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Why do B cells mutate their immunoglobulin receptors?

B cells have the unique ability to acquire large numbers of point mutations in the variable segment of rearranged immunoglobulin (Ig) genes during a germinal center reaction. It is broadly accepted that somatic hypermutation (SHM) and affinity maturation are required to generate memory B cells and to produce antibodies capable of accomplishing the host defense functions of the humoral component of the adaptive immune system. However, several studies illustrate that low-avidity interactions between antigen and the B-cell receptor can induce deletion, receptor editing and a T-dependent immune response, suggesting that the high-avidity binding of antigen is not essential. If enhanced antigen binding is not essential for immune responses, what is the purpose of SHM? An alternative benefit of SHM might be to enhance the ability of B cells to track antigens expressed by rapidly mutating microorganisms.

Antibodies↗

Isolation and tracking of a rare lymphoid progenitor cell which facilitates bone marrow transplantation in mice.

Bone marrow cells are composed of pluripotent stem cells to terminally differentiated cells, with a wide variety of abundance of each cell type. In the past, many of the cell types within this heterogeneous population have been characterized either by expression of specific proteins or using functional markers. In spite of promising results obtained with the latter method, various cell types within bone marrow have not been well characterized due to the low abundance of a specific cell type. Considering the demand for a reliable technique to enrich cell types, a wide variety of approaches, ranging from simple nylon wool columns to high-speed cell sorting, have evolved. Only limited success has been obtained with approaches ranging from the detection of MHC antigen to positron emission tomography to track the ontogeny of specific bone marrow-derived cells in studies of syngeneic or allogeneic transplantation. The present study describes a relatively simple method to enrich and track a rare bone marrow cell (facilitating cell, FC), which can facilitate allogeneic bone marrow stem cell transplantation in mice. The isolation technique is comprised of enrichment of FC by magnetic activated cell sorting (MACS) system followed by purification through high-speed cell sorter. An initial inoculation of 30,000 FC obtained from male mice was detected in the thymus, spleen, and bone marrow of allogeneic female recipients, by using 32P-labeled dCTP in a specific PCR for Y-chromosome. This technique may improve the efficiency of isolation of other rare cells from the bone marrow.

Animals↗

Inositol trisphosphate receptor calcium release is required for cerebral artery smooth muscle cell proliferation.

Vascular damage signals smooth muscle cells to proliferate, often exacerbating existing pathologies. Although the role of changes in "global" Ca2+ in vascular smooth muscle (VSM) cell dedifferentiation has been studied, the role of specific Ca2+ signals in determining VSM phenotype remains relatively unexplored. Earlier work with cultured VSM cells suggests that inositol 1,4,5-trisphosphate receptor (IP3R) expression and sarcoplasmic reticulum (SR) Ca2+ release may be linked to VSM cell proliferation in native tissue. Thus we hypothesized that SR Ca2+ release through IP3Rs in the form of discrete transient signals is necessary for VSM cell proliferation. To investigate this hypothesis, we used mouse cerebral arteries to design an organ culture system that permitted examination of Ca2+ dynamics in native tissue. Explanted arteries were cultured in normal medium with 10% FBS, and appearance of individual VSM cells migrating from explanted arteries (outgrowth cells) was tracked daily. Initial exposure to 10% FBS increased Ca2+ waves in myocytes in the arteries that were blocked by the IP3R antagonist 2-aminoethoxydiphenylborate (2-APB). Inhibition of IP3R opening (via 100 microM 2-APB, 10 microM xestospongin C, or 25 microM U-73122) dramatically reduced outgrowth cell number compared with untreated or ryanodine-treated (10 microM) arteries. Consistent with this finding, 2-APB inhibited cell proliferation, as measured by reduced proliferating cell nuclear antigen immunostaining within 48 h of culture but did not inhibit cell migration. These results indicate that activation of IP3R Ca2+ release is required for VSM cell proliferation in these arteries.

Animals↗

Directional cell migration establishes the axes of planar polarity in the posterior lateral-line organ of the zebrafish.

The proper orientation of mechanosensory hair cells along the lateral-line organ of a fish or amphibian is essential for the animal's ability to sense directional water movements. Within the sensory epithelium, hair cells are polarized in a stereotyped manner, but the mechanisms that control their alignment relative to the body axes are unknown. We have found, however, that neuromasts can be oriented either parallel or perpendicular to the anteroposterior body axis. By characterizing the strauss mutant zebrafish line and by tracking labeled cells, we have demonstrated that neuromasts of these two orientations originate from, respectively, the first and second primordia. Furthermore, altering the migratory pathway of a primordium reorients a neuromast's axis of planar polarity. We propose that the global orientation of hair cells relative to the body axes is established through an interaction between directional movement by primordial cells and the timing of neuromast maturation.

Animals↗

The bone marrow functionally contributes to liver fibrosis.

BACKGROUND & AIMS: Bone marrow (BM) cells may transdifferentiate into or fuse with organ parenchymal cells. BM therapy shows promise in murine models of cirrhosis, and clinical trials of bone marrow stem cell therapy for organ healing are underway. However, the BM may contribute to scar-forming myofibroblasts in various organs including the liver. We have studied this axis of regeneration and scarring in murine models of cirrhosis, including an assessment of the temporal and functional contribution of the BM-derived myofibroblasts. METHODS: Female mice were lethally irradiated and received male BM transplants. Carbon tetrachloride or thioacetamide was used to induce cirrhosis. BM-derived cells were tracked through in situ hybridization for the Y chromosome. BM transplants from 2 strains of transgenic mice were used to detect intrahepatic collagen production. RESULTS: In the cirrhotic liver, the contribution of BM to parenchymal regeneration was minor (0.6%); by contrast, the BM contributed significantly to hepatic stellate cell (68%) and myofibroblast (70%) populations. These BM-derived cells were found to be active for collagen type 1 transcription in 2 independent assays and could influence the fibrotic response to organ injury. These BM-derived myofibroblasts did not occur through cell fusion between BM-derived cells and indigenous hepatic cells but, instead, originated largely from the BM's mesenchymal stem cells. CONCLUSIONS: The BM contributes functionally and significantly to liver fibrosis and is a potential therapeutic target in liver fibrosis. Clinical trials of BM cell therapy for liver regeneration should be vigilant for the possibility of enhanced organ fibrosis.

Animals↗

Centripetal movement of corneal epithelial cells in the normal adult mouse.

PURPOSE: To study the natural movement of corneal epithelial cells in the normal adult mouse with histology and in vivo microscopy. METHODS: A transgenic mouse line that was engineered to ubiquitously express green fluorescent protein (GFP) was used to visualize corneal epithelial cells. For histology, epithelial GFP was imaged in a wholemounted cornea en face, and also in frozen cross-sections, under a fluorescence microscope. For in vivo studies, the anesthetized mouse was placed on a custom-made observation platform under a fluorescence microscope. Epithelial fluorescence was digitally recorded two to three times a week, and a rate of cell movement was determined from the time-lapse sequences. RESULTS: The GFP expression in the corneal epithelium was nearly ubiquitous up to about 1 week after birth, and thereafter it gradually became sporadic, resulting in a mosaic pattern of GFP positive cells, with the brightest cells present in the basal and suprabasal layer of the epithelium. Both high- and low GFP-cells formed radial streaks toward the central cornea, frequently displaying vortex patterns at the center. Clusters of several high-GFP cells were tracked in living mice for up to 7 weeks, and an analysis of time-lapse sequences revealed that they moved centripetally at an average rate of 26 micro m/d. CONCLUSIONS: Corneal epithelium of adult GFP mice exhibits a pattern of GFP expression that is suitable for studying cell movement in the normal cornea. Epithelial cells at the basal or suprabasal layers move centripetally in these mice at an average rate of 26 micro m/d.

Animals↗

Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons.

Recombinant adeno-associated viruses (rAAVs) are promising vectors for gene therapy since they efficiently and stably transduce a variety of tissues of immunocompetent animals. The major disadvantage of rAAVs is their limited capacity to package foreign DNA (< or =5 kb). Often, co-expression of two or more genes from a single viral vector is desirable to achieve maximal therapeutic efficacy or to track transduced cells in vivo by suitable reporter genes. The internal ribosome entry site (IRES) sequence of encephalomyocarditis virus has been widely used to construct bicistronic viral vectors. However, the IRES is rather long and IRES-mediated translation can be relatively inefficient when compared with cap-dependent translation. As an alternative to the IRES for in vivo gene expression, we studied the 16 amino-acid long 2A peptide of foot and mouth disease virus (FMDV). The 2A peptide mediates the primary cis-'cleavage' of the FMDV polyprotein in a cascade of processing events that ultimately generate the mature FMDV proteins. We have generated several different rAAV genomes in which two coding regions are fused in-frame via the FMDV 2A sequence. We show that FMDV 2A efficiently mediates the generation of the expected cleavage products from the artificial fusion proteins in cells. Furthermore, we find that both EGFP and alpha- synuclein are expressed at substantially higher levels from 2A vectors than from the corresponding IRES-based vectors, while SOD-1 is expressed at comparable or slightly higher levels. Finally, we demonstrate for the first time, that the 2A sequence results in effective bicistronic gene expression in vivo after injection of 2A-dependent rAAVs into the rat substantia nigra. We conclude that 2A-containing rAAVs may represent an attractive alternative to IRES-dependent vectors for ex vivo and in vivo gene expression and gene therapy.

Animals↗

Optical imaging of Renilla luciferase reporter gene expression in living mice.

Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imaging research. Studies have validated the use of reporter genes with positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI, fluorescence with wild-type and mutants of green fluorescent protein, as well as bioluminescence using Firefly luciferase enzyme/protein (FL). In the current study, we validate for the first time the ability to image bioluminescence from Renilla luciferase enzyme/protein (RL) by injecting the substrate coelenterazine in living mice. A highly sensitive cooled charge-coupled device camera provides images within a few minutes of photon counting. Cells, transiently expressing the Rluc were imaged while located in the peritoneum, s.c. layer, as well as in the liver and lungs of living mice tail-vein injected with coelenterazine. Furthermore, d-luciferin (a substrate for FL) does not serve as a substrate for RL, and coelenterazine does not serve as a substrate for FL either in cell culture or in living mice. We also show that both Rluc and Fluc expression can be imaged in the same living mouse and that the kinetics of light production are distinct. The approaches validated will have direct applications to various studies where two molecular events need to be tracked, including cell trafficking of two cell populations, two gene therapy vectors, and indirect monitoring of two endogenous genes through the use of two reporter genes.

Animals↗

Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling.

Luminous proteins include primary light producers, such as aequorin, and secondary photoproteins that in some organisms red-shift light emission for better penetration in space. When expressed in heterologous systems, both types of proteins may act as versatile reporters capable of monitoring phenomena as diverse as calcium homoeostasis, protein sorting, gene expression, and so on. The Ca(2+)-sensitive photoprotein aequorin was targeted to defined intracellular locations (organelles, such as mitochondria, endoplasmic reticulum, sarcoplasmic reticulum, Golgi apparatus and nucleus, and cytoplasmic regions, such as the bulk cytosol and the subplasmalemmal rim), and was used to analyse Ca(2+) homoeostasis at the subcellular level. We will discuss this application, reviewing its advantages and disadvantages and the experimental procedure. The applications of green fluorescent protein (GFP) are even broader. Indeed, the ability to molecularly engineer and recombinantly express a strongly fluorescent probe has provided a powerful tool for investigating a wide variety of biological events in live cells (e.g. tracking of endogenous proteins, labelling of intracellular structures, analysing promoter activity etc.). More recently, the demonstration that, using appropriate mutants and/or fusion proteins, GFP fluorescence can become sensitive to physiological parameters or activities (ion concentration, protease activity, etc.) has further expanded its applications and made GFP the favourite probe of cell biologists. We will here present two applications in the field of cell signalling, i.e. the use of GFP chimaeras for studying the recruitment of protein kinase C isoforms and the activity of intracellular proteases.

Aequorin↗

Rapid flow of passive neutrophils into a 4 microns pipet and measurement of cytoplasmic viscosity.

Neutrophils from five different individuals are isolated with a density separation technique. A total of 151 unactivated (passive) cells are rapidly aspirated at constant suction pressure and at room temperature into a pipet with a diameter of 4 microns. The suction pressures in excess of an initial yield threshold are 0.5, 1 and 2 kPa and are comparable to those encountered in the microcirculation. These pressures are well in excess of the small suction pressure of approximately 20 Pa that is required to form a static hemispherical bump on the cell. At a given aspiration pressure, the leading edge of an individual cell is "tracked" as it flows into the pipet. A theory based on the flow of a Newtonian liquid from either a hemisphere or a spherical segment into a cylinder is used to model the entry process. Both theory and experiment show that during most of the entry process the leading edge of the cell moves at a nearly constant velocity with a rapid acceleration at the end. For cells from five different individuals at the three different excess aspiration pressures, Newtonian theory gives a cytoplasmic viscosity of 135 +/- 54 Pa.s and overall entry times of 3.3s (0.5 kPa), 1.6s (1 kPa) and 0.82s (2 kPa). These results and those of Evans and Yeung at lower aspiration pressures indicate that the complex cytoplasm inside unactivated neutrophils behaves as a nearly Newtonian fluid with a viscosity on the order of 10(2) Pa.s over almost a two order of magnitude range in aspiration pressure and, thus, rate of deformation.

Cytoplasm↗

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering.

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34 + lin hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well can receive a single hematopoietic stem cell candidate. Sequential movement of each cell-containing well to a microscopic imaging system, serially over a several-day to several-week experiment, is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Bio-box built on the microscope stage. New image analysis software facilitates in the tracking of cell movement, recording of the time of cell division, and immunophenotyping of each of multiple individual or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and also may be applicable to a wide range of other systems of interest in tissue engineering.

Biotechnology↗

Orientation of the photosynthetic flagellate, Peridinium gatunense, in hypergravity.

The photosynthetic freshwater flagellate, Peridinium gatunense, uses both positive phototaxis and negative gravitaxis to move upwards in the water column. At higher fluence rates approaching those at the surface of their habitat, the cells tend to become unoriented and thus stop their upward movement. Orientation and motility of Peridinium gatunense has been studied in the slow rotating centrifuge microscope (NIZEMI), which allows observation of swimming behavior during centrifugation acceleration between 1 g and 5g. The movement vectors were analyzed by real time image analysis capable of tracking many cells simultaneously. At 1 g the orientation was not very precise, but the degree of orientation increased significantly at higher acceleration forces up to about 3 g. Most cells were capable of swimming even against an acceleration vector of 3.8 g; at higher acceleration forces the cells were not able to cope with the centrifugal force. The linear velocity of cells swimming against 1 g was about 20% lower than that of cells moving in other directions. The velocity decreased even more in cells swimming against higher acceleration forces.

Animals↗

Intramyocardial delivery of human CD133+ cells in a SCID mouse cryoinjury model: Bone marrow vs. cord blood-derived cells.

OBJECTIVE: The regenerative potential of endothelial and hematopoietic progenitor cells in the heart may vary according to their origin. This study was designed to compare the functional effects of CD133+ cells from human cord blood and bone marrow in a mouse model of myocardial injury. METHODS: 5 x 10(5) CD133+ cells from bone marrow (BM(CD133)) or cord blood (UCB(CD133)) were injected in the necrosis border zone of NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice with left ventricular cryoinjury (CI+). Transplanted cells were tracked by immunostaining for hNuclear antigen and by PCR for hDNA. Echocardiography was used to measure contractility. Scar size, capillary density, and cardiomyocyte apoptosis were evaluated by histology. In addition, the myogenic and endothelial differentiation capacity of BM(CD133) and UCB(CD133) was compared in vitro. RESULTS: DNA was detected 4 weeks after cell injection by PCR, but hNuc+ cells were found by immunostaining only after 48 h. Capillary density in both BM(CD133) and UCB(CD133) cell-treated CI+ mice was higher than in control CI+ mice, but not different between BM(CD133) and UCB(CD133) cell-treated hearts. There were no differences in scar size and myocardial mass among BM(CD133), UCB(CD133) and control CI+ mice, but cardiomyocyte apoptosis was reduced by both BM(CD133) and UCB(CD133) cells. The post-injury deterioration of shortening fraction (46.2+/-1% in sham-operated mice and 41.3+/-0.8% in control CI+ mice) was prevented by BM(CD133) cells (45.4+/-0.9%), but not by UCB(CD133) cells (40.8+/-0.7%). On the other hand, both BM(CD133) and UCB(CD133) cells abolished post-injury mortality. In vitro, neither cultivated BM(CD133) or UCB(CD133) cells developed into myocytes, but both readily differentiated towards an endothelial cell phenotype. CONCLUSIONS: While both cord blood and marrow CD133+ cells have some beneficial effects on post-injury angiogenesis and survival, only marrow cells appear to improve myocardial contractility.

AC133 Antigen↗

Understanding living clathrin-coated pits.

Most knowledge of clathrin-mediated endocytosis has been gained by biochemical fractionation and in vitro assays. Recently, the study of endocytosis has extended into the living cell. The tracking of individual clathrin-coated pits and vesicles (CCPs and CCVs) has provided new insight into understanding the dynamic nature of CCPs. The use of total internal reflection fluorescence microscopy (TIR-FM), also termed evanescent field microscopy, has enabled the direct observation of events occurring within a restricted area of the cell adjacent to and including the adherent plasma membrane. TIR-FM is now actively being pursued in the study of endocytic processes. The direct observation of CCP-associated proteins including clathrin itself, dynamin and, most recently, AP-2 has considerably challenged old models, confirming some points but raising very interesting new questions.

Adaptor Protein Complex 2↗

Multicolor Cell Lineage Tracing Using MAGIC Markers Strategies.

Multicolor MAGIC Markers strategies are useful lineage tracing tools to study brain development at a multicellular scale. In this chapter, we describe an in utero electroporation method to simultaneously label multiple neighboring progenitors and their respective progeny using these multicolor reporters. In utero electroporation enables the introduction of any gene of interest into embryonic neural progenitors lining the brain ventricles through a simple pipeline consisting of a micro-injection followed by the application of electrical pulses. Successful in utero electroporation requires a concise yet complete understanding of each step of the surgical protocol, spanning from the preoperative preparation to the postoperative care, as well as the MAGIC Markers tool outlined in this study. Besides a detailed protocol, we present non-integrative and integrative approaches to demonstrate the range of cell and lineage tracking possibilities of multicolored progenitors and their descent&#xa0;over time.

Cell Lineage↗

Compensatory and excess retrieval: two types of endocytosis following single step depolarizations in bovine adrenal chromaffin cells.

1. Endocytosis following exocytosis evoked by single step depolarizations was examined in bovine adrenal chromaffin cells using high resolution capacitance measurements in perforated-patch voltage clamp recordings. 2. Endocytosis was detected as a smooth exponential decline in membrane capacitance to either the pre-stimulus level ('compensatory retrieval') or far below the pre-stimulus level ('excess retrieval'). During excess retrieval, > 10% of the cell surface could be internalized in under 5 s. 3. Compensatory retrieval was equal in magnitude to stimulus-evoked exocytosis for membrane additions > 100 fF (about fifty large dense-cored vesicles). In contrast, excess retrieval surpassed both the stimulus-evoked exocytosis, and the initial capacitance level recorded at the onset of phase-tracking measurements. Cell capacitance was not maintained at the level achieved by excess retrieval but slowly returned to pre-stimulus levels, even in the absence of stimulation. 4. A large percentage of capacitance increases < 100 fF, usually evoked by 40 ms depolarizations, were not accompanied by membrane retrieval. 5. Compensatory retrieval could occur with any amount of Ca2+ entry, but excess retrieval was never triggered below a threshold Ca2+ current integral of 70 pC. 6. The kinetics of compensatory and excess retrieval differed by an order of magnitude. Compensatory retrieval was usually fitted with a single exponential function that had a median time constant of 5.7 s. Excess retrieval usually occurred with double exponential kinetics that had an extremely fast first time constant (median, 670 ms) and a second time constant indistinguishable from that of compensatory retrieval. 7. The speed of compensatory retrieval was Ca2+ dependent: the largest mono-exponential time constants occurred for the smallest amounts of Ca2+ entry and decreased with increasing Ca2+ entry. The Ca2+ dependence of mono-exponential time constants was disrupted by cyclosporin A (CsA), an inhibitor of the Ca(2+)- and calmodulin-dependent phosphatase calcineurin. 8. CsA also reduced the proportion of responses with excess retrieval, but this action was caused by a shift in Ca2+ entry values below the threshold for activation. The lower total Ca2+ entry in the presence of CsA was due to an increase in the rate of Ca2+ current inactivation rather than a reduction in peak amplitude. 9. Our data suggest that compensatory and excess retrieval represent two independent, Ca(2+)-regulated mechanisms of rapid membrane internalization in bovine adrenal chromaffin cells. Alternatively, there is a single membrane internalization mechanism that can switch between two distinct modes of behaviour.

Adrenal Glands↗

Cell cycle status affects coxsackievirus replication, persistence, and reactivation in vitro.

Enteroviral persistence has been implicated in the pathogenesis of several chronic human diseases, including dilated cardiomyopathy, insulin-dependent diabetes mellitus, and chronic inflammatory myopathy. However, these viruses are considered highly cytolytic, and it is unclear what mechanisms might permit their long-term survival. Here, we describe the generation of a recombinant coxsackievirus B3 (CVB3) expressing the enhanced green fluorescent protein (eGFP), which we used to mark and track infected cells in vitro. Following exposure of quiescent tissue culture cells to either wild-type CVB3 or eGFP-CVB3, virus production was very limited but increased dramatically after cells were permitted to divide. Studies with cell cycle inhibitors revealed that cells arrested at the G(1) or G(1)/S phase could express high levels of viral polyprotein and produced abundant infectious virus. In contrast, both protein expression and virus yield were markedly reduced in quiescent cells (i.e., cells in G(0)) and in cells blocked at the G(2)/M phase. Following infection with eGFP-CVB3, quiescent cells retained viral RNA for several days in the absence of infectious virus production. Furthermore, RNA extracted from nonproductive quiescent cells was infectious when transfected into dividing cells, indicating that CVB3 appears to be capable of establishing a latent infection in G(0) cells, at least in tissue culture. Finally, wounding of infected quiescent cells resulted in viral protein expression limited to cells in and adjacent to the lesion. We suggest that (i) cell cycle status determines the distribution of CVB3 during acute infection and (ii) the persistence of CVB3 in vivo may rely on infection of quiescent (G(0)) cells incapable of supporting viral replication; a subsequent change in the cell cycle status may lead to virus reactivation, triggering chronic viral and/or immune-mediated pathology in the host.

Cell Cycle↗

In vivo MR imaging of tissue-engineered human mesenchymal stem cells transplanted to mouse: a preliminary study.

Current progress integrating stem cell biology and tissue engineering techniques has been invaluable to clinical applications. Prior to the application of cellular transplantation technique to patients, we need to establish techniques that can monitor their tissue biodistribution non-invasively. In this study, we proposed an imaging modality using MRI to not only monitor implanted scaffold in vivo, but also to track transplanted cells and behavior around the implant. For this purpose, human bone marrow-derived mesenchymal stem cells (hMSCs) were labeled with superparamagnetic iron oxide (Feridex) and then labeled hMSCs were cultured in a gelatin sponge used as a scaffold to support cell growth and proliferation. Histological assessment and MTT assay showed that cell labeling with MR contrast agent did not harm cell viability. Also, Feridex-labeled hMSCs showed a significant decrease in T2 signal intensity, even within the gelatin sponge in vitro. After implanting the sponge/cell complex in vivo, we could visualize cellular behavior around the implant over time using a noninvasive MRI modality and this finding was correlated with histological study, which illustrates the potential of a new approach proposed here for in vivo monitoring of implanted cell-based tissue-engineered product.

Animals↗