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At least 487 records · Page 27Linked to original sources
PubMed1987

Lymphokine-activated killer cell activity in rheumatoid arthritis.

The lymphokine-activated killer (LAK) cell activity in the peripheral blood of 23 patients with rheumatoid arthritis has been studied. Two control groups comprised (a) nine patients with another chronic inflammatory disease (sarcoidosis) and (b) 19 normal healthy volunteers. The LAK activity induced by human recombinant IL-2 was very similar in controls and patients with rheumatoid arthritis but was significantly decreased in patients with sarcoidosis, although the frequency of LAK-cell precursors measured using a limiting dilution assay was comparable in all three groups. The DNA synthetic response of peripheral blood mononuclear (PBM) cells to IL-2 was slightly decreased in patients with both rheumatoid arthritis and sarcoidosis as compared to controls, but this decrease was not statistically significant. Spontaneous DNA synthesis in PBM cells cultured in the absence of IL-2 was essentially identical in all three groups. We conclude on the basis of these results that the higher risk of non-Hodgkin's lymphomas in patients with rheumatoid arthritis cannot be attributed to an impairment of LAK activity. Furthermore, the doses of gamma-irradiation, which abolished the 'background' cytotoxicity of PBM cells cultured without IL-2 and also blocked effectively both spontaneous and exogenous IL-2-dependent DNA synthesis, had little effect on the generation of LAK activity. These observations are discussed in regard to the role of non-specific cytotoxic cells and the therapeutic efficacy of antiproliferative drugs in rheumatoid arthritis.

Adult↗
PubMed1983

Frequencies of alloreactive and self-restricted cytotoxic T cell precursors during lymphatic regeneration.

The previous observation that during lymphatic regeneration precursors for MHC-restricted cytotoxic T cells appear before alloreactive precursors, was re-examined in macroculture and microculture experiments. Our macroculture experiments confirmed the observation that treatment with cyclophosphamide decreases the ratio of alloreactivity vs self-restricted reactivity. This shift in reactivity, however, did not result from a change in the T cell specificity repertoire, but was obviously mediated by regulatory effects in macrocultures. Spleen cells from cyclophosphamide-treated mice had normal ratios of CTL precursor frequencies for the three different specificities tested, but they contained a suppressive activity that inhibited the responses of normal spleen cells in macrocultures. Note Added: After submission of this paper we became aware of the paper of Sihvola and Hurme (J. Immunol. 130:1077, 1983) who also showed that although macrocultures from cyclophosphamide-treated mice were negative for the alloresponse, the same spleen cells showed only a approximately threefold decrease in CTL precursors when analyzed in limiting dilution cultures.

Animals↗
PubMed1994

Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34.

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

Animals↗
PubMed1996

Tumor-specific, cytotoxic T-lymphocyte response after idiotype vaccination for B-cell, non-Hodgkin's lymphoma.

Patients with non-Hodgkin's B-cell lymphoma who received an antitumor vaccine of idiotypic ig protein showed humoral and proliferative immune responses. Because immunity to some antigens, including tumor antigens and human pathogenic viruses, may be better correlated with the cytolytic cellular immune response, we evaluated 16 non-Hodgkin's lymphoma patients immunized with autologous idiotypic ig molecules for changes in tumor-specific cytotoxic T-lymphocyte precursor (CTLp) frequency using limiting dilution analysis. Eleven patients had a significant increase in tumor-specific CTLp. Eight of these 11 patients remain without evidence of disease or with stable minimal disease. In contrast, all five patients who did not have a significant change in tumor-specific CTLp have developed progressive disease. Patient vaccination with tumor associated protein antigens can increase tumor-specific CTLp frequencies. The correlation of increased tumor specific CTLp with freedom from progression is significant at P = .002. This study indicates that measurement of CTLp frequencies are relevant to the clinical evaluation of human tumor vaccines and suggests that cell-mediated cytolytic immune responses may be an important determinant of vaccine efficacy.

Adult↗
PubMed1996

The Use of Spectral Analysis of Limiting Dilution for Investigating the Immune Response.

In this study we present a method for characterizing a spectrum of cells engaged in the humoral response by two parameters: the frequency and potential activity of antigen specific precursor cells. Spleen cells from mice immunized with ovalbumin were placed in microcultures in a limiting dilution assay. The values derived from positive cultures in an activity/cell number plot were compatible with Poisson distributions for distinct families of precursor cells displaying either positive or negative (suppressor-like) activities. Proposed limiting dilution spectral analysis combines the potentialities of the standard limiting dilution analysis with those provided by histograms of distribution according to the activity of positive cultures. This method opens new opportunities in detecting and characterizing distinct groups of limiting precursor cells.

Journal Article↗
PubMed2004

Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors.

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.

Amino Acid Sequence↗
PubMed2005

Identification of Th0 cells responding to measles virus.

Mechanisms involved in the induction of immunity to measles virus (MV) are not well understood. In the present study, we assessed proliferation, interferon (IFN)-gamma, and interleukin (IL)-4 production of MV-specific T cells after secondary in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from human donors. Such secondary stimulation resulted in responses substantially higher than after primary in vitro exposure. Most study participants produced both IFN-gamma and IL-4 after secondary in vitro stimulation. Patterns of secondary in vitro responses that use genetically disparate antigen-presenting cells were consistent with T-cell recognition restricted to human leukocyte antigen class II molecules. Limiting dilution analyses indicated that precursor frequencies of cytokine secreting and proliferating cells ranged from about 0.001% to 0.1% among fresh PBMCs. Split-well analyses of limiting dilution cultures suggested that virtually all putative T-cell clones produced either IFN-gamma alone or both IFN-gamma and IL-4. Intracytoplasmic flow cytometric analysis of polyclonal MV-specific secondary in vitro responding T cells revealed a similar pattern of cytokine expression. These results suggest that memory T cells responding in vitro to MV generate cells that produce either IFN-gamma alone (and resemble Th1-like cells) or secreted both IFN-gamma and IL-4 (resembling Th0-like cells) in vitro with few cells expressing a Th2-like pattern.

Adolescent↗
PubMed2005

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae.

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.

Acyltransferases↗
PubMed1991

Structure-function mapping of interleukin 1 precursors. Cleavage leads to a conformational change in the mature protein.

The two interleukin 1 (IL-1) genes (IL-1 alpha and beta) encode 31-kDa precursor molecules, which are cleaved upon secretion to generate the mature, active, carboxyl-terminal 17-kDa proteins. The IL-1 beta precursor is inactive, whereas the IL-1 alpha precursor is as active as the mature IL-1 alpha. In this report, we demonstrate that when either of the recombinant precursors is processed to the mature form, the mature region undergoes a conformational change from a proteinase K-sensitive structure to one that is proteinase K-insensitive. In addition, cysteine residues that are exposed to solvent in the IL-1 beta precursor become buried in the mature protein. Limited structure-activity mapping of the IL-1 beta precursor indicates that the amino-terminal 76 residues are responsible for the conformational change, whereas the most dramatic change in biological activity occurs after further removal of residues 77-94. These findings suggest that the altered structure of the mature region in precursor IL-1s has been conserved for some function. Denaturation/renaturation experiments implicate the precursor domain in protein folding, and by analogy with signal-directed secretory proteins, the unique conformation of the precursors may play a role in IL-1 secretion.

Amino Acid Sequence↗
PubMed1996

L5-67 and LUQ-1 peptide precursors of Aplysia californica: distribution and localization of immunoreactivity in the central nervous system and in peripheral tissues.

Two genes (L5-67 and LUQ-1) that encode neuropeptide precursors have recently been shown to be expressed in a distinct and non-overlapping manner in the five left upper quadrant (LUQ) cells of the abdominal ganglion of Aplysia (Landry et al. [1992]. J. Neurobiol 23:89-101). By using wholemount immunohistochemistry and radioimmunoassay (RIA), the pattern of expression of these two genes was assessed at the protein level throughout the central nervous system (CNS) and in peripheral tissues of Aplysia californica. The distribution of LUQ-1 precursor-like immunoreactivity was fairly limited, occurring in the ventral LUQ cell (L5) and in a total of approximately 20 additional neurons in the abdominal and cerebral ganglia. L5-67 precursor-like immunoreactive material was more prevalent, appearing in a total of approximately 100 neurons distributed among each of the central ganglia. Identified L5-67-immunoreactive neurons included the four dorsal LUQ cells (L2-4 and L6) and two giant neurons (R2 and LPI1). In one group of cells, the H cluster of the cerebral ganglion, L5-67 immunofluorescence was substantially more intense in larger versus smaller animals, suggesting that this peptide precursor is subject to developmental regulation in certain neurons. Immunoelectron microscopic examination of the subcellular localization of L5-67 immunoreactivity in LUQ cell somata and axons revealed its association with dense-core vesicles (approximately 114 nm in diameter). In the periphery, L5-67-immunoreactive fibers were detected in specific regions of the circulatory system (auricle, ventricle, cristae aorta, anterior aorta) and the reproductive system (genital ganglion, large hermaphroditie duct, small hermaphroditie duct, ovotestis). The kidney and the intestine, two tissues in which considerable secretion and absorption occur, contained material immunoreactive to both L5-67 and LUQ-1 antisera. The localization of the two peptide precursors in these tissues differed substantially, with L5-67 occurring in widely ramifying varicose fibers, whereas LUQ-1 was found in restricted foci of fibers and in small spherical cells that appeared to lack processes. These results support previous findings concerning the heterogeneity of neurotransmitter phenotypes in the LUQ cells. Furthermore, they are indicative of a fairly broad role for the L5-67-derived neuropeptides, and a more limited role for the LUQ-1-derived neuropeptides, in the regulation of the visceral organ systems of Aplysia.

Animals↗
PubMed1994

Human immune response to HIV-1-Nef. I. CD45RO- T lymphocytes of non-infected donors contain cytotoxic T lymphocyte precursors at high frequency.

The immune response of peripheral blood lymphocytes (PBL) of non-exposed human individuals to the Nef protein of HIV-1 was studied. Nef is a regulatory protein of HIV which is immediately expressed after infection and which seems to be important in the pathogenicity of HIV. Nef may therefore serve as a potential target for effective immunity against HIV infection. Epstein-Barr (EBV)-transformed lymphoblastoid B cell lines (LCL) were established from four healthy young seronegative adults and transfected with the Nef gene. These cells served as stimulator cells for autologous PBL in vitro and as target cells for CTL. CTL responses were readily generated against Nef-transfected LCL, consisting of Nef-specific and putative EBV-specific CTL. Nef-specific CTL were generated exclusively from CD8+ cells and were MHC class I restricted. Since a vigorous Nef-specific CTL response in non-infected individuals was unexpected, CTL precursor frequencies were determined by limiting dilution analyses in non-fractionated PBL and in PBL separated into the CD45RO- (naive) and CD45RO+ (memory) T cell populations. As expected, the putative EBV-specific CTL precursors were predominantly found in the CD45RO+ subset at frequencies typical for memory T cells. Nef-specific CTL precursors, in contrast, were found predominantly in the CD45RO- population, at even higher frequencies of approximately 1/1000-1/3000. Nef may thus display either an unusually high number of immunogenic peptides or a limited number of peptides presented in a very efficient way, so that many T cells including low affinity cells, would be triggered.

Adult↗
PubMed1981

Pre-ornithine transcarbamylase. Properties of the cytoplasmic precursor of a mitochondrial matrix enzyme.

Biogenesis of the mitochondrial matrix enzyme, ornithine transcarbamylase, has been shown to begin with synthesis on cytoplasmic ribosomes of a precursor, designated pre-ornithine transcarbamylase, which is approximately 4000 daltons larger than its corresponding mitochondrial subunit, followed by post-translational uptake and proteolytic processing of the precursor to its mature counterpart by mitochondria. We now report initial studies on the structure and properties of preornithine transcarbamylase. When this precursor is labeled at the NH2 terminus with N-formyl[35S]methionine and processed by mitochondria, no label is recovered with the mature subunit. This demonstrates that the amino acid extension which is characteristic of the precursor and which is removed during mitochondrial processing is NH2-terminal. This NH2-terminal extension is found intact in two peptides produced by limited proteolysis of the labeled precursor. Moreover, this amino acid extension modifies the behavior of the precursor during immunoprecipitation in the presence of ionic detergents and plays a critical role in facilitating uptake of the precursor by mitochondria.

Animals↗
PubMed1976

An estimation of the frequency of precursor cells which generate cytotoxic lymphocytes.

The cell-mediated immune response has been generated in vitro with a polyacrylamide culture system which allows the segregation of foci (clones?) of cytotoxic lymphocytes. Using the method of limiting dilutions, the frequency of precursor cells in CBA spleen cells able to generate a cytotoxic response against DBA mastocytoma is estimated at 1 per 1,700 cells.

Animals↗
PubMed1989

Limiting dilution analysis for alloreactive, TCGF-secretory T cells. Two related LDA methods that discriminate between unstimulated precursor T cells and in vivo-alloactivated T cells.

We have developed a rapid limiting dilution analysis technique for quantitating alloantigen-specific TCGF-secretory T lymphocytes (operationally defined as helper T lymphocytes or HTL) in murine lymphoid populations. A simple permutation of this technique allows the distinction between HTL that have encountered antigen in vivo and the large population of naive precursor HTL (pHTL) with the same alloantigen specificity. We present evidence to validate these LDA techniques, and show that lymphoid tissue from mice bearing sponge matrix allografts, but not isografts, contain HTL that have been activated in vivo. In addition, we demonstrate a requirement for restimulation with alloantigen for secretion of lymphokine by the in vivo-activated cells. We suggest that this differential LDA technique could serve as a valuable tool in evaluating in vivo immunity and/or the in vivo efficacy of immunosuppressive, as well as immunopotentiating, therapies.

Animals↗
PubMed1984

Proliferation of peritoneal mast cells in the skin of W/Wv mice that genetically lack mast cells.

Presence of mast cell precursors in the mouse peritoneal cavity was demonstrated, and the precursors were characterized. When a cell suspension, containing mast cell precursor(s), was directly injected into the skin of genetically mast cell-deficient WBB6F1 (WB X C57BL/6)-W/Wv mice, a cluster composed of approximately 2,000 mast cells appeared at the injection site. By determining the proportion of injection sites at which the mast cell cluster appeared, the concentration of mast cell precursors can be calculated by limiting dilution analysis. The concentration in the peritoneal cavity was about five times as great as the concentration in the bone marrow. Although peritoneal mast cell precursors were shown to originate from the bone marrow, physical characterization revealed that the peritoneal precursors differed from the marrow precursors. The peritoneal precursors were less susceptible to irradiation than the marrow precursors; the former were heavier than the latter. When a 95% pure mast cell suspension was prepared from the peritoneal cells by the removal of phagocytes and the density gradient centrifugation, 1 out of 16 cells had the potentiality to make a mast cell cluster in the skin of the W/Wv mice. Moreover, when a single mast cell was identified under the phase contrast microscope and picked up with the micromanipulator, 1 out of 17 mast cells made the cluster. This indicated that some peritoneal mast cells kept extensive proliferative potentiality even after morphological differentiation. In other words, some peritoneal mast cells themselves may function as the committed precursors.

Animals↗
PubMed1987

Characteristics of mouse thymocyte-derived, interleukin 2-activated killer cells and their precursors.

Culture of hydrocortisone (HC)-resistant C57BL/6 mouse thymocytes with recombinant human interleukin 2 (IL 2) allowed the proliferation of the thymocytes and resulted in the generation of lymphokine-activated killer (LAK) cells cytotoxic to a variety of tumor cells. The cytotoxic activity of the LAK cells was greatly reduced by treatment with anti-Thy 1.2 or anti-Lyt 2.2 monoclonal antibody and complement but not with anti-asialoGM1 antibody plus complement. Fractionation of IL 2-stimulated thymocytes by means of Percoll density gradient centrifugation revealed that both cytotoxic activity and binding capacity to target cells were greater in the cells with lower density and larger size than in the cells with higher density and smaller size. These IL 2-activated thymocytes expressed higher levels of both Thy 1, Lyt 2 and lymphokine-activated cell-associated (LAA) antigens than unstimulated thymocytes, as indicated by a flow cytometric analysis. The frequency of LAK precursor cells was found to be 7.5 times greater in the HC-resistant thymocyte population than in total thymocytes, as determined by means of a limiting dilution method. The LAK precursor cells in HC-resistant thymocytes appeared to be Lyt1- (or dull Lyt 1+), L3T4-, Lyt 2-, asialoGM1-T cells, because elimination of bright Lyt 1+, Lyt 2+ or L3T4+ T cells from HC-resistant thymocytes had no effect on the generation of LAK cells. These results indicate that LAK cells from mouse thymocytes are Lyt 2+ T cells which are inducible from HC-resistant Lyt 2- thymocytes.

Animals↗
PubMed1986

Control of prostanoid synthesis: role of reincorporation of released precursor fatty acids.

Prostanoid synthesis is limited by the availability of free arachidonic acid. This polyunsaturated fatty acid is liberated by phospholipases and usually is an intermediate of the deacylation-reacylation cycle of membrane phospholipids. In rat peritoneal macrophages, ethylmercurisalicylate (merthiolate) or N-ethylmaleimide (NEM) dose dependently inhibited the incorporation of arachidonic acid into cellular phospholipids, at lower concentrations specifically into phosphatidylcholine. Furthermore, merthiolate could be shown to be a rather selective inhibitor of lysophosphatidylcholine acyltransferase. In contrast, phospholipase A2 activity was not affected over a wide dose range. Consequently, macrophages showed a large increase in prostanoid synthesis (prostaglandin E, prostacyclin and thromboxane) in the presence of both lysophosphatide acyltransferase inhibiting agents. Similar results were obtained with human platelets, in which merthiolate increased the release of thromboxane. Addition of free arachidonic acid also enhanced prostanoid synthesis in macrophages. At optimal concentrations, merthiolate had no further augmenting effect. It is concluded that the rate of prostanoid synthesis is not only controlled by phospholipase A2 activity, but rather by the activity of the reacylating enzymes, mainly lysophosphatide acyltransferase.

Animals↗
PubMed1982

Regulation of cathepsin D metabolism in rabbit heart: evidence for a role for precursor processing in the control of enzyme activity.

Production of active lysosomal enzymes may involve limited proteolysis of inactive high molecular weight precursors. Precursor processing potentially regulates lysosomal enzyme activity. To test whether rabbit cardiac cathepsin D is first synthesized as a precursor and whether prolonged fasting (a condition affecting both cathepsin D and total cardiac protein turnover) influences precursor processing, rates of cathepsin D synthesis and processing were compared in left ventricular slices of control and 3-d-fasted rabbits incubated in vitro with [(35)S]methionine. (35)S-labeled cathepsin D was isolated by butanol-Triton X-100 extraction, immunoprecipitation, and dodecyl sulfate-polyacrylamide gel electrophoresis. Total cardiac protein synthesis was measured by tracer incorporation and normalized for differences in precursor pool size by direct measurement of [(35)S]aminoacyl-tRNA-specific radioactivity. Relative cathepsin D synthetic rates were obtained by comparing (35)S incorporation into cathepsin D with (35)S incorporation into all cardiac proteins. Enzyme processing was assessed in pulse-chase experiments and assayed by autoradiography. The results indicate that (a) rabbit cardiac cathepsin D is synthesized as a precursor (53,000 mol wt) that is processed to a 48,000-mol wt form, (b) rates of both cathepsin D and total cardiac protein synthesis are similar in control and fasted rabbits, suggesting that decreased enzyme degradation rather than increased synthesis is responsible for the elevated levels of cardiac cathepsin D in starvation, and (c) cathepsin D processing in hearts of fasted animals is incomplete, with accumulation of the precursor during pulse-chase experiments of 6 h duration. Based upon these results, a three-stage model for the regulation of cathepsin D activity in rabbit heart is proposed.

Animals↗