Association of Lung Inflammatory Cells with Small Airways Function and Exhaled Breath Markers in Smokers – Is There a Specific Role for Mast Cells?

Background

Smoking is associated with a mixed inflammatory infiltrate in the airways. We evaluated whether airway inflammation in smokers is related to lung function parameters and inflammatory markers in exhaled breath.

Methods

Thirty-seven smokers undergoing lung resection for primary lung cancer were assessed pre-operatively by lung function testing including single-breath-nitrogen washout test (sb-N2-test), measurement of fractional exhaled nitric oxide (FeNO) and pH/8-isoprostane in exhaled breath condensate (EBC). Lung tissue sections containing cancer-free large (LA) and small airways (SA) were stained for inflammatory cells. Mucosal (MC_T) respectively connective tissue mast cells (MC_TC) and interleukin-17A (IL-17A) expression by mast cells was analysed using a double-staining protocol.

Results

The median number of neutrophils, macrophages and mast cells infiltrating the lamina propria and adventitia of SA was higher than in LA. Both MCTC and MC_T were higher in the lamina propria of SA compared to LA (MC_TC: 49 vs. 27.4 cells/mm²; MC_T: 162.5 vs. 35.4 cells/mm²; P<0.005 for both instances). IL-17A expression was predominantly detected in MC_TC of LA. Significant correlations were found for the slope of phase III % pred. of the sb-N2-test (r_s= -0.39), for the FEV₁% pred. (r_s= 0.37) and for FEV₁/FVC ratio (r_s=0.38) with MC_T in SA (P<0.05 for all instances). 8-isoprostane concentration correlated with the mast cells in the SA (r_s=0.44), there was no correlation for pH or FeNO with cellular distribution in SA.

Conclusions

Neutrophils, macrophages and mast cells are more prominent in the SA indicating that these cells are involved in the development of small airway dysfunction in smokers. Among these cell types, the best correlation was found for mast cells with lung function parameters and inflammatory markers in exhaled breath. Furthermore, the observed predominant expression of IL-17A in mast cells warrants further investigation to elucidate their role in smoking-induced lung injury, despite the lack of correlation with lung function and exhaled breath parameters.     

Mathematical modeling of laser based potato cutting and peeling

A mathematical model is developed and validated to predict the depth of cut in potato tuber slabs as a function of laser power and travel speed. The model considers laser processing parameters such as input power, spot size and exposure time as well as the properties of the material being cut such as specific heat, thermal conductivity, surface reflectance, etc. The model also considers the phase change of water in potato and the ignition temperature of the solid portion. The composition of the potato tuber is assumed to be of water and solid. The model also assumes that the ablation process is accomplished through ejection of liquid water, debris and water vapour, and combustion of solid. A CO₂ laser operating in c.w. mode was chosen for the experimental work because water absorbs laser energy highly at 10.6 μm, and CO₂ laser units with relatively high output power are available. Slabs of potato tuber were chosen to be laser processed since potato contains high moisture and large amounts of relatively homogeneous tissue. The results of the preliminary calculations and experiments concluded that the model is able to predict the depth of cut in potato tuber parenchyma when subjected to a CO₂ laser beam.     

Immunocytochemical identification of neuroendocrine markers in human cardiac paraganglion — like structures

Summary Paraganglion-like structures (PLS) containing chromaffin-positive cells have been reported to be present in the adult human heart. The present work was initiated in order to evaluate the densitity of these structures in the interatrial septum and to study the presence of immunoreactivity of their cells to NSE and PGP 9,5 antibodies, two neuroendocrine markers. Six hundred 6-m paraffin serial sections were obtained from the upper third of the interatrial septum from six adult human hearts. From 2 to 12 paraganglia were found in each case, and their principal cells stained positively with NSE and PGP 9,5 antibodies. Depending on how these PLS related to other cardiac structures, four different types were identified: Type I — True paraganglia (located adjacent to ganglia or nerve fibers); Type II — Free paraganglia (immersed in the interatrial adipose tissue, without evident connection to other structures); Type III — Intraganglionic paraganglia (located within the nervous ganglia); Type IV — Intramyocardic paraganglia (small nests of immunoreactive cells closely related to myocardiocyte bundles). These cardiac paraganglia, which probably belong to the visceral-autonomic group, may have a role in the regulation of the cardiac function and in the adaptive mechanisms of the heart. Its is also possible that they originate functioning and non-functioning tumours.Work supported by grants from FINEP and CNPq (Brazil)     

Renin-Angiotensin System Blockers Protect Pancreatic Islets against Diet-Induced Obesity and Insulin Resistance in Mice

Background

The associations between obesity, hypertension and diabetes are well established, and the renin-angiotensin system (RAS) may provide a link among them. The effect of RAS inhibition on type 2 diabetes is still unclear; however, RAS seems to play an important role in the regulation of the pancreas and glucose intolerance of mice fed high-fat (HF) diet.

Methods

C57BL/6 mice fed a HF diet (8 weeks) were treated with aliskiren (50 mg/kg/day), enalapril (30 mg/kg/day) or losartan (10 mg/kg/day) for 6 weeks, and the protective effects were extensively compared among groups by morphometry, stereological tools, immunostaining, Western blotting and hormonal analysis.

Results

All RAS inhibitors significantly attenuated the increased blood pressure in mice fed a HF diet. Treatment with enalapril, but not aliskiren or losartan, significantly attenuated body mass (BM) gain, glucose intolerance and insulin resistance, improved the alpha and beta cell mass and prevented the reduction of plasma adiponectin. Furthermore, enalapril treatment improved the protein expression of the pancreatic islet Pdx1, GLUT2, ACE2 and Mas receptors. Losartan treatment showed the greatest AT2R expression.

Conclusion

Our findings indicate that ACE inhibition with enalapril attenuated several of the deleterious effects of the HF diet. In summary, enalapril appears to be responsible for the normalization of islet morphology and function, of alpha and beta cell mass and of Pdx1 and GLUT2 expression. These protective effects of enalapril were attributed, primarily, to the reduction in body mass gain and food intake and the enhancement of the ACE2/Ang (1-7) /Mas receptor axis and adiponectin levels.     

Genome-wide identification and expression analysis of the molecular chaperone binding protein <Emphasis Type="Italic">BiP</Emphasis> genes in <Emphasis Type="Italic">Citrus</Emphasis>

Here, we report for the first time the genome-wide identification and expression analysis of the molecular chaperone BiP genes in Citrus. Six genes encoding the conserved protein domain family GPR78/BiP/KAR2 were identified in the genome of Citrus sinensis and C. clementina. Two of them, named here as CsBiP1 and CsBiP2, were classified as true BiPs based on their deduced amino acid sequences. Alignment of the deduced amino acid sequences of CsBiP1 and CsBiP2 with BiP homologs from soybean and Arabidopsis showed that they contain all the conserved functional motifs of BiPs. Analysis of the promoter region of CsBiPs revealed the existence of cis-acting regulatory sequences involved in abiotic, heat-shock, and endoplasmic reticulum (ER) stress responses. Publicly available RNA-seq data indicated that CsBiP1 is abundantly expressed in leaf, flower, fruit, and callus, whereas CsBiP2 expression is rarely detected in any tissues under normal conditions. Comparative quantitative real-time PCR (qPCR) analysis of expression of these genes between C. sinensis grafted on the drought-tolerant “Rangpur” lime (C. limonia) and -sensitive “Flying Dragon” trifoliate orange (Poncirus trifoliata) rootstocks showed that CsBiP1 was upregulated by drought stress on the former but downregulated on the latter, whereas the CsBiP2 mRNA levels were downregulated on drought-stressed “Flying Dragon,” but remained constant on “Rangpur.” CsBiP2 upregulation was only observed in C. sinensis seedlings subjected to osmotic and cold treatments. Taken together, these results indicate the existence of two highly conserved BiP genes in Citrus that are differentially regulated in the different tissues and in response to abiotic stresses.     

RB1 deletion in gonadoblastoma in an XY female

Cytogenetic studies of normal and tumor cells in a patient with gonadal dysgenesis and bilateral gonadoblastoma were performed. The karyotype was 46,XY in peripheral blood lymphocytes and skin fibroblasts. The conserved region of the SRY gene was detected by polymerase chain reaction amplification. Sequencing of this region did not reveal any alterations. A 46,XY chromosome constitution was observed in the right gonadoblastoma, but a partial deletion of chromosome 13 was present in the left tumor. This deletion included band 13q14, where the retinoblastoma gene is mapped. The study of the polymorphism of the variable number of tandem repeats region in intron 17 of the RB1 locus disclosed loss of heterozygosity in both the left tumor, which showed the deletion of chromosome 13, and in the right tumor, where no chromosome alterations of chromosome 13 were detected. In situ hybridization covering 130 kb of RB1 showed that a partial deletion of one of the RB1 alleles had occurred in the right tumor. Since the deletions affected different alleles in each tumor, independent events must have been involved in the development of the tumors. These findings point toward a significant role of RB1 in the development of gonadoblastoma. Received: 17 October 1995 / Accepted: 14 July 1997     

Expression of duck lens delta-crystallin cDNAs in yeast and bacterial hosts. Delta 2-crystallin is an active argininosuccinate lyase.

The major soluble protein in the lenses of most birds and reptiles is delta-crystallin. In chickens and ducks the delta-crystallin gene has duplicated, and in the duck both genes contribute to the protein in the lens, while in the chicken lens there is a great preponderance of the delta 1 gene product. Purified delta-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. In order to determine the enzymatic properties of the two duck delta-crystallins their corresponding cDNA molecules were placed in yeast and bacterial expression plasmids. In Saccharomyces cerevisiae, the activity of each crystallin was assessed by transformation of the expression plasmids into a strain deficient for argininosuccinate lyase activity. The ability of the resulting yeast to grow on arginine deficient medium was used as a measure of enzymatic activity. Yeast expressing the duck delta 2-crystallin protein grew rapidly, while those expressing delta 1-crystallin failed to grow. Enzyme activity measurements confirmed the presence of activity in the delta 2-crystallin-expressing yeast, and no detectable activity could be demonstrated in the delta 1-crystallin-expressing yeast. Northern blotting of RNA from the transformed yeast revealed equal levels of mRNA species from the two constructs. For further analysis, the delta 2-crystallin cDNA was placed in the bacterial expression plasmid, pET-3d. The delta 2-crystallin protein produced in Escherichia coli was purified to homogeneity and analyzed to determine the kinetic properties. A Km of 0.35 mM was determined for argininosuccinate and a Vm of 3.5 mumols/min/mg was determined. These data demonstrate that, following duplication of the primordial argininosuccinate lyase gene, one of the genes maintained its role as an enzyme (delta 2-crystallin) while also serving as a crystallin and the other has evolved to specialize as a structural protein in the lens (delta 1-crystallin), presumably losing most or all of its catalytic capacity.     

Purification and characterization of an exon 2-deleted human beta-tropomyosin constructed by the polymerase chain reaction.

We deleted exon 2 in human skeletal beta-tropomyosin (h beta-SK tropomyosin) using an improved adaptation of polymerase chain reaction (PCR) technology. The first PCR product was used to prime the full-length cDNA, leading to an exon 2-deleted h beta-SK tropomyosin. This new protein, des-(39-80)-tropomyosin, could then be expressed in Escherichia coli and purified to homogeneity. At the nucleotide level, the junction between exons 1 and 3 has been precisely made in the PCR product. The mutated protein was purified using high-performance liquid chromatography. Des-(39-80)-tropomyosin revealed new immunological properties but was still recognized by certain antitropomyosin antibodies. Furthermore, the structural characteristics of the mutated tropomyosin fit those of the full-length tropomyosin. This new adaptation of PCR technology appears to be suitable for every kind of mutation inside a cloned DNA molecule, and one mutation primer per mutation is sufficient.     

Genetic transformation of Rangpur lime (Citrus limonia osbeck) with thebO (bacterio-opsin) genen and its initial evaluation forPhytophthora nicotianae resistance

Transgenic plants expressing the bacterio-opsin (bO) gene can spontaneously activate programmed cell death (ped) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with thebO gene, viaAgrobacterium tumefaciens-mediated transformation, and evaluated these plants forPhytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation forPhytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.     

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.