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PubMedDate not supplied

Effect of interleukin-2 on peripheral blood mononuclear cells in severely injured patients.

The effect of human recombinant interleukin-2 (IL-2) on the proliferative and cytotoxic response of peripheral blood mononuclear cells was analyzed in 12 severely injured patients. The in vitro proliferative response (measured from tritiated thymidine uptake) was normal during the first post-injury week, but rapidly decreased from the second week. The cytotoxic response to natural killer cell-resistant target T24 was likewise diminished from the second week. To clarify if the defect in cytotoxic response was due to numerical reduction of precursors or to reduced responsiveness, limiting dilution assay was introduced. No post-trauma reduction in precursor numbers was observed. These data collectively suggest that, in addition to impaired IL-2 production, there is an endogenous defect in IL-2 responsiveness. In the context of high incidence of septic complications, the observations may be clinically important.

Adolescent↗
PubMed2000

Recent advances in the design and synthesis of carbon-14 labelled pharmaceuticals from small molecule precursors.

Over the past decade, the increased chemical complexity of new drug candidates has resulted in a parallel need to develop innovative syntheses of carbon-14 labelled pharmaceuticals. Faced with short time-lines and a limited number of labelled precursors, radiochemists have addressed this challenge by developing new reagents and adapting existing technology to labelled syntheses. Selected examples from the recent radiochemical literature illustrate some of the creative strategies used to rapidly solve these synthetic challenges. Examples describing the handling and use of common small molecule reagents, such as carbon-14 labelled carbon dioxide, methyl iodide, cyanide, acetic acids, sulfur and phosphorous stabilized ylides for the synthesis of labelled steroids, prostanoids, nucleosides, pyridines, quinolines, benzazepines and other heterocycles are presented. Several general strategies for radiolabelling are also discussed including the degradation strategy for accessing necessary intermediates and precursors, the radiolabelling of aromatic substrates, transition metal mediated cross-couplings, and the use of chiral auxiliaries for the enantioselective syntheses of radiolabelled pharmaceuticals.

Carbon Radioisotopes↗
PubMed2001

Lymphoid environment limits superantigen and antigen-induced T cell proliferation at high precursor frequency.

After superantigen challenge a significant proportion of superantigen-reactive T cells remain undivided. We provide evidence that the lymphoid environment limits T cell proliferation in the secondary lymphoid organs when the frequency of superantigen reactive T cells is unusually high. We monitored T cell proliferation and the percentage of undivided cells when the frequency of superantigen-reactive T cells was low (1%), intermediate (15%) or high (30-100%) by transferring fluorescently labeled cells into different recipients. When the frequency was low, practically all the reactive T cells entered cell cycle and proliferated maximally. At intermediate frequencies a large proportion of reactive T cells did not enter cell cycle and the whole population divided less. A further increase in reactive T cells did not alter the percentage of undivided cells but induced a further decrease in the number of cell divisions. Interestingly, the observations made with superantigens were confirmed with peptide antigen and TCR-transgenic mice. Moreover, in vivo and in vitro data suggest that dendritic cells are the most likely candidates in limiting T cell proliferation in the lymphoid environment. In conclusion, we show that the availability of APC in the lymphoid environment can quantitatively limit T cell priming.

Adoptive Transfer↗
PubMed1999

Posttherapy surveillance of B-cell precursor acute lymphoblastic leukemia. Value of polymerase chain reaction and limitations of flow cytometry.

Flow cytometric immunophenotypic analysis is critical in diagnosis and classification of acute leukemia and has been used after therapy to monitor for minimal residual disease. However, the presence of normal B-cell precursors, hematogones, particularly in the context of treated pediatric B-cell precursor acute lymphoblastic leukemia (BP-ALL), may confound such evaluation. In this study, the value of more specific genotypic markers (polymerase chain reaction evaluation of 2 antigen receptor genes) was assessed to resolve this issue. Flow cytometric analysis of enriched mononuclear cells revealed 1% to 20% precursor B cells (PBCs), based on expression of 1 or more pan-B cell antigens in addition to CD10, CD34, and terminal deoxynucleotidyl transferase in all 14 patients studied. Inasmuch as this mimicked the immunophenotype of the original leukemic clone, PBCs, in isolation, were considered suspicious for minimal residual disease. However, 11 of the 14 posttherapy specimens (79%) revealed no monoclonally rearranged antigen receptor genes, and 7 of these 11 patients had trackable genotypic markers at presentation. Accordingly, by PCR these 7 patients had complete molecular remission, supported by clinical follow up of 16 to 73 months. Among the remaining 4 patients with PCR-negative disease, 3 continue in remission, confirming the interpretation of false-positive flow cytometric analysis. In conclusion, flow cytometric monitoring of posttherapy bone marrow specimens from patients with BP-ALL may be misleading, if considered in isolation, in falsely suggesting the presence of minimal residual disease. Rather, PCR for antigen receptor gene rearrangements is a valuable and specific tool, helpful in differentiating hematogones from minimal residual disease in patients with treated BP-ALL whose bone marrow harbors increased PBCs.

Adolescent↗
PubMed1996

Primary alloreactive cytotoxic T-lymphocytes are not commonly restricted by self-HLA class I antigens.

To assess the role of HLA Class I molecules in the indirect presentation of alloantigen, we have investigated the fine specificity and MHC restriction of in vitro primary alloreactive cytotoxic T-lymphocytes (CTL), using limiting dilution analysis of CTL precursor frequencies in HLA-mismatched responder-stimulator pairs. By employing split-well analysis of limiting dilution (LD) microcultures and third-party target cells bearing a stimulatory HLA Class I antigen alone or in combination with a single responder HLA antigen, we demonstrate that self-Class I restriction of HLA-A- or HLA-B-specific CTL precursors is not a common feature of the primary in vitro alloresponse. Higher frequencies of alloantigen-specific CTL precursors in the presence of self-HLA antigens were only detected in 5 of 31 limiting dilution assays established from seven different responder-stimulator pairs. In two cases, the higher precursor frequencies could be explained on the basis of Class II-restricted presentation of Class I-derived antigenic peptide and are supported by flow cytometric analysis of HLA antigen expression on target cells. The remaining 3 assays of this type were suggestive of Class I restriction but revealed only marginally higher frequency estimates. All other LD assays revealed lower CTL precursor frequency estimates in the presence of self-HLA Class I antigens. A higher antigen-specific CTLp frequency was not detected when targets shared three HLA Class I antigens with the responder, demonstrating that we had not biased the responses by selecting single HLA antigen-sharing targets in the other assays. Analysis of reactivity against PHA blast targets at the single cell per well level demonstrated that CTL reactive only with the original stimulator comprised the majority of lytic reactions. Heteroclitic CTL (i.e., CTL that recognize single HLA targets only and not the original stimulator) formed only a small fraction of total reactivity. Our results confirm the role of Class II antigens in the indirect presentation of alloantigen in vitro but suggest that HLA Class I antigens play a limited role in this phenomenon.

Autoantigens↗
PubMed1990

Subtypes of HLA-A1 defined on the basis of CTL precursor frequencies.

Recently we have shown that limiting dilution analysis can be used to detect cytotoxic T-cell precursor frequencies directed against individual HLA class I antigens. Using the same protocol, we have been able to define two subtypes of HLA-A1, which are indistinguishable by conventional typing sera as well as by cell-mediated lympholysis. One-dimensional isoelectric focusing analysis of the variants did not show any overall charge differences. However, family studies indicated that these HLA-A1 subtypes are genetically determined and can be distinguished on the bases of T-cell precursor frequencies in HLA-A1-negative blood donors.

Binding, Competitive↗
PubMed1999

Quantifying the contribution of gluconeogenesis to glucose production in fasted human subjects using stable isotopes.

The contribution of gluconeogenesis to glucose production is estimated from the enrichment of the H bound to C-5 of glucose relative to either that bound to C-2 of glucose or the enrichment in body water on ingesting 2H2O in the fasted state. Contributions of all gluconeogenic substrates are included in the estimate and the limitation of an uncertain precursor enrichment removed. The half-life of 2H2O in body water precludes a repeat study for many weeks. Glycogen cycling could result in underestimation, but there is evidence that glycogen cycling does not occur in liver in the fasted state. Gluconeogenesis has been estimated by mass-isotopomer-distribution analyses, usually by administering 13C-labelled glycerol. Underestimates emphasize the major limitation of the method, i.e. the need to assume a single enrichment of the precursor pool. Estimates of gluconeogenesis from isotopomer distribution in arterial-blood glucose and lactate on infusing [U-13C6] glucose are unreliable, as a proportion of the glucose is formed from glycerol and from amino acids not converted to glucose via pyruvate. Loss of label in the Krebs cycle and relying on enrichment of arterial-blood lactate as a measure of hepatic pyruvate further add to the uncertainty. Estimates of the rate of gluconeogenesis by NMR are obtained by subtraction of the rate of glycogenolysis determined by NMR from the rate of glucose production. Estimates are then the mean rate for the period over which glycogen contents are measured. Technical considerations can limit the accuracy of analyses and result in overestimates.

Blood Glucose↗
PubMed1996

Use of monoclonal antibodies to study interleukin-1 beta-converting enzyme expression: only precursor forms are detected in interleukin-1 beta-secreting cells.

We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)-1 beta-converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross-reactivity with TX, the closest ICE homolog known to date. Two anti-p20 and one anti-p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE-transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67,000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL-1 beta was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL-1 beta cleavage. Altogether, these observations suggest that post-translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.

Amino Acid Sequence↗
PubMed1986

The immune response to bacterial dextrans. III. Ontogenic development and strain distribution of specific clonal precursors.

The frequencies of B512 dextran (Dex)-specific B cell precursors were determined by limiting dilution analysis in a number of mouse strains originally described as "high responder", "low responder" and "nonresponder" to this antigen. No significant difference in the frequencies of Dex-specific precursors was found in C57BL/6, B10.BR, C3H/Tif, BALB/c and A/Sn adult mice. Together with the large intra-strain variability in the magnitude of anti-Dex PFC responses in vivo, these results established that differential reactivity in vivo cannot be ascribed to genetically controlled absence or wide variation in the frequency of Dex-specific immunocompetent precursors. A similar analysis of the Dex-specific precursor frequency was carried out in C57BL/6 mice between 1 week and 3 months of age. While no Dex-specific antibody response was detected in vivo before the age of 3 weeks, clonal precursor analysis revealed that the appearance of these specificities parallels the development of competent (IgM-producing) B lymphocyte clonal precursors, such that no significant difference in absolute frequencies of Dex-specific precursors could be observed among these age groups. This is interpreted to suggest that the late development of the Dex-specific antibody responses is regulatory rather than due to late rearrangement and activation of the appropriate V genes and a sequential expression of antibody specificities in ontogenic development.

Age Factors↗
PubMed1997

Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation.

Many types of vertebrate precursor cells divide a limited number of times before they stop and terminally differentiate. In no case is it known what causes them to stop dividing. We have been studying this problem in the proliferating precursor cells that give rise to postmitotic oligodendrocytes, the cells that make myelin in the central nervous system. We show here that two components of the cell cycle control system, cyclin D1 and the Cdc2 kinase, are present in the proliferating precursor cells but not in differentiated oligodendrocytes, suggesting that the control system is dismantled in the oligodendrocytes. More importantly, we show that the cyclin-dependent kinase (Cdk) inhibitor p27 progressively accumulates in the precursor cells as they proliferate and is present at high levels in oligodendrocytes. Our findings are consistent with the possibility that the accumulation of p27 is part of both the intrinsic counting mechanism that determines when precursor cell proliferation stops and differentiation begins and the effector mechanism that arrests the cell cycle when the counting mechanism indicates it is time. The recent findings of others that p27-deficient mice have an increased number of cells in all of the organs examined suggest that this function of p27 is not restricted to the oligodendrocyte cell lineage.

Animals↗
PubMed2000

Self-limiting growth of metal fluoride thin films by oxidation reactions employing molecular precursors

FeF (2) films are grown by the reaction of XeF (2) and SeF (6) with iron foil. The growth initially follows the Mott-Cabrera parabolic rate law, indicating that the process is diffusion limited. At a certain film thickness, however, the growth abruptly stops, with the thickness using XeF (2) being nearly double that with SeF (6). It is suggested that the shutdown is due to the inability of the molecules to dissociate when too far from the substrate and that SeF (6) must approach more closely than XeF (2). This work suggests the use of molecular precursors to grow thin films via a self-limiting chemical process.

Journal Article↗
PubMed1998

p27Kip1 alters the response of cells to mitogen and is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation.

BACKGROUND: In many vertebrate cell lineages, precursor cells divide a limited number of times before they arrest and terminally differentiate into postmitotic cells. It is not known what causes them to stop dividing. We have been studying the 'stopping' mechanism in the proliferating precursor cells that give rise to oligodendrocytes, the cells that make myelin in the central nervous system. We showed previously that the cyclin-dependent kinase inhibitor p27Kip1 (p27) progressively accumulates in cultured precursor cells as they proliferate and that the time course of the increase is consistent with the possibility that p27 accumulation is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation at the appropriate time. RESULTS: We now provide direct evidence that p27 is part of the intrinsic timer. We show that although p27-/- precursor cells stop dividing and differentiate almost as fast as wild-type cells when deprived of mitogen, when stimulated by saturating amounts of mitogen they have a normal cell-cycle time but tend to go through one or two more divisions than wild-type cells before they stop and differentiate. Cells that are p27+/- behave in an intermediate way, going through at most one extra division, indicating that the levels of p27 matter in the way the timer works. We also show that p27-/- precursor cells are more sensitive than wild-type cells to the mitogenic effect of platelet-derived growth factor. CONCLUSIONS: These findings demonstrate that p27 is part of the normal timer that determines when oligodendrocyte precursor cells stop dividing and differentiate, at least in vitro. It seems likely that p27 plays a similar role in many other cell lineages, which could explain the phenotypes of the p27-/- and p27+/- mice.

Animals↗
PubMedDate not supplied

Terminal steps in JH biosynthesis in the honey bee (Apis mellifera L. ): developmental changes in sensitivity to JH precursor and allatotropin.

Juvenile hormone (JH) is considered the prime endogenous signal for the induction of queen development in honey bees (Apis mellifera L.). At the beginning of the last (5th) larval stadium, worker corpora allata synthesize less JH than queen corpora allata as a consequence of a limited production of JH precursors and a caste- and stage-specific block of the terminal step in JH biosynthesis. As previously shown, the Manduca sexta allatotropin stimulates JH biosynthesis in honey bee corpora allata in a dose-dependent and reversible manner, but can not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for worker early 5th instars. In experiments with M. sexta allatotropin and with the JH precursor farnesoic acid, we found characteristic stage-specific differences in their effects on JH biosynthesis. From the end of the spinning stage on, corpora allata could be stimulated by farnesoic acid to a much higher extent than in earlier developmental stages, suggesting a sudden increase in epoxidase activity. Manduca sexta allatotropin, however, stimulated corpora allata activity until the end of the spinning stage, at which time the corpora allata become suddenly insensitive. These data suggest that in worker larvae, important changes in the regulation of the terminal enzymatic steps in JH biosynthesis occur at the transition from the spinning stage to the prepupal stage. However, the analysis of in vitro activities of the involved enzymes, O-methyltransferase and methyl farnesoate epoxidase, remained inconclusive.

Animals↗
PubMed1994

Frequency of cytotoxic T lymphocyte precursors (CTLp) interacting with autologous tumor via the T-cell receptor: limiting dilution analysis of specific CTLp in peripheral blood and tumor-invaded lymph nodes of melanoma patients.

The frequencies of cytotoxic T-lymphocyte precursors (CTLp) that lyse autologous tumor by a T-cell receptor (TCR)-dependent mechanism (specific CTLp) were evaluated by limiting dilution analysis (LDA) using lymphocytes from peripheral blood (PBL) and from surgically resected, tumor-invaded lymph nodes (LNL) in 9 melanoma patients. The frequency of specific CTLp was determined in PBLs and/or LNIs of all patients by a modified LDA assay, enabling us to measure lytic activity on the autologous tumor that could be significantly inhibited by an anti-CD3 monoclonal antibody (MAb). This assay allowed us to detect frequencies of specific CTLp ranging from 1/720 to 1/32,037 in peripheral blood and from 1/328 to 1/22,061 in tumor-invaded lymph nodes. These frequencies indicated that lymphoid populations from PBLs or LNLs of melanoma patients may contain as low as 30 to as much as 3,000 specific CTLp/10(6) lymphocytes. In addition, comparison of wells containing specific CTLp with those showing no inhibition by anti-CD3 MAb indicated that specific CTLp represent between 3 and 88% of all precursors with lytic activity on the tumor. In 6 of 9 patients, no marked differences between PBLs and LNIs in specific CTLp frequencies were found. A 10-fold increase of specific CTLp, in comparison to PBL and LNL, was found only in lymphocytes isolated from a subcutaneous metastasis of one patient. Our results indicate that CTLp interacting with autologous tumor by a TCR-dependent mechanism exist in PBL and LNL of most melanoma patients, although a wide variation in their absolute number is evident among different patients.

Adult↗
PubMed1979

Proliferative capacity of erythropoietic stem cell lines and aging: an overview.

The earliest bone marrow precursor cell types, often called stem CELLS, have a very large capacity for self renewal. This makes them a useful model system in which to test the hypothesis that normal somatic cells have a limited proliferative capacity. Marrow precursor cells differentiate and multiply to replenish the supply of various blood cell types that constantly turn over. Especially with erythrocyte production, this function is well difined and can be tested rigorously to determine whether a significant amount of the stem cell proliferative capacity is exhausted. Functional tests generally show that marrow stem cell lines are exhausted after three to six serial transplantations into successive recipients; the few exceptions are cases in which functioning by cells from the irradiated recipients has not been ruled out. Genetic markers unambiguously identifying marrow stem cell lines from the original donor are necessary for clear cut interpretations of transplantation experiments. No significant differences are found when comparing erythrocyte production by marrow stem cell lines from old and young adult donors. This suggests that little or none of the erythropoietic stem cell's proliferative capacity is exhausted by a lifespan of normal functioning.

Aging↗
PubMed1991

Strain dependent production of ciguatoxin precursors (gambiertoxins) by Gambierdiscus toxicus (Dinophyceae) in culture.

Thirteen strains of Gambierdiscus toxicus isolated from Queensland (Australia), Hawaii, French Polynesia and the Virgin Islands were mass cultured and extracted for ciguatoxin. A biodetrital sample containing wild G. toxicus collected from the Republic of Kiribati was also extracted for ciguatoxin. Ciguatoxin, as characterized from moray eels, was not detected in any of the strains examined. Two Queensland strains and the wild G. toxicus produced putative ciguatoxin precursors named gambiertoxins. These gambiertoxins were less polar than ciguatoxin but produced bioassay signs in mice and in-vitro responses in isolated guinea pig atria and vas deferens which were similar (but not identical) to those produced by ciguatoxin. The gambiertoxins from cultured cells were also shown to competitively inhibit the binding of [3H]brevetoxin-3 to rat brain membranes in a dose-dependent manner. The gambiertoxins were more potent than ciguatoxin (on a per mouse unit basis) at stimulating neural elements of guinea pig atria. The two culture strains produced similar amounts of gambiertoxins, even when grown in nutrient media made from different seawater containing different concentrations of nutrients. Changes in nutrient media did not induce the other strains of G. toxicus to produce gambiertoxins. The production of these ciguatoxin precursors appears to be limited to only certain genetic strains of G. toxicus, with the majority of strains not producing these toxins. We propose that ciguatera occurs when blooms of G. toxicus strains genetically capable of producing these ciguatoxin precursors enter the marine food chain. These toxins could then become oxidatively metabolized in fishes to the major polar ciguatoxin. Wild cells produced approximately 100-fold greater quantities of gambiertoxins per cell than did the two culture strains indicating that there is considerable potential for increased production of these ciguatoxin precursors from G. toxicus in culture.

Animals↗
PubMed2006

The universal algorithm of maturation for secretory and excretory protein precursors.

During maturation, most proteins undergo different posttranslational modifications. In most simple cases, signal peptidases remove the signal or leader peptide from the precursors of the secretory proteins during their translocation across the ER membrane. For biologically active proteins, such as enzymes, regulatory and defense proteins, toxins, etc., additional maturation-regulating mechanisms were shown to proceed with limited proteolysis of inactive precursors by specific enzymes. A number of specific enzymes from different cell types selectively cleave proproteins at specific processing sites. In this work, we analyzed the sequences of protein precursors synthesized in the excretory glands of different animals and identified new, non-traditional processing sites. They differ from the motifs previously identified in secreted proteins' precursors and enabled us to reconstruct the sequence of events leading to the conversion of protein precursors into the final products (mature proteins). We also found that in animals, the maturation mechanism of secretory and excretory proteins and the set of enzymes involved are species specific. The processing sites identified in protein precursors in this study are useful for a more detailed genome analysis and more accurate mature protein sequence prediction.

Algorithms↗
PubMed1971

Cellular basis of the genetic control of immune responses to synthetic polypeptides. II. Frequency of immunocompetent precursors specific for two distinct regions within (Phe, G)-Pro--L, a synthetic polypeptide derived from multichain polyproline, in inbred mouse strains.

DBA/1 mice are high responders to the (Phe, G) determinant of the synthetic polypeptide (Phe, G)-Pro--L, whereas SJL mice respond well to the Pro--L region of this macromolecule (6). In order to determine whether the phenomenon described above is related to the number of antigen-sensitive units detected for both specificities, and whether responses to these determinants can be transferred independently, graded and limiting inocula of spleen cells from SJL, DBA/1, and F(1) donors were injected into X-irradiated, syngeneic, recipient mice with (Phe, G)-Pro--L. By this approach, one antigen-sensitive unit specific for (Phe, G) was detected in 1.7 x 10(6) and 8.5 x 10(6) spleen cells from immunized and nonimmunized DBA/1 donors, respectively. In contrast, one (Phe, G) relevant precursor was detected in 20 x 10(6) SJL spleen cells, irrespective of whether the donors had been immunized. On the other hand, for the Pro--L specificity, one limiting splenic precursor was found in 1.3 x 10(6) and in 3.4 x 10(6) cells for immunized and nonimmunized SJL donors, respectively; whereas one response unit was estimated for this determinant in 9.4 x 10(6) and in 38 x 10(6) spleen cells from immunized and nonimmunized DBA/1 mice. The findings reported here indicate that the phenotypic expression of the genetic control(s) for immune responsiveness to different immunopotent regions of (Phe, G)-Pro--L is directly correlated with the number of immunocompetent response units detected in two inbred mouse strains. In the spleens of immunized F(1) donors, similar frequencies of one limiting precursor in 3.0 x 10(6) and in 2.8 x 10(6) cells were detected for (Phe, G) and Pro--L, respectively. The results of a chi-square test for independence of (Phe, G) and Pro--L responses in F(1) animals is compatible with the hypothesis that the transferred spleen cells limiting the response to (Phe, G)-Pro--L are restricted to generate antibodies specific for only one of the two determinants of this macromolecule.

Animals↗