Expression level of a transgenic lambda2 chain results in isotype exclusion and commitment to B1 cells

Two new lambda2 chain-transgenic mouse lines were established, both of which showed stable transgene expression during aging of the mice. The line L23, which expressed the transgene at low levels, exhibited normal B cell development, antibody responses and serum Ig levels. Most of the B cells in this mouse line co-expressed the transgenic lambda2 chain together with an endogenous kappa chain, thus showing poor allelic exclusion of endogenous L chains. On the other hand, high expression of the transgenic lambda2 chain in the other mouse line, L2, resulted in nearly complete exclusion of endogenous L chain isotypes. In this line, the lambda2 transgene was already detectable in the cytoplasm of all preB-II cells and some pro/preB-I cells. Its expression during these early phases obviously inhibited development of conventional B2 cells, since the B cells in the periphery of these mice were almost exclusively of the B1 type. This finding was confirmed by adoptive transfer of transgenic bone marrow into lethally irradiated recipients. Very few B cells were present in the spleen of such recipients. The serum IgM levels of L2 mice were close to normal and the majority of these IgM were associated with the transgenic lambda2 chain. Antibody responses to thymus-dependent antigens in such mice were almost exclusively found to be of IgM class. Together, these findings indicate a developmental bias leading to a predominance of B1 cells in the L2 line.     

Precursor B cells showing H chain allelic inclusion display allelic exclusion at the level of pre-B cell receptor surface expression

Within the broader framework of facilitating investigations into the inherent responses of restricted neuronal phenotypes devoid of their in vivo afferents, serum- and steroid-free cultures enriched in corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), and beta-endorphin (beta-END) peptidergic neurons were prepared from the hypothalamic paraventricular (PVN: CRH and AVP) and/or arcuate (ARC: beta-END) nuclei of juvenile male rats. The functional viability of these ARC/PVN cultures was verified by their ability to synthesize and secrete CRH, AVP, and beta-END under basal and depolarizing (veratridine) conditions in vitro. Peptide secretion was shown to be Ca2+ and Na+ dependent in that it was blocked in the presence of verapamil and tetrodotoxin, respectively. Exposure of ARC/PVN cocultures to the glucocorticoid dexamethasone (DEX) resulted in a dose-dependent increase of CRH secretion and an inhibition of AVP and beta-END; the CRH responses deviated strikingly from predictions based on in vivo experiments. Steroid withdrawal or treatment with the glucocorticoid receptor antagonist RU38486 reversed these trends. Opposite effects of DEX on CRH secretion were observed in cultures consisting of PVN cells only. Supported by studies using an opioid receptor agonist (morphine) and antagonist (naloxone), these observations demonstrate that ARC-derived (beta-END) neurons modulate the responses of PVN neurons to DEX.     

Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci

CD19+CD10+ human B lineage bone marrow cells were separated into cycling or resting cells, which differ in their expression of CD34, VpreB, recombination activating gene (RAG-1), and terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for DHJH and VkJk, VkJkK(de) and VkK(de) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B lymphocyte pathway of development as follows: CD34+VpreB+RAG-1+TdT+, DHJH-rearranged, kL germline cycling pre-B I cells-->CD34-VpreB+microH chain+ (pre-B receptor+) RAG-1-TdT-, VHDHJH-rearranged, kL germline, cycling pre-B II cells-->CD34-VpreB-, intracytoplasmic microH chain+ (pre-B receptor-) RAG-1+/-TdT-, VHDHJH-rearranged, mainly kL germline cycling pre-B II cells-->CD34-VpreB-intracytoplasmic microH chain+, RAG-1+TdT-, VHDHJH-rearranged, VkJk-rearranged, IgM-, resting pre-B II cells CD34+VpreB-, sIgM+, RAG-1+TdT-, VHDHJH- and VkJk-rearranged IgM+ immature B cells-->CD34-, CD10-, sIgM+/sIgD+ mature B cells. This order, for the first time established for human B lineage cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.     

Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52(-/-) ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.     

Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity

The transit of immature to mature sIgM+ B cells, the life span, maturation kinetics and response to polyclonal activators have been analyzed with the help of a new mAb (493), that distinguishes immature, 493+ from mature, 493 B cells in a variety of mouse strains tested. Analysis of the turnover of immature 493+ B cells by bromodeoxyuridine (BrdU) labeling kinetics indicate that only 10-20 % of the cells reach the spleen as immature 493+ cells. The life span of 493+ B cells in bone marrow and spleen is around 4 days. BrdU chase experiments show that most of the immature cells in spleen enter the pool of mature, 493+ B cells where they gain a longer life span of 15-20 weeks. Immature and mature B cells respond equally well to LPS stimulation; anti-CD40, however, stimulates mature B cells better than immature B cells. IgM cross-linking of mature B cells results in proliferation, while it induces apoptosis in immature B cells. This apoptosis of immature cells can be inhibited by costimulation with anti-CD40 or by overexpression of bcl-2. We speculate that Ig receptor ligand-mediated apoptosis (negative selection) plays a major role in the transit of immature B cells from bone marrow to spleen, but only a minor role in the transit from immature B cells to mature B cells in the spleen.     

Investigation of Wear and Corrosion Protection of AlSi20 Coatings Produced by Plasma Spraying and Laser Cladding on AZ31B

Magnesium and magnesium alloys are the lightest structural materials with an approximate density of 1.7 g/cm² (density of aluminum ~2.7 g/cm²). Due to poor corrosion and wear resistance properties, they need to be coated for usage in service conditions under corrosive and tribological loads. AlSi20 was found to be a suitable coating material to improve the wear and corrosion protection properties of magnesium alloys. Within this work, AlSi20 coatings were applied by plasma spraying, laser cladding, and a combination of both processes. First, the coatings are characterized by their microhardness and residual stresses formed within the coating during the different coating processes. Then, these coatings were investigated regarding corrosion resistance in 3.5% sodium chloride solution in a three-electrode setup to obtain electrochemical corrosion characteristics. Abrasive wear was investigated using a pin-on-disk tribometer and the abrasion rate was calculated. Resistance against shock loads was tested by applying a cyclic load at 50 Hz to investigate the resistance against impact stresses.     

Precursor B cells of mouse bone marrow express two different complexes with the surrogate light chain on the surface

Two monoclonal antibodies raised against the complex of mu heavy (H) chain and Vpre-B/lambda 5 surrogate light (L) chains recognize surrogate L chain in different conformations on normal pre-B cells. One, LM34 recognizes free lambda 5 protein and free lambda 5/Vpre-B surrogate L chains and binds to surrogate L chains on the surface of early, pro-B and pre-B-I cells where the surrogate L chain is associated with a gp130/gp35-65 complex of proteins. It also recognizes the surrogate L chain associated with the mu H chain on pre-B-II cells. The other monoclonal antibody, SL156, does not recognize free surrogate L chain or its components, nor its complex with gp130/gp35-65 on pro-B and pre-B-I cells. However, it does bind to a conformational epitope on the surrogate light chain/mu H chain complex on a subpopulation of pre-B-II cells and on mu H chain-positive pre-B cell lines. On mouse precursor B cells prepared ex vivo on ice, expression of the surrogate L chain is very low and almost undetectable. Incubation of the precursor cells for 1 h at 37 degrees C up-regulates the surface expression of surrogate L chain associated with gp130/gp35-65 (early complex) as well as the mu H chain/surrogate L chain complex. These results reconcile some of the apparently discrepant results on surface expression of the surrogate L chain obtained with human and mouse bone marrow pre-B cells, and show that a surrogate L chain/mu H chain-containing pre-B cell receptor can be expressed also on the surface of mouse pre-B-II cells.     

Methodology for the systematic design of conical plain bearings for use as main bearings in wind turbines

With the possibility to replace sliding segments on the tower without disassembling the drivetrain, the use of segmented plain bearings with conical sliding surfaces as main bearing in wind turbines has a great potential to reduce the maintenance costs and thus the levelized cost of energy (LCOE). Furthermore, the short axial design leads to lower investment costs. Since this design is totally new and no design guidelines are available so far, the objective of this paper is to investigate the influence of the geometric parameters on the hydrodynamic pressure distribution of the bearing. In this context a parameter screening is performed using a suitable test field according to Plackett and Burman in order to determine the most relevant parameters. With the help of the simulations carried out after this test field, correlations between the geometric parameters and the hydrodynamic pressure distribution are evaluated. To be able to quantitatively analyze the three-dimensional pressure distribution, several key values are defined in this paper that describe the pressure distribution. The content of this paper is part of a methodology with the goal of developing a design guideline for conical plain bearings.

     

Activated murine B lymphocytes and dendritic cells produce a novel CC chemokine which acts selectively on activated T cells

Laboratory tests and imaging studies are often ordered for asymptomatic patients with malignant melanomas (MMs) in an effort to detect subclinical metastatic disease. However, their sensitivity and specificity for detecting cryptic metastases are not well established. A review of the literature on laboratory tests and imaging studies for MM metastases was undertaken to address the usefulness of such investigations in asymptomatic patients with MM in AJCC (American Joint Committee on Cancer system of classification) stages I, II, and III. A review of the pertinent literature since 1966 was conducted through MEDLINE, Medica, and Cancerlit. Laboratory tests and imaging studies revealed occult MM metastases in only a small number of the thousands of reported patients with putative AJCC stage I, II, and III MM. However, for those diagnosed with limited metastases, surgical removal with or without immunotherapy, chemotherapy, or radiotherapy can lead to long-term remissions in some patients. For patients with asymptomatic AJCC stage I or II disease, chest roentgenograms (CXR) and blood lactic dehydrogenase (LDH) levels may be obtained at low cost and prove to be of benefit if metastases are identified. For patients with AJCC stage III disease, computed tomographic (CT) scans of the thorax, abdomen, and pelvis (especially when the primary cutaneous site of the melanoma is below the waist) may be considered for detecting metastatic MM. Other tests, such as magnetic resonance imaging (MRI) scans of the brain, may be ordered based on symptoms or physical findings. In the future, technologically improved techniques and newer methods may prove cost-effective for detecting treatable asymptomatic MM metastases. Furthermore, improvement in treatments will also influence the indications for the search for occult MM metastases. At this time there is a need for an international consensus conference on laboratory tests and imaging studies for occult melanoma metastases.