Search

In the southern San Joaquin Valley (SJV) of California, an agriculturally productive region that relies on groundwater for irrigation and domestic water supply, the infiltration of produced water from oil reservoirs is known to impact groundwater due to percolation from unlined disposal ponds. However, previously documented impacts almost exclusively focus on salinity, while contaminant loadings commonly associated with produced water (e.g., radionuclides) are poorly constrained. For example, the infiltration of bicarbonate-rich produced waters can react with sediment-bound uranium (U), leading to U mobilization and subsequent transport to nearby groundwater. Specifically, produced water infiltration poses a particular concern for SJV groundwater, as valley-fill sediments are well documented to be enriched in geogenic, reduced U. Here, we analyzed monitoring well data from two SJV produced water pond facilities to characterize U mobilization and subsequent groundwater contamination. Groundwater wells installed within 2 km of the facilities contained produced water and elevated levels of uranium. There are >400 produced water disposal pond facilities in the southern SJV. If our observations occur at even a fraction of these facilities, there is the potential for widespread U contamination in the groundwaters of one of the most productive agricultural regions in the world., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The U.S. Environmental Protection Agency estimates that there are over 3.2 million abandoned wells in the United States. Studies conducted on gas emissions from abandoned wells have been limited to methane, a powerful greenhouse gas, due to concerns regarding climate change. However, volatile organic compounds (VOCs), including benzene, a known human carcinogen, are known to be associated with upstream oil and gas development and hence could also be released when methane is emitted to the atmosphere. In this investigation, we analyze gas from 48 abandoned wells in western Pennsylvania for fixed gases, light hydrocarbons, and VOCs and estimate associated emission rates. We demonstrate that (1) gas from abandoned wells contains VOCs, including benzene; (2) VOCs are emitted from abandoned wells, the magnitude of which depends on the flow rate and concentration of VOCs in the gas stream; and (3) nearly one-quarter of abandoned wells are located within 100 m of buildings, including residences, in Pennsylvania. Together, these observations indicate that further investigation is necessary to determine whether emissions from abandoned wells pose an inhalation risk to people living, working, or congregating near abandoned wells., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Oil and gas development generates large amounts of wastewater (i.e., produced water), which in California has been partially disposed of in unlined percolation/evaporation ponds since the mid-20th century. Although produced water is known to contain multiple environmental contaminants (e.g., radium and trace metals), prior to 2015, detailed chemical characterizations of pondwaters were the exception rather than the norm. Using a state-run database, we synthesized samples ( n = 1688) collected from produced water ponds within the southern San Joaquin Valley of California, one of the most productive agricultural regions in the world, to examine regional trends in pondwater arsenic and selenium concentrations. We filled crucial knowledge gaps resulting from historical pondwater monitoring by constructing random forest regression models using commonly measured analytes (boron, chloride, and total dissolved solids) and geospatial data (e.g., soil physiochemical data) to predict arsenic and selenium concentrations in historical samples. Our analysis suggests that both arsenic and selenium levels are elevated in pondwaters and thus this disposal practice may have contributed substantial amounts of arsenic and selenium to aquifers having beneficial uses. We further use our models to identify areas where additional monitoring infrastructure would better constrain the extent of legacy contamination and potential threats to groundwater quality.

People living near oil and gas development are exposed to multiple environmental stressors that pose health risks. Some studies suggest these risks are higher for racially and socioeconomically marginalized people, which may be partly attributable to disparities in exposures. We examined whether racially and socioeconomically marginalized people in California are disproportionately exposed to oil and gas wells and associated hazards. We longitudinally assessed exposure to wells during three time periods (2005-2009, 2010-2014, and 2015-2019) using sociodemographic data at the census block group-level. For each block group and time period, we assessed exposure to new, active, retired, and plugged wells, and cumulative production volume. We calculated risk ratios to determine whether marginalized people disproportionately resided near wells (within 1 km). Averaged across the three time periods, we estimated that 1.1 million Californians (3.0%) lived within 1 km of active wells. Nearly 9 million Californians (22.9%) lived within 1 km of plugged wells. The proportion of Black residents near active wells was 42%-49% higher than the proportion of Black residents across California, and the proportion of Hispanic residents near active wells was 4%-13% higher than their statewide proportion. Disparities were greatest in areas with the highest oil and gas production, where the proportion of Black residents was 105%-139% higher than statewide. Socioeconomically marginalized residents also had disproportionately high exposure to wells. Though oil and gas production has declined in California, marginalized communities persistently had disproportionately high exposure to wells, potentially contributing to health disparities., Competing Interests: The authors declare no conflicts of interest relevant to this study., (© 2023 The Authors. GeoHealth published by Wiley Periodicals LLC on behalf of American Geophysical Union.)

Toxic levels of trace metals from human activities accumulate in natural environments, yet these metal mixtures are rarely characterized or quantified. Metal mixtures accumulate in historically industrial urban areas and change as economies shift. Previous research has often focused on the sources and fate of a specific element, which limits our understanding of metal contaminant interactions in our environment. Here, we reconstruct the history of metal contamination in a small pond downstream of an interstate highway and downwind of fossil fuel and metallurgical industries that have been active since the middle of the nineteenth century. Metal contamination histories were reconstructed from the sediment record using metal ratio mixing analysis to attribute the relative contributions of contamination sources. Cadmium, copper, and zinc concentrations in sediments accumulated since the construction of major road arteries in the 1930s and 40s are, respectively, 3.9, 2.4, and 6.6 times more concentrated than those during industry-dominated time periods. Shifts in elemental ratios suggest these changes in metal concentrations coincide with increased contributions from road and parking lot traffic, and to a lesser extent, from airborne sources. The metal mixture analysis demonstrates that in near-road environments, contributions from modern surface water pathways can obscure historical atmospheric industrial inputs., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Accidental releases (i.e., spills) of produced water can occur at any point during oil and gas development operations, potentially resulting in chronic and/or catastrophic loadings of produced water to nearby ecosystems and exposures of human populations to toxic constituents including trace metals (e.g., arsenic), organic compounds (e.g., benzene), and/or radionuclides (e.g., radium). Despite California being one of the largest oil and gas producing states in the USA, no comprehensive reviews of produced water spills in the peer-reviewed literature have been published. To address this knowledge gap, produced water spill incident data contained within the California HazMat database were synthesized to elucidate trends in produced water spills in California. During the period of 2006-2020, a total of 1029 incidents involving produced water spills were reported. Despite the potential threat to environmental and human receptors, there are significant knowledge gaps concerning these incidents. Specifically, only ~ 6% of spill incidents contained geographic coordinates, greatly hindering assessments of the impacts of these events to public health and the environment. Moreover, updated spill volumes are not rapidly retrievable from the HazMat database, and during the years 2018-2020 volumes of produced water spilled were underreported in initial reports anywhere from 35-2750%. Further, it is unclear if groundwater monitoring is performed following spill events. This study highlights significant shortcomings in produced water spill reporting in California and recommends improvements to aid future investigations that assess the environmental and public health impacts of spill incidents., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The San Joaquin Valley (SJV) in California is one of the most agriculturally productive regions in the world relying in part on groundwater for irrigation and for domestic or municipal water supply for nearly 4 million residents. One area of growing concern in the SJV is potential impact to groundwater resources from ongoing and historical disposal of oilfield-produced water into unlined produced water ponds (PWPs). In this investigation, we utilized available information on composition of produced water disposed into unlined PWPs and levels of total dissolved solids in underlying groundwater to demonstrate that this disposal practice, both past and present, poses risks to groundwater resources, especially in the Tulare Basin in the southern SJV. Groundwater monitoring at unlined PWP facilities is relatively sparse, but where monitoring has occurred, impact to aquifers used for public and agricultural water supply has been observed and has proven to be too expensive to actively remediate. Results of this investigation should inform policy discussions in California and other locations where disposal of produced water into unlined impoundments occurs, especially at locations that overlie groundwater resources.

Soils are the largest dynamic stock of carbon (C) on Earth, and microbial respiration of soil organic C accounts for over 25% of global carbon dioxide (CO 2 ) emissions. Zones of oxygen depletion in upland soils (anaerobic microsites) are increasingly recognized as an important control on soil microbial respiration rates, but the factors governing the volume and distribution of anaerobic microsites are relatively unknown. We measured the dissolved oxygen (DO) content of porewater from incubated soil cores of varying moisture contents (<80% and >80% water saturation) and degrees of disturbance (undisturbed, conventionally tilled, and physically disturbed). Porewater was extracted sequentially from pores constrained by three effective pore diameters, ≥3.0 μm, 3.0-1.0 μm, and 1.0-0.6 μm, from cores incubated for 7, 14, or 28 days, using a modified Tempe cell extraction system. We observed a parabolic pattern in mean dissolved oxygen (DO) concentrations across pore sizes, independent of soil moisture and degree of disturbance. Specifically, DO values within the largest and smallest pore domains were relatively depleted (155 ± 10 μM and 160 ± 11 μM, respectively), while DO values within medium pores were closer to saturation (214 ± 8 μM). The observed DO pattern provides insight into the balance of microbial oxygen demand versus oxygen supply across pore domains within upland soils. Additionally, we observed iron and manganese reduction in all soils except samples subjected to disturbance and incubated at <80% water saturation, suggesting that disturbance enhances aeration and diminishes anaerobic metabolisms within upland soils. Our findings highlight the influence of soil moisture and management on soil redox and CO 2 efflux rates., Competing Interests: Declaration of competing interest The authors declare that they have no financial, or other conflicting, interests in the research presented here., (Copyright © 2021. Published by Elsevier B.V.)

Patients with systemic lupus erythematosus (SLE) display a complex blood transcriptome whose cellular origin is poorly resolved. Using single-cell RNA sequencing, we profiled ~276,000 peripheral blood mononuclear cells from 33 children with SLE with different degrees of disease activity and 11 matched controls. Increased expression of interferon-stimulated genes (ISGs) distinguished cells from children with SLE from healthy control cells. The high ISG expression signature (ISG hi ) derived from a small number of transcriptionally defined subpopulations within major cell types, including monocytes, CD4 + and CD8 + T cells, natural killer cells, conventional and plasmacytoid dendritic cells, B cells and especially plasma cells. Expansion of unique subpopulations enriched in ISGs and/or in monogenic lupus-associated genes classified patients with the highest disease activity. Profiling of ~82,000 single peripheral blood mononuclear cells from adults with SLE confirmed the expansion of similar subpopulations in patients with the highest disease activity. This study lays the groundwork for resolving the origin of the SLE transcriptional signatures and the disease heterogeneity towards precision medicine applications.

Differences in immune function and responses contribute to health- and life-span disparities between sexes. However, the role of sex in immune system aging is not well understood. Here, we characterize peripheral blood mononuclear cells from 172 healthy adults 22-93 years of age using ATAC-seq, RNA-seq, and flow cytometry. These data reveal a shared epigenomic signature of aging including declining naïve T cell and increasing monocyte and cytotoxic cell functions. These changes are greater in magnitude in men and accompanied by a male-specific decline in B-cell specific loci. Age-related epigenomic changes first spike around late-thirties with similar timing and magnitude between sexes, whereas the second spike is earlier and stronger in men. Unexpectedly, genomic differences between sexes increase after age 65, with men having higher innate and pro-inflammatory activity and lower adaptive activity. Impact of age and sex on immune phenotypes can be visualized at https://immune-aging.jax.org to provide insights into future studies.

Aging is linked to deficiencies in immune responses and increased systemic inflammation. To unravel the regulatory programs behind these changes, we applied systems immunology approaches and profiled chromatin accessibility and the transcriptome in PBMCs and purified monocytes, B cells, and T cells. Analysis of samples from 77 young and elderly donors revealed a novel and robust aging signature in PBMCs, with simultaneous systematic chromatin closing at promoters and enhancers associated with T cell signaling and a potentially stochastic chromatin opening mostly found at quiescent and repressed sites. Combined analyses of chromatin accessibility and the transcriptome uncovered immune molecules activated/inactivated with aging and identified the silencing of the IL7R gene and the IL-7 signaling pathway genes as potential biomarkers. This signature is borne by memory CD8 + T cells, which exhibited an aging-related loss in binding of NF-κB and STAT factors. Thus, our study provides a unique and comprehensive approach to identifying candidate biomarkers and provides mechanistic insights into aging-associated immunodeficiency., (© 2017 Ucar et al.)

Early industrial trace metal loadings are poorly characterized but potentially substantial sources of trace metals to the landscape. The magnitude of legacy contamination in southwestern Pennsylvania, the cradle of North American fossil fuel industrialization, is reconstructed from trace metal concentrations in a sediment core with proxies including major and trace metal chemistry, bulk density, and magnetic susceptibility. Trace metal chemistry in this sediment record reflects 19th and 20th century land use and industry. In particular, early 19th century arsenic loadings to the lake are elevated from pesticides used by early European settlers at a lakeside tannery. Later, sediment barium concentrations rise, likely reflecting the onset of acidic mine drainage from coal operations. Twentieth century zinc, cadmium, and lead concentrations are dominated by emissions from the nearby, infamous Donora Zinc Works yet record both the opening of a nearby coal-fired power plant and amendments to the Clean Air Act. The impact of early industry is substantial and rivals more recent metal fluxes, resulting in a significant potential source of contaminated sediments. Thus, modern assessments of trace metal contamination cannot ignore early industrial inputs, as the potential remobilization of legacy contamination would impact ecosystem and human health.

The Centers for Disease Control and Prevention have estimated that each year, two million people in the United States become infected with antibiotic-resistant bacteria, of which, approximately 23000 die as a direct result of these infections. Phage therapy, or the treatment of bacterial infection by specific, antagonistic viruses, provides one alternative to traditional antibiotics. Bacteriophages, or phages, are bacteria-specific viruses that possess biological traits that allow for not only the removal of bacterial infection, but also the evasion of bacterial resistance, which renders antibiotics ineffective. Previous research has shown the addition of iron-doped apatite nanoparticles (IDANPs) to bacteria prior to phage exposure results in increased bacterial plaques in vitro. Coupled with the biocompatible nature of apatite, these results provide promise for future use of IDANPs as adjuvants to phage therapy along with anti-bacterial applications yet to be explored. Although IDANP enhancement of phage infection has been replicated many times in gram-positive and gram-negative prokaryotic hosts as well as with the utilization of both RNA and DNA viruses, the specific mechanisms involved remain elusive. To further understand increased phage infections in a prokaryotic system, and to evaluate the safety of IDANPs as a treatment used in a eukaryotic system, we have replicated plaque assay experiments in an algal system using Chlorella variabilis NC64A and its virus, Paramecium bursaria chlorella virus 1 (PBCV-1). Statistical modeling was used to evaluate alteration in numbers of plaques observed after viral introduction in IDANP-exposed versus non-IDANP-exposed bacterial and algal cell cultures. While IDANPs synthesized between 25°C-45°C and doped with 30% iron have been shown to influence dramatic increases in phage-induced bacterial death, experiments replicated in an algal system indicated viral infections do not increase when C. variabilis cells are pre-exposed to IDANPs. It is essential to potential use of IDANPs as an antibacterial adjuvant that IDANPs do not increase viral infection of eukaryotic host cells during treatment.

The immune response of hemophilia A patients to administered FVIII is a major complication that obviates this very therapy. We have recently described the use of synthetic, biodegradable nanoparticles carrying rapamycin and FVIII peptide antigens, to induce antigen-specific tolerance. Herein we test the tolerogenicity of nanoparticles that contains full length FVIII protein in hemophilia A mice, focusing on anti-FVIII humoral immune response. As expected, recipients of tolerogenic nanoparticles remained unresponsive to FVIII despite multiple challenges for up to 6 months. Furthermore, therapeutic treatments in FVIII-immunized mice with pre-existing anti-FVIII antibodies resulted in diminished antibody titers, albeit efficacy required longer therapy with the tolerogenic nanoparticles. Interestingly, durable FVIII-specific tolerance was also achieved in animals co-administered with FVIII admixed with nanoparticles encapsulating rapamycin alone. These results suggest that nanoparticles carrying rapamycin and FVIII can be employed to induce specific tolerance to prevent and even reverse inhibitor formation., (Published by Elsevier Inc.)

Expansion of human regulatory T cells (Tregs) for clinical applications offers great promise for the treatment of undesirable immune responses in autoimmunity, transplantation, allergy, and antidrug antibody responses, including inhibitor responses in hemophilia A patients. However, polyclonal Tregs are nonspecific and therefore could potentially cause global immunosuppression. To avoid this undesirable outcome, the generation of antigen-specific Tregs would be advantageous. Herein, we report the production and properties of engineered antigen-specific Tregs, created by transduction of a recombinant T-cell receptor obtained from a hemophilia A subject's T-cell clone, into expanded human FoxP3(+) Tregs. Such engineered factor VIII (FVIII)-specific Tregs efficiently suppressed the proliferation and cytokine production of FVIII-specific T-effector cells. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production from FVIII-primed spleen cells in vitro were profoundly inhibited in the presence of these FVIII-specific Tregs, suggesting potential utility to treat anti-FVIII inhibitory antibody formation in hemophilia A patients.

Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.

A master control of both the innate and adaptive immune system is the body's ability to distinguish between self and foreign entities. This is accomplished by the elimination of autoreactive leukocytes through a series of checkpoints both in the thymus (central deletion) and in the circulating periphery (peripheral tolerance), thus establishing tolerance to self-antigens. When one or more of these controls is disrupted, there is the potential for the development of autoimmune disease. Current available therapies for these diseases often rely on global immune suppression or expensive treatments that are not affordable to all. Herein, we describe a novel therapeutic technique in which tolerance to self can be re-established via B-cell delivered antigen-specific tolerogenic gene constructs. Furthermore, this technique shows promise in the gene therapeutic treatment of monogenic disorders and the acceptance of tissue transplants.

Wood burning for residential heating is prevalent in the Rocky Mountain regions of the United States. Studies have shown that wood stoves can be a significant source of PM(2.5) within homes. In this study, the effectiveness of an electrostatic filter portable air purifier was evaluated (1) in a home where a wood stove was the sole heat source and (2) in a home where a wood stove was used as a supplemental heat source. Particle count concentrations in six particle sizes and particle mass concentrations in two particle sizes were measured for ten 12-hour purifier on and ten purifier off trials in each home. Particle count concentrations were reduced by 61-85 percent. Similar reductions were observed in particle mass concentrations. These findings, although limited to one season, suggest that a portable air purifier may effectively reduce indoor particulate matter concentrations associated with wood combustion during home heating.

Damaging inflammation arising from autoimmune pathology and septic responses results in severe cases of disease. In both instances, anti-inflammatory compounds are used to limit the excessive or deregulated cytokine responses. We used a model of robust T cell stimulation to identify new proteins involved in triggering a cytokine storm. A comparative proteomic mining approach revealed the differential mapping of Raf kinase inhibitory protein after T cell recall in vivo. Treatment with locostatin, an Raf kinase inhibitory protein inhibitor, induced T cell anergy by blocking cytokine production after Ag recall. This was associated with a reduction in Erk phosphorylation. Importantly, in vivo treatment with locostatin profoundly inhibited TNF-alpha production upon triggering the Ag-specific T cells. This effect was not limited to a murine model because locostatin efficiently inhibited cytokine secretion by human lymphocytes. Therefore, locostatin should be a useful therapeutic to control inflammation, sepsis, and autoimmune diseases.

LPS is a natural adjuvant that potentiates Ag-specific T cell survival and Th1 differentiation by stimulating MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) signaling pathways. In this study, we reveal the TRIF pathway is critical for amplifying murine effector T cell accumulation into nonlymphoid tissues following immunization with Ag plus LPS. Although LPS increased the accumulation of splenic T cells in TRIF-deficient mice, markedly fewer T cells were recovered from liver and lung in comparison to wild type. Most of the T cells primed in TRIF-deficient mice failed to up-regulate CXCR3 and had an overall reduced capacity to produce IFN-gamma, demonstrating effector T cell differentiation was linked to their migration. To investigate the role of TRIF-dependent cytokines, neutralization studies were performed in wild type mice. Although TNF neutralization reduced T cell numbers, its coneutralization with IL-10 unexpectedly restored the T cells, suggesting the balance between pro- and anti-inflammatory cytokines influences T cell survival rather than their magnitude. To investigate a role for costimulatory molecules, we tested whether the T cell defect in TRIF-deficient mice could be corrected with enforced costimulation. Boosting with a CD40 agonist in addition to LPS restored the effector CD8 T cell response in livers of TRIF-deficient mice while only partially restoring CD4 T cells, suggesting that LPS primes CD8 and CD4 T cell immunity through different mechanisms. Overall, our data support targeting TRIF for vaccines aimed to direct immune responses to nonlymphoid tissues.

Staphylococcus aureus, a primary source of bacterial superantigen (SAg), is known to colonize the human respiratory tract and has been implicated in airway inflammation. Studies have documented a role for SAgs in respiratory disorders, such as nasal polyps, chronic obstructive pulmonary disease, chronic rhinosinusitis, and asthma. However, cellular and molecular mediators involved in SAg-mediated pulmonary disease have not been clearly identified. In this study, we investigated the effect of intranasal staphylococcal enterotoxin A (SEA) exposure on murine lung. The pathological features in the lung resulting from SEA exposure had characteristics of both obstructive and restrictive pulmonary disorders. There was also an increase in bronchoalveolar lavage protein concentration and cellularity following SEA challenge. Massive CD8(+)Vbeta3(+) T cell accumulation observed in the lung was dependent on CD4 T cell help, both for recruitment and for IFN-gamma synthesis. The primary source of IFN-gamma synthesis was CD8 T cells, and depletion of these cells abrogated disease. IFN-gamma deficiency also prevented SEA-mediated disease, and this was by enhancing early recruitment of neutrophils as detected in the bronchoalveolar lavage. Thus, IFN-gamma appeared to selectively aid the recruitment of T cells to the lungs while preventing the neutrophil accumulation. Therefore, our results show that IFN-gamma-producing CD8 T cells mediated pulmonary alveolitis and inflammation, which were dependent upon CD4 T cells for their recruitment to the lung.

Background: Staphylococcusaureus, a primary source of bacterial superantigen, is known to colonize the human respiratory tract and has been implicated in airway inflammation. The potential pathological effect of staphylococcal enterotoxins on the respiratory tract necessitates a detailed understanding of how they regulate innate immune cells, particularly CD11c-expressing dendritic cells (DCs)., Methods: C57BL/6 mice were challenged intranasally with staphylococcal enterotoxin A (SEA) and at indicated time points lung tissue was perfused, digested and analyzed for CD11c+ expressing cells., Results: The pulmonary CD11c+ cells can be divided into two major populations based on their MHC II expression. One day following intranasal SEA challenge, there was rapid accumulation of CD11c+ cells expressing medium to high levels of MHC II. The peak accumulation of CD11c+ MHC II- population was observed 2 days after SEA challenge; however, careful examination of this cell population revealed that they were heterogeneous, being comprised of cells bearing CD3, CD19, NK1.1 and F4/80 along with varying levels of CD11c. Nevertheless, there was a 2-fold increase of CD11c+ MHC II- (CD3- CD19- NK1.1- F4/80-) cells in the lungs., Conclusion: The mechanism of increase in the CD11c+ MHC II- immune progenitor population was mainly due to cellular division rather than migration from blood to lung. In contrast, the early and rapid accumulation of CD11c+ MHC II(hi) cells, conventionally known as DCs, in the lung on day 1 was mostly due to migration from blood. Thus this study examines the pulmonary innate immune response to a powerful immune stimulus., (Copyright 2008 S. Karger AG, Basel.)

Bacterial LPS is a natural adjuvant that induces profound effects on T cell clonal expansion, effector differentiation, and long-term T cell survival. In this study, we delineate the in vivo mechanism of LPS action by pinpointing a role for MyD88 and CD11c(+) cells. LPS induced long-term survival of superantigen-stimulated CD4 and CD8 T cells in a MyD88-dependent manner. By tracing peptide-stimulated CD4 T cells after adoptive transfer, we showed that for LPS to mediate T cell survival, the recipient mice were required to express MyD88. Even when peptide-specific CD4 T cell clonal expansion was dramatically boosted by enforced OX40 costimulation, OX40 only synergized with LPS to induce survival when the recipient mice expressed MyD88. Nevertheless, these activated, but moribund, T cells in the MyD88(-/-) mice acquired effector properties, such as the ability to synthesize IFN-gamma, demonstrating that effector differentiation is not automatically coupled to a survival program. We confirmed this notion in reverse fashion by showing that effector differentiation was not required for the induction of T cell survival. Hence, depletion of CD11c(+) cells did not affect LPS-driven specific T cell survival, but CD11c(+) cells were paramount for optimal effector T cell differentiation as measured by IFN-gamma potential. Thus, LPS adjuvanticity is based on MyD88 promoting T cell survival, while CD11c(+) cells support effector T cell differentiation.

The TNFR superfamily members 4-1BB (CD137) and OX40 (CD134) are costimulatory molecules that potently boost CD8 and CD4 T cell responses. Concomitant therapeutic administration of agonist anti-CD137 and -CD134 mAbs mediates rejection of established tumors and fosters powerful CD8 T cell responses. To reveal the mechanism, the role of CD137 expression by specific CD8 T cells was determined to be essential for optimal clonal expansion and accumulation of effector cells. Nonetheless, dual costimulation induced production of supereffector CD8 T cells when either the specific T cells or the host alone bore CD137. Perhaps surprisingly, the total absence of CD137 prevented anti-CD134 augmentation of supereffector differentiation demonstrating an unappreciated link between these related pathways. Ultimately, it was reasoned that these powerful dual costimulatory responses involved common gamma family members, and we show substantial increases of CD25 and IL-7Ralpha-chain expression by the specific CD8 T cells. To investigate this further, it was shown that IL-7 mediated T cell accumulation, but importantly, a gradual and preferential effect of survival was directed toward supereffector CD8 T cells. In fact, a clear enhancement of effector differentiation was demonstrated to be proportional to the increasing amount of IL-7Ralpha expression by the specific CD8 T cells. Therefore, dual costimulation through CD137 and CD134 drives production and survival of supereffector CD8 T cells through a distinct IL-7-dependent pathway.

In general, TGFbeta is synthesized as a procytokine that requires proteolytic activation, release of the mature cytokine from its noncovalently associated latent-associated peptide, and binding to TGFbetaRII to mediate suppressive activity. We tracked this process in mice containing primed CD8 regulatory T cells (Tregs) by immunoblotting in primary whole cell lysates for pro-TGFbeta, latent-associated peptide and mature TGFbeta. Generation of CD8 Tregs promoted processing of the 50 kDa pro-TGFbeta protein into a 12.5 kDa mature TGFbeta species in vivo. Despite the inability to detect mature TGFbeta in the sera of mice with primed CD8 Tregs and in the synthetic culture medium of stimulated CD8 Tregs, we demonstrated engagement of TGFbetaRII through immunoblotting for Smad2 phosphorylation. This process relied on continual TCR triggering, which also induced Smad3 phosphorylation. To understand the movement of mature TGFbeta, we showed that in contrast to IFN-gamma, mature TGFbeta does not remain a soluble cytokine but is likely to be rapidly adsorbed by neighboring cells. These data show the exquisite local control directed toward TGFbeta by the immune system and underscore the fine specificity involved in its detection.

IL-18 induces inflammation resulting in either enhanced protection from pathogens or exacerbation of autoimmunity, and T cells are profoundly activated during these responses. How IL-18 influences T cell activation is unknown, but this study in mice shows that IL-18 boosted Ag-specific T cell clonal expansion of effector T cells and induced a subpopulation of IFN-gamma superproducing T cells. Commitment to IFN-gamma production through IL-18 was independent of NK cells and IL-12 but dependent on host-derived IFN-gamma. To determine how expansion of these effectors occurred, IL-18 was shown to induce OX40L on dendritic cells, whereas peptide stimulation induced CD134 (OX40) on specific T cells. CD134 blockade inhibited T cell effector expansion thereby reducing the number of IFN-gamma superproducers by 12-fold. Thus, independent of IL-12, IL-18 impacts T cell immunity throughout lymphoid and nonlymphoid tissue by bridging the innate and adaptive arms of the immune system through IFN-gamma and the CD134 costimulatory pathway.

In practice, vaccines should induce lasting and efficacious T cell immunity without promoting deleterious pathological consequences. To accomplish this goal we immunized mice with ovalbumin peptide, polyinosinic-polycytidylic and anti-CD137. Vaccinated mice retained a massive functional CD8 T cell memory pool in lymphoid and non-lymphoid tissues for >1 year. The memory T cells clonally expanded, produced substantial amounts of IFNgamma, and responded vigorously to vesicular stomatitis virus infection. To understand how the vaccine might function, we showed that the antigen-specific T cells must bear CD137 in order for optimal priming to occur. Thus, anti-CD137 agonist mAb directly stimulated peptide-specific CD8 T cells and conditioned them to survive. In contrast, CD137-deficient CD8 T cells did not survive despite CD137 expression by antigen presenting cells. Taken together, the data indicate that CD137 and adjuvant combined therapy is an efficacious vaccine strategy for immunization with non-replicating inert antigen.

In this report we examined overlap between superantigen (SAg) and Toll-like receptor 4 (TLR4) stimulation of the innate immune system. Before in vivo stimulation we found that mouse splenic DCs expressed unexpectedly low levels of surface TLR4 compared to macrophages. In response to LPS, TLR4 gene expression in fractionated spleen cells was downregulated. By comparison, surface TLR4 staining with the Sa15-21 mAb showed little downregulation, and the anti-TLR4 MTS510 mAb showed decreased staining, suggesting that LPS was bound to TLR4 at the time points examined. Interestingly, SAg stimulation induced decreased TLR4 staining as measured by the MTS510 mAb, even though the TLR4 gene was not downregulated. Nevertheless, LPS potently induced DCs to produce TNF and IL-12, but SAg did not, even though they efficiently activated DCs. Notwithstanding, in vivo stimulation with staphylococcal enterotoxin SAg conditioned the innate immune system to hyper-respond to various pathogen-associated molecular patterns (PAMPs). Specifically, pre-priming with SAg enhanced LPS-mediated DC synthesis of TNF and IL-12. Thus, SAgs may exert their pathogenesis on the host by conditioning DCs, in a T cell activation dependent manner to potentiate responses to PAMPs.

After in vivo immunization, Ag-specific T cells disappear from circulation and become sequestered in lymphoid tissue where they encounter Ag presented by dendritic cells. In the same site and just after Ag presentation, they "disappear" a second time and we investigated this process. Using a mouse model of T cell deletion (without Toll-like receptor (TLR) stimulation) vs survival (with TLR stimulation), Ag-specific T cells indeed became undetectable by flow cytometry, however were readily detected by immunohistochemistry. Thus, whether or not the activated T cells were destined to delete or survive, they were difficult to extract from lymphoid tissue and did not disappear but in fact were abundantly present. Nevertheless, profound differences were observed during this time period when tolerizing conditions were compared with immunizing conditions. TLR stimulation induced an increase in CD25 expression, acquisition of surface MHC class II, and abnormally high increases in forward and side scatter of the peptide-specific T cells. Using a modified adoptive transfer approach, we demonstrated by flow cytometry that in the presence of TLR stimulation the Ag-specific T cells were tightly coupled to dendritic cells, explaining the unusual increases in size and granularity. Ultimately, these events induced the specific T cells to differentiate into memory cells. We postulate that this is a stage where T cells are either conditioned to survive or to delete depending upon the activation status of the innate immune system.

To better understand how innate and adaptive immune responses interact with each other, we combined 4-1BB T cell costimulation with specific adjuvants to determine whether these treatments would influence specific T cell expansion and function in vivo. In the presence of 4-1BB ligation and Toll-like receptor 3 (TLR)3 and/or TLR4 triggering, CD8 T cell clonal expansion and survival was augmented profoundly. Specific T cells primed in vivo with TLR ligands responded normally to in vitro recall stimulus, but, surprisingly, copriming with 4-1BB costimulation significantly impaired the recall response even though many more specific effector T cells were rescued in vivo. Here, we demonstrate that the rescued CD8 T cells suppressed CD4 T cell proliferation via a type beta transforming growth factor-dependent mechanism. Thus, 4-1BB and TLR ligands induce survival of specific effector CD8 T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in mediating tumor clearance and prevention of autoimmune disease.

Thrombocytopenia is a relatively common side effect observed during glycoprotein (GP) IIb/IIIa antagonist therapy. With the oral antagonist roxifiban, we observed thrombocytopenia, defined as 50% reduction of platelets over predose values or below 90 000/microL (9 x 10(10)/L), with a frequency of 2% (8 of 386). Thrombocytopenia occurred either early (days 2 to 4) or delayed (days 11 to 16). No additional cases were observed with up to 6 months of treatment. Retrospective analysis provided evidence for drug-dependent antibodies (DDABs) to GP IIb/IIIa in 5 of 6 subjects, suggestive of an immune etiology of thrombocytopenia. The hypothesis that excluding patients based on positive DDAB reaction would reduce the frequency of thrombocytopenia was tested. Patients were screened for DDABs during the study qualification period and, overall, 3.9% of the patients were excluded based on pre-existing DDAB concentrations above a statistically defined medical decision limit. An additional 2.6% were excluded based on therapy-related antibody production during the first 2 weeks. With antibody testing, 0.2% of patients (2 of 1044) developed immune-mediated thrombocytopenia. One case developed a rapidly increasing antibody concentration and presented with thrombocytopenia despite discontinuation of roxifiban therapy. The second case was related to a false-negative test result. The frequency of thrombocytopenia was statistically significantly reduced from 2% to 0.2% (P =.0007) comparing nonscreened and screened patients. Testing for DDABs can reduce the frequency of thrombocytopenia in patients treated with roxifiban and, by analogy, other GP IIb/IIIa antagonists. Thus, DDAB testing may be employed to increase the safety of GP IIb/IIIa antagonists.

A study was conducted to determine the effects of strapped spectacles on the fit factors obtained during quantitative fit testing on three different brands of full facepiece negative pressure respirators. The three brands of respirators were evaluated with and without strapped spectacles worn by the test subjects. A total of 180 quantitative fit testing trials were conducted on ten male test subjects. For each test subject, three quantitative fit testing trials were performed with each brand of respirator with and without the spectacles. The average of the fit testing trials for each subject with each respirator was used for statistical analysis. The results demonstrated that the fit factor values were significantly lower during use of the spectacles (p < 0.05). The estimated percentage of test subjects who failed the American National Standards Institute pass/fail criteria for quantitative fit testing (1000) increased by 15-36% when spectacles were worn.

The binding parameters, the affinity constant (Ka) and binding capacity (Q), of a protein possessing ligand-protein complexes with a high dissociation rate (Sex Steroid Binding protein from Bufo arenarum) were determined using a solid-phase method. The technique is based upon the adsorption of the steroid-protein complex to DEAE-cellulose. This method was compared with a nonequilibrium method (charcoal adsorption of free ligand), and the latter resulted in underestimation of both binding parameters, Ka and Q. The solid-phase method reported here is appropriate to determine the binding parameters of proteins with high dissociation rates because the results are independent of the complex half-time. The method also possesses advantages compared to other equilibrium assays such as dialysis or steady-state electrophoresis. With minor modifications, it may be useful to characterize different proteins, particularly those possessing ligand-protein complexes with very high dissociation rates.