CD8+CD45RA+CCR7+FOXP3+ T Cells with Immunosuppressive Properties: A Novel Subset of Inducible Human Regulatory T Cells

CD8 T cells stimulated with a suboptimal dose of anti-CD3 Abs (100 pg/ml) in the presence of IL-15 retain a naive phenotype with expression of CD45RA, CD28, CD27, and CCR7 but acquire new functions and differentiate into immunosuppressive T cells. CD8(+)CCR7(+) regulatory T cells (Tregs) express FOXP3 and prevent CD4 T cells from responding to TCR stimulation and entering the cell cycle. Naive CD4 T cells are more susceptible to inhibition than memory cells. The suppressive activity of CD8(+)CCR7(+) Tregs is not mediated by IL-10, TGF-β, CTLA-4, CCL4, or adenosine and relies on interference with very early steps of the TCR signaling cascade. Specifically, CD8(+)CCR7(+) Tregs prevent TCR-induced phosphorylation of ZAP70 and dampen the rise of intracellular calcium in CD4 T cells. The inducibility of CD8(+)CCR7(+) Tregs is correlated with the age of the individual with PBLs of donors older than 60 y yielding low numbers of FOXP3(low) CD8 Tregs. Loss of CD8(+)CCR7(+) Tregs in the elderly host may be of relevance in the aging immune system as immunosenescence is associated with a state of chronic smoldering inflammation.     

In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells

The characterization of mesenchymal stem cells (MSCs) is of biological and clinical interest. We demonstrate that isolated MSCs, defined by CD34(low) c-kit(+) CD140a(+) Sca-1(high), are able to differentiate into cardiomyocytes, endothelial cells, and pericytes or smooth muscle cells by direct injection into adult heart. In skeletal muscle and lung, they also contributed to formation of the vasculature. MSCs did not transform into malignant cells or form excess extracellular matrix. This study suggests that MSCs may supply an ideal donor source of cardiovascular cells in patients with cardiopulmonary diseases.     

In vitro effects of dentin matrix protein-1 on hydroxyapatite formation provide insights into in vivo functions

Dentin matrix protein-1 (DMP1) is a mineralized tissue matrix protein synthesized by osteoblasts, hypertrophic chondrocytes, and ameloblasts as well as odontoblasts. DMP1 is believed to have multiple in vivo functions, acting both as a signaling molecule and a regulator of biomineralization. Using a cell-free system in vitro, we evaluated the action of DMP1 in the regulation of hydroxylapatite (HA) formation and crystal growth. The non-phosphorylated recombinant protein acted as an HA nucleator, increasing the amount of mineral formed in a gelatin gel HA growth system relative to protein-free controls. The recombinant protein phosphorylated in vitro had no detectable effect on HA formation and growth. In contrast, phosphorylated bovine DMP1 expressed in marrow stromal cells with an adenovirus vector containing 29.7 phosphates/mol was an effective inhibitor of HA formation and growth. The native full-length protein appeared to be absent or present in only small amounts in the extracellular matrix of bones and teeth. However, two highly phosphorylated fragments representing the N- and C-terminal portions of DMP1 have been identified, apparently arising from proteolytic cleavage of four X-Asp bonds. The highly phosphorylated C-terminal 57-kDa fragment (containing 42 phosphates/mol), like the non-phosphorylated DMP1, was an HA nucleator. These data suggest that, in its native form, DMP1 inhibits mineralization, but when cleaved or dephosphorylated, it initiates mineralization. These in vitro data are consistent with the findings in the DMP1 knockout mouse.     

“Silent” Hepatitis B Virus Mutants Are Responsible for Non-A,Non-B,Non-C,Non-D,Non-E Hepatitis

Since the recent introduction of diagnostic kits for hepatitis C and E, some cases of nonA, non-B, non-C, non-D, non-E hepatitis (so-called hepatitis F) have been revealed. We attempted to demonstrate that so-called hepatitis F is caused by hepatitis B virus (HBV) variants. Polymerase chain reaction (PCR) was used to amplify serum HBV DNAs from 20 patients with acute hepatitis and 20 patients with chronic hepatitis who had been diagnosed as having so-called hepatitis F on the basis of conventional serological markers. The PCR technique successfully amplified HBV DNAs in 18 (90%) cases of acute hepatitis and 17 (85%) cases of chronic hepatitis. Sequencing of HBV DNAs of six patients (acute 3, chronic 3) revealed equally a T-to-C mutation of DR2 and an 8-nucleotide deletion of the 3′-terminus of the X gene coding region, giving rise to the generation of a C-terminally truncated X protein and probable damage to the enhancer II/core promoter elements. These mutations of the X gene coding region may lead to suppression of replication and expression of HBV DNAs. Thus virtually all cases of so-called hepatitis F appear to be caused by “silent” HBV mutants, at least in Japan.     

Nonstructural protein of severe fever with thrombocytopenia syndrome phlebovirus targets STAT2 and not STAT1 to inhibit type I interferon-stimulated JAK-STAT signaling

The nonstructural protein NSs of severe fever with thrombocytopenia syndrome phlebovirus blocks type I interferon (IFN)-stimulated JAK-STAT signaling. However, there is continuing controversy as to whether NSs targets STAT1 or STAT2 or both for this blockade. The present study was designed to gain a further understanding of the blockade mechanism. Immunoprecipitation experiments revealed a stronger interaction of NSs with STAT2 than with any other component constituting the JAK-STAT pathway. Expression of NSs resulted in the formation of cytoplasmic inclusion bodies (IBs), and affected cytoplasmic distribution of STAT2. STAT2 was relocated to NSs-induced IBs. Consequently, NSs inhibited IFN-α-stimulated tyrosine phosphorylation and nuclear translocation of STAT2. These inhibitory effects as well as the signaling blockade activity were not observed in NSs mutant proteins lacking the STAT2-binding ability. In contrast, NSs affected neither subcellular distribution nor phosphorylation of STAT1 in response to IFN-α and IFN-γ, demonstrating that NSs has little physical and functional interactions with STAT1. Taken together, these results suggest that NSs sequesters STAT2 into NSs-induced IBs, thereby blocking type I IFN JAK-STAT signaling.     

Detection of the mcr‐1 gene in colistin‐resistant Escherichia coli from retail meat in Japan

In this study, the presence of the mcr‐1 gene in Escherichia coli from retail meat in Japan was investigated. Nine E. coli isolates (eight from chickens and one from pork) carried the mcr‐1 gene on the plasmid. In six isolates from domestic chickens, mcr‐1 was located on the IncI2 plasmid, which is approximately 60 kb in size. In the remaining three isolates from imported chicken and pork, mcr‐1 was located on the IncX4 plasmid (30 kb).

     

Recycling of the major thylakoid lipid MGDG and its role in lipid homeostasis in Chlamydomonas reinhardtii

Monogalactosyldiacylglycerol (MGDG), the most abundant lipid in thylakoid membranes, is involved in photosynthesis and chloroplast development. MGDG lipase has an important role in lipid remodeling in Chlamydomonas reinhardtii. However, the process related to turnover of the lysogalactolipid that results from MGDG degradation, monogalactosylmonoacylglycerol (MGMG), remains to be clarified. Here we identified a homolog of Arabidopsis thaliana lysophosphatidylcholine acyltransferase (LPCAT) and characterized two independent knockdown (KD) alleles in C. reinhardtii. The enzyme designated as C. reinhardtiiLysolipid Acyltransferase 1 (CrLAT1) has a conserved membrane-bound O-acyl transferase domain. LPCAT from Arabidopsis has a key role in deacylation of phosphatidylcholine (PC). Chlamydomonas reinhardtii, however, lacks PC, and thus we hypothesized that CrLAT1 has some other important function in major lipid flow in this organism. In the CrLAT1 KD mutants, the amount of MGMG was increased, but triacylglycerols (TAGs) were decreased. The proportion of more saturated 18:1 (9) MGDG was lower in the KD mutants than in their parental strain, CC-4533. In contrast, the proportion of MGMG has decreased in the CrLAT1 overexpression (OE) mutants, and the proportion of 18:1 (9) MGDG was higher in the OE mutants than in the empty vector control cells. Thus, CrLAT1 is involved in the recycling of MGDG in the chloroplast and maintains lipid homeostasis in C. reinhardtii.

A recycling system of the major thylakoid lipid monogalactosyldiacylglycerol in the Chlamydomonas reinhardtii chloroplast contributes to lipid homeostasis.     

Zebrafish gene knockdowns imply roles for human YWHAG in infantile spasms and cardiomegaly

Williams‐Beuren syndrome (WBS) is a neurodevelopmental disorder presenting with an elfin‐like face, supravalvular aortic stenosis, a specific cognitive‐behavioral profile, and infantile hypercalcemia. We encountered two WBS patients presenting with infantile spasms, which is extremely rare in WBS. Array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) analyses revealed atypical 5.7‐Mb and 4.1‐Mb deletions at 7q11.23 in the two patients, including the WBS critical region and expanding into the proximal side and the telomeric side, respectively. On the proximal side, AUTS2 and CALN1 may contribute to the phenotype. On the telomeric side, there are two candidate genes HIP1 and YWHAG. Because detailed information of them was unavailable, we investigated their functions using gene knockdowns of zebrafish. When zebrafish ywhag1 was knocked down, reduced brain size and increased diameter of the heart tube were observed, indicating that the infantile spasms and cardiomegaly seen in the patient with the telomeric deletion may be derived from haploinsufficiency of YWHAG. genesis 48:233–243, 2010. © 2010 Wiley‐Liss, Inc.     

Co-translational function of Cosmc, core 1 synthase specific molecular chaperone, revealed by a cell-free translation system

The core 1 structure of the mucin type O-glycan is synthesized by core 1 β1,3-galactosyltransferase (C1GalT). Core 1 synthase specific molecular chaperone (Cosmc), a molecular chaperone specific for C1GalT, is essential for the expression of functional C1GalT in mammalian cells. In this study, we have established a procedure for detecting the chaperone activity of Cosmc by using a wheat germ cell-free translation system. Active C1GalT was expressed following simultaneous translation with Cosmc or translation in the presence of recombinant Cosmc protein. Moreover, we show that recombinant Cosmc must be present during the translation of C1GalT, as it is ineffective when added after translation. These results indicate that Cosmc mediates the co-translational activation of C1GalT and that it may prevent the unfavorable aggregation of C1GalT.