Myofibroblast expression in airways and alveoli is affected by smoking and COPD

Background

Chronic obstructive pulmonary disease (COPD) is characterized by structural changes in alveoli and airways. Our aim was to analyse the numbers of alpha-smooth muscle actin (α-SMA) positive cells, as a marker of myofibroblasts, in different lung compartments in non-smokers and smokers with normal lung function or COPD.

Methods

α-SMA, tenascin-C (Tn-C) and EDA-fibronectin in alveolar level and airways were assayed by immunohistochemistry and quantified by image analysis. Immunohistochemical findings were correlated with clinical data. α-SMA protein was also analysed by Western blotting from fibroblastic cells cultured from peripheral lung of non-smokers, smokers without COPD and smokers with COPD.

Results

In many cases, the endings of the detached alveolar walls were widened, the structures of which were named as widened alveolar tips. Widened alveolar tips contained α-SMA positive cells, which were obviously myofibroblasts. There were less alveolar tips containing positive cells for α-SMA in alveoli and α-SMA positive cells in bronchioles in smokers and in COPD compared to non-smokers. The quantity of α-SMA positive cells was increased in bronchi in COPD. Tn-C was elevated in bronchi in COPD and smokers’ lung. The α-SMA protein level was 1.43-fold higher in stromal cells cultured from non-smokers than in those of smokers.

Conclusions

Myofibroblasts are localized variably in normal and diseased lung. This indicates that they have roles in both regeneration of lung and pathogenesis of COPD. The widened alveolar tips, these newly characterized histological structures, seemed to be the source of myofibroblasts at the alveolar level.     

Distinct localization of the complement C5b‐9 complex on Gram‐positive bacteria

The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo‐attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b‐9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram‐positive bacteria are protected from MAC‐dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram‐positive pathogens secrete small proteins that inhibit C5b‐9 formation. In this study, we found that complement activation on Gram‐positive bacteria in serum results in specific surface deposition of C5b‐9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS‐stable) forms, indicating the presence of ring‐structured C5b‐9. Surprisingly, confocal microscopy demonstrated that C5b‐9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b‐9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b‐9 complex and Gram‐positive bacteria, whichmight ultimately lead to a new model of MAC assembly and functioning.     

Yield and proliferation rate of adipose-derived stromal cells as a function of age,body mass index and harvest site—increasing the yield by use of adherent and supernatant fractions?

Background aimsAdipose-derived stem cells are easily accessed and have a relatively high density compared with other mesenchymal stromal cells. Isolation protocols of adipose-derived stem cells (ASC) rely on the cell's ability to adhere to tissue culture plastic overnight. It was evaluated whether the floating ASC fractions are also of interest for cell-based therapies. In addition, the impact of age, body mass index (BMI) and harvest site was assessed.MethodsThe surface protein profile with the use of flow cytometry, the cell yield and the doubling time of passages 4, 5 and 6 of ASC from 30 donors were determined. Adherent and supernatant fractions were compared. The impact of age, BMI and harvest site on cell yield and doubling times was determined.ResultsBoth adherent and supernatant fractions showed high mean fluorescence intensities for CD13, CD29, CD44, CD73, CD90 and CD105 and comparatively low mean fluorescence intensities for CD11b, CD62L, intracellular adhesion molecule-1 and CD34. Doubling times of adherent and supernatant fractions did not differ significantly. Whereas the old age group had a significantly lower cell yield compared with the middle aged group, BMI and harvest site had no impact on cell yield. Finally, doubling times for passages 4, 5 and 6 were not influenced by the age and BMI of the donors, nor the tissue-harvesting site.ConclusionsThe floating ASC fraction is an equivalent second cell source just like the adherent ASC fraction. Donor age, BMI and harvest site do not influence cell yield and proliferation rate.     

Insertional tagging of the Scheffersomyces stipitis gene HEM25 involved in regulation of glucose and xylose alcoholic fermentation

Amid known microbial bioethanol producers, the yeast Scheffersomyces (Pichia) stipitis is particularly promising in terms of alcoholic fermentation of both glucose and xylose, the main constituents of lignocellulosic biomass hydrolysates. However, the ethanol yield and productivity, especially from xylose, are still insufficient to meet the requirements of a feasible industrial technology; therefore, the construction of more efficient S. stipitis ethanol producers is of great significance. The aim of this study was to isolate the insertional mutants of S. stipitis with altered ethanol production from glucose and xylose and to identify the disrupted gene(s). Mutants obtained by random insertional mutagenesis were screened for their growth abilities on solid media with different sugars and for resistance to 3-bromopyruvate. Of more than 1,300 screened mutants, 17 were identified to have significantly changed ethanol yields during the fermentation. In one of the best fermenting strains (strain 4.6), insertion was found to occur within the ORF of a homolog to the Saccharomyces cerevisiae gene HEM25 (YDL119C), encoding a mitochondrial glycine transporter required for heme synthesis. The role of HEM25 in heme accumulation, respiration, and alcoholic fermentation in the yeast S. stipitis was studied using strain 4.6, the complementation strain Comp—a derivative from the 4.6 strain with expression of the WT HEM25 allele and the deletion strain hem25Δ. As hem25Δ produced lower amounts of ethanol than strain 4.6, we assume that the phenotype of strain 4.6 may be caused not only by HEM25 disruption but additionally by some point mutation.     

Spatial distribution of Culex mosquito abundance and associated risk factors in Hanoi,Vietnam

Japanese encephalitis (JE) is the major cause of viral encephalitis (VE) in most Asian-Pacific countries. In Vietnam, there is no nationwide surveillance system for JE due to lack of medical facilities and diagnoses. Culex tritaeniorhynchus, Culex vishnui, and Culex quinquefasciatus have been identified as the major JE vectors in Vietnam. The main objective of this study was to forecast a risk map of Culex mosquitoes in Hanoi, which is one of the most densely populated cities in Vietnam. A total of 10,775 female adult Culex mosquitoes were collected from 513 trapping locations. We collected temperature and precipitation information during the study period and its preceding month. In addition, the other predictor variables (e.g., normalized difference vegetation index [NDVI], land use/land cover and human population density), were collected for our analysis. The final model selected for estimating the Culex mosquito abundance included centered rainfall, quadratic term rainfall, rice cover ratio, forest cover ratio, and human population density variables. The estimated spatial distribution of Culex mosquito abundance ranged from 0 to more than 150 mosquitoes per 900m². Our model estimated that 87% of the Hanoi area had an abundance of mosquitoes from 0 to 50, whereas approximately 1.2% of the area showed more than 100 mosquitoes, which was mostly in the rural/peri-urban districts. Our findings provide better insight into understanding the spatial distribution of Culex mosquitoes and its associated environmental risk factors. Such information can assist local clinicians and public health policymakers to identify potential areas of risk for JE virus. Risk maps can be an efficient way of raising public awareness about the virus and further preventive measures need to be considered in order to prevent outbreaks and onwards transmission of JE virus.     

Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists

Heterotopic ossification consists of ectopic bone formation within soft tissues after surgery or trauma. It can have debilitating consequences, but there is no definitive cure. Here we show that heterotopic ossification was essentially prevented in mice receiving a nuclear retinoic acid receptor-γ (RAR-γ) agonist. Side effects were minimal, and there was no significant rebound effect. To uncover the mechanisms of these responses, we treated mouse mesenchymal stem cells with an RAR-γ agonist and transplanted them into nude mice. Whereas control cells formed ectopic bone masses, cells that had been pretreated with the RAR-γ agonist did not, suggesting that they had lost their skeletogenic potential. The cells became unresponsive to rBMP-2 treatment in vitro and showed decreases in phosphorylation of Smad1, Smad5 and Smad8 and in overall levels of Smad proteins. In addition, an RAR-γ agonist blocked heterotopic ossification in transgenic mice expressing activin receptor-like kinase-2 (ALK2) Q207D, a constitutively active form of the receptor that is related to ALK2 R206H found in individuals with fibrodysplasia ossificans progressiva. The data indicate that RAR-γ agonists are potent inhibitors of heterotopic ossification in mouse models and, thus, may also be effective against injury-induced and congenital heterotopic ossification in humans.     

Systemic and hepatic vein galectin-3 are increased in patients with alcoholic liver cirrhosis and negatively correlate with liver function

Recently we demonstrated higher galectin-3 in portal venous serum (PVS) compared to hepatic venous serum (HVS) in a small cohort of patients with normal liver function suggesting hepatic removal of galectin-3. Here, galectin-3 was measured by ELISA in PVS, HVS and systemic venous blood (SVS) of 33 patients with alcoholic liver cirrhosis and a larger cohort of 11 patients with normal liver function. Galectin-3 was cleared by the healthy but not the cirrhotic liver, and subsequently HVS and SVS galectin-3 levels were significantly increased in the patients with liver cirrhosis compared to controls. In healthy liver galectin-3 was produced by cholangiocytes and synthesis by hepatocytes was only observed in cirrhotic liver. Hepatic venous pressure gradient did not correlate with galectin-3 levels excluding hepatic shunting as the principal cause of higher SVS galectin-3. Galectin-3 was elevated in all blood compartments of patients with CHILD-PUGH stage C compared to patients with CHILD-PUGH stage A, and was higher in patients with ascites than patients without this complication. Galectin-3 was negatively associated with antithrombin-3 whose synthesis is reduced with worse liver function. Galectin-3 positively correlated with urea and creatinine, and PVS galectin-3 showed a negative association with creatinine clearance as an accepted measure of kidney function. To summarize in the current study systemic, portal and hepatic levels of galectin-3 were found to be negatively associated with liver function in patients with alcoholic liver cirrhosis and this may in part be related to impaired hepatic removal and/or increased synthesis in cirrhotic liver.     

Crystal structures of Trichoderma reesei β-galactosidase reveal conformational changes in the active site

We have determined the crystal structure of Trichoderma reesei (Hypocrea jecorina) β-galactosidase (Tr-β-gal) at a 1.2? resolution and its complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4? resolutions, respectively. Tr-β-gal is a potential enzyme for lactose hydrolysis in the dairy industry and belongs to family 35 of the glycoside hydrolases (GH-35). The high resolution crystal structures of this six-domain enzyme revealed interesting features about the structure of Tr-β-gal. We discovered conformational changes in the two loop regions in the active site, implicating a conformational selection-mechanism for the enzyme. In addition, the Glu200, an acid/base catalyst showed two different conformations which undoubtedly affect the pK(a) value of this residue and the catalytic mechanism. The electron density showed extensive glycosylation, suggesting a structure stabilizing role for glycans. The longest glycan showed an electron density that extends to the eighth monosaccharide unit in the extended chain. The Tr-β-gal structure also showed a well-ordered structure for a unique octaserine motif on the surface loop of the fifth domain.     

Intradermal injections of equine allogeneic umbilical cord-derived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions

BACKGROUND AIMS. The use of allogeneic mesenchymal stem cells (MSC) to treat acute equine lesions would greatly expand equine cellular therapy options; however, the safety and antigenicity of these cells have not been well-studied. We hypothesized that equine allogeneic umbilical cord tissue (UCT)-derived MSC would not elicit acute graft rejection or a delayed-type hypersensitivity response when injected intradermally. METHODS. Six Quarterhorse yearlings received 12 intradermal injections (autologous MSC, allogeneic MSC, positive control and negative control, in triplicate) followed by the same series of 12 injections, 3-4 weeks later, at another site. Wheals were measured and palpated at 0.25, 4, 24, 48, 72 h and 7 days post-injection. Biopsies were obtained at 48 and 72 h and 7 days post-injection. Mixed leukocyte reactions were performed 1 week prior to the first injections and 3 weeks after the second injections. RESULTS. There were no adverse local or systemic responses to two intradermal injections of allogeneic MSC. MSC injection resulted in minor wheal formation, characterized by mild dermatitis, dermal edema and endothelial hyperplasia, that fully resolved by 48-72 h. No differences were noted between allogeneic and autologous MSC. The second injection of MSC did not elicit more significant physical or histomorphologic alterations compared with the first MSC injection. Neither allogeneic nor autologous UCT-derived MSC stimulated or suppressed baseline T-cell proliferation in vitro prior to or after two MSC administrations. CONCLUSIONS. Equine allogeneic UCT MSC may be safely administered intradermally on multiple occasions without eliciting a measurable cellular immune response.