Subcutaneous immunotherapy suppresses Th2 inflammation and induces neutralizing antibodies, but sublingual immunotherapy suppresses airway hyperresponsiveness in grass pollen mouse models for allergic asthma.

BACKGROUND: Both subcutaneous and sublingual allergen immunotherapy (SCIT and SLIT) have been shown to effectively suppress allergic manifestations upon allergen exposure, providing long-term relief from symptoms in allergic disorders including allergic asthma. Clinical studies directly comparing SCIT and SLIT report a different kinetics and magnitude of immunological changes induced during treatment. Comparative studies into the mechanisms underlying immune suppression in SCIT and SLIT are lacking. OBJECTIVE: We aimed to establish an experimental model for grass pollen (GP) SCIT and SLIT that would allow a head-to-head comparison of the two treatments. METHODS: BALB/c mice were sensitized with GP extract, followed by SCIT and SLIT treatments with various GP dosages. Subsequently, we challenged mice with GP and measured airway responsiveness (AHR), GP-specific immunoglobulins, ear swelling tests (EST), eosinophilic inflammation in bronchoalveolar lavage fluid (BALF), and T cell cytokine release after restimulation of lung cells (IL-5, IL-10, and IL-13). RESULTS: We find that SLIT treatment was able to suppress allergen-induced AHR, while allergic inflammation was not effectively suppressed even at the highest GP dose in this model. In contrast, SCIT treatment induced higher levels of GP-specific IgG1, while SLIT was superior in inducing a GP-specific IgG2a response, which was associated with increased Th1 activity in lung tissue after SLIT, but not SCIT treatment. Interestingly, SCIT was able to suppress Th2-type cytokine production in lung cell suspensions, while SLIT failed to do so. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, GP-SCIT suppresses Th2 inflammation and induced neutralizing antibodies, while GP-SLIT suppresses the clinically relevant lung function parameters in an asthma mouse model, indicating that the two application routes depend on partially divergent mechanisms of tolerance induction. Interestingly, these data mirror observations in clinical studies, underscoring the translational value of these mouse models.

Subcutaneous immunotherapy with purified Der p1 and 2 suppresses type 2 immunity in a murine asthma model.

BACKGROUND: Allergen-specific immunotherapy can induce long-term suppression of allergic symptoms, reduce medication use, and prevent exacerbations of allergic rhinitis and asthma. Current treatment is based on crude allergen extracts, which contain immunostimulatory components such as ß-glucans, chitins, and endotoxin. Use of purified or recombinant allergens might therefore increase efficacy of treatment. AIMS: Here, we test application of purified natural group 1 and 2 allergens from Dermatophagoides pteronyssinus (Der p) for subcutaneous immunotherapy (SCIT) treatment in a house dust mite (HDM)-driven mouse model of allergic asthma. MATERIALS AND METHODS: HDM-sensitized mice received SCIT with crude HDM extract, a mixture of purified Der p1 and 2 (DerP1/2), or placebo. Upon challenges, we measured specific immunoglobulin responses, allergen-induced ear swelling response (ESR), airway hyperresponsiveness (AHR), and inflammation in bronchoalveolar lavage fluid (BAL) and lung tissue. RESULTS: ESR measurement shows suppression of early allergic response in HDM-SCIT- and DerP1/2-SCIT-treated mice. Both HDM-SCIT and DerP1/2-SCIT are able to suppress AHR and eosinophilic inflammation. In contrast, only DerP1/2-SCIT is able to significantly suppress type 2 cytokines in lung tissue and BAL fluid. Moreover, DerP1/2-SCIT treatment is uniquely able suppress CCL20 and showed a trend toward suppression of IL-33, CCL17 and eotaxin levels in lung tissue. DISCUSSION: Taken together, these data show that purified DerP1/2-SCIT is able to not only suppress AHR and inflammation, but also has superior activity toward suppression of Th2 cells and HDM-induced activation of lung structural cells including airway epithelium. CONCLUSIONS: We postulate that treatment with purified natural major allergens derived from HDM will likely increase clinical efficacy of SCIT.

Microsphere size influences the foreign body reaction.

Biodegradable poly-(DL-lactide-co-glycolide) (PLGA) microspheres (MSP) are attractive candidate vehicles for site-specific or systemic sustained release of therapeutic compounds. This release may be altered by the host's foreign body reaction (FBR), which is dependent on the characteristics of the implant, e.g. chemistry, shape or size. In this study, we focused on the characterisation of the influence of MSP size on the FBR. To this end we injected monodisperse MSP of defined size (small 5.8 µm, coefficient of variance (CV) 14 % and large 29.8 µm, CV 4 %) and polydisperse MSP (average diameter 34.1 µm, CV 51 %) under the skin of rats. MSP implants were retrieved at day 7, 14 and 28 after transplantation. The FBR was studied in terms of macrophage infiltration, implant encapsulation, vascularisation and extracellular matrix deposition. Although PLGA MSP of all different sizes demonstrated excellent in vitro and in vivo biocompatibility, significant differences were found in the characteristics of the FBR. Small MSP were phagocytosed, while large MSP were not. Large MSP occasionally elicited giant cell formation, which was not observed after implantation of small MSP. Cellular and macrophage influx and collagen deposition were increased in small MSP implants compared to large MSP. We conclude that the MSP size influences the FBR and thus might influence clinical outcome when using MSP as a drug delivery device. We propose that a rational choice of MSP size can aid in optimising the therapeutic efficacy of microsphere-based therapies in vivo.

Hand eczema­prognosis and consequences: a 7-year follow-up study.

BACKGROUND: Hand eczema is recognized as a long-lasting disease with personal and societal repercussions. Long-term studies are required to generate information on factors contributing to a poor outcome. OBJECTIVES: The aims of this 7-year follow-up study were to evaluate the clinical course of patients with hand eczema, the occupational consequences and to identify risk factors associated with a poor prognosis. MATERIALS AND METHODS: In all, 536 patients with hand eczema participated and were examined by a dermatologist. The clinical severity was assessed at baseline and 7 years later using a self-administrated photographic guide. Additional information was obtained from a questionnaire. RESULTS: Based on the photographic guide, 73% experienced a clinical improvement. Notably, 20% had moderate to very severe hand eczema at follow-up. Severe hand eczema or frequent eruptions at baseline and eczema in other body locations during the follow-up period were risk factors of a poor prognosis. The same factors, as well as being a woman, were associated with occupational consequences and low health-related quality of life. Of those with persistent hand eczema only 40% had visited a dermatologist during the follow-up period and 7% had oral treatment. CONCLUSIONS: The disease had improved 7 years later; nevertheless, many patients continued to have considerable symptoms. Patients with a greater risk of a poor outcome are characterized by frequent eruptions, severe hand eczema and more widespread eczema. It should be questioned if more aggressive therapy and closer medical follow-up would be beneficial.

Spatiotemporal expression of chemokines and chemokine receptors in experimental anti-myeloperoxidase antibody-mediated glomerulonephritis.

Myeloperoxidase (MPO)-anti-neutrophil cytoplasmic autoantibody (ANCA)-associated necrotizing crescentic glomerulonephritis (NCGN) is characterized by abundant leucocyte infiltration. Chemokines are chemotactic cytokines involved in receptor-mediated recruitment of leucocytes. Our objective was to analyse spatiotemporal gene expression of chemokines and chemokine receptors in anti-MPO-mediated NCGN, to find potential targets for intervening with leucocyte influx. NCGN was induced in mice by co-administration of anti-MPO immunoglobulin (Ig)G and lipopolysaccharide. mRNA expression levels of chemokines and chemokine receptors were analysed in whole kidney lysates as well as in laser microdissected glomeruli and tubulo-interstitial tissue 1 and 7 day(s) after NCGN induction. Several chemokines and chemokine receptors were induced or up-regulated in anti-MPO-mediated NCGN, both on day 1 (chemokines CCL3, 5; CXCL2, 5, 13; receptor CXCR2) and on day 7 (chemokines CCL2, 5, 7, 8, 17, 20; CXCL1, 2, 5, 10; CX(3)CL1; receptors CCR2, 8; CX(3)CR1). The expression levels of most chemokines and receptors were higher in glomeruli than in the tubulo-interstitium. Because of the temporal induction of CXCR2 on day 1, we hypothesized CXCR2 as a potential target for treatment in anti-MPO-induced NCGN. Inhibition of CXCR2 using a goat-anti-CXCR2 serum prior to NCGN induction increased glomerular neutrophil influx but did not affect crescent formation and albuminuria. In conclusion, expression levels of various chemokines and chemokine receptors were increased in anti-MPO NCGN, and expressed particularly in glomeruli. These chemokines and receptors may serve as potential targets for treatment. Inhibition of a single target, CXCR2, did not attenuate anti-MPO NCGN. Combinatorial interventions may be necessary to avoid redundancy.

Perivascular scaffolds loaded with adipose tissue-derived stromal cells attenuate development and progression of abdominal aortic aneurysm in rats.

Abdominal aortic aneurysm (AAA) is the pathological dilation and weakening of the abdominal aorta wall. Inflammation, degradation of the extracellular matrix (ECM) and loss of smooth muscle cells and skewing of their function are pivotal in AAA pathology. We developed a recombinant collagen-based patch (RCP) to provide structural integrity and deliver Adipose tissue-Derived Stromal Cells (ASC) for repair. Patches supported adhesion and function as well as proliferation of ASC. ASC-loaded RCPs or bare patches, applied around the aorta after AAA induction in rats, both maintained structural integrity of the aortic wall at time of explant (2w). However, wall thinning, accompanied by loss of elastin fibers and loss of medial SMC, was only attenuated in ASC-loaded RCP-treated AAA rats. Interestingly, this coincided with migration of ASC into the media and a reduced influx of macrophages. We hypothesize that the medially-migrated ASC dampened or skewed the adverse innate immunity and thus suppressed SMC apoptosis, phenotypic skewing and elastin degradation. We conclude that the periadventitial delivery of ASC with RCP suppresses development and progression of AAA, which is has an expected future clinical benefit in combination with an appropriate early screening program of patients at risk for aneurysms. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2494-2506, 2018.

Chromosome location, genomic organization of the porcine COL10A1 gene and model structure of the NC1 domain.

The porcine COL10A1 gene, encoding the alpha1(X) chain of type X collagen, has been sequenced. The gene structure is evolutionarily conserved, consisting of three exons and two introns spanning 7100 bp. Linkage mapping localized the gene to chromosome 1, which is in agreement with human-pig homology maps. Furthermore, protein structure comparison of the functionally important carboxyl domain between species revealed that amino acid changes were few and mainly situated in loop regions.

Factors determining prolongation of rat heart allograft survival by perioperative injection of donor spleen cells.

Previously, we have shown that a perioperative injection of donor mononuclear cells in combination with cyclosporine treatment on day 2 after transplantation prolongs heart allograft survival in rats. In this study we determined whether the efficacy of this treatment was influenced by the same factors that have been shown to affect the efficacy of preoperative administration of donor cells. The effect of the following factors were investigated: dosage and repetition of the donor cell injection, viability of the donor cells, immunosuppressive drugs other than cyclosporine, and the rat strain combination. We found that there was an optimal dosage of donor cells; dosages of 4 x 10(7) or 1 x 10(8) cells gave the best heart graft survival. Repetition of the donor cell injection was not useful. Reducing viability of the cells by irradiation did not abrogate the prolonged graft survival, whereas killing of the cells did. Methylprednisolone, azathioprine, or cyclophosphamide in combination with the perioperative donor cell injection did not prolong heart graft survival in comparison with treatment with the drug only. The efficacy of this treatment was also influenced by the rat strain combination. In some combinations, this treatment prolonged graft survival, whereas in others an effect was absent or undetectable. Importantly, this treatment never adversely affected graft survival. We conclude that the efficacy of this treatment is influenced by similar factors as found for preoperative treatment with donor cells. A major advantage of this treatment over preoperative blood transfusions is that it avoids sensitization.

The combi-effect--reduced rejection of the heart by combined transplantation with the lung or spleen.

The term combi-effect was introduced to describe the phenomenon of a reduction in rejection of heart grafts after combined transplantation with the lung. In this study in rats we investigated whether the combi-effect was an immunological process and whether it could also be induced by combined transplantation of the heart with the spleen or with a lymphocyte-depleted spleen. Heart and spleen grafts were transplanted into the abdomen; left lungs were transplanted into the thorax of recipient rats. To deplete spleens of their lymphocytes, prospective donor rats were irradiated. Cyclosporine was injected once, on day 2 after transplantation. All heart allografts transplanted alone and treated with cyclosporine were rejected acutely (median survival time [MST] of 14.5 days). In contrast, after combined transplantation of a donor lung or spleen with the heart, almost all heart grafts survived indefinitely. Transplantation of a syngeneic lung or third-party spleen had little effect on heart graft survival (MST of 22.5 days and 26.5 days, respectively). Without cyclosporine treatment, combined transplantation with a donor lung or spleen hardly prolonged heart graft survival. Transplantation of a lymphocyte-depleted spleen with the heart induced a combi-effect in cyclosporine-treated rats that was somewhat weaker: only two of six hearts survived indefinitely. We conclude that in the combi-effect an immunological mechanism reduces rejection of the heart. This mechanism is probably generated by the lymphoid tissue (bronchus-associated lymphoid tissue in lung and white pulp in spleen) in the combined transplant.

Expression of class II major histocompatibility complex antigens by bronchial epithelium in rat lung allografts.

Variations in expression of class II major histocompatibility complex antigens on bronchial epithelial cells and vascular endothelium were investigated in normal rat lungs and allografted lungs during acute rejection and after cyclosporine (CsA) treatment. BN (RT1n) left lungs were transplanted into LEW (RT1l) recipients. Lungs were excised during acute rejection in untreated rats on postoperative days 1 through 5, and after CsA treatment (25 mg/kg on days 2 and 3) on days 5 and 100. Cryostat sections were examined for class II antigen expression with an immunoperoxidase technique, using various monoclonal antibodies. In the normal lung, class II antigens were not expressed by epithelial or endothelial cells. In the allografts, induction of class II antigens closely correlated with the rejection process: on day 2, the ciliated bronchial epithelium was locally positive; it became uniformly positive with increasing cellular peribronchial infiltration on days 3 and 4. CsA treatment prevented class II antigen expression to a certain extent, leaving the bronchial epithelium weakly positive at 100 days. Endothelial cells were invariably negative for class II antigens in all allografted lungs. The class II antigens expressed on the bronchial epithelial cells were of graft origin, except for recipient-type class II molecules found on the ciliated surface in CsA-treated animals. We conclude that expression of class II antigens by bronchial epithelium is the result of a bronchus-directed rejection process, and hypothesize that such a rejection process may have caused bronchiolitis obliterans in several of the patients with combined heart-lung transplants. Important is the observation that class II molecules can be present on the membranes of cells that do not themselves produce these antigens.

The function of surfactant is impaired during the reimplantation response in rat lung transplants.

In this study we investigated the surfactant function in rat lung transplants at the peak of the reimplantation response in experimental groups with increasing warm ischemic times of the lung transplant. The left and right lungs in five groups of rats were assessed 24 hours after left lung transplantation: rats receiving transplants with lung graft ischemic times of 60 (n = 4), 90 (n = 5), and 120 (n = 5) minutes, donor rats with 120 minutes lung ischemia (n = 5) and normal (nonoperated) rats (n = 6). The reimplantation response was assessed by the ventilation score on chest roentgenograms, measurement of the static lung compliance, and the (serum) protein concentration in the bronchoalveolar lavage fluid. Surfactant in the bronchoalveolar lavage fluid was assessed by measuring the amount and the composition of surfactant phospholipids and the in vitro surfactant function in a pulsating bubble surfactometer. We found that longer ischemic times caused a more severe reimplantation response in the left lung grafts. Although the ventilation scores were equally low in the 60-, 90-, and 120-minute ischemia groups, the lung compliances decreased and the (serum) protein concentrations increased stepwise in correlation with longer ischemic times. The amount of surfactant phospholipids during the reimplantation response was not changed, but the percentage phosphatidyl choline decreased progressively in parallel with the severity of the reimplantation response. Finally, the in vitro function of surfactant from the lung transplants decreased in parallel with the prolongation of the ischemic time, whereas the function of surfactant from donor lungs with 120 minutes of ischemia and from native right lungs was not changed. We conclude that the surfactant function is impaired during the reimplantation response as a result of a high concentration of inhibiting serum proteins and a low percentage of phosphatidyl choline.

Evaluation of the inheritance of the complex vertebral malformation syndrome by breeding studies.

To investigate the congenital complex vertebral malformation syndrome (CVM) in Holstein calves, two breeding studies were performed including 262 and 363 cows, respectively. Cows were selected from the Danish Cattle Database based on pedigree and insemination records. Selected cows were progeny of sires with an established heterozygous CVM genotype and pregnant after insemination with semen from another sire with heterozygous CVM genotype. Following calving the breeders should state, if the calf was normal and was requested to submit dead calves for necropsy. In both studies, significantly fewer CVM affected calves than expected were obtained; a finding probably reflecting extensive intrauterine mortality in CVM affected foetuses. The findings illustrate increased intrauterine mortality as a major potential bias in observational studies of inherited disorders.