The role of Toll-like receptor-4 in pertussis vaccine-induced immunity.

BACKGROUND: The gram-negative bacterium Bordetella pertussis is an important causative agent of pertussis, an infectious disease of the respiratory tract. After introduction of whole-cell vaccines (wP) in the 1950's, pertussis incidence has decreased significantly. Because wP were found to be reactogenic, in most developed countries they have been replaced by acellular vaccines (aP). We have previously shown a role for Toll-like receptor 4 (Tlr4) in pertussis-infected mice and the pertussis toxin (Ptx)-IgG response in wP-vaccinated children, raising the issue of the relative importance of Tlr4 in wP vaccination of mice. Here we analyze the effects of wP and aP vaccination and B. pertussis challenge, in Tlr4-deficient C3H/HeJ and wild-type C3H/HeOuJ mice. aP consists of Ptx, filamentous hemagglutinin (FHA), and pertactin (Prn). RESULTS: We show an important role of Tlr4 in wP and (to a lesser extent) aP vaccination, induction of Th1 and Th17 cells by wP but not aP vaccination, and induction of Th17 cells by infection, confirming data by Higgins et al. (J Immunol 2006, 177:7980-9). Furthermore, in Tlr4-deficient mice, compared to wild-type controls (i) after vaccination only, Ptx-IgG (that was induced by aP but not wP vaccination), FHA-IgG, and Prn-IgG levels were similar, (ii) after infection (only), lung IL-1alpha and IL-1beta expression were lower, (iii) after wP vaccination and challenge, Prn-IgG level and lung IL-5 expression were higher, while lung IL-1beta, TNF-alpha, IFN-gamma, IL-17, and IL-23 expression were lower, and lung pathology was absent, and (iv) after aP vaccination and challenge, Prn-IgG level and lung IL-5 expression were higher, while Ptx-IgG level was lower. CONCLUSION: Tlr4 does not influence the humoral response to vaccination (without challenge), plays an important role in natural immunity, wP and aP efficacy, and induction of Th1 and Th17 responses, is critical for lung pathology and enhances pro-inflammatory cytokine production after wP vaccination and challenge, and diminishes Th2 responses after both wP and aP vaccination and challenge. wP vaccination does not induce Ptx-IgG. A role for LPS in the efficacy of wP underlines the usefulness of LPS analogs to improve bacterial subunit vaccines such as aP.

Toxicity of coarse and fine particulate matter from sites with contrasting traffic profiles.

Residence in urban areas with much traffic has been associated with various negative health effects. However, the contribution of traffic emissions to these adverse health effects has not been fully determined. Therefore, the objective of this in vivo study is to compare the pulmonary and systemic responses of rats exposed to particulate matter (PM) obtained from various locations with contrasting traffic profiles. Samples of coarse (2.5 microm-10 microm) and fine (0.1 microm-2.5 microm) PM were simultaneously collected at nine sites across Europe with a high-volume cascade impactor. Six PM samples from various locations were selected on the basis of contrast in in vitro analysis, chemical composition, and traffic profiles. We exposed spontaneously hypertensive (SH) rats to a single dose (3 mg PM/kg body weight or 10 mg PM/kg body weight) of either coarse or fine PM by intratracheal instillation. We assessed changes in biochemical markers, cell differentials, and histopathological changes in the lungs and blood 24 h postexposure. The dose-related adverse effects that both coarse and fine PM induced in the lungs and vascular system were mainly related to cytotoxicity, inflammation, and blood viscosity. We observed clear differences in the extent of these responses to PM from the various locations at equivalent dose levels. There was a trend that suggests that samples from high-traffic sites were the most toxic. It is likely that the toxicological responses of SH rats were associated with specific PM components derived from brake wear (copper and barium), tire wear (zinc), and wood smoke (potassium).

Tissue response in the rat and the mouse to degradable dextran hydrogels.

Two types of hydroxyethyl-methacrylated dextran (dex-HEMA) hydrogels differing in crosslink density were compared for local tissue responses and degradation characteristics in mice and rats. Implants (1 mm thick, rat: 10 mm diameter, mouse: 6 mm diameter) varying in degree of HEMA substitution (DS5 and DS13, meaning 5 or 13 HEMA groups per 100 glucose units of dextran) were subcutaneously implanted and tissue responses were evaluated at week 2, 6, and 13 after implantation. In the rat after 2 weeks a slight fibrous capsule was formed composed of macrophages and fibroblasts sometimes accompanied by a minimal infiltrate. Small fragments, surrounded by macrophages and giant cells indicated hydrogel degradation. After 13 weeks DS5 implants were resorbed while parts of the DS13 implants were still present. In the mouse a moderate to strong capsule formation was present at 2 weeks accompanied by inflammatory cells (macrophages and polymorphonuclear granulocytes) and debris. Draining lymph node activation was observed. Skin ulceration was present irrespective of the type of implant. Clear differences in the tissue responses between the rat and mouse were noted, as well as between implants of different degree of substitution. Mice showed a more pronounced early inflammatory response compared with rats, whereas the degradation was more complete in rats than in mice. The differences in histology between the hydrogels disappeared over time at 13 weeks after implantation and similar responses were noted for both types of hydrogels. Both in mice and rats the DS5 hydrogels showed a faster degradation rate than the DS13 hydrogels.

Lipopolysaccharide analogs improve efficacy of acellular pertussis vaccine and reduce type I hypersensitivity in mice.

Pertussis is an infectious disease of the respiratory tract that is caused by the gram-negative bacterium Bordetella pertussis. Although acellular pertussis (aP) vaccines are safe, they are not fully effective and thus require improvement. In contrast to whole-cell pertussis (wP) vaccines, aP vaccines do not contain lipopolysaccharide (LPS). Monophosphoryl lipid A (MPL) and Neisseria meningitidis LpxL2 LPS have been shown to display immune-stimulating activity while exerting little endotoxin activity. Therefore, we evaluated whether these LPS analogs could increase the efficacy of the aP vaccine. Mice were vaccinated with diphtheria-tetanus-aP vaccine with aluminum, MPL, or LpxL2 LPS adjuvant before intranasal challenge with B. pertussis. Compared to vaccination with the aluminum adjuvant, vaccination with either LPS analog resulted in lower colonization and a higher pertussis toxin-specific serum immunoglobulin G level, indicating increased efficacy. Vaccination with either LPS analog resulted in reduced lung eosinophilia, reduced eosinophil numbers in the bronchoalveolar lavage fluid, and the ex vivo production of interleukin-4 (IL-4) by bronchial lymph node cells and IL-5 by spleen cells, suggesting reduced type I hypersensitivity. Vaccination with either LPS analog increased serum IL-6 levels, although these levels remained well below the level induced by wP, suggesting that supplementation with LPS analogs may induce some reactogenicity but reactogenicity considerably less than that induced by the wP vaccine. In conclusion, these results indicate that supplementation with LPS analogs forms a promising strategy that can be used to improve aP vaccines.

Host transcription profiles upon primary respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract infection in children. Severe RSV disease is related to an inappropriate immune response to RSV resulting in enhanced lung pathology which is influenced by host genetic factors. To gain insight into the early pathways of the pathogenesis of and immune response to RSV infection, we determined the transcription profiles of lungs and lymph nodes on days 1 and 3 after infection of mice. Primary RSV infection resulted in a rapid but transient innate, proinflammatory response, as exemplified by the induction of a large number of type I interferon-regulated genes and chemokine genes, genes involved in inflammation, and genes involved in antigen processing. Interestingly, this response is much stronger on day 1 than on day 3 after infection, indicating that the strong transcriptional response in the lung precedes the peak of viral replication. Surprisingly, the set of down-regulated genes was small and none of these genes displayed strong down-regulation. Responses in the lung-draining lymph nodes were much less prominent than lung responses and are suggestive of NK cell activation. Our data indicate that at time points prior to the peak of viral replication and influx of inflammatory cells, the local lung response, measured at the transcriptional level, has already dampened down. The processes and pathways induced shortly after RSV infection can now be used for the selection of candidate genes for human genetic studies of children with severe RSV infection.

Effects of a diphtheria-tetanus-acellular pertussis vaccine on immune responses in murine local lymph node and lung allergy models.

We have previously shown that in mice, diphtheria-tetanus-acellular pertussis (DTaP) vaccination before Bordetella pertussis infection resulted in, besides effective clearance, immediate hypersensitivity (lung eosinophilia, increased total serum immunoglobulin E [IgE], and increased ex vivo Th2 cytokine production by cells from the bronchial lymph nodes). To better appreciate the extent of these findings, we measured DTaP vaccination effects in the local lymph node assay (LLNA) and an ovalbumin (OVA) lung allergy model. In the LLNA, mice were vaccinated or adjuvant treated before being sensitized with trimellitic anhydride (TMA; inducing a Th2-directed response) and dinitrochlorobenzene (DNCB; inducing a Th1-directed response). Compared to the adjuvant-treated controls, the vaccinated mice showed a decreased response to TMA and (to a much lesser extent) an increased response to DNCB. The decreased response to TMA coincided with increased transforming growth factor beta levels. With the exception of filamentous hemagglutinin, all vaccine constituents contributed to the decreased response to TMA. In the lung allergy model, sensitization induced OVA-specific IgE, lung pathology (peribronchiolitis, perivasculitis, and hypertrophy of the bronchiolar mucus cells) and increased the number of eosinophils, lymphocytes, and neutrophils in the bronchoalveolar lavage fluid. Vaccination failed to modulate these parameters. In conclusion, although DTaP vaccination may affect the LLNA response, we found no evidence of an effect on lung allergy.

Lung pathology and immediate hypersensitivity in a mouse model after vaccination with pertussis vaccines and challenge with Bordetella pertussis.

While evaluating vaccine efficacy against clinical Bordetella pertussis isolates in mice, after challenge vaccinated mice showed increased lung pathology with eosinophilia, compared to challenged, non-vaccinated animals. This led us to study bacterial clearance, lung pathology, lung TNF-alpha expression, and parameters of immediate hypersensitivity (IH), being serum IgE levels, eosinophil numbers in the bronchoalveolar lavage fluid, and ex vivo IL-4, IL-5, IL-10, IL-13, and IFN-gamma production by the bronchial lymph node cells. BALB/c mice received a combined Diphtheria (D), Tetanus (T), Poliomyelitis, and whole-cell Pertussis vaccine (WCV), a combined D, T, and three-component acellular Pertussis vaccine (ACV), aluminium hydroxide adjuvant, or PBS, 28 and 14 days before B. pertussis infection. Similarly treated non-infected mice were taken as a control. Infection induced pathology; this induction was stronger after (especially WCV) vaccination. WCV but not ACV vaccination induced TNF-alpha expression after challenge. After challenge, IH parameters were strongly increased by (especially ACV) vaccination. Vaccinated IL-4 KO mice showed similar clearance and pathology, in the absence of IgE and with reduced numbers of eosinophils. Vaccinated (Th1-deficient) T-bet KO mice showed reduced clearance and similar pathology. In summary, after challenge vaccination increased lung pathology, TNF-alpha expression (only WCV), and IH parameters. Th1 cells were critical for clearance.

Modulation of mammary tumor development in Tg.NK (MMTV/c-neu) mice by dietary fatty acids and life stage-specific exposure to phytoestrogens.

Breast cancer is a major public health problem among women worldwide. Phytoestrogens and dietary fat composition are being investigated to elucidate the role of nutrition in breast cancer risk. Both epidemiological and rodent studies suggest that the chemopreventive effect of phytoestrogens depends on timing of exposure. We investigated spontaneous mammary tumor development in female heterozygous MMTV/c-neu (Tg.NK) mice upon isoflavone exposure on background diets rich in either n-6 or n-3 polyunsaturated fatty acids (PUFAs). Three different exposure protocols were used, either from conception to weaning, or from weaning onwards, or lifelong. Mice fed diets high in n-3 PUFAs developed mammary tumors 15 weeks later than mice fed n-6 PUFA diets. In the latter mice, isoflavone exposure from weaning onwards resulted in a significant decrease in tumor incidence and a delay in tumor onset. Therefore, the effects of phytoestrogen exposure on tumor formation appear to depend on the composition of the background diet and on the timing of exposure within the life cycle.

Toxocara canis: effect of inoculum size on pulmonary pathology and cytokine expression in BALB/c mice.

Infection of mice with Toxocara canis results in pulmonary inflammation and the induction of a Th2 type of immune response. The aim of this study was to determine whether the effect of infection with this nematode depends on the inoculum size. Results indicate that mice infected with either a high or a low inoculum size showed, in a dose-dependent manner, pulmonary inflammation with eosinophil infiltration, increased levels of total IgE, and Toxocara-specific IgG1 that persisted up to 60 days p.i. Relative quantification of cytokine expression in lungs of mice infected with different doses showed proportional increased expression of the IL-4, IL-5, and IL-10 transcripts, whereas the expression of the IFN-gamma transcript was not different from that of uninfected controls. These results indicate that infection of BALB/c mice with T. canis results in chronic pulmonary inflammation and a dominant Th2 type of immune response, independent of the inoculum size.

Ozone induces clear cellular and molecular responses in the mouse lung independently of the transcription-coupled repair status.

The oxidant ozone is a well-known air pollutant, inhalation of which is associated with respiratory tract inflammation and functional alterations of the lung. It is well established as an inducer of intracellular oxidative stress. We investigated whether Cockayne syndrome B, transcription-coupled, repair-deficient mice (Csb(-/-)), known to be sensitive to oxidative stressors, respond differently to ozone than repair-proficient controls (Csb(+/-)). Mice were exposed to 0.8 parts/million ozone for 8 h, and we examined a wide range of biological parameters in the lung at the gene expression, protein, and cellular level 4 h after the ozone exposure. Relevant biological responses to ozone for both repair-deficient Csb(-/-) and repair-proficient Csb(+/-) mice, as determined by biochemical analysis of bronchoalveolar lavage fluid (e.g., increases of polymorphonuclear neutrophils, alkaline phosphatase, macrophage-inflammatory protein-2, and tumor necrosis factor-alpha), pathological examinations, and gene expression (upregulation of oxidative-stress-related genes) analyses were observed. The bronchoalveolar lavage fluid showed significantly more tumor necrosis factor-alpha in repair-deficient Csb(-/-) mice than in repair-proficient Csb(+/-) mice after ozone exposure. In addition, a clear trend was observed toward fewer differentially expressed genes with a lower fold ratio in repair-deficient Csb(-/-) mice than in repair-proficient Csb(+/-) mice. However, repair-deficient Csb(-/-) mice do not respond significantly more sensitively to ozone compared with repair-proficient Csb(+/-) mice at the level of gene expression. We conclude that, under the conditions employed here, although small differences at the transcriptional level exist between repair-proficient Csb(+/-) mice and transcription-coupled repair defective Csb(-/-) mice, these do not have a significant effect on the ozone-induced lung injury.

Response of spontaneously hypertensive rats to inhalation of fine and ultrafine particles from traffic: experimental controlled study.

BACKGROUND: Many epidemiological studies have shown that mass concentrations of ambient particulate matter (PM) are associated with adverse health effects in the human population. Since PM is still a very crude measure, this experimental study has explored the role of two distinct size fractions: ultrafine (<0.15 microm) and fine (0.15- 2.5 microm) PM. In a series of 2-day inhalation studies, spontaneously hypersensitive (SH) rats were exposed to fine, concentrated, ambient PM (fCAP) at a city background location or a combination of ultrafine and fine (u+fCAP) PM at a location dominated by traffic. We examined the effect on inflammation and both pathological and haematological indicators as markers of pulmonary and cardiovascular injury. Exposure concentrations ranged from 399 microg/m3 to 3613 microg/m3 for fCAP and from 269 microg/m3 to 556 microg/m3 for u+fCAP. RESULTS: Ammonium, nitrate, and sulphate ions accounted for 56 +/- 16% of the total fCAP mass concentrations, but only 17 +/- 6% of the u+fCAP mass concentrations. Unambiguous particle uptake in alveolar macrophages was only seen after u+fCAP exposures. Neither fCAP nor u+fCAP induced significant changes of cytotoxicity or inflammation in the lung. However, markers of oxidative stress (heme oxygenase-1 and malondialdehyde) were affected by both fCAP and u+fCAP exposure, although not always significantly. Additional analysis revealed heme oxygenase-1 (HO-1) levels that followed a nonmonotonic function with an optimum at around 600 microg/m3 for fCAP. As a systemic response, exposure to u+fCAP and fCAP resulted in significant decreases of the white blood cell concentrations. CONCLUSION: Minor pulmonary and systemic effects are observed after both fine and ultrafine + fine PM exposure. These effects do not linearly correlate with the CAP mass. A greater component of traffic CAP and/or a larger proportion ultrafine PM does not strengthen the absolute effects.

Inhalation of concentrated particulate matter produces pulmonary inflammation and systemic biological effects in compromised rats.

Although significant progress has been made over the past few years, there is still debate on the causal fractions that are responsible for particulate matter (PM)-associated adverse health effects. A series of 1-d inhalation exposures to concentrated ambient particles (CAPs) were performed in compromised rats, focusing on pulmonary inflammation and changes in blood factors as biological outcomes. Studies were carried out in The Netherlands at an urban background location in Bilthoven, an industrialized location in the city of Utrecht, as well as at a location that is heavily dominated by freeway emissions. It was hypothesized that exposure to CAPs resulted in oxidative stress in the lung, producing a release of inflammatory mediators, which in turn can result in cardiovascular effects. Both spontaneously hypertensive rats and rats preexposed to ozone were studied. The effects were studied at 2d postexposure, focusing on pathology and cell proliferation, bronchoalveolar lavage fluid (BALF) analysis (including cytokines, biochemistry, cell differentials, cell viability and proliferation, and Clara-cell 16 protein), and blood analyses (fibrinogen, Clara-cell 16 protein, Von Willebrand factor, and cell differentials). Using CAPs exposures as a binary term, mild inflammation (increased numbers of neutrophils) and increased lung permeability (protein and albumin leakage in BALF) were evident. In addition, CAPs also produced increased fibrinogen concentrations in blood of spontaneously hypertensive rats. In conclusion, inhalation up to 3700 microg/m3 CAPs in the size range of 0.15-2.5 microm did induce statistically significant effects in the lung and blood, but the effects observed may not potentially be very biologically relevant. PM mass concentrations and lung permeability were weakly associated. This suggests that other PM metrics might be more appropriate.

Effects of particulate matter on the pulmonary and vascular system: time course in spontaneously hypertensive rats.

BACKGROUND: This study was performed within the scope of two multi-center European Commission-funded projects (HEPMEAP and PAMCHAR) concerning source-composition-toxicity relationship for particulate matter (PM) sampled in Europe. The present study aimed to optimize the design for PM in vivo toxicity screening studies in terms of dose and time between a single exposure and the determination of the biological responses in a rat model mimicking human disease resulting in susceptibility to ambient PM. Dust in thoracic PM size-range (aerodynamic diameter <10 mum) was sampled nearby a road tunnel (RTD) using a high volume cascade impactor. Spontaneously hypertensive rats were exposed to urban dust collected in Ottawa, Canada (EHC-93 10 mg/kg of body weight; reference PM) or different RTD doses (0.3, 1, 3, 10 mg/kg of body weight) by intratracheal instillation. Necropsy was performed at 4, 24, or 48 hr after exposure. RESULTS: The neutrophil numbers in bronchoalveolar lavage fluid increased tremendously after exposure to the highest RTD doses or EHC-93. Furthermore, PM exposure slightly affected blood coagulation since there was a small but significant increase in the plasma fibrinogen levels (factor 1.2). Pulmonary inflammation and oxidative stress as well as changes in blood coagulation factors and circulating blood cell populations were observed within the range of 3 to 10 mg PM/kg of body weight without significant pulmonary injury. CONCLUSION: The optimal dose for determining the toxicity ranking of ambient derived PM samples in spontaneously hypertensive rats is suggested to be between 3 and 10 mg PM/kg of body weight under the conditions used in the present study. At a lower dose only some inflammatory effects were detected, which will probably be too few to be able to discriminate between PM samples while a completely different response pattern was observed with the highest dose. In addition to the dose, a 24-hr interval from exposure to sacrifice seemed appropriate to assess the relative toxic potency of PM since the majority of the health effects were observed one day after PM exposure compared to the other times examined. The aforementioned considerations provide a good basis for conducting PM toxicity screening studies in spontaneously hypertensive rats.

Pertussis toxin relaxes small arteries with no vascular lesions or vascular smooth muscle cell injury.

Previous studies showed that pertussis toxin (PT) decreased agonist-induced contractions of isolated rat small mesenteric resistance arteries independently from endothelium, nitric oxide-synthase or intracellular calcium concentrations. In this study, it was investigated if the PT-induced decreased contractile properties of small mesenteric resistance arteries could be a consequence of a PT-induced vascular and/or smooth muscle cell injury, leading to loss of contractile functionality. Male Wistar rats were treated with PT (30 microg/kg, intravenously) and sections of isolated small mesenteric resistance arteries were investigated with light- and electron microscopy. Light microscopic investigation of cross-sectioned small mesenteric resistance arteries of control animals clearly showed a contracted phase, while PT-pretreated animals showed a relaxed smooth inner surface of the vessel, indicating a vasodilated state. Electron microscopic investigation showed that PT-pretreatment neither induced vascular lesions nor caused morphological or numerical changes in cell organelles such as contractile elements of vascular smooth muscle cells. In conclusion, the PT-induced decreased contractile properties of isolated rat small resistance arteries are not caused by a PT-induced vascular and/or smooth muscle cell injury.

Association of Bordetella pertussis with host immune cells in the mouse lung.

Mouse models are frequently used to study immunity and pathogenesis to Bordetella pertussis infection. To improve the understanding of the mouse infection model, the influx of host cells and B. pertussis localisation in the lungs were evaluated. Furthermore, the roles of filamentous hemagglutinin (FHA) and fimbriae (Fim) in these processes were determined. B. pertussis infection stimulated the recruitment of polymorphonuclear granulocytes (PMN), alveolar macrophages, and lymphocytes. As determined by double immunofluorescence staining, 2 hr after infection most B. pertussis were free in the alveolar space, some were attached to alveolar epithelia, and some were associated with and phagocytosed by PMN. After 3 days, most bacteria were associated with and phagocytosed by macrophages, some by PMN. B. pertussis was shown not to be ingested by epithelial cells or associated with interstitial macrophages. B. pertussis mutants lacking expression of FHA or Fim were associated with and phagocytosed by the same cell types as parental bacteria. The Fim mutant, however, induced a more severe inflammation, and was cleared faster from the lungs compared to the parental strain and the FHA mutant. These results suggest that Fim does not affect bacterial localisation in the mouse lung, but does influence host immune mechanisms. Possibly, Fim may exert an anti-inflammatory function and thereby inhibit killing by macrophages.

Ultrastructural characterization of effector-target interactions for human neonatal and adult NK cells reveals reduced intercellular surface contacts of neonatal cells.

Limitations in neonatal natural killer (NK) cell responses may be associated with the less efficient newborn capacity to solve viral infections. Although these limitations have been extensively reported they are poorly characterized. Making use of the major histocompatibility complex (MHC) class I negative cell line K562, the parameters required for the initial events involved in neonatal NK/target cell interactions were determined and compared with adult blood NK cell/target cell interactions. Ultrastructural characterization of effector-target cell interactions revealed that neonatal NK cells are more strongly activated upon contact with K562 cells than adult blood NK cells. Furthermore, the neonatal capacity to establish contacts, in particular extensive contacts, is significantly reduced when compared with adult blood NK cells. However, no significant differences were found either in the cell surface expression levels or activation state of LFA-1, which could account for the reduced intercellular contacts. Because extensive contacts are crucial for effective immunologic synapse formation, these data suggest that a limited or nonsustained positive signaling may occur on neonatal NK cells, restricting their NK cell-mediated lysis capacity.

Respiratory syncytial virus enhances respiratory allergy in mice despite the inhibitory effect of virus-induced interferon-gamma.

In mice, respiratory syncytial virus (RSV) infection during allergic provocation aggravates the allergic Th2 immune response, characterised by production of interleukin (IL)-4, IL-5, and IL-13, and eosinophilic inflammation. This enhancement of the Th2 response occurs simultaneously with a strong RSV-induced Th1 cytokine response (IL-12 and IFN-gamma). The present study investigated whether IFN-gamma and IL-12 are critically involved in this RSV-enhanced OVA allergy. Therefore, IFN-gammaR- and IL-12-deficient mice (both on a 129/Sv/Ev background) were sensitised and challenged with ovalbumin (OVA) and infected with RSV during the OVA challenge period. Neither gene deletion affected the development of ovalbumin-induced allergic inflammation in mice. However, when OVA-allergic IFN-gammaR deficient mice were infected with RSV, an increased pulmonary eosinophilic infiltrate and increased IL-4 and IL-13 mRNA expression in lung tissue were observed compared with identically treated wild-type mice. In contrast, deficiency of IL-12 did not aggravate the Th2 immune and inflammatory response in OVA/RSV-treated mice, compared with wild-type. In conclusion, the virus-induced IFN-gamma response diminishes the Th2 inflammatory response during OVA allergy but fails to prevent totally the enhancement of the OVA allergy by RSV. In contrast, IL-12 is not involved in inhibiting nor increasing the RSV-enhanced allergy in 129/Sv/Ev mice.

Effect of lack of Interleukin-4, Interleukin-12, Interleukin-18, or the Interferon-gamma receptor on virus replication, cytokine response, and lung pathology during respiratory syncytial virus infection in mice.

RSV is an important cause of bronchiolitis in infants. Immunopathology may play a role in RSV-induced bronchiolitis and severe RSV-induced disease has been associated with a Th2 type immune response. The aim of the study was to identify cytokine pathways that are crucial in influencing RSV-induced disease. For that purpose we inoculated IFNgammaR-/-, IL-12-/-, IL-18-/-, or IL-4-/- mice with RSV. We observed that an RSV infection resulted in a predominant Th1 cytokine response associated with slight bronchiolitis and alveolitis. Pulmonary histopathology was only aggravated in IFN R-/- mice, characterised by eosinophilic influx around the bronchioles. Despite subtle changes in cytokine expression, no differences in histopathology were observed in IL-12-/- and IL-18-/- mice. Deficiency of IL-4 has no effect on RSV-induced Th1 cytokines and pulmonary histopathology. IFNgamma-receptor deficiency during primary RSV infection resulted in a disturbed Th1 response based on increased IL-4, IL-5, and IL-13 expression and the presence of eosinophils in the lungs. It is concluded that IFNgamma signalling is required for a pronounced Th1 response to RSV while IL-12 and IL-18 are not. A shift in the balance between Th1 and Th2 towards a Th2 response induced by missing IFNgamma signalling leads to aggravated pulmonary pathology. This is not caused by enhanced viral load.

Utility of rat liver slices to estimate hepatic clearance for application in physiologically based pharmacokinetic modeling: a study with tolbutamide, a compound with low extraction efficiency.

Liver slice experiments were performed to determine the slice intrinsic clearance and to extrapolate this to the in vivo liver intrinsic clearance in a physiologically based pharmacokinetic (PBPK)-like approach. Precision-cut liver slices were incubated with different initial concentrations of tolbutamide, and the time series of parent and metabolite concentrations were measured in slice and incubation medium. A mathematical model was built that modeled the uptake of tolbutamide and its metabolism in the liver slice. In addition, binding of tolbutamide to cellular constituents and partition over the water and lipid phase were accounted for by the model. Model analysis imposed sampling of parent compound in slice and of metabolites pooled from slice and medium. The model was calibrated to the data, fitting the intrinsic clearance, the parent compounds' free fraction in liver material, and a diffusion parameter describing medium-slice exchange of tolbutamide. In addition, to ensure a meaningful application of the theoretical model, slice viability parameters were monitored before and during the experiment. For the different incubations, the intrinsic clearance per unit of volume of slice ranged from 0.035 to 0.086 min(-1) when not correcting for slice viability and from 0.044 to 0.11 min(-1) when correcting for slice viability. The results were extrapolated to a PBPK model for tolbutamide in the rat. The value for the intrinsic clearance found by calibrating the PBPK model to previous in vivo data was 0.090 min(-1). This result suggests that liver slices are a valuable tool for predicting in vivo intrinsic clearance of low-extraction compounds.