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Immunomagnetic Separation (immunomagnetic + separation)
Kinds of Immunomagnetic Separation Selected AbstractsA Rapid Method for the Pre-Enrichment and Detection of Salmonella Typhimurium by Immunomagnetic Separation and Subsequent Fluorescence Microscopical TechniquesENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2005J. Steingroewer Abstract Detection of food-borne pathogens is of great importance in order to minimize the risk of infection for customers. These analyses should be as fast as possible. Any detection method requires enrichment and quantitative analysis of the enriched microbes. Conventional enrichment methods, which take several days, need to be replaced by faster techniques such as immunomagnetic separation (IMS). This technique is based on the use of paramagnetic microspheres coated with antibodies as ligands that have specific affinity to the microbes that have to be detected. In the studies reported here, a rapid method for the detection of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium), combining IMS and Direct Epifluorescence Filter Technique (DEFT), was developed. It was focused on releasing the target cells from the magnetic beads after IMS, because this is a premise for combining IMS, as an alternative pre-enrichment, with DEFT. Otherwise, the high number of beads form a layer on the filter membrane that makes the following microscopic analysis for the detection of the contaminants impossible. The CELLectionTM Dynabeads® used in this study, are coated with recombinant streptavidin (rSA) via a DNA linker. The rSA binds biotinylated antibodies that are able to capture target cells. The DNA linker provides the cleavable site, so that the beads can be removed from the captured cells after isolation. In this study a releasing procedure was developed. This procedure allows for an average 74,% ± 4,% of the bead-bound Salmonella Typhimurium cells to be released from the beads after IMS, so that the detection of the separated cells by DEFT will be possible. [source] Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniaeFEMS MICROBIOLOGY LETTERS, Issue 1 2009Wang Yang Abstract Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae. Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (,2 test, P<0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection. [source] Detection of Cryptosporidium parvum in lettuceINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 4 2007John E. Moore Summary Human cryptosporidiosis has emerged as an important gastrointestinal infection in the 1990s as a result of the ingestion of mainly contaminated water and to a lesser extent foodstuffs containing the protozoan parasite, Cryptosporidium parvum. This pathogen has particular clinical significance for immunocompromised persons, including AIDS patients and cancer patients receiving toxic chemotherapeutic drug regimens. There have been a limited number of studies performed examining the occurrence of the parasite on vegetables, including lettuce. Detection rates are very dependent on the laboratory isolation technique employed and has ranged from 1.2% to 14.5%. Current best practice of laboratory recovery, isolation and detection methods include detergent removal, oocysts concentration by immunomagnetic separation, followed by a combination of immunofluorescent microscopy and a nested PCR approach. Employment of contaminated non-potable water in the production of vegetables, particularly lettuce, may represent an important potential source of entry of pathogens into food processing and the human food chain. Given that lettuce is an important constituent of hamburger dressing, and the size of the fast-food industry, where lettuce is an important constituent, horticultural producers of lettuce should therefore place special emphasis on developing suitable and efficient Hazard Analysis Critical Control Point strategies for the critical control of oocysts depending on the type of unit operation employed and vegetable being processed. This review aims to examine (i) the incidence of C. parvum in vegetables, particularly lettuce and (ii) laboratory detection methods for the isolation and identification of this parasite from lettuce. [source] Diversity of Escherichia coli O157 in a longitudinal farm study using multiple-locus variable-number tandem-repeat analysisJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2008A.M. Urdahl Abstract Aims:, To perform a longitudinal study of the diversity of Escherichia coli O157 from a ruminant pasture/stream environment using multiple-locus variable-number tandem-repeat analysis (MLVA). Methods and Results:, Samples of faecal droppings from grazing ruminants and from an adjacent stream were tested longitudinally for E. coli O157 by enrichment and immunomagnetic separation (IMS). Using MLVA, 24 different profiles were identified from a total of 231 E. coli O157 isolates, of which 80 were included in a similarity analysis. Four main clusters with several subclusters were observed. Although there was close contact between sheep and cattle during the study period, E. coli O157 was surprisingly not detected from cattle faeces. Conclusions:, The cluster analysis indicated both unrelated and closely related E. coli O157 strains. The choice of loci to target in MLVA is important for the subtyping result, as loci with high diversities are essential for discriminating between closely related isolates. Significance and Impact of the Study:, There is a lack of data available on the use of MLVA to describe E. coli O157 diversity and changes over time in the animal reservoirs and the environment. Such data are needed in order to further develop MLVA as a typing method. [source] Occurrence of Cryptosporidium spp. oocysts in raw and treated sewage and river water in north-eastern SpainJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2005M. Montemayor Abstract Aims:, To determine the occurrence and levels of Cryptosporidium parvum oocysts in wastewater and surface waters in north-eastern Spain. Methods and Results:, Samples from five sewage treatment plants were taken monthly and quarterly during 2003. In addition, water was collected monthly from the River Llobregat (NE Spain) during the period from 2001 to 2003. All samples were analysed by filtration on cellulose acetate filters or through EnvirocheckTM using EPA method 1623, followed by immunomagnetic separation and examination by laser scanning cytometry. All raw sewage, secondary effluent and river water samples tested were positive for Cryptosporidium oocysts. Of the tertiary sewage effluents tested, 71% were positive for Cryptosporidium oocysts. The proportion of viable oocysts varied according to the sample. Conclusions:, Two clear maxima were observed during spring and autumn in raw sewage, showing a seasonal distribution and a correlation with the number of cryptosporidiosis cases and rainfall events. Significance and Impact of the Study:, This study provides the first data on the occurrence of Cryptosporidium oocysts in natural waters in north-eastern Spain. [source] Quantification and prevalence of Salmonella in beef cattle presenting at slaughterJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2004N. Fegan Abstract Aims:, A survey to determine the prevalence and numbers of Salmonella in beef cattle presented for slaughter at abattoirs across Australia was conducted between September 2002 and January 2003. Methods and Results:, Automated immunomagnetic separation (AIMS) was used for detection and isolation of Salmonella enriched from cattle faeces. Salmonella were enumerated from positive samples using a combination of the Most Probable Number (MPN) technique and AIMS. A total of 310 faecal samples were tested, 155 were from lot-fed cattle and 155 from grass-fed cattle. Salmonella spp. were isolated from 21 (6·8%) of the cattle and the prevalence amongst grass-fed cattle (4·5%) was not significantly different to that found in lot-fed cattle (9%). Counts of Salmonella in positive faeces varied from <3 MPN g,1 of faeces to 2·8 × 103 MPN g,1 and 71% of positive samples had counts <10 MPN g,1 faeces. There was no significant difference in the mean log10 number of Salmonella in faeces of cattle from each production system. Conclusion:, Low numbers of beef cattle were found to shed Salmonella at the time of slaughter and the prevalence and the associated faecal concentrations did not vary significantly with the pre-slaughter production system (grass or lot feeding). The faecal concentration of Salmonella in the majority of faeces was low (<10 MPN g,1) with few high concentrations up to 3 × 103 MPN g,1, suggesting there may be a low risk of carcase contamination. Significance and Impact of the Study:, Beef cattle do not appear to be a major source of entry of Salmonella into the human food chain and the quantitative information contained in this study can be used in quantitative assessments of the associated risk of human salmonellosis. [source] Intermittent and persistent shedding of Escherichia coli O157 in cohorts of naturally infected calvesJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2004S.E. Robinson Abstract Aims:, We conducted two short-term studies of cohorts of naturally infected calves to determine the prevalence and concentrations of Escherichia coli O157 shed in faeces. Methods and Results:, Two cohorts of calves were sampled; in the first study 14 calves were sampled up to five times a day for 5 days; in the second study a group of 16 separate calves were sampled once or twice a day for 15 days. All cattle within the two cohorts shed E. coli O157 at some point during the respective studies. In 18% of samples, E. coli O157 could only be isolated using immunomagnetic separation after an enrichment period, suggesting concentrations <250 CFU g,1. The highest concentrations recorded were 6·7 × 105 and 1·6 × 106 CFU g,1 for studies 1 and 2 respectively. Conclusions:, Persistent, high shedders (shedding >103 CFU g,1) were evident in both studies but, in the majority of calves, the pathogen was isolated intermittently. Significance and Impact of the Study:, The variable patterns of shedding have important implications for the design of appropriate sampling protocols and for gaining meaningful estimates of parameters used in mathematical models of transmission. [source] A comparison of two pre-enrichment media prior to immunomagnetic separation for the isolation of E. coli O157 from bovine faecesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2003G. Foster Abstract Aims: To compare the sensitivity of two pre-enrichment broth media prior to immunomagnetic separation for the isolation of Escherichia coli O157 from cattle faeces. Methods and Results: One-gram portions of 721 cattle faeces collected from 43 farms were pre-enriched in buffered peptone water containing vancomycin, cefixime and cefsulodin (BPW-VCC) and buffered peptone water without additives (BPW-WOA), respectively. A total of 137 samples were positive for E. coli O157: 127 pre-enriched with BPW-WOA and 89 pre-enriched in BPW-VCC. Representative isolates were tested for phage type, verotoxin and eae (E. coli attaching and effacing) gene sequences, resulting in the recognition of eight different types. All the E. coli O157 types recognized were isolated by both methods except for three different strains, each of which were isolated only on a single occasion: two by BPW-WOA and another by BPW-VCC. Conclusions: The results clearly demonstrate, under the conditions of this study, that BPW without antibiotics was the superior pre-enrichment medium for the isolation of E. coli O157 from cattle faeces. Significance and Impact of the Study: The use of BPW-WOA in preference to BPW-VCC for the isolation of E. coli O157 from cattle faeces in future research and outbreak studies should lead to a higher number of positive isolates. [source] Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milkJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2002R.D. Reinders Aim:,To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. Methods and Results:,Raw milk, pasteurized milk with 1·5% fat content and pasteurized milk with 3·5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37°C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFossÔ). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml,1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml,1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37°C) and mTSBn (16 h, 37°C), successively. Conclusions:,The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml,1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. Significance and Impact of the Study:,Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis. [source] Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cellsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2002W. Sun Aims:,To develop methods to assess the efficiency of immunomagnetic separation (IMS). Methods and Results:,The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence. Both methods were suitable for the enumeration of bacteria captured by the biosorbents. The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads. The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent. For cell concentrations of , 105 cfu ml,1, in a 1-ml sample volume, nearly 80,85% recovery of the pathogen was observed after 0·5 h of incubation. For an 11-ml sample containing 104 cfu ml,1, maximum recovery (50% of cells) was achieved only after 2 h incubation. Conclusions:,The detection limit of an ATP-based bioluminescent assay for E. coli O157:H7 was reduced by 1 log cycle after optimization of IMS. The bioluminescent methods could be used for screening and testing the affinity of antibodies or other affinity elements of biosorbents towards live bacterial cells. Significance and Impact of the Study:,Bioluminescent assays provide an easy way to optimize conditions for the capture of bacteria by biosorbents in real time. [source] Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation,polymerase chain reactionJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2000S. Hallier-Soulier Cryptosporidium parvum has emerged as one of the most important new contaminants found in drinking water. Current protocols for the detection of cryptosporidia are time-consuming and rather inefficient. We recently described an immunomagnetic separation,polymerase chain reaction (IMS,PCR) assay permitting highly sensitive detection of C. parvum oocysts in drinking water samples. In this study, a second IMS,PCR assay to detect all cryptosporidial oocysts was developed, and both IMS,PCR assays were optimized on river water samples. A comparative study of the two IMS,PCR assays and the classical detection method based on an immunofluorescence assay (IFA) was carried out on 50 environmental samples. Whatever the type of water sample, the discrepancy in C. parvum detection between the IFA and IMS,PCR took the form of IFA-negative/IMS,PCR-positive results, and was caused mainly by the greater sensitivity of IMS,PCR as compared with IFA. Of the 50 water samples, only five tested positive for C. parvum using IMS,PCR, and could constitute a threat to human health. These results show that both IMS,PCR assays provide a rapid (1 d) and sensitive means of screening environmental water samples for the presence of cryptosporidia and C. parvum oocysts. [source] Despite large-scale T cell activation, only a minor subset of T cells responding in vitro to Actinobacillus actinomycetemcomitans differentiate into effector T cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2000Homayoun H. Zadeh Recent studies in our laboratory have demonstrated that Actinobacillus actinomycetemcomitans has a potent T cell stimulatory effect, activating more than half of all T cells. However, since the fate of these activated T cells was not known, the present study sought to determine whether all of these T cells differentiate into effector cells. To that end, the intracellular expression of T cell cytokines (IL-2, IFN-,, IL-4 and IL-10) in response to A. actinomycetemcomitans was determined by flow cytometry. Results demonstrated a time-dependent increase in the expression of the cytokines, most reaching peak levels at 24,48 h. At 48 h, the proportion of T cells expressing each of the cytokines were as follows: IL-2 (1.7%±0.3), IFN-,(1.8%±0.5), IL-4 (1.0%±0.2) and IL-10 (1.5%±0.5). These data indicated that only 2,5% of all T cells stimulated with A. actinomycetemcomitans expressed any T cell cytokines. The finding of large-scale T cell activation in the absence of cytokine expression suggests that the activation of T cells in response to A. actinomycetemcomitans is incomplete. To investigate this phenomenon, peripheral blood mononuclear cells (PBMC) were cultured with A. actinomycetemcomitans for 24 h followed by sorting of the activated (CD69+) cells by immunomagnetic separation and restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Results demonstrated that nearly 90% of the T cells were unresponsive to further restimulation. A possible explanation for this unresponsiveness is the induction of clonal anergy among the responding T cells. To determine possible preferential effects of the stimulation on specific cytokines, the expression of each cytokine among T cells responding to A. actinomycetemcomitans was compared to the maximum levels achieved by PMA+ionomycin stimulation. Results showed that number of IL-2+ and IFN-,+ T cells observed in response to A. actinomycetemcomitans were between 2% and 7% of those seen in response to PMA+ionomycin. Conversely, the proportions of T cells expressing IL-4 or IL-10 were between 35% and 90% of those following stimulation with PMA+ionomycin. Hence, A. actinomycetemcomitans appears to more preferentially induce T cells [source] CD146-based immunomagnetic enrichment followed by multiparameter flow cytometry: a new approach to counting circulating endothelial cellsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 5 2008A. WIDEMANN Summary.,Background: Circulating endothelial cells (CECs) have emerged as non-invasive biomarkers of vascular dysfunction. The most widely used method for their detection is CD146-based immunomagnetic separation (IMS). Although this approach has provided consensus values in both normal and pathologic situations, it remains tedious and requires a trained operator. Objectives: Our objective was to evaluate a new hybrid assay for CEC measurement using a combination of pre-enrichment of CD146+ circulating cells and multiparametric flow cytometry measurement (FCM). Patients and methods: CECs were determined in peripheral blood from 20 healthy volunteers, 12 patients undergoing coronary angioplasty, and 30 renal transplant recipients, and blood spiked with cultured endothelial cells. CD146+ cells were isolated using CD146-coated magnetic nanoparticles and labeled using CD45,fluorescein isothiocyanate and CD146,PE or isotype control antibody and propidium iodide before FCM. The same samples were also processed using CD146-based immunomagnetic separation as the reference method. Results: The hybrid assay detected CECs, identified as CD45dim/CD146bright/propidium iodide+, with high size-related scatter characteristics, and clearly discriminated these from CD45bright/CD146dim activated T lymphocytes. The method demonstrated both high recovery efficiency and good reproducibility. Both IMS and the hybrid assay similarly identified increased CEC levels in patients undergoing coronary angioplasty and renal transplantation, when compared to healthy controls. In patients, CEC values from these two methods were of the same order of magnitude and highly correlated. Bland,Altman analysis revealed poor statistical agreement between methods, flerrofluid,FCM providing higher values than IMS. Conclusion: This new hybrid FCM assay constitutes an accurate alternative to visual counting of CECs following CD146-based IMS. [source] Detection, isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques, immunoassays and colony hybridizationLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2007F. Auvray Abstract Aims:, To provide information on detection of Shiga toxin-producing Escherichia coli (STEC) in retail-minced beef using an approach combining (i) PCR-based techniques and automated immunoassay for stx screening and detection of the five major serogroups associated with human infection, and (ii) immunomagnetic separation (IMS) and colony hybridization assays for bacterial strain isolation. Methods and Results:, Twenty-seven out of 164 minced beef samples were stx -positive by PCR-ELISA, nine of which were also positive by real-time PCR for at least one marker of the five main serogroups tested (O26, O103, O111, O145 and O157). Two E. coli O103 stx -negative strains were isolated from two out of 10 IMS and nine STEC strains that did not belong to the five main serogroups were isolated by colony hybridization. Conclusions:, PCR techniques are applicable for rapid screening of samples containing both an stx gene and an O-group marker of the five main pathogenic STEC serogroups. Isolation of STEC strains belonging to the main non-O157 serogroups remains difficult. Significance and Impact of the Study:, This study presents an evaluation of a multi-faceted approach for the detection of the most frequently reported human pathogenic STEC serogroups. The advantages and limits of this strategy are presented. [source] Enumeration of Escherichia coli O157 in cattle faeces using most probable number technique and automated immunomagnetic separationLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2004N. Fegan Abstract Aims:, To determine the numbers of Escherichia coli O157 present in the faeces of naturally infected cattle. Methods and Results:, A combination of the most probable number (MPN) technique and automated immunomagnetic separation (AIMS) was used to enumerate E. coli O157 in cattle faeces from both pasture-fed and grain-fed animals. A total of 22 E. coli O157 positive faecal samples were enumerated for E. coli O157 (10 from pasture-fed and 12 from grain-fed animals). The numbers of E. coli O157 in cattle faeces varied from undetectable (<3 MPN g,1 of faeces) to 2·4 × 104 MPN g,1. There was no significant difference (P = 0·06) between the numbers of E. coli O157 in pasture-fed or grain-fed cattle faeces, although the geometric mean (antilog of the mean of log10 transformed MPN values) was higher in grain-fed (130 MPN g,1) than in pasture-fed (13 MPN g,1). Conclusions:, Although the number of samples tested is small, the results indicate that E. coli O157 make up a small proportion of the total E. coli population present in cattle faeces. Significance and Impact of the Study:, Information on the numbers of E. coli O157 present in cattle will assist in developing more robust quantitative risk assessments and formulating intervention strategies. [source] Isolation of Shiga toxin-producing Escherichia coli O103 from sheep using automated immunomagnetic separation (AIMS) and AIMS-ELISA: sheep as the source of a clinical E. coli O103 case?LETTERS IN APPLIED MICROBIOLOGY, Issue 3 2002A.M. Urdahl Aims: To investigate whether a sheep flock was the original reservoir of a Shiga toxin-producing Escherichia coli (STEC) O103 strain causing a clinical human case and to compare the two diagnostic methods automated immunomagnetic separation (AIMS) and AIMS-ELISA. Methods and Results: AIMS detected Escherichia coli O103 in 36·5% of the samples and AIMS-ELISA detected E. coli O103 in 52·1% of the samples. Polymerase chain reaction detected stx1 and eae in three of 109 E. coli O103 isolates. Pulsed field gel electrophoresis showed that the sheep and human STEC O103 were characterized by distinctly different profiles. Conclusions: The sheep flock was shown to carry STEC O103, although an association between the sheep flock and the clinical human case could neither be proven nor eliminated. Substantial agreement was found between AIMS and AIMS-ELISA, but AIMS-ELISA was less time consuming and resulted in a higher recovery of E. coli O103. Significance and Impact of the Study: The study shows that sheep may be carriers of STEC that are associated with human disease and that the methods described can be used to increase the sensitivity of STEC detection. [source] A method for the isolation of glomerular and tubulointerstitial endothelial cells and a comparison of characteristics with the human umbilical vein endothelial cell modelNEPHROLOGY, Issue 4 2004STELLA MCGINN SUMMARY: Background: Abnormalities in the structure and function of glomerular endothelial cells play a pivotal role in the development of progressive renal disease. The vascular abnormalities observed in the renal tubulointerstitium, however, correlate more strongly with progressive renal failure. Therefore, the successful isolation and culture of human renal microvascular endothelial cells from both the glomerulus and tubulointerstitium are paramount in studying renal disease models. Methods and Results: This study describes a simple and reproducible method for the isolation of human tubulointerstitial and glomerular endothelial cells by using immunomagnetic separation with anti-platelet endothelial-cell adhesion (anti-PECAM-1) Dyna beads, followed by manual weeding of mesangial and fibroblast contamination. No significant changes in morphological or immunohistochemical characteristics were observed up to passage two of culture. The in vitro characteristics of the endothelial cells were compared to the renal cortical endothelial cells in vivo and the standard human umbilical vein endothelial cell model (HUVECs). Similar to HUVECs, both populations of renal microvascular endothelial cells had a classical cobblestone appearance, stained positively for von Willebrand Factor and PECAM-1 and negatively for antifibroblast surface antigen and anticytokeratin. Differences in the expression of von Willebrand Factor, Wiebel Palade bodies and Flk-1 staining were observed between glomerular and tubulointerstitial endothelial cells. These immunohistochemical characteristics suggested that tubulointerstital endothelial cells were more closely aligned to HUVECS than to the glomerular endothelial cells. This observation indicated that HUVECs may be a suitable model for determining the tubulointerstitial endothelial response to systemic injury. Conclusion: In conclusion, a unique and novel method for the differential isolation of both glomerular and tubulointerstitial endothelial cells has been developed. Significantly, characterization of these populations suggests a role for HUVECS in the study of renal tubulointerstitial disease. [source] Detection of Escherichia coli using immunomagnetic separation and bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 3 2003Angelo J. Madonna The application of whole cell analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a valuable tool for rapidly identifying/detecting bacteria. This technique requires minimal sample preparation and is simple to perform, but is generally limited to purified samples of bacteria at concentrations greater than 1.0,×,106 cells/mL. In this paper, we describe a bacterial detection method that integrates immunomagnetic separation with bacteriophage amplification prior to MALDI-MS analysis. The developed method consists of three main stages: (1) isolation of a target bacterium by immunomagnetic separation; (2) infection of the immuno-captured bacterium with a lytic bacteriophage; and (3) assay of infected medium for bacteriophage progeny using MALDI-MS to produce a molecular weight signal for the virus capsid protein. With this technique, the presence of Escherichia coli in broth was determined in less then 2 h total analysis time at a concentration of ,5.0,×,104 cells/mL. Copyright © 2002 John Wiley & Sons, Ltd. [source] Immunomagnetic detection of micrometastatic cells in bone marrow in uveal melanoma patientsACTA OPHTHALMOLOGICA, Issue 8 2009Nils Eide Abstract. Purpose:, Our objective was to introduce immunomagnetic separation (IMS) in ocular research by evaluating the possibility of detecting tumour cells in bone marrow (BM) and peripheral blood (PB) samples and validating the captured cells as melanocytic cells. Methods:, Mononuclear cell (MNC) fractions isolated from BM and PB in uveal melanoma patients were examined for tumour cells using our IMS method. Sheep-anti-mouse IgG antibody-coated super paramagnetic particles were conjugated to an anti-melanoma antibody. Microscopy of the magnetic fraction isolated from MNCs was performed to identify and count the number of bead-rosetted cells. The finding of at least two rosettes with coated beads in a 20-,l fraction of a sample was registered as a positive test. The melanocytic nature of the tumour cells was ascertained with a double labelling procedure using fluorescent microparticles. Results:, Using IMS in a study of 328 patients, tumour cells were at initial diagnosis found in BM and PB in 29.9% and 1.6% of cases, respectively. In positive samples, a median of 56 tumour cells (range 2,500) were identified. The captured cells were documented to be of melanocytic origin by the simultaneous binding of fluorescent beads coated with another melanoma-associated antibody. Conclusions:, The IMS method was sensitive and efficient in the detection of occult melanoma tumour cells in BM. The validity of the immunomagnetic technique was strengthened by verifying the melanocytic characteristics of the isolated cells. The IMS procedure identifies intact, vital tumour cells, permitting further molecular characterization, an advantage which makes this method attractive for extended use. The clinical relevance of the findings will be further investigated in follow-up studies with repeated sampling and characterization of the isolated tumour cells. [source] Activated T cell subsets in human type 1 diabetes: evidence for expansion of the DR+ CD30+ subpopulation in new-onset diseaseCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2007C. Baker Summary An important limitation in T cell studies of human autoimmune (type 1) diabetes is lack of direct access to cells infiltrating the pancreas. We hypothesized that cells recently released from the pancreas into the blood might express a characteristic combination of markers of activation. We therefore examined the recently activated circulating T cell population [CD3+, human leucocyte antigen D-related (HLA-DR+)] using cytokine production and 10 additional subset markers [CD69, CD25, CD122, CD30, CD44v6, CD57, CD71, CCR3 (CD193), CCR5 (CD195) or CXCR3 (CD183)], comparing newly diagnosed adult (ND) (age 18,40 years) patients (n = 19) to patients with diabetes for > 10 years [long-standing (LS), n = 19] and HLA-matched controls (C, n = 16). CD3+ DR+ cells were enriched by two-step immunomagnetic separation. No differences in basal or stimulated production of interleukin (IL)-4, IL-10, IL-13 or interferon (IFN)-, by CD3+ DR+ enriched cells were observed between the different groups of subjects. However, among the CD3+ DR+ population, significant expansions appeared to be present in the very small CD30+, CD69+ and CD122+ subpopulations. A confirmatory study was then performed using new subjects (ND = 26, LS = 15), three-colour flow cytometry, unseparated cells and three additional subset markers (CD38, CD134, CD4/CD25). This confirmed the expansion of the CD3+ DR+ CD30+ subpopulation in ND subjects. We conclude that a relative expansion in the T cell subpopulation with the activated phenotype CD3+ DR+ CD30+ is seen in the peripheral blood of subjects with newly diagnosed type 1 diabetes. This subpopulation represents less than 0·7% of circulating T cells and may provide a rich source of disease-specific T cells that can be isolated from blood. [source] | ||||||||||||||||