A Longitudinal Hepatitis B Vaccine Cohort Demonstrates Long-lasting Hepatitis B Virus (HBV) Cellular Immunity Despite Loss of Antibody Against HBV Surface Antigen.

BACKGROUND: Long-lasting protection resulting from hepatitis B vaccine, despite loss of antibody against hepatitis B virus (HBV) surface antigen (anti-HBs), is undetermined. METHODS: We recruited persons from a cohort vaccinated with plasma-derived hepatitis B vaccine in 1981 who have been followed periodically since. We performed serological testing for anti-HBs and microRNA-155 and assessed HBV-specific T-cell responses by enzyme-linked immunospot and cytometric bead array. Study subgroups were defined 32 years after vaccination as having an anti-HBs level of either ≥10 mIU/mL (group 1; n = 13) or <10 mIU/mL (group 2; n = 31). RESULTS: All 44 participants, regardless of anti-HBs level, tested positive for tumor necrosis factor α, interleukin 10, or interleukin 6 production by HBV surface antigen-specific T cells. The frequency of natural killer T cells correlated with the level of anti-HBs (P = .008). The proportion of participants who demonstrated T-cell responses to HBV core antigen varied among the cytokines measured, suggesting some natural exposure to HBV in the study group. No participant had evidence of breakthrough HBV infection. CONCLUSIONS: Evidence of long-lasting cellular immunity, regardless of anti-HBs level, suggests that protection afforded by primary immunization with plasma-derived hepatitis B vaccine during childhood and adulthood lasts at least 32 years.

A Repeat Pattern of Founder Events for SARS-CoV-2 Variants in Alaska.

Alaska is a unique US state because of its large size, geographically disparate population density, and physical distance from the contiguous United States. Here, we describe a pattern of SARS-CoV-2 variant emergence across Alaska reflective of these differences. Using genomic data, we found that in Alaska, the Omicron sublineage BA.2.3 overtook BA.1.1 by the week of 27 February 2022, reaching 48.5% of sequenced cases. On the contrary, in the contiguous United States, BA.1.1 dominated cases for longer, eventually being displaced by BA.2 sublineages other than BA.2.3. BA.2.3 only reached a prevalence of 10.9% in the contiguous United States. Using phylogenetics, we found evidence of potential origins of the two major clades of BA.2.3 in Alaska and with logistic regression estimated how it emerged and spread throughout the state. The combined evidence is suggestive of founder events in Alaska and is reflective of how Alaska's unique dynamics influence the emergence of SARS-CoV-2 variants.

Pattern of SARS-CoV-2 variant B.1.1.519 emergence in Alaska.

Alaska has the lowest population density in the United States (US) with a mix of urban centers and isolated rural communities. Alaska's distinct population dynamics compared to the contiguous US may have contributed to unique patterns of SARS-CoV-2 variants observed in early 2021. Here we examined 2323 SARS-CoV-2 genomes from Alaska and 278,635 from the contiguous US collected from December 2020 through June 2021 because of the notable emergence and spread of lineage B.1.1.519 in Alaska. We found that B.1.1.519 was consistently detected from late January through June of 2021 in Alaska with a peak prevalence in April of 77.9% unlike the rest of the US at 4.6%. The earlier emergence of B.1.1.519 coincided with a later peak of Alpha (B.1.1.7) compared to the contiguous US. We also observed differences in variant composition over time between the two most populated regions of Alaska and a modest increase in COVID-19 cases during the peak incidence of B.1.1.519. However, it is difficult to disentangle how social dynamics conflated changes in COVID-19 during this time. We suggest that the viral characteristics, such as amino acid substitutions in the spike protein, likely contributed to the unique spread of B.1.1.519 in Alaska.

A repeat pattern of founder events for SARS-CoV-2 variants in Alaska.

Alaska is a unique US state because of its large size, geographically disparate population density, and physical distance from the contiguous United States. Here, we describe a pattern of SARS-CoV-2 variant emergence across Alaska reflective of these differences. Using genomic data, we found that in Alaska the Omicron sublineage BA.2.3 overtook BA.1.1 by the week of 2022-02-27, reaching 48.5% of sequenced cases. On the contrary in the contiguous United States, BA.1.1 dominated cases for longer, eventually being displaced by BA.2 sublineages other than BA.2.3. BA.2.3 only reached a prevalence of 10.9% in the contiguous United States. Using phylogenetics, we found evidence of potential origins of the two major clades of BA.2.3 in Alaska and with logistic regression estimated how it emerged and spread throughout the state. The combined evidence is suggestive of founder events in Alaska and is reflective of how Alaska’s unique dynamics influence the emergence of SARS-CoV-2 variants.

Observed Changes in Natural Killer and T cell Phenotypes with Evaluation of Immune Outcome in a Longitudinal Cohort Following Sofosbuvir-Based Therapy for Chronic Hepatitis C Infection.

BACKGROUND: Chronic hepatitis C virus (HCV) infection diminishes immune function through cell exhaustion and repertoire alteration. Direct acting antiviral (DAA)-based therapy can restore immune cell subset function and reduce exhaustion states. However, the extent of immune modulation following DAA-based therapy and the role that clinical and demographic factors play remain unknown. METHODS: We examined natural killer (NK) cell, CD4+, and CD8+ T cell subsets along with activation and exhaustion phenotypes across an observational study of sofosbuvir-based treatment for chronic HCV infection. Additionally, we examined the ability of clinical variables and duration of infection to predict 12 weeks of sustained virologic response (SVR12) immune marker outcomes. RESULTS: We show that sofosbuvir-based therapy restores NK cell subset distributions and reduces chronic activation by SVR12. Likewise, T cell subsets, including HCV-specific CD8+ T cells, show reductions in chronic exhaustion markers by SVR12. Immunosuppressive CD4+ regulatory T cells decrease at 4-weeks treatment and SVR12. We observe the magnitude and direction of change in immune marker values from pretreatment to SVR12 varies greatly among participants. Although we observed associations between the estimated date of infection, HCV diagnosis date, and extent of immune marker outcome at SVR12, our regression analyses did not indicate any factors as strong SVR12 outcome predictors. CONCLUSION: Our study lends further evidence of immune changes following sofosbuvir-based therapy. Further investigation beyond SVR12 and into factors that may predict posttreatment outcome is warranted.

Exercise Increases Glucose Transporter-4 Levels on Peripheral Blood Mononuclear Cells.

PURPOSE: Glucose transporter 4 (GLUT4) plays a key role in the pathophysiology of type 2 diabetes. Glucose transporter 4 is upregulated in response to exercise, enhancing cellular glucose transport in skeletal muscle tissue. This mechanism appears to remain intact in individuals with insulin resistance. Details of the mechanism are poorly understood and are challenging to study due to the invasive nature of muscle biopsy. Peripheral blood mononuclear cells (PBMC) have documented insulin-sensitive GLUT4 activity and may serve as a proxy tissue for studying skeletal muscle GLUT4. The purpose of this study was to investigate whether GLUT4 in PBMC is affected by conditioning. METHODS: We recruited 16 student athletes from the cross-country running and skiing teams and fifteen sedentary students matched for age and sex from the University of Alaska Fairbanks. Peripheral blood mononuclear cells were collected with mononuclear cell separation tubes. The GLUT4 concentrations were measured using a commercially available enzyme linked immunosorbent assay. Additionally, correlations between PBMC GLUT4 and common indicators of insulin resistance were examined. RESULTS: Results indicate significantly higher PBMC GLUT4 levels in conditioned athletes than in their sedentary counterparts, similar to what has been documented in myocytes. Females were observed to have higher PBMC GLUT4 levels than males. Correlations were not detected between PBMC GLUT4 and hemoglobin A1c, glucose, insulin, homeostatic model assessment of insulin resistance, body mass index, or body fat. CONCLUSIONS: This study provides evidence to support exploration of PBMC as a proxy tissue for studying GLUT4 response to exercise or other noninsulin factors.

Cellular mechanisms of mainstream cigarette smoke-induced lung epithelial tight junction permeability changes in vitro.

Mainstream cigarette smoke increases the permeability of human airways; however, the mechanism for this increased permeability is poorly defined. Tight junctions between adjacent epithelial cells constitute the physiological barrier to fluid and macromolecules in epithelium. These structures are highly regulated by phosphorylation and their association with the cytoskeleton. The goal of these studies was to identify the signal transduction pathways that regulate smoke-induced permeability. Using a physiologically relevant air-liquid interface exposure system, electrically tight monolayers of the human bronchial epithelial cell-line Calu-3 were exposed to fresh, whole mainstream cigarette smoke. This exposure results in a regulated, dose-dependent loss of epithelial barrier function in the lung epithelial monolayers. With cigarette smoke exposure, transepithelial electrical resistance (TER) is decreased and albumin flux is increased, indicating a loss in barrier function to ions and macromolecules, respectively; however, both largely recover in 30 min. Smoke-induced losses of macromolecular barrier function are the result of multicellular junctional reorganization, resulting in increased leak volume rather than leak frequency. Inhibiting Rho kinase (ROCK) significantly reduces the smoke-induced permeability to both ions and macromolecules, while inhibiting protein tyrosine kinases (PTK) only reduces smoke-induced macromolecular permeability. Interestingly, inhibiting myosin light chain kinase (MLCK) exacerbates smoke-induced permeability, indicating that MLCK and ROCK have opposing regulatory roles. Our results demonstrate that the smoke-induced loss of epithelial barrier function in human bronchial epithelium is a regulated process rather than a cytotoxic response. Additionally, our results indicate that activation of PTK and ROCK and inactivation of MLCK contribute to the increased airway permeability caused by mainstream cigarette smoke.

CP-64131, an aminobenzazepine with cytokine-like properties, stimulates human neutrophil functions through the p38-MAPK pathway.

CP-64131 (CP), an aminobenzazepine with cytokine-like, physiologic effects similar to granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage (GM)-CSF, increases the number of neutrophils and stimulates marrow recovery after doxirubicin ablation. CP can also function as a neutrophil agonist, like formyl-Met-leu-Phe (fMLP). In these studies, we show that CP is unique in that it stimulates the p38-mitogen-activated protein kinase (MAPK) pathway but not extracellular signal-regulated kinase (ERK)1/2 or c-jun N-terminal kinase MAPKs in human neutrophils from peripheral blood. This is in contrast to other neutrophil agonists such as fMLP, interleukin (IL)-8, or GM-CSF, which stimulate multiple MAPK pathways. Like fMLP and IL-8, CP is capable of stimulating superoxide (O2-) production, CD11b expression, and cell polarization in human neutrophils. CP-stimulated O2- production is completely dependent on p38-MAPK activation, as determined by sensitivity to the p38-MAPK inhibitor SB203580. In contrast, SB203580 only partially inhibits expression of CD11b and has no effect on cell polarization stimulated by CP. Therefore, CP treatment of neutrophils activates p38-MAPK but has effects independent of p38-MAPK activation. In human embryonic kidney 293 cells, a human kidney epithelial cell line CP stimulates p38-MAPK and modestly activates ERK1/2. The findings define CP as a novel, small molecule, which has little cellular toxicity in vitro. CP has the ability to activate specific MAPK pathways in different cell types and should prove to be an effective agonist in combination with inhibitors to study biological responses regulated by MAPKs.

G-protein regulatory pathways: Rocketing into the twenty-first century.

Complex cellular responses involve the integration of heterotrimeric G protein systems with protein kinase signal transduction pathways. Key in this integration is the control of small GTP-binding proteins including Ras and Rho family members. In this paper, we discuss the control of signal transduction pathways by G proteins and their integration with specific tyrosine kinases. The integration of G proteins, kinases, and small GTP-binding proteins in controlling cellular responses is illustrated through the newly defined Gα12/13 -regulated pathways. Furthermore, the polymorphonuclear leukocyte provides a primary cell system for analyzing the integration of G proteins, kinases, and small GTP-binding proteins in controlling cellular functions such as superoxide production, adherence, chemotaxis, and granule secretion. J. Cell. Biochem. Suppls. 30/31:137-146, 1998. © 1998 Wiley-Liss, Inc.

Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins are associated with mainstream cigarette smoke-induced permeability of airway epithelium.

Cigarette smoke increases the permeability of the lung epithelium. Consequences of increased permeability include increased access of toxins and pathogens from the air spaces to the interstitium and even the blood stream, and leakage of fluids into the air spaces. The mechanisms for permeability alterations have not been elucidated for airway epithelia. By analogy with other types of epithelia, we hypothesized that changes in the phosphorylation status and function of tight junction (TJ) or cytoskeletal proteins might mediate the smoke-induced permeability changes. We investigated the effects of exposure to mainstream cigarette smoke (MS) on cultures of Calu-3 cells, an airway epithelial cell line. Specifically, MS exposure caused increases in phosphorylation of the myosin-binding subunit (MBS) of myosin phosphatase and myosin light chain (MLC), proteins involved in the regulation of actin polymerization. These results implicate activation of Rho kinase (ROCK), consistent with previously reported data indicating that inhibition of ROCK activation suppressed MS-induced increases in permeability. MS exposure also increased polymerized (filamentous) actin (f-actin) content and caused redistribution of the TJ proteins from the normal apical circumferential band to a more basal location. The translocation of the TJ proteins was spatially associated with local increases in both f-actin and macromolecular permeability. Finally, MS exposure increased tyrosine phosphorylation of occludin but not ZO-1 and decreased association between the two TJ proteins. These results indicate that MS exposure causes alterations in cytoskeletal and TJ structure and function, resulting in increased macromolecular permeability that may contribute to the adverse health effects of MS.

Diesel particulate material binds and concentrates a proinflammatory cytokine that causes neutrophil migration.

Exposure to combustion emissions is associated with adverse health effects, but the properties of the emissions that induce these effects are not fully understood. To examine the direct effects of diesel particulate material (DPM) on alveolar epithelial cells, A549 cells were exposed to DPM. Low concentrations of DPM increased the interleukin-8 (IL-8) detected in the conditioned medium. Higher doses appeared to suppress the response, although this suppression was not related to acute DPM toxicity. In a cell-free system, incubation of IL-8 with DPM resulted in loss of immunoreactive IL-8 from the supernatant of the reaction. In contrast, carbon black did not reduce the concentration of IL-8 in the mixture. The DPM-induced loss was only weakly blocked by a large excess of bovine serum albumin (BSA). High concentrations of salts partially prevented the loss, but extraction of the soot with organic solvents had no effect. To determine biological implications, human blood neutrophils were exposed to DPM that had been preincubated with IL-8, then washed to remove free IL-8. The neutrophils changed shape in a manner suggesting directed movement toward the particles. No morphological change was observed either with carbon black that had been incubated with IL-8 or with DPM alone. These results suggest that DPM not only induces the production of IL-8 by epithelial cells, but also binds biologically active chemokine in a particle- and protein-selective manner. DPM-induced inflammatory responses may therefore be more focused or sustained as a result of this binding of inflammatory mediators by DPM.

Chronic nicotine inhibits inflammation and promotes influenza infection.

Epidemiological data suggest an association between smoking, respiratory infections, and impaired wound healing. Inflammation is critical in the body's defense against pathogens and in the wound-healing process. Although nicotine is used to treat some inflammatory conditions, the mechanism of this action is largely unknown. To determine how nicotine affects inflammation, rats and mice were exposed to nicotine via miniosmotic pumps, and the inflammatory response to turpentine or influenza virus was assessed. Results showed that while nicotine suppressed the migration of leukocytes to the inflammation/infection site, it increased the influenza titer in the lung. The decreased inflammation correlated with lower chemotaxis/chemokinesis of peripheral blood mononuclear cells (PBMC) toward formyl-methionyl-leucyl-phenylalanine and monocyte chemoattractant protein-1 without affecting the density of their respective receptors. However, nicotine suppressed the chemokine-induced Ca(2+) response in PBMC, indicating impaired chemokine signaling. Thus, because nicotine suppresses leukocyte migration, it might contribute to the delayed wound healing and increased incidence of respiratory infections among smokers.