Pluripotent embryonic stem (ES) cells are capable of differentiating into all cell lineages, but the molecular mechanisms that regulate ES cell differentiation have not been sufficiently explored. In this study, we report that shear stress, a mechanical force generated by fluid flow, can induce ES cell differentiation. When Flk-1-positive (Flk-1(+)) mouse ES cells were subjected to shear stress, their cell density increased markedly, and a larger percentage of the cells were in the S and G(2)-M phases of the cell cycle than Flk-1(+) ES cells cultured under static conditions. Shear stress significantly increased the expression of the vascular endothelial cell-specific markers Flk-1, Flt-1, vascular endothelial cadherin, and PECAM-1 at both the protein level and the mRNA level, but it had no effect on expression of the mural cell marker smooth muscle alpha-actin, blood cell marker CD3, or the epithelial cell marker keratin. These findings indicate that shear stress selectively promotes the differentiation of Flk-1(+) ES cells into the endothelial cell lineage. The shear stressed Flk-1(+) ES cells formed tubelike structures in collagen gel and developed an extensive tubular network significantly faster than the static controls. Shear stress induced tyrosine phosphorylation of Flk-1 in Flk-1(+) ES cells that was blocked by a Flk-1 kinase inhibitor, SU1498, but not by a neutralizing antibody against VEGF. SU1498 also abolished the shear stress-induced proliferation and differentiation of Flk-1(+) ES cells, indicating that a ligand-independent activation of Flk-1 plays an important role in the shear stress-mediated proliferation and differentiation by Flk-1(+) ES cells.

The lectin complement pathway in innate immunity is closely related to the classical complement pathway in adaptive immunity, with respect to the structures and functions of their components. Both pathways are initiated by complexes consisting of collagenous proteins and serine proteases of the mannose-binding lectin (MBL)-associated serine protease (MASP)͞C1r͞C1s family. It has been speculated that the classical pathway emerged after the lectin pathway, and that the activation mechanism of the latter was partially conserved. The classical and lectin pathways can be traced back to at least cartilaginous fish and ascidian (urochordata), respectively. To elucidate the evolution of the complement system, we isolated and characterized a GlcNAc-binding lectin from sera of lamprey (agnathans), the most primitive vertebrate that lacks the classical pathway. Lamprey GlcNAc-binding lectin was an oligomer consisting of 24-kDa subunits. cDNA and phylogenetic analyses revealed that the lamprey GlcNAc-binding lectin is an orthologue of mammalian C1q, a collagenous subcomponent of the first component involved in binding to immunoglobulins in the classical pathway. Lamprey C1q copurified with MASP-A, a serine protease of the MASP͞C1r͞C1s family, which exhibited proteolytic activity against lamprey C3. Surface plasmon resonance analysis showed that lamprey C1q specifically bound to GlcNAc, but not various other carbohydrates tested. These results suggest that C1q may have emerged as a lectin and may have functioned as an initial recognition molecule of the complement system in innate immunity before the establishment of adaptive immunity such as immunoglobulins in the cartilaginous fish.

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.

Milk casein-derived tripeptides, valyl prolyl proline (VPP), and isoleucyl prolyl proline (IPP) inhibit angiotensin-converting enzyme (ACE) and both fermented milk and proteolytic hydrolysates of milk casein containing these peptides exert blood pressure-lowering effects in animals and humans. On the top of these results, we have recently reported that the hydrolysate of milk casein containing both VPP and IPP improved the vascular endothelial function of subjects with stage I hypertension, enforcing us to elucidate the mechanism of the improvement of endothelial dysfunction by these peptides. For this purpose, we examined the effect of VPP and IPP on induction of nitric oxide (NO) production using cultured vascular endothelial cells and isolated arterial vessels. When both VPP and IPP were added to the medium of cultured endothelial cells at final concentrations of more than 100 nmol/l, the NO(x) (NO(2) and NO(3)) concentration in the medium was significantly higher than that of the control. Moreover, both VPP and IPP induced endothelium-dependent relaxation of isolated aortic rings, and these effects were inhibited by NO synthase inhibitors, K channel inhibitors, and bradykinin B2 receptor antagonists. These lines of results suggested that both VPP and IPP induced production of vasodilative substances including NO.

Bacillus cereus is a growing concern as a cause of life-threatening infections in patients with hematologic malignancies. However, the risk factors for patients with unfavorable outcomes have not been fully elucidated. At our institution, we observed the growth of B. cereus in blood culture in 68 patients with (23) or without (45) hematologic malignancies treated from September 2002 to November 2009. We defined a case as having sepsis when more than two blood culture sets were positive for B. cereus or only a single set was positive in the absence of other microorganisms in patients who had definite infectious lesions. We determined 12 of 23 patients with hematologic malignancies as having sepsis, as well as 10 of 45 patients without hematologic malignancies (p = 0.012). Of the 12 patients with hematologic malignancies, four patients with acute leukemia died of B. cereus sepsis within a few days. In our cohort, risk factor analysis demonstrated that a neutrophil count of 0/mm(3), central venous (CV) catheter insertion, and the presence of central nervous system (CNS) symptoms were significantly associated with a fatal prognosis (p = 0.010, 0.010, and 0.010, respectively). Analysis of data from our cohort in conjunction with those from 46 previously reported patients with B. cereus sepsis identified similar risk factors, that is, acute leukemia, extremely low neutrophil count, and CNS symptoms (p = 0.044, 0.004, and 0.002, respectively). These results indicate that appropriate prophylaxis and early therapeutic intervention against possible B. cereus sepsis are crucially important in the treatment of hematologic malignancies.

A 64-year-old woman suffering from progressive amyloid A (AA) amyloidosis of the gastrointestinal (GI) tract, associated with active rheumatoid arthritis, was transferred to our hospital due to hypovolemic shock. Although intensive care, including treatment with prednisolone and methotrexate, improved the hypovolemic shock, paralytic ileus became dominant instead of the marked diarrhea, suggesting the terminal stage of AA amyloidosis of the GI tract. Thus, we administered tocilizumab, a humanized anti-interleukin 6 receptor antibody (8 mg/kg, repeated every 4 weeks). Two weeks after the first injection of tocilizumab, serum AA rapidly returned to their normal ranges in accordance with the amelioration of paralytic ileus and systemic joint pain. Surprisingly, after three courses of tocilizumab treatment, colon biopsy revealed no amyloid deposition. Tocilizumab is a promising agent to treat secondary AA amyloidosis by strongly suppressing serum AA levels.

A 55-year-old

Few studies have examined the prognostic impact of blood markers [other than the five factors in the enhanced International Prognostic Index (NCCN-IPI)] in elderly patients with diffuse large B cell lymphoma (DLBCL). We retrospectively analyzed 391 DLBCL patients receiving rituximab plus anthracycline-containing chemotherapy to examine the prognostic impact of simple blood markers. The NCCN-IPI was more accurate for discriminating prognoses than the original IPI. Multivariate analysis identified platelet count (<100,000/μl) and albumin (<3.5 g/dl) levels as significantly associated with lower overall survival (OS), independently of the NCCN-IPI. These parameters stratified patients into three risk groups: platelet-albumin (PA) score low (platelet count ≥100,000/μl, albumin ≥3.5 g/dl, n = 243); intermediate (platelet count <100,000/μl, albumin ≥3.5 g/dl or platelet count ≥100,000/μl, albumin <3.5 g/dl, n = 125); and high (platelet count <100,000/μl, albumin <3.5 g/dl, n = 23). The 5-year OS rates were 81.5, 48.6, and 20.2 %, respectively (p < 0.001). Notably, most patients with a low platelet count (n = 30) were stratified into the high-risk subgroup, suggesting that platelet count was prognostic for high-risk patients with a dismal outcome. In elderly patients (n = 291), the prognostic value of the NCCN-IPI might be diminished because the low-risk category was excluded; however, the PA score was predictive of survival: the 5-year OS rates for PA score low (n = 171), intermediate (n = 101), and high (n = 19) groups were 77.6, 47.9, and 19.0 %, respectively (p < 0.001). Platelet count and albumin levels are useful prognostic factors, and their combined use can predict survival, even in elderly patients.