Lung tissue extracellular matrix-derived hydrogels protect against radiation-induced lung injury by suppressing epithelial–mesenchymal transition

This study aimed to examine whether lung tissue extracellular matrix (ECM) hydrogels have protective effects on radiation-induced lung injury (RILI). The cytocompatibility and histocompatibility were tested for the obtained ECM-derived hydrogel. Sprague–Dawley rats were randomly divided into three groups (n = 18): control group (control); rats receiving irradiation and intratracheal injection of normal saline (IR + NS); and rats receiving irradiation and intratracheal injection of lung ECM-derived hydrogel (IR + ECM). The wet/dry weight ratio was used to evaluate the congestion and edema of the lungs. Histopathological analysis of lung tissues was performed using hemotoxylin and eosin staining and Masson's trichrome staining. Immunohistochemical staining and western blot analyses were carried out to determine the expression of epithelial–mesenchymal transition (EMT)-related proteins in lung tissues (E-cadherin, α-smooth muscle actin [α-SMA], and vimentin). In addition, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6), hydroxyproline, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were also evaluated. The ECM-derived hydrogels had good cytocompatibility and histocompatibility. ECM-derived hydrogel treatment improved lung histopathology injury and pulmonary edema. Higher expression of E-cadherin and lower expression of vimentin and α-SMA were found in the IR + ECM group compared with those in the IR + NS group. Hydroxyproline levels were reduced by ECM-derived hydrogel treatment compared with those in the IR + NS group. Obvious increases of TNF-α, IL-6, and TGF-β1 were identified following irradiation. Marked reductions in MDA content and increases in SOD were induced by ECM-derived hydrogel treatment in rats after radiation. ECM-derived hydrogels were shown to protect against RILI, potentially by reducing EMT, inflammation, and oxidative damage.     

Linked unresponsiveness: early cytokine gene expression profiles in cardiac allografts following pretreatment of recipients with bone marrow cells expressing donor MHC alloantigen

Linked unresponsiveness operates to induce specific unresponsiveness to fully mismatched vascularized allografts in recipients pretreated with anti-CD4 antibody and syngeneic bone marrow cells expressing a single donor MHC class I alloantigen. The aim of the study was to evaluate early post transplant cytokine expression in allografts where linked unresponsiveness was required for long term graft survival. CBA (H2(k)) mice were pretreated with CBK (H2(k)+K(b)) bone marrow cells under the cover of anti-CD4 antibody 28 days before transplantation of a CBK or a C57BL/10 (H2(b)) cardiac allograft. In both cases graft survival was prolonged (MST=100 days). Intragraft expression for interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-10, IL-12(p40), IL-18, iNOS, transforming growth factor (TGF)-beta(1) and C-beta was determined on day 1.5, 3, 7 and 14 after transplantation. Whereas rejecting allografts displayed a sharp peak in IFN-gamma, IL-2, IL-4 and IL-10 expression, non-rejecting allografts were characterized by an initial TGF-beta(1) and IFN-gamma production. An increasing IL-4 expression towards day 14 was a unique feature of linked unresponsiveness. All non-rejecting allografts were characterized by an increasing IL-12(p40) production towards day 14. In summary, the early cytokine expression pattern in allografts after bone marrow induced operational tolerance is influenced by the quantity of donor alloantigens expressed on the graft as well as on the bone marrow inoculum.     

Reporters for Single-Cell Analysis of Colicin Ib Expression in Salmonella enterica Serovar Typhimurium

Colicins are toxins that mediate interference competition in microbial ecosystems. They serve as a “common good” for the entire producer population but are synthesized by only few members which pay the costs of colicin production. We have previously shown that production of colicin Ib (cib), a group B colicin, confers a competitive advantage to Salmonella enterica serovar Typhimurium (S. Tm) over commensal E. coli strains. Here, we studied regulation of S. Tm cib expression at the single cell level. Comparative analysis of a single- and a multicopy gfp-reporter for the colicin Ib promoter (Pcib) revealed that the latter yielded optimal signal intensity for a diverse range of applications. We further validated this reporter and showed that gfp expression correlated well with colicin Ib (ColIb) protein levels in individual cells. Pcib is negatively controlled by two repressors, LexA and Fur. Only a small fraction of S. Tm expressed cib under non-inducing conditions. We studied Pcib activity in response to mitomycin C mediated DNA damage and iron limitation. Both conditions, if applied individually, lead to an increase in the fraction of GFP⁺ S. Tm, albeit an overall low fluorescence intensity. When both conditions were applied simultaneously, the majority of S. Tm turned GFP⁺ and displayed high fluorescence intensity. Thus, both repressors individually confine cib expression to a subset of the population. Taken together, we provide the first thorough characterization of a conventional gfp-reporter to study regulation of a group B colicin at the single cell level. This reporter will be useful to further investigate the costs and benefits of ColIb production in human pathogenic S. Tm and analyze cib expression under environmental conditions encountered in the mammalian gut.     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.     

Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4(+) T cell activation but is unable to prevent immune responses mediated by CD8(+) T cells. However, even in the absence of CD8(+) T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40(-/-) (H2(b)) recipients were transplanted with MHC-mismatched BALB/c (H2(d)) aortas. Transplant arteriosclerosis was evident in both CD40(-/-) and CD40(+/-) mice (intimal proliferation was 59 +/- 5% for CD40(-/-) mice vs 58 +/- 4% for CD40(+/-) mice) in the presence or absence of CD8(+) T cells (intimal proliferation was 46 +/- 7% for CD40(-/-) anti-CD8-treated mice vs 50 +/- 10% for CD40(+/-) anti-CD8-treated mice), confirming that CD8(+) T cells are not essential effector cells for the development of this disease. In CD40(-/-) recipients depleted of CD8(+) T cells, the number of eosinophils infiltrating the graft was markedly increased (109 +/- 24 eosinophils/grid for CD40(-/-) anti-CD8-treated mice vs 28 +/- 7 for CD40(+/-) anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40(-/-) recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 +/- 5 eosinophils/grid for CD40(-/-) anti-CD8(+), anti-IL-4-treated mice vs 109 +/- 24 for CD40(-/-) anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 +/- 5% for CD40(-/-) anti-CD8(+)-, anti-IL-4-treated mice vs 46 +/- 7% for CD40(-/-) anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8(+) T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.     

Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in combination with stable methotrexate therapy: a prospective single-centre study

Psoriasis and psoriatic arthritis are inflammatory diseases that respond well to anti-tumour necrosis factor-α therapy. To evaluate the effects of anti-tumour necrosis factor-α treatment on expression of adhesion molecules and angiogenesis in psoriatic lesional skin and synovial tissue, we performed a prospective single-centre study with infliximab therapy combined with stable methotrexate therapy. Eleven patients with both active psoriasis and psoriatic arthritis received infusions of infliximab (3 mg/kg) at baseline, and at weeks 2, 6, 14 and 22 in an open-label study. In addition, patients continued to receive stable methotrexate therapy in dosages ranging from 5 to 20 mg/week. Clinical assessments, including Psoriasis Area and Severity Index (PASI) and Disease Activity Score (DAS), were performed at baseline and every 2 weeks afterward. In addition, skin biopsies from a target psoriatic plaque and synovial tissue biopsies from a target joint were taken before treatment and at week 4. Immunohistochemical analysis was performed to detect the number of blood vessels, the expression of adhesion molecules and the presence of vascular growth factors. Stained sections were evaluated by digital image analysis. At week 16, the mean PASI was reduced from 12.3 ± 2.4 at baseline to 1.8 ± 0.4 (P ≤ 0.02). The mean DAS was reduced from 6.0 ± 0.5 to 3.6 ± 0.6 (P ≤ 0.02). We found some fluctuations in DAS response as compared with the change in PASI, with the latter exhibiting a steady decrease over time. After 4 weeks the cell infiltrate was reduced in both skin and synovium. There was a significant reduction in the number of blood vessels in dermis and synovium at week 4. A significant reduction in the expression of α_vβ₃ integrin, a marker of neovascularization, was also found in both skin and synovium at week 4. In addition, a significant reduction in the expression of adhesion molecules was observed in both skin and synovium at week 4. We also observed a trend toward reduced expression of vascular endothelial growth factor in both skin and synovium. In conclusion, low-dose infliximab treatment leads to decreased neoangiogenesis and deactivation of the endothelium, resulting in decreased cell infiltration and clinical improvement in psoriasis and psoriatic arthritis.