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PubMed2001

Kinematic comparison of Hybrid II test dummy to wheelchair user.

Hybrid test dummies provide a safe alternative to human subjects when investigating mechanisms of wheelchair tips and falls. The data that researchers acquire from these test dummies are more useful if the test dummy represents the population being studied. The goal of this study was to measure the validity of a 50th percentile Hybrid II test dummy (HTD) as an accurate representation of a wheelchair user. A test pilot with T8 paraplegia due to traumatic spinal cord injury served as a basis for validation. Simple modifications were made to the HTD to approximate the trunk stability characteristics of a person with a spinal cord injury. An HTD, a modified HTD, and a human test pilot were seated in an electric-powered wheelchair and several braking tests performed. The standard HTD underestimated the kinematics when compared to the test pilot. The modified HTD had less trunk stability than the standard HTD during all braking methods. The modified HTD and wheelchair test pilot had similar trunk stability characteristics during kill switch and joystick full-reverse braking conditions. The modified HTD is a satisfactory representation of a wheelchair user with a spinal cord injury; however, the modified test dummy underestimates the trunk dynamics during the less extreme joystick release braking. Work should continue on the development of a low-speed, low-impact test dummy that emulates the wheelchair user population.

Biomechanical Phenomena↗
PubMed2006

Regulated translation of listeriolysin O controls virulence of Listeria monocytogenes.

Listeria monocytogenes is an intracytosolic bacterial pathogen that escapes from the phagosome using a secreted cytolysin, listeriolysin O (LLO). In the host cytosol, LLO activity is minimized to prevent pore formation in the host plasma membrane; premature lysis of the infected host cell exposes the bacteria to extracellular immune defences of the host and is detrimental to infection. Here we identified nucleotide substitutions in the coding sequence of the LLO gene (hly) that did not alter the protein sequence, yet caused over-production of LLO, cytotoxicity and loss of virulence. These phenotypes were independent of the promoter and, under conditions in which the mutants produced more LLO protein than wild type, levels of hly mRNA were similar. Finally, negative regulation of LLO was maintained even when bacteria were engineered to produce elevated levels of the wild-type hly transcript. Together, our data demonstrate that translational regulation of LLO is critical for L. monocytogenes pathogenesis.

Animals↗
PubMed1987

Influence of the catBCE sequence on the phenotypic reversion of a pcaE mutation in Acinetobacter calcoaceticus.

Isofunctional beta-ketoadipate:succinyl coenzyme A transferases I and II are encoded by the pcaE and catE genes, respectively, of Acinetobacter calcoaceticus. The genes are under separate transcriptional control and genetically unlinked. Mutations in the pcaE gene result in a p-hydroxybenzoate-negative (POB-) phenotype, whereas catE mutations cause a benzoate-negative (Ben-) phenotype. A. calcoaceticus ADP125 carries the pcaE3125 mutation and gave rise to POB+ revertants with a frequency of 10(-4). A 5.0-kilobase-pair (kb) EcoRI restriction fragment containing the catBCDE genes possesses two SalI restriction sites separated by 1.5 kb. Removal of the DNA between the SalI sites created a deletion removing the terminal 35 base pairs of the catB gene, the 300-base-pair catC gene, and about 1.1 kb of the 1.2-kb catE gene. Transformation of strain ADP125 with the modified EcoRI fragment lacking the SalI segment produced natural transformants containing this designed deletion with a frequency of 20%. The frequency of POB+ phenotypic reversion of the pcaE3125 mutation in these transformants was more than 300-fold lower than the frequency of phenotypic reversion observed in genetic backgrounds containing the catBCE segment. Alleles created by pcaE phenotypic reversion in a wild-type cat genetic background were unlinked to the cat gene cluster, and revertant transferases were expressed inducibly with the pca genes. Alterations in the restriction pattern of the pca gene cluster in several revertants were observed, indicating that multiple sequence changes have occurred in the pca genes during reversion. Growth of the phenotypic revertants under nonselective conditions resulted in loss of either the POB+ phenotype or both the POB+ and Ben+ phenotypes at high frequency. Southern hybridizations revealed that loss of the POB+ of Ben+ phenotype was due to deletion of the entire pca or cat gene cluster, a loss of at least 16 kb in some strains. Revertants isolated in a catBCE deletion background were stable. These results suggest that enhanced phenotypic reversion of pcaE3125 in wild-type cat background is due to repair of the mutation by recombination between the catBCE and pcaE3125 sequences. Genetic instability of the phenotypic revertants may be attributed to deletion of pca and cat sequences by recombination between regions of homology created as a consequence of pcaE repair.

Acinetobacter↗
PubMed2004

Reengineering of aged Bruch's membrane to enhance retinal pigment epithelium repopulation.

PURPOSE: An earlier study showed that age-related changes in the inner collagen layer (ICL) inhibit RPE cell repopulation of human Bruch's membrane. The present study was undertaken to determine the effect of cleaning and/or an extracellular matrix (ECM) protein coating on the reattachment, apoptosis, proliferation, and final surface coverage of the transplanted RPE cells. METHODS: Explants of aged Bruch's membrane with ICL exposed were prepared from five human cadaveric eyes (donor ages, 69-84 years) and treated with Triton X-100 and/or coated with a mixture of laminin (330 microg/mL), fibronectin (250 microg/mL), and vitronectin (33 microg/mL). Viable human fetal and ARPE-19 cells (n = 15,000) were plated onto the surface and the RPE reattachment, apoptosis, and proliferation ratios were determined on the modified surfaces. Cells were cultured up to 17 days to determine the surface coverage. Ultrastructure of the modified Bruch's membrane and RPE morphology were studied with transmission and scanning electron microscopy. RESULTS: Reattachment ratios of fetal human RPE and ARPE-19 cells were similar on aged ICL (41.5% +/- 1.7% and 42.9% +/- 2.7%, P > 0.05). The reattachment ratio increased with ECM protein coating and decreased with detergent treatment. Combined cleaning and coating restored the reattachment ratio of the fetal RPE cells, but failed to increase the reattachment ratio of ARPE-19 cells. The highest apoptosis was observed on untreated ICL. Cleaning and the combined procedure of cleaning and ECM protein coating decreased fetal RPE cell apoptosis. Only RPE cells plated on cleaned or cleaned and ECM-coated ICL demonstrated proliferation that led to substantial surface coverage at day 17. CONCLUSIONS: Age-related changes that impair RPE repopulation of Bruch's membrane can be significantly reversed by combined cleaning and ECM protein coating of the ICL. Development of biologically tolerant techniques for modifying the ICL in vivo may enhance reattachment of the RPE and its repopulation of aged ICL.

Aged↗
PubMed2004

Osmophobic effect of glycerol on irreversible thermal denaturation of rabbit creatine kinase.

Protein stability plays an extremely important role not only in its biological function but also in medical science and protein engineering. Osmolytes provide a general method to protect proteins from the unfolding and aggregation induced by extreme environmental stress. In this study, the effect of glycerol on protection of the model enzyme creatine kinase (CK) against heat stress was investigated by a combination of spectroscopic method and thermodynamic analysis. Glycerol could prevent CK from thermal inactivation and aggregation in a concentration-dependent manner. The spectroscopic measurements suggested that the protective effect of glycerol was a result of enhancing the structural stability of native CK. A further thermodynamic analysis using the activated-complex theory suggested that the effect of glycerol on preventing CK against aggregation was consistent with those previously established mechanisms in reversible systems. The osmophobic effect of glycerol, which preferentially raised the free energy of the activated complex, shifted the equilibrium between the native state and the activated complex in favor of the native state. A comparison of the inactivation rate and the denaturation rate suggested that the protection of enzyme activity by glycerol should be attributed to the enhancement of the structural stability of the whole protein rather than the flexible active site.

Animals↗
PubMed1993

Functional expression of the uncomplexed serum retinol-binding protein in Escherichia coli. Ligand binding and reversible unfolding characteristics.

The serum retinol-binding protein solubilizes the lipophilic vitamin A alcohol and plays an important physiological role in the transport of this compound. The monomeric single-domain protein, the three-dimensional structure of which is known, constitutes a well-characterized member of the lipocalin family of proteins. We report here the functional expression of the apo-protein in Escherichia coli by secretion to the periplasm. The recombinant protein, purified in a single step by metal chelate affinity chromatography, exhibits the same ligand binding characteristics as described for the natural protein. Guanidinium chloride-induced unfolding and refolding experiments suggest that the recombinant retinol-binding protein adopts a stable conformation despite being expressed and purified in the absence of the large hydrophobic ligand. The expression system described here should also be useful for the recombinant production of other lipocalin proteins, thus permitting the elucidation of the structure-function relationships of ligand binding by protein engineering.

Amino Acid Sequence↗
PubMed2005

Invitro study of adherent mandibular osteoblast-like cells on carrier materials.

Augmentation of the craniofacial region is necessary for many aesthetic and reconstructive procedures. Tissue engineering offers a new option to supplement existing treatment regimens. In this procedure, materials composed of hydroxyapatite (HA), of synthetic or natural origin, are used as scaffolds. The aim of this study was to evaluate the effects of three HA materials on cultured human osteoblasts in vitro. Explant cultures of cells from human alveolar bone were established. Human osteoblasts were cultured on the surface of HA calcified from red algae (C GRAFT/Algipore), deproteinized bovine HA (Bio-Oss) and bovine HA carrying the cell binding peptide P-15 (Pep Gen P-15). Cultured cells were evaluated with respect to cell attachment, proliferation and differentiation. Cells were cultured for 6 and 21 days under osteogenic differentiation conditions, and tissue-culture polystyrene dishes were used as control. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay, protein synthesis analysis and by scanning electron microscopical examination. Osteogenic differentiation was screened by the expression of alkaline phosphatase. The osteoblastic phenotype of the cells was monitored using mRNA levels of the bone-related proteins including osteocalcin, osteopontin and collagen Type I. We found that cells cultured on C GRAFT/Algipore) and Pep Gen P-15 showed a continuous increase in DNA content and protein synthesis. Cells cultured on Bio-Oss showed a decrease in DNA content from Day 6 (P < 0.05) to Day 21 (P < 0.0001) and protein synthesis on Day 21 (P < 0.005). Alkaline phosphatase activity increased in cells grown on C GRAFT/Algipore and Pep Gen P-15 in contrast to cells grown on Bio-Oss, in which the lowest levels of activity could be observed on Day 21 (P < 0.05). Reverse transcriptase polymerase chain reaction analysis confirmed the osteoblastic phenotype of the cells grown on all three materials throughout the whole culture period. The results of our in vitro study show that the differences in metabolic activity of cells grown on HA materials are directly related to the substrate on which they are grown. They confirm the excellent properties of HA carrying the cell binding peptide P-15 and HA calcified from red algae as used in maxillofacial surgery procedures.

Alkaline Phosphatase↗
PubMed1991

Gating currents of inactivating and non-inactivating potassium channels expressed in Xenopus oocytes.

The Xenopus oocyte expression system in combination with patch-clamp techniques allows the measurement of ionic currents from a single class of genetically engineered ion channels. Ionic currents in the nanoampere range from oocytes injected with cRNA, corresponding to potassium channels, can be recorded in the inside-out patch configuration. These recordings have a high time resolution at low background noise. Substitution of impermeant ions for potassium and blocking of the channel conductance with tetraethylammonium allows the recording of potassium gating currents, Ig, which is hampered in natural excitable cells by the simultaneous presence of sodium channels and a variety of different potassium channels. The "on" transients, Ig(on), are fast and can have amplitudes of up to several tens of pA. Upon repolarization to -100 mV after small depolarizations, "off" gating currents, Ig(off)g, which reverse most of the "on" charge displacement, Q(on), within 1 ms, are readily observed. However, this fast recovery of the gating charge is drastically reduced upon increasing the amplitude of the depolarizing pulse. In contrast to sodium channels, this temporary charge immobilization is complete within a few milliseconds at positive membrane potentials. Furthermore, there seems to be no direct correlation between charge immobilization and inactivation because the same phenomenon occurs for channels that do not inactivate.

Animals↗
PubMed2006

High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris.

Dipetarudin is a potent direct thrombin inhibitor that was genetically engineered as a chimera between dipetalogastin II and hirudin. Dipetarudin was initially cloned and purified from Escherichia coli, but with a very low yield of about 0.3 mg/l of culture medium. In this study, we report the production of dipetarudin in the methylotrophic yeast Pichia pastoris using pPIC9 vector. The His+ transformants were screened for the best expression performances by prolongation of the ecarin clotting time. An optimal dipetarudin's expression was reached by addition of methanol in culture medium to a final concentration of 0.5%, every 8h during 4 days. Secreted dipetarudin was purified essentially using a two-step purification scheme: anion exchange chromatography in a Resource Q column, followed by C18-reversed phase HPLC. About 150 mg purified dipetarudin was obtained from 1l culture supernatant. This yield is 500-fold higher than the yield obtained with the E. coli system. The molecular mass of dipetarudin calculated by MALDI-TOF (7450 Da) was in agreement with the mass calculated by the amino acid composition (7454 Da), indicating correct processing of the signal sequence. The Ki value of dipetarudin was 399+/-83 fM, which is in agreement with that calculated for the inhibitor isolated from E. coli. This efficient and cost-effective expression system facilitates large-scale production and purification of dipetarudin for further structural, functional and pharmacological investigations.

Antithrombins↗
PubMed1996

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices.

Amino Acid Sequence↗
PubMedDate not supplied

Smart hydrogels for in situ generated implants.

The objective of this study was to explore the use of reverse thermo-responsive (RTG) polymers for generating implants at their site of performance, following minimally invasive surgical procedures. Aiming at combining syringability and enhanced mechanical properties, a new family of injectable RTG-displaying polymers that exhibit improved mechanical properties was created, following two different strategies: (1) to synthesize high-molecular-weight polymers by covalenty joining poly(ethylene glycol) and poly(propylene glycol) chains using phosgene as the coupling molecule and (2) to cross-link poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblocks after end-capping them with triethoxysilane or methacrylate reactive groups. While the methacrylates cross-linked rapidly, the triethoxysilane groups enabled the system to cross-link gradually over time. The chain-extended PEO/PPO copolymers had molecular weights in the 39 000-54 000 interval and exhibited improved mechanical properties. Reverse thermo-responsive systems displaying gradually increasing mechanical properties were generated by cross-linking triethoxysilane-capped (EO)(99)-(PO)(67)-(EO)(99) (F127) triblocks. Over time, the ethoxysilane groups hydrolyzed and created silanol moieties that subsequently condensated. With the aim of further improving their mechanical behavior, F127 triblocks were reacted with methacryloyl chloride and the resulting dimethacrylate was subsequently cross-linked in an aqueous solution at 37 degrees C. The effect of the concentration of the F127 dimethacrylate on the mechanical properties and the porous structure of the cross-linked matrixes produced was assessed. Rheometric studies revealed that the cross-linked hydrogels attained remarkable mechanical properties and allowed the engineering of robust macroscopic constructs, such as large tubular structures. The microporosity of the matrixes produced was studied by scanning electron microscopy. Monolayered conduits as well as structures comprising two and three layers were engineered in vitro, and their compliance and burst strength were determined.

Absorbable Implants↗
PubMed2006

Apolipoprotein A-I assumes a "looped belt" conformation on reconstituted high density lipoprotein.

Apolipoprotein A-I (apoA-I) plays a central role in the reverse cholesterol transport pathway; however, the structural basis for its antiatherogenic effects remains poorly understood. Here we employ EPR spectroscopy and fluorescence resonance energy transfer to elucidate the conformation and relative alignment of apoA-I monomers on discoidal (9.4 nm) reconstituted high density lipoprotein (rHDL). EPR spectroscopy provided evidence for an extended helical secondary structure. Position 139 since it was the only residue examined to display a dynamic motional character consistent with a flexible loop structure. The EPR spectra of nitroxide probes at positions 133 and 146 exhibit spin coupling, indicating that these positions are proximal to an apoA-I paired counterpart on the perimeter of rHDL. fluorescence resonance energy transfer studies employing engineered apoA-I variants possessing a single tryptophan (energy donor) and/or a single cysteine (whose thiol moiety was covalently labeled with an extrinsic energy acceptor) provided evidence that paired apoA-I molecules around the perimeter of rHDL align in an extended antiparallel conformation. Taken together with the observation that the EPR spectra of nitroxide probes positioned at intervening sequence positions (134-145) do not exhibit spin coupling, this has led us to propose a "looped belt" model, wherein residues 133-146 comprise a flexible loop segment that confers to apoA-I an intrinsic ability to adapt its structure to accommodate changing particle lipid content. Specifically, in the looped belt model, with the exception of amino acids 134-145, apoA-I aligns with its counterpart in a helix 5-helix 5 registry, centered at position 139.

Apolipoprotein A-I↗
PubMed1994

Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain.

Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.

Animals↗
PubMed2000

The conserved carboxyl-terminal half of herpes simplex virus type 1 regulatory protein ICP27 is dispensable for viral growth in the presence of compensatory mutations.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that regulates viral gene expression by poorly characterized mechanisms. Previous data suggest that its carboxyl (C)-terminal portion is absolutely required for productive viral infection. In this study, we isolated M16R, a second-site revertant of a viral ICP27 C-terminal mutant. M16R harbors an intragenic reversion, as demonstrated by the fact that its cloned ICP27 allele can complement the growth of an HSV-1 ICP27 deletion mutant. DNA sequencing demonstrated that the intragenic reversion is a frameshift alteration in a homopolymeric run of C residues at codons 215 to 217. This results in the predicted expression of a truncated, 289-residue molecule bearing 72 novel C-terminal residues derived from the +1 reading frame. Consistent with this, M16R expresses an ICP27-related molecule of the predicted size in the nuclei of infected cells. Transfection-based viral complementation assays confirmed that the truncated, frameshifted protein can partially substitute for ICP27 in the context of viral infection. Surprisingly, its novel C-terminal residues are required for this activity. To see if the frameshift mutation is all that is required for M16R's viability, we re-engineered the M16R ICP27 allele and inserted it into a new viral background, creating the HSV-1 mutant M16exC. An additional mutant, exCd305, was constructed which possesses the frameshift in the context of an ICP27 gene with the C terminus deleted. We found that both M16exC and exCd305 are nonviable in Vero cells, suggesting that one or more extragenic mutations are also required for the viability of M16R. Consistent with this interpretation, we isolated two viable derivatives of exCd305 which grow productively in Vero cells despite being incapable of encoding the C-terminal portion of ICP27. Studies of viral DNA synthesis in mutant-infected cells indicated that the truncated, frameshifted ICP27 protein can enhance viral DNA replication. In summary, our results demonstrate that the C-terminal portion of ICP27, conserved widely in herpesviruses and previously believed to be absolutely essential, is dispensable for HSV-1 lytic replication in the presence of compensatory genomic mutations.

Alleles↗
PubMed2004

Stem cells and kidney diseases.

The role of the stem cell (SC) in physiology and physiopathology has recently attracted much interest. SCs may originate from embryos, aborted fetuses, umbilical cord blood, and adult organs and tissues. In the adult, SCs were first defined in tissues with a high cell turnover, like skin and gut. Today, SCs have also been identified in tissues with no or low regenerative potential and turnover. However, the SC concept is changing rapidly: adult SCs not only reside locally in specific niches, but may also be recruited from the circulation to actively participate in the regeneration of various tissues. Furthermore, reverse differentiation has been demonstrated. In the kidney, both glomerular and tubular cells may differentiate into a range of phenotypes during the remodelling process in response to injury. The glomerular and tubulointerstitial scarring processes involve primarily interactions between infiltrating inflammatory cells and resident renal cells that culminate in loss of renal cells and their replacement by extracellular collagenous matrix. Reverse embryogenesis is a key step in renal healing and scarring: intrinsic renal cells regress to primitive/embryonic mesenchymal phenotype. In addition, it is clear that renal remodelling in health and disease involves the migration of hematopoietic SCs into the kidneys. These cells assume various glomerular and tubular epithelial phenotypes. Therefore, a better understanding of some of these key events in renal remodelling may open the way to new interventions based on their manipulations. Cell-based therapy may be a more successful strategy by providing a dynamic and individualized therapeutic approach that responds to the physiopathological condition of the patient. In fact, SCs may provide innovative methods for drug delivery, immunotherapy, tissue regenerative or replacement engineering.

Acute Kidney Injury↗
PubMed1992

Recombinant glycosylated human interleukin-6 accelerates peripheral blood platelet count recovery in radiation-induced bone marrow depression in baboons.

This report was aimed at confirming the potential clinical use for a genetically engineered glycosylated human interleukin-6 (rhIL-6) in hematopoiesis. Its tolerance and efficacy were assessed on hematopoietic restoration after neutron radiation-induced bone marrow injury on baboons, which represent an adequate model of parallelism for studying hematology in the human. The particular neutron radiation absorption pattern in the body allows the preservation of underexposed bone marrow areas that mimics an autotransplantation-like situation. An initial dose finding study (1 microgram up to 20 micrograms/kg/d for 8 consecutive days) in normal baboons established a dose-dependent response regarding the peripheral platelet count (range of increase, 1.5- to 4-fold). A significant elevation in white blood cell (WBC) count, as well as a substantial reversible normochromic normocytic anemia, were observed for the highest doses only (10 and 20 micrograms/kg/d). All rhIL-6 administered doses were clinically well tolerated. In myelosuppressed baboons, a selected dose of 10 micrograms/kg/d of rhIL-6 for 13 consecutive days significantly lessened the degree of induced thrombocytopenia as compared with the control group (P = .01) and shortened the time to occurrence of the nadir, showing that the onset of recovery occurs much earlier, ie, an average of 5 days (P = .003), in the treated group. Moreover, this accelerated platelet recovery is evidenced by an 8-day shorter mean time back to baseline values (P = .03) in the rhIL-6--treated animals. At this dose no effect was observed on the WBC recovery pattern. Importantly rhIL-6 did not accentuate the radiation-induced anemia and was clinically well tolerated. All tested monkeys recovered from their induced pancytopenia and no animal loss was recorded. IL-6, tumor necrosis factor, and IL-1 blood measurements are reported. In conclusion, rhIL-6 is a potent thrombopoietic factor for the treatment of induced thrombocytopenia in nonhuman primates at a clinically well-tolerated dose.

Animals↗
PubMed2002

Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8(+) cytotoxic T-cell activation and antitumor immunity.

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in their activation and is essential for induction of antigen-specific T-cell responses. In the present study, we investigated the efficacy of antitumor immunity induced by vaccination with DCs engineered to express CD40L and pulsed with Mut1 tumor peptide. Our data show that transfection of DCs with recombinant adenovirus AdV-CD40L resulted in activation of DCs with up-regulated expression of proinflammatory cytokines (IL-1beta and IL-12), chemokines (RANTES, IP-10, and MIP-1alpha), and immunologically important cell surface molecules (CD54, CD80, and CD86). Our data also demonstrate that DCs transfected with AdV-CD40L (DC(CD40L)) are able to stimulate enhanced allogeneic T-cell proliferation and Mut1-specific CD8(+) cytotoxic T-cell responses in vitro. Vaccination of mice with Mut1 peptide-pulsed control virus-transfected DC (DC(pLpA)) could only protect mice from challenge of a low dose (0.5 x 10(5) cells per mouse, 8/8 mice), but not a high dose (3 x 10(5) cells per mouse, 0/8 mice) of 3LL tumor cells. However, vaccination of Mut1 peptide-pulsed AdV-CD40L-transfected DC(CD40L) induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of 3LL tumor cells. Thus, DCs engineered to express CD40L by adenovirus-mediated CD40 ligand gene transfer may offer a new strategy in production of DC cancer vaccines.

Adenoviridae↗
PubMed2000

[A brief introduction to the methods for novel gene cloning].

There are a lot of methods for novel gene cloning, but how to clone candidate gene(s) quickly and correctly? This is a brief introduction to methods of novel gene cloning, these methods includes: differential display reverse transcriptase polymerase chain reaction(DD RT-PCR), suppression subtractive hybridization(SSH), RNA arbitrarily primed PCR(RAP-PCR), representational difference analysis(RDA), yeast two-hybrid system, cDNA capturation, et al. We not only introduced these methods, but also discussed the advantages and disadvantages of them. However, no single method is omnipotent, one should pick up the method most suitable for a special purpose.

Animals↗