Immunomagnetic Method Research Articles

Enrichment of Mesenchymal Progenitor Cells from Mouse Compact Bone.

Adult mammalian bone marrow (BM) contains at least two distinct stem cell populations; the stem cells of the hemopoietic lineage and a second population termed mesenchymal stem cells (MSC) whose function is to maintain the non-hemopoietic BM elements as well as skeletal homeostasis. MSC have been implicated as potential targets in a range of cellular therapies for treatment of defects of both the hemopoietic and skeletal systems, and as vehicles for gene therapy. In order to evaluate the potential of these cells in various therapies, a pre-clinical animal model in which both the biology and potential therapeutic applications of these cells can be assessed is of fundamental importance. The goal of the current study was to develop a robust and reproducible method for the isolation of MSC from murine hemopoietic tissues. Tibiae and femurs harvested from C57BL6/J mice were gently crushed with a pestle to release the marrow. The bone fragments were subsequently cut into small pieces with a scalpel and digested in a solution containing 3mg/ml Type I collagenase to yield a population of compact bone (CB) derived cells. Mesenchymal progenitor cells (MPC) were detected using an in vitro assay for fibroblast-colony forming cells (CFU-F). CB cells were plated at 1000 or 5000 cells per cm2 in complete MesenCult™ medium for 12 days, after which CFU-F-derived colonies were enumerated. We show that CFU-F are present at a significantly higher frequency in mouse CB than in the BM (433±225 vs 11.7±3.5 colonies/106 cells respectively, n=3). Based on these data we developed a simple and robust immunomagnetic selection method to highly enrich MPC from mouse CB by depleting essentially all nucleated hemopoietic cells (CD45+) and red blood cells (Ter119+) using magnetic particles and antibodies to CD45 and Ter119, respectively. Target CD45−Ter119− cells initially comprised 1.1±0.5% (n=9) of the total CB fraction as assayed by FACS. Following depletion, CD45−Ter119− cells comprised 74.5±16% (n=6) of the cells and were enriched 205 fold for CFU-F compared to the starting population, with a CFU-F frequency of 1 per 11 cells plated, and a total CFU-F recovery of 57.9 ± 18.5%. Analysis of CD45−Ter119−Sca-1+ cells, a phenotype previously shown to enrich for MPC, revealed that these cells were enriched 50 fold following depletion, from 0.53±0.5 to 26.5±8.23% (n=3). The enriched MPCs cultured at low O2 tension were devoid of hemopoietic contaminants at passage 1 and 2 as shown by lack of CD45, Ter119 and CD11b expression. The cultured CB-derived MPCs were capable of extensive in vitro proliferation and maintained the ability to differentiate into cells of the osteogenic, adipogenic and chondrogenic lineages. Furthermore, irradiated cultured mesenchymal cells supported long-term culture-initiating cells (LTC-IC) in 4-week cultures of Sca-1+ BM cells under limiting dilution conditions, at frequencies similar to those detected using irradiated primary BM feeders (i.e. 1 per 1600). These data provide a rapid, reproducible method by which multipotent mesenchymal cells devoid of contaminating hemopoietic cells can be readily obtained from limited numbers of mice to study the biology of MSC as well as the use of these cells as therapeutic agents in a preclinical animal model.

CD133 Immunomagnetic Separation: How Effective Is the Method for CD133+ Cell Isolation from Umbilical Cord Blood?.

Umbilical cord blood (UCB) is a rich source of stem cells. Characterisation of stem cell subpopulations residing in UCB requires specific stem cell markers and reliable and reproducible protocols. We assessed the feasibility of CD133 positive cells' isolation using immunomagnetic cell sorting protocols. The separation was based on the manufacturer's instructions (Myltenyi Biotec). We compared these results with the corresponding ones of CD34 isolation. Two additional protocols were also applied to increase the efficiency of the CD133+ yield. The first one included an extra-labeling of the selected CD133 subpopulation from the first column with CD133 microbeads before their application to the second column. The second protocol was an indirect labelling procedure, using CD133-PE monoclonal antibody and either anti-PE microbeads or goat anti-mouse IgG microbeads as well as a combination of them. The CD133 isolation was performed in 18 UCB samples, while the CD34 in 17 samples. The volume of the samples used for CD133+ isolation was 38.5 ± 2.1ml and the respective one for the CD34+ protocol was 40.4 ± 2.8ml. The mononuclear cell fraction had 1.29 ± 0.16 x108 cells and 0.53 ± 0.06% of them expressed CD133. The corresponding values for samples used for CD34 isolation were 2.8 ± 0.3 x108 mononuclear cells and 1.64 ± 0.15% CD34. Following the manufacturer's instructions for CD133 isolation, the number of cells obtained was 21.33 ± 3.3x104 (median 18 x104) and CD133 expression ranged from 10–85% (median=60%). Concerning the CD34 isolation, the number of cells obtained was 54.3 ± 2.1x104 and 93 ± 1.25% expressed CD34. Applying the extra-labeling method, the number of cells obtained was 8±4 x104 and these cells were CD133+ at a percentage of 32.2±4.1%. Applying the indirect immunomagnetic separation the results were as follow: using anti-PE microbeads only, 3 ± 3 x104 cells were retrieved and the percentage of CD133+ was 13.8±9.7. With the use of goat anti-mouse IgG microbeads the cell number obtained was 13±1 x104 of which CD133+ were just 2.0± 2%. In the combination method, the cells initially isolated with anti-PE microbeads and subsequently with goat anti-mouse IgG microbeads were 20 x 104, of which only 18.5% were CD133+. Finally, labeling the cells initially with anti-PE microbeads and consecutively with goat anti-mouse IgG microbeads followed by column isolation yielded 16x104 cells and 19.1% CD133 positive. In conclusion CD34 isolation by the immunomagnetic method results in highly pure CD34+ population confirming the reliability and reproducibility of the method. On the other hand, with the currently available CD133 isolation kit, efficient and reproducible yield of pure CD133 positive population is not feasible.

Comparison of Rapid Enzyme-Linked Immunosorbent Assay and Immunomagnetic Separation Methods for Detection of Escherichia coli O157 in Fecal, Hide, Carcass, and Ground Beef Samples

Rapid enzyme-linked immunosorbent assays (ELISAs) are approved for detection of Escherichia coli O157 in beef products. However, these kits have also been used in the industry to detect this pathogen on hides or in feces of cattle, although this use has not been validated. The objective of this study was to compare commercially available ELISAs (E. coli Now, Reveal, and VIP) with immunomagnetic separation along with selective media to detect E. coli O157 on hides, in feces, and in medium- and low-level-inoculated ground beef and carcasses (simulated by using briskets) samples. Naturally infected hide and fecal samples were subjected to both the immunomagnetic separation method and ELISAs for the detection of E. coli O157. Additionally, E. coli O157 inoculated and noninoculated ground beef and beef briskets were used to simulate meat and carcass samples. When comparing the detection results from the ELISAs (E. coli Now, Reveal, and VIP) to the immunomagnetic separation method, poor agreement was observed for fecal samples (kappa = 0.10, 0.02, and 0.03 for E. coli Now, Reveal, and VIP, respectively), and fair-to-moderate agreement was observed for hide samples (kappa = 0.30, 0.51, and 0.29 for E. coli Now, Reveal, and VIP, respectively). However, there was near-perfect agreement between the immunomagnetic separation method and ELISAs for ground beef (kappa = 1, 1, and 0.80 for E. coli Now, Reveal, and VIP, respectively) and brisket (kappa = 1, 1, and 1 for E. coli Now, Reveal, and VIP, respectively) samples. Assuming immunomagnetic separation is the best available method, these data suggest that the ELISAs are not useful in detecting E. coli O157 from hide or fecal samples. However, when ELISAs are used on ground beef and beef brisket samples they can be used with a high degree of confidence.

Enhanced interferon-γ secretion and antitumor activity of T-lymphocytes activated by dendritic cells loaded with glycoengineered myeloma antigens

Immunotherapy is emerging as a promising cure for cancer. However, a severe problem in this area is the immune tolerance to tumor cells and tumor-associated antigens, as evidenced by the ability of cancer to escape immune surveillance. To overcome this problem this work examined the potential of improving the antigenicity of myeloma by metabolic engineering of its cell surface carbohydrate antigens (i.e., glycoengineering) and presentation of the modified tumor antigens by dendritic cells (DCs) to generate cytotoxic T-lymphocytes (CTLs). CD138+ myeloma cells were isolated from 11 multipe myeloma (MM) patients by the immunomagnetic bead method. The MM cells were treated with N-propionyl-D-mannosamine (ManNPr), a synthetic analog of N-acetyl-D-mannosamine (ManNAc), the natural biosynthetic precursor of N-acetyl sialic acid (NeuNAc), to express unnatural N-propionylated sialoglycans. The glycoengineered cells were then induced to apoptosis, and the apoptotic products were added to cultured functional DCs that could present the unnatural carbohydrate antigens to autologous T-lymphocytes. It was found that the resultant DCs could activate CD4+ and CD8+ T-lymphocytes, resulting in increased expression of T cell surface markers, including CD8CD28 and CD4CD29. Moreover, upon stimulation by glycoengineered MM cells, these DC-activated T-lymphocytes could release significantly higher levels of IFN-gamma (P < 0.05). Lactate dehydrogenase (LDH) assays further showed that the stimulated T-lymphocytes were cytotoxic to glycoengineered MM cells. This work demonstrated that glycoengineered myeloma cells were highly antigenic and the CTLs induced by the DCs loaded with the unnatural myeloma antigens were specifically cytotoxic to the glycoengineered myeloma. This may provide a new strategy for overcoming the problem of immune tolerance for the development of effective immunotherapies for MM.

Monocyte enrichment from leukapheresis products by using the Elutra cell separator

Dendritic cells (DCs), used in clinical trials for cancer immunotherapy, require processing on an expanded scale to conform to current good manufacturing practice guidelines. This study evaluated a large-scale monocyte enrichment procedure with a commercially available cell separator (Elutra, Gambro BCT) and analyzed the capacity of enriched monocytes to differentiate into DCs. Mononuclear cells were collected in two patients with malignant melanoma and seven healthy donors by leukapheresis. Continuous-counterflow elutriation with the Elutra was performed to enrich and purify monocytes from leukapheresis products. Purity and recovery of enriched monocytes were analyzed by flow cytometry. DCs were generated from the elutriated monocytes and characterized by phenotypic surface marker and stimulatory capacity in an allogeneic mixed lymphocyte reaction. In the leukapheresis products, the total MNC count was 7.3 x 10(9) +/- 0.7 x 10(9) and the mean percentage of CD14+ monocytes was 16.5 +/- 3.8 percent, which increased to 68.9 +/- 7.4 percent after elutriation with the Elutra. The mean monocyte recovery was 94.3 percent. Elutriated monocytes were successfully cultured into phenotypically and functionally mature DCs. These results indicate that the Elutra cell separator allows for fast and easy enrichment of monocytes within a closed system. Furthermore, these monocytes can be differentiated into functionally mature DCs. Compared to plastic adherence and immunomagnetic selection methods, the elutriation procedure is inexpensive, efficient, and very effective.

Microbial community diversity in the phycosphere of natural populations of the toxic alga, Alexandrium fundyense

The dinoflagellate Alexandrium fundyense is the major causative organism of paralytic shellfish poisoning in the Gulf of Maine. While laboratory studies have shown that A. fundyense population dynamics can be affected dramatically by co-occurring bacteria, little is known about these interactions in nature. Because A. fundyense is typically a minor Gulf of Maine phytoplankton community member, analyses of the bulk community cannot be used to address bacterium-A. fundyense associations. Therefore, an immunomagnetic bead method was used to selectively capture A. fundyense cells, and the bacteria attached to them, from complex natural samples. Bulk particle-associated and free-living bacterial communities were collected simultaneously. DNA was extracted from all sample types and subjected to 16S rRNA gene fragment amplification, denaturing gradient gel electrophoresis (DGGE) and sequence analysis. Ordination analysis of DGGE profiles confirmed that A. fundyense-associated bacteria community profiles were distinct from bulk bacterial community profiles, indicating selection of specific phylotypes in the A. fundyense phycosphere. Phylogenetic analyses confirmed that Alexandrium-associates were distinct from bulk particle-associated bacteria and that they included a greater prevalence and broader diversity of Gammaproteobacteria than previously thought to be associated with toxic algae. Phylogenetic groups known to be associated with dinoflagellates were also found, including members of the families Alteromonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae and Flavobacteraceae.

THE COMPARATIVE ANALYSIS OF THE HEMATOPOIETIC STEM CELLS (CD34 +) EXTRACTION METHODS

In the short review article considered peculiarity of the various extraction and culture methods of the hematopoietic stem cells CD34+, carried out their comparative analysis. The main directions of the cellular therapy development detailed. An advantages of immunomagnetic separations method as compared with other methods described. The results of pilot experiences of stem cells extraction in Central Research Laboratory of Gomel’s state medical university represented.

Submucosal connective tissue-type mast cells contribute to the production of lysophosphatidic acid (LPA) in the gastrointestinal tract through the secretion of autotaxin (ATX)/lysophospholipase D (lysoPLD)

Lysophosphatidic acid (LPA) is involved in a broad spectrum of biological activities, including wound healing and cancer metastasis. Autotaxin (ATX), originally isolated from a melanoma supernatant as a tumor cell motility-stimulating factor, has been shown to be molecularly identical to lysophospholipase D (lysoPLD), which is the main enzyme in the production of LPA. Although ATX/lysoPLD is known to be widely expressed in normal human tissues, the exact distribution of ATX-producing cells has not been fully investigated. In this study, we evaluated ATX/lysoPLD expression by immunohistochemical staining using a rat anti-ATX mAb in the human gastrointestinal tract and found that submucosal mast cells (MC) highly expressed this enzyme. This was confirmed by immunofluorescent double staining using mAbs to tryptase and chymase. Then, we isolated MC from human gastric tissue by an immunomagnetic method using CD117-microbeads and showed that a subpopulation of CD203c-positive MC showed positive staining for intracellular ATX/lysoPLD on flowcytometry. This was confirmed by Western blotting of the isolated cells. Moreover, a significant level of ATX/lysoPLD release could be detected in the culture supernatants of human MC by Western blot analysis. Our data suggest that submucosal MC play significant roles in various aspects of pathophysiology in the gastrointestinal tract by locally providing bioactive LPA through the production of ATX/lysoPLD.

Detection of Toxoplasma gondii in water by an immunomagnetic separation method targeting the sporocysts

An immunomagnetic separation (IMS) method was developed to detect Toxoplasma gondii in fresh waters by using the monoclonal antibody 4B6 targeting the sporocyst wall of T. gondii, Hammondia hammondi, Hammondia heydorni, and Neospora caninum. Water concentrates obtained by filtering 10- to 20-l samples samples were spiked with Toxoplasma oocysts, sonicated to release the sporocysts, and analyzed by IMS-4B6. Mean sporocyst recoveries were 74.5 +/- 5.3% in drinking water, 30.6 +/- 2.4 and 37.1 +/- 3.2% in surface waters, and 81.6 +/- 2.1% in IMS buffer. Then, this IMS method was integrated in a multistep procedure (i.e., filtration, IMS, immunofluorescence and autofluorescence) to detect Toxoplasma in unspiked and spiked water samples (10-30 l) of various qualities. Sporocyst recoveries ranged from 14.4 to 44.7% in drinking water samples spiked with 1-10 oocysts/l, and from 17.8 to 32.5% in surface water samples spiked with 10 oocysts/l. Sporocysts were not detected in 25 unspiked water samples. A sporocyst-like structure was seen in one of these unspiked samples, but its coccidian nature could not be proved by three polymerase chain reaction (PCR) methods targeting sequences of coccidian small and large subunit rRNA genes and Toxoplasma repetitive elements. In conclusion, IMS-4B6 is relevant for the detection of Toxoplasma in water generating small concentrates (<1 ml). Due to 4B6 cross-reactions, a PCR would be useful to further characterize coccidian sporocysts found microscopically.

An evaluation of primers amplifying DNA targets for the detection of Cryptosporidium spp. using C. parvum HNJ-1 Japanese isolate in water samples

The performance of polymerase chain reaction (PCR) procedures for the detection of Cryptosporidium parvum HNJ-1 strain (genotype II) oocysts purified from mice using published protocols was evaluated. Oocysts were concentrated from fecal samples of infected severe combined immunodeficiency (SCID) mice by sucrose flotation and were then purified by immunomagnetic separation method. The genotype of C. parvum was established as type II by restriction fragment length polymorphism (RFLP) analysis. Water samples were spiked with different numbers of oocysts, determined by limiting dilution. Genomic DNA was extracted and used for PCR assays targeting various Cryptosporidium species genes (Beta-Tubulin, COWP, 70 kDa HSP, SSU rRNA, ITS1, TRAP-C1 and TRAP-C2 gene). DNA from oocyst numbers of more than 1 x 10(4) was detected using each of the primers. However, when using lower oocyst numbers, the tools based on 9 of the 16 different primer assays gave sufficient results. Assays using the remaining seven primers gave less than satisfactory results. A new primer set, named VKSS-F1/2 and VKSS-R1/2, that target the 18 SSU rRNA gene of C. parvum was constructed and applied. The VKSS-F1/2 and VKSS-R1/2 assays amplified DNA isolated from spiked samples in 206 of 211 trials (97.6%). This illustrates the difficulty of detecting low numbers of Cryptosporidium spp. oocysts by molecular methods when working with environmental samples.

A gene expression signature associated with metastatic cells in effusions of breast carcinoma patients

Malignant effusion in invasive breast carcinoma is associated with poor prognosis. To decipher molecular events leading to metastasis and to identify reliable markers for targeted therapies are of crucial need. Therefore, we have used cDNA microarrays to delineate molecular signatures associated with metastasis and relapse in breast carcinoma effusions. Taking advantage of an immunomagnetic method, we have purified to homogeneity EpCAM-positive cells from 34 malignant effusions. Immunopurified cells represented as much as 10% of the whole cell fraction and their epithelial and carcinoma features were confirmed by immunofluorescence labeling. Gene expression profiles of 19 immunopurified effusion samples, were analyzed using human pan-genomic microarrays, and compared with those of 4 corresponding primary tumors, 8 breast carcinoma effusion-derived cell lines, and 4 healthy mammary tissues. Principal component and multiple clustering analyses of microarray data, clearly identified distinctive molecular portraits corresponding to the 4 categories of specimens. Of uppermost interest, effusion samples were arranged in 2 subsets on the basis of their gene expression patterns. The first subset partly shares a gene expression signature with the different cell lines, and overexpresses CD24, CD44 and epithelial cytokeratins 8,18,19. The second subset overexpresses markers related to aggressive invasive carcinoma (uPA receptor, S100A4, vimentin, CXCR4). These findings demonstrate the importance of using pure cell fractions to accurately decipher in silico gene expression of clinical specimens. Further studies will lead to the identification of genes of oustanding importance to diagnose malignant effusion, predict survival and tailor appropriate therapies to the metastatic effusion disease in breast carcinoma patients.

Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell markers can be directly enriched by using the CD49a molecule

Bone marrow (BM) from human and rodent species contains a population of multipotential cells referred to as mesenchymal stem cells (MSCs). Currently, MSCs are isolated indirectly by using a culture step and then the generation of fibroblast colony-forming units (CFU-fs). Unprocessed or native BM MSCs have not yet been fully characterised. We have previously developed a direct enrichment method for the isolation of MSCs from human BM by using the CD49a protein (alpha1-integrin subunit). As the CD49a gene is highly conserved in mammals, we have evaluated whether this direct enrichment can be employed for BM cells from rodent strains (rat and mouse). We have also studied the native phenotype by using both immunodetection and immunomagnetic methods and have compared MSCs from mouse, rat and human BM. As is the case for human BM, we have demonstrated that all rodent multipotential CFU-fs are contained within the CD49a-positive cell population. However, in the mouse, the number of CFU-fs is strain-dependent. Interestingly, all rat and mouse Sca-1-positive cells are concentrated within the CD49a-positive fraction and also contain all CFU-fs. In human, the colonies have been detected in the CD49a/CD133 double-positive population. Thus, the CD49a protein is a conserved marker that permits the direct enrichment of BM MSCs from various mammalian species; these cells have been phenotyped as true BM stem cells.

Interferon γ Enhances the Anti-Myeloma Activity of the Fully Human Anti-HLA-DR Monoclonal Antibody 1D09C3.

The fully human anti-HLA-DR antibody 1D09C3 exerts a potent anti-lymphoma activity both in vitro and in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. 1D09C3 is currently tested in phase I clinical trials in patients with relapsed/refractory B-cell malignancies. To investigate whether 1D09C3 might represent a treatment modality to target malignant plasma cells, we re-evaluated HLA-DR expression on CD138+ plasma cells. Additionally, we investigated the capacity of interferon γ (IFN-γ ) to upregulate HLA-DR expression on myeloma cell lines, and tested in vitro and in vivo the anti-myeloma activity of 1D09C3 alone or in combination with IFN-γ . Bone marrow CD138+ cells were enriched using an immunomagnetic method from 60 multiple myeloma (MM) patients. Three-color flow cytometry revealed a highly heterogeneous HLA-DR expression on plasma cells. CD138+HLA-DR+ cells were detected in 31/60 patients (52%), with 15/60 patients (25%) having ≥ 20% CD138+HLA-DR+ cells (median, 50%; mean, 54%; range, 23 - 100), and 3 patients (5%) displaying 100% CD138+HLA-DR+ cells. Two thirds of HLA-DR+ patients expressed CD45 on CD138+ cells, suggesting that 1D09C3 might target self-renewing plasma cells. HLA-DR expression was not associated with distinct cytogenetic abnormalities. Since primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines (n = 6) with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin-V/propidium iodide double staining showed that 1D09C3-induced cell death strongly correlated with constitutive HLA-DR expression. Interestingly, induction of HLA-DR by IFN-γ restored the sensitivity of HLA-DR dim cell lines (i.e., RPMI-8226, KMS-11) to the cytotoxic activity of 1D09C3. As compared to controls, exposure of RPMI-8226 and KMS-11 cell lines to IFN-γ and 1D09C3 significantly increased cell death to 45% (P ≤ 0.0001) and 40% (P ≤ 0.0001), respectively. The in vivo activity of 1D09C3 was analyzed by xenografting NOD/SCID mice with the KMS-11 cell line. A significant increase of median survival over controls was detected in mice treated with 1D09C3 alone at either 3 mg/mouse (92 vs 48 days, P ≤ 0.0001) or 6 mg/mouse (89 vs 48 days, P ≤ 0.0001). The combined treatment with IFN-γ plus 1D09C3 (3 mg/mouse) resulted in a significant increase of median survival as compared to controls (147 vs 48 days, P ≤ 0.0001) or mice receiving 1D09C3 alone (147 vs 92 days, P ≤ 0.03). The better therapeutic activity of the combined IFN-γ /1D09C3 treatment over 1D09C3 alone was further demonstrated by a 2-fold increase of mice being disease-free at 150 days after xenograft (47% vs 25%). No mice experienced any apparent treatment-related toxicity. In conclusion, our data demonstrate that:one third of patients with MM express significant levels of HLA-DR on CD138+ cells;IFN-γ-induced upregulation of HLA-DR results in a potent enhancement of the in vivo anti-myeloma activity of 1D09C3.

Long Term Outcomes of Unrelated Donor Bone Marrow Transplantation in Children with Severe Aplastic Anemia: An Update of the Milwaukee Experience.

We demonstrated that partially T-cell depleted unrelated donor HSCT for Severe Aplastic Anemia (SAA) is a reasonable treatment option for children and young adults who fail immune suppression therapy. (Margolis et al., 1996, Br J Haematol.)We now report, long term follow up data for 40 patients transplanted for SAA between the years 1986 to 2002. The patient group consisted of 22 males and 18 females, ranging in age from 0.5–24.3 (median 8.5) yrs. Retrospective molecular HLA typing shows that donors were matched for nine patients, and mismatched for 31. Patients were conditioned with cytosine arabinoside, cyclophosphamide, and total body irradiation, as previously described. Some patients additionally received ATG to promote engraftment. The marrow product underwent partial T-cell depletion using an antibody and complement process as described. in the original report. GVHD prevention was with cyclosporine. Three patients did not engraft. All three with non-engraftment died within 60 days of BMT from infectious and hemorrhagic complications. Since employing ATG as part of the conditioning regimen, all patients have engrafted. Of the 37 patients who engrafted the median time to an ANC>500 was 16 (range 8–25) days. Eight patients developed Grade II AGvHD, 1 grade III and 2 grade IV. Of 29 evaluable patients, 12 developed limited chronic GVHD, and 3 developed extensive CGvHD. Twenty-one patients are currently surviving with a follow-up of 4 to 19 yrs. (median 12.7 yrs.). Overall survival is 52% at 12 yrs. Of the 19 patients that died, causes of death included infection n=7, (PCP n=1, CMV n=2, Aspergillus n=1, Adenovirus n=2, PTLD n=1); GVHD n=2; Graft failure n=3; Multiorgan system failure n=2; ARDS n=1; Hemorrhage n=2; VOD n=1; Secondary malignancy n=1 (Hodgkin's disease n=1). Of the 21 surviving patients, all patients have a Karnofsky score ≥ 90%. The late effects in our survivors include two secondary malignancies (osteosarcoma and vaginal carcinoma in situ); cataracts n=11; growth retardation n=11; gonadal dysfunction n=6; hypothyroidism n=5; cognitive problems n=4; musculoskeletal problems (AVN, osteoporosis) n=7; hyperlipidemia n=3; and renal disease n=2. One patient had a subsequent pregnancy that resulted in a preterm delivery at 26 weeks and a neonatal death. Our experience, now with long follow-up, shows that an intensive conditioning regimen to prevent graft rejection, coupled with partial T-cell depletion of an unrelated donor bone marrow graft to decrease the risk of GVHD, provides for durable survival with an acceptable incidence of acute and chronic GVHD. The relatively low incidence of GVHD is notable in view of the number of patients with donors who had identified HLA disparity. However, there are long-term risks associated with this regimen including secondary malignancies, delayed growth and development, metabolic problems, and musculoskeletal problems. We are encouraged by the recently reported short-term results using regimens for this disease which avoid or limit the use of TBI. Recognizing that many patients have donors with HLA disparity, which increases the risks of graft rejection and GVHD, we believe that combining partial T cell depletion with advanced immunomagnetic methods of graft manipulation and a fludarabine based regimen may allow us to balance the risks of graft rejection, GVHD, and late-effects that are unique to patients with SAA.

Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data.

IntroductionDiverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells.MethodsMalignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays.ResultsMPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours.ConclusionUsing highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer.

Increased expression of collagen receptors: α1β1 and α2β1 integrins on blood eosinophils in bronchial asthma

Eosinophils are one of the major effector cells in bronchial asthma. Their infiltration of airways correlates with the asthma severity. Recruitment and activation of eosinophils are partially mediated by integrins alpha4beta1 and alpha4beta7. Collagens type I and IV constitute important components of extracellular matrix and vascular basement membrane, respectively. Therefore, collagen-binding integrins (alpha1beta1 and alpha2beta1) may also play a role in eosinophil lung infiltration. To evaluate the possible presence of alpha1beta1 and alpha2beta1 integrins on peripheral blood eosinophils from asthmatic subjects. Collagen receptors were studied on eosinophils separated by immunomagnetic CD16-negative method from healthy donors (n=13) and patients with moderate persistent atopic bronchial asthma (n=15). Surface receptor identification was performed by flow cytometry and cell adhesion assay. Eosinophils isolated from the patients showed increased expression of both alpha1beta1 and alpha2beta1 integrins as compared with healthy controls. Moreover, adhesive function of eosinophils to collagen type IV was inhibited by snake venom disintegrins: viperistatin and obtustatin. These disintegrins contain KTS active motif and are specific inhibitors of alpha1beta1 integrin. We demonstrated for the first time that collagen receptors: alpha1beta1 and alpha2beta1 integrins are overexpressed on the surface of peripheral blood eosinophils of asthmatic subjects. Further studies may reveal potential application of KTS-disintegrins or their structural analogs for therapy of bronchial asthma.

Presence of Shiga toxin-producing Escherichia coli O157:H7 in living layer hens

To evaluate the presence of Shiga toxin-producing strains of Escherichia coli (STEC) of the O157:H7 serotype in living layer hens so as to analyse the role of this avian species as potential reservoir. Cloacal swabs were collected between November 2004 and November 2005 from four intensive management layer hen farms and analysed for STEC O157:H7 by immunomagnetic separation methods and multiplex polymerase chain reaction for stx1 and/or stx2, the E. coli attaching and effacing (eae) and hly genes. STEC was detected in 26 of the 720 samples. The layer hens analysed were shown to carry STEC O157:H7. The presence of this bacterium in living layer hen farms investigated did not result in any detectable increase in gastrointestinal disease in this species. Living layer hens are a novel potential reservoir of E. coli O157:H7.

Isolation and identification of vascular endothelial cells from hepatocellular carcinoma xenografts in nude mice

目的 分离肝癌血管内皮细胞.方法采用CD31抗体交联免疫磁珠分选法,并加以优化,对裸小鼠原位人肝癌移植瘤进行血管内皮细胞分选;采用形态学观察、功能实验及RT-PCR等方法进行验证.结果从肝癌组织中分离的细胞呈现典型的内皮细胞形态特征,免疫组化证实内皮细胞标记分子CD31及VE-Cadherin表达阳性,超过95%的阳性分选细胞呈乙酰化低密度脂蛋白摄取实验阳性,RT-PCR示获得的细胞中内皮细胞标记分子CD31、VE-Cadherin及CD146的mRNA含量明显高于分离前.结论采用优化的免疫磁分选法可有效分离纯化肝癌组织中的血管内皮细胞,分离的内皮细胞可用于肝癌血管生成的相关研究。

Maintenance of Bad Phosphorylation Prevents Apoptosis of Rat Hepatic Sinusoidal Endothelial Cells in Vitro and in Vivo

To elucidate the mechanism of apoptosis of liver sinusoidal endothelial cells (SECs), we examined the phosphorylation status of Bad and its upstream signaling molecules during apoptosis in culture and after ischemia-reperfusion injury. Rat SECs were isolated by the immunomagnetic method, and 2 days after culture, most SECs underwent apoptosis, which was associated with decreased tyrosine phosphorylation of cellular proteins. Addition of orthovanadate (OV), a protein tyrosine phosphatase inhibitor, sustained cellular protein phosphorylation and strongly inhibited apoptosis. Bad was dephosphorylated at Ser-112 and Ser-136 during apoptosis, but the phosphorylation status of Bad was maintained in the presence of OV. OV activated the Akt, extracellular signal-regulated protein kinase, and p38 mitogen-activated protein kinase pathways, which are involved in Bad phosphorylation. In the absence of OV, depletion of Bad by RNA interference conferred resistance to apoptosis. Hepatic injury after ischemia-reperfusion was alleviated by OV treatment, with significant inhibition of SEC apoptosis. SEC apoptosis in vivo was associated with dephosphorylation of Bad, Akt, and extracellular signal-regulated protein kinase, which was blocked by OV treatment. Our data suggest that maintenance of Bad phosphorylation is important in the prevention of SEC apoptosis and that the anti-apoptotic property of OV might have therapeutic utility.

Boosts with Highly Purified Stem Cells Can Improve Hematopoietic Regeneration after Allogeneic Transplantation from Alternative Donors.

In allogeneic transplantation it is a major goal to maintain a stable long term engraftment. However, several factors like viral infections, drug toxicity or low stem cell numbers may hamper hematopoietic recovery or may even cause a late graft failure after successful initial engraftment. Hematopoietic growth factors or additional stem cell donations (boosts) may help to overcome this problem. In this retrospective analysis, the efficacy of boosts with highly purified peripheral stem cells either from closely matched unrelated or from haploidentical related donors was evaluated in pediatric patients with stagnant hematopoiesis (<1500 leucocytes or <50 000 platelets between 30–90 days posttransplant, n=11) or with initially increasing and then decreasing hematopoiesis (n=3). Patients with Graft rejection or hemophagocytosis were excluded. Diagnoses were: acute leukemias (n=10), nonmalignatn diseases (n=4). Most boosts were taken from the original graft: if the grafts contained >10x106 (unrelated donors) or >20x106/kg BW CD34+ cells, surplus stem cells were purified with an immunomagnetic selection method using antiCD34 or antiCD133 coated microbeads and cryopreserved. Three haploidentical donors were asked for a second donation. Our aim was to maintain stable and complete hematopoiesis after transplantation without inducing acute or chronic GvHD.A total of 18 boosts were given between 30 and 290 days after transplantation with a median number of 5 (0.6–34) x 106 CD34+/CD133+ progenitor cells/kg and only 2200 (100–25000) residual T-cells/kg. All patients had a complete donor chimerism and no reconditioning or posttransplant pharmacological immunosuppression was administered. Patients who received G-CSF were excluded. Due to the low number of T cells, acute GvHD >grade I and also chronic GvHD could be completely avoided even in haploidentical donors.Leukocyte counts, neutrophile counts, lymphocyte counts (and platelet counts in patients with thrombocytopenia) within the week before infusion of the boosts were compared with cell counts 4 and 8 weeks after infusion, using the paired T-test. A significant increase of all cell counts was observed (medina numbers pre boosting: 900/μl, 312/μl, 142/μl; 4 weeks post boosting: 2065/μl, 1264/μl, 317/μl; 8 weeks post boosting:2570/μl, 1780/μl, 385/μl; p<0.05 for all comparisons). Furthermore, independence from platelet transfusion could be achieved.Conclusions: Boosts with purified stem cells and low T-cell contamination (<10000 T-cells/kg) can be administered after transplantation without the risk of inducing GvHD, even in mismatched donors. No pharmacological immunosuppression was necessary. Thus, the method represents a safe tool and can improve donor derived hematopoiesis.