishment, disease incidence, severity and yield. Harvested seed yams from asymptomatic and symptomatic plants were sampled for virus detections using Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA). Viral disease incidence (35.70%) and severity (1.51) observed on PST farms were significantly lower than non-PST farms with 54.11% and 2.80, respectively (p< 0.05). PST farms in the third season had higher plant establishment (66 plants/30 m2) and yield (48.32 kg/30 m2) with lower disease incidence (28%) and severity (1.50) compared to PST starters which respectively were 60 plants/30 m2, 33.14 kg/30 m2, 52% and 2.3 (p< 0.05). DAS-ELISA showed that, 85% of harvested seed yams from symptomatic plants were infected with YMV, CMV, or YMMV as against 35% seed yams from asymptomatic plants. YMV was highly accumulated in seed yams from symptomatic plants (0.344) than those from asymptomatic plants (0.269). The current findings show that PST if practised by farmers could minimise viral diseases in seed yams. However, poor observation of viral symptoms by farmers and latent viral infection of plants could be limitations which need to be considered in further studies. Key words: Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA), latent viral infection, virus accumulation, yam viruses.

Participatory Improved Potato Technology Promotion: The Case of Smallholder Farmers in North, West and Southwestern Highlands of Ethiopia

Utilization of healthy planting material is a key factor to improve potato yields to reduce the dissemination of diseases and pests. Quality seed is one of the major bottlenecks hindering the production and productivity of potato in major potato growing regions of the country. Therefore, participatory on-farm seed production and improved potato technologies promotion have been carried out during the year 2014-2016 at five districts, Jeldu, Dendi, Wolmera Degem and Kersana Malima of central Ethiopia. To produce quality potato seed various rapid multiplication techniques were used. Thus, improved potato technologies were demonstrated using different methodologies such as the farmers’ field schools, the farmers' research group and field days. To supply clean tubers about 138,886 high-quality mini-tubers of improved varieties of Belete, Gudanie, Jalenie and Awash were produced. Moreover, a total of 257.08 tons of seed tubers of 15 released potato varieties were produced and disseminated in different parts of the country for research and developments. The participant farmers produce 442.9 tons of relatively clean seed tubers and sold to different parts of the country. During, this activity positive selection technique was demonstrated and participant farmers applied the techniques that helped them to improve the yield and quality of seed potato. Farmers obtained a 40% yield increase and a 33% vigour improvement when positive selection techniques were applied. Training on improved potato production and postharvest handling technologies have also been given for farmers, development agents, agricultural experts and home agents. Therefore, decentralized quality seed production reduces the movement of dangerous diseases like bacterial wilt and viruses from invading areas with high potential for seed production due to purchase of seed only from within one’s same region.

Isolation and Characterization of a CD34+ Sub-Clone in B-Cell Lymphoma

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy in the US. Many types remain incurable despite response to initial therapy and achievement of complete remission (CR). Advanced laboratory techniques like multicolor flow cytometry (MCF) and polymerase chain reaction (PCR) have demonstrated persistence of rare malignant cell population post therapy referred to as minimal residual disease (MRD). However, the functional and biological characteristics of this population have not been fully elucidated. Established B-lymphoma cell lines (B-NHL) and patient-derived samples (PDS) were analyzed using 8-color FCM of leukemia and lymphoma antibody panels (28 antibodies). The CD34+ sub-population was enriched using in vitro exposure to 2-chlorodeoxyadenosine (2-CdA), and a CD34-coated magnetic beads isolation procedure (Miltenyi Biotech). Genetic analysis of CD34+ and CD34-/parent cell fractions was done by karyotyping, and by chromosomal microarray (CMA) using the oligonucleotide-single nucleotide polymorphism (Oligo-SNP), whole genome Agilent 180K GGXChip+SNP (Agilent Technologies, Inc). Sensitivity to chemotherapy was assayed by short-term in vitro exposure to drugs. Clonogenicity was determined by soft agar colony formation assay, and proliferation was determined using DNA staining with propidium iodide and flow cytometry. The side population was determined using the fluorescent vital dye Hoechst 33342 and flow cytometry. Analysis of three B-NHL cell lines revealed the presence of a minute sub-clone (&lt;1%) of monotypic B-cells that expresses CD34. This sub-population enriched several folds in response to exposure of the cells to 2-CdA. Enrichment was highest in the follicular lymphoma cell line, WSU-FSCCL reaching more than 50 folds compared with control (13.2% vs 0.26%), followed by WSU-WM, 10-fold (2.36% vs 0.23%), and least in WSU-DLCL2 (1.25% vs 0.71%). Using a CD34 microbead positive selection technique, we were able to enrich the CD34+ population up to 80% purity. Western blots confirmed higher expression of CD34 protein in the enriched population compared with parent cells. Using StemPro media (Gibco by Life Technologies), CD34+ cell fraction of WSU-WM showed more sustained growth and viability in culture over 9-day period compared with parent cells. This technique allowed the generation of large number of CD34+ cells for further characterization. Except for CD34 expression, this cell fraction expressed identical phenotype, karyotype, SNP and MCA profile to parent cells. However, the CD34+ fraction was more proliferative with an increase in S-phase cells (~60% vs 20% in control cells) and Hoechst 33342-positive cells (~40%). These cells were clonogenic even in presence of the cytotoxic agents Doxorubicin and 2-CdA that completely inhibited colony formation in parent cells. Resistance to chemotherapy was also noticed in liquid culture. A CD34+ population within clonal B-cells was also detected in 8 of 8 PDS. The lowest number of CD34+ cells was detected in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and the highest (5.35%) was seen in Richter's transformation of CLL. The isolated CD34+ cells in this study show many features of what is known as Lymphoma Stem Cells implicated in resistance to therapy and disease recurrence. Its relatedness to MRD cells is uncertain, however, since CD34 is not routinely included in MRD antibody panel. The observation that CD34+ cells are genetically identical to parent cells suggests an epigenetic modification. This conclusion is supported by our finding that Ehd3 expression is dramatically reduced in the CD34+ cell population of WSU-DLCL2 compared with parent cells. Ehd3 was recently identified as an epigenetically-silenced gene in solid tumors and in hematological malignancies including leukemia and lymphoma. The Ehd3-encoded protein belongs to the family of C-terminal Eps15 homology domain-containing (EHD) proteins, implicated in intracellular trafficking of tubular recycling endosomes (TRE), apoptosis, and cell cycle arrest. Further studies are underway to determine the contribution of Ehd3 to the biological and functional characteristics of the CD34+ cells in B-NHL. Utilizing the CD34+ monotypic B-cells in B-NHL as a model for screening new therapeutic agents and to identify potential new therapeutic strategies may lead to a more effective treatment of B-NHL. Disclosures No relevant conflicts of interest to declare.

Self-promotion of TV Stations from a Rhetorical Perspective. Persuasion Techniques

This paper discusses the persuasive techniques employed by media broadcasters in the self-promotional activities of television stations. A persuasive technique is understood as a specific manner of conduct of a broadcaster in the process of communication in which in order to increase their impact, a broadcaster emphasises or modifies some elements of the process. The starting point is the assumption that the selection of the persuasive techniques is related to such rhetoric determiners as the context of the operations of the media (consequences of technological changes: unification of media products, overwhelming increase in the amount of information, and ever growing global competition), the intended recipient, and the aim a broadcaster intends to achieve. The research material enabled the identification of the persuasive techniques in the self-promotional activities of media broadcasters emoployed at three levels: (1) mode of conveying information (e.g. repeating, and inherited audience); (2) content (e.g. positive selection technique, fragmentation, utilisation of the inaccessibility principle, emphasising the benefits of interacting with a medium, increasing the attractiveness of a communication by referring to broadly understood authority figures); and (3) form of communication (the technique of uniqueness).

Family profile mining in retailing

In the era of personalized marketing, the ability to leverage data analysis to recommend tailored products is a key competitive advantage for retailers. Family profiles are an essential aspect of customer information to boost the performance of knowledge-based recommendations. This study integrates positive and unlabeled learning and feature selection techniques to design a novel and flexible family profiling algorithm that tags target customers based on unlabeled transaction data. The empirical evaluation shows that our algorithm outperforms other algorithms in terms of the recall rate of families of the target tag. The knowledge of inferred family profiles also enhances the product recommendation performance. Our algorithm can help retailers efficiently target customers based on family profiles for better marketing performance.

Classification Software Engineering Documents Based on Hybrid Model

Care about automated documents classification has increased since the appearance of the digital documents and the wide diffusion of Internet. In the 1990's, the computer performance has greatly improved and has led to the methods of machine learning to establish automated classifiers. These methods have achieved good speed and classification's accuracy and researchers still investigate in this field to accomplish more accuracy and less time. Artificial immunologic systems have shown high performance in such as data clustering and anomaly detection which can be ascribed to the nature of the immunologic system in protecting the body. Some of the present methods and ways used in the training process of the document classification are time consuming and others have less accuracy rate concerned with the classification of the related document as software engineering document classes. For these reasons, this research deals with the study of Natural Immune System and using the dynamic process of the Adaptive Immune System work by hybridization Negative Selection (NS) and Positive Selection (PS) techniques and to propose a hybrid model called the Hybrid Positive Negative Selection Model (HPNS) to classify Software Engineering documents as they comprise information related to developing the software systems, that makes it easy for the software engineer who works in maintenance. HPNS has high classification's speed and accuracy besides easy and flexible use by designing interfaces that make it easy for the user to deal with the system. In order to improve the quality and the efficiency of HPNS method, it was compared to one of the best and well-known methods of classification referred to as, Naive Bayes(NB). After conducting several experiments on a various group of software engineering documents, evaluations results have shown that the accuracy of the Adaptive immunologic method (HPNS) has reached (HPNS) (95%), whereas Naïve classification method has reached (90 %) with training and classification speed that doesn’t exceed one minute. This shows the feasibility of using the algorithms of AIS systems in the field of information recovery and documents classification. This system was built and programmed in Java language and was implemented under an operating system environment Microsoft Windows7.

Nanoroughened adhesion-based capture of circulating tumor cells with heterogeneous expression and metastatic characteristics.

BackgroundCirculating tumor cells (CTCs) have shown prognostic relevance in many cancer types. However, the majority of current CTC capture methods rely on positive selection techniques that require a priori knowledge about the surface protein expression of disseminated CTCs, which are known to be a dynamic population.MethodsWe developed a microfluidic CTC capture chip that incorporated a nanoroughened glass substrate for capturing CTCs from blood samples. Our CTC capture chip utilized the differential adhesion preference of cancer cells to nanoroughened etched glass surfaces as compared to normal blood cells and thus did not depend on the physical size or surface protein expression of CTCs.ResultsThe microfluidic CTC capture chip was able to achieve a superior capture yield for both epithelial cell adhesion molecule positive (EpCAM+) and EpCAM- cancer cells in blood samples. Additionally, the microfluidic CTC chip captured CTCs undergoing transforming growth factor beta-induced epithelial-to-mesenchymal transition (TGF-β-induced EMT) with dynamically down-regulated EpCAM expression. In a mouse model of human breast cancer using EpCAM positive and negative cell lines, the number of CTCs captured correlated positively with the size of the primary tumor and was independent of their EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with detectable primary tumors independent of the cell lines’ metastatic ability.ConclusionsThe microfluidic CTC capture chip using a novel nanoroughened glass substrate is broadly applicable to capturing heterogeneous CTC populations of clinical interest independent of their surface marker expression and metastatic propensity. We were able to capture CTCs from a non-metastatic lung cancer model, demonstrating the potential of the chip to collect the entirety of CTC populations including subgroups of distinct biological and phenotypical properties. Further exploration of the biological potential of metastatic and presumably non-metastatic CTCs captured using the microfluidic chip will yield insights into their relevant differences and their effects on tumor progression and cancer outcomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2638-x) contains supplementary material, which is available to authorized users.

Abstract 4109: Unbiased detection of disseminated tumor cells in murine bone marrow

Abstract Approximately 20-30% of prostate cancer patients develop disease recurrence and metastasis years after initial therapy. This is thought to be largely due to the presence of growth-arrested and chemoresistant disseminated tumor cells (DTCs) in secondary sites, such as bone. Bone metastasis is found in 90-100% of prostate cancer patients who succumb to the disease. There are still many gaps in knowledge about the biological mechanisms by which DTCs home to bone, resist chemotherapy, become dormant, and escape dormancy to grow into clinical metastases. As such, it is important to be able to detect, quantify, and study bone marrow DTCs. In particular, it must be possible to do this in metastatic cancer mouse models, which are critical to study the process of tumor dissemination. DTC detection techniques currently exist, usually as either a positive selection or negative selection methodology. Positive selection techniques use markers or cell size to isolate and purify tumor cells out of the bone marrow. Positive selection markers are generally epithelial-specific, such as EpCam, E-cadherin, or Cytokeratin, and therefore may miss cells that lose epithelial marker expression and may gain mesenchymal markers. DTCs can also be as small as or smaller than white blood cells, meaning that positive selection based on size may miss some DTCs. Negative selection enriches for DTCs by removing blood and bone marrow cells from the population, usually using cell-specific markers. A popular strategy is CD45-based depletion, which removes white blood cells, and theoretically leaves behind DTCs. In our hands, this strategy causes loss of DTCs in the depletion process. To capture these heterogeneous and rare DTCs, we have developed a strategy to detect DTCs in murine bone marrow in an un-biased manner. The procedure entails removal of the bone marrow via centrifugation from the long bones (femur and tibia) of mice that have been injected with cancer cells (the injection site may vary depending on the experimental setup). The bone marrow then undergoes red blood cell lysis, and further centrifugation. The white blood cells are then counted, and the bone marrow is spread onto glass slides. The cells on the slide are fixed, permeabilized, and stained (immunofluorescence and RNA fluorescent in situ hybridization can be used). The staining can include any type of marker, including epithelial, mesenchymal, disease-specific, species-specific, or other biologically interesting markers, such as cell cycle markers. The unbiased nature of this procedure is based on the lack of positive or negative selection based on cell size or protein expression. Some DTC loss is noted in this protocol, due to the centrifugation and staining steps, but the cell population on the slide should include all DTC types. Notably, this protocol can be used to detect human or mouse cells in the mouse bone marrow and can thus be used in immune-compromise and immune-competent mouse models of metastasis. Citation Format: Kenneth C. Valkenburg, James R. Hernandez, Sarah R. Amend, James E. Verdone, Michael A. Gorin, Kenneth J. Pienta. Unbiased detection of disseminated tumor cells in murine bone marrow. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4109.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments.

Identification of Genes Preferentially Expressed by Highly Virulent Piscine Streptococcus agalactiae upon Interaction with Macrophages

Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood–brain barrier (BBB). The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS) was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB.

CK- and small nuclear size circulating tumor cell (CTCs) phenotypes in metastatic castration-resistant prostate cancer (mCRPC).

209 Background: Circulating tumor cell (CTCs) are traditionally defined as EpCAM/CK+ cells, CD45-, and morphologically distinct. However, recent evidence suggests that other populations of CTC candidates exist including cells that are EpCAM/CK- or smaller in size than traditional CTCs. CTC positive selection techniques that isolate CTCs based on size, density, or EpCam positivity may miss subpopulations. We aimed to molecularly characterize novel CTC candidates utilizing the Epic Sciences platform, which performs no physical selection. Methods: Blood from 10 healthy volunteers (HV) and 39 patients (pts) with metastatic castration-resistant prostate cancer (mCRPC) were collected and shipped to Epic Sciences, where all nucleated cells were plated onto glass slides and subjected to immunofluorescence (IF) staining and CTC identification by fluorescent scanners and algorithms. Traditional CTCs were identified as CK+CD45- cells with intact DAPI nuclei and after pathologist review of their morphology. Candidate CK- CTC populations were identified as CK-CD45- that were morphologically malignant. Small nuclear size candidate CTCs were identified as CK+CD45- cells with diameters similar to or smaller than that of a typical white blood cells (WBCs). All candidate CTC were evaluated with prostate cancer relevant biomarkers, including androgen receptor (AR) by IF and PTEN loss and ERG rearrangements by FISH. Results: Thrity eight out of 39 pts had one or more traditional CTCs/mL and 36 out of 39 pts had one or more CK- CTCs/mL of blood. Eight out of 39 samples had more than 10 CK- CTCs/mL. Fifteen out of 39 samples had evidence of small nuclear size CTCs at varying incidences and sizes of cells. We observed PCa biomarkers including high AR expression, PTEN deletion, and ERG rearrangements in both CK- and small nuclear size CTCs. These features were not observed in over 1,000 WBCs evaluated and HV. Conclusions: Candidate CTC that are CK-CD45- and/or with a small nucleus are identified on the Epic Sciences platform in blood from mCRPC pts. Studies are ongoing to determine their clinical relevance.

Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.

Isolation of Tissue Mast Cells

Located primarily in tissues, mast cells are one of the principal effector cells in allergic inflammation. Mast cells derive from mononuclear precursor cells which undergo their final phase of differentiation in the tissues. Mast cells express a unique set of proteases and display functional diversity depending on the tissue in which they differentiate--a phenomenon often referred to as mast cell heterogeneity. Enzymatic digestion and density centrifugation have often been used to isolate human mast cells from tissues such as lung and skin, frequently resulting in cells with low viability and purity. Here, we describe a protocol that combines gentle enzymatic digestion with positive selection techniques to isolate reasonably viable and substantially enriched preparations of tissue mast cells.

Embryonic stem cell—derived astrocytes expressing drug-inducible transgenes: differentiation and transplantion into the mouse brain

Embryonic stem cell (ESC)-derived astrocytes have many theoretical and practical advantages as vectors for delivery of gene therapy to the central nervous system (CNS). The aim of this study was to generate highly pure populations of ESC-derived astrocytes expressing drug-inducible transgenes, while minimizing contamination by undifferentiated ESCs Embryonic stem cells carrying a doxycycline-inducible green fluorescent protein (GFP) transgene were induced to differentiate into astrocytes by using feeder cell-free conditions that are completely defined. More than 95% of these cells expressed the astrocyte markers glial fibrillary acidic protein and GLT-1 glutamate transporter, and the morphological characteristics of these cells were typical of astrocytes. The expression of additional astrocyte markers was detected using reverse transcription-polymerase chain reaction. Undifferentiated ESCs comprised fewer than 0.1% of the cells after 10 days in this culture. Positive and negative selection techniques based on fluorescence-activated cell sorting were successfully used to decrease further the numbers of undifferentiated ESCs. Fully differentiated astrocytes expressed a GFP transgene under the tight control of a doxycycline-responsive promoter, and maintained their astrocytic phenotype 24 hours after transplantation into the mouse brain. This study shows that transgenic ESCs can be induced to differentiate into highly pure populations of astrocytes. The astrocytes continue to express the transgene under the tight control of a drug-inducible promoter and are suitable for transplantation into the mouse brain. The number of potentially hazardous ESCs can be minimized using cell-sorting techniques. This strategy may be used to generate cellular vectors for delivering gene therapy to the CNS.

Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS).

Salmonella enterica serovar Typhi (S. typhi) is a human-restricted pathogen which causes typhoid fever. Relatively little is known about S. typhi host interaction as animal models of this disease are severely limited by the lack of virulence of S. typhi in other hosts. The virulence of other Salmonella serovars in animal models is dependent on the abilities of these bacteria to survive within host macrophages. We have used selective capture of transcribed sequences (SCOTS) to identify S. typhi genes expressed during growth in human macrophages. This positive cDNA selection technique identified 28 distinct clones representing previously identified as well as novel, uncharacterized and hypothetical gene sequences that are expressed intracellularly. Transcripts for the Vi capsular antigen and genes whose products are involved in stress responses and nutrient acquisition were obtained from intracellular bacteria using SCOTS. Most of these clones are present in the S. typhimurium genome and are also expressed in murine macrophages. Nineteen of these gene sequences were disrupted insertionally in S. typhi, and most of the resulting mutants exhibited a lower level of survival within macrophages compared with the wild-type parent strain. Mutant strains, transformed with a copy of a wild-type gene, exhibited a macrophage survival level similar to that of the parental strain.

Purification of Hematopoietic Stem Cells for Further Biological Study

For many years, the hematopoietic system has provided a convenient and fascinating model for studies of the molecular processes regulating cell growth and differentiation. However, this system also poses considerable challenges because the most primitive “stem” cells as well as their initial differentiating progeny are normally present in hematopoietic tissues at extremely low frequencies and no unique, stable phenotype has yet been identified to allow hematopoietic cells with specific stem and progenitor functions to be measured directly. Rather, this requires the use of functional assays that detect their developmental properties and take several weeks to complete. Accordingly, many investigations of primitive hematopoietic cell behavior and their responses to molecular cues in the environment have relied on the development of cell separation techniques specifically designed for obtaining highly enriched populations of primitive hematopoietic cells. Key to these procedures is the use of a preenrichment step(s) in which differences in cell density, size, or sensitivity to pharmacological agents or surface phenotype are exploited to first “debulk” the sample. This step can then be followed by a more selective antibody-mediated procedure to generate useful numbers of highly purified cells. Batchwise immunoadsorption techniques offer many advantages for obtaining enriched populations of hematopoietic progenitors because they avoid the nonspecific toxicity seen with antibody-mediated cell killing and are suitable for rapidly processing large samples. For any cell separation procedure, a balance must be struck between the purity and the recovery of the desired cells because steps to increase cell purity usually reduce yields. Both the negative and the positive selection techniques are useful strategies but negative selection usually requires one less manipulation step and circumvents potential effects incurred by the presence of antibody on the surface of the cell being isolated. Specific details for the use and results obtained with an immunomagnetic negative column selection technique are then presented.