Sitting Postural Management to Prevent Migration Percentage Progression in Non-Ambulatory Children with Cerebral Palsy: Randomized Controlled Trial Preliminary Data.

Background/Objectives: To determine whether a sitting position with the femoral heads centered into the acetabulum is more effective than the usual sitting position in preventing migration percentage progression in non-ambulatory children with bilateral cerebral palsy. Methods: This was a multicenter, randomized controlled trial. INCLUSION CRITERIA: spastic or dyskinetic cerebral palsy, Gross Motor Function Classification System level IV-V, age 1-6 years, migration percentage <41%, and informed consent. EXCLUSION CRITERIA: contractures affecting the hip, anterior luxation, previous hip surgery, and lumbar scoliosis. The treatment group sat with their hips significantly abducted to reduce the head into the acetabulum in a customized system for at least five hours/day for two years. Controls sat with the pelvis and lower limbs aligned but the hips less abducted in an adaptive seating system. The primary outcome was migration percentage (MP) progression. Health-related quality of life and family satisfaction were among the secondary outcomes. The study was approved by the local ethics board and conducted in accordance with CONSORT reporting guidelines. CLINICALTRIALS: gov ID: NCT04603625. RESULTS: Overall median MP progression was 1.6 after the first year and 2.5 after the second year. No significant differences were observed between the groups. MP exceeded 40% and 50% in 1.8% and 0% of the experimental group and 5.4% and 3.6% of controls in years 1 and 2, respectively. Both groups expressed satisfaction with the postural system and stable health-related quality of life. Conclusions: MP remained stable over the two-year period in both groups. Considering outliers which progressed over 50%, a more protective trend of the hip-centering sitting approach emerged, but this needs to be confirmed in a final, larger dataset.

Anthocyanin-enriched extract from Ribes nigrum inhibits triglyceride and cholesterol accumulation in adipocytes.

Aim: Obesity is a chronic pathology of epidemic proportions. Mature adipocytes from a 3T3-L1 cell line were used as in vitro obesity model to test different bioactive compounds. We aim to evaluate cassis (Ribes nigrum) extract antioxidant activity and its antiadipogenic effect on mature adipocytes. Results: We produced an extract by using enzyme that combines cellulase and pectinase; we obtained high yield of the bioactive compound anthocyanin. Extract showed high antioxidant capacity. We conducted in vitro assays by adding the extract to adipocytes culture medium. Extract reduced intracellular levels of triglyceride by 62% and cholesterol by 32%. Conclusion: Enzymatic extract's high antioxidant activity was likely attributable to its high concentration of anthocyanin. This extract inhibits lipid accumulation in adipocytes.

Obesity is a disease all over the world. By 2030, nearly 20% of adults are predicted to be obese. The consumption of processed foods is related to obesity in some countries such as Argentina. More natural food is needed. There are many different anti-obesity medicines but there is no good one to lose weight. We took extracts from cassis fruits and tested whether they could decrease fats like cholesterol within fat cells. We found that these extracts could successfully reduce the fat levels in the cells. Our results indicate that natural compounds like cassis fruit extract may be helpful in preventing future obesity epidemics.

RSPO1-mutated keratinocytes from palmoplantar keratoderma display impaired differentiation, alteration of cell-cell adhesion, EMT-like phenotype and invasiveness properties: implications for squamous cell carcinoma susceptibility in patients with 46XX disorder of sexual development.

BACKGROUND: Secreted R-spondin (RSPO) proteins play a key role in reproductive organ development, epithelial stem cell renewal and cancer induction by reinforcing canonical Wnt signaling. We have previously reported that palmoplantar keratoderma (PPK), predisposition to cutaneous squamous cell carcinoma (SCC) development and sex reversal segregate as autosomal recessive trait in patients carrying RSPO1-mutations. Although our previous findings suggested that RSPO1 secreted from fibroblasts regulates keratinocyte growth or differentiation, the role of this protein in the epidermis remains largely unexplored. Our study was aimed at expanding the phenotypic, molecular and functional characterization of RSPO1-mutated skin and keratinocytes. RESULTS: Cultured primary keratinocytes from PPK skin of a RSPO1-mutated XX-sex reversed patient displayed highly impaired differentiation and epithelial-mesenchymal transition (EMT)-like phenotype. Interestingly, RSPO1-mutated PPK skin expressed markers of increased proliferation, dedifferentiation and altered cell-cell adhesion. Furthermore, all these signs were more evident in SCC specimens of the patient. Cultured PPK patient's keratinocytes exhibited increased expression of cell‒matrix adhesion proteins and extracellular matrix remodeling enzymes. Moreover, they showed invasiveness properties in an organotypic skin model in presence of PPK fibroblasts, which behave like cancer-associated fibroblasts. However, the co-culture with normal fibroblasts or treatment with the recombinant RSPO1 protein did not revert or reduce the EMT-like phenotype and invasion capability of PPK keratinocytes. Notably, RSPO1-mutated PPK fibroblasts induced a hyperproliferative and dedifferentiated phenotype of age-matched normal control plantar keratinocytes. Wnt signaling has a key role in both PPK promotion and SCC development. Accordingly, Wnt mediators were differentially expressed in both PPK keratinocytes and skin specimens of RSPO1-mutated patient compared to control. CONCLUSIONS: Altogether our data indicate that the absence of RSPO1 in patients with 46XX disorder of sexual development affects the skin microenvironment and epidermal integrity, thus contributing to the risk of SCC tumorigenesis in palmoplantar regions exposed to major frictional stresses.

RSPO1-mutated fibroblasts from non-tumoural areas of palmoplantar keratoderma display a cancer-associated phenotype.

R-spondin (RSPO)1 is a fibroblast-secreted protein that belongs to the R-spondin protein family which is essential for reproductive organ development, epithelial stem cell renewal and cancer induction or suppression. RSPO1 gene mutations cause palmoplantar hyperkeratosis with squamous cell carcinoma (SCC) of the skin, 46XX sex reversal and true hermaphroditism. To characterize RSPO1-deficient skin fibroblasts derived from two patients with mutations in RSPO1, with palmoplantar hyperkeratosis, recurrent SCC and 46XX sex reversal, to provide further insight into disease-related skin tumourigenesis. Fibroblast cultures from non-tumoural palmoplantar skin biopsies were established to evaluate features and properties that may be altered at cancer onset, i.e. proliferation, extracellular matrix contraction and invasion, as well as TGF-ß and matrix metalloproteinase (MMP) secretion. Fibroblasts demonstrated increased proliferative potential in vitro, a high level of collagen contraction and invasion by SCC cells, release of high levels of pro-inflammatory and pro-fibrotic TGF-ß, and increased expression of MMP1 and MMP3. Analysis of the expression of selected proteins associated with RSPO1-activated pathways confirmed sustained activation of the TGF-ß signalling pathway and indicated a loss of TGF-ß inhibitory feedback. Also, treatment of fibroblasts with a recombinant RSPO1 protein aggravated this pro-inflammatory phenotype, suggesting caution in designing therapeutic strategies based on restoration of protein function. Our findings indicate that fibroblasts from RSPO1-mutated patients behave similarly to cancer-associated fibroblasts. Chronic inflammation and fibrotic changes in palmoplantar skin may play a role in SCC development and recurrence, possibly by irreversibly activating the tumourigenic phenotype of fibroblasts.

N-acetylcysteine delivery with silica nanoparticles into 3T3-L1 adipocytes.

Background: The addition of 5 mM N-acetylcysteine (NAC) to 3T3-L1 adipocytes culture inhibits the accumulation of triglycerides (Tg) by 50%, but after 48 h uptake was only 16% of total NAC available. Based on these results, the aim of this study is to increase the NAC cellular uptake by encapsulating it in silica nanoparticles (NPs). Materials & methods: Silica NPs, 20 ± 4.5 nm in size, were developed, with an inner cavity loaded with 5 mM NAC. At 48 h after treatment, there was a dose-dependent cytotoxic effect. We attempted to reduce the cytotoxicity of silica NPs by coating them with bovine serum albumin. Results: While we obtained nontoxic bovine serum albumin coated NPs, their effect on Tg cellular accumulation was also reduced.

The Secretome of Aged Fibroblasts Promotes EMT-Like Phenotype in Primary Keratinocytes from Elderly Donors through BDNF-TrkB Axis.

Age-related changes in the dermis can play a primary role in tumor initiation promoting the unrestrained proliferation of precancerous keratinocytes (KCs) through cytokines and GF secretion. We found a high percentage of epithelial-to-mesenchymal transition-like colonies raising in primary human KC cultures from old subjects after treatment with aged fibroblast supernatants (SPNs). Continuous extracellular signals were required for maintaining these changes. Conversely, the secretome did not induce epithelial-to-mesenchymal transition-like colonies in KCs from young subjects. SPN-treated aged KCs displayed the activation of pathways involved in the disjunction of cell‒cell adhesion, extracellular matrix remodeling, manifestation of a mesenchymal phenotype, and dedifferentiation programs. Moreover, they recovered proliferation and clonogenic ability and showed enhanced migration. We identified an age-related increase of the BDNF secretion from fibroblasts as well as of the expression of its receptor TrkB in KCs. BDNF treatment of aged KCs induced TrkB phosphorylation and recapitulated the modifications promoted by aged fibroblast SPN. Furthermore, the treatment with a specific antibody against BDNF or a TrkB antagonist inhibited the paracrine signaling preventing SPN-mediated morphological and molecular changes. Finally, BDNF induced signs of matrix invasion in a three-dimensional organotypic model. Therefore, we demonstrate that aged fibroblast SPN promotes phenotypic plasticity in KCs from the elderly through BDNF-TrkB axis.

Multiple Skin Squamous Cell Carcinomas in Junctional Epidermolysis Bullosa Due to Altered Laminin-332 Function.

Variably reduced expression of the basement membrane component laminin-332 (α3aß3γ2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a ß3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to γ2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.

Cockayne Syndrome Type A Protein Protects Primary Human Keratinocytes from Senescence.

Defects in Cockayne syndrome type A (CSA), a gene involved in nucleotide excision repair, cause an autosomal recessive syndrome characterized by growth failure, progressive neurological dysfunction, premature aging, and skin photosensitivity and atrophy. Beyond its role in DNA repair, the CSA protein has additional functions in transcription and oxidative stress response, which are not yet fully elucidated. Here, we investigated the role of CSA protein in primary human keratinocyte senescence. Primary keratinocytes from three patients with CS-A displayed premature aging features, namely premature clonal conversion, high steady-state levels of reactive oxygen species and 8-OH-hydroxyguanine, and senescence-associated secretory phenotype. Stable transduction of CS-A keratinocytes with the wild-type CSA gene restored the normal cellular sensitivity to UV irradiation and normal 8-OH-hydroxyguanine levels. Gene correction was also characterized by proper restoration of keratinocyte clonogenic capacity and expression of clonal conversion key regulators (p16 and p63), decreased NF-κB activity and, in turn, the expression of its targets (NOX1 and MnSOD), and the secretion of senescence-associated secretory phenotype mediators. Overall, the CSA protein plays an important role in protecting cells from senescence by facilitating DNA damage processing, maintaining physiological redox status and keratinocyte clonogenic ability, and reducing the senescence-associated secretory phenotype-mediated inflammatory phenotype.

Microenvironmental influence on microtumour infiltration patterns: 3D-mathematical modelling supported by in vitro studies.

Mathematical modelling approaches have become increasingly abundant in cancer research. Tumour infiltration extent and its spatial organization depend both on the tumour type and stage and on the bio-physicochemical characteristics of the microenvironment. This sets a complex scenario that often requires a multidisciplinary and individually adjusted approach. The ultimate goal of this work is to present an experimental/numerical combined method for the development of a three-dimensional mathematical model with the ability to reproduce the growth and infiltration patterns of a given avascular microtumour in response to different microenvironmental conditions. The model is based on a diffusion-convection reaction equation that considers logistic proliferation, volumetric growth, a rim of proliferative cells at the tumour surface, and invasion with diffusive and convective components. The parameter values of the model were fitted to experimental results while radial velocity and diffusion coefficients were made spatially variable in a case-specific way through the introduction of a shape function and a diffusion-limited-aggregation (DLA)-derived fractal matrix, respectively, according to the infiltration pattern observed. The in vitro model consists of multicellular tumour spheroids (MTSs) of an epithelial mammary tumour cell line (LM3) immersed in a collagen I gel matrix with a standard culture medium ("naive" matrix) or a conditioned medium from adipocytes or preadipocytes ("conditioned" matrix). It was experimentally determined that both adipocyte and preadipocyte conditioned media had the ability to change the MTS infiltration pattern from collective and laminar to an individual and atomized one. Numerical simulations were able to adequately reproduce qualitatively and quantitatively both kinds of infiltration patterns, which were determined by area quantification, analysis of fractal dimensions and lacunarity, and Bland-Altman analysis. These results suggest that the combined approach presented here could be established as a new framework with interesting potential applications at both the basic and clinical levels in the oncology area.

Epidermolysis Bullosa (EB) Acquisita in an Adult Patient with Previously Unrecognized Mild Dystrophic EB and Biallelic COL7A1 Mutations.

Circulating anti-type VII collagen autoantibodies are frequently detected in patients with recessive dystrophic epidermolysis bullosa (RDEB). However, evidence supporting their pathogenic role in inducing epidermolysis bullosa acquisita (EBA) has been provided for only one individual with dominant dystrophic epidermolysis bullosa (DDEB). We describe here a patient who presented with dystrophic toenails since early childhood and developed trauma-induced skin blisters and oral erosions at age 26 years. Direct immunofluorescence showed IgG deposits with a u-serrated pattern along the cutaneous basement membrane zone, while no change in the expression of collagen VII could be detected by antigen mapping. High-titre anti-collagen VII antibodies were detected by enzyme-linked immunoassay (ELISA). In parallel, sequencing of epidermolysis bullosa (EB) genes identified compound heterozygous COL7A1 missense c.410G>A (p.Arg137Gln) and splicing c.3674C>T (p.Ala1225_Gln1241del) mutations, previously unrecognized in dystrophic epidermolysis bullosa (DEB). Thus, our patient had RDEB "nails-only" and developed mechanobullous EBA in adulthood. These data support a pathogenic role of circulating autoantibodies to collagen VII in inducing EBA in selected patients with DEB. Unforeseen worsening of skin symptoms in DEB should prompt laboratory investigations for EBA.

Stromal microenvironment in type VII collagen-deficient skin: The ground for squamous cell carcinoma development.

Recessive dystrophic epidermolysis bullosa (RDEB) is a skin fragility disease caused by mutations that affect the function and/or the amount of type VII collagen (C7), the major component of anchoring fibrils. Hallmarks of RDEB are unremitting blistering and chronic wounds leading to tissue fibrosis and scarring. Nearly all patients with severe RDEB develop highly metastatic squamous cell carcinomas (SCC) which are the main cause of death. Accumulating evidence from a murine RDEB model and human RDEB cells demonstrates that lack of C7 also directly alters the wound healing process. Non-healing RDEB wounds are characterized by increased inflammation, high transforming growth factor-ß1 (TGF-ß1) levels and activity, and are heavily populated by myofibroblasts responsible for enhanced fibrogenesis and matrix stiffness. These changes make the RDEB stroma a microenvironment prone to cancer initiation, where cells with features of cancer-associated fibroblasts are found. Here, we discuss recent knowledge on microenvironment alterations in RDEB, highlighting possible therapeutic targets to prevent and/or delay fibrosis and SCC development.

N-acetylcysteine inhibits kinase phosphorylation during 3T3-L1 adipocyte differentiation.

OBJECTIVES: Reports investigating the effects of antioxidants on obesity have provided contradictory results. We have previously demonstrated that treatment with the antioxidant N-acetylcysteine (NAC) inhibits cellular triglyceride (Tg) accumulation as well as total cellular monoamine oxidase A (MAOA) expression in 3T3-L1 mature adipocytes (Calzadilla et al., Redox Rep. 2013;210-218). Here we analyzed the role of NAC on adipogenic differentiation pathway. METHODS: Assays were conducted using 3T3-L1 preadipocytes (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC). We studied the effects of different doses of NAC (0.01 or 1 mM) on DC, to evaluate cellular expression of phospho-JNK½ (pJNK½), phospho-ERK½ (pERK½) and, mitochondrial expression of citrate synthase, fumarate hydratase and MAOA. RESULTS: Following the differentiation of preadipocytes, an increase in the expression levels of pJNK½ and pERK½ was observed, together with mitotic clonal expansion (MCE). We found that both doses of NAC decreased the expression of pJNK½ and pERK½. Consistent with these results, NAC significantly inhibited MCE and modified the expression of different mitochondrial proteins. DISCUSSION: Our results suggested that NAC could inhibit Tg and mitochondrial protein expression by preventing both MCE and kinase phosphorylation.

Mathematical modelling of microtumour infiltration based on in vitro experiments.

The present mathematical models of microtumours consider, in general, volumetric growth and spherical tumour invasion shapes. Nevertheless in many cases, such as in gliomas, a need for more accurate delineation of tumour infiltration areas in a patient-specific manner has arisen. The objective of this study was to build a mathematical model able to describe in a case-specific way as well as to predict in a probabilistic way the growth and the real invasion pattern of multicellular tumour spheroids (in vitro model of an avascular microtumour) immersed in a collagen matrix. The two-dimensional theoretical model was represented by a reaction-convection-diffusion equation that considers logistic proliferation, volumetric growth, a rim with proliferative cells at the tumour surface and invasion with diffusive and convective components. Population parameter values of the model were extracted from the experimental dataset and a shape function that describes the invasion area was derived from each experimental case by image processing. New possible and aleatory shape functions were generated by data mining and Monte Carlo tools by means of a satellite EGARCH model, which were fed with all the shape functions of the dataset. Then the main model is used in two different ways: to reproduce the growth and invasion of a given experimental tumour in a case-specific manner when fed with the corresponding shape function (descriptive simulations) or to generate new possible tumour cases that respond to the general population pattern when fed with an aleatory-generated shape function (predictive simulations). Both types of simulations are in good agreement with empirical data, as it was revealed by area quantification and Bland-Altman analysis. This kind of experimental-numerical interaction has wide application potential in designing new strategies able to predict as much as possible the invasive behaviour of a tumour based on its particular characteristics and microenvironment.

Frequent Occurrence of Aplasia Cutis Congenita in Bullous Dermolysis of the Newborn.

Bullous dermolysis of the newborn (BDN) is a subtype of dystrophic epidermolysis bullosa characterized by rapid improvement in skin fragility within the first months of life, associated with typical immunofluorescence and ultrastructural features. Inheritance can be autosomal dominant or recessive. We report here 4 cases of BDN, 2 of which presented with aplasia cutis congenita of the lower extremities. All patients improved rapidly and blister formation ceased by the third month of life in 3 cases. In these patients only residual milia, nail dystrophies and atrophic scarring at sites of aplasia cutis were visible by one year. Family history indicated dominant inheritance in 2 cases, confirmed by identification of COL7A1 mutation. Molecular analysis also revealed recessive inheritance in the 2 sporadic cases. A literature search identified several patients with BDN born with skin defects localized to the lower extremities. In conclusion, these findings indicate that aplasia cutis congenita is not an infrequent manifestation of BDN.

The role of oncogenic Ras in human skin tumorigenesis depends on the clonogenic potential of the founding keratinocytes.

The role of Ras in human skin tumorigenesis induction is still ambiguous. Overexpression of oncogenic Ras causes premature senescence in cultured human cells and hyperplasia in transgenic mice. Here, we investigated whether the oncogenic insult outcome might depend on the nature of the founding keratinocyte. We demonstrate that overexpression of the constitutively active Ras-V12 induces senescence in primary human keratinocyte cultures, but that some cells escape senescence and proliferate indefinitely. Ras overexpression in transient-amplifying- or stem-cell-enriched cultures shows that p16 (encoded by CDKN2A) levels are crucial for the final result. Indeed, transient-amplifying keratinocytes expressing high levels of p16 are sensitive to Ras-V12-induced senescence, whereas cells with high proliferative potential, but that do not display p16, are resistant. The subpopulation that sustains the indefinite culture growth exhibits stem cell features. Bypass of senescence correlates with inhibition of the pRb (also known as RB1) pathway and resumption of telomerase reverse transcriptase (TERT) activity. Immortalization is also sustained by activation of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) and Akt pathways. Moreover, only transduced cultures originating from cultures bearing stem cells induce tumors in nude mice. Our findings demonstrate that the Ras overexpression outcome depends on the clonogenic potential of the recipient keratinocyte and that only the stem cell compartment is competent to initiate tumorigenesis.