A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon’s fibroblasts

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon’s fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2–3 cm² undamaged Tenon’s biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon’s fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon’s and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.     

Diverging trends of chronic bronchitis and smoking habits between 1998 and 2010

Background

No study has been carried out on the time trend in the prevalence of chronic bronchitis (CB) in recent years, despite its clinical and epidemiological relevance. We evaluated the trend in CB prevalence during the past decade among young Italian adults.

Methods

A screening questionnaire was mailed to general population samples of 20–44 year-old subjects in two cross-sectional surveys: the Italian Study on Asthma in Young Adults (ISAYA) (1998/2000; n = 18,873, 9 centres) and the screening stage of the Gene Environment Interactions in Respiratory Diseases (GEIRD) study (2007/2010; n = 10,494, 7 centres). CB was defined as having cough and phlegm on most days for a minimum of 3 months a year and for at least 2 successive years. The prevalence rates and the risk ratios (RRs) for the association between CB and each potential predictor were adjusted for gender, age, season of response, type of contact, cumulative response rate, and centre.

Results

CB prevalence was 12.5% (95% CI: 12.1-12.9%) in 1998/2000 and 12.6% (95% CI: 11.7-13.7%) in 2007/2010; it increased among never smokers (from 7.6 to 9.1%, p = 0.003), current light smokers (<15 pack-years; from 15.1 to 18.6%, p < 0.001), and unemployed/retired subjects (from 14.3 to 19.1%, p = 0.001). In this decade, the prevalence of current smoking decreased (from 33.6 to 26.9%, p < 0.001), whereas the prevalence of unemployment/premature retirement (from 5.3 to 6.0%, p = 0.005), asthma (from 5.0 to 6.2%, p = 0.003), and allergic rhinitis (from 19.5 to 24.5%, p < 0.001) increased. In both 1998/2000 and 2007/2010, the likelihood of having CB was significantly higher for women, current smokers, asthmatic patients, and subjects with allergic rhinitis. During this period, the strength of the association between CB and current heavy smoking (≥15 pack-years) decreased (RR: from 4.82 to 3.57, p = 0.018), whereas it increased for unemployment/premature retirement (from 1.11 to 1.53, p = 0.019); no change was observed for gender, asthma, and allergic rhinitis.

Conclusions

Despite the significant reduction in current smoking, CB prevalence did not vary among young Italian adults. The temporal pattern of CB prevalence can only be partly explained by the increase of unemployment/premature retirement, asthma and allergic rhinitis, and suggests that other factors could have played a role.     

Fetal mesenchymal stromal cells from cryopreserved human chorionic villi: cytogenetic and molecular analysis of genome stability in long-term cultures

Background aimsFirst-trimester chorionic villi (CV) are an attractive source of human mesenchymal stromal cells (hMSC) for possible applications in cellular therapy and regenerative medicine. Human MSC from CV were monitored for genetic stability in long-term cultures.MethodsWe set up a good manufacturing practice cryopreservation procedure for small amounts of native CV samples. After isolation, hMSC were in vitro cultured and analyzed for biological end points. Genome stability at different passages of expansion was explored by karyotype, genome-wide array-comparative genomic hybridization and microsatellite genotyping.ResultsGrowth curve analysis revealed a high proliferative potential of CV-derived cells. Immunophenotyping showed expression of typical MSC markers and absence of hematopoietic markers. Analysis of multilineage potential demonstrated efficient differentiation into adipocytes, osteocytes, chondrocytes and induction of neuro-glial commitment. In angiogenic experiments, differentiation in endothelial cells was detected by in vitro Matrigel assay after vascular endothelial growth factor stimulation. Data obtained from karyotyping, array-comparative genomic hybridization and microsatellite genotyping comparing early with late DNA passages did not show any genomic variation at least up to passage 10. Aneuploid clones appeared in four of 14 cases at latest passages, immediately before culture growth arrest.ConclusionsOur findings indicate that hCV-MSC are genetically stable in long-term cultures at least up to passage 10 and that it is possible to achieve clinically relevant amounts of hCV-MSC even after few stages of expansion. Genome abnormalities at higher passages can occasionally occur and are always associated with spontaneous growth arrest. Under these circumstances, hCV-MSC could be suitable for therapeutic purposes.     

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow–derived mesenchymal stromal cells

Background aimsA medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed.MethodsPlatelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells.ResultsAfter 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations.ConclusionsThe proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.     

Oxidative Stress and Erythrocyte Membrane Alterations in Children with Autism: Correlation with Clinical Features

It has been suggested that oxidative stress may play a role in the pathogenesis of Autism Spectrum Disorders (ASD), but the literature reports somewhat contradictory results. To further investigate the issue, we evaluated a high number of peripheral oxidative stress parameters, and some related issues such as erythrocyte membrane functional features and lipid composition. Twenty-one autistic children (Au) aged 5 to 12 years, were gender and age-matched with 20 typically developing children (TD). Erythrocyte thiobarbituric acid reactive substances, urinary isoprostane and hexanoyl-lysine adduct levels were elevated in Au, thus confirming the occurrence of an imbalance of the redox status of Au, whilst other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma radical absorbance capacity and carbonyl groups, erythrocyte superoxide dismutase and catalase activities) were unchanged. A very significant reduction of Na⁺/K⁺-ATPase activity (−66%, p<0.0001), a reduction of erythrocyte membrane fluidity and alteration in erythrocyte fatty acid membrane profile (increase in monounsaturated fatty acids, decrease in EPA and DHA-ω3 with a consequent increase in ω6/ω3 ratio) were found in Au compared to TD, without change in membrane sialic acid content. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity. Oxidative stress and erythrocyte membrane alterations may play a role in the pathogenesis of ASD and prompt the development of palliative therapeutic protocols. Moreover, the marked decrease in NKA could be potentially utilized as a peripheral biomarker of ASD.     

NT Pro BNP Plasma Level and Atrial Volume Are Linked to the Severity of Liver Cirrhosis

Background and Aims

Plasma levels of NT-pro-BNP, a natriuretic peptide precursor, are raised in the presence of fluid retention of cardiac origin and can be used as markers of cardiac dysfunction. Recent studies showed high levels of NT pro BNP in patients with cirrhosis. We assessed NT pro-BNP and other parameters of cardiac dysfunction in patients with cirrhosis, with or without ascites, in order to determine whether the behaviour of NT pro BNP is linked to the stage of liver disease or to secondary cardiac dysfunction.

Methods

Fifty eight consecutive hospitalized patients mostly with viral or NAFLD-related cirrhosis were studied. All underwent abdominal ultrasound and upper GI endoscopy. Cardiac morpho-functional changes were evaluated by echocardiography and NT-pro-BNP plasma levels determined upon admission. Twenty-eight hypertensive patients, without evidence of liver disease served as controls.

Results

Fifty eight cirrhotic patients (72% men) with a median age of 62 years (11% with mild arterial hypertension and 31% with type 2 diabetes) had a normal renal function (mean creatinine 0.9 mg/dl, range 0.7–1.06). As compared to controls, cirrhotic patients had higher NT pro-BNP plasma levels (365.2±365.2 vs 70.8±70.6 pg/ml; p<0.001). Left atrial volume (LAV) (61.8±26.3 vs 43.5±14.1 ml; p = 0.001), and left ventricular ejection fraction (62.7±6.9 vs. 65.5±4%,; p = 0.05) were also altered in cirrhotic patients that in controls. Patients with F2-F3 oesophageal varices as compared to F0/F1, showed higher e'' velocity (0.91±0.23 vs 0.66±0.19 m/s, p<0.001), and accordingly a higher E/A ratio (1.21±0.46 vs 0.89±0.33 m/s., p = 0.006).

Conclusion

NT-pro-BNP plasma levels are increased proportionally to the stage of chronic liver disease. Advanced cirrhosis and high NT-pro-BNP levels are significantly associated to increased LAV and to signs of cardiac diastolic dysfunction. NT pro-BNP levels could hence be an useful prognostic indicators of early decompensation of cirrhosis.     

Different Permeability of Potassium Salts across the Blood-Brain Barrier Follows the Hofmeister Series

The passage of ions across biological membranes is regulated by passive and active mechanisms. Passive ion diffusion into organs depends on the ion-pairing properties of salts present in the serum. Potassium ions could affect brain activity by crossing the blood-brain barrier (BBB) and its accumulation in the extracellular cerebral space could precipitate seizures. In the present study, we analyze passive diffusion of a series of potassium salts in the in vitro isolated guinea pig brain preparation. Different potassium counter-anions confer ion-pairing and lipophilicity properties that modulate membrane diffusion of the salt. Extracellular recordings in different cortical areas demonstrated the presence of epileptiform activities that strongly relate to anion identity, following the qualitative order of the Hofmeister series. Indeed, highly lipophilic salts that easily cross the BBB enhanced extracellular potassium concentration measured by ion-selective electrodes and were the most effective pro-epileptic species. This study constitutes a novel contribution for the understanding of the potential epileptogenicity of potassium salts and, more generally, of the role of counter-anions in the passive passage of salts through biological membranes.