Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate-bead culture model.

Background: Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model. Methods: After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 µg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis. Results: Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment. Conclusions: WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.

Atezolizumab Retains Cellular Binding to Programmed Death Ligand 1 Following Aerosolization via Mesh Nebulizer.

BACKGROUND/AIM: Cytotoxic inhalable drugs were shown to be advantageous in treating malignancies of the respiratory tract. However, these drugs have not always presented a safe profile and were reported to induce local adverse events. Protein-based anticancer drugs, such as immune checkpoint and vascular endothelial growth factor inhibitors, do not induce tissue injury, nor do they exhibit vesicant properties upon direct contact with tissues. Protein drugs are susceptible to the heat and stress encountered during droplet generation for delivery by nebulization. The aim of this study was to investigate the capacity of atezolizumab, an antibody to programmed death ligand 1, to bind target cells after nebulization with a vibrating mesh (VM) nebulizer. MATERIALS AND METHODS: We compared Fourier-transformed infrared (FTIR) and Raman spectra of native atezolizumab (60 mg/ml) and its nebulized form following 10-min nebulization in a piezoceramic VM nebulizer. The binding of atezolizumab to DU-145 prostate cancer cells was evaluated using competitive blocking of anti-CD274 staining. RESULTS: Nebulization did not induce Raman or FTIR spectral modification nor did it affect the binding capacity of atezolizumab. Conversely, heat-inactivated atezolizumab lost its cell-binding capacity and did not reduce anti-CD274 immunostaining. Native and nebulized atezolizumab displayed identical spectra, whereas the FTIR spectra of the heat-inactivated drug was significantly altered. CONCLUSION: VM nebulization does not obliterate the functionality of the drug atezolizumab. The integrity of a nebulized form can be rapidly assessed by FTIR and Raman spectrometry.

Inflammation Control and Immunotherapeutic Strategies in Comprehensive Cancer Treatment.

Tumor growth and expansion are determined by the immunological tumor microenvironment (TME). Typically, early tumorigenic stages are characterized by the immune system not responding or weakly responding to the tumor. However, subsequent tumorigenic stages witness the tumor promoting its growth and metastasis by stimulating tumor-protective (pro-tumor) inflammation to suppress anti-tumor immune responses. Here, we propose the pivotal role of inflammation control in a successful anti-cancer immunotherapy strategy, implying that available and novel immunotherapeutic modalities such as inflammation modulation, antibody (Ab)-based immunostimulation, drug-mediated immunomodulation, cancer vaccination as well as adoptive cell immunotherapy and donor leucocyte transfusion could be applied in cancer patients in a synergistic manner to amplify each other's clinical effects and achieve robust anti-tumor immune reactivity. In addition, the anti-tumor effects of immunotherapy could be enhanced by thermal and/or oxygen therapy. Herein, combined immune-based therapy could prove to be beneficial for patients with advanced cancers, as aiming to provide long-term tumor cell/mass dormancy by restraining compensatory proliferation of surviving cancer cells observed after traditional anti-cancer interventions such as surgery, radiotherapy, and metronomic (low-dose) chemotherapy. We propose the Inflammatory Prognostic Score based on the blood levels of C-reactive protein and lactate dehydrogenase as well as the neutrophil-to-lymphocyte ratio to effectively monitor the effectiveness of comprehensive anti-cancer treatment.

Thymus Subset Alterations Accompanying Concomitant Tumor Immunity Mimics Phenotypic Patterns of Cytotoxic Drug Doxorubicin.

BACKGROUND/AIM: Concomitant immunity (CIM) is a phenomenon that elicits an antitumor response not sufficient enough to destroy the primary tumor but prevents a secondary implant from growing and spreading. This study aimed to develop a method of identification of serum tumoricidal factors released into circulation during CIM and to compare the CIM-related effect to the effect elicited by the cytotoxic drug doxorubicin. MATERIALS AND METHODS: SL2 tumor-bearing mice were studied at three time points - day 4, day 7, and day 11 following i.p. 5×105 cell implantation. Hematological effects and thymocyte immunophenotyping (CD4/CD8) data were compared to the effects induced by intravenous 10 mg/kg doxorubicin (DOX) administration to intact DBA 2 mice. The level of plasma colony stimulating factor-granulocyte macrophage (CSF-GM) was evaluated by ELISA. RESULTS: Identical thymus histopathology and an extent of double-positive CD4+CD8+ subset depletion was found in day 11 tumor-bearing mice (TBM-11) and in DOX-administered animals. TBM-11 exhibited a leukemoid reaction with an increase in monocyte and granulocyte counts. Conversely, DOX administration was followed by severe leukocytopenia at the 72-h time point. No increase in CSF-GM was observed in mice with or without a leukemoid reaction. CONCLUSION: The complexity of CIM can be examined by tracking alterations in the most fragile cortical CD8+CD4+ double positive population. Thymocyte apoptosis induced by DOX and TBM-11 might be associated with different mechanisms. TBM-11 did not exhibit severe myelotoxicity as DOX did. CIM-related serum factors can be assessed and screened via thymocyte subset analysis.

Comparative Evaluation of Cellular Uptake of Free and Liposomal Doxorubicin Following Short Term Exposure.

BACKGROUND/AIM: Liposomal Doxorubicin (lipDOX) and free Doxorubicin (DOX) are reported to exhibit similar antitumor efficacy. However, cellular internalization mechanisms of lipDOX are still a subject of controversy. MATERIALS AND METHODS: Intact and permeabilized cells were exposed for short time to lipDOX and free DOX and drug intracellular content was evaluated by flow cytometry. Then, the antiproliferative capacities of lipDOX and free DOX were compared by the leukocyte nadir test in mice in vivo. RESULTS: The fluorescence increase was 11.2-fold higher in intact cells and 19.7-fold higher in permeabilized cells after exposure to free DOX as compared to lipDOX. Mice injected with DOX showed pronounced antiproliferative activity with a leukocyte count decrease to 2.8±0.65 k/µl (p<0.01) - an effect significantly stronger than that in the lipDOX group. CONCLUSION: Intact and permeabilized cells internalize free DOX manifold faster than lipDOX. The LipDOX formulation does not induce a remarkable leukocyte nadir effect in vivo.

Osteochondral Repair and Electromechanical Evaluation of Custom 3D Scaffold Microstructured by Direct Laser Writing Lithography.

OBJECTIVE: The objective of this study was to assess a novel 3D microstructured scaffold seeded with allogeneic chondrocytes (cells) in a rabbit osteochondral defect model. DESIGN: Direct laser writing lithography in pre-polymers was employed to fabricate custom silicon-zirconium containing hybrid organic-inorganic (HOI) polymer SZ2080 scaffolds of a predefined morphology. Hexagon-pored HOI scaffolds were seeded with chondrocytes (cells), and tissue-engineered cartilage biocompatibility, potency, efficacy, and shelf-life in vitro was assessed by morphological, ELISA (enzyme-linked immunosorbent assay) and PCR (polymerase chain reaction) analysis. Osteochondral defect was created in the weight-bearing area of medial femoral condyle for in vivo study. Polymerized fibrin was added to every defect of 5 experimental groups. Cartilage repair was analyzed after 6 months using macroscopical (Oswestry Arthroscopy Score [OAS]), histological, and electromechanical quantitative potential (QP) scores. Collagen scaffold (CS) was used as a positive comparator for in vitro and in vivo studies. RESULTS: Type II collagen gene upregulation and protein secretion was maintained up to 8 days in seeded HOI. In vivo analysis revealed improvement in all scaffold treatment groups. For the first time, electromechanical properties of a cellular-based scaffold were analyzed in a preclinical study. Cell addition did not enhance OAS but improved histological and QP scores in HOI groups. CONCLUSIONS: HOI material is biocompatible for up to 8 days in vitro and is supportive of cartilage formation at 6 months in vivo. Electromechanical measurement offers a reliable quality assessment of repaired cartilage.

Bone regeneration in rabbit calvarial defects using PRGF and adipose-derived stem cells: histomorphometrical analysis.

Aim: The aim of this study was to evaluate the osteogenic potential of adipose-derived stem cells (ADSCs) and to assess the influence of plasma rich in growth factors (PRGF) on bone regeneration using ADSCs. Materials & methods: Bone defects were randomly allocated to the five treatment modalities: spontaneous healing, natural bovine bone mineral (BBM), BBM loaded with PRGF, BBM loaded with ADSCs and BBM loaded with a combination of ADSCs and PRGF. Results: The PRGF significantly enhanced the biomaterial-to-bone contact. Defects treated with ADSCs and PRGF or a combination of both showed the greatest bone regeneration. Conclusion: Combining PRGF and ADSCs boosts the bone graft regenerative potential at the earliest period of healing.

Retraction Note to: Stromal vascular fraction cells for the treatment of critical limb ischemia: a pilot study.

The Editor-in-Chief and the publisher have retracted this article [1]. An investigation by the Lithuanian Bioethics Committee concluded that, contrary to the statements in the article, the study described was not conducted in the Vilnius City Clinical Hospital and the Commission of Medical Ethics did not issue any approval for such a study.

Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy.

BACKGROUND: The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool. METHODS: Vcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice. RESULTS: 5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 µM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals. CONCLUSIONS: We have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy.

Post-operative unadjuvanted therapeutic xenovaccination with chicken whole embryo vaccine suppresses distant micrometastases and prolongs survival in a murine Lewis lung carcinoma model.

Immunotherapy in the form of anticancer vaccination relies on the mobilization of the patient's immune system against specific cancer antigens. Instead of focusing on an autologous cell lysate, which is not always available in clinical practice, the present study investigates vaccines utilizing xenogeneic foetal tissue that are rich in oncofoetal antigens. Lewis lung carcinoma (LLC)-challenged C57BL/6 mice were treated with either a xenogeneic vaccine made from chicken whole embryo, or a xenogeneic vaccine made from rat embryonic brain tissue, supplemented with a Bacillus subtilis protein fraction as an adjuvant. Median and overall survival, size of metastatic foci in lung tissue and levels of circulating CD8a+ T cells were evaluated and compared with untreated control mice. Following primary tumour removal, a course of three subcutaneous vaccinations with xenogeneic chicken embryo vaccine led to significant increase in overall survival rate (100% after 70 days of follow-up vs. 40% in untreated control mice), significant increase in circulating CD8a+ T cells (18.18 vs. 12.6% in untreated control mice), and a significant decrease in the area and incidence of metastasis foci. The xenogeneic rat brain tissue-based vaccine did not improve any of the investigated parameters, despite promising reports in other models. We hypothesize that the proper selection of antigen source (tissue) can constitute an effective immunotherapeutic product.

Scaffold design for artificial tissue with bone marrow stem cells.

OBJECTIVE: The aim of this study was to test polymeric materials (collagen, fibrin, polyimide film, and polylactic acid) for single- and multi-layer scaffold formation. MATERIALS AND METHODS: In our study, we used rabbit bone marrow stem cells (rBMSCs) and human mesenchymal stem cells (hMSCs) with materials of a different origin for the formation of an artificial scaffold, such as a collagen scaffold, fibrin scaffold produced from clotted rabbit plasma, electrospun poly(lactic acid) (PLA) mats, polyimide film (PI), and the combination of the latter two. Cell imaging was performed 3-14 days after cell cultivation in the scaffolds. Time-lapse imaging was used to determine hMSC mobility on the PI film. RESULTS: Cell incorporation in collagen and clotted fibrin scaffolds was evaluated after 2-week cultivation in vitro. Histological analysis showed that cells penetrated only external layers of the collagen scaffold, while the fibrin clot was populated with rBMSCs through the entire scaffold thickness. As well, cell behavior on the laser micro-structured PI film was analyzed. The mobility of hMSCs on the smooth PI film and the micro-machined surface was 20±2µm/h and 18±4µm/h, respectively. After 3-day cultivation, hMSCs were capable of spreading through the whole 100±10µm-thick layer of the electrospun PLA scaffold and demonstrated that the multilayer scaffold composed of PI and PLA materials ensured a suitable environment for cell growth. CONCLUSIONS: The obtained results suggest that electrospinning technology and femtosecond laser micro-structuring could be employed for the development of multi-layer scaffolds. Different biopolymers, such as PLA, fibrin, and collagen, could be used as appropriate environments for cell inhabitation and as an inner layer of the multi-layer scaffold. PI could be suitable as a barrier blocking cell migration from the scaffold. However, additional studies are needed to determine optimal parameters of inner and outer scaffold layers.

Stromal vascular fraction cells for the treatment of critical limb ischemia: a pilot study.

BACKGROUND: Cell-based therapy is being explored as an alternative treatment option for critical limb ischemia (CLI), a disease associated with high amputation and mortality rates and poor quality of life. However, therapeutic potential of uncultured adipose-derived stromal vascular fraction (SVF) cells has not been evaluated as a possible treatment. In this pilot study, we investigated the efficacy of multiple injections of autologous uncultured adipose-derived SVF cells to treat patients with CLI. METHODS: This study included 15 patients, from 35 to 77 years old, with rest pain and ulceration. SVF cells were injected once or twice in the ischemic limb along the arteries. Digital subtraction angiography was performed before and after cell therapy. The clinical follow up was carried out for the subsequent 12 months after the beginning of the treatment. RESULTS: Multiple intramuscular SVF cell injections caused no complications during the follow-up period. Clinical improvement occurred in 86.7% of patients. Two patients required major amputation, and the amputation sites healed completely. The rest of patients achieved a complete ulcer healing, pain relief, improved ankle-brachial pressure index and claudication walking distance, and had ameliorated their quality of life. Digital subtraction angiography performed before and after SVF cell therapy showed formation of numerous vascular collateral networks across affected arteries. CONCLUSION: Results of this pilot study demonstrate that the multiple intramuscular SVF cell injections stimulate regeneration of injured tissue and are effective alternative to achieve therapeutic angiogenesis in CLI patients who are not eligible for conventional treatment. Trial registration number at ISRCTN registry, ISRCTN13001382. Retrospectively registered at 26/04/2017.

Bacterial ghosts as adjuvants in syngeneic tumour cell lysate-based anticancer vaccination in a murine lung carcinoma model.

Instead of relying on external anticancer factors for treatment, immunotherapy utilizes the host's own immune system and directs it against given tumour antigens. This study demonstrated that it is possible to overcome the documented immunosuppressive properties of tumour cell lysate by supplementing it with appropriate adjuvant. Lewis lung carcinoma (LLC)­challenged C57BL/6 mice were treated with LLC cryo­lysate mixed with either bacterial ghosts (BGs) generated from E. coli Nissle 1917 or B. subtilis 70 kDa protein as adjuvants. Median and overall survival, the size of metastatic foci in lung tissue and levels of circulating CD8a+ T cells were evaluated and compared to the untreated control mice or mice treated with LLC lysate alone. After primary tumour removal, a course of three subcutaneous vaccinations with LLC lysate supplemented with BGs led to a significant increase in overall survival (80% after 84 days of follow­up vs. 40% in untreated control mice), a significant increase in circulating CD8a+ T cells (16.57 vs. 12.6% in untreated control mice) and a significant decrease in metastasis foci area and incidence. LLC lysate supplemented with B. subtilis protein also improved the inspected parameters in the treated mice, when compared against the untreated control mice, but not to a significant degree. Therefore, whole cell lysate supplemented with BGs emerges as an immunostimulatory construct with potential clinical applications in cancer treatment.

Preclinical study of SZ2080 material 3D microstructured scaffolds for cartilage tissue engineering made by femtosecond direct laser writing lithography.

Over the last decade DLW employing ultrafast pulsed lasers has become a well-established technique for the creation of custom-made free-form three-dimensional (3D) microscaffolds out of a variety of materials ranging from proteins to biocompatible glasses. Its potential applications for manufacturing a patient's specific scaffold seem unlimited in terms of spatial resolution and geometry complexity. However, despite few exceptions in which live cells or primitive organisms were encapsulated into a polymer matrix, no demonstration of an in vivo study case of scaffolds generated with the use of such a method was performed. Here, we report a preclinical study of 3D artificial microstructured scaffolds out of hybrid organic-inorganic (HOI) material SZ2080 fabricated using the DLW technique. The created 2.1 × 2.1 × 0.21 mm(3) membrane constructs are tested both in vitro by growing isolated allogeneic rabbit chondrocytes (Cho) and in vivo by implanting them into rabbit organisms for one, three and six months. An ex vivo histological examination shows that certain pore geometry and the pre-growing of Cho prior to implantation significantly improves the performance of the created 3D scaffolds. The achieved biocompatibility is comparable to the commercially available collagen membranes. The successful outcome of this study supports the idea that hexagonal-pore-shaped HOI microstructured scaffolds in combination with Cho seeding may be successfully implemented for cartilage tissue engineering.

WITHDRAWN: Autologous adipose tissue-derived stromal vascular fraction cells application in patients with osteoarthritis.

Ahead of Print article withdrawn by publisher

Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: to use or not to use?

Accumulation of firm evidence that clinically apparent cancer develops only when malignant cells manage to escape immunosurveillance led to the introduction of tumor immunotherapy strategies aiming to reprogramm the cancer-dysbalanced antitumor immunity and restore its capacity to control tumor growth. There are several immunotherapeutical strategies, among which specific active immunotherapy or therapeutic cancer vaccination is one of the most promising. It targets dendritic cells (DCs) which have a unique ability of inducing naive and central memory T cell-mediated immune response in the most efficient manner. DCs can be therapeutically targeted either in vivo/in situ or by ex vivo manipulations followed by their re-injection back into the same patient. The majority of current DC targeting strategies are based on autologous or allogeneic tumor-associated antigens (TAAs) which possess various degrees of inherent tolerogenic potential. Therefore still limited efficacy of various tumor immunotherapy approaches may be attributed, among various other mechanisms, to the insufficient immunogenicity of self-protein-derived TAAs. Based on such an idea, the use of homologous xenogeneic antigens, derived from different species was suggested to overcome the natural immune tolerance to self TAAs. Xenoantigens are supposed to differ sufficiently from self antigens to a degree that renders them immunogenic, but at the same time preserves an optimal homology range with self proteins still allowing xenoantigens to induce cross-reactive T cells. Here we discuss the concept of xenogeneic vaccination, describe the cons and pros of autologous/allogeneic versus xenogeneic therapeutic cancer vaccines, present the results of various pre-clinical and several clinical studies and highlight the future perspectives of integrating xenovaccination into rapidly developing tumor immunotherapy regimens.

Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells.

Mesenchymal stem/stromal cells (MSCs) comprise a heterogeneous population of cells with multilineage differentiation potential, the ability to modulate oxidative stress, and secrete various cytokines and growth factors that can have immunomodulatory, angiogenic, anti-inflammatory and anti-apoptotic effects. Recent data indicate that these paracrine factors may play a key role in MSC-mediated effects in modulating various acute and chronic pathological conditions. MSCs are found in virtually all organs of the body. Bone marrow-derived MSCs (BM-MSCs) were discovered first, and the bone marrow was considered the main source of MSCs for clinical application. Subsequently, MSCs have been isolated from various other sources with the adipose tissue, serving as one of the alternatives to bone marrow. Adipose tissue-derived MSCs (ASCs) can be more easily isolated; this approach is safer, and also, considerably larger amounts of ASCs can be obtained compared with the bone marrow. ASCs and BM-MSCs share many biological characteristics; however, there are some differences in their immunophenotype, differentiation potential, transcriptome, proteome, and immunomodulatory activity. Some of these differences may represent specific features of BM-MSCs and ASCs, while others are suggestive of the inherent heterogeneity of both BM-MSC and ASC populations. Still other differences may simply be related to different isolation and culture protocols. Most importantly, despite the minor differences between these MSC populations, ASCs seem to be as effective as BM-MSCs in clinical application, and, in some cases, may be better suited than BM-MSCs. In this review, we will examine in detail the ontology, biology, preclinical, and clinical application of BM-MSCs versus ASCs.

Protein oligomerization induced by oleic acid at the solid-liquid interface--equine lysozyme cytotoxic complexes.

Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein alpha-lactalbumin, known as human alpha-lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4-30 lysozyme molecules. Each lysozyme molecule is able to bind 11-48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8-anilinonaphthalene-1-sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin-T dye, shows a spherical morphology, assembles into ring-shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well-populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.

Engrafting fetal liver cells into multiple tissues of healthy adult mice without the use of immunosuppressants.

We have shown the fetal liver cell engraftments into multiple tissues of adult healthy mice, achieved without suppressing the animals' immune systems. Fetal cells from the livers of male C57Bl/6J Black lineage mice at day 13 to 15 of gestation were injected intravenously into female adult CC57W/MY White mice. The grafting was evaluated by Y-chromosome-specific PCR, cytometric analysis of fluorescently stained donor cells, and histological analysis. All the methods consistently showed the presence of multiple engraftments randomly distributed through the various organs of the recipients. After 60 days, the grafts still constituted 0.1 to 2.75% of the tissues. The grafted cells did not change their appearance in any of the organs except the brain, where they became enlarged. Inflammatory reactions were not detected in any of the histological preparations. The frequency of engraftments was higher in the liver, indicating that similarity between the donor and recipient cells facilitates engraftment. The high inherent plasticity of fetal liver cells underlies their ability to integrate into healthy recipient organs, which can be governed by environmental conditions and connections with neighboring cells rather than by the initial cellular developmental programs. The fact that fetal liver cells can be grafted into multiple tissues of healthy animals indicates that they can be used to replace the natural loss of cells in adult organisms.