Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies

Background aimsBone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34⁺-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI.MethodsThe need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34⁺ cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34⁺ cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells.ResultsGene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14⁺ subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively.ConclusionsOur data suggest that combining basal and expanded BM-derived CD34⁺ cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.     

OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion

Mitochondria amplify activation of caspases during apoptosis by releasing cytochrome c and other cofactors. This is accompanied by fragmentation of the organelle and remodeling of the cristae. Here we provide evidence that Optic Atrophy 1 (OPA1), a profusion dynamin-related protein of the inner mitochondrial membrane mutated in dominant optic atrophy, protects from apoptosis by preventing cytochrome c release independently from mitochondrial fusion. OPA1 does not interfere with activation of the mitochondrial "gatekeepers" BAX and BAK, but it controls the shape of mitochondrial cristae, keeping their junctions tight during apoptosis. Tightness of cristae junctions correlates with oligomerization of two forms of OPA1, a soluble, intermembrane space and an integral inner membrane one. The proapoptotic BCL-2 family member BID, which widens cristae junctions, also disrupts OPA1 oligomers. Thus, OPA1 has genetically and molecularly distinct functions in mitochondrial fusion and in cristae remodeling during apoptosis.     

Process-wide control and automation of an integrated continuous manufacturing platform for antibodies

Integrated continuous manufacturing is entering the biopharmaceutical industry. The main drivers range from improved economics, manufacturing flexibility, and more consistent product quality. However, studies on fully integrated production platforms have been limited due to the higher degree of system complexity, limited process information, disturbance, and drift sensitivity, as well as difficulties in digital process integration. In this study, we present an automated end-to-end integrated process consisting of a perfusion bioreactor, CaptureSMB, virus inactivation (VI), and two polishing steps to produce an antibody from an instable cell line. A supervisory control and data acquisition (SCADA) system was developed, which digitally integrates unit operations and analyzers, collects and centrally stores all process data, and allows process-wide monitoring and control. The integrated system consisting of bioreactor and capture step was operated initially for 4 days, after which the full end-to-end integrated run with no interruption lasted for 10 days. In response to decreasing cell-specific productivity, the supervisory control adjusted the loading duration of the capture step to obtain high capacity utilization without yield loss and constant antibody quantity for subsequent operations. Moreover, the SCADA system coordinated VI neutralization and discharge to enable constant loading conditions on the polishing unit. Lastly, the polishing was sufficiently robust to cope with significantly increased aggregate levels induced on purpose during virus inactivation. It is demonstrated that despite significant process disturbances and drifts, a robust process design and the supervisory control enabled constant (optimum) process performance and consistent product quality.     

“Timing” of arrival and in-hospital mortality in a cohort of patients under anticoagulant therapy presenting to the emergency departments with cerebral hemorrhage: A multicenter chronobiological study in Italy

Therapy with oral anticoagulants (OACs) is a risk factor for cerebral hemorrhage (CH). Although different studies have been undertaken to investigate the timing of the onset of major cardiovascular events, no data exist on temporal patterns of the onset of CH in subjects treated with OACs. The aim of this study is to evaluate the timing of CH in patients treated with OACs. All patients who developed CH under OACs therapy and admitted to 28 Italian Emergency Departments (EDs) between September 2011 and July 2013 were enrolled. Age, sex, time and location of the hemorrhagic lesion, type of the bleeding events (idiopathic or post-traumatic), anticoagulant therapy (warfarin or new oral anticoagulants - NOAs) and time of ED admission (i.e., hour, day, month and season) were recorded. Five hundred and seventeen patients (63.2% male aged 80 ± 7.9 yrs) with CH were involved. Warfarin was taken by 494 patients (95.6%), and NOAs by 23 (4.4%). In-hospital mortality (IHM) was recorded in 208 cases (40.2%). Cosinor analysis showed a peak of CH arrival between 12:00 and 14:00 h both in the whole population (PR 73.9%, p = 0.002) and the male subgroup (PR 65.2%, p = 0.009), whereas females showed an anticipated morning peak between 08:00 and 10:00 h (PR 65.7%, p = 0.008). A further analysis between idiopathic and post-traumatic CH confirmed the presence of a 24 h pattern with a peak between 12:00 and 14:00 h (PR 58.5%, p = 0.019) and between 08:00 and 10:00 h (PR80.1%, p < 0.001) for idiopathic events and post-traumatic hemorrhages, respectively. Moreover, a seasonal winter peak was identified for idiopathic forms (PR 74%, p = 0.035), and a summer peak for post-traumatic forms (PR 77%, p = 0.025). The present study suggests the presence of a temporal pattern of ED arrivals in CH patients treated with OACs.     

Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

Pancreatic cancer has a 5-year survival rate of less than 4%. Despite advances in diagnostic technology, pancreatic cancer continues to be diagnosed at a late and incurable stage. Accurate biomarkers for early diagnosis and to predict treatment response are urgently needed. Since alteration of glucose metabolism is one of the hallmarks of cancer cells, we proposed that pyruvate kinase type M2 (M2PK) and lactate dehydrogenase A (LDHA) enzymes could represent novel diagnostic markers and potential therapeutic targets in pancreatic cancer. In 266 tissue sections from normal pancreas, pancreatic cystic neoplasms, pancreatic intraepithelial neoplasia (PanIN) and cancer, we evaluated the expression of PKM2, LDHA, Ki-67 and CD8+ by immunohistochemistry and correlated these markers with clinicopathological characteristics and patient survival. PKM2 and LDHA expression was also assessed by Western blot in 10 human pancreatic cancer cell lines. PKM2 expression increased progressively from cyst through PanIN to cancer, whereas LDHA was overexpressed throughout the carcinogenic process. All but one cell line showed high expression of both proteins. Patients with strong PKM2 and LDHA expression had significantly worse survival than those with weak PKM2 and/or LDHA expression (7.0 months vs. 27.9 months, respectively, p = 0.003, log rank test). The expression of both PKM2 and LDHA correlated directly with Ki-67 expression, and inversely with intratumoral CD8+ cell count. PKM2 was significantly overexpressed in poorly differentiated tumours and both PKM2 and LDHA were overexpressed in larger tumours. Multivariable analysis showed that combined expression of PKM2 and LDHA was an independent poor prognostic marker for survival. In conclusion, our results demonstrate a high expression pattern of two major glycolytic enzymes during pancreatic carcinogenesis, with increased expression in aggressive tumours and a significant adverse effect on survival.     

Lack of TNF‐alpha receptor type 2 protects motor neurons in a cellular model of amyotrophic lateral sclerosis and in mutant SOD1 mice but does not affect disease progression

Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1‐G93A co‐cultures. Deleting TNFR2 from SOD1‐G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1‐G93A/TNFR2?/? mice showed high phospho‐TAR DNA‐binding protein 43 (TDP‐43) accumulation and low levels of acetyl‐tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane‐bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology.

     

Multiple signaling events in amyloid beta-induced,oxidative stress-dependent neuronal apoptosis

Current evidence suggests that amyloid beta peptides (Abeta) may play a major role in the pathogenesis of Alzheimer's disease by eliciting oxidative stress and neuronal apoptosis. In this study we have used differentiated SK-N-BE neurons to investigate molecular mechanisms and regulatory pathways underlying apoptotic neuronal cell death elicited by Abeta(1-40) and Abeta(1-42) peptides as well as the relationships between apoptosis and oxidative stress. Abeta peptides, used at concentrations able to induce oxidative stress, elicit a classic type of neuronal apoptosis involving mitochondrial regulatory proteins and pathways (i.e. affecting Bax and Bcl-2 protein levels as well as release of cytochrome c in the cytosol), poly-ADP rybose polymerase cleavage and activation of caspase 3. This pattern of neuronal apoptosis, that is significantly prevented by alpha-tocopherol and N-acetylcysteine and completely abolished by specific inhibitors of stress-activated protein kinases (SAPK) such as JNKs and p38(MAPK), involved early elevation of p53 protein levels. Pretreatment of neurons with alpha-pifithrin, a specific p53 inhibitor, resulted in a 50-60% prevention of Abeta induced apoptosis. These results suggest that oxidative stress - mediated neuronal apoptosis induced by amyloid beta operates by eliciting a SAPK-dependent multiple regulation of pro-apoptotic mitochondrial pathways involving both p53 and bcl-2.     

Identification of early secretory antigen target-6 epitopes for the immunodiagnosis of active tuberculosis

The early secretory antigenic target (ESAT)-6 purified protein and peptides from Mycobacterium tuberculosis were evaluated as antigens for the immunodiagnosis of tuberculosis (TB). Because the control of TB requires improved diagnostic procedures, efforts have increased to identify Mycobacterium tuberculosis-specific epitopes for the immunodiagnosis of active TB and to discriminate between active and latent states of infection. Two multiepitopic peptides from ESAT-6 protein were selected by computational analysis. Patients with active TB (7 HIV(+) and 20 HIV(-)) and control patients (17 HIV(+) and 28 HIV(-)) were enrolled. Enzyme-linked immunospot assay analysis for interferon-g expression by peripheral blood mononuclear cells was quantified after stimulation with selected ESAT-6 peptides, purified protein derivative, or the intact ESAT-6 protein. During active TB, 20 of 27 patients responded in vitro to ESAT-6 peptides and 23 of 27 patients to purified protein derivative. None of the controls without active TB, including individuals with latent TB infection, recognized ESAT-6 peptides. By contrast, latently infected individuals did respond in vitro to both intact ESAT-6 protein and purified protein derivative. Thus, high T-cell response frequencies to ESAT-6 peptides are present only during active TB and can be used to discriminate between active and latent forms of infection.     

Highly variable rates of genome rearrangements between hemiascomycetous yeast lineages

Hemiascomycete yeasts cover an evolutionary span comparable to that of the entire phylum of chordates. Since this group currently contains the largest number of complete genome sequences it presents unique opportunities to understand the evolution of genome organization in eukaryotes. We inferred rates of genome instability on all branches of a phylogenetic tree for 11 species and calculated species-specific rates of genome rearrangements. We characterized all inversion events that occurred within synteny blocks between six representatives of the different lineages. We show that the rates of macro- and microrearrangements of gene order are correlated within individual lineages but are highly variable across different lineages. The most unstable genomes correspond to the pathogenic yeasts Candida albicans and Candida glabrata. Chromosomal maps have been intensively shuffled by numerous interchromosomal rearrangements, even between species that have retained a very high physical fraction of their genomes within small synteny blocks. Despite this intensive reshuffling of gene positions, essential genes, which cluster in low recombination regions in the genome of Saccharomyces cerevisiae, tend to remain syntenic during evolution. This work reveals that the high plasticity of eukaryotic genomes results from rearrangement rates that vary between lineages but also at different evolutionary times of a given lineage.     

Structural Alteration of the Dorsal Visual Network in DLB Patients with Visual Hallucinations: A Cortical Thickness MRI Study

Visual hallucinations (VH) represent one of the core features in discriminating dementia with Lewy bodies (DLB) from Alzheimer’s Disease (AD). Previous studies reported that in DLB patients functional alterations of the parieto-occipital regions were correlated with the presence of VH. The aim of our study was to assess whether morphological changes in specific cortical regions of DLB could be related to the presence and severity of VH. We performed a cortical thickness analysis on magnetic resonance imaging data in a cohort including 18 DLB patients, 15 AD patients and 14 healthy control subjects. Relatively to DLB group, correlation analysis between the cortical thickness and the Neuropsychiatric Inventory (NPI) hallucination item scores was also performed. Cortical thickness was reduced bilaterally in DLB compared to controls in the pericalcarine and lingual gyri, cuneus, precuneus, superior parietal gyrus. Cortical thinning was found bilaterally in AD compared to controls in temporal cortex including the superior and middle temporal gyrus, part of inferior temporal cortex, temporal pole and insula. Inferior parietal and supramarginal gyri were also affected bilaterally in AD as compared to controls. The comparison between DLB and AD evidenced cortical thinning in DLB group in the right posterior regions including superior parietal gyrus, precuneus, cuneus, pericalcarine and lingual gyri. Furthermore, the correlation analysis between cortical thickness and NPI hallucination item scores showed that the structural alteration in the dorsal visual regions including superior parietal gyrus and precuneus closely correlated with the occurrence and severity of VH. We suggest that structural changes in key regions of the dorsal visual network may play a crucial role in the physiopathology of VH in DLB patients.