HBcAg-specific IL-21-producing CD4+ T cells are associated with relative viral control in patients with chronic hepatitis B.

Function exhaustion of specific cytotoxic CD8+ T cell in chronic virus infection partly results from the low levels of CD4 help, but the mechanisms by which CD4 help T cell required to control hepatitis B virus infection are not well understood. In this study, we investigated the role of interleukin-21-producing CD4+ T cell response in viral control of hepatitis B virus infection. HBcAg-specific interleukin-21-producing CD4+ T cells in blood were detected in patients with hepatitis B virus infection. Patients with acute hepatitis B had greater HBcAg-specific interleukin-21-producing CD4+ T cells in blood compared with chronic hepatitis B patients, and there was no statistical significance between immune active chronic hepatitis B patients and inactive healthy carrier patients for these cells, whereas frequencies of these cells negatively correlated with HBV DNA levels but positively correlated with HBc18-27-specific IFN-γ-producing CD8+ T cells. Moreover, interleukin-21 sustained HBc18-27-specific CD8+ T cells in vitro, and interleukin-21 production by HBcAg-specific IL-21-producing CD4+ T cells of acute hepatitis B patients enhanced IFN-γ and perforin expression by CD8+ T cells from chronic hepatitis B patients. Our results demonstrate that HBcAg-specific interleukin-21-producing CD4+ T cell responses might contribute to viral control by sustaining CD8+ T cell antiviral function.

Junction temperature measurement of light emitting diode by electroluminescence.

Junction temperature (JT) is a key parameter of the performance and lifetime of light emitting diodes (LEDs). In this paper, a mobile instrument system has been developed for the non-contact measurement of JTs of LED under LabVIEW control. The electroluminescence (EL) peak shift of the LED is explored to measure the JT. Commercially available high power blue LEDs are measured. A linear relation between emission peak shift and JT is found. The accuracy of the JT is about 1 °C determined by the precision of the emission peak shift, ±0.03 nm, at 3σ standard deviation for blue LED. Using this system, on-line temperature rise curves of LED lamps are determined.

Regulated secretion of YopN by the type III machinery of Yersinia enterocolitica.

During infection, Yersinia enterocolitica exports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 microM (low-calcium response). Yersiniae lacking yopN (lcrE), yscB, sycN, or tyeA do not inactivate the type III pathway even when the concentration of calcium is above 100 microM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of yopN gene sequences to the 5' end of the npt reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of yopN. Expression of plasmid-borne yopN, but not of yopN(1-294)-npt, complements the calcium-blind phenotype of yopN mutants. Surprisingly, yopN mutants respond to environmental changes in calcium concentration and secrete YopN(1-294)-Npt in the absence but not in the presence of calcium. tyeA is required for the low-calcium regulation of YopN(1-294)-Npt secretion, whereas sycN and yscB mutants fail to secrete YopN(1-294)-Npt in the presence of calcium. Experiments with yopN-npt fusions identified two other signals that regulate the secretion of YopN. yopN codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of yscB and sycN. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional tyeA. These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.

LcrQ/YscM1, regulators of the Yersinia yop virulon, are injected into host cells by a chaperone-dependent mechanism.

Pathogenic Yersinia species employ type III machines to secrete YopBDR into the extracellular milieu. After attaching to host cells, yersiniae transform the type III machinery into an injection device and target YopEHMNOPT into eukaryotic cells. Yersinia pseudotuberculosis LcrQ is a transcriptional regulator that prevents the expression of yop genes. We report that LcrQ is injected into eukaryotic cells. YscM1, the transciptional regulator of Yersinia enterocolitica, is also injected into eukaryotic cells, whereas the related YscM2 protein remains associated with bacterial cells. Type III targeting of YscM1 requires binding to the SycH chaperone. Chaperone binding as well as depletion of YscM1 and YscM2 from the cytoplasm of Y. enterocolitica causes an increase in yop expression, whereas a block in regulator export reduces expression. We propose a model whereby the chaperone-mediated injection of LcrQ/YscM1 functions as a regulatory switch for bacteria that are attached to host cells, triggering the expression of Yops that travel the type III targeting pathway.

Yersinia enterocolitica TyeA, an intracellular regulator of the type III machinery, is required for specific targeting of YopE, YopH, YopM, and YopN into the cytosol of eukaryotic cells.

Pathogenic Yersinia species employ type III machines to target effector Yops into the cytosol of eukaryotic cells. Yersinia tyeA mutants are thought to be defective in the targeting of YopE and YopH without affecting the injection of YopM, YopN, YopO, YopP, and YopT into the cytosol of eukaryotic cells. One model suggests that TyeA may form a tether between YopN (LcrE) and YopD on the bacterial surface, a structure that may translocate YopE and YopH across the plasma membrane of eukaryotic cells (M. Iriarte, M. P. Sory, A. Boland, A. P. Boyd, S. D. Mills, I. Lambermont, and G. R. Cornelis, EMBO J. 17:1907-1918, 1998). We have examined the injection of Yop proteins by tyeA mutant yersiniae with the digitonin fractionation technique. We find that tyeA mutant yersiniae not only secreted YopE, YopH, YopM, and YopN into the extracellular medium but also targeted these polypeptides into the cytosol of HeLa cells. Protease protection, cell fractionation, and affinity purification experiments suggest that TyeA is located intracellularly and binds to YopN or YopD. We propose a model whereby TyeA functions as a negative regulator of the type III targeting pathway in the cytoplasm of yersiniae, presumably by preventing the export of YopN.

Type III machines of Gram-negative bacteria: delivering the goods.

Many Gram-negative pathogens use a type III secretion machine to translocate protein toxins across the bacterial cell envelope. Pathogenic Yersinia spp. export at least 14 Yop proteins via a type III machine, which recognizes secretion substrates by signals encoded in yop mRNA or chaperones bound to unfolded Yop proteins. During infection, substrate recognition appears to be regulated in a manner that allows the Yersinia type III pathway to direct Yops to the bacterial envelope, the extracellular medium or into the cytosol of host cells.

An unusual alkaline phosphatase isoenzyme associated with gastric carcinoma.

We describe a 50-year-old man with a diagnosis of gastric carcinoma made on gastroscopy after X-rays of the thoracolumbar spine had revealed multiple lytic metastases. A bone marrow aspirate showed adenocarcinoma cells. Polyacrylamide gel electrophoresis incorporating wheat germ lectin was used to separate the serum alkaline phosphatase isoenzymes. Isoenzyme separation showed a markedly increased amount of bone isoenzyme, a normal amount of liver isoenzyme and a considerable amount of an intestinal-like isoenzyme running cathodic to the bone isoenzyme. There was also some immunoglobulin-complexed alkaline phosphatase, which, when digested, showed more of the intestinal-like isoenzyme. This was a variant alkaline phosphatase isoenzyme found in a patient with a gastric carcinoma with a super bone scan. There have been two previous reports of patients with a variant alkaline phosphatase isoenzyme and a super bone-scan.

Yersinia enterocolitica type III secretion. On the role of SycE in targeting YopE into HeLa cells.

Yersinia enterocolitica inject toxic proteins (effector Yops) into the cytosol of eukaryotic cells by a mechanism requiring the type III machinery. Previous work mapped a signal sufficient for the targeting of fused reporter proteins to amino acids 1-100 of YopE. Targeting requires the binding of SycE to YopE residues 15-100 in the bacterial cytoplasm. We asked whether SycE functions only to stabilize YopE in the bacterial cytoplasm, or whether the secretion chaperone itself contributes to substrate recognition by the type III machinery. Fusions of glutathione S-transferase to either the N or C terminus of SycE resulted in hybrid proteins that bound YopE but prevented targeting of the export substrate into HeLa cells. As compared with wild-type SycE, glutathione S-transferase-SycE bound and stabilized YopE in the bacterial cytoplasm but failed to release the polypeptide for export by the type III machinery. Thus, it appears that SycE functions to deliver YopE to the type III secretion machinery. A model is presented that accounts for substrate recognition of effector Yops, a group of proteins that do not share amino acid sequence or physical similarities.

Two independent type III secretion mechanisms for YopE in Yersinia enterocolitica.

Pathogenic Yersinia species escape the infected host's defense mechanisms by targeting cytotoxic Yop proteins into the cytoplasm of macrophages via a type III secretion pathway. Two separate secretion signals contained in YopE were identified, each of which were sufficient but not necessary for the secretion of reporter molecules. One signal is located within the coding sequence of the first 15 amino acids and is sufficient for the secretion of fusion proteins but not required for YopE secretion. The second signal is located downstream at residues 15-100 of YopE and is only recognized by the type III machinery when it is bound to SycE. We propose the existence of two independent mechanisms that allow for the secretion of Yop proteins.

Active efflux of bile salts by Escherichia coli.

Enteric bacteria such as Escherichia coli must tolerate high levels of bile salts, powerful detergents that disrupt biological membranes. The outer membrane barrier of gram-negative bacteria plays an important role in this resistance, but ultimately it can only retard the influx of bile salts. We therefore examined whether E. coli possessed an energy-dependent efflux mechanism for these compounds. Intact cells of E. coli K-12 appeared to pump out chenodeoxycholate, since its intracellular accumulation increased more than twofold upon deenergization of the cytoplasmic membrane by a proton conductor. Growth inhibition by bile salts and accumulation levels of chenodeoxycholate increased when mutations inactivating the acrAB and emrAB gene clusters were introduced. The AcrAB system especially appeared to play a significant role in bile acid efflux. However, another efflux system(s) also plays an important role, since the accumulation level of chenodeoxycholate increased strongly upon deenergization of acrA emrB double mutant cells. Everted membrane vesicles accumulated taurocholate in an energy-dependent manner, apparently consuming delta pH without affecting delta psi. The efflux thus appears to be catalyzed by a proton antiporter. Accumulation by the everted membrane vesicles was not decreased by mutations in acr and emrB genes and presumably reflects activity of the unknown system seen in intact cells. It followed saturation kinetics with Vmax and Km values in the neighborhood of 0.3 nmol min(-1) mg of protein(-1) and 50 microM, respectively.