Mesenchymal stromal cells for articular cartilage repair: preclinical studies.

Rheumatic diseases such as osteoarthritis (OA) are a major social and economic burden because of the population aging and the lack of curative solutions. An effective cell therapy may be the best treatment option for OA and other cartilage diseases. However, the main cellular strategy used to repair articular cartilage, the transplantation of autologous chondrocytes, is limited to a small number of patients with traumatic lesions. The use of joint replacement after years of disease progression proves the great medical need in current practice. Mesenchymal stromal/stem cells (MSCs) provide an alternative cell source for cartilage regeneration due to numerous advantages, comprising relative ease to isolate and culture, chondrogenic capacity, and anti-inflammatory effects. Initial clinical trials with MSCs have led to encouraging results, but many variables have to be considered to attain true amelioration of disease or repair (type and status of cartilage disease, source and conditions of cells, administration regime, combinatorial approaches). Particularly, allogeneic MSCs are an advantageous cellular product. The animal models chosen for preclinical evaluation are also relevant for successful translation into clinical practice. Considering the limitations in the field, rigorous comparative and validating studies in well-established animal models (including large animals) are still needed to set up the bases for additional clinical trials. The present review of studies performed in small and large animal models should help clarify the applicability of MSC-based therapies for articular cartilage repair.

Corrigendum: Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

This corrects the article DOI: 10.1038/srep43923.

Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

Stem cells possess significant age-dependent differences in their immune-response profile. These differences were analysed by Next-Generation Sequencing of six age groups from bone marrow mesenchymal stem cells. A total of 9,628 genes presenting differential expression between age groups were grouped into metabolic pathways. We focused our research on young, pre-pubertal and adult groups, which presented the highest amount of differentially expressed genes related to inflammation mediated by chemokine and cytokine signalling pathways compared with the newborn group, which was used as a control. Extracellular vesicles extracted from each group were characterized by nanoparticle tracking and flow cytometry analysis, and several micro-RNAs were verified by quantitative real-time polymerase chain reaction because of their relationship with the pathway of interest. Since miR-21-5p showed the highest statistically significant expression in extracellular vesicles from mesenchymal stem cells of the pre-pubertal group, we conducted a functional experiment inhibiting its expression and investigating the modulation of Toll-Like Receptor 4 and their link to damage-associated molecular patterns. Together, these results indicate for the first time that mesenchymal stem cell-derived extracellular vesicles have significant age-dependent differences in their immune profiles.

Influence of age on rat bone-marrow mesenchymal stem cells potential.

Mesenchymal stem cells promising role in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age. Six age groups from bone marrow mesenchymal stem cells of Wistar rats were studied (newborn, infant, young, pre-pubertal, pubertal and adult). Quantitative proteomic assay was performance by iTRAQ using an 8-plex iTRAQ labeling and the proteins differentially expressed were grouped in pluripotency, proliferative and metabolism processes. Proliferation makers, CD117 and Ki67 were measure by flow cytometry assay. Real time polymerase chain reaction analysis of pluripotency markers Rex1, Oct4, Sox2 and Nanog were done. Biological differentiation was realized using specific mediums for 14 days to induce osteogenesis, adipogenesis or chondrogenesis and immunostain analysis of differentiated cell resulting were done. Enzimoimmunoassay analysis of several enzymes as L-lactate dehydrogenase and glucose-6-phosphate isomerase were also done to validate iTRAQ data. Taking together these results indicate for the first time that mesenchymal stem cells have significant differences in their proliferative, pluripotency and metabolism profiles and those differences are age depending.

[Quality improvement: recommendations for risks and irregularities management]. / Amélioration continue : recommandations pour la gestion des risques et des anomalies.

This document describes the different steps to implement for the risk management system in a medical laboratory. The risk management and the treatment of non-conformities are two essential pieces in the continuous improvement of quality system. Taking into account, according to the requirements, the non-conformities leads to immediately remedial action, with corrective action to avoid recurrence is a real task for information and continuous improvement.

[False negative diagnosis of HIV-1]. / Falso negativo en el diagnóstico de VIH-1.

We report a case of a false negative diagnosis of HIV-1 infection in an African girl. Two HIV-1 DNA polymerase chain reaction (PCR) tests were negative at the second and fourth months of life. Because anti-HIV antibodies persisted when the patient was 18 months old, the HIV-1 RNA PCR test was performed with a positive result, confirming HIV-1 non-B subtype, recombinant A-G. The prevalence of non-B HIV-1 subtypes are increasing in Spain, which could be related to the phenomenon of immigration. Approximately one-third of HIV-infected foreigners have non-B subtypes and the percentage increases to 70 % of the African population in Spain. In non-B HIV-1 subtypes, false negative results and inconsistencies between viral load and CD4 count are more frequent. These subtypes also show a higher rate of resistance to protease inhibitors, which can have therapeutic implications.

Falso negativo en el diagnóstico de VIH-1 / False negative diagnosis of HIV-1

Se presenta un caso de falso negativo en el diagnóstico de infección por virus de la inmunodeficiencia humana tipo 1 (VIH-1) en una niña de origen africano. Las pruebas de reacción en cadena de la polimerasa (PCR) de ADN proviral realizadas a los 2 y 4 meses de vida fueron negativas. A los 18 meses, por persistencia de los anticuerpos anti-VIH-1, se realizó una PCR de ARN que resultó positiva, confirmándose la infección por VIH-1, subtipo no B, forma recombinante A-G. La prevalencia de los subtipos no B está en aumento en nuestro medio en relación con el fenómeno de la inmigración, ya que un tercio de los extranjeros infectados lo están por subtipos no B y asciende al 70 % de los pacientes africanos. En estos subtipos son más frecuentes los resultados falsos negativos y las discrepancias entre la carga viral y el recuento de linfocitos CD4. Los subtipos no B muestran una mayor tasa de resistencias a los inhibidores de la proteasa, lo que puede tener implicaciones terapéutica

We report a case of a false negative diagnosis of HIV-1 infection in an African girl. Two HIV-1 DNA polymerase chain reaction (PCR) tests were negative at the second and fourth months of life. Because anti-HIV antibodies persisted when the patient was 18 months old, the HIV-1 RNA PCR test was performed with a positive result, confirming HIV-1 non-B subtype, recombinant A-G. The prevalence of non-B HIV-1 subtypes are increasing in Spain, which could be related to the phenomenon of immigration. Approximately one-third of HIV-infected foreigners have non-B subtypes and the percentage increases to 70 % of the African population in Spain. In non-B HIV-1 subtypes, false negative results and inconsistencies between viral load and CD4 count are more frequent. These subtypes also show a higher rate of resistance to protease inhibitors, which can have therapeutic implications

Las rabietas en la infancia: qué son y cómo aconsejar a los padres / Temper tantrums. What are they and how to help parents to overcome them

Las rabietas son conductas de oposición o terquedad que se consideran parte del desarrollo normal entre los 18 meses y los 4 años. Se manifiestan como enfado, llanto, gritos, pataleo, insultos, improperios; el niño puede llegar a dañar objetos e incluso a sí mismo. Se deben a la frustación que les genera el conflicto entre sus deseos de independencia y autonomía, y los límites impuestos por los cuidadores. También intervienen de forma importante la limitación en el lenguaje y el fuerte egocentrismo propios de los niños alrededor de los 2-3 años. Ante una rabieta el primer paso es no condecer al niño aquello que se ha desencadenado el enfado. Es necesario mantener la calma e ignorar al niño; es decir, aplicar la extinción, que consiste en retirar la atención de aquellos comportamientos inadecuados que se quieren eliminar. Una variante de la extinción es el tiempo fuera. Para que la extinción funcione es necesario reforzar todas las conductas positivas que se dan fuera de las rabietas; de esta manera aprenden la forma correcta de formular las peticiones. Hay distintos reforzadores que se comentan en el artículo

Temper tantrums are opposition or obstinacy behaviors that are normal in the developing child, between 18 months and 4 years old. A temper tantrum shows with anger, screams, crying or insults; it´s possible that the child tries to damage anything, even himself. Temper tantrums haver their origin in the conflict between child´s independent wishes and caretakers limits. In the other hand, it´s very important language limits and the strong egocentric feeling in 2-3 years old children are also present. When a child starts a temper tantrum, the most important thing to do is not to give him what he or she expects just because he´s angry. It´s necessary to keep calm and to be ignorant of the child. This is called extinction, what means withdrawal of reinforcements following target behavior. The time out technique is an extinction variant. In order that extinction works, it´s necessary to reinforce appropiate behaviors; in this way, the child learns the right way to ask for what he wants. There are several kinds of reinforcements that are discussed in the article

Detection of Ki-ras gene point mutations in bile specimens for the differential diagnosis of malignant and benign biliary strictures.

BACKGROUND AND AIM: The present study was undertaken to determine if detection of Ki-ras gene point mutations in bile specimens could differentiate between benign and malignant biliary strictures. PATIENTS: Bile specimens were obtained from 117 patients exhibiting a stricture of the main bile duct, the nature of which was assessed by cholangiography, histology, and follow up. METHODS: DNA from frozen bile specimens was extracted, amplified, and tested for codon 12 point mutations of Ki-ras gene using sequence specific oligonucleotide hybridisation and mutant allele specific amplification. RESULTS: DNA amplification was successful in 110/117 bile specimens (94%). Detection of Ki-ras gene mutations in bile specimens was positive in 24.4% (22/90) of patients with malignant strictures, in 31.4% (22/70) when only primary malignant tumours were considered, and in 4% (1/25) of patients with benign strictures. Of the 49 patients with histological specimens obtained before surgery, the sensitivity of histology, Ki-ras mutation analysis, and combined methods was 59.2%, 28.6%, and 73.5% respectively. CONCLUSIONS: Our study showed that Ki-ras mutations may be detected in about one third of bile specimens from patients with primary tumours invading the main bile duct. Detection of such mutations appears to be specific and may help to differentiate between benign and malignant biliary strictures.

Expression of cell cycle regulatory proteins in chronic lymphocytic leukemias. Comparison with non-Hodgkin's lymphomas and non-neoplastic lymphoid tissue.

The expression of certain cell cycle regulatory proteins: cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin E, Bcl2 and PCNA was examined in peripheral blood lymphocytes (PBL) from 25 cases of chronic lymphocytic leukemias (CLL) in order to analyze a possible cell cycle involvement of CLL lymphocytes. For comparison, we also studied the expression of these proteins in: 23 samples of non-Hodgkin's lymphoma (NHL) tissue of different histological types, 10 samples of non-neoplastic lymphoid tissue (NLT), non-stimulated PBL (NS-PBL) and PHA-stimulated PBL (PHA-PBL) from three healthy donors. Samples were lysed and proteins were resolved on polyacrylamide gel followed by Western blot. The expression of cdk4 and cyclin E, both known to act in early cell cycle stage, was approximately on the same level in all groups of lymphoid pathology examined. In particular, we found that that 19 out of 24 CLL cases were cyclin E positive and all but one were cdk4 positive, ie they expressed these markers over twice the level of non-stimulated healthy PBL. The cdk1 expression was above the level seen in NS-PBL in 14 (56%) cases, but the average expression was significantly lower than in the other tissues examined, including low-grade lymphomas. Cdk2 expression was comparable in CLL and in low malignancy grade NHL, but weaker than in other NHL and in NLT. Cyclins A and B, normally observed in advanced cell cycle phases, were not seen in any CLL case. The presence of cdk4 and cyclin E in the blood cells of the majority of CLL cases studied, as well as cdk1 and cdk2 in some cases, indicate that the CLL cells are not quiescent, but are blocked in an early stage of the G1 cell cycle phase, and/or that the expression of these proteins is pathologically deregulated.

Phosphorylation of herpes simplex virus type 1 Us11 protein is independent of viral genome expression.

The Us11 protein is a true late gene product of herpes simplex virus type 1 (HSV-1), whose exact function is unknown but which exhibits RNA-binding properties and which is phosphorylated on serine residues. In order to determine whether the Us11 protein is phosphorylated by cellular kinase(s) or by virally encoded kinase(s), the Us11 gene has been cloned and transiently expressed in HeLa cells. In addition, HeLa-derived cell lines have been selected for their ability to express Us11 protein constitutively. 32P-Labeling and analysis by two-dimensional electrophoresis of transiently and constitutively expressed Us11 protein demonstrated that, indeed, multiple phosphorylation of the protein occurs in absence of HSV-1 genome expression, indicating that the protein behaves as a natural substrate for cellular kinase(s). In addition, a sequence heterogeneity of the Us11 protein, due to a difference in the number of SPREPR repeats, has been characterized between different strains of HSV-1.

Type II phospholipase A2 recombinant overexpression enhances stimulated arachidonic acid release.

The coding sequence of type II phospholipase A2 from human placenta was cloned in a bovine papilloma virus-derived eukaryotic expression vector under the control of the metallothionein promoter. Stably transfected C127 mouse fibroblast lines were obtained with this vector. These transfected cells overexpressed a functional 14 kDa phospholipase A2, which was bulky secreted. However, a significant phospholipase A2 activity was measured in cell homogenates. The involvement of this 14 kDa phospholipase A2 in mechanisms related to stimulated arachidonic acid release was investigated. We could parallel the overexpression of phospholipase A2 with an increase in phorbol ester and fluoroaluminate-stimulated arachidonic acid release. Pertussis toxin inhibited this stimulation. These results suggest that the 14 kDa type II phospholipase A2 might contribute to stimulation of arachidonic acid release, and therefore to eicosanoid production.

[Cellular phospholipases A2, structure-function relationship]. / Les phospholipases A2 cellulaires, relations structures-fonction.

Two PLA2 are involved in cell signaling and in phospholipid homeostasis in mammalian cells. The first one is a 14 kDa protein whose cDNA was cloned in 1989. This enzyme exhibits a strong homology with pancreatic PLA2 but is more related to type II PLA2. This PLA2 is secreted by different tissues in response to inflammatory processes. Their main function seems to be the hydrolysis of membranes of altered cells or of bacteria and the stimulation of lipid mediator synthesis. The first cDNA of an another important PLA2 group was cloned in 1991. The protein deduced is a 88 kDa cytoplasmic protein. It is involved in cell signaling by stimulating the production of free fatty acids and of their oxygenated products. These products might in turn either activate transducing proteins or stimulate membrane receptors.

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor.

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.

Phospholipid synthesis by extracellular phospholipase A2 in organic solvents.

The catalytic activity of extracellular phospholipase A2 was studied in low polarity solvents where hydrolytic enzymes have been demonstrated to catalyze synthesis reactions. It was demonstrated that extracellular phospholipase A2 can catalyze the esterification of lysophosphatidylcholine with oleic acid. Up to 6.5% of lysophosphatidylcholine can be esterified into phosphatidylcholine. This activity requires a preincubation of the enzyme in a pH 9 aqueous solution containing calcium, before the incubation in the non-aqueous solvent. No transfer of fatty acid between a phospholipid and a lysophospholipid or between two phospholipids was observed. These results may be useful in understanding the function of the membrane phospholipase A2 which may catalyze acylation or deacylation depending on the local physico-chemical environment.

Evaluation of protocols for purification of mouse monoclonal antibodies. Yield and purity in two-dimensional gel electrophoresis.

Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-to-day reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.