Cellular responses and cytokine production in post-treatment hookworm patients from an endemic area in Brazil

Human hookworm infections are distributed widely in tropical areas and have a significant impact on host morbidity and human health. In the present study, we investigated the cellular responsiveness and cytokine production in peripheral blood mononuclear cells (PBMC) from Necator americanus-infected schoolchildren who had recently received chemotherapy, and compared them with non-infected endemic controls. Hookworm patients and treated, egg-negative individuals showed a lower cellular reactivity against phytohaemagglutinin (PHA) and hookworm antigen when compared with egg-negative endemic controls. The baseline production of proinflammatory tumour necrosis factor-alpha (TNF-alpha) in PBMC from infected patients and treated, egg-negative individuals was elevated. On the other hand, PHA- or hookworm antigen-induced interleukin (IL)-12 and interferon (IFN)-gamma secretion was higher in endemic controls than in hookworm patients, who either continued egg-positive or were egg-negative after treatment. Also, PBMC from endemic controls secreted more IL-5 and IL-13 than the other patient groups. Opposite to that, the spontaneous as well as the antigen-driven IL-10 secretion was lower in endemic controls when compared with the other groups. In summary, patently hookworm-infected as well as egg-negative treated patients disclosed an elevated spontaneous cellular secretion of proinflammatory TNF-alpha, a prominent secretion of regulatory Th2-type IL-10 and an impaired production of IL-12, IFN-gamma, IL-5 and IL-13.     

Immune responses following experimental infection with Ascaridia galli and necrotic enteritis in broiler chickens

Broilers commonly suffer from necrotic enteritis (NE). Other gastrointestinal infectious diseases affect poultry, including nematode infections which are considered a re-emerging disease in barn and free-range systems. The aim of this study was to characterize the immune response of broilers after artificial infection with NE and contrast these with responses to the nematode Ascaridia galli and determine whether immune parameters measured during the course of infection can be used to distinguish infected from uninfected birds.

A total of 96 one-day-old male Ross 308 broiler chickens were used in this study. At 10 days of age, broilers were randomly assigned to one of the following treatment groups: control birds (n?=?32), A. galli infected birds (n?=?32), or NE infected birds (n?=?32) and inoculated with the appropriate infective agents. The immune response of birds was monitored through evaluation of haematology parameters, acute phase protein production, and intraepithelial intestinal lymphocyte population changes at 11, 16, 20, and 32 days of age.

T-helper cells (CD4⁺CD8^?) increased significantly over time, and were significantly higher in A. galli and NE compared to day 10 controls. In conclusion, α-1 glycoprotein levels can distinguish birds with NE from other birds, including those infected with A. galli; also T-helper cell numbers can distinguish both NE and A. galli from uninfected birds and thirdly, 10 days post infection is the best time point to evaluate the bird’s immune response for A. galli infections.     

Immune responses in pregnant cattle and bovine fetuses following experimental infection with Neospora caninum

Humoral and cell-mediated immune (CMI) responses [i.e. proliferative responses and gamma interferon (IFN-gamma) production], were elicited in five cows infected between 159 and 169 days of gestation by a combined intravenous-intramuscular inoculation of Neospora caninum tachyzoites. Analysis of antigen-specific immunoglobulin (IgG) subclasses revealed a predominant IgG2 response in two cows, a mixed IgG1-IgG2 response in two other cows and a predominant IgG1 response in one cow. No correlation was found between IgG2 titers and IFN-gamma levels. CD4-T cells were responsible for the CMI responses in peripheral blood mononuclear cells from three infected cows. All five fetuses removed from infected dams at week 9 post-infection (219-231 days of gestation) mounted strong Neospora-specific humoral responses and had a predominant IgG1 response, regardless of their ability to produce IFN-gamma. However, CMI responses were highly variable between fetuses. These data indicate the complexity of the immune mechanisms associated with Neospora infection in both the dams and their fetuses.     

Cytokine release and its modulation by dexamethasone in whole blood following exercise

Glucocorticoids (GC) play an important role in the treatment of inflammatory diseases like asthma. However, in selected patients a relative resistance to GC has been reported. Recently, it has been suggested that GC sensitivity of peripheral blood leucocytes may be regulated in a dynamic fashion during exercise, in association with activation of the hypothalamic–pituitary–adrenal (HPA) axis. The aim of the present study was to explore changes in the GC sensitivity of cytokine production by leucocytes following strenuous exercise by well trained oarsmen. These changes were studied using lipopolysaccharide (LPS)-induced and anti-CD2/anti-CD28 MoAb-stimulated cytokine release in whole blood and its modulation by dexamethasone. Following exercise, significant decreases in LPS-induced release of IL-6, tumour necrosis factor-alpha (TNF-α) and IL-10 and anti-CD2/anti-CD28 MoAb-stimulated secretion of interferon-gamma (IFN-γ) were observed. In addition, the inhibitory effect of dexamethasone on both IL-6 and TNF-α secretion was significantly reduced following exercise, whereas that on IL-10 and IFN-γ release was not affected. These exercise-induced changes were accompanied by activation of the HPA axis, as indicated by an increase in circulating adrenocorticotropic hormone (ACTH) levels immediately following exercise. The results from the present study suggest that GC sensitivity of whole blood cytokine release can be regulated in a dynamic fashion and that this can be assessed using an ex vivo stimulation assay. Moreover, since dexamethasone responsiveness of anti-CD2/anti-CD28 MoAb-induced IFN-γ secretion in whole blood is not affected by exercise, it may suggest that exercise differentially affects monocytes and lymphocytes. The dynamic regulation of steroid responsiveness of leucocytes, as observed in the present study, could have important consequences for the effectiveness of GC treatment in inflammatory diseases.     

Experimental hookworm infection: a randomized placebo‐controlled trial in asthma

Background Epidemiological studies suggest that hookworm infection protects against asthma, and therefore that hookworm infection may have a direct or an indirect therapeutic potential in this disease. We now report the first clinical trial of experimental hookworm infection in people with allergic asthma. Objectives To determine the effects of experimental hookworm infection in asthma. Methods Thirty‐two individuals with asthma and measurable airway responsiveness to adenosine monophosphate (AMP) were randomized and double blinded to cutaneous administration of either ten Necator americanus larvae, or histamine solution (placebo), and followed for 16 weeks. The primary outcome was the change in provocation dose of inhaled AMP required to reduce forced expiratory volume in 1 s by 20% (PD₂₀AMP) from baseline to week 16. Secondary outcomes included change in several measures of asthma control and allergen skin sensitivity and the occurrence of adverse effects. Results Mean PD₂₀AMP improved in both groups, more in the hookworm [1.49 doubling doses (DD)] than the placebo group (0.98 DD), but the difference between groups was not significant (0.51 DD; 95% confidence interval: ?1.79 to 2.80; P=0.65). There were no significant differences between the two groups for other measures of asthma control or allergen skin sensitization. Infection was generally well tolerated. Conclusions Experimental infection with ten hookworm larvae in asthma did not result in significant improvement in bronchial responsiveness or other measures of asthma control in this study. However, infection was well tolerated and resulted in a non‐significant improvement in airway responsiveness, indicating that further studies that mimic more closely natural infection are feasible and should be undertaken. Cite this as: J. R. Feary, A. J. Venn, K. Mortimer, A. P Brown, D. Hooi, F. H. Falcone, D. I. Pritchard and J. R. Britton, Clinical & Experimental Allergy, 2010 (40) 299– 306.     

The synthetic antimicrobial peptide LTX21 induces inflammatory responses in a human whole blood model and a murine peritoneum model

The global spread of antimicrobial resistance and the increasing number of immune‐compromised patients are major challenges in modern medicine. Targeting bacterial virulence or the human host immune system to increase host defence are important strategies in the search for novel antimicrobial drugs. We investigated the inflammatory response of the synthetic short antimicrobial peptide LTX21 in two model systems: a human whole blood ex vivo model and a murine in vivo peritoneum model – both reflecting early innate immune response. In the whole blood model, LTX21 increased the secretion of a range of different cytokines, decreased the level of tumour necrosis factor (TNF) and activated the complement system. In a haemolysis assay, we found 2.5% haemolysis at a LTX21 concentration of 500 mg/L. In the murine model, increased influx of white blood cells (WBCs) and polymorphonuclear neutrophils (PMNs) in the murine peritoneal cavity was observed after treatment with LTX21. In addition, LTX21 increased monocyte chemoattractant protein‐1 (MCP‐1). In conclusion, LTX21 affected the inflammatory response; the increase in cytokine secretion, complement activation and WBC influx indicates an activated inflammatory response. The present results indicate the impact of LTX21 on the host–pathogen interplay. Whether this will also affect the course of infection has to be investigated.     

Safety of hookworm infection in individuals with measurable airway responsiveness: a randomized placebo-controlled feasibility study

Background Epidemiological evidence suggests that hookworm infection protects against asthma. However, for ethical and safety reasons, before testing this hypothesis in a clinical trial in asthma it is necessary to establish whether experimental hookworm infection might exacerbate airway responsiveness during larval lung migration. Objective To determine whether hookworm larval migration through the lungs increases airway responsiveness in allergic individuals with measurable airway responsiveness but not clinical asthma, and investigate the general tolerability of infection and effect on allergic symptoms. Methods Thirty individuals with allergic rhinoconjunctivitis and measurable airway responsiveness to adenosine monophosphate (AMP) but not clinically diagnosed asthma were randomized, double‐blind to cutaneous administration of either 10 hookworm larvae or histamine placebo, and followed for 12 weeks. The primary outcome was the maximum fall from baseline in provocative dose of inhaled AMP required to reduce 1‐s forced expiratory volume by 10% (PD₁₀AMP) measured at any time over the 4 weeks after active or placebo infection. Secondary outcomes included peak flow variability in the 4 weeks after infection, rhinoconjunctivitis symptom severity and adverse effect diary scores over the 12‐week study period, and change in allergen skin test responses between baseline and 12 weeks. Results Mean maximum change in PD₁₀AMP from baseline was slightly but not significantly greater in the hookworm than the placebo group (?1.67 and ?1.16 doubling doses; mean difference ?0.51, 95% confidence interval ?1.80 to 0.78, P=0.42). Symptom scores of potential adverse effects were more commonly reported in the hookworm group, but infection was generally well tolerated. There were no significant differences in peak‐flow variability, rhinoconjunctivitis symptoms or skin test responses between groups. Conclusion Hookworm infection did not cause clinically significant exacerbation of airway responsiveness and was well tolerated. Suitably powered trials are now indicated to determine the clinical effectiveness of hookworm infection in allergic rhinoconjunctivitis and asthma.     

Immune interventions in HIV infection

Immune-based therapy (IBT) interventions have found a window of opportunity within some limitations of the otherwise successful combined antiretroviral therapy (cART). Two major paradigms drove immunotherapeutic research to combat human immunodeficiency virus (HIV) infection. First, IBTs were proposed either to help restore CD4⁺ T-cell counts in cases of therapeutic failures with cytokines, interleukin-2 (IL-2) or IL-7, or to better control HIV and disease progression during treatment interruptions with anti-HIV therapeutic candidate vaccines. The most widely used candidates were HIV-recombinant live vector-based alone or combined with other vaccine compounds and dendritic cell (DC) therapies. A more recent and current paradigm aims at achieving HIV cure by combining IBT with cART using either cytokines to reactivate virus production in latently infected cells and/or therapeutic immunization to boost HIV-specific immunity in a ‘shock and kill’ strategy. This review summarizes the rationale, hopes, and mechanisms of successes and failures of these cytokine-based and vaccine-based immune interventions. Results from these first series of IBTs have been so far somewhat disappointing in terms of clinical relevance, but have provided lessons that are discussed in light of the future combined strategies to be developed toward an HIV cure.     

Nicotinamide does not influence cytokines or exhaled NO in human experimental endotoxaemia

This study examined the hypothesis that nicotinamide could attenuate endotoxin-induced inflammatory responses in humans as indicated by levels of cytokines and nitric oxide. Ten healthy male volunteers participated in a randomised, double-blind, cross-over design with regard to the effects of nicotinamide. The volunteers received orally 4 g nicotinamide or placebo at 14 h and at 2 h preceding the experiment (total dose of 8 g). Endotoxin (E. coli, 2 ng/kg), was administered intravenously. Blood samples and haemodynamic data were collected prior to and up to 6 h after the endotoxin infusion. Orally exhaled NO was measured hourly. Following endotoxin, body temperature increased from baseline 36.3 +/- 0.09 degrees C to a maximum of 38.0 +/- 0.1 degrees C for all (mean +/- SEM, P < 0.001) and heart rate increased from 59 +/- 1.9 to 87.0 +/- 2.6 beats/min after 3 h (mean +/- SEM, P < 0.001). Endotoxin challenge also markedly elevated the TNF-alpha, IL-6, IL-8 and IL-10 concentrations (P < 0.001 versus baseline for all) during the study period. Orally exhaled NO also increased (P < 0.01) compared to baseline. Nicotinamide treatment did not influence the patterns of cytokine and NO response to endotoxin. In conclusion, there was no effect on the inflammatory parameters by oral nicotinamide at a dose of 8 g, limiting the potential use of this agent for anti-inflammatory purpose in man.     

Identification of human cell responses to hexavalent chromium

Hexavalent chromium [Cr(VI)] is a recognized environmental toxin with ubiquitous distribution in industrialized societies. Its concentration in ambient air derives from several sources including but not limited to chemical processes, the burning of fossil fuels and the production of cement. It is a food contaminant because of its deposition into bodies of water. The majority of published studies on the effects of Cr(VI) concern animal models and these studies have shown that it can induce a variety of cytotoxic and genotoxic reactions that affect the immune system. In order to identify the specific cellular impact of Cr(VI) on humans, we studied its effect on protein production and gene expression in human peripheral blood mononuclear cells (PBMC) obtained from both men and women of each major ethnic group including Caucasians, Hispanics, Asians and African-Americans. High-throughput protein profiling using bead-based protein arrays showed a concentration-dependent biphasic effect of Cr(VI) on the expression of many cytokines and chemokines by activated PBMC. High-density oligonucleotide microarray analysis identified several functional families of genes including those involved in immune response, intracellular signaling, cell cycle, apoptosis, RNA transport and binding, organelle organization and biogenesis that were strongly affected by Cr(VI). Cr(VI) suppressed many cellular receptor genes involved in immune response and activated many cell cycle-related and proapoptotic genes. These results defined responses that were unique to Cr(VI). This methodology defined an effective manner for identifying injurious/toxic human exposures to Cr(VI) at the cellular level that may facilitate the identification and monitoring of efficacious treatments for Cr(VI)-related maladies.     

Long-term antibody responses following human infection with Campylobacter jejuni

Epidemiological evidence suggests prior infection of humans by Campylobacter jejuni leads to protection against disease following further exposure. It is known that infections elicit strong antibody responses following the onset of disease and that antibody levels are elevated in putatively immune populations. To determine if systemic and mucosal antibodies induced by a confirmed infection remain at elevated levels for prolonged periods, repeat serum, saliva and urine samples were taken from campylobacter patients from 1 week and up to a year postinfection. Antibodies were monitored by ELISAs using three different antigen preparations: acid-glycine extracts (AE) of C. jejuni strain 81116 and an aflagellate mutant (R2), and a whole-cell R2 sonicate, and by Western blotting. Levels of serum IgG antibodies against 81116AE and R2 sonicate, but not R2AE, remained significantly raised over time when compared to a comparison population. Serum anti-sonicate IgA antibody levels were initially significantly raised but decreased over time to levels similar to the comparison group. There were no significant differences in levels of salivary IgA against the AEs. Anti-sonicate salivary IgA and IgG levels were initially significantly higher than in the comparison group. Both declined over time but the IgG levels remained significantly higher. Significant correlations were seen between serum IgG levels and age and duration of illness. Serum antibodies against flagellin, 40 kDa and 29 kDa antigens were still detectable in most patients up to a year postinfection, as were salivary antibodies to flagellin, the major outer-membrane protein and a 40 kDa antigen.     

人全血热原试验细胞因子法研究进展

当免疫细胞尤其是血液中的单核细胞和巨噬细胞与进入体内的热原物质接触后会分泌一些信号分子,这些信号分子包括白介素-1、白介素-6、肿瘤坏死因子-α等细胞因子,它们可直接和间接引起机体的体温升高,因此被称为内源性热原。在这组内源性热原中,研究得最清楚的是IL-1。利用这一反应机理作为一种检测热原的新方法,将被测样品与健康供血者提供的少量血液共同孵育,任何能引起产生IL-1的热原都能用ELISA方法检测出来。本文综述了人全血热原试验细胞因子法的背景、原理、方法与验证、应用、影响因素及展望。     

Analysis of the costimulatory requirements for generating human virus-specific in vitro T helper and effector responses

The present study analyzes the role of CD28-B7-mediated costimulation during in vitro human peripheral blood memory T cell activation by influenza A virus. Inhibition studies using the B7-binding fusion protein CTLA4Ig and antibodies against CD80 and CD86 demonstrate that CTLA4Ig and anti-CD86 inhibited influenza-specific T cell proliferation, interleukin (IL)-2 and interferon (IFN)- production, and generation of influenza-specific CD8+ CTL. The production of IL-10 and IL-18, which are known to modulate T cell immune responses, were not affected by blocking the CD28-B7 costimulatory pathway. Inhibition of diverse influenza-specific T cell functions could be reversed by the addition of exogenous IL-2 or IL-12 but not by the addition of IFN- or IL-18. Although IL-2 is known to overcome CD28-B7 costimulatory requirements, this is the first report showing that exogenous IL-12 is able to bypass CD28-B7 costimulatory blockade induced by CTLA4Ig in vitro. The induction of IFN- production with the recently described IFN- inducing cytokine IL-18 was not detected. In conclusion, these results demonstrate that CD86 represents a major costimulatory signal for the activation of resting peripheral blood memory T cells with recall antigens. These observations may have important implications for the development of immunotherapeutic strategies in diverse immunodeficiency diseases as well as in tumor immunotherapy.     

Leishmanin skin test lymphoproliferative responses and cytokine production after symptomatic or asymptomatic Leishmania major infection in Tunisia

Resistance to Leishmania parasite infection requires the development of a cellular immune response that activates macrophage leishmanicidal activity. In this study we have investigated the lymphoproliferative responses and in vitro cytokine production of peripheral blood mononuclear cells (PBMC) from individuals living in an endemic area for L. major infection in Tunisia. The results were compared with the DTH reaction of the leishmanin skin test (LST). Sixty-seven individuals were included in the study: 22 persons (age range 9-60 years) who developed, 2 years before the present study, a parasitologically confirmed localized cutaneous leishmaniasis (LCL) that healed spontaneously, and 45 individuals (age range 18-20 years) born and living in the same area, with no previous history of LCL. LST was positive (skin induration > or = 5 mm) in 20/22 cured cases of LCL and in 75% of healthy individuals without history of LCL. LST+ individuals expressed vigorous Leishmania-specific lymphoproliferative responses associated with in vitro production of interferon-gamma (IFN-gamma) but not IL-4. Interestingly, IL-10 was detected in parallel with the highest levels of IFN-gamma in PBMC supernatants from 3/20 cured LCL and 8/25 individuals without history of LCL. Our results showed a 98% concordance between the DTH reaction assessed by LST and the in vitro proliferative assay induced by soluble leishmanial antigens. Moreover, proliferative assays as well as cytokine analysis did not show any significant difference of the immune memory to parasite antigens developed by patients who had overt cutaneous leishmaniasis and those who had apparently asymptomatic infection.     

Fatty acids modulate cytokine and chemokine secretion of stimulated human whole blood cultures in diabetes

Fatty acids, uric acid and glucose are thought to contribute to subclinical inflammation associated with diabetes mellitus. We tested whether co‐incubation of free fatty acids and uric acid or glucose influences the secretion of immune mediators from stimulated human whole blood in vitro. Fresh whole blood samples from 20 healthy subjects, 20 patients with type 1 diabetes and 23 patients with type 2 diabetes were incubated for 24 h with palmitic acid (PAL), linolenic acid (LIN) or eicosapentaenoic acid (EPA) alone or together with elevated concentrations of uric acid or glucose. Concentrations of proinflammatory cytokines interleukin (IL)‐1β, IL‐2, IL‐12(p70), IL‐18, IFN‐γ, of regulatory cytokines IL‐4, IL‐10, IL‐17 and chemokine CCL2 (MCP‐1) were measured by multiplex‐bead technology from supernatants. Co‐incubation of fatty acids with uric acid resulted in a significant reduction of IL‐10, IL‐12(p70), IFN‐γ and CCL2 (MCP‐1) concentrations in supernatants compared to incubation with uric acid alone (P < 0·0001). In contrast, IL‐18 was up‐regulated upon co‐stimulation with fatty acids and uric acid. Similarly, co‐incubation of fatty acids with glucose diminished secretion of IL‐10, IFN‐γ and CCL2 (monocyte chemotactic protein‐1), while IL‐8 was up‐regulated (P < 0·001). Samples from healthy and diabetic subjects did not differ after adjustment for age, sex, body mass index and diabetes type. All three fatty acids similarly influenced whole blood cytokine release in vitro and modulated uric acid or glucose‐stimulated cytokine secretion. Although the ω‐3‐fatty acid EPA showed slightly stronger effects, further studies are required to elaborate the differential effects of PAL, LIN and EPA on disease risk observed previously in epidemiological studies.     

Activation of human AML14.3D10 eosinophils by nanoparticles: Modulatory activity on apoptosis and cytokine production

Eosinophilic inflammation is frequently observed in response to nanoparticle (NP) exposure in airway rodent models of allergies where the number of eosinophils is increased in lungs. Despite this, it is surprising that the potential cytotoxic effect of NP, as well as their direct role on eosinophils is poorly documented. The present study investigated how different NP can alter the biology of the human eosinophilic cell line AML14.3D10. It was found that among NP forms of CeO₂, ZnO, TiO₂, and nanosilver of 20?nm (AgNP₂₀) or 70?nm (AgNP₇₀) diameters, only ZnO and AgNP₂₀ induced apoptosis. Caspases-7 and -9 were not activated by the tested NP while caspase-3 was activated by AgNP₂₀ only. However, both ZnO and AgNP₂₀ induced cytoskeletal breakdown as evidenced by the cleavage of lamin B₁. Using an ELISArray approach for the simultaneous detection of several analytes (cytokines/chemokines), it was found that only ZnO and AgNP₂₀ increased the production of different analytes including the potent pro-inflammatory CXCL8 (IL-8) chemokine. From the data here, we conclude that toxic effects of some NP could be observed in human eosinophil-like cells and that this could be related, at least partially, by induction of apoptosis and production of cytokines and chemokines involved in inflammation. The results of this study also indicate that distinct NP do not activate similarly human eosinophils, since ZnO and AgNP₂₀ induce apoptosis and cytokine production while others such as TiO₂, CeO₂, and AgNP₇₀ do not.     

人IL-6、TNF-α对登革病毒感染人树突状细胞的影响

目的:探讨IL-6和TNF-α对登革病毒感染人树突状细胞(DC)的影响。方法:从人外周血中常规分离单核细胞,以GM-CSF和IL-4诱导成DC并进行形态学特征、细胞表型和淋巴细胞刺激能力的鉴定。用不同浓度的IL-6、TNF-α作用于Ⅱ型登革病毒(DV2)感染的DCs,于感染后6、24、48、72、96h收集上清,采用甲基纤维素微量病毒空斑法检测病毒的滴度,以MTT比色法测定IL-6和TNF-α对DV感染DC及正常DC数量的变化。结果:中、低浓度的IL-6对DC中DV2的增殖均有增强作用;高、中浓度的TNF-α对DC中DV2的增殖具有抑制作用。IL-6和TNF-α对DV感染DC及正常DC数量无明显影响。结论:IL-6和TNF-α通过对DC的影响在DV2感染中具有重要作用。