An immunoassay for the detection of circulating antigens in human echinococcosis.

An immunoassay (double-antibody-sandwich-ELISA) was developed to detect circulating antigens (CAg) in patients with cystic (Echinococcus granulosus) echinococcosis. Echinococcus antigens derived from heterologous intermediate hosts were used to immunize rabbits and to purify the rabbit-IgG-fraction obtained by affinity-chromatography, thus avoiding major interference with host components. The purified rabbit anti-hydatid IgG was immunosorbed with bovine and human sera. One part of the resulting IgG served as coating agent in a double antibody sandwich-ELISA; the other part, coupled to alkaline phosphatase, as detecting conjugate. The specificity of the antibody reaction was demonstrated by immunoelectrophoresis. Sera of 21 patients with cystic echinococcosis were examined with this test system. In seven of the patients' sera CAg were detected in concentrations ranging between 310 ng and 680 ng protein per ml serum. Comparing pre- and postoperative serum samples obtained from nine patients operated on for cystic echinococcosis, four sera were found to be CAg-positive before and three after operation.

Echinococcus metacestode: in search of viability markers

Epidemiological studies have demonstrated that most humans infected with Echinococcus spp. exhibit resistance to disease. When infection leads to disease, the parasite is partially controlled by host immunity: in case of immunocompetence, the normal alveolar echinococcosis (AE) or cystic echinococcosis (CE) situation, the metacestode grows slowly, and first clinical signs appear years after infection; in case of impaired immunity (AIDS; other immunodeficiencies), uncontrolled proliferation of the metacestode leads to rapidly progressing disease. Assessing Echinococcus multilocularis viability in vivo following therapeutic interventions in AE patients may be of tremendous benefit when compared with the invasive procedures used to perform biopsies. Current options are F18-fluorodeoxyglucose-positron emission tomography (FDG-PET), which visualizes periparasitic inflammation due to the metabolic activity of the metacestode, and measurement of antibodies against recEm18, a viability-associated protein, that rapidly regresses upon metacestode inactivation. For Echinococcus granulosus, similar prognosis-associated follow-up parameters are still lacking but a few candidates may be listed. Other possible markers include functional and diffusion-weighted Magnetic Resonance Imaging (MRI), and measurement of products from the parasite (circulating antigens or DNA), and from the host (inflammation markers, cytokines, or chemokines). Even though some of them have been promising in pilot studies, none has been properly validated in an appropriate number of patients until now to be recommended for further use in clinical settings. There is therefore still a need to develop reliable tools for improved viability assessment to provide the sufficient information needed to reliably withdraw anti-parasite benzimidazole chemotherapy, and a basis for the development of new alternative therapeutic tools.

New findings on echinococcosis

The following new aspects of cystic and alveolar echinococcosis (= infections with the metacestode stages of Echinococcus granulosus and E. multilocularis respectively) are reported: identification of a Swiss E. granulosus isolate as "cattle strain" which differs from the "sheep strain"; new observations on proliferation and metastasis formation of larval E. multilocularis; information on chemotherapy of human echinococcosis; recent developments in immunoserology. The latter includes a new technique for serological differential diagnosis of cystic and alveolar echinococcosis, the determination of parasite-specific immunoglobulin classes (IgG, IgM, IgA and IgE) and circulating antigens in ELISA, and the introduction of arc-5 detection in routine serodiagnosis. A highly purified, species-specific antigen from E. multilocularis is now available for seroepidemiological studies.

Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells

T-cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen-specific T-cells are thought to be less powerful. However, synthetic T-cell vaccines composed of Melan-A/MART-1 peptide, CpG and IFA can induce high frequencies of tumor-specific CD8 T-cells in PBMC of melanoma patients. Here we analyzed the functionality of these T-cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL-2) and upregulation of LAMP-1 (CD107a) by tumor (Melan-A/MART-1) specific T-cells was comparable to virus (EBV-BMLF1) specific CD8 T-cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor- and virus-specific T-cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T-cells were induced irrespective of patient's age or gender. Finally, CD8 T-cell function correlated with disease-free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor-specific CD8 T-cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.

Self-efficacy buffers the relationship between dementia caregiving stress and circulating concentrations of the proinflammatory cytokine interleukin-6

the proinflammatory cytokine interleukin (IL)-6 has been linked with health morbidity, particularly risk for cardiovascular disease (CVD). The purpose of this study was to investigate the potential protective role of coping self-efficacy on the relationship between caregiving stress and circulating concentrations of IL-6.

High levels of circulating CD34+ cells at autologous stem cell collection are associated with favourable prognosis in multiple myeloma

High-dose chemotherapy with autologous stem cell transplantation is a cornerstone in the first-line treatment of multiple myeloma patients. However, only few factors have been identified affecting the outcome in such patients. We hypothesised that varying levels of mobilised CD34+ cells confer prognostic information in myeloma patients undergoing high-dose chemotherapy.

Monomeric and dimeric IgG fractions show differential reactivity against pathogen-derived antigens

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

Meta-analysis and dose-response metaregression: circulating insulin-like growth factor I (IGF-I) and mortality

Context: IGF-I plays a central role in metabolism and growth regulation. High IGF-I levels are associated with increased cancer risk and low IGF-I levels with increased risk for cardiovascular disease. Objective: Our objective was to determine the relationship between circulating IGF-I levels and mortality in the general population using random-effects meta-analysis and dose-response metaregression. Data Sources: We searched PubMed, EMBASE, Web of Science, and Cochrane Library from 1985 to September 2010 to identify relevant studies. Study Selection: Population-based cohort studies and (nested) case-control studies reporting on the relation between circulating IGF-I and mortality were assessed for eligibility. Data Extraction: Data extraction was performed by two investigators independently, using a standardized data extraction sheet. Data Synthesis: Twelve studies, with 14,906 participants, were included. Overall, risk of bias was limited. Mortality in subjects with low or high IGF-I levels was compared with mid-centile reference categories. All-cause mortality was increased in subjects with low as well as high IGF-I, with a hazard ratio (HR) of 1.27 (95% CI = 1.08–1.49) and HR of 1.18 (95% CI = 1.04–1.34), respectively. Dose-response metaregression showed a U-shaped relation of IGF-I and all-cause mortality (P = 0.003). The predicted HR for the increase in mortality comparing the 10th IGF-I with the 50th percentile was 1.56 (95% CI = 1.31–1.86); the predicted HR comparing the 90th with the 50th percentile was 1.29 (95% CI = 1.06–1.58). A U-shaped relationship was present for both cancer mortality and cardiovascular mortality. Conclusions: Both low and high IGF-I concentrations are associated with increased mortality in the general population.

Effect of Alzheimer Caregiving on Circulating Levels of C-Reactive Protein and Other Biomarkers Relevant to Cardiovascular Disease Risk: A Longitudinal Study

Providing care to a spouse with Alzheimer's disease (AD) may contribute to cardiovascular disease (CVD). The acute phase reactant C-reactive protein (CRP) is a well-established biomarker of an increased CVD risk.

Association between circulating cytokine levels, diabetes and insulin resistance in a population-based sample (CoLaus study)

OBJECTIVE: The associations between inflammation, diabetes and insulin resistance remain controversial. Hence, we assessed the associations between diabetes, insulin resistance (using HOMA-IR) and metabolic syndrome with the inflammatory markers high sensitivity C-reactive protein (hs-CRP), interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). DESIGN: CROSS-SECTIONAL STUDY: PARTICIPANTS: 2884 MEN AND 3201 WOMEN AGED 35 TO 75: METHODS: CRP was assessed by immunoassay and cytokines by multiplexed flow cytometric assay. In a subgroup of 532 participants an oral glucose tolerance test was performed to screen for impaired glucose tolerance (IGT). RESULTS: IL-6, TNF-α and hs-CRP were significantly and positively correlated with fasting plasma glucose, insulin and HOMA-IR. Participants with diabetes had higher IL-6, TNF-α and hs-CRP levels than participants without diabetes; this difference persisted for hs-CRP after multivariate adjustment. Participants with metabolic syndrome had increased IL-6, TNF-α and hs-CRP levels; these differences persisted after multivariate adjustment. Participants in the highest quartile of HOMA-IR had increased IL-6, TNF-α and hs-CRP levels; these differences persisted for TNF-α and hs-CRP after multivariate adjustment. No association was found between IL-1β levels and all diabetes and insulin resistance markers studied. Finally, participants with IGT had higher hs-CRP levels than participants with a normal OGTT, but this difference disappeared after controlling for body mass index (BMI). CONCLUSION: subjects with diabetes, metabolic syndrome and increased insulin resistance present with increased levels of IL6, TNF-α and hs-CRP, while no association was found with IL-1β. The increased inflammatory state of subjects with IGT is partially explained by increased BMI. © 2012 Blackwell Publishing Ltd.

In vitro induction of lymph node cell proliferation by mouse bone marrow dendritic cells following stimulation with different Echinococcus multilocularis antigens

The immune response of mice experimentally infected with Echinococcus multilocularis metacestodes becomes impaired so as to allow parasite survival and proliferation. Our study tackled the question on how different classes of E. multilocularis antigens (crude vesicular fluid (VF); purified proteinic rec-14-3-3; purified carbohydrate Em2(G11)) are involved in the maturation process of bone-marrow-derived dendritic cells (BMDCs) and subsequent exposure to lymph node (LN) cells. In our experiments, we used BMDCs cultivated from either naïve (control) or alveolar echinococcosis (AE)-infected C57BL/6 mice. We then tested surface markers (CD80, CD86, MHC class II) and cytokine expression levels (interleukin (IL)-10, IL-12p40 and tumour necrosis factor (TNF)-α) of non-stimulated BMDCs versus BMDCs stimulated with different Em-antigens or lipopolysaccharide (LPS). While LPS and rec-14-3-3-antigen were able to induce CD80, CD86 and (to a lower extent) MHC class II surface expression, Em2(G11) and, strikingly, also VF-antigen failed to do so. Similarly, LPS and rec-14-3-3 yielded elevated IL-12, TNF-α and IL-10 expression levels, while Em2(G11) and VF-antigen didn't. When naïve BMDCs were loaded with VF-antigen, they induced a strong non-specific proliferation of uncommitted LN cells. For both, BMDCs or LN cells, isolated from AE-infected mice, proliferation was abrogated. The most striking difference, revealed by comparing naïve with AE-BMDCs, was the complete inability of LPS-stimulated AE-BMDCs to activate lymphocytes from any LN cell group. Overall, the presenting activity of BMDCs from AE-infected mice seemed to trigger unresponsiveness in T cells, especially in the case of VF-antigen stimulation, thus contributing to the suppression of clonal expansion during the chronic phase of AE infection.

M-ficolin concentrations in cord blood are related to circulating phagocytes and to early-onset sepsis

The pattern-recognition molecule M-ficolin is synthesized by monocytes and neutrophils. M-ficolin activates the complement system in a manner similar to mannan-binding lectin (MBL), but little is known about its role in host defense. Neonates are highly vulnerable to bacterial sepsis, in particular, due to their decreased phagocytic function.

Circulating DNA and myeloperoxidase indicate disease activity in patients with thrombotic microangiopathies

Thrombotic microangiopathies (TMAs) are a group of life-threatening disorders characterized by thrombocytopenia, fragmentation of erythrocytes, and ischemic organ damage. Genetic disorders, autoimmune disease, and cancer are risk factors for TMAs, but an additional, unknown trigger is needed to bring about acute disease. Recent studies suggest that DNA and histones are released during inflammation or infection and stimulate coagulation, thrombosis, thrombocytopenia, and organ damage in mice. We show that extracellular DNA and histones as well as markers of neutrophils are present in acute TMAs. Analysis of plasma from TMA patients of different clinical categories revealed elevated levels of DNA-histone complexes and myeloperoxidase (MPO) from neutrophil granules as well as S100A8/A9, a heterocomplex abundant in neutrophil cytosol. During therapy of thrombotic thrombocytopenic purpura, a subtype of TMAs often associated with severe ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13) deficiency, plasma DNA and MPO were inversely correlated with platelet counts, and their levels indicated amelioration or exacerbation of the disease. ADAMTS13 deficiency together with increased levels of plasma DNA and MPO were characteristic for acute thrombotic thrombocytopenic purpura. A minor infection often precedes acute TMA and extracellular DNA and histones released during the inflammatory response could provide the second hit, which precipitates acute TMA in patients with pre-existing risk factors.