An inhibitor of NADPH oxidase-4 attenuates established pulmonary fibrosis in a rodent disease model.

Idiopathic pulmonary fibrosis is a chronic progressive disease of increasing prevalence for which there is no effective therapy. Increased oxidative stress associated with an oxidant-antioxidant imbalance is thought to contribute to disease progression. NADPH oxidases (Nox) are a primary source of reactive oxygen species within the lung and cardiovascular system. We demonstrate that the Nox4 isoform is up-regulated in the lungs of patients with IPF and in a rodent model of bleomycin-induced pulmonary fibrosis and vascular remodeling. Nox4 is constitutively active, and therefore increased expression levels are likely to contribute to disease pathology. Using a small molecule Nox4/Nox1 inhibitor, we demonstrate that targeting Nox4 results in attenuation of an established fibrotic response, with reductions in gene transcripts for the extracellular matrix components collagen 1α1, collagen 3α1, and fibronectin and in principle pathway components associated with pulmonary fibrosis and hypoxia-mediated vascular remodeling: transforming growth factor (TGF)-ß1, plasminogen activator inhibitor-1, hypoxia-inducible factor, and Nox4. TGF-ß1 is a principle fibrotic mediator responsible for inducing up-regulation of profibrotic pathways associated with disease pathology. Using normal human lung-derived primary fibroblasts, we demonstrate that inhibition of Nox4 activity using a small molecule antagonist attenuates TGF-ß1-mediated up-regulation in expression of profibrotic genes and inhibits the differentiation of fibroblast to myofibroblasts, that is associated with up-regulation in smooth muscle actin and acquisition of a contractile phenotype. These studies support the view that targeting Nox4 may provide a therapeutic approach for attenuating pulmonary fibrosis.

Lung volume quantified by MRI reflects extracellular-matrix deposition and altered pulmonary function in bleomycin models of fibrosis: effects of SOM230.

Idiopathic pulmonary fibrosis is a progressive and lethal disease, characterized by loss of lung elasticity and alveolar surface area, secondary to alveolar epithelial cell injury, reactive inflammation, proliferation of fibroblasts, and deposition of extracellular matrix. The effects of oropharyngeal aspiration of bleomycin in Sprague-Dawley rats and C57BL/6 mice, as well as of intratracheal administration of ovalbumin to actively sensitized Brown Norway rats on total lung volume as assessed noninvasively by magnetic resonance imaging (MRI) were investigated here. Lung injury and volume were quantified by using nongated or respiratory-gated MRI acquisitions [ultrashort echo time (UTE) or gradient-echo techniques]. Lung function of bleomycin-challenged rats was examined additionally using a flexiVent system. Postmortem analyses included histology of collagen and hydroxyproline assays. Bleomycin induced an increase of MRI-assessed total lung volume, lung dry and wet weights, and hydroxyproline content as well as collagen amount. In bleomycin-treated rats, gated MRI showed an increased volume of the lung in the inspiratory and expiratory phases of the respiratory cycle and a temporary decrease of tidal volume. Decreased dynamic lung compliance was found in bleomycin-challenged rats. Bleomycin-induced increase of MRI-detected lung volume was consistent with tissue deposition during fibrotic processes resulting in decreased lung elasticity, whereas influences by edema or emphysema could be excluded. In ovalbumin-challenged rats, total lung volume quantified by MRI remained unchanged. The somatostatin analog, SOM230, was shown to have therapeutic effects on established bleomycin-induced fibrosis in rats. This work suggests MRI-detected total lung volume as readout for tissue-deposition in small rodent bleomycin models of pulmonary fibrosis.

Primary healthcare delivery models in African conflict-affected settings: a systematic review.

BACKGROUND: In conflict-affected settings, access to primary healthcare for displaced populations is constrained by multiple challenges. These include geographical, cultural, communication, logistical and financial barriers, as well as risks posed to health workers and the population by insecurity. Different models of care are used to provide primary healthcare to affected communities. However, there is a paucity of evidence on how these models are selected and implemented by organisations working in conflict and displacement-affected settings. Our aim was to explore the different primary healthcare delivery models used in conflict-affected settings to understand gaps in existing healthcare delivery models. METHODS: We conducted a systematic review using the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. The review protocol was registered with the International Prospective Register of Systematic Reviews. We searched six databases for manuscripts published from January 1992 to December 2020. Publications were included if they reported primary healthcare models of care in conflict-affected settings of Africa. Data was analyzed descriptively and thematically using tables, charts and text. RESULTS: Forty-eight primary research articles were included for analysis from which thirty-three were rated as "high" quality. The results showed that the models of care in place in these conflict-affected settings include health facility-based, community-based, mobile clinics, outreach and home visits. Primary healthcare for internally displaced persons and refugees is provided by a wide range of actors including national and international organisations. A range of services is offered, most commonly nutrition, mental health and sexual/reproductive health. Some organisations offer vertical (stand-alone) services, while others use an integrated service delivery model. Multiple cadres of healthcare workers provide services, frequently lay healthcare workers such as Community Health Workers. CONCLUSION: Understanding the different modalities of primary healthcare delivery in conflict-affected settings is important to identify existing practices and gaps in service delivery. Service delivery using community health workers in conflict-affected settings is a low-cost primary care delivery strategy that may help optimize contributions of existing personnel through task shifting.

Oseltamivir is protective for in-patient mortality in PCR confirmed influenza B and influenza A(H3N2) infections in an historic cohort of 1,048 patients hospitalised during the 2016-17 and 2017-18 influenza seasons.

Standard course oseltamivir 75mg two times daily for five days was associated with an 82% reduction of odds of in-patient death (OR 0.18 (0.07,0.51)) compared to no oseltamivir treatment (OR 1.0 Reference) in a final multivariable logistic regression model of a retrospective cohort of PCR confirmed influenza B and influenza A (H3N2) infected patients admitted to a large UK teaching hospital in influenza seasons 2016-17 and 2017-18. No difference of protective odds for standard course oseltamivir was observed between influenza B and influenza A (H3N2) nor between influenza seasons. These observations strongly support clinical guidelines for molecular testing for respiratory viruses on admission to hospital and prompt treatment of confirmed seasonal influenza B and A with oseltamivir 75mg twice daily for five days.

Humanitarian led community-based surveillance: case study in Ekondo-titi, Cameroon.

BACKGROUND: Community-based surveillance (CBS) has been used successfully in many situations to strengthen existing health systems as well as in humanitarian crises. The Anglophone crisis of Northwest Southwest Cameroon, led to burning of villages, targeting of health personnel and destruction of health facilities which, in combination with distrust for the government services led to a collapse of surveillance for outbreak prone diseases. METHODS: We evaluated the ability of the CBS system to identify suspected cases of outbreak prone diseases (OPD) as compared to the facility-based surveillance, evaluated the timeliness of the CBS system in identifying an OPD, reporting of OPD to District Health Service (DHS) and timeliness in outbreak response. The paper also assessed the collaboration with the DHS and contribution of the CBS system with regards to strengthening the overall surveillance of the health district and also determine the interventions undertaken to contain suspected/confirmed outbreaks. RESULTS: In total 9 alerts of suspected OPDs were generated by the CBS system as compared to 0 by the DHS, with 8 investigated, 5 responses and 3 confirmed outbreaks. Average time from first symptoms to alert generation by the CBS system was 7.3 days. Average time lag from alert generation from the CBS to the DHS was 0.3 days which was essentially within 24 h. There was extensive and synergistic collaboration with the DHS. DISCUSSION: CBS generated a higher number of alerts than traditional outbreak reported used in the region, and had timely investigations and if appropriate, responses. Careful selection of CHWs with strong community engagement led to the success of the project, and the use of the mobile health team in situ allowed for rapid responses to potential outbreaks, as well as for feedback to CHWs and communities. CBS was also well utilized for identification of other events, such as displacement and malnutrition. CONCLUSION: In conflict settings, CBS can help in outbreak identification as well as other events, and a mobile health team is crucial to the success of the CBS due to the ability to rapidly response to generated alerts. The mobile health team provided timely investigation of 8 of 9 alerts generated. Collaboration with existing DHS structures is important for systems strengthening in such settings.

Mobile clinics in conflict-affected communities of North West and South West regions of Cameroon: an alternative option for differentiated delivery service for internally displaced persons during COVID-19.

INTRODUCTION: The guidelines for differentiated service delivery (DSD) for HIV treatment became operational in Cameroon in 2017 with the Test and Treat national strategy elaborating services that can be decentralized and task shifted at community level, but with little to no guidelines for DSD in fragile and conflict-affected settings. Since 2016, more than 680,000 Cameroonians have been internally displaced due to the conflict in the North West and South West regions (NWSW). This conflict has impacted on the health system with numerous attacks on health facilities and staff, reducing access to health care for internally displaced persons. The outbreak of COVID-19 further reduced humanitarian responses for fear of spreading COVID-19. Mobile clinics were utilized as a model of care in piloting DSD for HIV in conflict-affected settings within the COVID-19 context. METHODS: The HIV DSD framework was used to evaluate a project that used mobile clinics in 05 divisions across the NWSW to provide primary health care to internally displaced persons in hard-to-reach areas. These mobile clinics were operated in the COVID-19 context and integrated HIV services in the benefit package. The mobile clinics mainstreamed HIV and COVID-19 sensitization during community mobilization, HIV consultations, HIV testing and referrals, and in some cases antiretroviral (ARV) dispensation. The project ran from March to October 2020. The results from the evaluation of this model of HIV care delivery were analysed in 06 of 08 mobile clinics. RESULTS: In 07 months, a total of 14,623 persons living in conflict-affected settings were sensitized on HIV, 1979 received HIV testing from which 122 were positive and 33 placed on ARVs. 28 loss-to-follow up people living with HIV were relinked to treatment and 209 consultations for persons living with HIV were conducted. Despite the good collaboration at regional and field level, there was distrust by ARV centers for humanitarian organizations. CONCLUSION: Mobile clinics are a model of care which could be leveraged in fragile and conflict-affected settings as an alternative model of care for HIV DSD to ensure continuum of HIV care and treatment. However this should be integrated within the benefit package of primary health care services offered by mobile clinics.

Effects of the fibroblast activation protein inhibitor, PT100, in a murine model of pulmonary fibrosis.

Bleomycin (BLM) induced lung injury is detectable in C57BL/6 mice using magnetic resonance imaging (MRI). We investigated the effects of the fibroblast activation protein (FAP) inhibitor, PT100, in this model. BLM (0.5mg/kg/day) was administered on days -7, -6, -5, -2, -1, 0 in the nostrils of male mice. PT100 (40µg/mouse) or vehicle (0.9%NaCl) was dosed per os twice daily from day 1-14. MRI was performed before BLM and at days 0, 7 and 14. After the last MRI acquisition, animals were euthanised and the lungs harvested for histological and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. As evidenced longitudinally by MRI, the BLM-elicited lesions in the lungs of vehicle-treated mice progressed over time. In contrast, responses elicited by BLM did not progress in animals receiving PT100. Histology demonstrated significant less fibrosis in PT100- than in vehicle-treated, BLM-challenged mice. Significant correlation (R=0.91, P<0.001, N=24) was found between the volumes of BLM-induced lesions detected in vivo by MRI and the collagen content determined histologically (picrosirius staining). FAP was overexpressed in the lungs of BLM-challenged mice. Upon PT100 treatment, FAP expression was reduced. Significant differences in the MMP-12, MIP-1α, and MCP-3 mRNA expression levels in the lungs of PT100- compared to vehicle-treated mice were also revealed by qRT-PCR. The IBA-1 level determined histologically was higher in the lungs of PT100- compared to vehicle-treated mice. Taken together, these observations suggest that treatment with PT100 in this murine model of pulmonary fibrosis had an anti-fibro-proliferative effect and increased macrophage activation.

Induction of regulatory T cells by leflunomide in a murine model of contact allergen sensitivity.

Allergic contact dermatitis and contact hypersensitivity (CHS) are characterized by allergen-specific activation of CD8+ and CD4+ T cells and the production of cytokines resulting in an inflammatory response and tissue damage. We show here that the immunosuppressive compound leflunomide (N-[4-trifluoro-methylphenyl]-5-methylisoxazol-4 carboxamide, HWA 486) (LF) inhibited the contact allergic response induced in mice by epicutaneous application of the haptens dinitrofluorobenzene (DNFB) and oxazolone. The extent of ear swelling remained significantly reduced following repeated challenge with DNFB for up to 18 weeks. LF and DNFB had to be applied simultaneously for inhibition to occur. The loss of CHS responses was shown to be antigen-specific. Adoptive transfer of leukocytes from LF-treated mice into naïve mice resulted in a loss of CHS responsiveness. Transfer of both CD4+ and CD8+ cells was required for maximal loss of CHS responses, with CD8+ cells playing a major role. Significantly enhanced levels of IL-10 mRNA were detected in CD8+ T cells, but not in CD4+ T cells, following LF treatment of mice. LF also suppressed CHS responses in mice previously sensitized and challenged with hapten, when administered together with the hapten. Our data suggest that LF induces a long-lived tolerance in mice by inducing CD8+ and CD4+ regulatory T cells.

Reduced interleukin-8 response to Streptococcus pneumoniae by alveolar macrophages from adults with HIV/AIDS.

BACKGROUND: HIV-infected adults are highly susceptible to pneumococcal disease. OBJECTIVE: To examine if alveolar macrophages from HIV-infected subjects exhibited a failure of cytokine production in response to Streptococcus pneumoniae in vitro. DESIGN: Case-control comparison of alveolar macrophages from 11 HIV-infected and 13 non-infected adults. METHODS: Type 1 opsonized S. pneumoniae were used to challenge the alveolar macrophages in vitro. Cell supernatant fluid was collected from unstimulated cells, and cells challenged with bacteria for 0, 6, 12 and 24 h. Cytokine production (interleukins 1beta, 6 and 8) was measured in all fluids using an enzyme-linked immunosorbent assay. RESULTS: All the cytokines tested increased over time in both HIV-infected and uninfected subjects. Interleukin-8 release was significantly lower in HIV-infected than in non-HIV-infected subjects (P = 0.02). CONCLUSION: Reduced interleukin-8 production may result in decreased neutrophil recruitment, and hence increased susceptibility to pneumococcal infection in HIV-infected subjects.

A translational preclinical model of interstitial pulmonary fibrosis and pulmonary hypertension: mechanistic pathways driving disease pathophysiology.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease, in which a decline in patient prognosis is frequently associated with the onset of pulmonary hypertension (PH). Animal models exhibiting principle pathophysiological features of IPF and PH could provide greater insight into mechanistic pathways underlying disease progression and a means for evaluating novel therapeutic approaches for intervention. Here, we describe an in vivo disease model, in which animals develop progressive interstitial pulmonary fibrosis and associated PH, as defined by the presence of fibrotic foci adjacent to areas of alveolar injury and remodeling of the pulmonary vasculature. Associated changes in physiological parameters included a decline in lung function and increase in mean pulmonary arterial pressure (mPAP) >25 mmHg. The early fibrotic pathology is associated with a profibrogenic microenvironment, elevated levels of the matrix metalloproteases, MMP-2, MMP-7, and MMP-12, TIMP-1, the chemoattractant and mitogen, PDGF-ß, and the chemokines CCL2 and CXCL12, that are associated with the recruitment of macrophages, mast cells, and fibrocytes. Principle mechanistic pathways associated with disease pathogenesis are upregulated in the lungs and pulmonary arteries, with sustained increases in gene transcripts for the profibrotic mediator TGF-ß1 and components of the TGF-ß signaling pathway; PAI-1, Nox-4, and HIF-1α. Therapeutic treatment with the ALK-5/TGF-ß RI inhibitor SB-525334 reversed established pulmonary fibrosis and associated vascular remodeling, leading to normalization in clinically translatable physiological parameters including lung function and hemodynamic measurements of mPAP. These studies highlight the application of this model in validating potential approaches for targeting common mechanistic pathways driving disease pathogenesis.

Administration of bleomycin via the oropharyngeal aspiration route leads to sustained lung fibrosis in mice and rats as quantified by UTE-MRI and histology.

Pulmonary fibrosis can be experimentally induced in small rodents by bleomycin. The antibiotic is usually administered via the intratracheal or intranasal routes. In the present study, we investigated the oropharyngeal aspiration of bleomycin as an alternative route for the induction of lung fibrosis in rats and mice. The development of lung injury was followed in vivo by ultrashort echo time magnetic resonance imaging (UTE-MRI) and by post-mortem analyses (histology of collagen, hydroxyproline determination, and qRT-PCR). In C57BL/6 mice, oropharyngeal aspiration of bleomycin led to more prominent lung fibrosis as compared to intranasal administration. Consequently, the oropharyngeal aspiration route allowed a dose reduction of bleomycin and, therewith, a model refinement. Moreover, the distribution of collagen after oropharyngeal aspiration of bleomycin was more homogenous than after intranasal administration: for the oropharyngeal aspiration route, fibrotic areas appeared all over the lung lobes, while for the intranasal route fibrotic lesions appeared mainly around the largest superior airways. Thus, oropharyngeal aspiration of bleomycin induced morphological changes that were more comparable to the human disease than the intranasal administration route did. Oropharyngeal aspiration of bleomycin led to a homogeneous fibrotic injury also in rat lungs. The present data suggest oropharyngeal aspiration of bleomycin as a less invasive means to induce homogeneous and sustained fibrosis in the lungs of mice and rats.

The alveolar microenvironment of patients infected with human immunodeficiency virus does not modify alveolar macrophage interactions with Streptococcus pneumoniae.

We tested the hypothesis that HIV infection results in activation of alveolar macrophages and that this might be associated with impaired defense against pneumococcus. We compared alveolar macrophages and lymphocytes in 131 bronchoalveolar lavage samples from HIV-infected and healthy controls using inflammatory gene microarrays, flow cytometry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA) to determine the pattern of macrophage activation associated with HIV infection and the effect of this activation on defense against pneumococcus. We used gamma interferon (IFN-γ) priming to mimic the cellular milieu in HIV-infected lungs. InnateDB and BioLayout 3D were used to analyze the interactions of the upregulated genes. Alveolar macrophages from HIV-infected adults showed increased gene expression and cytokine production in a classical pattern. Bronchoalveolar lavage from HIV-infected subjects showed excess CD8(+) lymphocytes with activated phenotype. Toll-like receptor 4 (TLR4) expression was increased in macrophages from HIV-infected subjects, but function was similar between the groups; lung lavage fluid did not inhibit TLR function in transfected HeLa cells. Alveolar macrophages from HIV-infected subjects showed normal binding and internalization of opsonized pneumococci, with or without IFN-γ priming. Alveolar macrophages from HIV-infected subjects showed classical activation compared to that of healthy controls, but this does not alter macrophage interactions with pneumococci.

Inhaled delivery of 23-valent pneumococcal polysaccharide vaccine does not result in enhanced pulmonary mucosal immunoglobulin responses.

We compared the effect of intramuscular vs. inhaled 23-valent pneumococcal capsular polysaccharide vaccine (23-PPV) on pulmonary mucosal immunoglobulin levels. Bronchoalveolar lavage (BAL) and serum were collected from 33 adults before and 1 month after injected (n=16) or inhaled (n=17) 23-PPV. Levels of pneumococcal capsule-specific IgG and IgA to types 1, 9V and 14 were measured in each sample. Injected 23-PPV produced a significant increase in types 1, 9V and 14 capsule-specific IgG and type 1 IgA in both serum and BAL (type 1 geometric mean BAL IgG 9.8 ng/ml post-vaccine vs. 5 ng/ml pre-vaccine, p=0.01; type 9V geo mean 5.6 ng/ml vs. 2.7 ng/ml, p=0.001; type 14 geo mean 23.6 ng/ml vs. 6.2 ng/ml, p=0.02). Inhaled vaccine produced no response in either BAL or serum.

Reversal of established CD4+ type 2 T helper-mediated allergic airway inflammation and eosinophilia by therapeutic treatment with DNA vaccines limits progression towards chronic inflammation and remodelling.

Immunostimulatory DNA-based vaccines can prevent the induction of CD4(+) type 2 T helper (Th2) cell-mediated airway inflammation in experimental models, when administered before or at the time of allergen exposure. Here we demonstrate their efficacy in limiting the progression of an established response to chronic pulmonary inflammation and airway remodelling on subsequent allergen challenge. Mice exhibiting Th2-mediated airway inflammation induced following sensitization and challenge with group 1 allergen derived from Dermatophagoides pteronyssinus group species (Der p 1), a major allergen of house dust mite, were treated with pDNA vaccines. Their airways were rechallenged and the extent of inflammation assessed. In plasma DNA (pDNA)-vaccinated mice, infiltration of inflammatory cells, goblet cell hyperplasia and mucus production were reduced and subepithelial fibrosis attenuated. The reduction in eosinophil numbers correlated with a fall in levels of the profibrotic mediator transforming growth factor (TGF)-beta1 in bronchoalveolar lavage (BAL) and lung tissue. In addition to lung epithelial cells and resident alveolar macrophages, infiltrating eosinophils, the principle inflammatory cells recruited following allergen exposure, were a major source of TGF-beta1. Protection, conferred irrespective of the specificity of the pDNA construct, did not correlate with a sustained increase in systemic interferon (IFN)-gamma production but in a reduction in levels of the Th2 pro-inflammatory cytokines. Notably, there was a reduction in levels of interleukin (IL)-5 and IL-13 produced by systemic Der p 1 reactive CD4(+) Th2 cells on in vitro stimulation as well as in IL-4 and IL-5 levels in BAL fluid. These data suggest that suppression of CD4(+) Th2-mediated inflammation and eosinophilia were sufficient to attenuate progression towards airway remodelling. Immunostimulatory DNA may therefore have a therapeutic application in treatment of established allergic asthma in patients.

Kinetics and mode of peptide delivery via the respiratory mucosa determine the outcome of activation versus TH2 immunity in allergic inflammation of the airways.

BACKGROUND: Specific immunotherapy involving systemic injection of allergen, though highly effective, can cause severe side effects due to IgE-mediated activation of effector cells. Allergen-derived peptides might provide a safer alternative. We have investigated the use of mucosally delivered peptide to induce CD4(+) T(H)2 cell tolerance and thus protect against allergen-induced airway inflammation. OBJECTIVE: The purpose of this study was to investigate whether intranasal administration of an allergen-derived peptide, either alone or adsorbed to chitosan, can prevent the induction of T(H)2-mediated pulmonary inflammation after sensitization and challenge of the airways with allergen. METHODS: Mice were given (intranasally) a peptide containing an immunodominant epitope of the Dermatophagoides pteronyssinus (Der p) 1 allergen, either as soluble antigen or adsorbed to chitosan, before sensitization and allergen challenge. Pulmonary inflammation, antigen-specific CD4(+) T-cell responses, and antibody levels in sera were then determined. RESULTS: Mice given peptide adsorbed to chitosan had significant reductions in airway eosinophilia, which correlated with reduced levels of IL-4 and IL-5 in the bronchoalveolar lavage fluid. There was decreased recruitment of activated CD4(+) T cells into the airways after allergen challenge, which correlated with a loss of Der p 1-specific T-cell cytokine responses in the periphery and the localized production of IL-10 by antigen-specific T cells in bronchial lymph nodes. Induction of peripheral T-cell tolerance was preceded by transient T-cell activation and IFN-gamma production. CONCLUSION: Our data demonstrate that suppression of airway inflammation by intranasal administration of peptide antigen adsorbed to chitosan is initiated by transient T-cell activation and maintained by the production of IL-10 by antigen-specific T cells in the draining lymph nodes.

Suppression of allergen reactive Th2 mediated responses and pulmonary eosinophilia by intranasal administration of an immunodominant peptide is linked to IL-10 production.

The potential to induce systemic tolerance following exposure of the airway mucosa to soluble antigen, may be applied therapeutically for the treatment of allergic disease. Since the use of allergen can trigger IgE mediated inflammation, we investigated whether mucosal delivery of a peptide, containing an immunodominant epitope of the Der p1 allergen of house dust mite, can lead to CD4(+) Th2 cell tolerance and thus protect against airway inflammatory responses to inhalant allergen. The administration of microencapsulated peptide to the nasal mucosa of mice, protected against airway inflammation, with significant reductions in eosinophil infiltration into the airways following allergen challenge. Der p1 specific antibody levels in sera were not modulated. Allergen reactive CD4(+) T cells expressed a tolerized phenotype, with reduction in levels of the cytokines, IL-5, IL-13 and IFN-gamma although IL-10 levels were increased. The mucosal administration of a peptide containing an immunodominant region of an allergen can protect against the induction of systemic and local inflammatory responses to allergen challenge.

Different profile of CD8+ effector T cells induced in Der p 1-allergic and naïve mice by DNA vaccination.

DNA vaccination holds great promise in both prophylactic and therapeutic vaccines. Recent evidence suggests that DNA vaccines could be powerful therapies countering Th2-mediated disorders such as allergies. Here, we studied the allergen-specific CD4+ and CD8+ T cell populations induced following immunization of allergic and non-allergic mice with DNA vaccine vectors encoding discrete epitopes of the house dust mite (HDM) Dermatophagoides pteronyssinus group I (Der p 1) allergen. Specifically, mice were sensitized to Der p 1 and exhibited a strong Th2/allergic response. Sensitized and non-allergic mice were then compared for their responses to DNA immunization. Using Elispot analysis, we demonstrate that allergic/vaccinated mice generate a mixed Th1/Th2 response against the allergen with high numbers of allergen-specific CD4+ T cells secreting IFN-gamma or IL-4, whereas in non-allergic/vaccinated mice a polarized Th1 response was dominant. Allergen-specific CD8+ T cells secreting IFN-gamma were induced at equal frequencies in both allergic and non-allergic mice. However, the CD8+ T cells from allergic mice were markedly deficient in their cytotoxic potential when compared to their counterparts in non-allergic mice. These results indicate that during an ongoing Th2 response, DNA vaccination leads to the generation of a distinct population of non-cytotoxic/regulatory CD8+ T cells.

Scavenger receptor-specific allergen delivery elicits IFN-gamma-dominated immunity and directs established TH2-dominated responses to a nonallergic phenotype.

BACKGROUND: Immunotherapeutic approaches to allergy consist of reliably changing allergen-specific T(H)2 immunity associated with secretion of IL-4, IL-5, and IL-13, along with IgE antibodies in atopic individuals to T(H)1 immunity. Our earlier data show that targeting of protein antigens to antigen-presenting cells (APCs), such as macrophages, by means of scavenger receptors (SRs) results in a pronounced T(H)1 immunity. Here we demonstrate a novel experimental approach for the conversion of T(H)2 immunity to T(H)1 immunity by using SR delivery of allergens. OBJECTIVES: We sought to show that targeting of allergens by means of SRs to APCs triggers T(H)1 immunity and that an established T(H)2 immunity to the Der p 1-immunodominant peptide 111-139 (p1, 111-139) can be modulated to a nonallergic T(H)1 phenotype. METHODS: Analysis of the T cell-derived cytokines IL-4, IL-5, IL-13, and IFN-gamma in response to p1, 111-139 in C57BL/6 mice 7 to 42 days after immunization, measurement of specific antibody responses, eosinophilic infiltrate, and skin hypersensitivity in response to allergen challenge constitute the parameters of in vivo immunity. RESULTS: We show that p1, 111-139, when delivered to APCs by means of SR, elicits a T(H)1-dominant immunity. If it is delivered to APCs either after chemical coupling to SR ligands or by means of mere coadsorption on alum in the presence of an SR ligand, the established T(H)2 immunity can be modified to T(H)1 immunity. CONCLUSIONS: SR-mediated delivery of allergens has immunotherapeutic potential that may be usable in atopic individuals.

CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells.

We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (approximately 98% CCR3(+)). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19(+) (approximately 40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.

Expression of the developmental Sonic hedgehog (Shh) signalling pathway is up-regulated in chronic lung fibrosis and the Shh receptor patched 1 is present in circulating T lymphocytes.

During pulmonary development, Sonic hedgehog (Shh) and transforming growth factor beta1 (TGF-beta1) signalling both contribute to branching morphogenesis. In interstitial lung disease, the complex alveolar structure of the lung is disrupted and remodelled, which leads to fibrosis, loss of respiratory surface, morbidity, and mortality. It is well documented that TGF-beta1 is involved in fibrosis. However, little is known about Shh signalling in damaged epithelia. This study examined whether or not components of the Shh signalling pathway, as well as TGF-beta1, are expressed in human fibrotic lung disease (cryptogenic fibrosing alveolitis and bronchiectasis) and in murine experimental models of fibrotic and non-fibrotic chronic pulmonary inflammation. Using immunohistochemistry, it was observed that Shh, like TGF-beta1, is up-regulated in epithelial cells at sites of fibrotic disease but not non-fibrotic inflammation. The Shh receptor patched was detected in infiltrating mononuclear cells and alveolar macrophages, as well as normal resting peripheral blood T lymphocytes. Neither Shh nor patched is expressed by hyperproliferative goblet cells in inflammatory epithelium. This study demonstrates that patched is present in human peripheral CD4 and CD8 lymphocytes at both protein and mRNA levels. Taken together, these results suggest that components of the highly conserved Shh signalling pathway may play a role in the remodelling of damaged pulmonary epithelium and that damaged epithelium and cells of the immune system may communicate via this pathway.