Comparison of growth factor and interleukin content of adult peripheral blood and cord blood serum eye drops for cornea and ocular surface diseases.

INTRODUCTION: Various blood-derived products have been proposed for the topical treatment of ocular surface diseases. The aim of the study was to compare the different content of Growth Factors (GFs) and Interleukins (ILs) in peripheral blood (PB-S) and Cord Blood (CB-S) sera. MATERIALS AND METHODS: Sera were obtained from 105 healthy adult donors (PB-S) and 107 umbilical/placental veins at the time of delivery (CB-S). The levels of epithelial-GF (EGF), fibroblast-GF (FGF), platelet-derived-GF (PDGF), insulin-GF (IGF), transforming-GF alpha (TGF-α,) and beta 1-2-3 (TGF-ß1-ß2-ß3), vascular endothelial-GF (VEGF), nerve-GF (NGF), Interleukin (IL)-1ß,IL-4,IL-6,IL-10, and IL-13 were assessed by Bio-Plex Protein Array System (Bio-Rad Laboratories, CA, USA). The Mann-Whitney test for unpaired data was applied to compare GFs and ILs levels in the two sources. The associations among each GF/IL level and the obstetric data for CB-S and hematological characteristics for PB-S were also investigated. RESULTS: The levels of EGF, TGF-α, TGF-ß2, FGF, PDGF, VEGF, NGF, IL-1B, IL-4, IL-6, IL-10, and IL-13 were significantly higher in CB-S compared to PB-S. Conversely, the levels of IGF-1, IGF-2, and TGF-ß1 were significantly higher in PB-S. The female sex and the weight of the child showed a significant association in predicting EGF and PDGF levels. CONCLUSION: A significantly different content in those GFs and ILs was demonstrated in the two blood sources. Since each GF/IL selectively regulates different cellular processes involved in corneal healing, the use of PB-S or CB-S should be chosen on the basis of the cellular mechanism to be promoted in each clinical case.

Chondroprotective activity of N-acetyl phenylalanine glucosamine derivative on knee joint structure and inflammation in a murine model of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed in vitro. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). DESIGN: 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2 weeks with one intra-articular injection of NAPA (2.5 mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. RESULTS: The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. CONCLUSIONS: NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo.

Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins.

Failures in bipolar fresh osteochondral allograft for the treatment of end-stage knee osteoarthritis.

PURPOSE: Bipolar fresh osteochondral allografts (BFOA) recently became a fascinating option for articular cartilage replacement, in particular in those young patients non-suitable for traditional replacement because of age. While the use of osteochondral allografts for the treatment of focal osteochondral lesions in the knee is well established, their use in the treatment of end-stage arthritis is far more controversial. The purpose of this paper is to describe our experience in a series of seven patients who underwent a resurfacing of both tibio-femoral and patello-femoral joints by BFOA. METHODS: From 2005 to 2007, seven patients (mean age 35.2 ± 6.3 years) underwent BFOA for end-stage arthritis of the knee. Patients were evaluated clinically, radiographically and by CT scan preoperatively and at established intervals up to the final follow-up. RESULTS: No intra-operative complications occurred. Nevertheless, joint laxity and aseptic effusion, along with a progressive chondrolysis, lead to early BFOA failure in six patients, which were revised by total knee arthroplasty at 19.5 ± 3.9 months follow-up. Only one patient, who received the allograft to convert a knee arthrodesis, gained a satisfactory result at the last follow-up control. CONCLUSIONS: BFOA in the knee joint still remains an inapplicable option in the treatment of post-traumatic end-stage arthritis of the young patient, due to the high rate of failure. Further studies are necessary in order to investigate the causes of failure and improve the applicability of this method. Still, after extensive counselling with the patient, BFOA may represent a salvage procedure aimed to revise scarcely tolerated knee arthrodesis. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

Bone marrow concentrated cell transplantation: rationale for its use in the treatment of human osteochondral lesions.

Bone marrow is one of the best characterized stem cell microenvironments that contains Mesenchymal Stem Cells (MSCs), a rare population of non-hematopoietic stromal cells. MSCs have been indicated as a new option for regenerative medicine because of their ability to differentiate into various lineages such as bone, cartilage and adipose tissue. However, isolation procedures are crucial for the functional activity of the transplanted cells. The use of concentrated bone marrow cells (BMCs) enables a cell population surrounded by its microenvironment (niche) to be implanted while avoiding all the complications related to the in vitro culture. The cells of the niche are able to regulate stem cell behavior through direct physical contact and secreting paracrine factors. The aim of this study was to characterize BMCs in vitro to evaluate their ability to differentiate toward mature cells and try to understand whether there are differences in the chondrogenic and osteogenic potential of cells from patients of different ages. Mononuclear Cells (MNCs) isolated by Ficoll were used as control. Both cell populations were grown in monolayers and differentiated with specific factors and analyzed by histological and molecular biology assays to evaluate the expression of some specific extracellular matrix molecules. The present investigations revealed the ability of BMCs to act as isolated cells. They are able to form colonies and differentiate toward chondrogenic and osteogenic lineages, the latter pathway appearing to be influenced by donor age. The results obtained by this study support the use of BMCs in clinical practice for the repair of osteochondral damage, which might be particularly useful for the one-step procedure allowing cells to be directly implanted in operating room.

Treatment of human cartilage defects by means of Nd:YAG Laser Therapy.

Articular cartilage lesions represent a challenging problem for orthopaedic surgeons. The purpose of this study was to evaluate the effect of a new pulsed Nd:YAG High Intensity Laser Therapy on the regeneration of cartilage tissue in patients with traumatic lesions. Clinical, histological and immunohistochemical evaluations were performed. Ten patients affected by chondral lesions scheduled for ACI procedure, were enrolled into the study. During the chondrocyte expansion for ACI procedure, cartilage from five patients was treated by Nd:YAG High Intensity Laser Therapy (HILT group). No laser treatment was performed in the remaining patients, who were used as controls. Cartilage repair was assessed by clinicians using two different scores: Cartilage Repair Assessment (CRA) and Overall Repair Assessment (ORA). Cartilage biopsy specimens were harvested to perform histological and immunohistochemical analyses at T0 (before laser treatment) and T1 (at the end of the treatment). A significant decrease in cartilage depth was noticed in the HILT group at T1. Histological and immunohistochemical evaluations showed some regenerative processes in cartilaginous tissue in terms of high amount of proteoglycans, integration with adjacent articular cartilage and good cellular arrangement in the HILT group. By contrast, a not well organized cartilaginous tissue with various fibrous features in the control group at T0 and T1 was observed. In conclusion, the use of this new pulsed Nd:YAG HILT resulted promising in the treatment of moderate cartilage lesions markedly in the young patients.

Evaluation of chondrocyte behavior in a new equine collagen scaffold useful for cartilage repair.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. An ideal scaffold should possess a preformed three-dimensional shape, fix the cells to the damaged area and prevent their migration into the articular cavity. Furthermore, the constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and a phenotype re-differentiation process. The aim of this study was to verify the ability of an equine collagen membrane to support the growth of human chondrocytes and to allow the re-expression of their original phenotype. This ability was assessed by the evaluation of collagen type I, II and aggrecan mRNA expression by Real-Time PCR. Immunohistochemical analyses were performed to evaluate collagen type I, II and proteoglycans synthesis. Electron microscopy was utilized to highlight the structure of the biomaterial and its interactions with the cells. Our data indicate that human chondrocytes seeded onto a collagen membrane express and produce collagen type II and aggrecan and downregulate the production of collagen type I during the experimental times analyzed. These results provide an in vitro demonstration for the therapeutic potential of autologous chondrocyte transplantation by an equine collagen membrane as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects.

Chemical-physical properties and in vitro cell culturing of a novel biphasic bio-mimetic scaffold for osteo-chondral tissue regeneration.

The requirements for a successful regeneration of an osteo-chondral defect could effectively be met by using a bi-layered composite scaffold, able to support proliferation and differentiation of mesenchymal stem cells, while providing a biochemical environment promoting the formations of the two distinct tissues. The novel strategy here presented consists of developing a bio-mimetic scaffolds obtained by the combination of two integrated organic compounds (type I collagen and chitosan) with or without bioactive Mg-doped hydroxyapatite (Mg-HA) nanocrystals, depending on the specific layer, reproducing cartilaginous or subchondral bone tissue. An innovative patented methodology for scaffolds production, called - pH-dependent 3-phasic assembling -, allowed to development of a highly homogenous and chemically stable scaffold, presenting a very good integration among all three components, as confirmed by extensive SEM and thermogravimetric analyses. A preliminary in vitro evaluation was also carried out by seeding bi-layered scaffold with human bone marrow stromal cells (h-MSCs), by giving particular emphasis to cell viability and distribution at day 0, 7 and 14. Cells were viable and uniformly colonized the whole scaffold until day 14, indicating that the scaffold contributed to the maintenance of cell behaviour.

Prospects on the Potential In Vitro Regenerative Features of Mechanically Treated-Adipose Tissue for Osteoarthritis Care.

Gathering a better grasp on the adipose stromal vascular fraction (SVF) is demanding among clinicians for osteoarthritis (OA) care because of its promising but multifaceted clinical outcomes. The aim of this preclinical in vitro study was to test whether the mechanical approach with Hy-Tissue SVF system, a class IIa CE marked device of adipose tissue micro-fragmentation, influences the biological features and functions of SVF. We compared mechanical generated-SVF (mSVF) with the enzymatic generated-SVF (eSVF) by testing cell survival, phenotype, differentiation, and paracrine properties using ELISA assays. Both adipose SVF showed 80% viable cells and enrichment for CD-44 marker. The mSVF product preserved the functions of cell populations within the adipose tissue; however, it displayed lowered nucleated cell recovery and CFU-F than eSVF. As for multipotency, mSVF and eSVF showed similar differentiation commitment for osteochondral lineages. Both adipose SVF exhibited an increased release of VEGF, HGF, IGF-1 and PDGF-bb, involved in pathways mediating osteochondral repair and cell migration. Both mSVF and eSVF also displayed high release for the anti-inflammatory cytokine IL-10. After in vitro culture, supernatants from both mSVF and eSVF groups showed a low release of cytokines except for IL-10, thereby giving evidence of functional changes after culture expansion. In this study, mSVF showed active cell populations in the adipose tissue comparable to eSVF with excellent survival, differentiation and paracrine properties under a new mechanical adipose tissue micro-fragmentation system; thereby suggesting its potential use as a minimally invasive technique for OA treatment.

Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold.

OBJECTIVES: Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology. METHODS: A 3D model of bioengineered medial meniscus tissue was created, based on MRI scans of a human volunteer. The Digital Imaging and Communications in Medicine (DICOM) data from these MRI scans were processed using dedicated software, in order to obtain an STL model of the structure. The chosen 3D Discovery printing tool was a microvalve-based inkjet printhead. Primary mesenchymal stem cells (MSCs) were isolated from bone marrow and embedded in a collagen-based bio-ink before printing. LIVE/DEAD assay was performed on realized cell-laden constructs carrying MSCs in order to evaluate cell distribution and viability. RESULTS: This study involved the realization of a human cell-laden collagen meniscus using 3D bioprinting. The meniscus prototype showed the biological potential of this technology to provide an anatomically shaped, patient-specific construct with viable cells on a biocompatible material. CONCLUSION: This paper reports the preliminary findings of the production of a custom-made, cell-laden, collagen-based human meniscus. The prototype described could act as the starting point for future developments of this collagen-based, tissue-engineered structure, which could aid the optimization of implants designed to replace damaged menisci.Cite this article: G. Filardo, M. Petretta, C. Cavallo, L. Roseti, S. Durante, U. Albisinni, B. Grigolo. Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold. Bone Joint Res 2019;8:101-106. DOI: 10.1302/2046-3758.82.BJR-2018-0134.R1.

Ligament repair: a molecular and immunohistological characterization.

The anterior cruciate ligament (ACL) is the most commonly injured tissue of the human knee. Its poor ability to regenerate after injury represents a challenge to ligament tissue engineering. An understanding of the molecular composition of the structures used for its repair is essential for clinical assessments and for the implementation of tissue engineering strategies. The objective of this study was to evaluate, both at gene and protein levels, the expression of characteristic molecules in human ACL, patellar, semitendinosus and gracilis tendons and in the ligament reconstructed with patellar or semitendinosus and gracilis tendons. We demonstrated that primary ACL and tendon tissues all express collagen I, II, Sox-9, tenascin-C and aggrecan. Collagen X expression was detected at very low levels or undetectable. Cathepsin B, MMP-1 and MMP-13 were expressed at higher levels in the ACL reconstructed by the two tendons, showing that a remodeling process occurs during "ligamentization". Both our molecular and immunohistochemical evaluations did not reveal significative differences between the tendons and ligaments analyzed. However, ACL reconstructed with semitendinosus and gracilis tendon seems to present a higher expression of collagen type II when compared to that reconstructed with patellar tendon. This study could give a reasonable identification of genetic and protein markers specific to tendon/ligament tissues and be helpful in testing tissue engineering approaches for ACL reconstruction.

Cultures of a human synovial cell line to evaluate platelet-rich plasma and hyaluronic acid effects.

Synovial inflammation plays an important role in osteoarthritis (OA) pathogenesis. Different biological compounds have been tested mainly on chondrocytes, to treat early stages of OA. However, because OA has been recently defined as "an organ" pathology, investigation on synoviocytes is also needed. Therefore, the aim of the present study was to validate a human fibroblast-like synoviocytes cell line (K4IM) to test the effects of platelet-rich plasma (PRP) and hyaluronan (HA) on anabolic and catabolic gene expression and on HA secretion from cell cultures. In order to determine the effect of PRP and HA, K4IM cells were maintained in culture with or without TNF-α stimulation. In the presence of PRP, unstimulated K4IM cells presented the same expression of IL1B, IL6, CXCL8, VEGF, TIMP1, and hyaluronic synthase isoform HAS3 as primary human synoviocytes, while HA addition did not change their expression pattern, which was similar to control cells. Stimulated cells expressed significantly higher values of IL1B, CXCL8, and VEGF compared with unstimulated ones. PRP did not show any modification, except for VEGF, while HA addition modulated IL1B expression. PRP did not modulate HA release of both stimulated and unstimulated cells. Our study showed the possibility to use K4IM synoviocytes as an in vitro model to test biological compounds useful for the treatment of early OA. Primary cells reflect the phenotype of cells in vivo, but limited recovery from biopsies and restricted lifespan makes experimental manipulation challenging. Therefore, despite cell lines present some limitations, they could be used as an alternative for preliminary experiments.

Glucosamine and its N-acetyl-phenylalanine derivative prevent TNF-alpha-induced transcriptional activation in human chondrocytes.

OBJECTIVES: Glucosamine (GlcN) is used in the treatment of osteoarthritis as symptomatic slow-acting drug, but its mode of action is not completely known. We analyzed the influence of GlcN and its N-acetyl-phenylalanine derivative (NAPA) on mRNA transcription level of TNF-alpha-stimulated genes in cell culture. METHODS: Human immortalized chondrocyte cell line lbpva55 was stimulated with TNF-alpha and treated with GlcN and NAPA. mRNA transcription level of several genes, identified by complementary DNA microarray (cDNA microarray), was validated by Quantitative Real-Time Polymerase Chain Reaction (Q-RT-PCR). RESULTS: Several genes, whose mRNA level was increased by TNF-alpha treatment and significantly reduced by GlcN and NAPA in lbpva55 cells, were identified. These include cytokine receptors TNF-R1 and TNF-R2, their associated factor TRAF-6, signaling intermediates IGFB-6 and Rnd1, as well as cell cycle regulating proteins CUL-2 and G1S protein 1. Down- regulation of mRNA expression level of some of these genes is in accordance with inactivation of NF-kB transcription factor. Moreover, we found down-regulation of c-jun mRNA level, a component of AP-1 transcription factor. CONCLUSIONS: Our study suggests that GlcN and NAPA interfere with activation of NF-kB and AP-1 transcription factors, which are responsible for the expression of genes involved in diverse biological processes, such as cell growth and death, inflammatory and stress responses, accounting for the beneficial effects of GlcN in osteoarthritis.

Subchondral bone response to injected adipose-derived stromal cells for treating osteoarthritis using an experimental rabbit model.

Although articular cartilage is the target of osteoarthritis (OA), its deterioration is not always clearly associated with patient symptoms. Because a functional interaction between cartilage and bone is crucial, the pathophysiology of OA and its treatment strategy must focus also on subchondral bone. We investigated whether adipose-derived stromal cells (ASCs) injected into a joint at two different concentrations could prevent subchondral bone damage after the onset of mild OA in a rabbit model. We measured both volumetric and densitometric aspects of bone remodeling. Although OA can stimulate bone remodeling either catabolically or anabolically over time, the accelerated turnover does not allow complete mineralization of new bone and therefore gradually reduces its density. We measured changes in morphometric and densitometric bone parameters using micro-CT analysis and correlated them with the corresponding parameters in cartilage and meniscus. We found that ASCs promoted cartilage repair and helped counteract the accelerated bone turnover that occurs with OA.

Specific inductive potential of a novel nanocomposite biomimetic biomaterial for osteochondral tissue regeneration.

Osteochondral lesions require treatment to restore the biology and functionality of the joint. A novel nanostructured biomimetic gradient scaffold was developed to mimic the biochemical and biophysical properties of the different layers of native osteochondral structure. The present results show that the scaffold presents important physicochemical characteristics and can support the growth and differentiation of mesenchymal stromal cells (h-MSCs), which adhere and penetrate into the cartilaginous and bony layers. H-MSCs grown in chondrogenic or osteogenic medium decreased their proliferation during days 14-52 on both scaffold layers and in medium without inducing factors used as controls. Both chondrogenic and osteogenic differentiation of h-MSCs occurred from day 28 and were increased on day 52, but not in the control medium. Safranin O staining and collagen type II and proteoglycans immunostaining confirmed that chondrogenic differentiation was specifically induced only in the cartilaginous layer. Conversely, von Kossa staining, osteocalcin and osteopontin immunostaining confirmed that osteogenic differentiation occurred on both layers. This study shows the specific potential of each layer of the biomimetic scaffold to induce chondrogenic or osteogenic differentiation of h-MSCs. These processes depended mainly on the media used but not the biomaterial itself, suggesting that the local milieu is fundamental for guiding cell differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

Thermodynamic features of bile salt-human serum albumin interaction.

Interaction with human serum albumin is responsible for important aspects of the physiological behaviour of bile salts, although this factor has not been adequately examined. The nature of the binding is investigated here by means of thermodynamic parameters determined by equilibrium dialysis and microcalorimetric measurements. The positive enthalpy and entropy values obtained indicate the presence of a poorly specific hydrophobic bonding.

Analysis of mesenchymal stem cells grown on a three-dimensional HYAFF 11-based prototype ligament scaffold.

Ligaments are complex structures that maintain the mechanical stability of the joint. Healing of injured ligaments involves the interactions of different cell types, local cellular environment, and the use of devices. To gain new information on the complex interactions between mesenchymal stem cells (MSCs) and a specific hyaluronan-based prototype scaffold (HYAFF, useful for ligament tissue engineering, short time-course experiments were performed to analyze the proliferation, vitality, and phenotype of MSCs grown on the scaffold. MSC proliferation was analyzed using the MTT test, during the early time points (2, 4, 6, days). Viability was assessed using calcein/acetyloxymethylester immunofluorescence dye and confocal microscopy analysis. Hyaluronic acid receptor (CD44), typical matrix ligament proteins (collagen type I, type III, laminin, fibronectin, actin), and chondrogenic/osteogenic markers (collagen type II and bone sialoprotein) were evaluated by immunohistochemistry. Our data demonstrated that MSC growth and viability were cell density-dependent. MSCs completely wrapped the fibers of the scaffold, expressed CD44, collagen type I, type III, laminin, fibronectin, and actin, and were negative to collagen type II and bone sialoprotein. These data demonstrate that MSCs survive well in the hyaluronan-based prototype ligament scaffold, as assessed after 2 days from seeding, and express CD44, a receptor important for scaffold interaction, and proteins responsible for the functional characteristics of the ligaments.

Intracellular Cu/Zn superoxide dismutase levels in T and non-T cells from normal aged subjects.

The decreased immune response associated with aging may, in part, reflect intrinsic age-related biochemical alteration in lymphocytes from older subjects. The 'reactive oxygen species hypothesis' of aging postulates that these molecules are involved in the modifications leading to cellular senescence. Superoxide dismutase (SOD), and in particular the Cu/Zn-dependent intracellular form, plays a critical role in the defense against these species, but it is controversial whether this function declines in lymphocytes in old age. We utilized two different methods to evaluate Cu/Zn SOD levels in T and non-T cells (CD3+, CD3-, CD4+, CD8+, CD16+) from young and old individuals: a specific and sensitive enzyme immunoassay performed on extracts of sorted cells, and a flow cytometry double fluorescence technique with monoclonal antibodies against Cu/Zn SOD and the different lymphocyte subsets. The Cu/Zn SOD cell content was assayed both in basal conditions and after peripheral blood lymphocyte stimulation with Concanavalin A, anti-CD3 monoclonal antibody and phorbol myristate acetate. In basal conditions, and considering the various subsets, no differences were found between young and old individuals, although data analysis revealed high and low responders in both groups. Taking all the subjects together, higher levels of this enzyme were found in CD3+ T lymphocytes, in particular in the CD4+ cells. After peripheral blood lymphocyte stimulation, Cu/Zn SOD concentration was higher than levels in unstimulated cells, both in young and old individuals, and particularly using Concanavalin A with respect to anti-CD3 and phorbol myristate acetate. In conclusion, the synthesis of Cu/Zn SOD does not seem to be affected by aging in proliferating cells. The highest levels of Cu/Zn SOD present in CD4+ cells, both from young and old individuals, may prevent the oxidant stress of these elements which play a major role in the inflammation sites.

Bile acids with cyclopropane-containing side chain. 2. Synthesis and properties of 3 alpha,7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid (2-sulfoethyl)amide.

The preparation of 3 alpha,7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid (2-sulfoethyl)amide (5) by the one-step EEDQ-induced conjugation between ursodeoxycholic acid "cyclopropylog" (4) and taurine is described. The presence of a cyclopropyl ring adjacent to the amide bond is shown to make it resistant to degradation by intestinal bacteria. This new cyclopropylog is neither deconjugated at the C-24 amide bond nor 7-dehydroxylated when incubated with human stools in anaerobic conditions.

Bile acids with a cyclopropyl-containing side chain. 3. Separation, identification, and properties of all four stereoisomers of 3 alpha,7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid.

3 alpha,7 beta-Dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid (CUDCA) (2a), a side-chain cyclopropylog of ursodeoxycholic acid (UDCA) was shown to be a mixture of four stereoisomers (CUDCA A-D). The 22S,23R, and 22R,23S diastereoisomers have been separated, their respective configurations assigned by 13C NMR spectroscopy, and original synthetic schemes for their preparation elaborated. Moreover, theoretical models of the structure of UDCA and CUDCA A-D were built by using molecular computer graphic techniques. It was shown that the four diastereoisomers greatly differ in hydrophilicity, in critical micellar concentration (CMC) in water, and exhibit a different interaction with intestinal bacterial enzymes. It was also shown that CUDCA A-C are not conjugated with glycine or taurine in the liver, while CUDCA D is secreted into bile predominantly as taurine and glycine conjugate.