Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.     

Natural immunity in mice to the envelope glycoprotein of endogenous ecotropic type C viruses: neutralization of virus infectivity.

The ability of naturally immune mouse sera to neutralize ecotropic AKR murine leukemia virus (MuLV) was examined by using unfrozen virus preparations harvested for 1 h. In this assay several mouse sera significantly and consistently neutralized MuLV infectivity. The ability of these sera to neutralize was correlated with the presence of antibodies against MuLV detectable in a radioimmune precipitation assay using radioactively labeled intact virions. This neutralization was specific, in that either N- or B-tropic viruses, but not Friend MuLV, were neutralized. In addition, neutralization could be abrogated with purified AKR MuLV gp71 at concentrations that do not interfere with virus infectivity but could not be abrogated with Rauscher MuLV gp71. Neutralizing activity could be removed by absorption with intact AKR MuLV, but not by absorption with Friend MuLV, a BALB/c xenotropic virus, or with NZB xenotropic virus. All the neutralizing activity of (B6C3)F1 mouse sera was associated with the immunoglobulin G fraction.     

Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance

The MUC1 mucin represents a prime target antigen for cancer immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas. Attempts to generate strong humoral immunity to MUC1 by immunization with peptides have generally failed partly because of tolerance. In this study, we have developed chemoenzymatic synthesis of extended MUC1 TR glycopeptides with cancer-associated O-glycosylation using a panel of recombinant human glycosyltransferases. MUC1 glycopeptides with different densities of Tn and STn glycoforms conjugated to KLH were used as immunogens to evaluate an optimal vaccine design. Glycopeptides with complete O-glycan occupancy (five sites per repeat) elicited the strongest antibody response reacting with MUC1 expressed in breast cancer cell lines in both Balb/c and MUC1.Tg mice. The elicited humoral immune response showed remarkable specificity for cancer cells suggesting that the glycopeptide design holds promise as a cancer vaccine. The elicited immune responses were directed to combined glycopeptide epitopes, and both peptide sequence and carbohydrate structures were important for the antigen. A MAb (5E5) with similar specificity as the elicited immune response was generated and shown to have the same remarkable cancer specificity. This antibody may hold promise in diagnostic and immunopreventive measures.     

Mitochondrial phylogeny, taxonomy and biogeography of the silvered langur species group (Trachypithecus cristatus)

With a distribution ranging from mainland Southeast Asia to the Sunda region, the silvered langur species group is the most widely distributed species complex of the genus Trachypithecus. However, the systematic classification of its members and the phylogenetic relationships among them are less understood, leading to different classification schemes and proposed distribution zones. To address these issues, we sequenced a 573 bp long fragment of the mitochondrial cytochrome b gene from 115 silvered langurs (68 individuals from known origin). According to our data, five monophyletic clades were detected, which refer to the five taxa auratus, cristatus, germaini, margarita and mauritius. The phylogenetic relationships among them are not well resolved, indicating a radiation-like splitting event, which was estimated to have occurred about 0.95-1.25 mya. Within T. cristatus, two major clades were detected, with one comprising specimens from Sumatra, Borneo and the Natuna archipelago, and the other solely individuals from the Malaysian peninsula. According to our findings, we propose to rank all five taxa as distinct species. While T. auratus, T. germaini, T. margarita and T. mauritius seem to be monotypic, T. cristatus should be split into two subspecies, with the Malaysian form being described as new form here. From a phylogeographic perspective, the species group most likely originated on Java. During the early Pleistocene, its range was expanded to the Malaysian peninsula and to the Southeast Asian mainland. Later on, the Malaysian form colonised further regions of the Sunda region, including Sumatra, Borneo and the Natuna archipelago.     

Negative regulation of Jak2 by its auto-phosphorylation at tyrosine 913 via the Epo signaling pathway

Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y(913)) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y(913) rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y(1007/1008) in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y(913) (Y(913)F) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y(913) (Y(913)E) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing Y(913)F mutant generated many mature erythroid BFU-E and CFU-E colonies, while Y(913)E mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y(939), a corresponding tyrosine residue with Y(913), negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family.     

Preventive antibacterial therapy in acute ischemic stroke: a randomized controlled trial

Background

Pneumonia is a major risk factor of death after acute stroke. In a mouse model, preventive antibacterial therapy with moxifloxacin not only prevents the development of post-stroke infections, it also reduces mortality, and improves neurological outcome significantly. In this study we investigate whether this approach is effective in stroke patients.

Methods

Preventive ANtibacterial THERapy in acute Ischemic Stroke (PANTHERIS) is a randomized, double-blind, placebo-controlled trial in 80 patients with severe, non-lacunar, ischemic stroke (NIHSS>11) in the middle cerebral artery (MCA) territory. Patients received either intravenous moxifloxacin (400 mg daily) or placebo for 5 days starting within 36 hours after stroke onset. Primary endpoint was infection within 11 days. Secondary endpoints included neurological outcome, survival, development of stroke-induced immunodepression, and induction of bacterial resistance.

Findings

On intention-to treat analysis (79 patients), the infection rate at day 11 in the moxifloxacin treated group was 15.4% compared to 32.5% in the placebo treated group (p = 0.114). On per protocol analysis (n = 66), moxifloxacin significantly reduced infection rate from 41.9% to 17.1% (p = 0.032). Stroke associated infections were associated with a lower survival rate. In this study, neurological outcome and survival were not significantly influenced by treatment with moxifloxacin. Frequency of fluoroquinolone resistance in both treatment groups did not differ. On logistic regression analysis, treatment arm as well as the interaction between treatment arm and monocytic HLA-DR expression (a marker for immunodepression) at day 1 after stroke onset was independently and highly predictive for post-stroke infections.

Interpretation

PANTHERIS suggests that preventive administration of moxifloxacin is superior in reducing infections after severe non-lacunar ischemic stroke compared to placebo. In addition, the results emphasize the pivotal role of immunodepression in developing post-stroke infections.

Trial Registration

Controlled-Trials.com ISRCTN74386719     

Dopamine/serotonin receptor ligands. Part 17: a cross-target SAR approach: affinities of azecine-styled ligands for 5-HT(2A) versus D1 and D2 receptors

Dibenzo- and benzindolo-azecines represent a novel class of high-affinity dopamine receptor antagonists. To further characterize these drugs as potential neuroleptics, we selected a set of azecine derivatives and ring expanded homologues and we measured their antagonist activity at the 5-HT(2A) receptor in the porcine coronary artery. SARs found for the 5-HT(2A) receptor resemble those for the D1 but not the D2 receptor. The protein-ligand interactions were discussed with respect to the different binding pockets.     

Fibronectin fibril pattern displays the force balance of cell–matrix adhesion

Formation of fibrillar patterns of fibronectin on polymer substrates with gradated physicochemical surface properties was analysed during early stages of endothelial cell adhesion. Fibronectin was pre-adsorbed onto three maleic anhydride copolymer thin films with distinct differences in the protein adsorption strength as verified by heteroexchange experiments. The evolved micrometer scale fibrillar patterns of fibronectin on the compared polymer surfaces were characterized after 50 min of cellular reorganization by an auto-correlation analysis using fluorescence microscopy data. Statistical analysis revealed a decrease of the typical spacings of the fibronectin fibrils from 2.6 to 1.8 m with decreasing fibronectin adsorption strength to the substrate. Size and density of focal adhesions correlated with this dependence of the fibronectin fibril pattern. From these data a model was developed relating the fibronectin fibril pattern to the fibronectin-substrate adsorption strength through the cytoskeletal force regulation mechanism of the cell.