Immunomagnetic Method Research Articles

Isolation of Platelet Granules

Functional analysis of platelet intracellular structures requires isolation and purification of these cellular compartments. With regard to the function of platelets, both, dense (delta) and alpha granules are relevant target structures. However, the availability of sufficient purification protocols for these structures is rather limited. This unit describes two protocols for isolation and purification of platelet granule structures. The Basic Protocol describes a new technique based on immunolabeling with target-specific antibodies followed by magnetic sorting, whereas the Alternate Protocol describes the more traditional procedure based on differential centrifugation and density-based sedimentation. For both methods, the degree of granule purification can be most easily determined by immunoblotting using various antibodies that recognize structure-specific proteins. The immunomagnetic sorting method is especially good for studies requiring highly purified material (e.g., for the identification of specific transporters and receptors).

Cuantificación inmunomagnética de células tumorales circulantes en pacientes con cáncer de próstata: correlación clínica y patológica

To detect and enumerate circulating prostatic tumor cells (CTC) in the peripheral blood of patients with prostate cancer (PC) and study the relationship between CTCs and clinical-pathological parameters. Prospective three-arm study: 26 patients (p) with localised PC (LPC); 24 P with metastatic PC (MPC) and 30 healthy volunteer controls. A single 7.5 ml sample of peripheral blood was retrieved; CTCs were isolated using an immunomagnetic method based on the CellSearch system (Veridex). CTCs were identified as nucleated cells negative for CD45 (leukocytes) and positive for cytokeratins. (8, 18 y 19) The relationship between CTC numbers and PSA levels, Gleason score and TNM classification was studied. Only 10% of the healthy controls had 1 CTC/7.5 mL, none of the patients with localised PC had more than 3 CTCs (88% < or = 2 CTCs), and patients with MPC had significantly higher CTC levels [m: 29 (1-178)] compared with the other two groups (P: 0.000). A positive correlation was demonstrated between the CTC count and PSA levels, tumor size, and presence or absence of enlarged lymph nodes. Gleason score was the only parameter that did not show any correlation with CTC levels, and although the number of CTCs was higher in patients with visceral metastases [m: 297 (0-416)] compared with bone metastases patients [m: 68 (9.5-168)] , these differences were not significant. Immunomagnetic analysis permits CTCs to be enumerated in peripheral blood and could be a possible way to correctly stage and make a reasonable prognosis of metastatic disease.

Isolation of Human Adrenocortical Aldosterone-Producing Cells by a Novel Immunomagnetic Beads Method

We detected intense CD56 immunostaining in the zona glomerulosa (ZG) and medulla of the normal human adrenal gland and therefore identified CD56, the neural cell adhesion molecule, as a membrane antigen specific for the ZG, aldosterone-producing adenoma (APA), and chromaffin cells. The APA and pheochromocytoma cells, which are histogenetically derived from the ZG and medulla, respectively, also showed intense CD56 immunostaining. Based on these findings we developed a strategy for isolating cells from the ZG and APA using CD56 immunobinding to magnetic beads. Morphology, gene expression studies, and aldosterone measurement confirmed that CD56 positive (+) cells were ZG and APA cells. Analysis of CD56+ cells under light and phase contrast microscopy evidenced that these cells formed clumps, as the ZG cells usually do; with electron microscopy they showed multiple features typical of a steroidogenic phenotype. Expression levels of the CD56 and the aldosterone synthase (CYP11B2) gene were markedly higher in CD56+ cells than CD56- cells (+1600 and +2100% increase, respectively). Moreover, aldosterone secretion was higher (+1380%) from CD56+ cells than from CD56- cells. Hence, this novel methodology allows isolation of a pure population of ZG and APA cells exhibiting multiple characteristics of the aldosterone-producing cells.

Circulating tumor cells: Determining its number and what it means

WHEN Steven Paget published his theory of ‘‘seed and soil’’ in 1889 (1), the idea of hematogenous tumor cell dissemination was born. In many breast cancer patients, circulating cells with the characteristics of tumor cells can be identified in the peripheral blood that are known as circulating tumor cells (CTCs). These cells are present not only in patients with metastatic disease but also in those whose tumors are apparently localized (2). It has been reported that tumor cells shed into the circulation in response to microinvasive events within the tumor (3). Many of the current research works are therefore focused on the detection of CTCs in the peripheral venous blood. Techniques used to detect CTCs can be generally divided into cytometric and nucleic acid-based approaches. Cytometric approaches use immunocytochemical methods to identify and characterize individual tumor cells. For instance, Cristofanilli et al. (4) successfully used the CellSearch System to detect CTCs. Since then, this method has been used by many other researchers on breast cancer (5,6). It was also used by the researchers in the other area. Hristov et al. (7) analyzed angiogenic monocytes and endothelial progenitor cells (EPC) for vascular homeostasis in peripheral blood by flow cytometry. Nucleic acid-based approaches detect DNA or RNA sequences that are differentially expressed in tumor cells and in normal blood components (8). In addition, other techniques have been developed to analyze CTCs. For instance, Swennenhuis et al. (9) characterized CTCs by fluorescence in situ hybridization. The technology of multiparameter flow cytometry described by Hu et al. (10) in their report published in this issue is found interesting, because it represents a significant improvement over the technology that was used by Rehse et al. (11). In their report, Hu et al. isolated the mononucleocytes and labeled them with monoclonal antibodies antiCD45-PerCP, Ep-CAM-PE, and Cytokeratin 8,18,19-FITC in a similar manner as used by Cristofanilli et al. (4). However, the enrichment procedure was quite different. By now, most researchers use immunomagnetic method to enrich mononucleocytes (12). This method often results in significant loses of target cells. It is expensive, and it involves multiple processes. All these have made its clinical application difficult. Hu’s group demonstrated the usefulness and specificity of multiparameter flow cytometry in detecting human breast cancer cells (SKBR-3) in their report. The specificity was significantly higher compared with traditional RT-PCR analyses, and the reported high sensitivity limit of 10 is sensitive enough to detect the target cells in peripheral blood (12). Hu et al. also investigated the correlation of overall survival (OS) with the number of CTCs detected (advanced breast cancer patients: CTCs 5; limited breast cancer patients: CTCs < 5). The median OS was 95 weeks and 65.5 weeks for patients with CTCs < 5 and CTCs 5, respectively. The results of the retrospective study also demonstrated that the OS was independent of clinical pathology and the tumor size but was closely correlated with CTC levels as well as age and metastasis. In addition, this article shows that multiparameter flow cytometry technique is atraumatic and could be used to detect disease progression more accurately than imaging. Together, the results presented in this study using multiparameter flow cytometry to measure CTCs show great promises for its clinical usage and may there-

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Study on peripheral blood T lymphocyte cell membrane in thyroid associated ophthalmopathy by using atomic force microscopy

To study the cell membrane of T-lymphocytes in thyroid associated ophthalmopathy (TAO) by using atomic force microscopy (AFM). Case-control study. Forty-two patients were selected as the subjects, and divided into two groups: acute stage (18) and stationary stage (24) ones on the basis of symptom. Ninety-two healthy persons were in the normal groups. Dynal immunomagnetic beads isolation method was used to separate the T cells. CD3+ was chosen as a surface molecule marker of T cells to determine the purity of cells isolated. Cultivated T cells were observed by AFM. Amplitude and height images were obtained in the tapping mode with a scan rate of 2 Hz and an integral gain of 0.3 to 0.5. Statistical analysis was performed using single-factor analysis of variance and the P value was calculated. The topographies of these three groups of T cells showed significant difference (F = 28.809, 58.213, 169.789, 35.933, 121.325; P < 0.05. Average diameter and roughness of T cells in acute stage of TAO were significantly greater than those of the other two groups. One way analysis of variance showed that significant differences in various parameters (Ra, Peak count, Rpm, Rvm, Surface area diff) were found between these three groups (P = 0.047, 0.002). The morphology and ultrastructure of lymphocytes in TAO are different from the normal subjects by observation with AFM. Lymphocytes in acute stage of TAO are also different from those in stationary stage.

HEMATOPOIETIC STEM CELL SEPARATION FOR EXPERIMENTAL PURPOSES - METHODIC LIMITATIONS

Protooncogene CD117 is a cytokine receptor important for hematopoietic element development. Currently, we are not able to routinely separate a sufficient quantity of bone marrow CD117+ cells for experimental purposes. The aim of this study was to establish an immunomagnetic separation method for CD117+ hematopoietic stem cell isolation and to estimate the expression of chosen BCl-2 family protein members in these elements. 120 samples of human and murine bone marrow were acquired using the magnetic separation system. The cells were stained for CD117, BCl-2, BAX, and CD33 by an indirect fluorescent immunocytochemistry. The flow cytometry analysis showed only 2.6% CD117+ cells from human as well as mouse bone marrow which is insufficient for further experiments. Cytospin was not good for morphologic characterization and immunophenotyping due to the fragility and destruction of the studied cells. Therefore, cell suspension staining was selected and by this method we found CD117 positivity in 70% of the mononuclerar (CD33 positive) elements in the case of chronic myeloid leukaemia. Labelling of the BCl-2 family in this case showed antiapoptotic BCl-2 expression in 80 %, proapoptotic BAX expression in approximately 5%. Our results show that CD117 immunomagnetic separation from bone marrow material is not acceptable for experimental purposes. They demonstrate that the only practical useful for the bone marrow cell examination (morphology and immunophenotype) is cell suspension staining which uncovers the distribution of both cytoplasmic proteins and surface antigens of immature blood elements.

Effects on activity of mouse NK cells by inhibitory Ly49 receptors blockade

To observe the effects on antitumor activity of mouse NK cells and the mixed lymphocyte reaction with blocking the interaction between the inhibitory Ly49 receptor on mouse NK cell and MHC-I molecules on target cells with monoclonal antibody. NK cells were isolated from splenocytes of C57BL/6J mouse with immunomagnetic method. YAC-1 and M1 cells were used as target cells, the cytotoxicity of NK cells was examined with mixed lymphocyte reaction before and after treatment of anti-Ly49 monoclonal antibody. The cytotoxicity of NK cells to M1 cell was significantly augmented (10.7% +/- 0.5% compared with 84.4 % +/- 2.9%), and the clone-forming capacity of M1 cell was inhibited remarkably (8.2 % +/- 2.2% compared with 94.0% +/- 3.3%) with the increase of concentration of the antibody (0 approximate, equals 60 microg/ml) (P <0.05). The difference of cytotoxicity to YAC-1 cells was only found between 0 microg/ml and 60 microg/ml groups (P <0.05). In mixed lymphocyte reaction, after pretreated with antibody, the proliferation of splenocytes of BALB/C mouse was inhibited (0.398 +/-0.025 compared with 0.128 +/- 0.014), and statistical difference was observed only between antibody groups of 0 microg/ml and 60 microg/ml (P<0.05). Blocking the inhibitory Ly49 receptor on NK cells with monoclonal antibody augments the cytotoxicity to target cells.

Clinical Significance of Cytokeratin 20-Positive Circulating Tumor Cells Detected by a Refined Immunomagnetic Enrichment Assay in Colorectal Cancer Patients

Current immunomagnetic enrichment method can only detect general epithelial antigens of circulating tumor cells (CTC). Further characterization of the CTCs to provide specific information on the tumor type is not possible. We attempted to overcome this drawback by developing the methodology for using a gastrointestinal-specific anti-cytokeratin (CK) 20 antibody to detect CTCs in colorectal cancer patients' blood. The protocol was validated using a colorectal cancer SW480 cell line. The clinical significance of findings in colorectal cancer was investigated by detecting CK20-positive CTCs (pCTC) in patients with colorectal cancer, other common cancers, colorectal adenoma, benign colorectal diseases, and normal subjects. Moreover, the malignant nature of CK20 pCTCs was examined by comparing chromosome 17 aberration patterns with those from the corresponding primary tumors. The assay successfully showed CK20-positive SW480 cells. When applied in patient samples, the detection rates were 62% (132 colorectal cancer patients; median number = 11 CTCs), 0% (120 patients with other common cancers), 6% (50 colorectal adenoma patients), 0% (120 patients with benign colorectal diseases), and 0% (40 normal subjects). Furthermore, statistical analysis showed that CK20 pCTC numbers were associated with tumor-node-metastasis stage and lymph node status. Using the median CK20 pCTC numbers as the cutoff points, stratified groups of colorectal cancer patients had significant differences in their recurrence, metastasis, and survival. Finally, chromosome 17 aneusomy in 90% of colorectal cancer patients with CK20 pCTCs matched with those from the primary tumors. Detection of CK20 pCTCs using the new protocol could generate clinically important information for colorectal cancer patients.

Immunomagnetic quantitative immuno-PCR for detection of less than one HIV-1 virion

Methods that allow the accurate and reliable detection of ultra-low molecular levels of proteins using techniques such as quantitative immuno-PCR (qIPCR) have demonstrated numerous technical difficulties. Protein detection methods lose specificity when the protein target is immersed within a matrix of thousands of molecules having wide ranges of concentrations. In addition, sensitivities are limited because of high background signals. To validate the performance of an immunomagnetic bead qIPCR method designed to remove the ‘matrix’ effect for HIV-1 p24 antigen detection, regression analyses were performed using samples from patients infected with HIV-1 diluted to approximately 100–1000, 10–100, 1–10, and 0.1–1.0 HIV-1 p24 Ag molecules/reaction. The number of HIV-1 p24 Ag molecules was derived from quantified HIV-1 RNA determinations. The modified immunomagnetic qIPCR bead assay demonstrated a limit of quantification of 10–100 HIV-1 p24 molecules per reaction, with an average correlation coefficient of 0.948 ± 0.028 over a 4-log dynamic range. This method detects less than one HIV-1 virion (a limit of detection unreported previously for HIV-1), and thus, has the potential to identify HIV-1 infection and monitor the dynamics of the disease course earlier than nucleic acid methods. The immunomagnetic qIPCR bead assay is a simple and inexpensive method for ultra-low protein detection of infectious agents, toxins, and cancer markers at a level unrecognized previously using any enzymatic or molecular method.

Cryptosporidium Oocyst Detection in Water Samples: Floatation Technique Enhanced with Immunofluorescence Is as Effective as Immunomagnetic Separation Method

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, 10(1), 10(2), and 10(3) per 10 microl were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < 10(2) per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.

Monoclonal Antibody CC188 Binds a Carbohydrate Epitope Expressed on the Surface of Both Colorectal Cancer Stem Cells and their Differentiated Progeny

Recently, cancer stem cells (CSC), undifferentiated cancer progenitor cells, have been successfully isolated from colorectal tumors. Targeting both CSCs and differentiated, rapidly proliferating tumor cells with therapeutic drugs provides a focused strategy to treat cancer. In this study, we isolated the monoclonal antibody (mAb) CC188 and characterized the epitope recognized by mAb CC188, which is useful for developing biological reagents that target CSCs. We used a hybridoma technique to generate mAbs and an immunomagnetic method to isolate colon CSCs. We characterized mAb CC188 binding epitope and examined the epitope distribution in normal and tumor tissues, particularly in CSCs using tissue arrays and immunofluorescence staining method. We also evaluated the effect of mAb CC188 on invasiveness of NSY tumor cells. mAb CC188 was generated and 98.9% (187 of 189 cases) of colon cancer were positively stained by mAb CC188. "+", "++," and "+++" staining were 25.9%, 28.6%, and 43.4%, respectively. The mAb CC188 binding epitope was identified as a carbohydrate, which was expressed on the surface of colon CSCs (CD133+), differentiated colon cancer cells (CD133-), and cells from various types of epithelial tumors. In contrast, the expression of the carbohydrate epitope was low in normal prostate muscle and pancreatic acinar cells, as well as in some normal epithelial cells of the breast duct, cervix, and skin. A functional study indicated that mAb CC188 suppressed the invasiveness of NSY tumor cells. mAb CC188 selectively targets a carbohydrate epitope expressed on cancer cells, providing a viable method for specific tumor imaging and targeted therapy.

THE PREVALENCE OF E. COLI O157/O157:H7, L. MONOCYTOGENES AND SALMONELLA SPP. ON BOVINE CARCASSES IN TURKEY

ABSTRACTEscherichia coli, O157, E. coli O157:H7, Listeria monocytogenes and Salmonella spp. are important foodborne pathogens, that some studies have demonstrated that cattle are a major reservoir of these pathogens. The main purpose of this study was to evaluate the prevalence of E. coli O157/O157:H7, L. monocytogenes and Salmonella spp. on slaughtered bovine carcasses. In this study, five abattoirs in Afyonkarahisar were visited between March and August in 2005 in the center of Afyonkarahisar Province, Turkey. A total of 250 bovine carcasses were sampled by surface swabbing the carcasses at four points. The presence of E. coli O157/O157:H7, L. monocytogenes and Salmonella spp. in the samples collected from 250 cattle was determined with the application of the immunomagnetic separation method. Overall, the prevalence of E. coli O157/O157:H7, L. monocytogenes and Salmonella spp. was 3.2, 0.8, 6.8 and 10%, respectively. Our results demonstrated that bovine carcasses can also be a source for E. coli O157/O157:H7, L. monocytogenes and Salmonella spp. infections for humans. The widespread occurrence of these pathogens in slaughterhouses reinforces the need for the implementation of effective control measures.PRACTICAL APPLICATIONSThis study indicates that relatively high values, particularly Salmonella spp., and widespread occurrence of these pathogens in bovine carcasses require the need for the implementation of effective public health measures.

Seasonality of Cryptosporidium oocyst detection in surface waters of Meru, Kenya as determined by two isolation methods followed by PCR

Meru, Kenya has watersheds which are shared by wildlife, humans and domesticated animals. These surface waters can be contaminated by the waterborne pathogen Cryptosporidium. To quantify the seasonality and prevalence of Cryptosporidium in Meru regional surface waters, we used a calcium carbonate flocculation (CCF) and sucrose floatation method, and a filtration and immunomagnetic bead separation method, each of which used PCR for Cryptosporidium detection and genotyping. Monthly water samples were collected from January through June in 2003 and 2004, bracketing two April-May rainy seasons. We detected significant seasonality with 8 of 9 positive samples from May and June (p<0.0014), which followed peak rainy season precipitation and includes some of the subsequent dry season. Six of 9 positive samples revealed C. parvum, and 3 contained C. andersoni. None contained C. hominis. Our results indicate that Meru surface waters are Cryptosporidium-contaminated at the end of rainy seasons, consistent with the timing of human infections reported by others from East Africa and contrasting with the onset of rainy season peak incidence reported from West Africa.

Quantitative Real-time RT-PCR Detection for Survivin, CK20 and CEA in Peripheral Blood of Colorectal Cancer Patients

To establish a sensitive method for the early detection of circulating tumor cells (CTCs) in peripheral blood (PB) of colorectal cancer (CRC) patients. PB samples were collected from 156 CRC patients, 40 benign colorectal disease patients, 40 healthy individuals and 45 patients with other solid tumors. The combination of negative and positive immunomagnetic bead method was used to enrich cancer cells. Then, cytokeratin-20 (CK20), survivin and carcinoembryonic antigen (CEA) mRNA were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). In addition, analyses were carried out for their correlation with patients' clinicopathologic features. The positive rates of survivin, CK20 and CEA mRNA in the PB of CRC patients were 57.7, 47.4 and 39.1%, respectively, and the sensitivity increased from 39.1% of CEA mRNA alone to 60.9% of the combined panel. The expression of the three mRNAs in CRC patients was significantly higher than that in benign control and healthy volunteers, and the expression of survivin and CK20 was not significantly higher than that of patients with other solid tumors. However, the expression of CEA mRNA was significantly higher than that of patients with other solid tumors. The expression of survivin, CK20 and CEA mRNA was significantly correlated with Dukes stages and lymph node metastasis. The combined use of negative and positive immunomagnetic beads followed by amplification of survivin, CK20 and CEA mRNA by means of qRT-PCR is a non-invasive and sensitive assay for the detection of circulating CRC cells. The combined panel improved the sensitivity of detection in CRC patients.

Isolation of monocytes from leukapheretic products for large‐scale GMP‐grade generation of cytomegalovirus‐specific T‐cell lines by means of an automated elutriation device

Dendritic cells (DC) act as antigen-presenting cells in immune response-mediated mechanisms against malignant cells and/or viral or fungal pathogens. CD14+ monocytes have been so far isolated by techniques of plastic adherence or by using immunomagnetic methods. Here the effectiveness of a commercially available cell separation system (Elutra, Gambro BCT) in the separation of monocytes and the large-scale production of cytomegalovirus (CMV)-specific T-cell lines were investigated. Six mononuclear cell (MNC) collections were processed with the Elutra system. Monocyte-enriched fraction was differentiated into DCs by addition of granulocyte-macrophage-colony-stimulating factor and interleukin (IL)-4. After 6 days of culture, DCs were matured in the presence of interferon (IFN)-gamma, IFN-alpha, IL-1beta, tumor necrosis factor-alpha, and poly(I:C) and pulsed with a pool of 48 MHC Class I and II-binding CMV peptides. Lymphocytes were then stimulated with mature autologous CMV peptide-pulsed DCs. After elutriation, the mean monocyte yield was 0.89 x 10(9) +/- 0.65 x 10(9), with a 51.0 +/- 31.6 percent recovery and a 51.1 +/- 35.4 percent purity. A significant correlation was observed when basal monocyte content was related to the postelutriation recovery (p < 0.0116). More than 60 percent of plated monocytes were differentiated into DCs, which after pulsing with CMV peptides, were able to stimulate a robust enrichment in CMV antigen-specific T cells in all tested samples (mean percentage of pentamer-positive CD8+ cells, 35% compared to the initial 2%). Our findings might be helpful for an appropriate MNC collection, to maximize the efficiency of the elutriation system and subsequently obtain an optimal monocyte-enriched yield for further DC generation and T-cell stimulation.

OCCURRENCE OF ESCHERICHIA COLIO157:H7/O157, LISTERIA MONOCYTOGENES AND SALMONELLA SPP. IN BEEF SLAUGHTERHOUSE ENVIRONMENTS, EQUIPMENT AND WORKERS

ABSTRACTEscherichia coli O157/O157:H7, Listeria monocytogenes and Salmonella spp. are major foodborne pathogens and the emergence of these pathogens has been reported in many countries. The aim of this study was to investigate contamination of the beef slaughterhouse environment, equipment and workers with E. coli O157/O157:H7, L. monocytogenes and Salmonella spp. For this study, 500 swab samples were taken from 19 different points in five privately owned slaughterhouses, their periphery, slaughterhouse equipment and slaughterhouse employees. The presence of E. coli O157:H7/O157, L. monocytogenes and Salmonella spp. was determined with the application of the immunomagnetic separation method. Our study showed that the swabs taken from the five slaughterhouses contained E. coli O157:H7 in the environment, equipment, abattoir workers and water with a frequency 0.31, 1, 1.42 and 0%, respectively; while E. coli O157 was evident in the environment, equipment, abattoir workers and water with a ratio of 15, 10, 10 and 0%, respectively; L. monocytogenes was detected in the environment, equipment, abattoir workers and water at a ratio of 4.37, 15, 5.71 and 0%, respectively; and Salmonella spp. occurrence in the environment, equipment, abattoir workers and water at a ratio of 3.43, 16, 11.42, and 0%, respectively. Implementing hazard analysis critical control point principles in work procedures would definitely reduce the gross contamination occurring in abattoirs.PRACTICAL APPLICATIONSThis study has revealed the effect of personnel and equipment on the contamination routes of E. coli O157:H7, Listeria monocytogenes and Salmonella spp. in meat in slaughterhouses and showed that especially L. monocytogenes and Salmonella spp. may pose a higher risk than E. coli O157:H7 in slaughterhouses.

Bone marrow micrometastases in advanced stage non-small cell lung carcinoma patients

The clinical relevance of bone marrow micrometastases in non-small cell lung cancer (NSCLC) is undetermined, and the value of such analyses in advanced stage patients has not been assessed previously. Immunomagnetic selection with the MOC31 (anti-EpCam) antibody was performed to isolate and detect tumor cells in bone marrow aspirates obtained from 196 patients with NSCLC, and the patients were subjected to follow-up for the assessment of survival. Repeated bone marrow samples, 2-7 samples per patient, were obtained from 13 long-term survivors. MOC31 positive tumor cells were detected in 107 of 196 (55%) samples, a frequency similar to results reported in low-stage patients prior to curative surgical resection for NSCLC. No association was found between the presence of bone marrow micrometastases and disease stage or histological subgroup, and survival was similar in patients with and without detectable tumor cells in bone marrow. Repeated bone marrow analysis revealed, for the first time in this group of patients, the continued presence of tumor cells regardless of the given therapy and treatment response. The immunomagnetic method utilized is a feasible strategy for the detection of bone marrow micrometastases in NSCLC. In advanced stage patients, the presence of MOC31 positive cells in bone marrow does not predict survival. Repeated analyses of bone marrow samples from long-term survivors revealed tumor cells in bone marrow which may represent dormant cells of particular interest for further characterization and follow-up.

Evaluation of an immunomagnetic separation method to capture Candida yeasts cells in blood

BackgroundCandida species have become the fourth most-frequent cause of nosocomial bloodstream infections in immunocompromised patients. Therefore, rapid identification of pathogenic fungi to species level has been considered critical for treatment. Conventional diagnostic procedures such as blood culture or biochemical tests are lacking both sensitivity and species specificity, so development of rapid diagnostic is essential.ResultsAn immunomagnetic method involving anti-Candida monoclonal antibodies was developed to capture and concentrate in human blood four different species of Candida cells responsible for invasive yeast infections. In comparison with an automated blood culture, processing time of immunomagnetic separation is shorter, saving at least 24 hours to obtain colonies before identification.ConclusionThus, this easy to use method provides a promising basis for concentrating all Candida species in blood to improve sensitivity before identification.

Functional characterization of CD4+CD25+ regulatory T cells differentiated in vitro from bone marrow-derived haematopoietic cells of psoriasis patients with a family history of the disorder

In psoriasis CD4+CD25+ regulatory T cells are functionally deficient. The imbalance between regulatory and effector T-cell functions is important for inducing psoriasis. It is reasonable to speculate that the dysfunctional activity of CD4+CD25+ regulatory cells may originate partly from the abnormal haematopoietic cells determined mainly by genetic background. To test the hypothesis that haematopoietic stem cells are responsible for dysfunctional CD4+CD25+ regulatory cells in psoriasis. Bone marrow-derived CD34+ haematopoietic cells from patients with psoriasis (with a family history of psoriasis) and from normal controls were differentiated into T cells in vitro. CD4+CD25+ T cells were isolated by an immunomagnetic bead method, and proliferation activity and capacity for cytokine secretion were determined. Furthermore, the ability of CD4+CD25+ T cells to suppress the proliferative responses of allogeneous peripheral blood CD4+CD25- effector T cells was assessed in vitro. The differentiated CD4+CD25+ T cells of psoriatic origin showed similar characteristics to those of normal volunteers, including proliferation activity and secretion profile of the cytokines interleukin (IL)-2, IL-4, IL-8, IL-10 and interferon (IFN)-gamma. However, proliferation and secretion levels of the cytokines IL-2 and IL-10 for CD4+CD25+ cells of psoriatic CD34+ cell origin were significantly lower than those of normal controls in response to streptococcal superantigen (Strep-A). In particular, CD4+CD25+ T cells differentiated from psoriatic CD34+ cells were functionally insufficient to restrain effector T-cell proliferation. CD4+CD25+ T cells differentiated in vitro from haematopoietic cells of patients with psoriasis are impaired in regulatory function. The dysfunction of psoriatic CD4+CD25+ T cells may be due to inherent genetic programming passed down from bone marrow-derived haematopoietic cells.