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PubMed2000

Limited neuropeptide Y precursor processing in unfavourable metastatic neuroblastoma tumours.

Neuropeptide Y (NPY) is found at high concentrations in neural crest-derived tumours and has been implicated as a regulatory peptide in tumour growth and differentiation. Neuroblastomas, ganglioneuromas and phaeochromocytomas with significant concentrations of NPY-like immunoreactivity were investigated for different molecular forms of NPY and for significance of proNPY processing. Gel-permeation chromatography identified intact NPY (1-36) in all tumours, whereas proNPY (69 amino acids) was detected only in control adrenal tissue and malignant neuroblastomas. Purification of NPY-like immunoreactivity in tumour extracts and structural characterization revealed that both NPY (1-36) and the truncated form NPY (3-36) was present. The degree of processing of proNPY to NPY in tumour tissue was lower in advanced neuroblastomas with regional or metastatic spread (stage 3 and 4) (n = 6), (41%, 12-100%, median, range), compared to the less aggressive stage 1, 2 and 4S tumours (n = 12), (93%; 69-100%), (P= 0.012). ProNPY processing of less than 50% was correlated with poor clinical outcome (P = 0.004). MYCN oncogene amplification was also correlated to a low degree of proNPY processing (P = 0.025). In summary, a low degree of proNPY processing was correlated to clinical advanced stage and poor outcome in neuroblastomas. ProNPY/NPY processing generated molecular forms of NPY with known differences in NPY-receptor selectivity, implicating a potential for in vivo modulation of NPY-like effects in tumour tissue.

Child↗
PubMed2002

Periodontal Screening and Recording (PSR) Index: precursors, utility and limitations in a clinical setting.

Periodontal disease is a widespread condition ranging from simple gingival inflammation to severe periodontal breakdown. Early detection and diagnosis are key elements in the prevention of this oral pathology. Diagnostic aids available to dentists, such as the Periodontal Screening and Recording or PSR Index, accelerate the screening of periodontal conditions. While many studies provide support for the PSR Index as a valuable tool because it is reproducible, reliable, and quick, others have suggested that it may under or overestimate existing periodontal conditions. Some authors have suggested modifications to the recording method to improve its use either for screening purposes or for monitoring the outcome of periodontal therapy. This literature review briefly describes the PSR method, and compares studies based on both PSR and its counterpart, CPITN, in order to evaluate its usefulness and establish its limitations in a clinical setting.

Disease Progression↗
PubMed1996

Distinct developmental patterns of short-term and long-term functioning lymphoid and myeloid precursors defined by competitive limiting dilution analysis in vivo.

Functional abilities of individual marrow precursor cells were defined by competitive limiting dilution without enrichment, tissue culture, or induced marking, manipulations that might affect cell functions. We directly measured long-term repopulating abilities in limiting doses (0.25-1.0 x 10(5)) of genetically marked congenic marrow cells. These were mixed with a standard dose of 4 or 5 x 10(5) competitor marrow cells, which contained a predictable distribution of precursor cells and allowed quantitative assays. Percentages of donor type T and B lymphocytes, granulocytes, platelets, and erythrocytes were measured in recipient blood. Applying the maximum likelihood statistic, concentrations (per 10(5)) of precursors repopulating at least one lineage were: 4.7 and 6.0 after 6 wk, 1.6 and 2.7 after 14 to 15 wk, and 1.2 and 1.9 after 30 to 32 wk; concentrations repopulating at least three lineages were 2.3 and 3.4 after 6 wk, 0.9 and 1.7 after 14 to 15 wk, and 0.9 and 1.3 after 32 wk. Almost all precursors functioning after 14 wk repopulated all lineages. At 6 wk, similar levels of donor cells were produced in recipients of both short- and long-term precursors. However, after 14 to 32 wk, contributions by short-term precursors (about two-thirds of the precursors) dropped to zero, while contributions by long-term precursors (about one-quarter of the precursors) expanded severalfold. The latter permanently repopulated all lineages after 30 to 32 wk, functioning as the most primitive stem cells (PSC) in the immune and myeloid systems. Nearly all the variance in long-term repopulated recipients was explained using the Poisson distribution to calculate donor percentages in a model where each donor and competitor PSC contributed equally.

Animals↗
PubMed1977

Histocompatibility antigen-activated cytotoxic T lymphocytes. II. Estimates of the frequency and specificity of precursors.

Using limiting dilutions of responding cells in mouse mixed leukocyte cultures, we obtained direct estimates of the minimum frequency of precursors of cytotoxic T lymphocytes (CTL.P) for a variety of antigens. Depending on the strain combination, there were as many as 4-15 CTL.P reactive to DBA/2 among 10(4) lymph node cells. Taking into account that only 5-10% of peripheral T lymphocytes have the potential to develop into cytotoxic T lymphocytes (CTLs) (6), this implies that at least 1-2% of all CTL.P are responsive to any given H-2 haplotype difference. Precursors of cytotoxic cells thus have the same high frequency of cells reactive to alloantigens of the major histocompatibility complex as found among proliferating cells in graft-vs.-host reactions and mixed lymphocyte interactions. The frequencies of CTL.P reactive to xenoantigens (rat) or trinitrophenyl-modified self were less than half the frequency of alloreactive CTL.P. A minority of the CTL.P specific for one H-2 haplotype were also reactive to a third party H-2 haplotype, presumably on the basis of recognition of shared determinants. By dilution of sensitized cells from single microcultures, it was shown that a single CTL.P undergoes a minimum of three to four cell divisions and generates at least 8-16 CTLs after antigenic activation.

Animals↗
PubMed1987

Human cytotoxic T lymphocytes. II. Frequency analysis of cyclosporin A-sensitive alloreactive cytotoxic T-lymphocyte precursors.

A limiting dilution (LD) culture system was used to investigate the effect of cyclosporin A (CsA) on the activation and differentiation of human alloreactive cytotoxic T-lymphocyte precursors (CTL-p). CsA reduced in a dose-dependent fashion the frequency of alloantigen-inducible CTL-p. With most normal individuals tested there was a 20- to 50-fold reduction of alloreactive CTL-p frequencies in the presence of 500-1000 ng/ml CsA. Both unseparated T cells and CD8+ T cells were CsA-sensitive under LD culture conditions. Importantly, however, alloreactive CTL-p from two out of 21 normal individuals were found to be largely CsA-resistant. CsA did not affect the growth of MLR-primed CTL in secondary LD culture. Furthermore, CsA slightly inhibited the cytolytic activity of some alloantigen-specific CTL clones. These results are discussed with respect to the clinical use of CsA in transplantation medicine.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Comparison of the response to phytohemagglutinin, of peripheral blood mononuclear cells and cerebrospinal fluid lymphocytes in multiple sclerosis and other neurological diseases. Determination of the frequency of the precursor cells by a limiting dilution analysis.

Proliferative response to phytohemagglutinin (PHA) of cerebrospinal fluid lymphocytes (CSF-L) and peripheral blood mononuclear cells (PMBC) from patients with multiple sclerosis (MS) and other neurological diseases (OND) was tested using either bulk culture conditions or limiting dilution techniques in the presence of additional exogenous IL2. CSF-L response to PHA was found significantly lower than that of PBMC in both MS and OND patients. There was no significant difference in the response of CSF-L between the two groups. The limiting dilution analysis of the frequency of the precursors of PHA-responsive cells showed a strikingly decreased frequency of these precursors among CSF-L.

Adolescent↗
PubMed1992

Quantitation of glioma-reactive cytolytic T lymphocyte precursors by means of limiting dilution analysis.

The purpose of the present study was to define the usefulness of limiting dilution analysis (LDA) to enumerate glioma-reactive cytolytic T lymphocytes (CTL) as a constituent of tumor infiltrating lymphocytes (TIL) isolated from rat gliomas. Optimum LDA microculture conditions were defined by co-cultivating graded numbers of responder TIL together with 10(5) irradiated syngeneic rat splenocytes, 10(3) irradiated glioma cells, and 10 U/well of recombinant interleukin-2 incubated for 8 days. Antigenic specificity of the anti-tumor response was demonstrated by high levels of [3H]thymidine incorporation by TIL derived from F98 gliomas following stimulation with irradiated F98 glioma cells compared to low levels following stimulation with the antigenically distinct D74 glioma cells. Limiting dilution analysis showed that cytolytic T lymphocyte-precursors were present in TIL of F98 gliomas of immunized rats at an approximate frequency of 300 CTL/10(6) TIL, indicating that less than 1% of the TIL were tumor-reactive CTL. As determined by cell depletion experiments using various MoAbs and complement, the majority of the cytolytic activity detected against glioma targets was mediated by OX-8+ TIL. Split culture experiments revealed that high levels of glioma-reactive CTL activity and low levels of NK activity, which are simultaneously detected among TIL, were mediated by separate cell populations. Our data demonstrate that LDA microcultures can be used as a powerful tool to differentiate tumor-reactive CTL from other effector cell populations.

Animals↗
PubMed1994

Periplasmic aggregation limits the proteolytic maturation of the Escherichia coli penicillin G amidase precursor polypeptide.

The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26 degrees C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotropic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.

Biotechnology↗
PubMed1982

Quantitative studies on T cell diversity. III. Limiting dilution analysis of precursor cells for T helper cells reactive to xenogeneic erythrocytes.

Splenic T cells exposed to concanavalin A (Con A), and subsequently to factors produced by rat spleen cells in response to Con A (Con A sup), acquire the ability to function as helper T (TH) cells in response to xenogeneic erythrocytes (RBC). Help is measured as the reconstitution of the plaque-forming cell response of a spleen cell population depleted of T cells by treatment with anti-Thy-1 serum and complement. We propose that precursor TH cells differentiate during the in vitro treatment into mature TH cells. As differentiation occurs under limiting dilution conditions, an estimation of the precursor frequency should in principle be possible. However, a single-hit Poisson distribution does not fit our data. Instead, we observe, dependent on the T cell concentration, three separate "peaks" of response. In many experiments, using sheep, horse, and chicken RBC as antigens, we reproducibly find these "peaks" at 40-190, 600-3,000, and 20,000-100,000 T cells, placed into limiting dilution cultures, respectively. By various experiments we can show that the helper activity is not due to passively transferred rat factors, but to the titrated cells themselves. The active cell is a T cell that appears to function in an antigen-specific way and to require direct cell contact to do so. It thus resembles the classical helper T cell. As we find precursor TH cells already at very low concentrations of T cells, we titrated the range between 0 and 100 T cells/well carefully. The bent shape of the titration curves does not always allow a statistically satisfying regression analysis, and we therefore cannot estimate precise precursor frequencies from every experiment. However, a common sense argument can be made that these frequencies must be on the order of 1/10-1/100 T cells. We propose that the limiting dilution curves obtained in this system most likely reflect fundamentally important cellular interactions that regulate immunological effector functions. We favor a concept of independently interacting sets of helper and suppressor T cells of various frequencies, but other models are possible.

Animals↗
PubMed1998

Interphase cytogenetics of esophageal adenocarcinoma and precursor lesions.

Limited information is currently available on chromosomal abnormalities in esophageal adenocarcinoma and associated premalignant lesions. In this study, numeric changes affecting chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 17, 18, X, and Y were analyzed by using fluorescence in situ hybridization (FISH) with chromosome-specific centromere DNA probes in 12 esophageal adenocarcinomas. In addition, TP53 overexpression, measured by immunohistochemistry, and amplification of HER-2/neu and C-MYC, detected by FISH, were analyzed within the same tumors. The most common numeric abnormalities detected were gains of chromosomes 12 (8 cases), 6 (7 cases), 7 (7 cases), and 11 (6 cases). The total number of abnormal chromosomes varied from 0 to 10, with an average of 4.6 per case. Overexpression of TP53 was present in 9 of 12 cases. No correlation was noted between the degree of aneusomy and TP53 overexpression. In contrast, HER-2/neu amplification was present in two cases, both with large numbers of aneusomic chromosomes. Amplification of C-MYC was detected in only one case that had a moderate number of numeric abnormalities. In a subset of cases in which premalignant lesions were examined, aneusomy was found to be an early change, frequently present in both Barrett's esophagus and dysplastic regions. In contrast, gene amplification and TP53 overexpression were restricted to more advanced areas of dysplasia and malignancy. Screening larger cohorts of patients with Barrett's esophagus or dysplasia for numeric abnormalities of chromosomes 6, 7, 11, and 12 may determine whether any of these abnormalities are predictive markers of progression to malignancy.

Adenocarcinoma↗
PubMed1987

In vitro and in vivo synthesis of dolichol and other main mevalonate products in various organs of the rat.

The relative rate of biosynthesis of dolichol from [3H]mevalonate in nine rat organs was studied in slices and in the whole animal. This biosynthesis was also compared to that of cholesterol and ubiquinone. All tissues examined are able to synthesize dolichol, as well as ubiquinone and cholesterol. Comparison of the data from slices in vitro with the in vivo studies demonstrated relatively good agreement for dolichol and ubiquinone synthesis. Although dolichol of high specific radioactivity was recovered in the blood, redistribution between organs, such as occurs with cholesterol, appears to be insignificant. The highest rates of dolichol biosynthesis were found in kidney, spleen and liver. On the other hand, muscle makes the largest contribution to total body dolichol synthesis. Newly synthesized dolichol also appears in the bile, but excretion by this route is far from sufficient to account for dolichol turnover. Incorporation of mevalonate into the final products is mainly dependent on biosynthetic activity. For comparison of the biosynthetic rates in different organs, possible sources of errors (such as variations in the size of the precursor pool, limitation by the rate of precursor uptake or non-linear incorporation) were investigated the size of the mevalonate pool in various organs. Equilibration of this pool with exogenous mevalonate is a rapid and passive process. The size of the mevalonate pool does not determine the rates of cholesterol and dolichol biosynthesis, indicating the presence of regulatory steps in the terminal portion of these biosynthetic pathways.

Adipose Tissue↗
PubMed1985

Differentiation of murine B cell precursors in agar culture. II. Response of precursor-enriched populations to growth stimuli and demonstration that the clonable pre-B cell assay is limiting for the B cell precursor.

We established culture conditions under which fetal liver-derived B cell progenitors divide and differentiate in semisolid agar, forming colonies containing antibody-secreting cells. An important feature of this assay system is that, under certain conditions, it is limiting for only one component. This was revealed by determining the slope of the least-squares log-log regression line generated when the initial seeding density was varied. When support for the growth of the clonable pre-B cells was provided by fetal liver-derived adherent accessory cells, the slope of the regression line was 1, consistent with the interpretation that only one component, the pre-B cell, was limiting under these conditions. This interpretation was tested in the present report by positive selection for cells expressing B220, Lyb-2, or AA4.1, surface markers known to be present on cell lines of the B lineage. In all cases, an increased incidence of clonable pre-B cells was observed. Furthermore, regression analysis of titration experiments undertaken with these enriched populations revealed that the assay was still limiting for a single component. A second set of growth conditions have been defined in which the clonable pre-B assay appears to be limiting for more than one component. These conditions are obtained when the adherent accessory cells are replaced by one of three distinct hemopoietic growth factors, including CSF-1 (macrophage growth factor), GM-CSF (neutrophil/macrophage growth factor), or Multi-HGF (multi-lineage hemopoietic growth factor, also called BPA or IL 3). The most straightforward interpretation of these data is that a second cell type, distinct from the B cell precursor and dependent on the growth factors, was limiting under these conditions. In the present report, this hypothesis was confirmed because growth factor responsiveness was completely absent in B220 and Lyb-2-enriched populations. Factor responsiveness was found in unseparated fetal liver and in AA4-enriched populations. However, the AA4-enriched populations, in contrast to the B220- or Lyb-2-enriched populations, also contained a large number of factor-responsive neutrophil/macrophage colonies, raising the possibility that the effect of factors on AA4+ clonable pre-B cells was accessory cell-mediated.

Agar↗
PubMed1979

Structural characterization of the murine fourth component of complement and sex-limited protein and their precursors: evidence for two loci in the S region of the H-2 complex.

The S region of the murine major histocompatibility complex controls the expression of two related, serum substance-positive proteins; one (C4) has functional complement activity, whereas the other, the sex-limited protein (Slp), is hemolytically nonfunctional. The structural relationships of these molecules to each other and to their putative intracellular precursors have been examined. Radiolabeled intracellular C4 and Slp precursors were isolated from lysates of cultured peritoneal cells. The C4 and Slp precursors and their processed subunits were purified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Antigenically distinct precursors for C4 and Slp were demonstrated by sequential immunoprecipitation experiments in which anti-Slp-reactive molecules were precleared by exhaustive immunoprecipitation and residual C4 molecules were precipitated by antibody to serum substance. Both molecules had apparent molecular weights of 185,000. Their molecular identities as precursors of the mature C4 and Slp proteins were established in pulse-chase studies and by comparisons of their tryptic peptide profiles with those of isolated subunits from the processed proteins. When isolated alpha- or beta-subunits from C4 and Slp proteins were compared by peptide mapping, it was possible to detect multiple distinct and multiple shared peptides. This evidence indicates that the C4 and Slp proteins derive from distinct precursor polypeptides and suggests that the primary structures of the C4 and Slp alpha- and beta-subunits are different. These results support the postulate that the S region contains two discrete structural loci that specify discrete C4 and Slp proteins.

Animals↗
PubMed1984

Long term survival of allografted muscle precursor cells following a limited period of treatment with cyclosporin A.

Previous work (Watt et al., 1982) has shown that, in the mouse, skeletal muscle can be transplanted successfully in the form of a suspension of it's mononucleate precursors. Eventual therapeutic application of this technique by the implantation of precursor cells derived from normal muscle into myopathic individuals would require a means of preventing allograft rejection applicable to man. We have therefore investigated the use of the drug cyclosporin A (CyA) as a means of prolonging the survival in mice of allografts of mononucleate muscle cells made into a region of regenerating host muscle. We have administered CyA to the hosts at doses of either 75 or 150 mg/kg body weight/day for 42 days from the day of grafting. By using isoenzyme allotypes as markers of host and donor tissues, we have shown that allografted mononucleate cells become incorporated in host muscle fibres and that the mosaic host/donor muscle fibres so formed survive for as long as we continued the experiment, a maximum of 107 days after grafting, or 65 days after the end of CyA treatment.

Animals↗
PubMed1985

Human cytotoxic T lymphocytes. I. Limiting-dilution analysis of alloreactive cytotoxic T-lymphocyte precursor frequencies.

A limiting-dilution system was established to measure the frequency of alloreactive cytotoxic T-lymphocyte precursors (CTL-p) in human peripheral blood T cells. Culture medium supplemented with recombinant interleukin-2 enabled clonal expansion of all CTL-p stimulated by allogeneic peripheral blood or spleen cells. The range of CTL-p frequencies in fully HLA-mismatched responder-stimulator combinations was 1:240 to 1:1230. Split-well analysis of individual microwells showed that the cytotoxic T-cell clones generated under limiting-dilution conditions showed exquisite specificity for the stimulating alloantigens. Alloreactive CTL-p were enriched in the OKT4- T-cell subset. This limiting-dilution system was highly reproducible and can thus be applied to investigate human cytotoxic T-cell precursor frequencies in various clinically relevant situations.

Cells, Cultured↗
PubMed1985

Functional characterization of human thymocytes: a limiting dilution analysis of precursors with proliferative and cytolytic activities.

In this report we have analyzed the pool size of human thymocytes capable of proliferating or mediating cytolytic activity. Furthermore, the relationship between these functional capabilities and cell surface antigen expression was studied. Graded numbers of human thymocytes were plated under limiting dilution conditions with irradiated human spleen cells (as a source of feeder cells) and phytohemagglutinin (PHA) in the presence of a saturating concentration of interleukin 2. Cell proliferation, which was evaluated after 20 days of culture, was usually compared with the proliferation of peripheral blood T cell populations cultured under identical conditions. Although essentially all peripheral blood T cells were clonogenic, only 3 to 8% of thymocytes proliferated. Of proliferating microcultures, 48 to 86% showed cytolytic activity in a PHA-dependent assay, whereas 26 to 83% killed the NK-sensitive target cell K-562 in the absence of added lectin. Similar frequency analysis of functional precursors was performed on thymocyte subsets selected according to their expression of T3, T6, T4, and T8 antigens. All precursors of proliferating cells (PTL-P) were found in the T3+ subset. From the comparison of the percentages of total thymocytes capable of proliferation and the proportion of cells brightly stained with anti-T3 antibody, it was evident that only a fraction of T3+ cells was clonogenic. Although the large majority of PTL-P was found in the T6- subpopulation, a small fraction of functional precursors was detected in the T6+ subset. When thymocytes were fractionated according to T4 or T8 antigen expression, it was found that 80 to 90% of the recovered PTL-P were confined to the T4+ fraction, whereas only 20 to 28% of the recovered PTL-P were present in the T8+ subset. Analysis of the precursors of cytolytic T lymphocytes (CTL-P) in thymocyte populations fractionated according to T4 or T8 antigen expression showed that 70 to 90% of the recovered CTL-P were found in the T4+ fraction and 17 to 30% were in the T8+ subset. Because approximately 80% of proliferating T4+ thymocytes had CTL activity (as compared with less than 5% in peripheral blood T4+ lymphocytes), it appears that the subset distribution of thymic CTL-P differs from that of peripheral blood T cells.

Antigens, Differentiation, T-Lymphocyte↗