Inflammatory and psychological consequences of chronic high exposure firefighting.

OBJECTIVES: This study aimed to examine the impact of extreme heat exposure frequency on inflammation and well-being in UK Fire Service personnel. METHODS: 136 Fire personnel and 14 controls (CON) were recruited [92 Firefighters (FF), 44 Breathing Apparatus Instructors (BAI)]. BAI were split into low (LBAI; ≤15 exposures per month) and high (HBAI; ≥20 exposures per month) categories. Measures of inflammation, mood and fatigue were collected at 0, 3 and 6 month times points. These variables were analysed for differences between groups and association with frequency of exposure. RESULTS: HBAI exhibited raised IL-1ß, IL-6, IL-10, IgE and lower IgM (p < 0.05). In addition, IL-1ß, IL-6, IL-10 and IgM were associated with monthly exposure number, with exposures accounting for 15.4% of the variance in IL-6, 11.8% of IL-1ß and 25.2% of IL-10. No differences in mood or fatigue were reported (p > 0.05). CONCLUSION: High exposure firefighting consistently causes systemic inflammation without perceptual recognition of potential health risks.

CD28null pro-atherogenic CD4 T-cells explain the link between CMV infection and an increased risk of cardiovascular death.

An increased risk of cardiovascular death in Cytomegalovirus (CMV)-infected individuals remains unexplained, although it might partly result from the fact that CMV infection is closely associated with the accumulation of CD28null T-cells, in particular CD28null CD4 T-cells. These cells can directly damage endothelium and precipitate cardiovascular events. However, the current paradigm holds that the accumulation of CD28null T-cells is a normal consequence of aging, whereas the link between these T-cell populations and CMV infection is explained by the increased prevalence of this infection in older people. Resolving whether CMV infection or aging triggers CD28null T-cell expansions is of critical importance because, unlike aging, CMV infection can be treated. Methods: We used multi-color flow-cytometry, antigen-specific activation assays, and HLA-typing to dissect the contributions of CMV infection and aging to the accumulation of CD28null CD4 and CD8 T-cells in CMV+ and CMV- individuals aged 19 to 94 years. Linear/logistic regression was used to test the effect of sex, age, CMV infection, and HLA-type on CD28null T-cell frequencies. Results: The median frequencies of CD28null CD4 T-cells and CD28null CD8 T-cells were >12-fold (p=0.000) but only approximately 2-fold higher (p=0.000), respectively, in CMV+ (n=136) compared with CMV- individuals (n=106). The effect of CMV infection on these T-cell subsets was confirmed by linear regression. Unexpectedly, aging contributed only marginally to an increase in CD28null T-cell frequencies, and only in CMV+ individuals. Interestingly, the presence of HLA-DRB1*0301 led to an approximately 9-fold reduction of the risk of having CD28null CD4 T-cell expansions (OR=0.108, p=0.003). Over 75% of CMV-reactive CD4 T-cells were CD28null. Conclusion: CMV infection and HLA type are major risk factors for CD28null CD4 T-cell-associated cardiovascular pathology. Increased numbers of CD28null CD8 T-cells are also associated with CMV infection, but to a lesser extent. Aging, however, makes only a negligible contribution to the expansion of these T-cell subsets, and only in the presence of CMV infection. Our results open up new avenues for risk assessment, prevention, and treatment.

Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses.

Background: Interleukin (IL)-7 promotes the generation, expansion, and survival of memory T cells. Previous mouse and human studies showed that IL-7 can support immune cell reconstitution in lymphopenic conditions, expand tumor-reactive T cells for adoptive immunotherapy, and enhance effector cytokine expression by autoreactive T cells. Whether pathogen-reactive T cells also benefit from IL-7 exposure remains unknown. Methods: In this study, we investigated this issue in cultures of peripheral blood mononuclear cells (PBMCs) derived from patients infected with various endemic pathogens. After short-term exposure to IL-7, we measured PBMC responses to antigens derived from pathogens, such as Mycobacterium tuberculosis, Candida albicans, and cytomegalovirus, and to the superantigen Staphylococcus aureus enterotoxin B. Results: We found that IL-7 favored the expansion and, in some instances, the uncovering of pathogen-reactive CD4 T cells, by promoting pathogen-specific interferon-γ, IL-2, and tumor necrosis factor recall responses. Conclusions: Our findings indicate that IL-7 unveils and supports reactivation of pathogen-specific T cells with possible diagnostic, prognostic, and therapeutic significance of clinical value, especially in conditions of pathogen persistence and chronic infection.

CMV-Specific T-cell Responses at Older Ages: Broad Responses With a Large Central Memory Component May Be Key to Long-term Survival.

Cytomegalovirus (CMV) infection sometimes causes large expansions of CMV-specific T cells, particularly in older people. This is believed to undermine immunity to other pathogens and to accelerate immunosenescence. While multiple different CMV proteins are recognized, most publications on age-related T-cell expansions have focused on dominant target proteins UL83 or UL123, and the T-cell activation marker interferon-γ (IFN-γ). We were concerned that this narrow approach might have skewed our understanding of CMV-specific immunity at older ages. We have, therefore, widened the scope of analysis to include in vitro-induced T-cell responses to 19 frequently recognized CMV proteins in "young" and "older" healthy volunteers and a group of "oldest old" long-term survivors (>85 years of age). Polychromatic flow cytometry was used to analyze T-cell activation markers (CD107, CD154, interleukin-2 [IL-2], tumor necrosis factor [TNF], and IFN-γ) and memory phenotypes (CD27, CD45RA). The older group had, on average, larger T-cell responses than the young, but, interestingly, response size differences were relatively smaller when all activation markers were considered rather than IFN-γ or TNF alone. The oldest old group recognized more proteins on average than the other groups, and had even bigger T-cell responses than the older group with a significantly larger central memory CD4 T-cell component.

Functional Diversity of Cytomegalovirus-Specific T Cells Is Maintained in Older People and Significantly Associated With Protein Specificity and Response Size.

BACKGROUND: Parallel upregulation of several T-cell effector functions (ie, polyfunctionality) is believed to be critical for the protection against viruses but thought to decrease in large T-cell expansions, in particular at older ages. The factors determining T-cell polyfunctionality are incompletely understood. Here we revisit the question of cytomegalovirus (CMV)-specific T-cell polyfunctionality, including a wide range of T-cell target proteins, response sizes, and participant ages. METHODS: Polychromatic flow cytometry was used to analyze the functional diversity (ie, CD107, CD154, interleukin 2, tumor necrosis factor, and interferon γ expression) of CD4+ and CD8+ T-cell responses to 19 CMV proteins in a large group of young and older United Kingdom participants. A group of oldest old people (age >85 years) was included to explore these parameters in exceptional survivors. Polyfunctionality was assessed for each protein-specific response subset, by subset and in aggregate, across all proteins by using the novel polyfunctionality index. RESULTS: Polyfunctionality was not reduced in healthy older people as compared to young people. However, it was significantly related to target protein specificity. For each protein, it increased with response size. In the oldest old group, overall T-cell polyfunctionality was significantly lower. DISCUSSION: Our results give a new perspective on T-cell polyfunctionality and raise the question of whether maintaining polyfunctionality of CMV-specific T cells at older ages is necessarily beneficial.

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

Induction of Human Regulatory T Cells with Bacterial Superantigens.

Regulatory T cells (Tregs) that suppress the activation of immune effector cells limit immunopathology and are fast emerging as therapeutic targets for autoimmune and cancer disease. Tools enabling Treg in vitro-induction, expansion, and characterization and manipulation will help future clinical developments. In this chapter, we describe in detail how to use bacterial superantigens to induce human Tregs efficiently from peripheral blood mononuclear cells. How to assess human Treg phenotype and suppressive capacity are also described. Technical details, variations, and alternative experimental conditions are provided.

Health profile and disease determinants among asylum seekers: a cross-sectional retrospective study from an Italian reception centre.

BACKGROUND: Data on diseases' determinants and health status of asylum seekers (ASs) are limited. METHODS: We performed a cross-sectional retrospective study in a large ASs centre in Italy. Data were collected during a 1-year period. Descriptive statistics were calculated. A χ(2) test was used to assess the association between socio-demographics characteristics of ASs and screening test results. A multiple logistic regression analysis was performed to identify diseases' predictors by using ICD-10 diagnoses classification as outcome variable, socio-demographic characteristics as independent variable and visits' number as confounding variable. RESULTS: Overall, data on 792 ASs (mean age 27 years, 80% males, 58% from Africa) were assessed, 43% underwent voluntary infectious diseases screening and 2843 diagnoses were recorded. The most frequent diagnoses were: respiratory diseases, symptoms/signs not elsewhere classified, digestive diseases and infectious diseases. Gender was the most frequent predictor of ICD-10 diagnoses, while African origin, civil status and education were, respectively, predictive of cardiovascular and infectious diseases, genitourinary diseases and pregnancy-related disorders. Higher mean age was associated with syphilis, HIV and HCV infection and African origin with HIV infection. CONCLUSIONS: Communicable diseases were not prevalent in the ASs population we analysed. A stronger cultural mediation support is needed to facilitate prevention, access and continuity of care for ASs.

WITHDRAWN: Gender differences and age-specific associations between Body Mass Index and other cardiovascular risk factors in CMV infected and uninfected people.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.imlet.2014.09.010. The duplicate article has therefore been withdrawn.

Gender differences and age-specific associations between body mass index and other cardiovascular risk factors in CMV infected and uninfected people.

Body mass index (BMI) is a known risk factor for cardiovascular disease and cancer. It is also related to white blood count (WBC) and inflammation. The effects of age and gender on these associations have not been explored. Here we have examined the relationships between BMI and inflammatory parameters/cardiovascular risk factors including WBC/neutrophil count (NC), CRP and mean arterial blood pressure (MAP), in young (20-35 years) and older (60-85 years) healthy donors with respect to gender and CMV IgG serology. In young but not older people significant associations between BMI and WBC were observed, however, with opposite directions in the two genders. Only in CMV+ older women a positive trend was preserved. Across the population, there was no significant association between NC and MAP; however, among older men we saw a positive correlation between the two parameters. Linear regression confirmed that across the whole population, age group (young versus older) and also the interaction between gender and age group but not gender alone had significant effects on this association. When analysing CMV+ older people separately we established that both NC and its interaction with gender had a significant effect on MAP. This study reveals that the correlations between common inflammatory markers/cardiovascular risk factors depend on age, gender, and CMV status in a complex fashion. Our findings support the need to evaluate risk factors independently in men and women and to take into account CMV infection status. More focused studies will be required to shed light on these novel findings.

Cell-mediated immunity to human CMV infection: a brief overview.

The cellular immune response to human cytomegalovirus (HCMV) has different components originating from both the adaptive and innate immune systems. There is a significant global interest in understanding how the immune system keeps HCMV under control, in particular with a view to situations where HCMV infection causes severe damage. Such settings include HIV infection, transplantation, and maybe most importantly perinatal medicine, HCMV being a major cause of sometimes catastrophic birth defects. The development of an active HCMV vaccine has proven very difficult but some recent successes raise hope that this might be available in the future. However, adoptive transfer of HCMV-specific T cells has been successfully used to prevent CMV disease after bone marrow transplantation for many years. In fact, the CD8 T cell response has been thought to be the most important effector response, with numerous reports focusing on specific T cell subsets recognizing select peptides in select human leukocyte antigen (HLA) contexts. However, it is becoming increasingly clear now that other cells, first and foremost CD4 T cells, but also gamma/delta (γ/δ) T cells and natural killer cells, are critically involved in the cellular immune response to HCMV. This commentary aims to provide a brief overview of the field.

A novel cytomegalovirus-induced regulatory-type T-cell subset increases in size during older life and links virus-specific immunity to vascular pathology.

BACKGROUND: Cytomegalovirus (CMV) infection directly targets vascular endothelium and smooth muscle and at older ages is associated with accelerated vascular pathology and mortality. CMV-specific cellular immunity might directly contribute to this process. METHODS: Conventional ex vivo activation-induced T-cell responses to 19 dominant CMV antigens, along with CMV-specific inducible regulatory-type CD4+ T cells (iTregs), were measured in healthy older people, using a novel protocol that included classic Treg markers alongside the activation marker CD134. Measurements were correlated with diastolic, systolic, and mean arterial blood pressure, a surrogate marker for arterial stiffness. RESULTS: CMV-specific iTregs recognized the same antigens as conventional CD4+ T cells and were significantly more frequent at older ages. They suppressed antigen-specific and nonspecific proliferation and in large part expressed Foxp3. Frequencies of CMV-specific iTregs and CD8+ T cells (summated response) were significantly associated with diastolic and mean arterial pressures. Confounders, including age, body mass index, smoking, antihypertensive medication use, or C-reactive protein levels, did not explain these observations. CONCLUSIONS: A novel CMV-induced regulatory-type CD4+ T-cell subset is readily detectable in CMV-infected people and, like the aggregate CD8+ T-cell response to the most dominant CMV antigens, is quantitatively associated with arterial stiffness in older life. Whereas CD8+ effector T cells might directly cause vascular injury, iTregs may attenuate this response.

Cytomegalovirus infection modulates the phenotype and functional profile of the T-cell immune response to mycobacterial antigens in older life.

Infection with Cytomegalovirus is associated with accelerated immunosenescence. Expansions of CMV-specific T cell responses have previously been demonstrated to affect the ability of T cells to respond to other infections. Most people above 60years of age display M. tuberculosis-specific immunity because of vaccination, exposure, or both. T-cell responses can be assessed by measuring intracellular IFN-γ in vitro after tuberculin stimulation. Here we investigated tuberculin-specific CD4 T-cell responses in independently living healthy older people in the South of England using flow-cytometry. Individuals were investigated for tuberculin and CMV-specific T-cell immunity using in vitro antigen stimulation followed by intracellular staining for IFN-γ, TNF-α, IL2, as well as degranulation and CD154 upregulation. We also examined a control group of younger individuals (20-35years of age). There was no significant difference between older and young people in regards to tuberculin responsiveness of CD4 T-cells; however, older people seemed to show more outliers. Increased responsiveness to tuberculin was significantly correlated to CMV responsiveness but not age. In older donors, the memory phenotype of tuberculin-induced T-cells was significantly skewed towards a more terminal differentiation phenotype in CMV-infected compared to uninfected individuals and the degree of skewing correlated quantitatively with the size of the CMV-specific CD4 T-cell response. This is a fundamental advance over previous reports of changes of the tuberculin-specific CD4 T-cell response with CMV serostatus. Our results show that how the immune system responds to CMV has a fundamental impact on the phenotype and function of the immune response to mycobacterial antigens in older life.

The TCR Vbeta signature of bacterial superantigens spreads with stimulus strength.

Superantigens (Sags) induce large-scale stimulation of T lymphocytes by a mechanism distinct from conventional antigen presentation, involving direct MHC binding and stimulation of TCR families based on Vbeta gene usage. The specific Vbeta targets of a given Sag have, since the earliest studies in murine models, been considered a hallmark of that toxin. Bacterial Sags are implicated in the aetiology of a wide range of human diseases, although their role has been most clearly defined in toxic shock syndrome. While Sags have been defined by the Vbeta-specific changes in T cell repertoire they induce, human studies of in vitro stimulation or analysis of cells from infected patients have produced inconsistent findings. Here we have evaluated the contribution of HLA allelic polymorphisms and strength of stimulus to this response. We show that there are differences in binding and presentation of the staphylococcal Sag, staphylococcal enterotoxin A (SEA), by different HLA-DR alleles. We also show that the TCR Vbeta response, previously thought to be a fixed property defining a given Sag, varies with stimulus strength such that a broader repertoire of response is seen at higher concentrations or following presentation by high-binding class II types. Responses of human Vbeta8 and Vbeta1 to SEA, Vbeta5 to SEB and of Vbeta12 and Vbeta13 to streptococcal pyrogenic exotoxin A are absolutely dependent on stimulus strength. These findings have important implications for heterogeneity in the response to Sags and the consequent differences in susceptibility to severe toxic shock.

The expression and function of cathepsin E in dendritic cells.

Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells.

The human chorionic gonadotropin-beta arginine68 to glutamic acid substitution fixes the conformation of the C-terminal peptide.

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG(beta)) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCG(beta)-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG(beta)-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG(beta)-specific and anti-hCG(beta)/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCG(beta)-R68E was eliminated, mAbs reacting with hCG(beta)-specific epitopes bound to hCG(beta) and hCG(beta)-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCG(beta)-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG(beta)-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed.

DNA immunization with plasmids expressing hCGbeta-chimeras.

Human chorionic gonadotropin has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of DNA immunization with the aim of improving the immunogenicity of this hormone. Stimulating the muscle with electric pulses following intramuscular injection of plasmids expressing hCGbeta resulted in higher levels of human chorionic gonadotropin (hCG)-specific antibodies, which could be further enhanced following a protein boost with hCG mixed with adjuvant. DNA vaccines encoding a membrane attached or a secreted form of hCGbeta produced similar-albeit relatively modest-antibody responses. Providing hCGbeta with additional T cell help by vaccinating with a plasmid encoding a hCGbeta-hFc fusion protein did not further increase the antibody levels in the immunized animals. However, immunization of mice with a construct encoding hCGbeta fused to C3d(3) produced significantly lower antibody levels relative to mice immunized with the hCGbeta-alone expression plasmid, even though the hCGbeta-C3d(3) chimera was expected to facilitate cross-linking of the antigen-specific B-cell receptor and CR2 thereby lowering the threshold of activation. Thus the limiting factor determining the antibody levels following hCGbeta immunization, at least for DNA immunization, is related to the amount of protein available rather than the form of protein produced or lack of T cell epitopes.