Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol.

Injuries to the peripheral nerves represent a frequent cause of permanent disability in adults. The repair of large nerve lesions involves the use of autografts, but they have several inherent limitations. Overcoming these limitations, the use of decellularized nerve matrix has emerged as a promising treatment in tissue regenerative medicine. Here, we generate longer human decellularized nerve segments with a novel decellularization method, using nonionic, zwitterionic, and enzymatic incubations. Efficiency of decellularization was measured by DNA quantification and cell remnant analysis (myelin, S100, neurofilament). The evaluation of the extracellular matrix (collagen, laminin, and glycosaminoglycans) preservation was carried out by enzyme-linked immunosorbent assay (ELISA) or biochemical methods, along with histological and immunofluorescence analysis. Moreover, biomechanical properties and cytocompatibility were tested. Results showed that the decellularized nerves generated with this protocol have a concentration of DNA below the threshold of 50 ng/mg of dry tissue. Furthermore, myelin, S100, and MHCII proteins were absent, although some neurofilament remnants could be observed. Moreover, extracellular matrix proteins were well maintained, as well as the biomechanical properties, and the decellularized nerve matrix did not generate cytotoxicity. These results show that our method is effective for the generation of decellularized human nerve grafts. The generation of longer decellularized nerve segments would allow the understanding of the regenerative neurobiology after nerve injuries in both clinical assays and bigger animal models. Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol. Combination of zwitterionic, non-ionic detergents, hyperosmotic solution and nuclease enzyme treatment remove cell remnants, maintain collagen, laminin and biomechanics without generating cytotoxic leachables.

Translation and validation of the Visual Function and Corneal Health Status (V-FUCHS) questionnaire into Spanish language.

PURPOSE: To translate and validate the V-FUCHS questionnaire into Spanish in a population of patients with Fuchs endothelial dystrophy (DEF). METHODS: The V-FUCHS consists of 15 short, easily understandable questions that assess visual aspects of quality of life in patients with DEF, which can be gathered into a group of seven that assess the "Visual Difficulty" factor and another group of eight that assess the "Glare Factor". For the translation and cultural adaptation, the standardized norms for this process were followed, among other phases, a translation, a back-translation and an application in patients with DEF. RESULTS: In the first phase, consensus was reached on the Spanish translation of the V-FUCHS. Subsequently, 25 patients were included to carry out the pre-test phase with the aim of assessing the applicability and feasibility of the test. The score obtained a minimum value of -0.88 and a maximum value of +2.44, according to the Rasch probabilistic scale. The mean value obtained from the Visual Difficulty factor was 0.61 (±0.71), while the mean for the Glare Factor was 0.41 (±0.51). CONCLUSION: The validation of the V-FUCHS questionnaire, after its translation and adaptation into Spanish, proved to be a useful tool for assessing the visual quality of patients with DEF. Patients with a more advanced stage of the disease presented a greater severity in the test result. Likewise, the Glare Factor (Glare) correlates better with the pachymetric increase than with the visual acuity of the patient.

Protocol for the treatment of cystoid macular edema secondary to retinitis pigmentosa and other inherited retinal dystrophies.

Inherited retinal dystrophies (IRD) are the leading cause of legal blindness in the working population. Cystic macular edema (CME) is one of the treatable causes of visual loss, affecting up to 50% of the patients. A bibliographic review has been carried out combining "inherited retinal dystrophy", "retinitis pigmentosa", "macular oedema" and a diagnostic-therapeutic protocol according to the levels of evidence and recommendations of the "US Agency for Healthcare Research and Quality". This protocol has been discussed in the monthly meetings of the XAREA DHR group with the participation of more than 25 ophthalmologists, creating a consensus document. The etiology of CME is multifactorial: dysfunction of the blood-retinal barrier, retinal pigment epithelium, and Müller cells, inflammation, and vitreous traction. OCT is the test of choice for the diagnosis and follow-up of CME associated with IRD. The drugs with the highest degree of scientific evidence are carbonic anhydrase inhibitors (IAC). Intravitreal corticosteroids, anti-VEGF, and vitrectomy with peeling of the internal limiting membrane do not have sufficient evidence. A treatment scheme is proposed for the CME in IRD in adults, another for pediatric patients and another for IRD and cataract surgery. Oral and topical IACs are effective in the treatment of CME secondary to IRD. Treatment with corticosteroids, anti-VEGF, and vitrectomy are second-line options. Randomized clinical trials are required to establish the therapeutic scale in these patients.

[Clinical indications for the use of cardiac MRI. By the SIRM Study Group on Cardiac Imaging]. / Indicazioni cliniche per l'utilizzo della cardio RM. A cura del Gruppo di lavoro della Sezione di Cardio-Radiologia della SIRM.

Cardiac magnetic resonance (CMR) is considered an useful method in the evaluation of many cardiac disorders. Based on our experience and available literature, we wrote a document as a guiding tool in the clinical use of CMR. Synthetically we describe different cardiac disorders and express for each one a classification, I to IV, depending on the significance of diagnostic information expected.

Pterygium surgery with lyophilized versus cryopreserved amniotic membrane graft.

PURPOSE: To evaluate surgical outcomes (recurrence rate, aesthetics and symptoms) of pterygium surgery with two different amniotic membrane preservation approaches - lyophilized (LAM) and cryopreserved (CAM). METHODS: Primary pterygium patients were randomized to either LAM or CAM surgery. Demographic data, ocular surface disease index (OSDI), aesthetic grading (1 to 4), recurrences and complications were recorded over a 6-month follow-up period. RESULTS: Twenty-nine patients were recruited. Recurrence at month 6 was detected in 11 cases (37.9%) and was more prevalent with CAM grafts, without reaching statistical significance (P=0.196). Aesthetic outcome grading showed no differences between LAM and CAM at month 6 (P=0.124). Aesthetic results were mostly unsatisfactory (grade 3 and 4) without statistical differences between groups (P=0.514). Baseline OSDI was similar in both groups (P=0.888), and it significantly decreased by the last follow-up visit (P<0.001) for both the LAM and CAM groups. This decrease did not significantly differ between amniotic membrane preservation approach surgery groups (P=0.714). CONCLUSION: LAM might be considered a legitimate alternative to CAM, showing no inferiority in outcomes, since clinical and aesthetic outcomes were similar for both groups.

Role of first pass and delayed enhancement in assessment of segmental functional recovery after acute myocardial infarction.

PURPOSE: Assessing myocardial viability is crucial in decision making and prognostic restratification after acute myocardial infarction (MI). A number of noninvasive imaging modalities have been employed in viability identification, but contrast-enhanced magnetic resonance (MR) imaging has been shown to be extremely accurate because of its transmural resolution and precise definition of microvascular obstruction. Our purpose was to assess functional recovery after acute MI, with special focus on the role of infarct transmurality and microvascular obstruction. MATERIALS AND METHODS: Forty-six consecutive patients with first acute MI, reperfused by primary percutaneous transluminal coronary angioplasty (PTCA) (n=40) or fibrinolysis (n=6), underwent MR imaging within the first week to assess oedema, microvascular obstruction, function and viability and then again after 4-6 months to assess functional recovery and scar. RESULTS: At first MR examination, postcontrast images were analysed according to three patterns, based on a combination of first-pass and delayed-enhancement data: pattern 1 (normal first pass and late hyperenhancement <50% thickness) identified viable myocardium, whereas pattern 2 (late hyperenhancement >50% thickness, with or without first-pass perfusion defect) and pattern 3 (perfusion defect at first pass and late hypoenhancement) recognised nonviable myocardium, with 93% sensitivity, 75% specificity, 92% positive predictive value and 78% negative predictive value for identifying viable tissue. Furthermore, by dividing pattern 2 into two subpatterns, 2A and 2B, based on absence or presence of microvascular obstruction in >50% transmural infarcts, we were able to better identify the segments without recovery or that were nonviable with a 1.39 relative risk of failed recovery. CONCLUSIONS: After acute MI, not all infarcts with transmurality >50% can be considered nonviable; microvascular obstruction detected at first pass can help to better stratify these cases.

Clinical indications for cardiac computed tomography. From the Working Group of the Cardiac Radiology Section of the Italian Society of Medical Radiology (SIRM).

Cardiac computed tomography (CCT) has grown as a useful means in different clinical contexts. Technological development has progressively extended the indications for CCT while reducing the required radiation dose. Even today there is little documentation from the main international scientific societies describing the proper use and clinical indications of CCT; in particular, there are no complete guidelines. This document reflects the position of the Working Group of the Cardiac Radiology Section of the Italian Society of Radiology concerning the indications for CCT.

Fast protocol for the processing of split-thickness skin into decellularized human dermal matrix.

BACKGROUND: Dermal scaffolds for tissue regeneration are nowadays an effective alternative in not only wound healing surgeries but also breast reconstruction, abdominal wall reconstruction and tendon reinforcement. The present study describes the development of a decellularization protocol applied to human split-thickness skin from cadaveric donors to obtain dermal matrix using an easy and quick procedure. METHODS: Complete split-thickness donor was decellularized through the combination of hypertonic and enzymatic methods. To evaluate the absence of epidermis and dermal cells, and ensure the integrity of the extracellular matrix (ECM) structure, histological analysis was performed. Residual genetic content and ECM biomolecules (collagen, elastin, and glycosaminoglycan) were quantified and tensile strength was tested to measure the effect of the decellularization technique on the mechanical properties of the tissue. RESULTS: Biomolecules quantification, residual genetic content (below 50 ng/mg dry tissue) and histological structure assessment showed the efficacy of the decellularization process and the preservation of the ECM. The biomechanical tests confirmed the preservation of native properties in the acellular tissue. CONCLUSIONS: The acellular dermal matrix obtained from whole split-thickness skin donor with the newly developed decellualrization protocol, maintains the desired biomechanical and structural properties and represents a viable treatment option for patients.

Rab27a: A key to melanosome transport in human melanocytes.

Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes.

[Acute posterior multifocal placoid pigment epitheliopathy. Study of 16 cases]. / Epiteliopatía pigmentaria placoide posterior multifocal aguda. Estudio de 16 casos.

OBJECTIVE: Acute Posterior Multifocal Placoid Pigment Epitheliopathy (APMPPE) is a rare disease with a probable inflammatory component which mostly affects young patients. The aim of our study was to analyse the demographic and clinical features of this disease in a group of 16 patients. METHODS: Sixteen patients with APMPPE were included in this study. We analyzed their demographic data (age, sex) and the most relevant clinical findings: visual acuity and retinal disease outcome, association with other systemic diseases and response to treatment. We also collected data from fluorescence angiography, autofluorescence and optical coherence tomography (OCT) in some of the patients. RESULTS: Average age at diagnosis was 26.75 years with no sex predilection. Average final visual acuity (Snellen Scale) in our study was 0.73. Four patients presented with a systemic disease related to the APMPPE. Eleven patients were treated with oral steroids (one patient with steroids and cytotoxic agents) while the remaining 5 patients received no treatment. CONCLUSIONS: In our patients, the average age at diagnosis was less than 30 years, with no sex predilection, as previously described by many authors. The visual outcome is usually good regardless of the treatment given, although there are cases with a bad visual outcome, especially those with foveal involvement when initially seen.

Determination of the Culture Time Point to Induce Corneal Epithelial Differentiation in Induced Pluripotent Stem Cells.

BACKGROUND: Limbal stem cells (LSC) are progenitor cells in the ocular surface that renew the corneal epithelium. Limbal stem cell deficiency usually induces blindness through the loss of corneal transparency, and bilateral cases do not an accurate treatment because of the lack of an autologous source of stem cells. METHODS: Induced pluripotent stem cells (iPSC) are promising for use in cell therapy because of their autologous origin and the capability to differentiate into corneal epithelial cells. However, there are not standardized protocols to achieve a complete corneal epithelial differentiation. We examined the expression of several markers in a human episomal iPSC line after an induction period from embryoid bodies. RESULTS: Progenitor LSC and corneal epithelial differentiation markers, some extracellular matrix protein adhesion molecules, and wingless signaling pathway were studied. Overall, LSC progenitor and corneal epithelium differentiation markers increased after maintaining cell culture in specific conditions for 14 days, whereas pluripotency markers decreased. CONCLUSIONS: Our approach indicated that the optimal time point to initiate iPSC differentiation into LSC and corneal phenotypes, with the use of specific medium, is from 14 days after initial embryoid bodies treatment induction.

An improved method using densitometry for evaluating severity of laser photocoagulation induced CNV.

Laser photocoagulation induced choroidal neovascularization currently is the most effective model available for the study of this disease in terms of efficacy of new drugs and therapies. Previously, evaluating the extent of choroidal neovascularization using this model was time-consuming and required the use of experienced personnel. We describe a new method for simple and rapid evaluation of laser induced choroidal neovascularization using densitometry. Fluorescein angiograms of a laser photocoagulated rat eye were scanned into a computer. Densitometry software subsequently was used to calculate the severity of the laser lesions. The densitometry method proved effective for calculating the extent of laser induced choroidal neovascularization. In addition, this method was more rapid than visual evaluations and less likely to produce errors.

[Efficacy of sodium carboxymethylcellulose in the treatment of dry eye syndrome]. / Eficacia de la carboximetilcelulosa sódica para el tratamiento del síndrome del ojo seco.

AIM: To assess the efficacy of sodium carboxymethylcellulose in the treatment of dry eye. MATERIAL AND METHODS: We carried out a prospective, randomized, masked-observer, control/problem group, single-center clinical assay during a period of 12 months in 19 patients that presented mild or moderate forms of dry eye. Patients were clinically evaluated each 3 months and treated with a 0.5% isotonic solution of sodium carboxymethylcellulose (CMC) or balanced salt solution. Subjective symptoms, functional tests and conjunctival impression cytology were performed according preexistent schedule study visits. To compare data between groups chi squared (chi2) analysis was applied. RESULTS: We observed a significant (p<0.05) decrease in the frequency of subjective symptoms and a significant (p<0.05) improvement of tearfilm interface stability after CMC treatment. There was a tendency to improve the degree of corneal surface wettability and the tearfilm integrity with higher percentage improvements in the CMC group compared to controls. Improved baseline values in at least one of the objective functional tests carried out (p<0.05) was also observed in an elevated percentage of patients in the CMC group (83.3%) compared with controls (34%). Furthermore, we observed a tendency to diminish the frequency of associated subjective symptoms after treatment. Conjunctival impression cytology did not provide significant differences related with therapeutic response. CONCLUSIONS: The results show a significant beneficial effect of CMC to improve clinical parameters in mild and moderate forms of dry eye.

Alpha v integrin antagonists induce the disassembly of focal contacts in melanoma cells.

In recent years, several antagonists of alpha(v)beta3 have been used to develop therapeutic approaches to the treatment of melanoma neoplasia. We studied the effects of anti-alpha(v)-integrin-blocking antibodies on attached M21 melanoma cells, the cellular distribution of alpha(v)-integrin and the molecular organization of focal structures. Anti-alpha(v)-integrin-blocking antibodies 17E6 and LM609, and an anti-alpha(v)beta3-integrin antagonist peptide cRGD 85189 induced detachment of M21 melanoma cells cultured for 24 hours on various substrates. cRGD was the most effective antagonist, reducing the number of adherent cells by 80%, while 17E6 reduced adhesion by only 30%. Light- and electron microscopy revealed attached cells with a flat shape and well-formed actin cytoskeleton. After treatment, cells became rounded and detached from the culture dish. alpha(v)-Integrins and focal-contact proteins were observed at adhesion sites in focal structures by immunocytochemistry. After treatment, however, cell rounding was accompanied by disorganization of the actin filaments and redistribution of alpha(v)-integrins and most of the focal proteins studied, except vinculin and tensin. Our results indicate that treatment of M21 melanoma cells with a(v)-integrin antagonists disrupts the actin cytoskeleton, redistributes a(v)-integrin and induces molecular disassembly of focal contacts.

The role of O-linked sugars in determining the very low density lipoprotein receptor stability or release from the cell.

The very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O-linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O-linked sugar domain presented a rapid cleavage from the cell and that a large amino-terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O-linked sugar domain may be responsible for blocking the access to the protease-sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O-glycosylation, was used. The instability of the O-linked sugar-deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O-linked sugar domain. Moreover, our data suggest that the O-linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O-linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O-linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor.

The role of fibronectin, laminin, vitronectin and their receptors on cellular adhesion in proliferative vitreoretinopathy.

PURPOSE: To examine the possible role of some adhesion multifunctional glycoproteins of the extracellular matrix, such as fibronectin (FN), laminin (LN), vitronectin (VN) and their receptors (beta 1-subunit complex and alpha v beta 3 integrins) in events of cell migration and adhesion in proliferative vitreoretinopathy (PVR). METHODS: Optical and electron-immunocytochemical techniques were carried out on epiretinal membranes. Electrophoretic immunoblotting methods and densitometric analysis of normal and PVR vitreous were also undertaken. Chi-square (chi 2) and unbalanced analysis of variance were employed for statistical analysis. RESULTS: FN was detected as a major component in the extracellular matrix in both fibrillar and pericellular arrangement. A change in pericellular distribution to more fibrillous organization was related to the time of intraocular proliferative tissue development (P < 0.001). LN and VN were observed as minor components in extracellular matrix. A colocalized pattern between VN and FN in collagenic bundles of the matrix was often observed. Beta-1 subunit and alpha v beta 3 receptors were usually localized in a position that could mediate the interaction of FN, VN, and/or LN to the cell plasma membrane. Increased levels of FN concentration were observed in both subretinal fluid and pathologic vitreous; intravitreal FN concentration tends to increase with clinical stages of the evolution of PVR, whereas intravitreal VN levels tend to decrease. CONCLUSIONS: Results suggest that FN could mediate the initial events involved in epiretinal membrane formation, and VN could modulate the adhesion mechanisms in established membranes.

Epithelial-mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells.

PURPOSE: To improve our understanding of how retinal pigment epithelial (RPE) cells behave in vivo and to establish similarities with dedifferentiation and adaptive events observed in RPE cells cultured under simulated intraocular pathologic conditions. At the same time, to examine the origin of epithelioid-shaped and fibroblast/fusiform-shaped cells in epiretinal membranes (ERM) from proliferative vitreoretinopathy (PVR). METHODS: Cells of ERM were studied by electron-immunocytochemical techniques, using simple, double, and triple immunostaining for cytokeratins (CK), vimentin (Vim), and glial fibrillary acidic protein (GFAP). Ultrastructural morphology analysis was also carried out. Adult human RPE cells were obtained and cultured with normal and pathologic vitreous from proliferative vitreoretinal disorders, subretinal fluid aspirates from retinal detachment, and normal human serum. Their cytoskeleton was fractionated at 7 (early cultures) and 24 (late cultures) days of culture, electrophoresed, immunoblotted for intermediate filament proteins, and quantified by densitometric analysis for each condition. Changes in phenotype characteristics were also evaluated. RESULTS: Epithelioid-shaped and fibroblast/fusiform-shaped cells, resembling RPE cells, expressed CK-Vim-GFAP simultaneously as intermediate filament proteins in their cytoskeleton. RPE cells in culture also expressed CK-Vim-GFAP and changed from an epithelial shape to a migratory fibroblast/fusiform-shaped phenotype in the presence of subretinal fluid aspirates and pathologic vitreous from proliferative intraocular disorders. In simulated cultures of proliferative intraocular disorders, cells decreased or retained their CK7, CK8, and CK18, retained Vim, and increased CK19 and GFAP, while their mesenchymal morphology became clearer over time. CONCLUSIONS: Studies of intermediate filament proteins in vivo suggest that dedifferentiation occurs in RPE cells in ERM. Dedifferentiated RPE cells may be responsible for epithelioid-like and fibroblast/fusiform-like cells. Furthermore, changes in intermediate filament protein levels were observed in RPE cells in simulated cultures of proliferative intraocular disorders. These changes were linked to cells acquiring a mesenchymal migratory, phenotype. Results indicate that the dedifferentiation of RPE cells occurs both in vivo and in vitro and that it can be explained as an epithelial-mesenchymal transition.

High-resolution genetic map of Nb, a gene that confers hypersensitive resistance to potato virus X in Solanum tuberosum.

Nb is a single dominant gene in potato that confers hypersensitive resistance to potato virus X (PVX) isolates from strain groups 1 and 2. Genetic and molecular analyses showed that Nb is located on the upper arm of chromosome V and forms part of a cluster of resistance genes encoding specificities to many different pathogens. We describe the genetical localisation of molecular markers tightly linked to the Nb locus and the development PCR-based markers suitable for isolation of the Nb resistance gene by positional cloning. A bulked segregant approach was applied to identify polymorphic AFLP markers tightly linked to the Nb locus. These markers were mapped in a population of segregating S1 progeny (1,300 plants) from a self-pollinated potato cultivar, Pentland Ivory. From this analysis, Nb was placed in an interval of 0.76 cM, flanked by the AFLP markers GM339 and GM637. Recombinant PVX strains carrying different combinations of avirulence genes were used in biological assays to show that Nb was also present in potato cv. Cara but was masked by the extreme PVX resistance conferred by the Rx gene. PCR-based screening of a Cara genomic BAC library with markers closest to the Nb locus identified a new marker tightly linked to Nb.