Cryopreservation based on freezing protocols for the long-term storage of microencapsulated myoblasts

One important challenge in biomedicine is the ability to cryogenically preserve not only cells, but also tissue-engineered constructs. In the present paper, alginate-poly-l-lysine-alginate (APA) microcapsules containing erythropoietin (Epo)-secreting C₂C₁₂ myoblasts were elaborated, characterized and tested both in vitro and in vivo. Dimethylsulfoxide (DMSO) was selected as cryoprotectant to evaluate the maintenance of physiological activity of cryopreserved microencapsulated myoblasts employing procedures based on freezing protocols up to a 45-day cryopreservation period. High chemical resistance of the cryopreserved microcapsules was observed using 10% DMSO as cryoprotectant following a standard slow-cooling procedure. Although a 42% reduction in Epo release from the microencapsulated cells was observed in comparison with the non-cryopreserved group, the in vivo biocompatibility and functionality of the encapsulated cells subcutaneously implanted in Balb/c mice was corroborated by high and sustained hematocrit levels over 194 days and lacking immunosuppressive protocols. No major host reaction was observed. Based on the results obtained in our study, a slow-cooling protocol using 10% DMSO as cryoprotectant (confirmed for cryopreservation periods up to 45 days) might be considered a suitable therapeutic strategy if the long-term storage of microencapsulated cells, such as C₂C₁₂ myoblasts is pretended.     

Longitudinal study of a mouse model of chronic pulmonary inflammation using breath hold gated micro-CT

Objectives  

To evaluate the feasibility of using automatic quantitative analysis of breath hold gated micro-CT images to detect and monitor disease in a mouse model of chronic pulmonary inflammation, and to compare image-based measurements with pulmonary function tests and histomorphometry.     

NADPH oxidase-mediated oxidative stress: genetic studies of the p22(phox) gene in hypertension

Increased vascular production of reactive oxygen species, especially superoxide anion, significantly contributes to the oxidative stress associated with hypertension. An enhanced superoxide production causes an increased inactivation of nitric oxide that diminishes nitric oxide bioavailability, thus contributing to endothelial dysfunction and hypertrophy of vascular cells. It has been shown that NADPH oxidases play a major role as the most important sources of superoxide anion in phagocytic and vascular cells. Several experimental observations have described an enhanced superoxide generation as a result of NADPH oxidase activation in hypertension. Although these enzymes respond to stimuli such as vasoactive factors, growth factors, and cytokines, recent data suggest a significant role of the genetic background in the modulation of the expression of its different components. Several polymorphisms have been identified in the promoter and in the coding region of CYBA, the gene that encodes the essential subunit of the NADPH oxidase p22phox, some of which seem to influence significantly the activity of these enzymes in the context of cardiovascular diseases. Among CYBA polymorphisms, genetic investigations have provided a novel marker, the -930(A/G) polymorphism, which determines the genetic susceptibility of hypertensive patients to oxidative stress.     

Association between neural reactivity and startle reactivity to uncertain threat in two independent samples

Prior studies indicate that anxiety disorders are associated with heightened sensitivity to uncertain threat (U threat). Individual differences in reactivity to U threat have been measured in the laboratory with two methodologies—startle eyeblink potentiation and fMRI. While startle and fMRI are purported to relate to each other, very little research exists on whether individual differences in one measure are associated with individual differences in another and, thus, whether startle and fMRI capture shared mechanisms. Therefore, the current study was designed to investigate if and where in the brain measures of startle potentiation and fMRI BOLD signal correlate during response to U threat across two independent samples. Participants in both studies completed two threat anticipation tasks—once during collection of startle potentiation and once during fMRI. In Study 1 (n = 43), the startle and fMRI tasks both used electric shock as the threat. As an extension, in Study 2 (n = 38), the startle task used electric shock but the fMRI task used aversive images. Despite these methodological differences, greater startle potentiation to U threat was associated with greater dorsal anterior cingulate, caudate, and orbitofrontal cortex reactivity to U threat in both samples. The findings suggest that startle and fMRI measures of responding to U threat overlap, and points toward an integrated brain‐behavior profile of aberrant U threat responding.     

Cell heterogeneity in clear cell renal cell carcinoma

The aim of this study was to define the histological spectrum, frequency and significance of nonconventional tumour cells in clear cell renal cell carcinomas (CCRCC). Fifty‐one totally sampled CCRCC were studied histologically to evaluate the spectrum of cell morphology variability, its frequency and significance, and their correlation with tumour grade and stage, and other histological parameters of aggressive behaviour like necrosis. Aside from conventional clear/eosinophilic granular cells, three additional cellular types were identified and considered in this study: small clear cells, syncytial cells and rhabdoid cells. Small clear cells were detected in 11 cases (21.5%), syncytial cells in 8 (15.6%) and rhabdoid cells in 5 (9.8%). The presence of syncytial and rhabdoid cells statistically correlated with grade (p = 0.003 and p = 0.006) and stage (p = 0.049 and p = 0.05) in CCRCC. Necrosis correlated with stage (p = 0.018) and grade (p = 0.004), but not with syncytial, rhabdoid or small clear cells. The presence of syncytial and rhabdoid cells in CCRCC is a relatively frequent event that significantly correlates with high‐grade tumours and high stage status.     

Prefrontal Responses during Proactive and Reactive Inhibition Are Differentially Impacted by Stress in Anorexia and Bulimia Nervosa

Binge eating is a distressing, transdiagnostic eating disorder symptom associated with impulsivity, particularly in negative mood states. Neuroimaging studies of bulimia nervosa (BN) report reduced activity in frontostriatal regions implicated in self-regulatory control, and an influential theory posits that binge eating results from self-regulation failures under stress. However, there is no direct evidence that psychological stress impairs self-regulation in binge-eating disorders, or that any such self-regulatory deficits generalize to binge eating in underweight individuals (i.e., anorexia nervosa bingeing/purging subtype; AN-BP). We therefore determined the effect of acute stress on inhibitory control in 85 women (BN, 33 women; AN-BP, 22 women; 30 control participants). Participants underwent repeated functional MRI scanning during performance of the Stop-signal anticipation task, a validated measure of proactive (i.e., anticipation of stopping) and reactive (outright stopping) inhibition. Neural and behavioral responses to induced stress and a control task were evaluated on 2 consecutive days. Women with BN had reduced proactive inhibition, while prefrontal responses were increased in both AN-BP and BN. Reactive inhibition was neurally and behaviorally intact in both diagnostic groups. Both AN-BP and BN groups showed distinct stress-induced changes in inferior and superior frontal activity during both proactive and reactive inhibition. However, task performance was unaffected by stress. These results offer novel evidence of reduced proactive inhibition in BN, yet inhibitory control deficits did not generalize to AN-BP. Our findings identify intriguing alterations of stress responses and inhibitory function associated with binge eating, but they counsel against stress-induced failures of inhibitory control as a comprehensive explanation for loss-of-control eating.SIGNIFICANCE STATEMENT Binge eating is a common psychiatric syndrome that feels uncontrollable to the sufferer. Theoretically, it has been related to reduced self-regulation under stress, but there remains no direct evidence for this link in binge-eating disorders. Here, we examined how experimentally induced stress affected response inhibition in control participants and women with anorexia nervosa and bulimia nervosa. Participants underwent repeated brain scanning under stressful and neutral conditions. Although patient groups had intact action cancellation, the slowing of motor responses was impaired in bulimia nervosa, even when the likelihood of having to stop increased. Stress altered brain responses for both forms of inhibition in both groups, yet performance remained unimpaired. These findings counsel against a simple model of stress-induced disinhibition as an adequate explanation for binge eating.     

Phagocytic NADPH oxidase overactivity underlies oxidative stress in metabolic syndrome

Oxidative stress plays a critical role in the pathogenesis of atherosclerosis in patients with metabolic syndrome. This study aimed to investigate whether a relationship exists between phagocytic NADPH oxidase activity and oxidative stress and atherosclerosis in metabolic syndrome patients. The study was performed in 56 metabolic syndrome patients (metabolic syndrome group), 99 patients with one or two cardiovascular risk factors (cardiovascular risk factor group), and 28 healthy subjects (control group). NADPH oxidase expression and activity was augmented (P < 0.05) in metabolic syndrome compared with cardiovascular risk factor and control groups. Insulin was enhanced (P < 0.05) in metabolic syndrome patients compared with cardiovascular risk factor and control groups and correlated with NADPH oxidase activity in the overall population. Insulin stimulated NADPH oxidase activity; this effect was abolished by a specific protein kinase C inhibitor. Oxidized LDL and nitrotyrosine levels and carotid intima-media thickness were increased (P < 0.05) in the metabolic syndrome group compared with cardiovascular risk factor and control groups and correlated with NADPH oxidase activity in the overall population. These findings suggest that phagocytic NADPH oxidase overactivity is involved in oxidative stress and atherosclerosis in metabolic syndrome patients. Our findings also suggest that hyperinsulinemia may contribute to oxidative stress in metabolic syndrome patients through activation of NADPH oxidase.     

Evaluation of micro-CT for emphysema assessment in mice: comparison with non-radiological techniques

Objectives

To define the potential, limitations and synergies of micro-CT and other non-radiological techniques for the quantification of emphysema and related processes in mice, by performing a complete characterization of the elastase-induced emphysema model.

Materials and methods

Ninety A/J mice (45 treated and 45 controls) were studied at different time points using breath-hold gated micro-CT, functional test parameters, RT-PCR for RNA cytokine expression, Luminex technology for cytokine plasma concentration and histomorphometry.

Results

Both histomorphometry and micro-CT imaging reflect rapid initial emphysema progression followed by steady-state development at decreasing rates. Cytokine measurements reveal an acute inflammatory response within the first 24?h that disappears after the first week. Limited systemic effect was observed based on plasma cytokine concentration. Lung compliance decreases during the acute inflammation phase and increases afterwards.

Conclusion

Histomorphometry is the most sensitive technique since it detects airspace enlargement before the other methods (1?h after treatment). Micro-CT correlates well with histology (r2?=?0.63) proving appropriate for longitudinal studies. Functional test parameters do not necessarily correlate with the extent of emphysema, as they can be influenced by acute inflammation. Finally, cytokine measurements correlate with the presence of inflammation in histology but not with emphysema.