A fast and highly sensitive method for detecting freemartinism in bovine twins using immunomagnetic beads and Y-specific PCR primers

A method for detecting male cells in the blood of the female calf in bovine heterosexual twin pregnancies has been established. Nucleated cells were isolated from full blood by immunomagnetic separation, lysed by boiling and then subjected to polymerase chain reaction (PCR) amplification with Y chromosome specific primers. Diagnosis was achieved within one day. The method was successfully used on blood samples that had been stored at +4°C for more than one month. Dilution of male blood in female blood showed that XY cells were detectable down to a concentration of 0.1%. This method should be amenable to automatization and can be adapted to any PCR-based genetic test.     

Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array

Here we report a new method for isolating antigen-specific antibody-secreting cells (ASCs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ASCs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. This protocol can be completed in less than 7 h, including 3 h of cell culture. The method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (ELISPOT) but it also overcomes the limitations of ELISPOT in recovering ASCs that can be used to produce antigen-specific human monoclonal antibodies. This method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis.     

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.     

Construction of a panel of glucose indicator proteins for continuous glucose monitoring

The development of implantable glucose sensors for use in diabetes treatment has been pursued for decades. However, enzyme-based glucose sensors often fail in vivo. In our previous work, we engineered a novel glucose indicator protein (GIP) that can sense glucose without relying on any enzymes and cofactors. Nevertheless, this GIP is unsuitable for blood glucose monitoring due to its low dissociation constant. Here, we report a novel approach to creating a new GIP that can be used to monitor blood glucose level. By disrupting pi-pi stacking around GIP's glucose binding site through site-directed mutagenesis, we showed that GIP's dissociation constant can be manipulated from 0.026 mM to 7.86 mM. This approach yielded four GIP mutants. We showed that one of the mutants can be used to detect glucose from 0 to 32 mM, while another mutant can be employed to visualize intracellular glucose (0-200 μM) within living cells through FRET imaging microscopy measurement.     

Cell differentiation and malignancy

An understanding of the mechanism that controls growth and differentiation in normal cells would seem to be an essential requirement to elucidate the origin and reversibility of malignancy. For this approach I have mainly used normal and leukemic blood cells, and in most studies have used myeloid blood cells as a model system. Our development of systems for the in vitro cloning and clonal differentiation of normal blood cells made it possible to study the controls that regulate growth (multiplication) and differentiation of these normal cells and the changes in these controls in leukemia. Experiments with normal blood cell precursors have shown that normal cells require different proteins to induce growth and differentiation. We have also shown that in normal myeloid precursors, growth-inducing protein induces both growth and production of differentiation-inducing protein so this ensures the coupling between growth and differentiation that occurs in normal development. The origin of malignancy involves uncoupling of growth and differentiation. This can be produced by changes from inducible to constitutive expression of specific genes that result in asynchrony to the coordination required for the normal developmental program. Normal myeloid precursors require an external source of growth-inducing protein for growth, and we have identified different types of leukemic cells. Some no longer require and other constitutively produce their own growth-inducing protein. But addition of the normal differentiation-inducing protein to these malignant cells still induces their normal differentiation, and the mature cells are then no longer malignant. Genetic changes that produce blocks in the ability to be induced to differentiate by the normal inducer occur in the evolution of leukemia. But even these cells can be induced to differentiate by other compounds, including low doses of compounds now being used in cancer therapy, that induce the differentiation program by other pathways. This differentiation of leukemic cells has been obtained in vitro and in vivo, and our in vivo results indicate that this may be a useful approach to therapy. In some tumours, such as sarcomas, reversion from a malignant to a non-malignant phenotype can be a result of chromosome changes that suppress malignancy. But in myeloid leukemia, the stopping of growth in mature cells by induction of differentiation bypasses the genetic changes that produce the malignant phenotype. These conclusions can also be applied to other types of normal and malignant cells.     

Co-cultivation of whole blood from male and female muntjacs and the cell proliferation kinetics in vitro

A mixed blood culture (MBC) of heparinized whole blood from male and female Indian muntjac has been done using the BrdU-Hoechst-sunlight-Giemsa method to study the cell-cycle kinetics in vitro. Blood lymphocytes of both male and female muntjacs show a much shorter cell cycle time, roughly, 10-12 h for the initial but only 8 h for the subsequent cycles. There is a significant difference in the rate of cell proliferation between male and female cells. The male blood cells constitute a majority of the 'slow'-dividing cells which reach a peak at the first cycle of mitosis at 40 h, whereas a similar peak of first cycle mitosis is reached by female cells at 32 h, indicating the occurrence of a high frequency of 'fast' dividing female cells as compared to those of males. This novel sex-based differential cell proliferation kinetics is observed both in mixed and separate cultures. This type of MBC method which is free of interculture variations can be reliably used for comparative studies where two genomes can be distinguished.     

Rapid isolation of viable circulating tumor cells from patient blood samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of >50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.     

Delivering cellular therapies: lessons learned from ex vivo culture and clinical applications of hematopoietic cells

Advances in stem cell biology and cellular therapy have led to promising treatments in a range of incurable diseases. However, it is unclear whether primitive stem cells can be delivered to damage tissue for regeneration of functional mature cells or stem cells must be stimulated to differentiate into mature cells in vitro and these cells delivered to patients. A range of other questions remains to be determined including how to formulate cellular products for in vivo delivery and how to undertake pharmacological testing of cellular products. Insights into these questions can be obtained from hematopoietic stem cells (HSC) which have been used for the past 50 years in bone marrow transplantation for regeneration of blood cells in patients undergoing high dose chemotherapy to treat cancer. The differentiation of HSC into mature blood cells is controlled by proteins called hematopoietic growth factors and these factors have been used to generate cellular products in vitro for clinical applications. This chapter will review some of the results of cellular therapies performed with HSC and the lessons that can be learned from these studies.     

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-γ). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.     

Prisoner's dilemma in cancer metabolism

As tumors outgrow their blood supply and become oxygen deprived, they switch to less energetically efficient but oxygen-independent anaerobic glucose metabolism. However, cancer cells maintain glycolytic phenotype even in the areas of ample oxygen supply (Warburg effect). It has been hypothesized that the competitive advantage that glycolytic cells get over aerobic cells is achieved through secretion of lactic acid, which is a by-product of glycolysis. It creates acidic microenvironment around the tumor that can be toxic to normal somatic cells. This interaction can be seen as a prisoner's dilemma: from the point of view of metabolic payoffs, it is better for cells to cooperate and become better competitors but neither cell has an incentive to unilaterally change its metabolic strategy. In this paper a novel mathematical technique, which allows reducing an otherwise infinitely dimensional system to low dimensionality, is used to demonstrate that changing the environment can take the cells out of this equilibrium and that it is cooperation that can in fact lead to the cell population committing evolutionary suicide.     

Plasmodium-infected blood cells analyzed and sorted by flow fluorimetry with the deoxyribonucleic acid binding dye 33258 Hoechst.

Red cells from Plasmodium berghei infected mouse blood can be sorted on the basis of their DNA content with the bisbenzimidazole dye 33258 Hoechst. The optimal conditions for dye uptake have been established and with these conditions uninfected cells are nonfluorescent and can be completely separated from infected cells which exhibit fluorescence in almost direct proportion to the number of parasite nuclei (i.e. DNA) they contain. The number of fluorescent cells detected and their fluorescence intensity is shown to be dependent on the dye concentration and the incubation medium being used. At least a proportion of the infected cells sorted from each fluorescence peak in the cell distribution retain their infectivity in vivo with some, but not all, conditions of labeling. This technique is being used to separate minor cell populations from infected blood for biochemical and immunochemical analyses and to screen human samples for malaria infected cells.     

Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.     

From anhydrobiosis to freeze-drying of eukaryotic cells

Using what has been learned from nature, it has become possible to stabilize biological structures, including intact cells, in the dry state. Stabilization of cells or tissues in the dried state is of considerable practical significance, as is described in this review. The need for stabilization of cells in the dried state is particularly urgent in bloodbanks, where proper storage of blood cells (platelets and erythrocytes) is still a major problem. Human blood platelets are stored in blood banks for 5 days, after which they are discarded according to Federal regulation. This short lifetime has led to a chronic shortage of platelets. We report here that platelets can be preserved by freeze-drying them with trehalose, a sugar found at high concentrations in organisms that naturally survive drying. We suggest that this finding will obviate the storage problem with platelets and that the principles established here may be extended to more complex eukaryotic cells.     

RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication.

A key feature of the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection is the gradual loss of CD4-positive T cells. A number of gene therapy strategies have been designed with the intent of inhibiting HIV replication in mature T cells. As T cells are products of hematolymphoid differentiation, insertion of antiviral genes into hematopoietic stem cells could serve as a vehicle to confer long-term protection in progeny T cells derived from transduced stem cells. One such "cellular immunization" strategy utilizes the gene coding for the HIV-1 rev trans-dominant mutant protein RevM10 which has been demonstrated to inhibit HIV-1 replication in T-cell lines and in primary T cells. In this study, we used a Moloney murine leukemia virus-based retrovirus encoding a bicistronic message coexpressing RevM10 and the murine CD8-alpha' chain (Lyt2). This vector allows rapid selection of transgene-expressing cells as well as quantitation of transgene expression. We demonstrate that RevM10-transduced CD34-enriched hematopoietic progenitor-stem cells (HPSC) isolated from human umbilical cord blood or from granulocyte colony-stimulating factor-mobilized peripheral blood can give rise to mature thymocytes in the SCID-hu thymus/liver mouse model. The phenotypic distribution of HPSC-derived thymocytes is normal, and expression of the transgene can be detected by flow cytometric analysis. Moreover, we demonstrate that RevM10 can inhibit HIV replication in T cells derived from transduced HPSC after expansion in vitro. This is the first demonstration of anti-HIV efficacy in T cells derived from transduced human HPSC.     

华南虎血常规两种测定方法的比较应用

通过全自动血细胞分析仪与人工镜检法分别对华南虎(Panthera tigris amoyensis)血常规进行分析,并对获得结果做比较分析,探究建立适合华南虎血细胞分析的方法。收集临床华南虎血液标本40份,分别使用全自动血液分析仪和人工显微镜检法对红细胞、白细胞进行计数检测,并对白细胞进行分类分析。如人工计数和仪器计数获得的数据均符合正态分布,则应用配对样本T检验比较差异性,否则应用非参数检验(两个相关样本:Wilcoxon带符号秩检验)。相关性采用Spearman分析,以P <0.05为差异有统计学意义。结果表明,通过两种方法获得的红细胞计数、白细胞计数、中性粒细胞比率、嗜酸性粒细胞比率结果差异均不显著(P> 0.05);淋巴细胞比率、单核细胞比率和嗜碱性粒细胞比率,两种方法检测结果具有显著性差异(P <0.05)。红细胞计数(r=0.915)、白细胞计数(r=0.832)、淋巴细胞比率(r=0.832)、中性粒细胞比率(r=0.481)应用两种方法获得的结果相关性均较好。单核细胞比率(r=0.283)、嗜酸性粒细胞比率(r=0.309)、嗜碱性粒细胞比率(r=0....     

Use of some snake venoms in the diagnostics of paroxysmal nocturnal hemoglobinuria (PNH)

The crude toxin of Agkistrodon piscivorus was found to produce hemolysis of PNH erythrocytes by way of complement activation, however the degree of PNH blood cell lysis was lower than in other techniques used. The crude toxins of Naja naja and Naja oxiana cause much higher hemolysis though lower hemolysis takes place in normal blood cells. In some normal persons and different blood diseases it is responsible for high hemolysis even when the complement is absent. From Naja naja toxin a fraction can be isolated which has a "specific" complement effect only on PNH blood cells. This fraction can thus be utilized in a specific test for this disease.     

Flow cytometric determination of PMCA-mediated Ca2+-extrusion in individual red blood cells.

BACKGROUND: Differences among red blood cells in the activity of the plasma membrane Ca2+-ATPase (PMCA) can impact cell signaling and survival. However, no method has been reported that measures this activity directly in individual cells. METHODS: We have designed a novel assay for PMCA activity that uses the fluorescent Ca2+-reporter Fluo4 and flow cytometric analysis. The method recognizes the extrusion of Ca2+ from the cell after a short Ca2+-loading pulse, which avoids the problem of ATP depletion and ascertains activity at Vmax capacity. RESULTS: Our assay is responsive to known PMCA inhibitors, and while not intended for quantitative kinetic analysis of Ca2+-pumping, it can be used to determine qualitative differences between red blood cell populations that vary in PMCA activity. Using this assay, we confirmed that a normal red blood cell population shows heterogeneity with respect to the PMCA Vmax. CONCLUSION: We report a novel assay of PMCA activity in red blood cells that can provide qualitative information on PMCA activity in individual cells.     

Transplantation of hematopoietic stem cells from the peripheral blood

Hematopoietic stem cells can be collected from the peripheral blood. These hematopoietic stem cells (HSC), or better progenitor cells, are mostly expressed as the percentage of cells than react with CD34 antibodies or that form colonies in semi-solid medium (CFU-GM). Under steady-state conditions the number of HSC is much lower in peripheral blood than in bone marrow. Mobilization with chemotherapy and/or growth factors may lead to a concentration of HSC in the peripheral blood that equals or exceeds the concentration in bone marrow. Transplantation of HSC from the peripheral blood results in faster hematologic recovery than HSC from bone marrow. This decreases the risk of infection and the need for blood-product support. For autologous stem-cell transplantation (SCT), the use of peripheral blood cells has completely replaced the use of bone marrow. For allogeneic SCT, on the other hand, the situation is more complex. Since peripheral blood contains more T-lymphocytes than bone marow, the use of HSC from the peripheral blood increases the risk of graft-versus-host disease after allogeneic SCT. For patients with goodrisk leukemia, bone marrow is still preferred, but for patients with high-risk disease, peripheral blood SCT has become the therapy of choice.     

Dendritic cells derived from pluripotent stem cells:Potential of large scale production

Human pluripotent stem cells(hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are promising sources for hematopoietic cells due to their unlimited growth capacity and the pluripotency. Dendritic cells(DCs), the unique immune cells in the hematopoietic system, can be loaded with tumor specific antigen and used as vaccine for cancer immunotherapy. While autologous DCs from peripheral blood are limited in cell number, hPSC-derived DCs provide a novel alternative cell source which has the potential for large scale production. This review summarizes recent advances in differentiating hPSCs to DCs through the intermediate stage of hematopoietic stem cells. Step-wise growth factor induction has been used to derive DCs from hPSCs either in suspension cultureof embryoid bodies(EBs) or in co-culture with stromal cells. To fulfill the clinical potential of the DCs derived from hPSCs, the bioprocess needs to be scaled up to produce a large number of cells economically under tight quality control. This requires the development of novel bioreactor systems combining guided EB-based differentiation with engineered culture environment. Hence, recent progress in using bioreactors for hPSC lineage-specific differentiation is reviewed. In particular, the potential scale up strategies for the multistage DC differentiation and the effect of shear stress on hPSC differentiation in bioreactors are discussed in detail.     

Predicting therapy response in live tumor cells isolated with the flexible micro spring array device

Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response. Here, we demonstrate how the flexible micro spring array (FMSA) device—a novel microfluidic device that enriches CTCs by two physical parameters: size and deformability—could be used in the rational development of treatment intervention and as a method to study the fundamental biology of CTCs. Cancer cells of different origins were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked human blood was filtered using the FMSA device, and the recovered cells were successfully expanded in vitro and in a novel in vivo system. A series of experiments were performed to characterize these cells and to investigate the effect of chemotherapy on the resulting cultures. As few as 20 colon cancer cells in 7.5 mL blood could be isolated with the FMSA device, expanded both in vitro and in vivo and used at 25 cells per well to obtain significant and reliable chemosensitivity data. We also show that isolating a low number of viable patient CTCs and maintaining them in culture for a few weeks is possible. The isolation of viable cancer cells from human blood using the FMSA device provides a novel and realistic means for studying the biology of viable CTCs and for testing drug efficacy on these rare cells—a hypothesis that can be tested in future clinical trials.