Antibody response and intra-host viral evolution after plasma therapy in COVID-19 patients pre-exposed or not to B-cell-depleting agents.

Administration of plasma therapy may contribute to viral control and survival of COVID-19 patients receiving B-cell-depleting agents that impair humoral immunity. However, little is known on the impact of anti-CD20 pre-exposition on the kinetics of SARS-CoV-2-specific antibodies. Here, we evaluated the relationship between anti-spike immunoglobulin G (IgG) kinetics and the clinical status or intra-host viral evolution after plasma therapy in 36 eligible hospitalized COVID-19 patients, pre-exposed or not to B-cell-depleting treatments. The majority of anti-CD20 pre-exposed patients (14/17) showed progressive declines of anti-spike IgG titres following plasma therapy, contrasting with the 4/19 patients who had not received B-cell-depleting agents (p = 0.0006). Patients with antibody decay also depicted prolonged clinical symptoms according to the World Health Organization (WHO) severity classification (p = 0.0267) and SARS-CoV-2 viral loads (p = 0.0032) before complete virus clearance. Moreover, they had higher mutation rates than patients able to mount an endogenous humoral response (p = 0.015), including three patients with one to four spike mutations, potentially associated with immune escape. No relevant differences were observed between patients treated with plasma from convalescent and/or mRNA-vaccinated donors. Our study emphasizes the need for an individualized clinical care and follow-up in the management of COVID-19 patients with B-cell lymphopenia.

[The Internal medicine articles that struck us the most in 2021]. / Année 2021 mise en perspective par les internistes hospitaliers.

The year 2021 has seen many breakthroughs in general internal medicine, despite the ongoing COVID-19 pandemic, with multiple implications in our daily clinical practice. From shorter antibiotic treatment duration in community-acquired pneumonia, to new indications for colchicine treatment, without forgetting better targets of hemoglobin for transfusion, questioning of the interest of high dose vitamin D substitution when preventing falls in older patients and finally disappointing hopes for new indications of albumin substitution in cirrhosis, the literature is full of new evidence. Each year, the chief residents of the internal medicine ward in Lausanne university hospital (CHUV) in Switzerland meet up to share their readings: here is a selection of ten articles, chosen, summarized, and commented for you.

L'année 2021, malgré la pandémie de Covid-19, a vu de nombreux progrès en médecine interne générale, avec de multiples implications pour notre pratique quotidienne. D'une durée diminuée d'antibiothérapie pour le traitement de la pneumonie communautaire à de nouvelles indications au traitement de colchicine, en passant par des précisions sur les cibles de transfusion érythrocytaire, ainsi qu'une remise en question de l'intérêt de la vitamine D à haute dose dans la prévention des chutes chez la personne âgée, et pour finir des espoirs déçus de nouvelle indication à la substitution d'albumine dans la cirrhose, les nouveautés abondent dans la littérature. Chaque année, les cheffes et chefs de clinique du Service de médecine interne du CHUV se réunissent pour partager leurs lectures : voici une sélection de dix articles choisis, revus et commentés pour vous.

Preliminary Study of New Gallium-68 Radiolabeled Peptide Targeting NRP-1 to Detect Brain Metastases by Positron Emission Tomography.

Due to their very poor prognosis and a fatal outcome, secondary brain tumors are one of the biggest challenges in oncology today. From the point of view of the early diagnosis of these brain micro- and macro-tumors, the sensitivity and specificity of the diagnostic tools constitute an obstacle. Molecular imaging, such as Positron Emission Tomography (PET), is a promising technique but remains limited in the search for cerebral localizations, given the commercially available radiotracers. Indeed, the [18F]FDG PET remains constrained by the physiological fixation of the cerebral cortex, which hinders the visualization of cerebral metastases. Tumor angiogenesis is recognized as a crucial phenomenon in the progression of malignant tumors and is correlated with overexpression of the neuropilin-1 (NRP-1) receptor. Here, we describe the synthesis and the photophysical properties of the new gallium-68 radiolabeled peptide to target NRP-1. The KDKPPR peptide was coupled with gallium-68 anchored into a bifunctional NODAGA chelating agent, as well as Cy5 for fluorescence detection. The Cy5 absorbance spectra did not change, whereas the molar extinction coefficient (ε) decreased drastically. An enhancement of the fluorescence quantum yield (φF) could be observed due to the better water solubility of Cy5. [68Ga]Ga-NODAGA-K(Cy5)DKPPR was radiosynthesized efficiently, presented hydrophilic properties (log D = -1.86), and had high in vitro stability (>120 min). The molecular affinity and the cytotoxicity of this new chelated radiotracer were evaluated in vitro on endothelial cells (HUVEC) and MDA-MB-231 cancer cells (hormone-independent and triple-negative line) and in vivo on a brain model of metastasis in a nude rat using the MDA-MB-231 cell line. No in vitro toxicity has been observed. The in vivo preliminary experiments showed promising results, with a high contrast between the healthy brain and metastatic foci for [68Ga]Ga-NODAGA-K(Cy5)DKPPR.

[Albumin substitution : A review of the indications in general internal medicine]. / Substitution en albumine : revue des indications en médecine interne générale.

Albumin has multiple physiological functions such as maintenance of oncotic pressure, binding of plasmatic molecule, immunomodulation and endothelial stabilization. Considering potential negative effects of hypoalbuminemia, the question of albumin substitution arises. For cirrhotic patients, indications for substitution are well established with a scientific consensus for spontaneous bacterial peritonitis, paracentesis-induced circulatory dysfunction and hepatorenal syndrome. However, in the case of hepatic encephalopathy or of hypoalbuminemia in non-cirrhotic patients, the scientific consensus is absent, and the results of clinical studies differ. In this article, we present the different indications of albumin substitution by examining the most recent studies.

L'albumine exerce de multiples fonctions physiologiques, notamment : maintien de la pression oncotique, liaison à d'autres molécules plasmatiques, immunomodulation et stabilisation endothéliale. Face aux potentiels effets délétères de l'hypoalbuminémie, la question d'une substitution se pose. Chez le patient cirrhotique, les indications à la substitution sont bien définies avec un consensus scientifique pour la péritonite bactérienne spontanée, la dysfonction circulatoire postparacentèse de grand volume et le syndrome hépatorénal. Néanmoins, en cas d'encéphalopathie hépatique ou d'hypoalbuminémie chez le patient non cirrhotique, le consensus est absent et les résultats des études divergent. Dans cet article, nous présentons les différentes indications à sa substitution, en mettant l'accent sur les études récentes en la matière.

[The Internal medicine articles that struck us the most in 2020]. / L'année 2020 mise en perspective par les internistes hospitaliers.

2020 has continued to bring important progress in all areas of internal medicine, impacting our daily practice. From a shift to oral stepdown antibiotics in infectious endocarditis, to new indications for apixaban, SLGT-2 inhibitors and macrolide antibiotics, passing by the catheter-based renal denervation in hypertension, early paracentesis in cirrhosis and new diagnostic criteria in pulmonary embolism, internal medicine journals are full of novelties. Every year, the chief residents of internal medicine at the Swiss University Hospital of Lausanne meet up to share their readings: here is their selection of eleven articles, summarized and commented for you.

L'année 2020 a vu de notables progrès dans tous les domaines de la médecine interne, avec un impact important sur notre pratique quotidienne. Du passage à une antibiothérapie orale dans l'endocardite infectieuse, aux nouvelles indications pour l'apixaban, les inhibiteurs du SGLT2 (sodium-glucose co-transporteur de type 2) et les macrolides en passant par la dénervation rénale dans l'hypertension artérielle, la ponction d'ascite et les changements de critères diagnostiques de l'embolie pulmonaire, les nouveautés abondent dans la littérature. Chaque année, les chefs de clinique du Service de médecine interne du CHUV se réunissent pour partager leurs lectures : voici une sélection de onze articles choisis, revus et commentés pour vous.

Live cell imaging reveals actin-cytoskeleton-induced self-association of the actin-bundling protein WLIM1.

Crosslinking of actin filaments into bundles is essential for the assembly and stabilization of specific cytoskeletal structures. However, relatively little is known about the molecular mechanisms underlying actin bundle formation. The two LIM-domain-containing proteins define a novel and evolutionarily conserved family of actin-bundling proteins whose actin-binding and -crosslinking activities primarily rely on their LIM domains. Using TIRF microscopy, we describe real-time formation of actin bundles induced by tobacco NtWLIM1 in vitro. We show that NtWLIM1 binds to single filaments and subsequently promotes their interaction and zippering into tight bundles of mixed polarity. NtWLIM1-induced bundles grew by both elongation of internal filaments and addition of preformed fragments at their extremities. Importantly, these data are highly consistent with the modes of bundle formation and growth observed in transgenic Arabidopsis plants expressing a GFP-fused Arabidopsis AtWLIM1 protein. Using two complementary live cell imaging approaches, a close relationship between NtWLIM1 subcellular localization and self-association was established. Indeed, both BiFC and FLIM-FRET data revealed that, although unstable NtWLIM1 complexes can sporadically form in the cytosol, stable complexes concentrate along the actin cytoskeleton. Remarkably, disruption of the actin cytoskeleton significantly impaired self-association of NtWLIM1. In addition, biochemical analyses support the idea that F-actin facilitates the switch of purified recombinant NtWLIM1 from a monomeric to a di- or oligomeric state. On the basis of our data, we propose a model in which actin binding promotes the formation and stabilization of NtWLIM1 complexes, which in turn might drive the crosslinking of actin filaments.

PAMAM G4.5-chlorin e6 dendrimeric nanoparticles for enhanced photodynamic effects.

There is currently great interest in the development of efficient and specific carrier delivery platforms for systemic photodynamic therapy. Therefore, we aimed to develop covalent conjugates between the photosensitizer chlorin e6 (Ce6) and PAMAM G4.5 dendrimers. Singlet oxygen generation (SOG) efficiency and fluorescence emission were moderately affected by the covalent binding of the Ce6 to the dendrimer. Compared to free Ce6, PAMAM anchored Ce6 displays a much higher photodynamic effect, which is ascribable to better internalization in a tumor cell model. Intracellular fate and internalization pathway of our different compounds were investigated using specific inhibition conditions and confocal fluorescence microscopy. Free Ce6 was shown to enter the cells by a simple diffusion mechanism, while G4.5-Ce6-PEG internalization was dependent on the caveolae pathway, whereas G4.5-Ce6 was subjected to the clathrin-mediated endocytosis pathway. Subcellular localization of PAMAM anchored Ce6, PEGylated or not, was very similar suggesting that the nanoparticles behave similarly in the cells. As a conclusion, we have demonstrated that PEGylated G4.5 PAMAM-Ce6 dendrimers may offer effective biocompatible nanoparticles for improved photodynamic treatment in a preclinical tumor model.

Deciphering Natural Killer Cell Cytotoxicity Against Medulloblastoma in vitro and in vivo: Implications for Immunotherapy.

Purpose: Medulloblastoma (MB) is the most prevalent paediatric brain tumour. Despite improvements in patient survival with current treatment strategies, the quality of life of these patients remains poor owing to the sequelae and relapse risk. An alternative, or, in addition to the current standard treatment, could be considered immunotherapy, such as Natural Killer cells (NK). NK cells are cytotoxic innate lymphoid cells that play a major role in cancer immunosurveillance. To date, the mechanism of cytotoxicity of NK cells, especially regarding the steps of adhesion, conjugation, cytotoxic granule polarisation in the cell contact area, perforin and granzyme release in two and three dimensions, and therapeutic efficacy in vivo have not been precisely described. Materials and Methods: Each step of NK cytotoxicity against the three MB cell lines was explored using confocal microscopy for conjugation, Elispot for degranulation, flow cytometry, and luminescence assays for target cell necrosis and lysis and mediators released by cytokine array, and then confirmed in a 3D spheroid model. Medulloblastoma-xenografted mice were treated with NK cells. Their persistence was evaluated by flow cytometry, and their efficacy in tumour growth and survival was determined. In addition, their effects on the tumour transcriptome were evaluated. Results: NK cells showed variable affinities for conjugation with MB target cells depending on their subgroup and cytokine activation. Chemokines secreted during NK and MB cell co-culture are mainly associated with angiogenesis and immune cell recruitment. NK cell cytotoxicity induces MB cell death in both 2D and 3D co-culture models. NK cells initiated an inflammatory response in a human MB murine model by modulating the MB cell transcriptome. Conclusion: Our study confirmed that NK cells possess both in vitro and in vivo cytotoxic activity against MB cells and are of interest for the development of immunotherapy.

Characterization of an Innovative Biomaterial Derived From Human Wharton's Jelly as a New Promising Coating for Tissue Engineering Applications.

The extracellular matrix (ECM) offers the opportunity to create a biomaterial consisting of a microenvironment with interesting biological and biophysical properties for improving and regulating cell functions. Animal-derived ECM are the most widely used as an alternative to human tissues that are of very limited availability. However, incomplete decellularization of these tissues presents a high risk of immune rejection and disease transmission. In this study, we present an innovative method to extract human ECM derived from the Wharton's jelly (WJ-ECMaa) of umbilical cords as a novel biomaterial to be used in tissue engineering. WJ-ECMaa was very efficiently decellularized, suggesting its possible use in allogeneic conditions. Characterization of its content allowed the identification of type I collagen as its main component. Various other matrix proteins, playing an important role in cell adhesion and proliferation, were also detected. WJ-ECMaa applied as a surface coating was analyzed by fluorescent labeling and atomic force microscopy. The results revealed a particular arrangement of collagen fibers not previously described in the literature. This biomaterial also presented better cytocompatibility compared to the conventional collagen coating. Moreover, it showed adequate hemocompatibility, allowing its use as a surface with direct contact with blood. Application of WJ-ECMaa as a coating of the luminal surface of umbilical arteries for a use in vascular tissue engineering, has improved significantly the cellularization of this surface by allowing a full and homogeneous cell coverage. Taking these results together, our novel extraction method of human ECM offers a very promising biomaterial with many potential applications in tissue engineering such as the one presented direct in vascular tissue engineering. Further characterization of the composition and functionality will help explore the ways it can be used in tissue engineering applications, especially as a scaffold or a surface coating.

Impact of Lacticaseibacillus rhamnosus GG on the Emulsion Stability of Raw Milk.

Lactic acid bacteria (LAB) have been studied for several decades to understand and determine their mechanism and interaction within the matrix into which they are introduced. This study aimed to determine the spatial distribution of Lacticaseibacillus rhamnosus GG (LGG) in a dairy matrix and to decipher its behaviour towards milk components, especially fat globules. Two strains of this widely studied bacterium with expected probiotic effects were used: LGG WT with pili on the cell surface and its pili-depleted mutant-LGG ΔspaCBA-in order to determine the involvement of these filamentous proteins. In this work, it was shown that LGG ΔspaCBA was able to limit creaming with a greater impact than the wild-type counterpart. Moreover, confocal imaging evidenced a preferential microbial distribution as aggregates for LGG WT, while the pili-depleted strain tended to be homogenously distributed and found as individual chains. The observed differences in creaming are attributed to the indirect implication of SpaCBA pili. Indeed, the bacteria-to-bacteria interaction surpassed the bacteria-to-matrix interaction, reducing the bacterial surface exposed to raw milk. Conversely, LGG ΔspaCBA may form a physical barrier responsible for preventing milk fat globules from rising to the surface.

Case Report: Stepwise Anti-Inflammatory and Anti-SARS-CoV-2 Effects Following Convalescent Plasma Therapy With Full Clinical Recovery.

In these times of COVID-19 pandemic, concern has been raised about the potential effects of SARS-CoV-2 infection on immunocompromised patients, particularly on those receiving B-cell depleting agents and having therefore a severely depressed humoral response. Convalescent plasma can be a therapeutic option for these patients. Understanding the underlying mechanisms of convalescent plasma is crucial to optimize such therapeutic approach. Here, we describe a COVID-19 patient who was deeply immunosuppressed following rituximab (anti-CD20 monoclonal antibody) and concomitant chemotherapy for chronic lymphoid leukemia. His long-term severe T and B cell lymphopenia allowed to evaluate the treatment effects of convalescent plasma. Therapeutic outcome was monitored at the clinical, biological and radiological level. Moreover, anti-SARS-CoV-2 antibody titers (IgM, IgG and IgA) and neutralizing activity were assessed over time before and after plasma transfusions, alongside to SARS-CoV-2 RNA quantification and virus isolation from the upper respiratory tract. Already after the first cycle of plasma transfusion, the patient experienced rapid improvement of pneumonia, inflammation and blood cell counts, which may be related to the immunomodulatory properties of plasma. Subsequently, the cumulative increase in anti-SARS-CoV-2 neutralizing antibodies due to the three additional plasma transfusions was associated with progressive and finally complete viral clearance, resulting in full clinical recovery. In this case-report, administration of convalescent plasma revealed a stepwise effect with an initial and rapid anti-inflammatory activity followed by the progressive SARS-CoV-2 clearance. These data have potential implications for a more extended use of convalescent plasma and future monoclonal antibodies in the treatment of immunosuppressed COVID-19 patients.

Adhesive Interactions Between Lactic Acid Bacteria and ß-Lactoglobulin: Specificity and Impact on Bacterial Location in Whey Protein Isolate.

In the last decade, there has been an increasing interest in the potential health effects associated with the consumption of lactic acid bacteria (LAB) in foods. Some of these bacteria such as Lactobacillus rhamnosus GG (LGG) are known to adhere to milk components, which may impact their distribution and protection within dairy matrices and therefore is likely to modulate the efficiency of their delivery. However, the adhesive behavior of most LAB, as well as its effect on food structuration and on the final bacterial distribution within the food matrix remain very poorly studied. Using a recently developed high-throughput approach, we have screened a collection of 73 LAB strains for their adhesive behavior toward the major whey protein ß-lactoglobulin. Adhesion was then studied by genomics in relation to common bacterial surface characteristics such as pili and adhesion-related domain containing proteins. Representative adhesive and non-adhesive strains have been studied in further depth through biophysical measurement using atomic force microscopy (AFM) and a relation with bacterial distribution in whey protein isolate (WPI) solution has been established. AFM measurements have revealed that bacterial adhesion to ß-lactoglobulin is highly specific and cannot be predicted accurately using only genomic information. Non-adhesive strains were found to remain homogeneously distributed in solution whereas adhesive strains gathered in flocs. These findings show that several LAB strains are able to adhere to ß-lactoglobulin, whereas this had only been previously observed on LGG. We also show that these adhesive interactions present similar characteristics and are likely to impact bacterial location and distribution in dairy matrices containing ß-lactoglobulin. This may help with designing more efficient dairy food matrices for optimized LAB delivery.

In vitro and in vivo evaluation of an electrospun-aligned microfibrous implant for Annulus fibrosus repair.

Annulus fibrosus (AF) impairment is associated with reherniation, discogenic pain, and disc degeneration after surgical partial discectomy. Due to a limited intrinsic healing capacity, defects in the AF persist over time and it is hence necessary to adopt an appropriate strategy to close and repair the damaged AF. In this study, a cell-free biodegradable scaffold made of polycaprolactone (PCL), electrospun, aligned microfibers exhibited high levels of cell colonization, alignment, and AF-like extracellular matrix deposition when evaluated in an explant culture model. The biomimetic multilayer fibrous scaffold was then assessed in an ovine model of AF impairment. After 4 weeks, no dislocation of the implants was detected, and only one sample out of six showed a partial delamination. Histological and immunohistochemical analyses revealed integration of the implant with the surrounding tissue as well as homogeneously aligned collagen fiber organization within each lamella compared to the disorganized and scarcer fibrous tissue in a randomly organized control fibrous scaffold. In conclusion, this biomimetic electrospun implant exhibited promising properties in terms of AF defect closure, with AF-like neotissue formation that fully integrated with the surrounding ovine tissue.

Photodynamic therapy with intratumoral administration of Lipid-Based mTHPC in a model of breast cancer recurrence.

BACKGROUND AND OBJECTIVES: Generalized skin sensitization is a main drawback of photodynamic therapy with systemic administration of photosensitizers. We have evaluated the potential use of an intratumoral injection of a liposomal formulation of mTHPC (Foslip) in a mouse model of local recurrence of breast cancer. MATERIALS AND METHODS: Mice were directly injected into the tumor (IT) with 25 microl of a Foslip suspension (0.15 mg/ml) and illumination (652 nm, 20 J/cm(2)) was performed at different time points with pathological assessment after 48 hours. In a parallel mice series plasma samples were obtained at different endpoints after IT Foslip injection for HPLC analysis and the tumors were subjected in toto to macrofluorescence imaging. Fluorescence polarization measurements were conducted in vitro to estimate the rate of sensitizer redistribution from liposomes. RESULTS: Optimal, albeit partial, cure rates were obtained at 24 hours post-sensitizer and uninistration. Inhomogeneous and weak fluorescence was observed at early time points and became maximal at 24 hours. Plasma levels of mTHPC increased until 15 hours. Fluorescence polarization measurements showed a slow sensitizer transfer from liposomes to model membranes. DISCUSSION AND CONCLUSION: The weak intratumoral fluorescence at early time points could be explained by concentration quenching within the liposomes as evidenced from fluorescence polarization studies. Progressive mTHPC redistribution from liposomes and its further incorporation into tumor tissue resulted in fluorescence build-up over time with a maximum at 24 hours post-injection. This correlates perfectly with the best therapeutic effect at this time point. The absence of total cure can be attributed to inhomogeneous photosensitizer distribution. mTHPC is reabsorbed into the blood stream but the total administered amount is much reduced as opposed to systemic administration so that repeated PDT sessions might be favorable in terms of side effects and tumor response.

Influence of incubation time and sensitizer localization on meta-tetra(hydroxyphenyl)chlorin (mTHPC)-induced photoinactivation of cells.

The present study addresses the impact of different aggregation states of meta-tetra(hydroxyphenyl)chlorin (mTHPC) on the photoinactivation of cells. Measurements of the photophysical properties of mTHPC in MCF-7 cells showed progressive sensitizer aggregation with increasing incubation time. Reconstructed absorption spectra of intracellular mTHPC showed a significant decrease in the molar extinction coefficient and broadening of the Soret band at 24 h incubation compared to 3 h. Intracellular photobleaching of mTHPC slowed down, and the profile changed from mono- to bi-exponential upon incubation. Fluorescence lifetime imaging (FLIM) measurements revealed a substantial decrease in the lifetime of mTHPC fluorescence at 24 h compared to 3 h. In addition, the intracellular localization of mTHPC as observed by fluorescence microscopy changed from a diffuse homogeneous fluorescence pattern at short incubation times to a punctiform pattern at 24 h. The efficiency of photodynamic therapy (PDT) assessed by a clonogenic assay was three times greater at 24 h. However, when the survival curves were replotted as a function of the number of absorbed photons, the efficiency was 1.8 times greater at 3 h than at 24 h. The loss of photosensitizing efficiency at higher mTHPC concentrations was attributed to self-quenching of the triplet states of the sensitizers.

Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis.

Osteoarthritis is characterized by a gradual degradation of extracellular matrix, resulting from an excess of chondrocyte cell death, mainly due to an increase in apoptotis. Recent studies have revealed the essential role of HSP70 in protecting cells from stressful stimuli. Therefore, overexpressing HSP70 in chondrocytes could represent a good strategy to prevent extracellular matrix destruction. To this end, we have developed a vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to cell death induced by mono-iodoacetate (MIA). To overcome the barrier-effect of matrix, we investigated the efficacy of plasmid delivery by electroporation (EP) in rat patellar cartilage. Two days after EP, 50% of patellar chondrocytes were HSP/GFP+. After 3 months, long-term expression of transgene was only depicted in the deep layer (20-30% positive cells). HSP70 overexpression inhibited the natural endochondral ossification in the deep layer, thus leading to a lesser decrease in chondrocyte distribution. Moreover, overexpression of HSP70, after a preventive EP transfer in rat patella, was sufficient to decrease the severity of osteoarthritis-induced lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of HSP70, through EP delivery, could protect chondrocytes from cellular injuries and thus might be a novel chondroprotective modality in rat OA.

New tools for non-invasive exploration of collagen network in cartilaginous tissue-engineered substitute.

In tissue engineering approaches, the quality of substitutes is a key element to determine its ability to treat cartilage defects. However, in clinical practice, the evaluation of tissue-engineered cartilage substitute quality is not possible due to the invasiveness of the standard procedure, which is to date histology. The aim of this work was to validate a new innovative system performed from two-photon excitation laser adapted to an optical macroscope to evaluate at macroscopic scale the collagen network in cartilage tissue-engineered substitutes in confrontation with gold standard histologic techniques or immunohistochemistry to visualize type II collagen. This system permitted to differentiate the quality of collagen network between ITS and TGF-ß1 treatments. Multiscale large field imaging combined to multimodality approaches (SHG-TCSPC) at macroscopical scale represent an innovative and non-invasive technique to monitor the quality of collagen network in cartilage tissue-engineered substitutes before in vivo implantation.

Endothelial cell--interactions with polyelectrolyte multilayer films.

The seeding of endothelial cells (ECs) on biomaterial surfaces became a major challenge, allowing to improve the non-thrombogenic properties of these surfaces. Recently, the use of polyelectrolyte films has been suggested as a new versatile technique of surface modification aimed at tissue engineering. In this study, we evaluate the adhesion properties of ECs on two types of polyelectrolyte films ending either by poly(D-lysine) (PDL), or poly(allylamine hydrochloride) (PAH), and compared them to data obtained on PDL or PAH monolayers, glass and fibronectin (Fn)-coated glass. ECs seeded on polyelectrolyte films showed a good morphology, allowing ECs to resist physiological shear stress better compared to ECs seeded on glass or Fn. The expression of beta1 integrins was slightly lower on polyelectrolyte films than on control surfaces. However, the phosphorylation of focal adhesion kinase, involved in the transduction of adhesion signal, was not modified on PAH ending films compared to control surfaces; whereas it became lower on PDL ending films. Finally, PAH ending films improve strongly ECs adhesion without disturbing the adhesion mechanism, necessary for the development of a new endothelium. These types of films or similar build-ups could thus be used in the future as a way to modify surfaces for vascular tissue engineering.

Direct gene transfer into rat articular cartilage by in vivo electroporation.

To establish a system for efficient direct in vivo gene targeting into rat joint, we have evaluated a strategy of gene transfer by means of the delivery of external electric pulses (EP) to the knee after intra-articular injection of a reporter gene (GFP). Rats were killed at various times after the electro gene-therapy to analyze GFP gene expression by immunohistochemistry. GFP staining was detected in the superficial, middle, and deep zones of the patellar cartilage at days 2 and 9, and thereafter only in the deep zone (months 1 and 2). The average percentage of GFP-positive cells was estimated at 30% both one and 2 months after the gene transfer. Moreover, no pathologic change caused by the EP was detected in the cartilage. The level and stability of the long-term GFP expression found in this study demonstrate the feasibility of a treatment of joint disorders (inflammatory or degenerative, focal or diffuse) using electric gene transfer.

The 2-aminoglucosamide motif improves cellular uptake and photodynamic activity of tetraphenylporphyrin.

Several strategies have been proposed to improve the efficiency of photosensitizers used in photodynamic therapy (PDT). In this context, the synthesis of mono- (1) and di-glucosylated (2) porphyrins, and mono-glucosylated chlorin (3) was performed. HT29 human adenocarcinoma cells were significantly more sensitive to asymmetric and less hydrophobic glucosylated photosensitizers-mediated PDT (1, 3), compared to tetraphenylporphyrin (TPP). The lowest photosensitivity observed for TPP was consistent with the lowest uptake. Moreover, the most pronounced photodynamic activity measured for 3 was in relation with the improvement of cellular uptake, the singlet oxygen quantum yield and the high extinction coefficient value at 650 nm compared to porphyrins. Cellular localization analysis showed that 1 and 3 accumulated mainly inside the endoplasmic reticulum.