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PubMed1982

Recovery of humoral and cellular immunity by soluble mediators after 5-fluorouracil-induced immunosuppression.

5-Fluorouracil (5-FU) administered in daily injections to mice (15-60 mg/kg; subcutaneous) was differentially toxic to helper T cells. Precursors for both antibody forming cells and cytotoxic T lymphocytes (CTL) were spared. 5-FU suppressed the in vitro T cell-dependent antibody response to sheep red blood cells (SRBC). This low response was restored to normal levels by the addition of T cell replacing factor (TRF) or mixed lymphocyte culture (MLC) supernatants to the culture system. T cell-independent antibody responses to TNP-Ficoll or TNP-LPS were not eliminated by 5-FU but, in contrast, were elevated two-four-fold. These results indicate that precursors for antibody forming cells for T cell-dependent and -independent antibody responses were not eliminated by 5-FU, 5-FU administered in the same regimen did not reduce the number of CTL precursors as shown by limiting dilution analysis, but did cause a reduction in the capacity of lymphocytes from pre-treated mice to generate a CTL response in vitro. This low CTL response was restored to control levels by adding Lyt 1+2- T cells or sources of interleukin 2 (IL2) to the culture system, indicating that 5-FU similarly eliminated helper cells for CTL precursor differentiation as well as helper cells for antibody synthesis.

Animals↗
PubMed1982

Genetic analysis of the processing of a spliced tRNA.

We analyzed the effect of 18 single nucleotide changes on the processing of the transcripts produced by cloned yeast tRNATyr genes after microinjection into the nucleus of living Xenopus oocytes. The processing step most easily blocked by mutation is the early maturation of the 5' and 3' termini of the tRNATyr primary transcript, involving removal of 5'-leader and 3'-trailer sequences and CCA addition. The enzymes seem to recognize the whole tRNA cloverleaf structure since mutations in all regions of the molecule can stop processing. Mutations that affect splicing of the 92-nucleotide precursor (which has mature ends but still contains the intervening sequence, and is the normal substrate for the splicing enzymes), are located in the vicinity of the intervening sequence. Base modification enzymes that add pseudouridine, 1-methyladenosine and 5-methylcytosine appear rather insensitive to changes in secondary and tertiary structure of early transcripts in the 16 mutants examined. These enzymes may recognize only limited regions of the precursor RNA. RNA polymerase III behaves as if able to count the number of Us added before termination; and aberrant termination products in two mutants suggest that the secondary structure of the nascent transcript can be very imortant in eukaryotic transcription termination.

Animals↗
PubMed1994

Myogenic cells in the rat embryonic brain stem.

The principal finding of this study is the establishment of culture conditions that permit the survival of a very limited population of muscle precursor cells from the embryonic and neonatal rat brain stem, with these cells displaying the capacity to differentiate into contractile striated fibers under these in vitro conditions. Examination of desmin and troponin T expression by immunoperoxidase analysis revealed the presence of the two muscle proteins in the mononucleated precursors and in differentiated multinucleated myotubes. In vivo, examination of sections at corresponding stages revealed the simultaneous presence of troponin T, desmin, and lectin binding sites--a property of endothelial cells--association which appears to be transitory, since it is no longer detectable in the adult brain. The presence of the choroid plexus neuroepithelium, in close vicinity with TnT+, desmin+cells located in a limited zone of the brain stem, as well as the stage of expression for these markers suggest an interaction between brain myogenic cells and the onset of the embryonic circulation. The question of the embryological origin of the brain myogenic cells is discussed.

Animals↗
PubMed1984

Differential staining of cytoid bodies and skin-limited amyloids with monoclonal anti-keratin antibodies.

The authors have used 5 different monoclonal antikeratin antibodies to study the antigenic profiles of cytoid bodies and skin-limited amyloids. Monoclonal antibodies AE1 (which stains the basal cell layer in normal human epidermis), AE2 (suprabasal layers), AE3 (whole epidermis), EKH4 (lower 2-3 layers), and EKH1 (recognizes all classes of intermediate filaments) were used to stain frozen skin sections by the indirect immunofluorescent or indirect immunoperoxidase technique. Cytoid bodies in lichen planus (LP) and discoid lupus erythematosus (DLE) were strongly stained with AE1, AE3, EKH4, and EKH1 antibodies but were negative with AE2. In contrast, amyloids in lichen amyloidosus and macular amyloidosis were stained strongly with EKH4 but only weakly or not at all with AE1, AE2, AE3, and EKH1. Amyloid associated with epithelial tumors showed closer immunologic profiles to cytoid body. These findings suggest that epidermal keratins are the major precursor substance of skin-limited amyloids as well as cytoid bodies in LP and DLE. Sequential changes in antigenic profiles from basal cells to amyloids through cytoid bodies further suggest that cytoid bodies may represent one of the precursor substances of skin-limited amyloids.

Amyloid↗
PubMed1983

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. II. Mechanisms, specificity, and cellular analysis of 2,4,6-trinitrophenol (TNP)-specific cytolytic response priming by intravenous versus subcutaneous injection with TNP-modified syngeneic cells.

We have examined the underlying mechanisms accounting for the enhanced in vitro TNP-specific cytotoxic T-lymphocyte (CTL) response following the parenteral injection of syngeneic hapten-modified lymphoid cells. Augmented CTL activity noted following parenteral injection (iv vs sc) of 2,4,6-trinitrophenol-modified syngeneic spleen cells (TNP-SC) is most apparent when limiting numbers of TNP-modified stimulator cells are used in the in vitro sensitization phase. Enhanced CTL responses seen following sc and iv priming is due to distinct mechanisms. Spleen and lymph node (LN) cells from sc primed mice were found to contain significant levels of radioresistant helper activity upon coculture with either viable normal spleen cells in bulk culture or with thymocytes as the source of precursor CTLs in a limiting dilution assay. The helper activity was found to be mediated by a Lyt 1+2- T cells. In addition, Lyt 2-depleted spleen and LN cells from sc primed BALB/c mice could restore the ability of tolerant spleen cells from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-injected BALB/c mice to generate TNP-specific CTLs. Conversely, Lyt 2-depleted spleen and LN cells from iv primed mice provided no measurable helper activity either in bulk culture or in the limiting dilution assay and did not restore the ability of TNBS-tolerant BALB/c spleen cells to generate TNP-specific CTLs. CTL priming via the iv route was found to be completely antigen specific as iv injection of either 2,4-dinitrophenol (DNP)- or fluorescein isothiocyanatel (FITC)-modified cells caused no enhanced CTL activity. Priming via the sc route exhibited a unique specificity pattern as it was shown that sc injection of both TNP-SC and DNP-SC, but not FITC-SC, resulted in enhanced TNP-specific CTL responses. CTL T-helper (Th)-cell induction via the sc route was correlated with (1) the presence of H-2 I region determinants on the inducer cells as the sc injection of TNP-modified erythrocytes led to no enhanced CTL responses or CTL Th activity (while iv injection of TNP-erythrocytes did lead to enhanced CTL responses without detectable helper activity) and (2) the detection of both hapten-specific T-cell proliferation and Interleukin 2 (IL-2) production upon restimulation in culture. We conclude that the sc injection of TNP-SC leads preferentially to an increase of specific Lyt 1+ helper activity, while iv injection leads preferentially to an apparent expansion of Lyt 2+ prelytic effector CTLs.

Animals↗
PubMed1983

Analysis of true anti-hapten cytotoxic clones in limit dilution microcultures after correction for "anti-self" activity: precursor frequencies, Ly-2 and Thy-1 phenotype, specificity, and statistical methods.

By the use of split culture techniques we have been able to demonstrate conclusively that the "anti-self" activity and the spontaneous anti-hapten activity within an apparent anti-hapten cytotoxic T cell response is a clonal phenomenon and is not caused by cross-reactivity between anti-self and true anti-hapten clones. With the knowledge of this clonality we have been able formally to prove that the true hapten-generated, hapten-specific response can be obtained by subtracting the response generated by "self" but directed against modified targets from the response generated by modified self and directed against modified targets. Unbiased statistical estimators, which do not make assumptions about the origin (0 responder cells, 100% negative cultures), have been developed to plot accurately limit dilution data (maximal likelihood estimator and minimal chi-square estimator), and the analysis demonstrates that all the components of an apparent anti-hapten response obey zero order (single hit) kinetics. By specifically identifying true anti-hapten and true anti-self clones, we have been able to study the phenotype of their precursors as well as the effectors themselves at the clonal level. Precursors of true anti-hapten and anti-self clones are Thy-1+, Ly-2+. However, anti-hapten and anti-self effector cells show marked clonal variation with respect to Ly-2, as some clones are almost completely insensitive to anti-Ly-2 and complement whereas others show minimal to complete sensitivity. All anti-hapten clones are completely sensitive to anti-Thy-1 and complement, whereas about one-third of anti-self clones show only partial sensitivity, with the most lytic clones showing the most sensitivity. Hapten-specificity and anti-self-specificity have been examined clonally. Ten percent of anti-TNP clones recognize NIP, 10% of anti-NIP clones recognize TNP, and 20% of anti-self clones recognize an allo-target. These figures are in accordance with the overall specificity of effectors generated in bulk culture in the presence of CAS (concanavalin A-stimulated spleen cell conditioned medium). However, hapten specificity and H-2 restriction of bulk generated effectors are improved if CAS is omitted from cultures.

Animals↗
PubMed1990

Cardiovascular and ventilatory effects of various acidic, basic and neutral L-amino acids in normotensive rats.

Cardiovascular effects have been attributed to the amino acids which are precursors of catecholamines or other neurotransmitters. To study if even other amino acids may exert cardiovascular or ventilatory effects, a number of various acidic, basic and neutral L-amino acids were injected intravenously to anaesthetised normotensive rats at the doses of 0.2-1.6 mmol/kg. All amino acids were able to produce either blood pressure, heart rate or ventilation rate changes. Hence, in this study the production of cardiovascular or ventilatory effects was not limited to the known precursors of neurotransmitters. Therefore, in addition to increased formation and release of neurotransmitters, other mechanisms are apparently involved in the cardiovascular and ventilatory effects of various L-amino acids.

Amino Acids↗
PubMed1994

High frequency of Gag- and envelope-specific cytotoxic T lymphocyte precursors in children with vertically acquired human immunodeficiency virus type 1 infection.

Circulating human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) are seen less frequently in unstimulated peripheral blood mononuclear cells (PBMC) from children with vertically acquired HIV infection than in PBMC from HIV-infected adults. HIV-1 Gag-, reverse transcriptase (RT)-, and envelope (Env)-specific cytotoxic activity was studied in PBMC from HIV-infected children. Only 9% of subjects had Gag- or RT-specific CTL in unstimulated PBMC. However, in PBMC studied after CD3 stimulation, Gag- and Env-specific CTL were found in PBMC from 91% and 78% of HIV-infected children, respectively. Limiting dilution analysis of precursor CTL (pCTL) frequencies in PBMC from children > 12 months old demonstrated Gag- and Env-specific pCTL frequencies from 0.5 to 6.3/10,000 PBMC and from 0.66 to 33.0/10,000 PBMC, respectively. Thus, children with vertically acquired HIV infection have high frequencies of HIV-specific pCTL.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Limiting dilution analysis of human IL-2 producing T-cell precursors. Determination of hierarchical alloantigenic potential of human leukocyte antigens and evaluation of alloimune responses to single HLA-DP molecules.

Twenty nine healthy unrelated individuals were carefully selected and divided into three groups according to their HLA (Human Leukocyte Antigens) phenotypes. A sensitive and reproducible limiting dilution analysis (LDA) based bioassay using CTLL-20 cells for detection of human IL-2 was set up and used to assess the hierarchical impact of highly polymorphic HLA molecules on individual's alloreactivity. Our main interest was to evaluate the role of HLA-DP molecules in this process. By calculating frequencies of IL-2 producing helper T cell precursors (HTLp) and amounts of IL-2 produced in each experiment, we were able to confirm that HLA-DR molecules are the most potent alloantigens. In 29 different combinations where a single HLA-DP mismatch between stimulating and responding cells was evaluated, some were reasonably tolerant, while the other ones evoked moderate to relatively strong alloimmune responses. Finally, two groups with statistically significant difference in alloimmune responses to stimulating HLA-DP molecules carrying D,E,A,V or G,G,P,M amino acid sequences at positions 84,85,86 and 87 in the sixth variable region F of the molecule could be formed, according to HTLp frequencies and amounts of IL-2 detected. Data presented are of great importance for the selection of unrelated as well as related bone marrow donors for haematological patients.

Cell Line↗
PubMedDate not supplied

Analysis of responses of peripheral blood lymphocytes from sarcoidosis patients to purified protein derivative.

PURPOSE: Negative skin test reaction against purified protein derivative (PPD) is one of the important diagnostic criteria for sarcoidosis. The purpose of this study was to investigate the relationship between negative skin test reaction against PPD and the responses of peripheral blood lymphocytes (PBLs) against PPD in sarcoidosis patients. METHODS: Sarcoidosis patients (n = 14) with ocular changes were selected for this study. As a control, blood was collected from volunteers without uveitis (n = 10). All subjects in both groups had a history of bacille de Calmette-Guerin vaccination, and 8 of the 14 patients underwent the PPD skin test. Peripheral blood lymphocytes were obtained from all patients and separated by gradient centrifugation. Peripheral blood lymphocytes were tested for their proliferative responses to PPD. To determine the frequency of PPD-specific precursor cells in blood, limiting dilution analysis was used. The frequency was calculated following the Poisson model. The culture supernatants were harvested 3 days after incubation and assayed for interferon (IFN)-gamma production by ELISA. RESULTS: Four of the 8 patients showed a negative skin test reaction against PPD, while all the healthy volunteers had a positive reaction. Compared to PBLs from healthy volunteers, PBLs from sarcoidosis patients showed weaker responses to PPD. There was no clear difference between these two groups about frequency of precursor cells against PPD and production of IFN-gamma. CONCLUSIONS: Negative skin test for PPD in sarcoidosis patients does not seem to have any relationship to the low precursor frequency of PPD-specific cells in patient blood.

Adult↗
PubMed2001

frizzled 9 is expressed in neural precursor cells in the developing neural tube.

The wnt signaling pathway has important functions in nervous system development. To better understand this process we have cloned and analyzed the expression of the wnt receptor, frizzled 9, in the developing nervous system in mouse, chick and zebrafish. The earliest expression of mouse frizzled 9 mRNA expression begins at E8.5 with expression throughout the entire rostral-caudal neuraxis. This early expression pattern within the neural tube appears to be conserved between chick and zebrafish. Expression becomes restricted to a ventral domain in the mouse ventricular zone at E11.5, a region specified to give rise to neurons and glia. Using a polyclonal antibody to MFZ9 further shows expression limited to neural restricted precursors cells.

Animals↗
PubMed2004

Regulation of thymus size by competition for stromal niches among early T cell progenitors.

Thymic T cell production is characterized by differentiating waves of non-self-renewing, bone marrow-derived progenitors. The factors constraining new progenitor recruitment, intrathymic precursor expansion, and thymus size remain enigmatic, but are believed to be controlled by a feedback loop responding to lymphoid cellularity and competition for stromal niches. In this study, we show that competition for stromal niches does occur, but is solely limited to cells at the early CD4(-)8(-) precursor stages of differentiation. The overall size of the organ is determined both by this limitation on early precursor expansion, and by a second, cell-intrinsic limit on expansion of progenitor cells transiting to the CD4(+)8(+) stage. Together with asymmetric use of marrow-derived progenitors to reconstitute the intrathymic pool, these processes facilitate continuous generation of new T cells while maintaining a relatively stable organ size.

Animals↗
PubMed1997

Effects of thyroid hormone on embryonic oligodendrocyte precursor cell development in vivo and in vitro.

The oligodendrocyte precursor cell divides a limited number of times before terminal differentiation. The timing of differentiation depends on both intracellular mechanisms and extracellular signals, including mitogens that stimulate proliferation and signals such as thyroid hormone (TH) and retinoic acid (RA) that help trigger the cells to stop dividing and differentiate. We show here that, both in vivo and in vitro, TH is required for the normal development of rodent optic nerve oligodendrocytes, although in its absence some oligodendrocyte development still occurs, perhaps promoted by signals from axons. We also demonstrate that TH from both mother and pup plays a part in oligodendrocyte development in vivo. Finally, we show that precursors in embryonic nerve cultures differ from those in postnatal cultures in two ways: they respond much better to TH than to RA, and they respond more slowly to TH, suggesting that oligodendrocyte precursor cells mature during their early development.

Animals↗
PubMed2000

Protein tyrosine phosphatases from amphioxus, hagfish, and ray: divergence of tissue-specific isoform genes in the early evolution of vertebrates.

Since separation from fungi and plants, multicellular animals evolved a variety of gene families involved in cell-cell communication from a limited number of ancestral precursors by gene duplications in two separate periods of animal evolution. In the very early evolution of animals before the separation of parazoans and eumetazoans, animals underwent extensive gene duplications by which different subtypes (subfamilies) with distinct functions diverged. The multiplicity of members (isoforms) in the same subtype increased by further gene duplications (isoform duplications) in the first half of chordate evolution before the fish-tetrapod split; different isoforms are virtually identical in structure and function but differ in tissue distribution. From cloning and phylogenetic analyses of four subfamilies of the protein tyrosine kinase (PTK) family, we recently showed extensive isoform duplications in a limited period around or just before the cyclostome-gnathostome split. To obtain a reliable estimate for the divergence time of vertebrate isoforms, we have conducted isolation of cDNAs encoding the protein tyrosine phosphatases (PTPs) from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Potamotrygon motoro, a ray. We obtained 33 different cDNAs in total, most of which belong to known PTP subfamilies. The phylogenetic analyses of five subfamilies based on the maximum likelihood method revealed frequent isoform duplications in a period around or just before the gnathostome-cyclostome split. An evolutionary implication was discussed in relation to the Cambrian explosion.

Animals↗
PubMed1979

Trypsin liberates an arginine vasopressin-like peptide and neurophysin from a Mr 20,000 putative common precursor.

Although the hypothesis that vasopressin and its associated neurophysin are synthesized together in one macromolecular common precursor was put forward more than a decade ago, direct conformation of this hypothesis has been lacking. A [35S]cysteine-labeled putative precursor for vasopressin-related neurophysin (Mr 20,000, pI 6.1) has been isolated from the supraoptic nuclei of rats. This precursor was subjected to limited proteolysis with trypsin which produced a Mr 10,000 protein and peptide products. The former was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and its behavior in isoelectric focusing systems (pI 4.6-4.8). The tryptic peptides proved to be vasopressin-like because they: (i) were rich in cysteine, (ii) comigrated with vasopressin on gel filtration columns in 6 M guanidine HCl, (iii) bound to a neurophysin-Sepharose affinity column at pH 5.7, and (iv) were recognized by antibodies against vasopressin. These data on the Mr 20,000, pI 6.1 protein represent direct experimental evidence for a candidate for the common precursor of vasopressin and neurophysin. We propose that this common precursor be called "propressophysin."

Animals↗
PubMed1988

Frequency of human alloantigen-reactive T lymphocytes. III. Evidence that cyclosporine has an inhibitory effect on human CTL and CTL precursors, independent of CsA-mediated helper T cell dysfunction.

We have used limiting dilution analysis to study the behavior of alloantigen-reactive cytolytic T lymphocytes derived from human peripheral blood. During these studies, we found that the presence of cyclosporine in limiting dilution microcultures significantly impairs the subsequent development of alloantigen-reactive cytolytic T cell activity. As a result, CsA reduces the estimate of CTL precursor frequency by limiting dilution analysis. CTL frequency estimates are reduced by CsA in a dose-dependent manner, and concentrations of CsA that are readily achieved in human peripheral blood (100-1000 ng/ml) are capable of reducing estimates of CTL frequency by 90% to 100%. Further studies revealed that (1) human CTL derived either from fresh peripheral blood or from primary mixed lymphocyte cultures are sensitive to the suppressive effects of cyclosporine in limiting dilution microcultures, indicating that CsA influences both alloantigen-primed CTL and CTL precursors; (2) CsA impairs an immunologic event or events, that occurs for at least the first four days of limiting dilution microculture incubation; (3) CsA-mediated suppression is eliminated by separation of CTL from cyclosporine; (4) CsA blocks development of CTL generation, but not cell proliferation in limiting dilution microcultures; and (5) the CsA-mediated suppression is not reversed by supraoptimal concentrations of IL-2, high concentrations of gamma-IFN, or supplementation with the multiple lymphokines present in MLC supernatants. These data suggest that CsA may have a direct inhibitory influence on the differentiation of human CTL precursors that is independent of helper T cell dysfunction.

Cell Differentiation↗
PubMed1990

Detection of the hybrid BCR/ABL messenger RNA in single CFU-GM colonies using the polymerase chain reaction.

In order to study which hemopoietic precursor cells express the hybrid BCR/ABL fusion mRNA we have developed a technique based on the polymerase chain reaction (PCR) for the examination of single hemopoietic colonies grown on semi-solid agar. The technique was developed by examining single CFU-GM colonies grown from newly diagnosed patients with chronic myeloid leukaemia (CML). RNA was isolated from individual 14 day colonies and reverse transcribed to a complementary DNA (cDNA) copy which formed the substrate for a PCR. We have studied 3 cases of CML using this method and have found that 5 out of 5, 9 out of 10 and 20 out of 23 colonies examined were positive. Thus we describe a simple and useful technique for the study of gene expression in a limited number of hemopoietic precursor cells.

Bone Marrow↗
PubMed1992

Isolation of IFAPa-400 cDNAs: evidence for a transient cytostructural gene activity common to the precursor cells of the myogenic and the neurogenic cell lineages.

Differentiation of neural and muscle cells is characterized by a switch in the expression of the type of intermediate filament protein subunit. In these lineages, vimentin is transiently expressed in the initial stages of development and is gradually replaced by a tissue specific protein. We have identified a giant developmentally regulated antigen (IFAPa-400) which colocalizes with vimentin in the precursor cells of the neurogenic and myogenic lineages of the chick embryo [Chabot and Vincent (1990) Dev. Brain Res. 54, 195-204; Cossette and Vincent (1991) J. Cell Sci. 98, 251-260]. Based on the expression of this protein during neurogenesis and myogenesis, we hypothesize that IFAPa-400 and vimentin define a special intermediate filament network, common to the non-differentiated cells derived from the neuroectoderm and those of the myogenic tissues. We report here the isolation and sequence of partial cDNAs encoding more than 400 amino acids of the carboxy-terminus of this protein. RNA blot analysis and in situ hybridization indicate that IFAPa-400 represents a bona fide developmentally regulated gene product. These results further confirm that IFAPa-400 mRNA transcripts are limited to the early precursor cells of both neurogenic and myogenic lineages.

Amino Acid Sequence↗