Influence of cigarette smoke on the arginine pathway in asthmatic airways: increased expression of arginase I

BACKGROUND: Up to 30% of asthmatic subjects are smokers, and smoking might be an important contributor to asthma pathology. Inducible nitric oxide synthase (iNOS), ornithine decarboxylase (ODC), and arginase I are involved in the arginine pathway. We have shown that arginase I and iNOS are upregulated in asthma. Smoking asthmatic subjects are reported to have low exhaled nitric oxide levels. The effect of cigarette smoking on the expression of arginase I in asthma is unknown. OBJECTIVES: The aims of this study were to investigate the expression of arginase I, ODC, and iNOS in asthmatic airways of smokers and nonsmokers and in vitro after nicotine stimulation. METHODS: Endobronchial biopsies were performed on 24 steroid-naive subjects with mild asthma: 12 smokers and 12 nonsmokers. Arginase I, ODC, and iNOS levels were assessed by means of immunohistochemistry and in situ hybridization (arginase I). In vitro stimulation of airway cells with nicotine was performed, followed by real-time PCR. RESULTS: Arginase I, ODC, and iNOS were expressed in the epithelium and smooth muscle bundles of both subgroups of asthmatic subjects. There was an increase of arginase I and ODC immunoreactivities in smoking compared with nonsmoking asthmatic subjects. There was no significant difference in immunoreactivity for iNOS between groups. Nicotine induced a 2-fold increase in arginase I and ODC expression in airway epithelial cells and fibroblasts. CONCLUSION: This study demonstrates that the expression of arginase I and ODC is increased in airways of smoking compared with nonsmoking asthmatic subjects and in vitro by nicotine. CLINICAL IMPLICATIONS: Increased expression of arginase I might lead to low exhaled nitric oxide and chronic obstructive pulmonary disease-like airway remodeling in smoking asthmatic subjects.     

hSNF5/INI1-deficient tumours and rhabdoid tumours are convergent but not fully overlapping entities

Rhabdoid tumours (RTs) are rare but highly aggressive tumours of childhood. Their rarity and their miscellaneous locations make the diagnosis particularly challenging for pathologists. Central nervous system and peripheral RTs have been associated with biallelic inactivation of the hSNF5/INI1/SMARCB1 (hSNF5/INI1) tumour suppressor gene. Immunohistochemistry (IHC) with a monoclonal anti-hSNF5/INI1 antibody has recently been proposed as an efficient diagnostic tool for RTs. We have conducted a retrospective study of 55 tumours referred to our institution with a suspicion of RT. This analysis included pathological review, IHC with anti-hSNF5/INI1 antibody, and molecular investigation using quantitative DNA fluorescent analysis and sequencing of the nine exons of hSNF5/INI1. The molecular lesion could be detected in 37 of the 39 cases exhibiting negative staining for hSNF5/INI1. In the two discrepant cases, the lack of detection of genetic abnormality was probably owing to the presence of a high number of non-tumour cells in the samples. This indicates that hSNF5/INI1 IHC is very sensitive and highly specific for the detection of hSNF5/INI1 loss-of-function. Among the 38 cases with typical RT histological features, six failed to exhibit hSNF5/INI1 mutation and stained positive for hSNF5/INI1. This strongly supports the evidence of a second genetic locus, distinct from hSNF5/INI1, associated with RT. Conversely, seven tumours with histological features poorly compatible with RT stained negative for hSNF5/INI1; they nevertheless exhibited an age of onset and a clinical behaviour similar to RT. This suggests that hSNF5/INI1 inactivation is not strictly limited to typical RT but characterizes a wider family of hSNF5/INI1-deficient tumours. Consequently, we believe that anti-hSNF5/INI1 IHC should be performed widely, even when the pathological characteristics are not typical. The molecular investigation should be performed in infants when a rhabdoid predisposition syndrome is suspected.     

Specific magnetic bead based capture of genomic DNA from clinical samples: application to the detection of group B streptococci in vaginal/anal swabs

BACKGROUND: Group B streptococci (GBS) are a leading cause of sepsis and meningitis in newborns. We previously developed a rapid diagnostic system for GBS detection from vaginal/anal samples obtained from pregnant women during delivery. To facilitate the adaptation of this method for point-of-care testing, we have developed a specific and efficient GBS DNA capture method that is compatible with both PCR and nonamplification detection technologies. METHODS: Superparamagnetic beads were functionalized with oligonucleotide capture probes of different lengths and used to capture GBS genomic DNA (gDNA). A rapid extraction procedure was used to provide DNA from GBS cultures or vaginal/anal samples with added GBS. Hybridization reactions consisting of functionalized beads and target DNA in 30 muL of hybridization buffer were performed for 1 h at room temperature, followed by washing and resuspension in water. Captured DNA was then detected using quantitative PCR. RESULTS: A 25-mer capture probe allowed detection of 1000 genome copies of purified GBS DNA. The ability to detect GBS was improved by use of a 50-mer (100 copies) and a 70-mer capture probe (10 copies). Detection of approximately 1250 CFU/mL was achieved for diluted GBS broth culture and for vaginal/anal swab samples with added GBS. CONCLUSION: Oligonucleotide-functionalized superparamagnetic microbeads efficiently capture GBS gDNA from both bacterial cultures and vaginal/anal samples with added GBS. Efficiency of gDNA capture increases with oligonucleotide length. This technology could be combined with sample preparation and detection technologies in a microfluidic system to allow point-of-care testing for GBS.     

Propranolol Promotes Bone Formation and Limits Resorption Through Novel Mechanisms During Anabolic Parathyroid Hormone Treatment in Female C57BL/6J Mice

Although the nonselective β-blocker, propranolol, improves bone density with parathyroid hormone (PTH) treatment in mice, the mechanism of this effect is unclear. To address this, we used a combination of in vitro and in vivo approaches to address how propranolol influences bone remodeling in the context of PTH treatment. In female C57BL/6J mice, intermittent PTH and propranolol administration had complementary effects in the trabecular bone of the distal femur and fifth lumbar vertebra (L₅), with combination treatment achieving microarchitectural parameters beyond that of PTH alone. Combined treatment improved the serum bone formation marker, procollagen type 1 N propeptide (P1NP), but did not impact other histomorphometric parameters relating to osteoblast function at the L₅. In vitro, propranolol amplified the acute, PTH-induced, intracellular calcium signal in osteoblast-like cells. The most striking finding, however, was suppression of PTH-induced bone resorption. Despite this, PTH-induced receptor activator of nuclear factor κ-B ligand (RANKL) mRNA and protein levels were unaltered by propranolol, which led us to hypothesize that propranolol could act directly on osteoclasts. Using in situ methods, we found Adrb2 expression in osteoclasts in vivo, suggesting β-blockers may directly impact osteoclasts. Consistent with this, we found propranolol directly suppresses osteoclast differentiation in vitro. Taken together, this work suggests a strong anti-osteoclastic effect of nonselective β-blockers in vivo, indicating that combining propranolol with PTH could be beneficial to patients with extremely low bone density. © 2022 American Society for Bone and Mineral Research (ASBMR).     

A novel immune competent murine hypertrophic scar contracture model: A tool to elucidate disease mechanism and develop new therapies

Hypertrophic scar (HSc) contraction following burn injury causes contractures. Contractures are painful and disfiguring. Current therapies are marginally effective. To study pathogenesis and develop new therapies, a murine model is needed. We have created a validated immune‐competent murine HSc model. A third‐degree burn was created on dorsum of C57BL/6 mice. Three days postburn, tissue was excised and grafted with ear skin. Graft contraction was analyzed and tissue harvested on different time points. Outcomes were compared with human condition to validate the model. To confirm graft survival, green fluorescent protein (GFP) mice were used, and histologic analysis was performed to differentiate between ear and back skin. Role of panniculus carnosus in contraction was analyzed. Cellularity was assessed with 4′,6‐diamidino‐2‐phenylindole. Collagen maturation was assessed with Picro‐sirius red. Mast cells were stained with Toluidine blue. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. RNA for contractile proteins was detected by quantitative real‐time polymerase chain reaction (qRT‐PCR). Elastic moduli of skin and scar tissue were analyzed using a microstrain analyzer. Grafts contracted to ～45% of their original size by day 14 and maintained their size. Grafting of GFP mouse skin onto wild‐type mice, and analysis of dermal thickness and hair follicle density, confirmed graft survival. Interestingly, hair follicles disappeared after grafting and regenerated in ear skin configuration by day 30. Radiological analysis revealed that panniculus carnosus doesn't contribute to contraction. Microscopic analyses showed that grafts show increase in cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in collagen maturation over time. CD31 stain revealed increased vascularity. Macrophages and mast cells were increased. qRT‐PCR showed up‐regulation of transforming growth factor beta, alpha smooth muscle actin, and rho‐associated protein kinase 2 in HSc. Tensile testing revealed that human skin and scar tissues are tougher than mouse skin and scar tissues.     

Percutaneous single- and double-balloon aortic valvotomy in adolescents and young adults with congenital aortic stenosis

Percutaneous balloon aortic valvotomy was attempted in six consecutiveadolescents and young adults, (meanage 18 ± 5 years),with severe congenital aortic valvular stenosis. Peak systolicaortic valve gradient measurement and aortography was performedbefore and immediately after valvotomy, and a 1–6 monthsfollow-up. The single-balloon technique was used in two patientsand the double-balloon technique in four patients. After balloonvalvotomy the mean peak systolic aortic valve gradient (PSG)decreased from 122 ± 53 to 43 ± 20 mmHg (P >001). The haemodynamic improvement persisted at follow-up (PSG= 47 ±31 mmHg), except in one patient who had previouslyundergone aortic surgical valvotomy. One balloon valvotomy wasunsuccessful, presumably because of the selection of an inappropriatelysmall balloon. Two patients with critical aortic stenosis (PSG>165mmHg), who exhibited a thick aortic valve on echocardiographywith a relatively small aortic root and annulus, had a dramaticreduction in PSG. However, the degree of aortic regurgitationincreased significantly in both cases. The theoretical haemodynamicadvantage of the double versus the single balloon techniqueswas confirmed in one patient. We have demonstrated that percutaneousballoon aortic valvotomy using either the single- or double-balloontechnique is feasible in young adults with severe congenitalaortic stenosis. Further studies are required both to defineappropriate patients and the selection of balloon diameter andlength.     

No evidence of maternal cell colonization in reverted liver nodules of tyrosinemia type I patients

BACKGROUND AND AIMS: Hereditary tyrosinemia type I (HTI) is a recessively inherited disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine catabolic pathway. The mosaic pattern of FAH expression observed in the livers of >85% of studied patients was shown to result from the correction of the mutation in one of the FAH alleles. Bilateral cell trafficking can occur between mother and fetus and such an event could be responsible for the chimerism observed in some diseases. It also has been reported that the liver repopulation observed in a HTI murine model by serial transplantation of bone marrow-derived cells was caused by a fusion of these cells to host hepatocytes. These observations led us to test the possibility that the transfer of nucleated heterozygous maternal cells in the fetal circulation could be responsible for the mosaic liver expression of FAH in HTI patients. METHODS: We used polymorphic markers of short cytosine-adenine DNA repeats to compare DNA from corrected liver sections of 4 HTI patients with DNA from their parents' blood. RESULTS: Genotyping showed that only one maternal allele is present in DNA isolated from FAH-expressing liver nodules of each proband for at least 1 marker. CONCLUSIONS: The corrected liver nodules in HTI patients are not of maternal origin and do not support cell trafficking and cell fusion as mechanisms of correction of the gene defect in hepatocytes of tyrosinemia patients.