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PubMed2004

Advances in cell therapy for renal failure.

Cell therapy is one of the most exciting fields in translational medicine. It stands at the intersection of a variety of rapidly developing scientific disciplines: stem cell biology, immunology, tissue engineering, molecular biology, biomaterials, transplantation biology, regenerative medicine and clinical research. Cell-based therapy may develop into a new therapeutic platform to treat a vast array of clinical disorders. Blood transfusions and bone marrow transplantation are prime examples of the successful application of cell-based therapeutics; but recent advances in cellular and molecular biology have expanded the potential applications of this approach. Although recombinant genetic engineering to produce a variety of therapeutics, such as human erythropoietin and insulin has proven successful, these treatments are unable to completely correct or reverse disease states, because most common disease processes are not due to the deficiency of a single protein but develop due to alterations in the complex interactions of a variety of cell components. In these complex situations, cell-based therapy may be a more successful strategy by providing a dynamic, interactive and individualized therapeutic approach that responds to the pathophysiological condition of the patient. In this regard, cells may provide innovative methods for drug delivery of biologics, immunotherapy, and tissue regenerative or replacement engineering [Nature 392 (1998) 518-524, Nat Biotechnol 20 (2002) 339-343]. The translation of this discipline to medicinal practice has tremendous potential, but in many applications technological issues need to be overcome. Since many cell-based indications are already being evaluated in the clinic, the field appears to be on the threshold of a number of successes. This review will focus on our group's use of human stem/progenitor cells in the treatment of acute and chronic renal failure as extensions to current successful renal substitution processes of hemodialysis and hemofiltration.

Acute Kidney Injury↗
PubMed2005

Stem cells: the next therapeutic frontier.

Cell therapy is one of the most exciting fields in translational medicine. It stands at the intersection of a variety of rapidly developing scientific disciplines: stem cell biology, immunology, tissue engineering, molecular biology, biomaterials, transplantation biology, regenerative medicine, and clinical research. Cell-based therapy may develop into a new therapeutic platform to treat a vast array of clinical disorders. Blood transfusions and bone marrow transplantation are prime examples of the successful application of cell-based therapeutics; but recent advances in cellular and molecular biology have expanded the potential applications of this approach. Although recombinant genetic engineering to produce a variety of therapeutics such as human erythropoietin and insulin has proven successful, these treatments are unable to completely correct or reverse disease states, because most common disease processes are not due to the deficiency of a single protein but develop due to alterations in the complex interactions of a variety of cell components. In these complex situations, cell-based therapy may be a more successful strategy by providing a dynamic, interactive, and individualized therapeutic approach that responds to the pathophysiological condition of the patient. In this regard, cells may provide innovative methods for drug delivery of biologics, immunotherapy, and tissue regenerative or replacement engineering (1,2). The translation of this discipline to medical practice has tremendous potential, but in many applications technological issues need to be overcome. Since many cell-based indications are already being evaluated in the clinic, the field appears to be on the threshold of a number of successes. This review will focus on our group's use of human stem/progenitor cells in the treatment of acute and chronic renal failure as extensions to the current successful renal substitution processes of hemodialysis and hemofiltration.

Animals↗
PubMed2005

Meniscal regeneration using tissue engineering with a scaffold derived from a rat meniscus and mesenchymal stromal cells derived from rat bone marrow.

The purpose of this study was to regenerate a meniscus using a scaffold from a normal meniscus and mesenchymal stromal cells derived from bone marrow (BM-MSCs). Thirty Sprague-Dawley rat menisci were excised and freeze-thawed three times with liquid nitrogen to kill the original meniscal cells. Bone marrow was aspirated from enhanced green fluorescent protein transgenic Sprague-Dawley rats. BM-MSCs were isolated, cultured for 2 weeks, and 2 x 10(5) cells were then seeded onto the meniscal scaffolds. Using a fluorescent microscope and immunohistochemical staining, repopulation of enhanced green fluorescent protein positive cells was observed in the superficial zone of the scaffold after 1 week of culture, and then in the deep zone after 2 weeks. At 4 weeks, expression of extracellular matrices was detected histologically and expression of mRNA for aggrecan and type X collagen was detected. Stiffness of the cultured tissue, assessed by the indentation stiffness test, had increased significantly after 2 weeks in culture, and approximated the stiffness of a normal meniscus. From this study, we conclude that a scaffold derived from a normal meniscus seeded with BM-MSCs can form a meniscus approximating a normal meniscus.

Animals↗
PubMed2005

The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells.

Advances in tissue engineering require biofunctional scaffolds that can not only provide cells with structural support, but also interact with cells in a biological manner. To achieve this goal, a frequently used cell adhesion peptide Arg-Gly-Asp (RGD) was covalently incorporated into poly(ethylene glycol) diacrylate (PEODA) hydrogel and its dosage effect (0.025, 1.25 and 2.5 mm) on osteogenesis of marrow stromal cells in a three-dimensional environment was examined. Expression of bone-related markers, osteocalcin (OCN) and Alkaline phosphatase (ALP), increased significantly as the RGD concentration increased. Compared with no RGD, 2.5 mm RGD group showed a 1344% increase in ALP production and a 277% increase in OCN accumulation in the medium. RGD helped MSCs maintain cbfa-1 expression when shifted from a two-dimensional environment to a three-dimensional environment. Soluble RGD was found to completely block the mineralization of marrow stromal cells, as manifested by quantitative calcium assay, phosphorus elemental analysis and Von Kossa staining. In conclusion, we have demonstrated that RGD-conjugated PEODA hydrogel promotes the osteogenesis of MSCs in a dosage-dependent manner, with 2.5 mm being optimal concentration.

Alkaline Phosphatase↗
PubMed2006

Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide.

Human adipose derived adult stem (hADAS) cells have the ability to differentiate into a chondrogenic phenotype in three-dimensional culture and media containing dexamethasone and TGF-beta. The current study examined the potential of a genetically engineered elastin-like polypeptide (ELP) to promote the chondrocytic differentiation of hADAS cells without exogenous chondrogenic supplements. hADAS cells were cultured in ELP hydrogels in either chondrogenic or standard medium at 5% O2 for up to 2 weeks. By day 14, constructs cultured in either medium exhibited significant increases in sulfated glycosaminoglycan (up to 100%) and collagen contents (up to 420%). Immunolabeling confirmed that the matrix formed consisted mainly of type II and not type I collagen. The composition of the constructs cultured in either medium did not differ significantly. To assess the effect of oxygen tension on the differentiation of the above constructs, samples were cultured in standard medium at either 5% or 20% O2 for 7 days and their gene expression profile was evaluated using real time RT-PCR. In both cases, the hADAS-ELP constructs upregulated SOX9 and type II collagen gene expression, while type I collagen was downregulated. However, constructs cultured in 20% O2 highly upregulated type X collagen, which was not detected in the 5% O2 cultures. The study suggests that ELP can promote chondrogenesis for hADAS cells in the absence of exogenous TGF-beta1 and dexamethasone, especially under low oxygen tension conditions.

Adipose Tissue↗
PubMed2001

Desiccation tolerance in human cells.

The ability to desiccate mammalian cells while maintaining a high degree of viability would have implications for many areas of biological science, including tissue engineering. Previously, we reported that introduction of the genes for trehalose biosynthesis allowed human cells in culture to be reversibly desiccated for up to 5 days. Here, we have further investigated the factors that allow human cells to survive in the desiccated state. The most important finding is that vacuum greatly enhances the ability of human cells in culture to withstand desiccation. In fact, cells dried slowly and stored under vacuum are able to withstand desiccation even in the absence of added carbohydrates or polyols. In addition to vacuum, the rate of desiccation, the temperature at which cells are maintained, the degree of confluence when dried, and the presence or absence of light have a large effect on the ability to retain viability in the desiccated state. Our data are consistent with a model in which cells can retain viability if they are desiccated in such a way that cellular structures are maintained. However, gradual loss of viability may be due to damage that occurs over time in the desiccated state, perhaps due to free radicals. Further optimization of the process for desiccating and maintaining cells is required before long-term storage of desiccated cells can be achieved.

Carbohydrates↗
PubMed2005

Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of gamma-Gliadin.

The gene encoding the repetitive domain located in the N-terminal half of gamma-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of gamma-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypeptides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.

Chromatography, High Pressure Liquid↗
PubMed2004

Molecular-level description of proteins from saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics.

For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions by top down mass spectrometry holds promise but has yet to be achieved on a large scale. Using lysate from Saccharomyces cerevisiae, 117 gene products were identified with 100% sequence coverage revealing 26 acetylations, 1 N-terminal dimethylation, 1 phosphorylation, 18 duplicate genes, and 44 proteolytic fragments. The platform for this study combined continuous-elution gel electrophoresis, reversed-phase liquid chromatography, automated nanospray coupled with a quadrupole-FT hybrid mass spectrometer, and a new search engine for querying a custom database. The proteins identified required no manual validation, ranged from 5 to 39 kDa, had codon biases from 0.93 to 0.083, and were primarily associated with glycolysis and protein synthesis. Illustrations of gene-specific identifications, PTM detection and subsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database) show how larger scale proteome projects incorporating top down may proceed in the future using commercial Q-FT instruments.

Acetylation↗
PubMed2005

Disulfide trapping to localize small-molecule agonists and antagonists for a G protein-coupled receptor.

A key question regarding the signaling mechanism for G protein-coupled receptors (GPCRs) is what triggers agonism versus antagonism. Peptide analogs derived from the chemokine, complement fragment 5 anaphylatoxin (C5a), can act as agonists or antagonists to the C5a receptor, a member of the GPCR family [Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J. & Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-4000]. Recently, we showed that two Cys residues engineered near a proposed binding site in the C5a receptor on transmembrane helices III and VI can selectively and reversibly trap short Cys-containing 3-mer peptides derived from C5a by disulfide bond formation [Buck, E. A., Bourne, H. & Wells, J. A. (November 18, 2004) J. Biol. Chem., 10.1074/jbc.C400500200]. Here, a library of 10,000 compounds, each containing an exchangeable thiol, was screened to identify specific small-molecule mimics that block binding of C5a. Some of the selected compounds acted as agonists and were as potent as the natural C5a ligand, and some acted as antagonists. A residue near these compounds, Ile-116 in helix III, functions as a "gatekeeper" to modulate these effects. A small substitution, Ile-116-Ala, enhanced affinity for some compounds and allowed antagonists to function as agonists; a larger substitution, Ile-116-Trp, decreased affinity and agonism. Thus, subtle changes in either the structure of the ligand or the receptor at the site between helix III, VI, and VII can switch the receptor on or off. This ligand binding and activation site may be similarly positioned in other members of the chemokine receptor family. Selective ligand trapping by reversible disulfide formation may serve to nucleate the development of small-molecule mimics.

Binding Sites↗
PubMed1995

A double mutant of sperm whale myoglobin mimics the structure and function of elephant myoglobin.

The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.

Amino Acid Sequence↗
PubMed2006

Hypertonic saline resuscitation prevents hydrostatically induced intestinal edema and ileus.

OBJECTIVE: We have shown that acute edema induced by mesenteric venous hypertension (MV-HTN) impairs intestinal transit and reduces the standardized engineering measures of intestinal stiffness (elastic modulus) and residual stress (opening angle). We hypothesized that hypertonic saline (7.5%) would reverse these detrimental effects of acute edema. DESIGN: Laboratory study. SETTING: University laboratory. SUBJECTS: Male Sprague Dawley rats (270-330 g). INTERVENTIONS: Rats were randomized to five groups: sham, MV-HTN alone, MV-HTN with 4 mL/kg normal saline resuscitation (equal volume), MV-HTN with 33 mL/kg normal saline resuscitation (equal salt), and MV-HTN with 4 mL/kg hypertonic saline. Intestinal edema was measured by wet to dry tissue weight ratio. A duodenal catheter was placed and, 30 mins before death, fluorescein isothiocyanate Dextran was injected. At death, dye concentrations were measured to determine intestinal transit. Segments of distal ileum were hung to a fixed point in a tissue bath and to a force displacement transducer and stretched in increments of 1 mm; we recorded the new length and the corresponding force from the force displacement transducer to determine elastic modulus. Next, two transverse cuts were made yielding a 1- to 2-mm thick ring-shaped segment of tissue which was then cut radially to open the ring. Then the opening angle was measured. MEASUREMENTS AND MAIN RESULTS: MV-HTN, MV-HTN with 4 mL/kg normal saline, and MV-HTN with 33 mL/kg normal saline caused a significant increase in tissue edema and a significant decrease in intestinal transit, stiffness, and residual stress compared with sham. Hypertonic saline significantly lessened the effect of edema on intestinal transit and prevented the changes in stiffness and residual stress. CONCLUSIONS: Hypertonic saline prevented intestinal tissue edema. In addition, hypertonic saline improved intestinal transit, possibly through more efficient transmission of muscle force through stiffer intestinal tissue.

Animals↗
PubMed2005

PbSe nanocrystal solids for n- and p-channel thin film field-effect transistors.

Initially poorly conducting PbSe nanocrystal solids (quantum dot arrays or superlattices) can be chemically "activated" to fabricate n- and p-channel field effect transistors with electron and hole mobilities of 0.9 and 0.2 square centimeters per volt-second, respectively; with current modulations of about 10(3) to 10(4); and with current density approaching 3 x 10(4) amperes per square centimeter. Chemical treatments engineer the interparticle spacing, electronic coupling, and doping while passivating electronic traps. These nanocrystal field-effect transistors allow reversible switching between n- and p-transport, providing options for complementary metal oxide semiconductor circuits and enabling a range of low-cost, large-area electronic, optoelectronic, thermoelectric, and sensing applications.

Journal Article↗
PubMed1993

Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.

Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.

Amino Acid Sequence↗
PubMed2006

Live, attenuated influenza A H5N1 candidate vaccines provide broad cross-protection in mice and ferrets.

BACKGROUND: Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. METHODS AND FINDINGS: Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 10(6) 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. CONCLUSIONS: The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans.

Animals↗
PubMed2001

DNA polymerases as engines for biotechnology.

This review examines the DNA polymerases, a class of enzymes that has been an essential tool for molecular biology research. Several families of DNA polymerases have been defined based on amino acid sequence comparisons, and new enzymes are continually being discovered, driving the expansion of the current classifications. Structural similarities among the enzymes are examined, as well as the functions of the various subunits and enzyme domains. The natural variety of polymerase activities has been harnessed for applications such as amplification, labeling, and detection of DNA sequences. In addition, enhancements to DNA polymerases by genetic engineering will be described, such as enzymes specifically designed for DNA sequencing by improving the incorporation of dideoxynucleotide terminators. Reverse transcription, the ability to use RNA as a template for DNA synthesis, is described for the application of making cDNA. We believe that new and unanticipated applications will emerge as new polymerases and mutated polymerases are created and characterized.

Biotechnology↗
PubMed1996

Chemoenzymatic synthesis: application to the study of carbohydrate recognition.

Reported here are useful strategies recently developed for the large-scale synthesis of complex and polyfunctional molecules using native or engineered enzymes as catalysts. Several important issues in the field regarding the problems of substrate specificity, product inhibition, reaction reversibility, enzyme stability and catalytic efficiency are addressed in the representative synthesis of carbohydrates and carbohydrate mimetics designed for use to study carbohydrate-mediated cell adhesion.

Carbohydrates↗
PubMed2001

[Assisted circulation. I. Systems with short-term mechanical circulatory support].

The concept of artificial circulatory support has been established almost 200 years ago. It has only been within the last four decades that physicians and engineers have developed mechanical assist devices that can temporarily support the circulation until the native heart recovers from a reversible injury. If the heart does not recover sufficient function to maintain adequate hemodynamics, long-term circulatory support or permanent replace (biologically--heart transplant or permanent mechanical circulatory support) is needed. In this paper we describe the devices (intraaortic balloon pump, roller, centrifugal and axial pumps), that are in current clinical use for temporary (short-term) mechanical circulatory support.

Assisted Circulation↗
PubMed2001

[Assisted circulation II--systems for intermediate and long-term mechanical circulatory support].

The concept of artificial circulatory support has been established almost 200 years ago. It has only been within the last four decades that physicians and engineers have developed mechanical assist devices that can temporarily support the circulation until the native heart recovers from a reversible injury. In patients who do not regain native heart function, long-term circulatory support or permanent replace (biologically--heart transplant or permanent mechanical circulatory support) is indicated. In this paper we describe the devices (ABIOMED BSV 5000, Thoratec, HeartMate, Novacor and CardioWest TAH), that are in current clinical use for intermediate and long-term mechanical circulatory support.

Assisted Circulation↗