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PubMed1996

Functional heterogeneity of the hematopoietic microenvironment: rare stromal elements maintain long-term repopulating stem cells.

It has been hypothesized that distinct stromal cells from niches within the microenvironment that selectively regulate stem cell functions. To test this hypothesis, we derived a panel of matched stromal cell lines from murine fetal liver. The lines were immortalized with a retroviral vector encoding a temperature sensitive SV40 T antigen, to provide a snapshot of potential heterogeneity of the in vivo stroma compartment. All the stromal cell lines tested, supported the proliferation and differentiation of myeloid cells in Dexter type bone marrow cultures. Furthermore, RT-PCR analysis indicates that these lines are similar with respect to the production of an array of cytokines. However, the stromal cell lines differed markedly in their ability to maintain in vitro stem cells with in vivo repopulating capacity. Stem cell levels were measured in the competitive repopulation assay, following 3 weeks of coculture on individual stromal cell lines. Three classes of stromal cell lines were identified: (1) lines that did not support stem cells, (2) lines that sustained low levels of stem cells that often showed limited persistence in vivo, and (3) an infrequent line (1 out of 16 lines tested) that maintained high levels of primitive, long-term repopulating stem cells. This suggests that stromal cells that can support primitive stem cells are rare in the hematopoietic microenvironment. Taken together, these data substantiate the hypothesis that distinct stromal cells interact selectively with stem cells.

Animals↗
PubMed2005

Empirical codon substitution matrix.

BACKGROUND: Codon substitution probabilities are used in many types of molecular evolution studies such as determining Ka/Ks ratios, creating ancestral DNA sequences or aligning coding DNA. Until the recent dramatic increase in genomic data enabled construction of empirical matrices, researchers relied on parameterized models of codon evolution. Here we present the first empirical codon substitution matrix entirely built from alignments of coding sequences from vertebrate DNA and thus provide an alternative to parameterized models of codon evolution. RESULTS: A set of 17,502 alignments of orthologous sequences from five vertebrate genomes yielded 8.3 million aligned codons from which the number of substitutions between codons were counted. From this data, both a probability matrix and a matrix of similarity scores were computed. They are 64 x 64 matrices describing the substitutions between all codons. Substitutions from sense codons to stop codons are not considered, resulting in block diagonal matrices consisting of 61 x 61 entries for the sense codons and 3 x 3 entries for the stop codons. CONCLUSION: The amount of genomic data currently available allowed for the construction of an empirical codon substitution matrix. However, more sequence data is still needed to construct matrices from different subsets of DNA, specific to kingdoms, evolutionary distance or different amount of synonymous change. Codon mutation matrices have advantages for alignments up to medium evolutionary distances and for usages that require DNA such as ancestral reconstruction of DNA sequences and the calculation of Ka/Ks ratios.

Amino Acid Substitution↗
PubMed1992

Detection of hematogenous micrometastasis in patients with prostate cancer.

The goal of this study was to determine if patients with stage D0-3 prostatic adenocarcinoma have detectable hematogenous micrometastasis. Polymerase chain reaction amplification of prostate-specific antigen mRNA, which is exclusively expressed by prostatic epithelial cells, was used to detect circulating prostatic cells. Peripheral venous blood was obtained from 17 control and 12 prostate cancer patients with stage D0-3 prostatic adenocarcinoma. Of the 12 cancer cases, four patients (stage D1-3) tested positive for prostate-specific antigen RNA, indicating the presence of circulating micrometastasis. The 17 negative controls all tested negative. Contrary to a long held hypothesis, these data point to the possibility that hematogenous metastasis may be a relatively early event in the natural history of human prostate cancer. These findings may have an important impact on our understanding and treatment of prostate cancer.

Base Sequence↗
PubMed1993

Effect of amplicon size on PCR detection of bacteria exposed to chlorine.

The effect of amplicon size on the PCR detection of Legionella pneumophila after chlorine inactivation was investigated. Two amplicons specific to the L. pneumophila mip gene were used for the PCR analyses: a 650-bp amplicon and smaller 168-bp amplicon within the 650-bp amplicon; a 108-bp amplicon specific to species rRNA coding sequence also was used. After exposure to chlorine, viable agar grown cells were not detected by plate counts or direct counts with p-iodonitrotetrazolium (INT) after 1 min for treatment at 10 mg/l, after 2 min for treatment at 5 mg/l, and after 4 min for treatment at 2.5 mg/l; viable water grown cells were present at least 4 min after biocide addition even with a chlorine dose of 5 mg/l. At the 10-mg/l dosage, PCR products from the 168-bp amplicon were detected on agarose gels up to 16 min after chlorination; even after 24 hr of PCR the 168-bp products were detectable using a capture probe hybridization assay. However, the 650-bp target was not detected after 4 min chlorine contact time at the same biocide dosage using agarose gels, and PCR products could not be detected by hybridization after 32 min. At lower chlorine concentrations, a similar pattern was seen with the 168-bp amplicon detectable longer after biocide addition than the 650-bp mip amplification target. On the basis of these data, larger amplicons appear to correlate better with viability of L. pneumophila in water samples.

Bacterial Proteins↗
PubMed1998

Scintillation proximity assay for E-, P-, and L-selectin utilizing polyacrylamide-based neoglycoconjugates as ligands.

In this study, a novel scintillation proximity assay (SPA) that uses radiolabeled soluble neoglycoconjugates as synthetic alternatives to the natural E-, P-, and L-selectin counterligands was developed. The neoglycoconjugates contained sialyl LewisX or sialyl LewisA attached via a three-carbon spacer to a poly[N-(hydroxyethyl)acrylamide] backbone, thus presenting the carbohydrates in a multivalent form. Selectin-ZZ fusion proteins were immobilized on anti-rabbit IgG-coated SPA beads via a rabbit IgG bridge. The neoglycoconjugate ligands bound to all three bead-immobilized selectins, with the highest binding levels apparent with E-selectin. Saturation binding studies with E-selectin revealed a complex interaction indicative of two or more binding affinities. The response to carbohydrate inhibitors was comparable in E-selectin assays that used either the neoglycoconjugates or the tritium-labeled HL60 cells as selectin counterligands. The incorporation of tyrosine sulfate groups into the backbone of the neoglycoconjugate resulted in enhanced binding avidity to both P- and L-selectin, indicating that the sulfate-containing neoglycoconjugates are viable synthetic mimics of the natural P- and L-selectin counterligands. The use of these radiolabeled neoglycoconjugates in conjunction with SPA results in a format ideally suited for the high-throughput screening for selectin antagonists. Furthermore, this approach can potentially be used to measure other low-avidity lectin-carbohydrate interactions.

Acrylic Resins↗
PubMed1996

Vanadium-induced chemokine mRNA expression and pulmonary inflammation.

Occupational exposure to vanadium is common in petrochemical, mining, steel, and utilities industries and results in toxic effects largely confined to the respiratory system. Vanadium exposure has been associated with inflammatory changes in the upper and lower respiratory tracts in addition to changes in pulmonary function. We investigated the abilities of several vanadium compounds to increase mRNA levels for selected cytokines in bronchoalveolar lavage (BAL) cells and also to induce pulmonary inflammation. Rats (200-250 g) were intratracheally instilled with either sodium metavanadate (NaVO3), vanadyl sulfate (VOSO4), vanadium pentoxide (V2O5) at several concentrations, or vehicle alone. Pulmonary inflammation was assessed by cytologic analysis of cells recovered from the respiratory tract (1 hr to 10 days postexposure). All three vanadium compounds were capable of inducing pulmonary inflammation in a dose-dependent manner. Neutrophil influx was greatest following exposure to VOSO4 (peaked at approximately 40% of cell population) and lowest following exposure to V2O5 (peaked at approximately 20 %). Significant neutrophil influx was detected as early as 4 hr following the instillation of NaVO3 and VOSO4 but not until 24 hr upon exposure to V2O5. The VOSO4-induced inflammatory response persisted longer (5 days) than that induced by NaVO3 and V2O5. Analysis of inflammatory cytokine mRNA expression closely followed these cytologic observations. Levels of mRNA for macrophage inflammatory protein-2 (MIP-2) and KC, considered the principal neutrophil chemotactic factors expressed in the rat, were rapidly induced as early as 1 hr following exposure, continued to be expressed throughout 48 hr, and were low but detectable at 5 and 10 days. NaVO3 and VOSO4, both very soluble forms of vanadium, tended to induce pulmonary inflammation and inflammatory cytokine mRNA expression more rapidly and more intensely than the less soluble form, V2O5. Analysis of KC mRNA expression in BAL cells 24 hr after instillation of NaVO3 by PCR in situ hybridization confirmed the increase in KC mRNA levels and indicated that alveolar macrophages have the highest expression level observed. Vanadium content of lavage fluid, BAL cells, and lung indicated rapid clearance of the metal from the lung surface and substantial accumulation by BAL cells and lung tissue. The rapid expression of MIP-2 and KC mRNA in BAL cells prior to the observed neutrophilia implicate them as important in the initiation of inflammation.

Animals↗
PubMed1998

Experimental induction of mucosal disease: consequences of superinfection of persistently infected cattle with different strains of cytopathogenic bovine viral diarrhea virus.

Mucosal disease (MD) can be induced in cattle persistently infected with noncytopathogenic bovine viral diarrhea virus (ncp BVD virus) by superinfecting them with antigenically related cytopathogenic (cp) BVD virus strains. While some of these animals succumb to early onset MD after 2 to 3 weeks post infectionem (p.i.), others only react by producing neutralizing antibodies against the cp BVD virus strain and may develop late onset MD after longer incubation periods. The aim of this study was to determine if an increasing degree of antigenic homology between the ncp and the superinfecting cp BVD virus strains as determined by their comparative reactivity with E2 glycoprotein specific monoclonal antibodies (mabs) increases the probability of inducing early or late onset MD, respectively. For this, each two of eight clinically healthy animals from the same herd and persistently infected with the same ncp BVD viruses were superinfected with four different cp BVD virus strains. As only two of these animals developed late onset MD, one animal from a different herd that developed early onset MD was included in the study. Besides clinical observation and testing for antibody production, virus isolation and characterization of the cp BVD virus isolates were performed. The results indicate that antigenic similarity as determined by comparative mab analysis alone is not sufficient to allow prediction of the outcome of the disease.

Amino Acid Sequence↗
PubMed1993

Zidovudine susceptibility testing of human immunodeficiency virus type 1 (HIV) clinical isolates.

Traditional antiviral susceptibility testing methods using cell lines can be applied to no more than about 30% of clinical HIV isolates (Larder et al., 1989a; Fenyo et al., 1989). We tested the cell-free supernatant from low passage clinical HIV isolates using donor peripheral blood mononuclear cells (PBMC). Drug susceptibility was assessed by measuring the effect of increasing zidovudine (ZDV) concentrations on HIV P24 antigen production. Susceptibility results were obtained on 24/27 consecutive clinical isolates and 6/6 laboratory isolates. The mean IC90 of isolates from untreated patients was 0.008 microM ZDV (range: 0.002-0.038). The IC90s of isolates from ZDV-treated patients ranged from 0.007 to greater than 10 microM ZDV. All isolates with an IC90 < 0.1 microM ZDV had a wild type sequence at codon 215 of the HIV pol gene; 11/12 isolates with an IC90 > 0.1 microM ZDV had a mutation at codon 215 (P < 0.001). Among 16 ZDV-treated patients, there was a modest correlation between the change in CD4 count from the start of ZDV treatment and the IC90 of the patient's isolate following treatment (r = 0.51). Susceptibility testing using donor PBMC can be a sensitive means of testing a broad range of clinical HIV isolates.

Base Sequence↗
PubMed2005

Effects of in-situ ozonation on indigenous microorganisms in diesel contaminated soil: survival and regrowth.

Soil column experiments were conducted to investigate the effects of chemical oxidation on the survival of indigenous microbes (i.e., heterotrophic microbes, phenanthrene-degrading microbes, and alkane-degrading microbes) for field soil contaminated with diesel fuel. Rapid decreases of total petroleum hydrocarbons (TPH) and aromatics of diesel fuel were observed within the first 60 min of ozone injection; after 60 min, TPH and aromatics decreased asymptotically with ozonation time. The three types of indigenous microbes treated were very sensitive to ozone in the soil column experiment, hence the microbial population decreased exponentially with ozonation time. The numbers of heterotrophic, alkane-degrading, and phenanthrene-degrading bacteria were reduced from 10(8) to 10(4), 10(7) to 10(3), and 10(6) CFU g soil(-1) to below detection limit after 900 min of ozonation, respectively. Except for the soil sample ozonated for 900 min, incubation of ozone-treated soil samples that were not limited by oxygen diffusion showed further removal of TPH. The soil samples that were ozonated for 180 min exhibited the lowest concentration of TPH and the highest regrowth rate of the heterotrophic and alkane-degrading populations after the 9 weeks of incubation.

Bacteria↗
PubMed2004

Intra- and intercellular variations in the repair efficiency of O6-methylguanine, and their contribution to kinetic complexity.

Following administration to rats of various doses of N-nitrosodimethylamine (NDMA), O(6)-methylguanine (O(6)-meG) was lost from the DNA of four tissues (liver, white blood cells, lymph nodes, bone marrow) over two, sharply demarcated phases with substantially differing repair rates. Repair during each phase followed approximately first-order kinetics in O(6)-meG, even after a high dose of NDMA which caused substantial depletion of O(6)-alkylguanine-DNA alkyltransferase (AGT), a suicide repair protein. This is compatible with rate-determining adduct repair being brought about by a distinct, minor pool of AGT molecules which is rapidly replenished by de novo AGT synthesis. Similar biphasic repair kinetics were also observed in HepG2 cells treated in vitro with NDMA. In this case, the first phase of repair was inhibited by alpha-amanitin, an inhibitor of RNA polymerase II-mediated transcription. However, no dependence on transcriptional activity was found when O(6)-meG repair in specific gene sequences with different transcriptional status in rat liver was examined, suggesting that the effects of alpha-amanitin in HepG2 cells did not reflect inhibition of preferential repair of transcribed sequences. Repair was also examined in rat liver hepatocytes and non-parenchymal cells separately after administration of NDMA at non-AGT depleting doses. Within each cell-population, the repair followed single phase, first-order kinetics, with adduct loss from AGT-rich hepatocytes being significantly faster than from the relatively AGT-deficient non-parenchymal cells. In conclusion, differences in the AGT content of different cell subpopulations in the liver (and probably in other tissues), as well as additional cellular factors affecting repair efficiency, appear to determine the observed variation in the kinetics of repair of O(6)-meG. The additional cellular factors involved appear not to be related to the transcriptional state of the sequences being repaired, but may reflect different states of chromatin condensation.

Alkyl and Aryl Transferases↗
PubMed2005

Protection against multiple influenza A subtypes by vaccination with highly conserved nucleoprotein.

Influenza epidemic and pandemic strains cannot be predicted with certainty. Current vaccines elicit antibodies effective against specific strains, but new strategies are urgently needed for protection against unexpected strains. DNA vaccines encoding conserved antigens protect animals against diverse subtypes, but their potency needs improvement. We tested DNA prime-recombinant adenoviral boost immunization to nucleoprotein (NP). Strong antibody and T cell responses were induced. Protection against challenge was T cell-dependent and substantially more potent than DNA vaccination alone. Importantly, vaccination protected against lethal challenge with highly pathogenic H5N1 virus. Thus, gene-based vaccination with NP may contribute to protective immunity against diverse influenza viruses through its ability to stimulate cellular immunity.

Animals↗
PubMed1987

Point mutations as an optimal search process in biological evolution.

Point mutations are pictured as jumps in a phase space representing the sequences of amino acids or nucleotides as discrete points. It is shown that this space can be given a natural metric by quantifying common physical and chemical properties of amino acid constituents in terms of a natural measure. Evolution through point mutations is simulated by the search for points in the phase space representing amino acid sequences of high survival fitness. Due to the local compactness of the distribution of these functionally allowed points in phase space any successful search procedure has characteristics qualitatively different from those in the case of a random distribution. This is demonstrated by model calculations. A specified distribution of allowed points is generated with subsequent evaluation of the success of the retrieval process as a function of the jump probabilities between lattice sites. The results of such simulations are compared with data obtained from the analysis of the DNA or mRNA sequences coding related proteins. By counting silent and expressed nucleotide replacement frequencies one can draw conclusions as to the efficacy of the natural evolutionary search processes in the phase space of amino acid sequences. There are cases, where the highest possible information gain of one bit per accepted point mutation is achieved. In general the information gain is found to be somewhat sub-maximal due to functional requirements.

Amino Acid Sequence↗
PubMed2003

A L.E.A.P.S. heteroconjugate vaccine containing a T cell epitope from HSV-1 glycoprotein D elicits Th1 responses and protection.

The L.E.A.P.S. heteroconjugate vaccine antigen (JgD), composed of a T cell epitope from glycoprotein D (gD(8-23)) of herpes simplex virus (HSV) linked with a peptide sequence from beta-2-microglobulin (aa38-50), elicited protection against lethal intraperitoneal (IP) challenge and prevented disease signs in most, and limited disease progression, for the rest of BALB/c mice challenged in the epidermal abrasion-zosteriform spread mouse infection model. JgD elicited a Th1 response in vaccinated mice as indicated by delayed type hypersensitivity (DTH) responses to HSV antigen, and gD and virion specific antibodies with an IgG2a/IgG1 >1. Vaccination with the JgD peptide delayed the onset of disease signs, reduced severity of the disease and reduced mortality rates in mice with different MHC backgrounds as compared to their respective control mice. CD8 cells were demonstrated as important for initiation of the immune response to JgD and CD4 cells and interferon gamma (IFN-gamma) for delivering immune protection in BALB/c mice, as indicated in monoclonal antibody ablation studies. JgD, and other J-L.E.A.P.S. vaccine antigens, appear to prime T cells to initiate a Th1 response, which is subsequently boosted upon viral challenge to result in protection.

Amino Acid Sequence↗
PubMed1996

Hybridization assays using an expressible DNA fragment encoding firefly luciferase as a label.

We report the use of a new label, an expressible enzyme-coding DNA fragment, for nucleic acid hybridization assays. The DNA label contains a firefly luciferase coding sequence downstream from a T7 RNA polymerase promoter. The target DNA (200 bp) is denatured and hybridized simultaneously with two oligonucleotide probes. One of the probes is immobilized in microtiter wells, via the digoxigenin/anti-digoxigenin interaction, and the other probe is biotinylated. After completion of the hybridization, the hybrids are reacted with a streptavidin-luciferase DNA complex. Subsequently, the solid-phase bound DNA is expressed by coupled transcription/ translation. The synthesized luciferase catalyzes the luminescent reaction of luciferin with O2 and ATP. The luminescence is linearly related to the amount of target DNA in the range of 5-5000 amol. The CVs obtained for 20 and 100 amol of target are 6.5% and 10.8%, respectively (n = 4).

Animals↗
PubMed1990

Homing of a gamma delta thymocyte subset with homogeneous T-cell receptors to mucosal epithelia.

In mice gamma delta T-cell populations with distinct T-cell receptor (TCR) repertoires and homing properties have been identified. Diversified populations are found in lymphoid organs and intestinal epithelia. By contrast, the gamma delta T-cells that have been found in the murine skin are homogeneous. They express a TCR consisting of one particular V gamma 5 and one particular V delta 1 chain and seem to originate from early fetal thymocytes. We have now systematically analysed many tissues by immunohistochemistry and TCR gene sequencing aided by the polymerase chain reaction. These studies revealed a second homogeneous gamma delta T-cell subset in epithelia not of the intestine and skin, but of the vagina, uterus and tongue. The TCR expressed by this gamma delta T-cell subset consists of the same V delta 1 chain. Cells that express this particular TCR have previously been shown to be positively selected in the late fetal thymus.

Animals↗
PubMed2000

TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease.

B cells are important in the development of autoimmune disorders by mechanisms involving dysregulated polyclonal B-cell activation, production of pathogenic antibodies, and co-stimulation of autoreactive T cells. zTNF4 (BLyS, BAFF, TALL-1, THANK) is a member of the tumour necrosis factor (TNF) ligand family that is a potent co-activator of B cells in vitro and in vivo. Here we identify two receptors for zTNF4 and demonstrate a relationship between zTNF4 and autoimmune disease. Transgenic animals overexpressing zTNF4 in lymphoid cells develop symptoms characteristic of systemic lupus erythaematosus (SLE) and expand a rare population of splenic B-Ia lymphocytes. In addition, circulating zTNF4 is more abundant in NZBWF1 and MRL-lpr/lpr mice during the onset and progression of SLE. We have identified two TNF receptor family members, TACI and BCMA, that bind zTNF4. Treatment of NZBWF1 mice with soluble TACI-Ig fusion protein inhibits the development of proteinuria and prolongs survival of the animals. These findings demonstrate the involvement of zTNF4 and its receptors in the development of SLE and identify TACI-Ig as a promising treatment of autoimmune disease in humans.

Amino Acid Sequence↗
PubMed2006

Efficient amplification of melanoma-specific CD8+ T cells using artificial antigen presenting complex.

In vitro large amplification of tumor-specific cytotoxic T lymphocytes (CTLs) and adoptive transfer of these cells is one of the most promising approaches to treat malignant diseases in which an effective immune response is not achieved by active immunization. However, generating sufficient numbers of tumor-specific CTLs stimulated with autologous antigen presenting cells (APCs) in vitro is one of the most problematic steps in the adoptive cell transfer (ACT) therapy. To circumvent this problem, we have developed an artificial antigen presenting complex (aAPCs) using MHC class I molecules loaded with a melanoma-specific TRP-2 peptide epitope. Our results show that TRP-2-specific CD8+ T cells elicited by immunization with recombinant adenovirus expressing the mini-gene epitope are efficiently stimulated and amplified in vitro to a greater extent by aAPCs than by natural splenic APCs. These aAPC-induced CTLs recognized endogenously processed antigens present on B16F10 melanoma cells. Efficient stimulation and proliferation of antigen- specific T cells was also confirmed using ovalbumin peptide-loaded aAPCs and OT-I TCR transgenic cells. These results demonstrate that prior in vivo immunization, which increases the precursor frequency, simplifies posterior expansion of tumor- specific CD8+ T cells, and aAPCs is superior to autologous APC for in vitro amplification. This "prime and expand" regimen can be an alternative method for large amplification of rare tumor-specific CTLs and aAPCs should be a useful tool for ACT immunotherapy.

Adoptive Transfer↗