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.     

WIP Regulates Persistence of Cell Migration and Ruffle Formation in Both Mesenchymal and Amoeboid Modes of Motility

The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.     

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL,MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.     

Citrus Allergy from Pollen to Clinical Symptoms

Allergy to citrus fruits is often associated with pollinosis and sensitization to other plants due to a phenomenon of cross-reactivity. The aims of the present study were to highlight the cross-reactivity among citrus and the major allergenic pollens/fruits, throughout clinical and molecular investigations, and to evaluate the sensitization frequency to citrus fruits in a population of children and adults with pollinosis. We found a relevant percentage of sensitisation (39%) to citrus fruits in the patients recruited and in all of them the IgE-mediated mechanism has been confirmed by the positive response to the prick-to-prick test. RT-PCR experiments showed the expression of Cit s 1, Cit s 3 and a profilin isoform, already described in apple, also in Citrus clementine pollen. Data of multiple sequence alignments demonstrated that Citrus allergens shared high percentage identity values with other clinically relevant species (i.e. Triticum aestivum, Malus domestica), confirming the possible cross-allergenicity citrus/grasses and citrus/apple. Finally, a novelty of the present work has been the expression of two phospholipaseA2 isoforms (PLA2 α and β) in Citrus as well as in Triticum pollens; being PLA2 able to generate pro-inflammatory factors, this enzyme could participate in the activation of the allergenic inflammatory cascade.     

Adenosine-Mediated Enteric Neuromuscular Function Is Affected during Herpes Simplex Virus Type 1 Infection of Rat Enteric Nervous System

Adenosine plays an important role in regulating intestinal motility and inflammatory processes. Previous studies in rodent models have demonstrated that adenosine metabolism and signalling are altered during chronic intestinal inflammatory diseases. However, the involvement of the adenosinergic system in the pathophysiology of gut dysmotility associated to a primary neurodysfunction is still unclear. Recently, we showed that the neurotropic Herpes simplex virus type-1 (HSV-1), orally inoculated to rodents, infects the rat enteric nervous system (ENS) and affects gut motor function without signs of systemic infection. In this study we examined whether changes in purinergic metabolism and signaling occur during permanent HSV-1 infection of rat ENS. Using isolated organ bath assays, we found that contraction mediated by adenosine engagement of A₁ or A_2A receptors was impaired at 1 and 6 weeks post-viral administration. Immunofluorescence studies revealed that viral infection of ENS led to a marked redistribution of adenosine receptors: A₁ and A_2B receptors were confined to the muscle layers whereas A_2A and A₃ receptors were expressed mainly in the myenteric plexus. Viral-induced ENS neurodysfunction influenced adenosine metabolism by increasing adenosine deaminase and CD73 levels in longitudinal muscle-myenteric plexus with no sign of frank inflammation. This study provides the first evidence for involvement of the adenosinergic system during HSV-1 infection of the ENS. As such, this may represent a valid therapeutic target for modulating gut contractility associated to a primary neurodysfunction.     

Removal of Vanadium(III) and Molybdenum(V) from Wastewater Using Posidonia oceanica (Tracheophyta) Biomass

The use of dried and re-hydrated biomass of the seagrass Posidonia oceanica was investigated as an alternative and –low-cost biomaterial for removal of vanadium(III) and molybdenum(V) from wastewaters. Initial characterisation of this biomaterial identified carboxylic groups on the cuticle as potentially responsible for cation sorption, and confirmed the toxic-metal bioaccumulation. The combined effects on biosorption performance of equilibrium pH and metal concentrations were investigated in an ideal single-metal system and in more real-life multicomponent systems. There were either with one metal (vanadium or molybdenum) and sodium nitrate, as representative of high ionic strength systems, or with the two metals (vanadium and molybdenum). For the single-metal solutions, the optimum was at pH 3, where a significant proportion of vanadium was removed (ca. 70%) while there was ca. 40% adsorption of molybdenum. The data obtained from the more real-life multicomponent systems showed that biosorption of one metal was improved both by the presence of the other metal and by high ionic strength, suggesting a synergistic effect on biosorption rather than competition. There data ware used for the development of a simple multi-metal equilibrium model based on the non-competitive Langmuir approach, which was successfully fitted to experimental data and represents a useful support tool for the prediction of biosorption performance in such real-life systems. Overall, the results suggest that biomass of P. oceanica can be used as an efficient biosorbent for removal of vanadium(III) and molybdenum(V) from aqueous solutions. This process thus offers an eco-compatible solution for the reuse of the waste material of leaves that accumulate on the beach due to both human activities and to storms at sea.     

Opposite Roles of NMDA Receptors in Relapsing and Primary Progressive Multiple Sclerosis

Synaptic transmission and plasticity mediated by NMDA receptors (NMDARs) could modulate the severity of multiple sclerosis (MS). Here the role of NMDARs in MS was first explored in 691 subjects carrying specific allelic variants of the NR1 subunit gene or of the NR2B subunit gene of this glutamate receptor. The analysis was replicated for significant SNPs in an independent sample of 1548 MS subjects. The C allele of rs4880213 was found to be associated with reduced NMDAR-mediated cortical excitability, and with increased probability of having more disability than the CT/TT MS subjects. MS severity was higher in the CC group among relapsing-remitting MS (RR-MS) patients, while primary progressive MS (PP-MS) subjects homozygous for the T allele had more pronounced clinical worsening. Mean time to first relapse, but not to an active MRI scan, was lower in the CC group of RR-MS patients, and the number of subjects with two or more clinical relapses in the first two years of the disease was higher in CC compared to CT/TT group. Furthermore, the percentage of relapses associated with residual disability was lower in subjects carrying the T allele. Lesion load at the MRI was conversely unaffected by the C or T allele of this SNP in RR-MS patients. Axonal and neuronal degeneration at the optical coherence tomography was more severe in the TT group of PP-MS patients, while reduced retinal nerve fiber thickness had less consequences on visual acuity in RR-MS patients bearing the T allele. Finally, the T allele was associated with preserved cognitive abilities at the Rao’s brief repeatable neuropsychological battery in RR-MS. Signaling through glutamate NMDARs enhances both compensatory synaptic plasticity and excitotoxic neurodegeneration, impacting in opposite ways on RR-MS and PP-MS pathophysiological mechanisms.     

Age and sex differences in numerical responses,dietary shifts,and total responses of a generalist predator to population dynamics of main prey

Oecologia - Fluctuations in the abundance of main prey species might shape animal communities, by inducing numerical responses and dietary shifts in predators. Whether numerical responses and...