Autologous Mesenchymal Stem Cells for Treatment of Chronic Active Antibody-Mediated Kidney Graft Rejection: Report of the Phase I/II Clinical Trial Case Series.

Mesenchymal stem cell (MSCs) therapy has already been studied in kidney transplant recipients (KTRs), and the available data showed that it is safe and well tolerated. The aim of this study was to evaluate the safety and efficacy of autologous MSCs in combination with standard therapy in KTRs with biopsy-proven chronic active antibody-mediated rejection (AMR). Patients with biopsy-proven chronic active AMR received treatment with autologous bone marrow-derived MSCs (3 × 106 cells/kg iv) after completion of standard therapy and were followed for up to 12 months. The primary endpoints were safety by assessment of adverse events. Secondary endpoints included assessment of kidney graft function, immunological and histological changes related to AMR activity and chronicity assessed by conventional microscopy and molecular transcripts. A total of 3 patients were enrolled in the study before it was terminated prematurely because of adverse events. We found that AMR did not improve in any of the patients after treatment with MSCs. In addition, serious adverse events were observed in one case when autologous MSCs therapy was administered in the late phase after kidney transplantation, which requires further elucidation.

Autologous chondrocytes versus filtered bone marrow mesenchymal stem/stromal cells for knee cartilage repair-a prospective study.

PURPOSE: To document clinical, radiologic, and cellular data of a prospective patient series treated by a tri-layer collagen-hydroxyapatite biomimetic osteochondral scaffold (CHAS) intra-operatively seeded with cultivated autologous chondrocytes (AC) or with filtered bone marrow stem/stromal cells (fBMSC) to address chronic osteochondral knee lesions. METHODS: Thirty-six consecutive patients (15 to 59 years) with chronic osteochondral lesions (1.8-10 cm2) in the condylar or patellofemoral knee surfaces were enrolled. Lesions were covered with CHAS fixed with a fibrin glue. The superficial layer of CHAS was intra-operatively injected with active cells: in initial five patients, ACs were put directly onto dry CHAS (dry-AC); next, eight AC patients had CHAS moistened with cell culture media (media-AC), while the tourniquet was released allowing blood soaking of CHAS in the rest (14 blood-AC, 9 blood-fBMSC). Seventeen (50%) patients required different concomitant procedures. All patients were followed for serious adverse events (SAE) or graft failures; clinical, radiographic, and MRI evaluation was conducted. Cellular data on the injected cells were assessed. RESULTS: At a follow-up of 39 months (16-81), 17 patients required an additional surgical intervention: seven graft-related SAE (early post-operative synovitis and/or arthrofibrosis) were registered (3 dry-AC, 3 media-AC, 1 blood-fBMSC). There were two graft failures (1 dry-AC, 1 blood-fBMSC) for secondary reasons. All clinical scores significantly improved from pre- to post-operative values: IKCD subjective 44 to 65; IKDC examination (9/17/5/5) to (20/10/5/1); KOOS (P61/S59/ADL67/Sp32/QoL31) to (P79/S75/ADL84/Sp55/QoL51); Tegner activity scale 3.3 to 4.4. There was evidence of radiographic osteoarthritis progression-Kellgren-Lawrence 1.0 to 1.5. MOCART scores at the final follow-up averaged 71 (10 to 95). Graft-type analysis demonstrated an increased rate of graft-related SAE in dry-AC and media-AC, but their final outcomes were equivalent. Cellular data of AC at the implantation were as follows: cells in suspension 9.2 × 106, viability 95%. In blood-fBMSC group, a cell suspension with 87% viability was injected, which contained 1156 CFU-Fs. CONCLUSION: CHAS with intra-operative seeding of active cells, either AC or fBMSC, led to an overall successful outcome for the treatment of chronic osteochondral lesions in the knee. Blood soaking of CHAS in situ before cell seeding significantly decreased early post-operative adverse events, such as synovitis and arthrofibrosis.

Compassionate mesenchymal stem cell treatment in a severe COVID-19 patient: a case report.

COVID-19 presentations range from cold-like symptoms to severe symptoms with the development of acute respiratory distress syndrome (ARDS). We report on a severe COVID-19 patient who was mechanically ventilated and who developed ARDS and bacterial infection. Because of rapid clinical deterioration and the exhaustion of other treatment options, the family and attending physicians requested a compassionate use of adult allogeneic bone marrow-derived mesenchymal stem cells (MSC) in addition to commonly used immunosuppressive, antiviral, and supportive therapy. The clinical course is discussed thoroughly, with a special emphasis on the safety and effect of MSC therapy. Compassionate MSC treatment, given in three rounds, affected ARDS regression. The patient was discharged from the intensive care unit after 31 days and from hospital after 49 days in a good general condition. MSC treatment was not associated with any side effects and was well tolerated in a three-week period; therefore, it should be studied in larger trials and considered for compassionate use.

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars.

Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me2SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me2SO- and serum-free biopreservation strategies due to safety concerns over Me2SO-induced side effects and immunogenicity of animal serum. In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 µs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay). Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me2SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at -80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me2SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.

Cryopreservation of Human Adipose-Derived Stem Cells in Combination with Trehalose and Reversible Electroporation.

New cryopreservation approaches for medically applicable cells are of great importance in clinical medicine. Current protocols employ the use of dimethyl sulfoxide (DMSO), which is toxic to cells and causes undesirable side effects in patients, such as cardiac arrhythmias, neurological events, and others. Trehalose, a nontoxic disaccharide, has been already studied as a cryoprotectant. However, an efficient approach for loading this impermeable sugar into mammalian cells is missing. In our study, we assessed the efficiency of combining reversible electroporation and trehalose for cryopreservation of human adipose-derived stem cells. First, we determined reversible electroporation threshold by loading of propidium iodide into cells. The highest permeabilization while maintaining high cell viability was reached at 1.5 kV/cm, at 8 pulses, 100 µs, and 1 Hz. Second, cells were incubated in 250 or 400 mM trehalose and electroporated before cryopreservation. After thawing, 83.8 ± 1.8 % (mean ± SE) cell recovery was obtained at 250 mM trehalose. By using a standard freezing protocol (10 % DMSO in 90 % fetal bovine serum), cell survival after thawing was about 91.5 ± 1.6 %. We also evaluated possible effects of electroporation on cells' functionality before and after thawing. Successful cell growth and efficient adipogenic and osteogenic differentiation were achieved. In conclusion, electroporation seems to be an efficient method for loading nonpermeable trehalose into human adipose-derived stem cells, allowing long-term cryopreservation in DMSO-free and xeno-free conditions.

Applicability of human osteoarthritic chondrocytes for in vitro efficacy testing of anti-TNFα drugs.

In vitro cell-based models are important tools for assessing efficacies of new leads in early phases of drug development. Human osteoarthritic chondrocytes (OACs), obtained from biomedical waste material, represent a valuable, relatively accessible cellular source that could be used for this purpose. By employing reverse transcription-polymerase chain reaction (qRT-PCR) we compared gene expression profiles of key anabolic, catabolic and inflammatory genes of freshly isolated vs. monolayer cultured OACs (passages P0-P2) and non-stimulated vs. tumor necrosis factor alpha (TNF-α) stimulated P2 OACs. After expansion of OACs in monolayer cultures, the expression of almost all analyzed genes significantly decreased. The subsequent addition of TNF-α to OACs at P2 significantly increased expressions of all catabolic and inflammatory genes, leaving the anabolic profile almost unchanged. TNF-α-treated OACs were later utilized for efficacy testing of anti-TNF-α drugs infliximab and etanercept and both significantly reduced the expressions of all catabolic and inflammatory genes tested.

Gene expression of cultured human chondrocytes as a model for assessing neutralization efficacy of soluble TNFα by TNFα antagonists.

Tumor necrosis factor-alpha (TNFα) antagonists are efficacious in the treatment of various immune-mediated inflammatory diseases. Because of rapidly growing demand for developing new or biosimilar versions of these biologicals, the need to create in vitro testing models that best represent physiological conditions is increasing. Primary human chondrocytes were used for potency evaluation and comparison between the molecular effects of anti-TNFα biologicals. Infliximab and etanercept were chosen to assess the suitability of chondrocyte cell culture for determination of anti-TNFα neutralization efficacy employing quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technology. Use of both anti-TNFα biologics resulted in decrease of TNFα-stimulated expression of various matrix metalloproteinases, interleukins and other inflammation-related genes in our cell model. Significant differences in inhibition efficacy of etanercept and infliximab were observed, which were confirmed also on protein level. To evaluate the potency of anti-TNFα biologicals, a selection of TNFα-responsive target genes was made from the gene array data. The selected genes were employed in development of statistical model, which enables comparability of anti-TNFα biologicals. The presented analytical approach is suitable for assessment of the neutralization efficacy of various anti-TNFα biologicals. As such, it can be used for additional comprehensive characterization and comparability of TNF antagonists in preclinical drug testing.

Effect of intra-articular local anesthesia on articular cartilage in the knee.

PURPOSE: To evaluate the hypothetical toxic effect of local anesthetics on the articular cartilage using patient data from autologous chondrocyte cultivation with different anesthesia types used for arthroscopic cartilage biopsy specimen procurement. METHODS: A retrospective analysis of patient data from the national autologous chondrocyte implantation registry and the corresponding hospital records was approved by the National Medical Ethics Committee. Articular cartilage biopsy specimens from the knees of 49 consecutive patients assigned for autologous chondrocyte implantation (aged 14 to 44 years) were procured from the non-weight-bearing articular surface during arthroscopy under general anesthesia (12 patients), spinal anesthesia (18 patients), or local anesthesia (intra-articular injection of 15 to 20 mL of 2% lidocaine hydrochloride) (19 patients). All the biopsy specimens were further manipulated following the same chondrocyte cultivation protocol. General patient data and surgery-related parameters, together with chondrocyte viability, population doublings, and chondrocyte morphology in biopsy specimens and primary cell cultures, were analyzed and compared across different types of anesthesia. RESULTS: Patients in the general, spinal, and local anesthesia groups showed no statistical differences in age (27 years, 29 years, and 32 years, respectively), duration of surgery (36 minutes, 37 minutes, and 39 minutes, respectively), weight of biopsy specimens (110 mg, 178 mg, and 130 mg, respectively), cell viability in cartilage biopsy specimens (67%, 69%, and 78%, respectively) or primary cultures (95%, 95%, and 95%, respectively), and population doublings (5.2, 5.2, and 5.2, respectively). Similar chondrocyte morphology in primary cell cultures was observed among the 3 groups. CONCLUSIONS: This retrospective study showed that a single intra-articular injection of lidocaine hydrochloride used for knee arthroscopy did not influence the viability, morphology, and cultivation potential of chondrocytes in articular cartilage biopsy specimens assigned for autologous chondrocyte implantation. LEVEL OF EVIDENCE: Level IV, retrospective comparative study.

Hyaluronic acid conjugates of glycine peptides and L-tryptophan.

This work reports about the conjugation of glycine C-terminal ethyl and methyl ester peptides and L-tryptophan methyl ester with sodium hyaluronate in aqueous solutions using the peptide coupling agent DMTMM (or short DMT, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride). Detailed infrared (IR) absorbance and 1H and 13C (2D) NMR studies (heteronuclear multi-bond correlation spectroscopy, HMBC) confirmed covalent and regioselective amide bonds with the D-glucuronate, but also proves the presence of DMT traces in all conjugates. The ethyl ester`s methyl protons on the peptides` C-terminal could be used to quantify the degree of substitution of the peptide on the hyaluronate scaffold by NMR. The ester group also proved stable during conjugation and work-up, and could in some cases be selectively cleaved in water whilst leaving the amide bond intact as shown by potentiometric charge titration, NMR and IR. The conjugates did not influence the capability of human umbilical vein endothelial cells (HUVECs) to reduce MTS (5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2-thiazolyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) to a formazan dye, which points towards a low cytotoxicity for the obtained products. The conjugation method and products could be tested for tissue engineering gels or drug delivery purposes with alternative, biologically active peptides.

Intracellular delivery of trehalose renders mesenchymal stromal cells viable and immunomodulatory competent after cryopreservation.

Trehalose is a nontoxic disaccharide and a promising cryoprotection agent for medically applicable cells. In this study, the efficiency of combining trehalose with reversible electroporation for cryopreservation of two types of human mesenchymal stromal cells was investigated: adipose-derived stromal cells, and umbilical-cord-derived stromal cells. Comparable results to standard dimethyl sulfoxide cryopreservation protocols were achieved, even without extensive electroporation parameters and protocol optimization. The presence of high extracellular trehalose resulted in comparable cell viabilities without and with electroporation. According to the determination of trehalose concentrations, 250 mM extracellular trehalose resulting in, 20 mM to 50 mM intracellular trehalose were sufficient for successful cryopreservation of cells. With electroporation, higher (i.e. 50 mM to 90 mM) intracellular trehalose was achieved after cryopreservation, although cell survival was not improved significantly. To evaluate the impact of electroporation and cryopreservation on cells, stress and immune-activation-related gene expression were analyzed. Electroporation and/or cryopreservation resulted in increased SOD2 and HSPA1A expression. Despite the increased stress response, the high up-regulation by mesenchymal stromal cells of immunomodulatory genes in the inflammatory environment was not affected. Highest expression was seen for the IDO1 and TSG6 genes. In conclusion, cryopreservation of mesenchymal stromal cells in trehalose results in comparable characteristics to their cryopreservation using dimethyl sulfoxide.

Combination of Filtered Bone Marrow Aspirate and Biomimetic Scaffold for the Treatment of Knee Osteochondral Lesions: Cellular and Early Clinical Results of a Single Centre Case Series.

BACKGROUND: Osteochondral injury is a very common orthopaedic pathology, mainly affecting young, active population, with limited current treatment options. Herein we are presenting cellular and early clinical data of a patient series treated for chronic osteochondral lesions in the knee with a filter-based intra-operative bone marrow aspirate (BMA) separation device. METHODS: Fifteen patients with chronic knee osteochondral lesions (60% females, 19-59 years) were included in this prospective case series. Filtered BMA (f-BMA), containing mesenchymal stem/stromal cells (MSCs), was combined with a biomimetic collagen-hydroxyapatite scaffold (CHAS) and implanted into the site of the lesion. Harvested BMA and post-separation f-BMA were analysed for blood cell counts, flow cytometry, and fibroblast colony forming units (CFU-Fs). Patients were followed for serious adverse events and graft failures. Clinical evaluation was assessed using the knee injury and osteoarthritis outcome score (KOOS). In 8 patients a magnetic resonance imaging (MRI)/arthroscopy were performed. RESULTS: Cell suspension contained 0.027% CD271+ CD45- 7-AAD- cells, 0.15% CD73+ CD90+ CD105+ cells and 0.0012% CFU-Fs of all nucleated cells with 86% viability. Filtration process resulted in 12.8 (4.0-40.8) fold enrichment in terms of CFU-F content in comparison to initial BMA. No serious adverse events related directly to the osteochondral treatment were reported. After an average follow-up of 20 months (14-25) all KOOS subscales (Symptoms/Pain/Daily activities/Sport and recreation/Quality of life) increased significantly from pre-operative 55/56/67/30/30 to post-operative 73/76/79/51/52 (p values < 0.05), respectively. MRI or arthroscopic evaluation revealed nearly normal to normal overall International Cartilage Repair Society assessment in 7/8 patients. CONCLUSION: The filter-based BMA separation procedure significantly increased the frequency of mesenchymal stem/stromal cells (MSCs), however their concentration was not increased. The clinical evaluation revealed high safety profile of the treatment and resulted in improved clinical status of the patients.

Human mesenchymal stromal cells from different tissues exhibit unique responses to different inflammatory stimuli.

BACKGROUND: Mesenchymal stromal cell (MSC) - based therapies are emerging as promising treatment of various autoimmune diseases, however the utility of different MSC tissue sources remains elusive. We aimed to characterize MSC from different origins, namely bone marrow (BM), adipose tissue (AT) and umbilical cord (UC) and determine their functional effects on normal human lung fibroblasts (NHLF). METHODS: BM-, AT- or UC-MSC were isolated each from 3 different healthy donors. The gene expression and protein secretion were analyzed at basal level, along with TNFα-, IL-1ß- and SAA- stimulated cells using real-time PCR and Luminex technology. Effect of conditioned medium (CM) from different MSC sources on migration was determined with wound scratch assay, while mitotic and apoptotic rates were studied using immunofluorescence microscopy. RESULTS: BM-MSC expressed highest basal mRNA levels of SDF1 and VCAM-1, while other genes were similarly expressed between MSC origins. TNFα priming of AT-MSC gained a prominent increase in IDO1 and CCL5 gene expression, with 928-fold and 4396-fold changes, respectively. Among all tissue sources, basal UC-MSC released highest protein levels of most measured analytes, including IL-6, IL-8, MCP-1, ICAM1, HGF, MMP1 and CH3L1. BM- and AT-MSC derived CM enhanced wound closure in NHLF, while an opposite effect was observed with UC-MSC derived CM. Our data also suggests that MSC-CM could contribute to decreased mitotic potential and increased apoptotic rate in lung fibroblasts. CONCLUSIONS: Our study highlights origin-specific MSC profile differences and emphasizes a heterogenic response of different MSC to inflammatory stimuli.

Testing the potency of anti-TNF-α and anti-IL-1ß drugs using spheroid cultures of human osteoarthritic chondrocytes and donor-matched chondrogenically differentiated mesenchymal stem cells.

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor-alpha (TNF-α) and interleukin 1ß (IL-1ß). New in vitro testing systems are needed to evaluate efficacies of new anti-inflammatory biological drugs, ideally in a patient-specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti-inflammatory drugs, spheroids were exposed to TNF-α, IL-1ß, or to supernatant containing secretome from activated macrophages (MCM). The anti-inflammatory efficacies of anti-TNF-α biologicals adalimumab, infliximab, and etanercept, and the anti-IL-1ß agent anakinra were assessed in short-term microspheroid and long-term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF-α or IL-1ß. The differences in potency of anti-TNF-α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short-term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti-TNF-α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045-1058, 2018.

Mesenchymal Stem Cells in the Musculoskeletal System: From Animal Models to Human Tissue Regeneration?

The musculoskeletal system includes tissues that have remarkable regenerative capabilities. Bone and muscle sustain micro-damage throughout the lifetime, yet they continue to provide the body with the support that is needed for everyday activities. Our current understanding is that the regenerative capacity of the musculoskeletal system can be attributed to the mesenchymal stem/ stromal cells (MSCs) that reside within its different anatomical compartments. These MSCs can replenish various tissues with progenitor cells to form functional cells, such as osteoblasts, chondrocytes, myocytes, and others. However, with aging and in certain disorders of the musculoskeletal system such as osteoarthritis or osteoporosis, this regenerative capacity of MSCs appears to be lost or diverted for the production of other non-functional cell types, such as adipocytes and fibroblasts. In this review, we shed light on the tissue sources and subpopulations of MSCs in the musculoskeletal system that have been identified in animal models, discuss the mechanisms of their anti-inflammatory action as a prerequisite for their tissue regeneration and their current applications in regenerative medicine. While providing up-to-date evidence of the role of MSCs in different musculoskeletal pathologies, in particular in osteoporosis and osteoarthritis, we share some thoughts on their potential as diagnostic markers in musculoskeletal health and disease.

Effect of sphingomyelin and cholesterol on the interaction of St II with lipidic interfaces.

Sticholysin II (St II) is a cytolysin produced by the sea anemone Stichodactyla helianthus, characterized by forming oligomeric pores in natural and artificial membranes. In the present work the influence of the membrane lipidic components sphingomyelin (SM) and cholesterol (Cho) on binding and functional activity of St II, was evaluated using ELISA, lipid monolayers and liposomes. The aim of this work was to establish the promoting role of Cho and SM, both in St II binding and pore formation efficiency. In general the association (evaluated by ELISA and incorporation to phospholipid monolayers) of St II to lipids mixtures was better than to any one of the single components. Regarding the unique role of SM, it was found that, albeit inefficiently, St II binds to phosphatidylcholine (PC):Cho monolayers and liposomes, and is able to form active pores in these bilayers. The results in monolayers and liposomes show that the presence of SM and large amounts of Cho leads to the highest values of critical pressure and rate of association to monolayers, the most favorable interaction with liposomes, and the fastest rate of pore formation, in spite of the rigidity of the layers as suggested by the high generalized polarization (GP) of Laurdan incorporated to liposomes and FTIR data. Taken together, the present results show that the joint presence of SM and Cho, both in binary and ternary (PC containing) mixtures provide conditions particularly suitable for St II binding and function. We suggest that microdomains present in the bilayers could be important for toxin-membrane association.

Membrane insertion of the N-terminal alpha-helix of equinatoxin II, a sea anemone cytolytic toxin.

Equinatoxin II (Eqt-II) is a member of the actinoporins, a unique family of cytotoxins comprising 20 kDa pore-forming proteins isolated from sea anemones. Actinoporins bind preferentially to lipid membranes containing sphingomyelin, and create cation-selective pores by oligomerization of three to four monomers. Previous studies have shown that regions of Eqt-II crucial for its cytolytic mechanism are an exposed aromatic cluster and the N-terminal region containing an amphipathic alpha-helix. In the present study, we have investigated the transfer of the N-terminal alpha-helix into the lipid membrane by the use of three mutants containing an additional tryptophan residue in different positions within the amphipathic alpha-helix (Ile18-->Trp, Val22-->Trp and Ala25-->Trp). The interaction of the mutants with different model systems, such as lipid monolayers, erythrocytes and ghost membranes, was extensively characterized. Intrinsic fluorescence measurements and the use of vesicles containing brominated phospholipids indicated a deep localization of the N-terminal amphipathic helix in the lipid bilayer, except for the case of Val22-->Trp. This mutant is stabilized in a state immediately prior to final pore formation. The introduction of additional tryptophan residues in the sequence of Eqt-II has proved to be a suitable approach to monitor the new environments that surround defined regions of the molecule upon membrane interaction.

Comparison of articular and auricular cartilage as a cell source for the autologous chondrocyte implantation.

Articular (medial femoral condyle) and auricular cartilage (anithelix) was compared as a cell source for the autologous joint repair. Cells isolated from five human cadaveric donors were cultured parallel in the monolayer cultures and in the 3D alginate hydrogel constructs for 1 week. Cell morphology was controlled by the fluorescent microscopy and gene expressions of type I collagen (COL1), type II collagen (COL2), aggrecan (AGR), versican (VER), and elastin (ELS) were analyzed by the real-time polymerase chain reaction. COL1 and ELS, predominant in the phenotype of auricular biopsy, were statistically lower in the articular biopsies. Even though COL2 and AGR decreased in monolayers of both cell sources, the dedifferentiation process affected auricular cells intensely. Cells embedded in the alginate hydrogel directly after the isolation did not exhibit the dedifferentiated phenotype. Additionally, COL1, COL2, AGR, and VER were comparable between the two sources. ELS however, remained higher in the auricular cells regardless of the culture type. The study indicates that auricular chondrocytes cultured in a 3D environment immediately after the isolation have a neo-cartilage potential for the articular surface reconstruction.

Purification, cloning and characterization of fragaceatoxin C, a novel actinoporin from the sea anemone Actinia fragacea.

Actinia fragacea is commonly called the "strawberry" anemone because of the distinctive yellow or green spots displayed on its red column. Its venom contains several haemolytic proteins with a molecular mass of approximately 20 kDa that can be separated by ion-exchange column chromatography. One of them was purified to homogeneity and was named fragaceatoxin C (FraC). Its 15 N-terminal residues were identified by Edman degradation and served to obtain its complete DNA coding sequence by RT-PCR. The coding region of FraC was amplified and cloned in the expression vector pBAT-4. Purified recombinant FraC consists of 179 amino acids and multiple sequence alignment with other actinoporins clearly indicates that FraC belongs to this protein family. The secondary structure, thermal stability and lytic activity of native and recombinant FraC were practically identical and exhibit the same basic features already described for equinatoxin-II and sticholysin-II.

Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation.

Autologous chondrocyte implantation (ACI) relies on the implantation of in vitro expanded cells. The aim was to study the dedifferentiation of human articular chondrocytes under different cultivating conditions [days 0-10 in the primary culture (P0); passages in a monolayer from P0 to P3; monolayer vs. alginate and monolayer vs. alginate/agarose hydrogels] using real-time PCR analysis. The relative gene expressions for collagen type I and II, aggrecan and versican were quantified and the corresponding differentiation indexes (Col2/Col1, Agr/Ver) were calculated. The values of both differentiation indexes decreased exponentially with time in the P0 monolayer culture, and continued with a significant decrease over the subsequent monolayer passages. On the contrary, the chondrocytes seeded in either of the hydrogels significantly increased the indexes compared to their parallel monolayer cultures. These results indicate that alginate and alginate/agarose hydrogels offer an appropriate environment for human articular chondrocytes to redifferentiate after being expanded in vitro. Therefore the three-dimensional (3D) hydrogel chondrocyte cultures present not only surgical, but also biological advantage over the classic suspension-periosteum chondrocyte implantation.

Lipid phase coexistence favors membrane insertion of equinatoxin-II, a pore-forming toxin from Actinia equina.

Equinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with model membranes is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II. However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such a mechanism in the assembly of a toroidal pore are discussed.