Dysregulation of serum gamma interferon levels in vascular chronic Q Fever patients provides insights into disease pathogenesis.

A large community outbreak of Q fever occurred in the Netherlands in the period 2007 to 2010. Some of the infected patients developed chronic Q fever, which typically includes pathogen dissemination to predisposed cardiovascular sites, with potentially fatal consequences. To identify the immune mechanisms responsible for ineffective clearance of Coxiella burnetii in patients who developed chronic Q fever, we compared serum concentrations of 47 inflammation-associated markers among patients with acute Q fever, vascular chronic Q fever, and past resolved Q fever. Serum levels of gamma interferon were strongly increased in acute but not in vascular chronic Q fever patients, compared to past resolved Q fever patients. Interleukin-18 levels showed a comparable increase in acute as well as vascular chronic Q fever patients. Additionally, vascular chronic Q fever patients had lower serum levels of gamma interferon-inducible protein 10 (IP-10) and transforming growth factor ß (TGF-ß) than did acute Q fever patients. Serum responses for these and other markers indicate that type I immune responses to C. burnetii are affected in chronic Q fever patients. This may be attributed to an affected immune system in cardiovascular patients, which enables local C. burnetii replication at affected cardiovascular sites.

Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol.

Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecular interaction of compounds with the neural differentiation process. Within the 11-day differentiation protocol of the assay, embryonic stem cells lost their pluripotency, evidenced by the reduced expression of stem cell markers Pou5F1 and Nanog. Moreover, stem cells differentiated into neural cells, with morphologically visible neural structures together with increased expression of neural differentiation-related genes such as ßIII-tubulin, Map2, Neurogin1, Mapt and Reelin. Valproic acid (VPA) and carbamazepine (CBZ) exposure during hESTn differentiation led to concentration-dependent reduced expression of ßIII-tubulin, Neurogin1 and Reelin. In parallel VPA caused an increased gene expression of Map2 and Mapt which is possibly related to the neural protective effect of VPA. These findings illustrate the added value of gene expression analysis for detecting compound specific effects in hESTn. Our findings were in line with and could explain effects observed in animal studies. This study demonstrates the potential of this assay protocol for mechanistic analysis of specific compound-induced inhibition of human neural cell differentiation.

Effects of prophylactic and therapeutic paracetamol treatment during vaccination on hepatitis B antibody levels in adults: two open-label, randomized controlled trials.

UNLABELLED: Worldwide, paracetamol is administered as a remedy for complaints that occur after vaccination. Recently published results indicate that paracetamol inhibits the vaccination response in infants when given prior to vaccination. The goal of this study was to establish whether paracetamol exerts similar effects in young adults. In addition, the effect of timing of paracetamol intake was investigated. In two randomized, controlled, open-label studies 496 healthy young adults were randomly assigned to three groups. The study groups received paracetamol for 24 hours starting at the time of (prophylactic use) - or 6 hours after (therapeutic use) the primary (0 month) and first booster (1 month) hepatitis B vaccination. The control group received no paracetamol. None of the participants used paracetamol around the second booster (6 months) vaccination. Anti-HBs levels were measured prior to and one month after the second booster vaccination on ADVIA Centaur XP. One month after the second booster vaccination, the anti-HBs level in the prophylactic paracetamol group was significantly lower (p = 0.048) than the level in the control group (4257 mIU/mL vs. 5768 mIU/mL). The anti-HBs level in the therapeutic paracetamol group (4958 mIU/mL) was not different (p = 0.34) from the level in the control group. Only prophylactic paracetamol treatment, and not therapeutic treatment, during vaccination has a negative influence on the antibody concentration after hepatitis B vaccination in adults. These findings prompt to consider therapeutic instead of prophylactic treatment to ensure maximal vaccination efficacy and retain the possibility to treat pain and fever after vaccination. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN03576945.

A quantitative approach to assess the potency of skin sensitizers in the elicitation phase.

The concept that thresholds exist for the induction of allergic contact dermatitis by chemicals with skin sensitizing properties has been used for a quantitative risk assessment approach. In this approach the potency of skin sensitizers as determined in the Local Lymph Node Assay is used to calculate the threshold for induction of sensitization. These are then used to estimate safe exposure levels for consumers. Whether these exposure levels will protect subjects that are already sensitized is unknown. The elicitation of allergic contact dermatitis supposedly occurs above a certain threshold as well and this threshold is most likely lower than that for the induction. It is unclear if induction thresholds can be extrapolated to elicitation thresholds. The aim of this study was to assess the potency of sensitizers with different sensitizing potencies in the elicitation phase in a mouse model for elicitation. Mice were sensitized by topical application on days 0 and 7 using equipotent concentrations of oxazolone, 2,4-dinitrochlorobenzene (DNCB) and eugenol to ensure that the sensitization strength would not influence the elicitation potency. Mice were challenged on day 21 by topical application on the ears in a dose-dependent manner and dose-response data were used to calculate the elicitation potency. Unexpectedly, sensitizers with different sensitizing potencies induced not the same dose-response curves in sensitized mice. The most potent sensitizer in the elicitation phase was oxazolone, followed by DNCB and eugenol. Similar to the induction phase, under equipotent sensitization conditions strong sensitizers such as oxazolone and DNCB elicit allergic reactions at lower concentrations than weak sensitizers such as eugenol. Our results indicate that elicitation thresholds cannot be readily deduced from sensitization thresholds.

Proteins involved in extracellular matrix dynamics are associated with respiratory syncytial virus disease severity.

Severity of respiratory syncytial virus (RSV) infection ranges widely. To what extent the local immune response is involved in RSV disease pathogenesis and which markers of this response are critical in determining disease severity is still a matter of debate. The local immune response was studied in nasopharyngeal aspirates (NPAs) during RSV infection. 47 potential markers of disease severity were analysed in a screening cohort of RSV-infected infants with mild disease at home (n = 8), hospitalised infants (n = 10) and infants requiring mechanical ventilation (n = 7). Results were confirmed in a cohort of infants hospitalised for RSV infection (n = 200). Finally, genetic validation was studied in a cohort of infants hospitalised for RSV infection (n = 465) and healthy controls (n = 930). The concentration of TIMP-1 (tissue inhibitor of metalloproteinase) was higher in the NPAs of hospitalised infants compared with the NPAs of infants at home (1,199 versus 568 ng · mL(-1); p<0.0001). Similar results were found for matrix metalloproteinase (MMP)-3 (765 versus 370 pg · mL(-1); p = 0.004). MMP-3 was confirmed as a marker of disease severity in a larger cohort and MMP3 gene polymorphism rs522616 was associated with severe RSV infection (OR 0.82, p<0.05). In conclusion, extracellular matrix proteinases play an important role in the pathogenesis of RSV bronchiolitis.

Local interleukin-10 production during respiratory syncytial virus bronchiolitis is associated with post-bronchiolitis wheeze.

BACKGROUND: Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants. Following RSV bronchiolitis, 50% of children develop post-bronchiolitis wheeze (PBW). Animal studies have suggested that interleukin (IL)-10 plays a critical role in the pathogenesis of RSV bronchiolitis and subsequent airway hyperresponsiveness. Previously, we showed that ex vivo monocyte IL-10 production is a predictor of PBW. Additionally, heterozygosity of the single-nucleotide polymorphism (SNP) rs1800872 in the IL10 promoter region was associated with protection against RSV bronchiolitis. METHODS: This study aimed to determine the in vivo role of IL-10 in RSV pathogenesis and recurrent wheeze in a new cohort of 235 infants hospitalized for RSV bronchiolitis. IL-10 levels in nasopharyngeal aspirates (NPAs) were measured at the time of hospitalization and the IL10 SNP rs1800872 genotype was determined. Follow-up data were available for 185 children (79%). RESULTS: Local IL-10 levels during RSV infection turned out to be higher in infants that later developed physician diagnosed PBW as compared to infants without PBW in the first year after RSV infection (958 vs 692 pg/ml, p = 0.02). The IL10 promoter SNP rs1800872 was not associated with IL-10 concentration in NPAs. CONCLUSION: The relationship between high local IL-10 levels during the initial RSV infection and physician diagnosed PBW provides further evidence of the importance of the IL-10 response during RSV bronchiolitis.

Contact and respiratory sensitizers can be identified by cytokine profiles following inhalation exposure.

There are currently no validated animal models that can identify low molecular weight (LMW) respiratory sensitizers. The Local Lymph Node Assay (LLNA) is a validated animal model developed to detect contact sensitizers using skin exposure, but all LMW respiratory sensitizers tested so far were also positive in this assay. Discrimination between contact and respiratory sensitizers can be achieved by the assessment of cytokine profiles. In a LLNA using the inhalation route, both contact and respiratory sensitizers enhanced proliferation in the draining lymph nodes. The question was if their cytokine profiles were affected by the route of exposure. Male BALB/c mice were exposed head/nose-only during 3 consecutive days to the respiratory sensitizers trimellitic anhydride, phthalic anhydride, toluene diisocyanate, hexamethylene diisocyanate (HDI), and isophorone diisocyanate; the contact sensitizers dinitrochlorobenzene (DNCB), oxazolone (OXA) and formaldehyde (FA), and the irritant methyl salicylate (MS). Three days after the last exposure the draining lymph nodes were excised and cytokine production was measured after ex vivo stimulation with Concanavalin A. Skin application was used as a positive control. After inhalation exposure the respiratory sensitizers induced more interleukin-4 (IL-4) and interleukin (IL-10) compared to the contact sensitizers, whereas the contact sensitizers, except formaldehyde, induced relatively more interferon-gamma (IFN-gamma) production. When IL-4 and IFN-gamma were plotted as a function of the proliferative response, it was shown that IL-4 could be used to identify respiratory sensitizers, except HDI, at concentration levels inducing intermediate stimulation indices. HDI could be distinguished from DNCB and OXA at high SI values. In contrast, contact sensitizers could only be identified when IFN-gamma was measured at high stimulation indices. The skin positive control, tested at high concentrations, showed comparable results for IL-4 and IL-10, whereas IFN-gamma levels could not be used to discriminate between respiratory and contact sensitizers. The contact sensitizer FA and the irritant MS did not induce significant cytokine production after inhalation and skin exposure. In conclusion, the respiratory LLNA is able to identify and distinguish strong contact and respiratory sensitizers when simultaneously proliferation and cytokine production are assessed in the upper respiratory tract draining LNs.

The respiratory local lymph node assay as a tool to study respiratory sensitizers.

The local lymph node assay (LLNA) is used to test the potential of low molecular weight (LMW) compounds to induce sensitization via the skin. In the present study, a respiratory LLNA was developed. Male BALB/c mice were exposed head/nose-only during three consecutive days for 45, 90, 180, or 360 min/day to various LMW allergens. Ear application (skin LLNA) was used as a positive control. Negative controls were exposed to the vehicle. Three days after the last exposure, proliferation was determined in the draining mandibular lymph nodes, and the respiratory tract was examined microscopically. Upon inhalation, the allergens trimellitic anhydride, phthalic anhydride, hexamethylene diisocyanate, toluene diisocyanate, isophorone diisocyanate (IPDI), dinitrochlorobenzene, and oxazolone were positive and showed stimulation indices (SIs) up to 11, whereas trimeric IPDI, formaldehyde, and methyl salicylate were negative (viz. SI < 3). All compounds, except trimeric IPDI, induced histopathological lesions predominantly in the upper respiratory tract. Exposure by inhalation is a realistic approach to test respiratory allergens. However, based on the local toxicity, the dose that can be applied is (generally) much lower than can be achieved by skin application. It is concluded that strong LMW allergens, regardless their immunological nature, besides the skin can also sensitize the body via the respiratory tract. In addition, the contact allergens were as potent as the respiratory allergens, although the potency ranking differed from that in a skin LLNA.

Effect of Prolonged Repeated Exposure to Formaldehyde Donors with Doses Below the EC3 Value on Draining Lymph Node Responses.

In the Local Lymph Node Assay (LLNA), a stimulation index of 3 (SI = 3) is established as a threshold value for hazard identification of sensitization. The corresponding EC3 value, the effective concentration inducing a threefold increase compared to controls, can possibly predict threshold levels for sensitization in humans. Exposure to a dose below the threshold dose would not result in an induction of an immune response. Each repeated contact would be considered and viewed as a new contact and as long as the dose is below the threshold there will be no response, even after repeated exposures. However, repeated exposure may result in local accumulation eventually resulting in a dose that induces a response above the threshold for immunization. We investigated lymph node responses after short and prolonged exposure to formaldehyde donors, chemicals that are highly reactive with proteins and may thus persist in the skin. The studies were performed with formaldehyde and formaldehyde releasers (formaldehyde, paraformaldehyde, Quaternium-15, 2-Chloro-N-(hydroxymethyl)acetamide, and hexamethylenetetramine), at concentrations that induce a SI = 2, i.e., below the threshold for hazard identification. For all test chemicals investigated enhanced lymph node responses were obtained when comparing long-term prolonged exposure to short-term exposure, while three of five chemicals induced responses above SI = 3. Our results show that repeated and prolonged exposure to doses below the EC3 value can induce reactions above the SI = 3, the hazard identification threshold for sensitization in mice. So, when discussing the possible use of the EC3 as benchmark for risk assessment, one should consider duration of exposure and the possibility of local accumulation of the chemical under investigation.

Evaluation of immunomodulation by Lactobacillus casei Shirota: immune function, autoimmunity and gene expression.

Lactic acid bacteria are claimed to have immunomodulating effects. Stimulation as well as suppression of T helper (Th)1 mediated immune responses, have been described for various strains. Experiments involving Lactobacillus casei Shirota (LcS) detected mainly enhancement of innate immune responses and promotion of Th1 mediated immune reactivity. To confirm and further investigate modulation of Th1 responses and development of autoimmune disease by LcS, the consequences of oral administration of LcS were assessed in several experiments. The effect of LcS varied between the different models. No modulation was found in the mitogen-induced cell proliferation and cytokine release assays in mesenteric lymph nodes of Wistar rats. LcS inhibited the Th1 mediated immune response in an adapted murine Local Lymph Node Assay (LLNA) in BALB/c mice, whereas experimental autoimmune encephalomyelitis (EAE) in Lewis rats was aggravated. These varying effects on Th1 responses indicate that beneficial as well as harmful effects on immune related disorders could occur after LcS consumption. Since microarray analysis is suggested to be more sensitive and predictive than functional tests, gene expression profiling was included as an alternative endpoint in the testing of immunomodulation. The detected gene expression profiles did not reflect the effects of LcS on the immune system. Microarray analysis may therefore have no more predictive value than immune function assays when investigating immunomodulation by probiotics. To gain further insight into effects of probiotics on immune function, experiments including cytokine assays and gene expression analysis combined with disease models could be useful.

Application of a systemic herpes simplex virus type 1 infection in the rat as a tool for sunscreen photoimmunoprotection studies.

The application of a novel model for sunscreen photoimmunoprotection studies was assessed using a systemic infection of rats with herpes simplex virus type 1 (HSV-1). Rats were irradiated daily with 1 minimal erythemal/oedematous dose of UVB for 7 consecutive days on their shaved backs with or without application of a broad-spectrum sunscreen (containing TiO2) with a sun protection factor of 10. Subsequently, rats were infected intranasally with HSV. UV exposure prior to HSV infection induced increased severity and incidence of clinical signs of disease, suppression of cellular immune responses as assessed by delayed type hypersensitivity and increased viral load in the brain. The sunscreen provided protection against all these UV-induced effects. We conclude that this novel model is a promising way of testing the immunoprotective qualities of sunscreens, based on the response to a common infectious agent of human subjects.