A Single-Center, Randomized Double-Blind Placebo-Controlled Study Evaluating the Effects of Poly-Gamma-Glutamate on Human NK Cell Activity after an 8-Week Oral Administration in Healthy Volunteers

A randomized double-blind placebo-controlled immunity study involving 99 healthy volunteers was performed to investigate the effect of poly-γ-glutamate (γ-PGA) on human natural killer (NK) cell activity in peripheral blood. The volunteers were randomly assigned to one of three groups and orally treated with solutions (25 mL) containing 0 mg (placebo), 250 mg (low dosage), or 500 mg (high dosage) of γ-PGA. Each volunteer took one dose every 12 hours for 8 weeks. Blood samples were drawn before the initial treatment and at the 4th and the 8th weeks of treatment. NK cell activity was assessed by measuring its degranulation, cytokine production, and cytotoxicity against the K562 cell line. Our results revealed that the cytotoxic activities of NK cells from the high-dosage γ-PGA group were significantly higher (P<0.05 for all comparisons) compared to the low dosage and placebo groups at weeks 4 and 8 after the initial treatment. This increase in the NK cell activity among peripheral blood mononuclear cells (PBMCs) of healthy individuals was also confirmed in vitro (as assessed by the degranulation and cytokine production). These results suggest that the oral administration of γ-PGA induces a cell-mediated immunity by increasing the NK cell activity in humans

The Volatile Anesthetic Isoflurane Increases Endothelial Adenosine Generation via Microparticle Ecto-5′-Nucleotidase (CD73) Release

Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5′-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation

Class-Switch Recombination in the Absence of the IgH 3′ Regulatory Region

The ~28 kb 3′-regulatory region (3′RR) which is located at the most distal 3′ region of the immunoglobulin (Ig) heavy (H) chain locus has multiple regulatory functions that control IgH expression, class switch recombination (CSR) and somatic hypermutation (SHM). Here, we report that deletion of the entire 3′RR in a mouse B cell line that is capable of robust cytokine-dependent CSR to IgA results in reduced but not abolished CSR. These data suggest that the 3′RR is not absolutely required for CSR, and thus is not essential for targeting AID to S regions as has been suggested. Moreover, replacing the 3′RR with a DNA fragment including only its four DNase I hypersensitive sites (lacking the large spacer regions), restores CSR to a level equivalent to or even higher than wild type cells, suggesting that the four HS sites contain most of the CSR-promoting functions of the 3′RR. Stimulated cells express abundant germline transcripts, with the presence or absence of the 3′RR, providing evidence that the 3’RR has a separate role in promoting CSR, that is unique from enhancing S region transcription

Epigenetic modification and antibody-dependent expansion of memory-like NK cells in human cytomegalovirus-infected individuals.

Long-lived "memory-like" NK cells have been identified in individuals infected by human cytomegalovirus (HCMV), but little is known about how the memory-like NK cell pool is formed. Here, we have shown that HCMV-infected individuals have several distinct subsets of memory-like NK cells that are often deficient for multiple transcription factors and signaling proteins, including tyrosine kinase SYK, for which the reduced expression was stable over time and correlated with epigenetic modification of the gene promoter. Deficient expression of these proteins was largely confined to the recently discovered FcRγ-deficient NK cells that display enhanced antibody-dependent functional activity. Importantly, FcRγ-deficient NK cells exhibited robust preferential expansion in response to virus-infected cells (both HCMV and influenza) in an antibody-dependent manner. These findings suggest that the memory-like NK cell pool is shaped and maintained by a mechanism that involves both epigenetic modification of gene expression and antibody-dependent expansion

Isoflurane transiently increases CD73 activity without changing CD73 synthesis in cultured endothelial cells.

<p>A. Human umbilical vein endothelial (EA.hy926) cells treated with 2.5% isoflurane showed a significant but transient induction of CD73 activity. CD73 activity peaked at 3 hr and then decreased to near baseline at 6–16 hr after isoflurane treatment (N = 6–8). B. Isoflurane treatment for 3 hr caused dose-dependent increase in CD73 activity in EA.hy926 cells compared to carrier gas-treated cells (N = 4–5). Data are presented as means ± SEM. *P<0.05 vs. CD73 activity measured at baseline (A) or in cells treated with 0% isoflurane (B). C and D. Representative images for CD73 mRNA (RT-PCR) and protein (immunoblotting) expression in EA.hy926 cells. EA.hy926 cells were treated with carrier gas or with 2.5% isoflurane for 6 hr (C) or for 16 hr (D). Isoflurane treatment did not increase CD73 mRNA or protein expression in EA.hy926 cells. Representative of 3–4 experiments.</p

Epigenetic modification and antibody-dependent expansion of memory-like NK cells in human cytomegalovirus-infected individuals.

Long-lived "memory-like" NK cells have been identified in individuals infected by human cytomegalovirus (HCMV), but little is known about how the memory-like NK cell pool is formed. Here, we have shown that HCMV-infected individuals have several distinct subsets of memory-like NK cells that are often deficient for multiple transcription factors and signaling proteins, including tyrosine kinase SYK, for which the reduced expression was stable over time and correlated with epigenetic modification of the gene promoter. Deficient expression of these proteins was largely confined to the recently discovered FcRγ-deficient NK cells that display enhanced antibody-dependent functional activity. Importantly, FcRγ-deficient NK cells exhibited robust preferential expansion in response to virus-infected cells (both HCMV and influenza) in an antibody-dependent manner. These findings suggest that the memory-like NK cell pool is shaped and maintained by a mechanism that involves both epigenetic modification of gene expression and antibody-dependent expansion

Primers used to amplify cDNAs based on published GenBank sequences for human.

<p>bp, base pairs; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ICAM-1, intercellular adhesion molecule-1; TNF-α, tumor necrosis factor-alpha; VCAM-1, vascular cell adhesion molecule-1; CD73, Ecto-5′-nucleotidase. Respective anticipated PCR product size (bp, base pairs), PCR cycle number for linear amplification, and annealing temperatures used for each primer are also provided.</p

Flow cytometric analyses of endothelial cell culture media microparticles.

<p>A. Representative flow cytometric analyses of microparticles isolated from EA.hy926 endothelial cell culture media. EA.hy926 cells were treated with 2.5% isoflurane or with carrier gas for 1 hr and isolated microparticles were incubated with CD73 antibody and Annexin V. B. EA.hy926 cells treated with 2.5% isoflurane for 1 hr had significantly higher CD73+ Annexin V+ microparticles compared to microparticles isolated from carrier gas-treated endothelial cells (N = 5). *P<0.05 vs. carrier gas group. Error bars represent 1 SEM.</p

Isoflurane releases CD73 containing microparticles in cultured endothelial cells.

<p>A. Human umbilical vein endothelial (EA.hy926) cells were treated with 0–2.5% isoflurane for 1 hr and endothelial cell culture media microparticles (MP) were isolated and assayed for CD73 activity. Isoflurane caused a significant increase in human endothelial cell microparticle CD73 activity (N = 5–8). B. Microparticles isolated from mouse glomerular endothelial cells (GENC) treated with 2.5% isoflurane for 1 hr also had higher CD73 activity compared to carrier gas-treated cells (N = 4–6). C and D. Representative CD73 immunoblotting images (C) and band intensity quantifications (D) from microparticles isolated from EA.hy926 cells. Beta-actin protein expression was also quantified to normalize lane loading. Isoflurane treatment (2.5% for 1 hr) significantly increased CD73 protein expression in EA.hy926 cell microparticles compared to carrier gas-treated cells. *P<0.05 vs. carrier gas group. Error bars represent 1 SEM.</p

Isoflurane stimulates Rho kinase activity in human endothelial cells.

<p>A. Rho kinase activity in human endothelial (EA.hy9262) cells was measured by detecting myosin phosphatase target protein-1 phosphorylation after treatment with 2.5% isoflurane or with carrier gas for 30 min. Isoflurane significantly increased Rho kinase activity in EA.hy9262 cells compared to carrier gas-treated cells (N = 5). *P<0.05 vs. carrier gas group. Error bars represent 1 SEM. B. EA.hy9262 endothelial Rho kinase activity was also assessed by detecting myosin light chain (MLC) phosphorylation in EA.hy9262 cells with immunoblotting. MLC phosphorylation increased in EA.hy9262 cells treated with 2.5% isoflurane for 30 min compared to carrier gas-treated cells. Total MLC immunoreactivity did not change with isoflurane treatment. Representative of 2 experiments performed in triplicate.</p