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PubMed2006

Analysis of natural killer cells isolated from human decidua: Evidence that 2B4 (CD244) functions as an inhibitory receptor and blocks NK-cell function.

While during the first trimester of pregnancy natural killer (NK) cells represent the most abundant lymphocyte population in the decidua, their actual function at this site is still debated. In this study we analyzed NK cells isolated from decidual tissue for their surface phenotype and functional capability. We show that decidual NK (dNK) cells express normal surface levels of certain activating receptors, including NKp46, NKG2D, and 2B4, as well as of killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A inhibitory receptor. In addition, they are characterized by high levels of cytoplasmic granules despite their CD56(bright) CD16- surface phenotype. Moreover, we provide evidence that in dNK cells, activating NK receptors display normal triggering capability whereas 2B4 functions as an inhibitory receptor. Thus, cross-linking of 2B4 resulted in inhibition of both cytolytic activity and interferon-gamma (IFN-gamma) production. Clonal analysis revealed that, in the majority of dNK cell clones, the 2B4 inhibitory function is related to the deficient expression of signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) mRNA. Moreover, biochemical analysis revealed low levels of SAP in the dNK polyclonal population. This might suggest that dNK cells, although potentially capable of killing, are inhibited in their function when interacting with cells expressing CD48.

Antigens, CD↗
PubMed1998

Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group.

Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.

Codon↗
PubMed1994

Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins.

The protein and antigen profiles of 11 isolates of Mycoplasma bovis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of whole organisms. The isolates examined included the type strain PG45 and 10 other filter-cloned strains or purified isolates both from animals without clinical signs and from clinical cases of bovine mastitis, arthritis, or pneumonia. While the overall protein patterns visualized by silver staining were very similar, marked differences in the antigen banding profiles were detected by rabbit antiserum prepared against whole organisms from one of the strains analyzed. This antigenic heterogeneity was shown to be independent of the geographical origin, the type of clinical disease, and the site of isolation and was also observed among serial isolates from a single animal. Antigen profiles were further monitored throughout sequentially subcloned populations of the PG45 strain. This clonal analysis revealed a high-frequency variation in the expression levels of several prominent antigens. All of these variable antigens were defined by detergent-phase fractionation with Triton X-114 as amphiphilic integral membrane proteins. A subset of different-sized membrane proteins was identified by a monoclonal antibody raised against a PG45 subclone expressing a 63- and a 46-kDa variant antigen within that set. The selective susceptibility of these proteins to trypsin treatment of intact organisms and their ability to bind the monoclonal antibody in colony immunoblots demonstrated that they were exposed on the cell surface. In addition, their preferential recognition by serum antibodies from individual cattle with naturally induced M. bovis mastitis or arthritis confirmed that they were major immunogens of this organism. These studies establish that the apparent antigenic heterogeneity among M. bovis isolates reported here does not represent stable phenotypic strain differences generated from accumulated mutational events but reflects distinct expression patterns of diverse, highly variable membrane surface proteins.

Animals↗
PubMed2005

Hedgehog signaling controls Soma-Germen interactions during Drosophila ovarian morphogenesis.

The genetic analysis of Drosophila adult oogenesis has provided insights into the molecular mechanisms that control cell proliferation, differentiation, migration, and intercellular signaling. However, little is known about the larval and pupal cellular events leading to the formation of the highly organized adult ovary, which is composed of ovarioles each containing germline cells enveloped by specialized somatic cells. We describe here the presence of ovarioles devoid of any germ cells in adult females mutant for fused, which encodes a Hedgehog signal transducing serine/threonine kinase. We show that this phenotype corresponds to a requirement for fused function for the organization of germ cells with respect to ovarian somatic cells during ovariole formation specifically during pupal stages and provide some evidence by means of clonal analysis suggesting that fused function may be necessary in the germline. hedgehog is expressed specifically in somatic terminal filament cells in pupal ovaries, and females bearing hedgehog strong loss-of-function mutations also exhibit aberrant germ cell distribution and formation of agametic ovarioles. These results indicate a positive role for Fused in the transduction of somatic Hedgehog signaling instructing ovariole morphogenesis. We also provide evidence for the use of noncanonical Hedgehog signal transducer(s) within germline cells.

Alleles↗
PubMed1998

The Polycomb-group gene eed is required for normal morphogenetic movements during gastrulation in the mouse embryo.

We have characterized an induced mutation, called embryonic ectoderm development or eed, that disrupts A-P patterning of the mouse embryo during gastrulation. Positional cloning of this gene revealed it to be the highly conserved homologue of the Drosophila gene extra sex combs, which is required for maintenance of long-term transcriptional repression of homeotic gene expression. Mouse embryos homozygous for loss-of-function alleles of eed initiate gastrulation but display abnormal mesoderm production. Very little embryonic mesoderm is produced; in contrast, extraembryonic mesoderm is relatively abundant. These observations, along with mRNA in situ hybridization analyses, suggested a defect in the anterior primitive streak, from which much of the embryonic mesoderm of the wild-type embryo is derived. To analyse this defect, we initiated clonal analysis of the pre-streak epiblast in eed mutant embryos, using the lineage tracer horseradish peroxidase (HRP). The results of these studies indicate that epiblast cells ingress through the anterior streak, but the newly formed mesoderm does not migrate anteriorly and is mislocalized to the extraembryonic compartment. Abnormal localization of mesoderm to the extraembryonic region did not appear to be due to a restriction and alteration of distal epiblast cell fate, since the majority of clones produced from regions fated to ingress through the anterior streak were mixed, displaying descendants in both embryonic and extraembryonic derivatives. eed mutant embryos also fail to display proper epiblast expansion, particularly with respect to the A-P axis. Based on patterns of clonal spread and calculated clone doubling times for the epiblast, this does not appear to be due to decreased epiblast growth. Rather, epiblast, which is normally fated to make a substantial contribution to the axial midline, appears to make mesoderm preferentially. The data are discussed in terms of global morphogenetic movements in the mouse gastrula and a disruption of signalling activity in the anterior primitive streak.

Animals↗
PubMed1988

In situ characterization, clonogenic potential, and antitumor cytolytic activity of T lymphocytes infiltrating human brain cancers.

Mononuclear cells infiltrating human brain tumors were isolated from seven of nine surgical biopsy specimens. These cells were small T11+, T3+ lymphocytes that did not express DR antigens or the receptor for interleukin-2. In addition, large granular lymphocytes were recovered from two of these tumors. The clonogenic potential of tumor-infiltrating lymphocytes (TIL's) was assessed by limiting-dilution analysis (LDA) using a microculture system that permits proliferation of virtually 100% of normal peripheral blood T lymphocytes (PBL-T's). In comparison to normal and autologous PBL-T's, TIL's had a strikingly reduced proliferative potential revealed by a decrease in the frequency of proliferating T lymphocyte precursors calculated by LDA. On average, only one of every 100 T cells from TIL's was able to proliferate, as compared to one of every two or all of the T cells from the patient's peripheral blood or from normal donors. Furthermore, the TIL populations showed depressed proliferative responses to the lectins phytohemagglutinin and concanavalin A and to the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate. Clonal analysis performed on the proliferating microcultures from three tumors demonstrated that the majority of these clones possessed cytolytic activity against various tumor cell targets. Among clones tested for cytolytic activities with glioma cells, four lysed cultured autologous tumor cells, and the specific lysis was greater than 50% in all cases. Numerous clones with natural killer (NK)-like activity were obtained from two TIL preparations, and the frequency of cytolytic T lymphocyte precursors with NK-like activity was determined for one of these preparations and was found to be higher than that in the patient's peripheral blood. Glioma cells grown in culture and then mixed with normal peripheral blood lymphocytes (PBL's) were capable of inhibiting the PBL's response to lectins. This inhibitory property may account in part for the observed poor clonogenicity of TIL's from brain tumors. Nevertheless, nearly all proliferating clones displayed cytotoxicity against either autologous or allogeneic tumor cell targets and may imply selective accumulation of cytolytic effector cells at the tumor site.

Adult↗
PubMedDate not supplied

[Mutant gene expression in mouse aggregation chimeras. 2. The dominant cataract-FR gene].

An analysis of dominant cataract-Fr (CatFr) gene in chimaeric mice CatFr/CatFr c/c in equilibrium +/+C/C has shown that CatFr gene is active in the differentiation lens fibres. The pycnosis of nuclei was observed in the primary lens fibres both in 14 day old chimaeric and CatFr/CatFr embryos. In the central part of lens of the 17 day old chimaeric embryos, there are, besides the fragmented pycnotic nuclei, nuclei with normal morphology which are not observed in CatFr/CatFr embryos. The pycnosis of nuclei was observed in some deep cortical lens fibres of both 17 day old chimaeric and CatFr/CatFr embryos. In the newborn chimaeric mice practically all centrally located lens fibres had destructive changes. The effect of gene CatFr, pycnosis of nuclei, was used as cell marker in clonal analysis of lens development. The count of pycnotic nuclei in the primary and secondary lens fibres of, respectively, 14 and 17 day old chimaeric embryos has shown a high degree of correlation in the ratio of normal and mutant cells between the left and right eye lenses. A high degree of correlation was also established in the ratio of pigmented and unpigmented cells in the retinal pigment epithelium between the left and right eye. A positive correlation was also shown in the ratio of mutant and normal cells between the lens and retinal pigment epithelium in both 14 and 17 day old chimaeric embryos.

Animals↗
PubMed1992

Clonal segregation of oligodendrocytes and astrocytes during in vitro differentiation of glial progenitor cells.

To study the clonal lineage of the glial progenitor population, isolated from newborn rat brain (Lubetzki et al. J Neurochem 56:671, 1991), we combined somatic transgenesis using a retroviral vector encoding a modified bacterial beta-galactosidase with nuclear localization, and triple immunofluorescence labeling with A2B5, anti-galactosylceramide, and anti-glial acidic fibrillary protein antibodies. This allowed clonal analysis of the postnatal glial lineage with precise phenotypic identification of each cell within the lacZ-positive clones. When infected cells were cultivated under constant conditions, in the presence of either 1% or 10% fetal calf serum (FCS)-containing medium, all the 250 lacZ-positive clusters examined were homogeneous, i.e., either oligodendroglial or astroglial. Mixed astrocyte-oligodendroglial clones were observed when cells cultivated in the presence of 1% FCS were switched to a 10% FCS-containing medium, confirming the bipotentiality of glial progenitor cells (Temple and Raff Nature 313:223, 1985). However, even under the switch culture conditions, segregation into homogeneous clones of either oligodendrocytes or astrocytes still predominated, and the percentage of mixed clones dropped from 25 to 8 or to 3, when the switch took place at 8, 16, or 22 days in vitro, respectively. Two additional observations lead us to suggest that microenvironmental factors are responsible for the clonal segregation of glial progenitor cells: 1) the uneven distribution of oligodendrocyte and astrocyte clusters, the latter being seen mostly on the edge of the coverslips; and 2) the presence, in the vicinity of an homogeneous lacZ-positive clone, of some lacZ-negative cells expressing the same phenotype.

Animals↗
PubMed2004

Clonal diversity and stability of subgingival Eikenella corrodens.

Eikenella corrodens is a commensal subgingival bacterium commonly found in both periodontally nondiseased and diseased subjects. The present study examined the clonal diversity and stability of subgingival E. corrodens over time. Ninety-five subjects were enrolled at the baseline examination, including 44 periodontally nondiseased subjects and 51 subjects with aggressive periodontitis. Twenty-two nondiseased subjects and 11 subjects with aggressive periodontitis were subsequently reexamined after an average interval of 14 months. Two subgingival plaque samples were obtained from each subject to determine the total cultivable bacteria. In addition, multiple E. corrodens isolates from each sample were recovered for clonal analysis by arbitrarily primed PCR. The mean numbers of distinct E. corrodens clones harbored by nondiseased subjects and subjects with aggressive periodontitis were 1.3 and 3.0, respectively. Thirty-nine percent of the nondiseased subjects and 63% of the subjects with aggressive periodontitis harbored multiple clones of E. corrodens. The numbers of distinct E. corrodens clones increased significantly (Mann-Whitney ranking test, P < 0.05) in sites from patients with aggressive periodontitis, in sites with pocket depths of 4 mm or greater, in sites with a clinical attachment loss of 2 mm or greater, and in sites coinfected with Porphyromonas gingivalis. Comparison of E. corrodens clones recovered at the baseline and those recovered at the follow-up examination showed that E. corrodens colonization was not stable. Thirty-eight of the 66 follow-up samples (58%) showed a complete change (including de novo colonization of the sites or complete elimination of the organism from the sites) of the subgingival E. corrodens clonal types between the baseline and the follow-up examinations. Our results suggest a complexity of subgingival microbiota not seen previously.

Adolescent↗
PubMed1997

Detection of clonality and genetic alterations in endometrial pipelle biopsy and its surgical specimen counterpart.

Carcinoma of the endometrium is the most frequently diagnosed gynecologic malignancy in the western world. Because endometrial carcinoma is monoclonal in origin, the small samples obtained in endometrial pipelle biopsies can be used in PCR clonal studies to distinguish cancerous from noncancerous lesions. The method used for clonal analysis was based on RFLP of the X chromosome-linked phosphoglycerokinase gene and random inactivation of one X chromosome by methylation in women. Among 50 endometrial pipelle biopsies, 26 (52%) were found to be heterozygous for the above-mentioned polymorphism. Of the samples taken from these informative (ie, heterozygous) patients, six were monoclonal including five cases of endometrial carcinoma and one of endometrial atypical hyperplasia. In each case, the same pattern of monoclonality was present in the surgical specimen counterpart. All of the remaining samples were polyclonal and, when the anatomical pathology data were contrasted, they correlated with nonmalignant endometrium (five secretory, five proliferative, seven atrophic, and three simple hyperplasias). In addition, genetic alterations study of monoclonal endometrial samples revealed a K-ras point mutation and a c-erbB2/neu gene amplification in two different endometrial carcinomas. Both alterations were also detected in the surgical specimens. In addition, a diagnosed set of 10 samples of simple hyperplasia and 5 of atypical hyperplasia were subjected to clonal assay. Among eight informative cases, the three that showed that showed the monoclonal pattern corresponded with cases of atypical hyperplasia. No other genetic alterations were detected in these samples. In conclusion, our data indicate that the detection of clonality in endometrial biopsy samples obtained by pipelle would be a useful application for the early diagnosis of endometrial cancer.

Adenocarcinoma↗
PubMed1985

Phenotypic and functional analysis of T cells cloned directly from the blood and cerebrospinal fluid of patients with multiple sclerosis.

A single-cell cloning technique was used to analyze both phenotype and function of individual T cells in patients with multiple sclerosis (MS). Blood and cerebrospinal fluid (CSF) lymphocytes were plated at 1 cell per well, stimulated with phytohemagglutinin followed by interleukin 2, and expanded to 3 X 10(6) cells per "clone." More than 90% of the T8 clones generated from patients with MS and controls in both blood and CSF were cytotoxic precursors. There was also a slight decrease in cytotoxic T4 clones in the blood of patients with MS. The cytotoxic precursor frequencies of T cells in the CSF generally reflected those in the blood. In separate experiments, antigen reactivity was examined in lines established from blood or CSF. No reactivity to myelin basic protein or white matter was found in patients with MS or controls. Myelin basic protein-reactive clones could, however, be generated after first stimulating lymphocytes with antigen before cloning. These results suggest that changes in the T8 population from the blood of patients with MS involve cytotoxic as well as suppressor cells. Sequestration of myelin basic protein- or white matter-reactive T cells was not seen in the CSF of patients with MS, unlike reports of viral meningoencephalitis, in which large numbers of antigen-specific cells were found in the CSF. Direct single-cell clonal analysis of the CSF should provide a more sophisticated approach to the study of T cell abnormalities in patients with MS.

Brain↗
PubMed1999

Cells with TP53 mutations in low grade astrocytic tumors evolve clonally to malignancy and are an unfavorable prognostic factor.

It is important to understand how low grade tumors recur and progress to malignant lesions since this dramatically shortens patient survival. Here, we evaluated the concept that malignant progression and poor prognosis of low grade astrocytic tumors are TP53 dependent through clonal expansion of mutated cells. TP53 status was established in primary and recurrent tumors from 36 patients with WHO grade II astrocytic tumors and two tumor types were found. Tumors from 14 patients (39%; type 1) had TP53 mutated cells, and 92% of these recurred with 57% progressing to malignancy. The evolution of TP53 mutated cells before and after progression was examined using a clonal analysis procedure in yeast. Malignant progression was accompanied by an increased percentage of mutant TP53 (red) yeast colonies resulting from monoclonal expansion of cells with mutated TP53. The presence of TP53 mutations in WHO grade II astrocytic tumors was associated with malignant progression (P=0. 034, chi2 test) and shorter progression-free survival (PFS; 47.6+/-9. 6 months for TP53-mutated tumors vs 67.8+/-8.2 months for TP53-wild type tumors, P<0.05, log-rank test). Tumors from 22 patients (61%; type 2) were without TP53 mutations, and 64% of these recurred without a change in TP53 status, although 41% progressed to malignancy. This suggests that TP53 mutation is not an initiating or progression event in the majority of low grade astrocytic tumors. Our study also indicates that irradiation for WHO grade II astrocytic tumors might be associated with poor outcome (P<0.0001) and this was independent of TP53 status. These findings have important implications in the clinical management of patients with low grade astocytoma and provide new support to the clonal evolution model for tumor progression.

Adolescent↗
PubMed1982

Multiple cytolytic T-cell clones with distinct cross-reactivity patterns coexist in anti-self + hapten cell lines.

C57BL/10 T cells sensitized with TNBS-treated syngeneic cells and maintained in culture by repeated stimulations exhibit high cytolytic activity toward syngeneic TNBS-treated target cells with marked cross-reactivity on TNBS-treated target cells from mice of independent H-2 haplotypes (ten tested). The analysis of the reactivities of 48 T-cell clones derived by limiting dilution from such T-cell populations revealed three types of cytotoxic T-cell clones: (1) clones restricted by H-2Kb + TNP without cross-reaction on TNBS-treated or untreated target cells of other tested mouse haplotypes; (2) clones that lysed H-2b + TNP and also TNBS-treated target cells from not more than one, two or three different H-2 haplotypes; (3) clones that lysed untreated H-2k target cells. No T-cell clone was found to exhibit the wide pattern of cross-reactivity on any TNBS-treated mouse cell, characteristic of the original T-cell populations, indicating that these were composed of individual T-cell clones specific for TNP + private or for TNP + distinct "public" H-2 determinants. Correlation with described serological public H-2 specificities was possible for some cytotoxic T-cell clone reactivity, but not for others. The general pattern of T-cell reactivity as revealed by clonal analysis in this study, as well as in published work, includes cross-reactions between self H-2a + X and allogeneic H-2n + X, between self H-2a + X and unmodified allogeneic H-2n, or between allogeneic H-2n and allogeneic H-2m + X, and is consistent with the hypothesis that MHC-class restriction is the main rule in T-cell recognition.

Animals↗
PubMedDate not supplied

The cortical ancestry of oligodendrocytes: common principles and novel features.

Studies on the development of cortical oligodendrocytes indicate that although general principles that apply to other parts of the CNS are applicable, there are important differences that appear to be critical to the analysis of this lineage in the cortex. Herein, we review previous studies demonstrating that oligodendrocyte-type-2 astrocyte progenitor cells (or oligodendrocyte precursor cells; aka O-2A/OPCs) of the developing postnatal cortex exhibit a striking cell-intrinsic bias towards undergoing prolonged self-renewal in the relative absence of oligodendrocyte generation [Power et al., Dev Biol 2002;245:362-375]. This phenotype is quite distinct from that observed in comparable cells isolated from the optic tract. This predilection for self-renewal is associated with a lessened response to inducers of oligodendrocyte generation and of possible mechanistic importance in regards to these other properties. We also review studies on stem/progenitor cells isolated from the embryonic cortex that are able to generate oligodendrocytes. As for the studies on O-2A/OPCs, important differences also distinguish these early cells from those studied in other CNS regions in their response to signaling molecules and expression of the Dlx family of transcriptional regulators [He et al., J Neurosci 2001;21:8854-8862; Yung et al., Proc Natl Acad Sci USA 2002;99:16273-16278]. We also present new data on clonal analysis of A2B5+ precursor cells isolated from the E13.5 cortex, demonstrating that this tissue appears to contain a cell similar in properties to the tripotential glial-restricted precursor cell that has been isolated from embryonic spinal cord [Rao et al., Proc Natl Acad Sci USA 1998;95:3996-4001]. Moreover, the A2B5+ precursor cells isolated from embryonic cortex are much more heterogeneous than is seen in the spinal cord at this age, even to the point of including an A2B5/PSA-NCAM double-positive cell that can generate neurons.

Animals↗
PubMed2026

Evidence for the prognostic value of TP53 mutations in circulating tumor DNA across solid malignancies: a systematic review and meta-analysis.

BACKGROUND: The purpose of this meta-analysis study is to provide evidence for the clinical utility of TP53 mutations in circulating tumor DNA (ctDNA) as a prognostic biomarker. METHODS: We searched the PubMed, Embase, Cochrane, and Web of Science databases (last update May 2025) for studies on TP53 mutations in ctDNA or cfDNA as prognosis overall survival and in solid tumors. A total of 21 studies that met the criteria were utilized and data was collected regarding the authors, year of publication, study design, site of the study, number of patients, detection, mutation sample size and outcome measures were collected. The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of the study, and meta-analysis was done by using STATA 16.0. Effect sizes were in the form of hazard ratios (HR) that had 95% confidence intervals (CI). The models used were fixed-effects and random-effects based on heterogeneity. Funnel plots, and Egger's test was used to measure publication bias, and sensitivity analysis conducted through a leave-one-out method. RESULTS: A total of 21 studies (2,685 TP53-mutated patients, one unreported) showed: Mutated patients had worse progression-free survival (PFS) (HR=2.10, p=0.000; 12 studies, heterogeneity resolved after excluding Yoshida 2023), shorter OS (HR=1.74, p=0.014; 9 studies), and reduced DFS (HR=1.73, p=0.007; 3 studies), but RFS (2 items) showed no statistically significant differences. Subgroup analyses revealed: Prospective studies showed stronger PFS (HR=2.14 vs retrospective 1.90) with Japanese subgroup HR=4.90; Lung/liver cancers had higher HRs than breast. Prospective OS HR=2.25 (lung 3.14, endometrial 0.75). Retrospective DFS HR=1.89 vs Japanese breast RFS HR=4.00. Heterogeneity originated from study design, region, and cancer type variations, with no significant publication bias (Egger's test p>0.05). CONCLUSION: Current evidence suggests that TP53 mutations detected in ctDNA are significantly associated with poor prognosis in various solid tumors, particularly lung cancer. The association is robust for PFS and OS, though high heterogeneity and biological complexity warrant cautious interpretation. These findings support the potential incorporation of ctDNA-based TP53 mutation status into clinical prognostic assessment systems as an adjunctive parameter; however, further standardization of detection protocols, functional annotation of mutation types (e.g., LOF vs. GOF), incorporation of VAF and clonality analysis, and validation in large prospective multicenter cohorts are needed before routine clinical implementation. PROSPERO REGISTRATION NUMBER: CRD420251021095.

Humans↗
PubMed1995

Characterization of simple and complex hepatitis C virus quasispecies by heteroduplex gel shift analysis: correlation with nucleotide sequencing.

In infected humans, hepatitis C virus (HCV) exists as a quasispecies typically characterized by multiple nucleotide substitutions within the second envelope gene hypervariable region 1 (HVR1). In the current study, we used heteroduplex gel shift analysis (GSA) of HVR1 sequences amplified directly from patients' sera to define two patterns of HCV quasispecies: (i) simple quasispecies, which gave a mostly homogeneous gel shift profile with a single predominant band and (ii) complex quasispecies, which gave a gel shift profile with multiple bands. Recombinant HVR1 libraries were generated from two patients with complex HCV quasispecies (cases 1 and 2) and two patients with simple HCV quasispecies (cases 3 and 4), and 129 individual clones were analysed by either GSA, nucleotide sequencing or both techniques. In case 1 we identified a highly complex HCV quasispecies with 11 distinct HVR1 variants differing by 1-51 nucleotide changes. We found a general but not absolute correlation between GSA pattern and the number or position of nucleotide changes within HVR1. In case 2, the complex HCV quasispecies consisted of three distinct major variants; GSA of individual HVR1 clones allowed us to reconstruct the complex quasispecies pattern in vitro. In case 3, the simple quasispecies comprised 66% homogeneous clones and 33% unique minor variants differing by 1-3 nucleotides from the consensus sequence. In case 4, the simple quasispecies was 84% homogeneous, but six unique major shift variants were identified among 31 clones by GSA. In summary, HCV quasispecies can be characterized based on GSA profiles following direct PCR amplification of HVR1 sequences from patients' serum; the GSA profiles approximate the clonal population of HCV as determined by clonal analysis. GSA of HVR1 clones showed a strong correlation with nucleotide sequencing.

Base Sequence↗
PubMed1997

Cell size control and a cell-intrinsic maturation program in proliferating oligodendrocyte precursor cells.

We have used clonal analysis and time-lapse video recording to study the proliferative behavior of purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve growing in serum-free cultures. First, we show that the cell cycle time of precursor cells decreases with increasing concentrations of PDGF, the main mitogen for these cells, suggesting that PDGF levels may regulate the cell cycle time during development. Second, we show that precursor cells isolated from embryonic day 18 (E18) nerves differ from precursor cells isolated from postnatal day 7 (P7) or P14 nerves in a number of ways: they have a simpler morphology, and they divide faster and longer before they stop dividing and differentiate into postmitotic oligodendrocytes. Third, we show that purified E18 precursor cells proliferating in culture progressively change their properties to resemble postnatal cells, suggesting that progressive maturation is an intrinsic property of the precursors. Finally, we show that precursor cells, especially mature ones, sometimes divide unequally, such that one daughter cell is larger than the other; in each of these cases the larger daughter cell divides well before the smaller one, suggesting that the precursor cells, just like single-celled eucaryotes, have to reach a threshold size before they can divide. These and other findings raise the possibility that such stochastic unequal divisions, rather than the stochastic events occurring in G1 proposed by "transition probability" models, may explain the random variability of cell cycle times seen within clonal cell lines in culture.

Age Factors↗
PubMed2001

Combined study of prostatic carcinoma by classical cytogenetic analysis and comparative genomic hybridization.

Conventional cytogenetic analysis of prostatic carcinoma (PC) is characterized by inefficient growth of tumor cells during in vitro culture, leading to a lack of aberrant karyotypes in many of investigated tumors. In this study we have combined a modified short-term tissue culture method for conventional banding analysis and comparative genomic hybridization (CGH) to examine genetic changes in PC, and to evaluate the effect of the in vitro culture on chromosomal changes by comparing results of the two methods. Cytogenetic analysis was performed on 34 PCs using both, conventional and molecular methods. Tumor tissues were obtained predominantly from untreated primary tumors from 48 patients. For karyotyping all tumor samples were short-term cultured using a feeder layer technique. Additionally DNA from uncultured tumor material from 17 of those patients was isolated and screened for copy number changes using CGH. Conventional banding analysis: clonal aberrations were detected in 65% of the tumor samples. Most of the chromosomal findings were numerical changes, including loss of chromosomes Y (32%), 18, 19 and 21 (each 12%). Less frequent, trisomy of chromosome 7 and monosomy of chromosomes 9, 12 and 22 (each 9%) was found. Additionally an inversion of chromosome 9p and a deletion at chromosome 7q was found in two cases. In 35% no clonal aberrations could be detected. CGH: DNA copy number changes were detected in 65% of the analyzed tumors. Predominantly losses of DNA sequences were found. The most common losses were found at chromosome regions 13q21q33 (29%), 6q11q23 (24%), 16q, and 18 (each 18%), and the most common gains at 19 (18%). In six tumors no copy number changes were found. Both methods showed a similar aneuploidy rate, suggesting that the feeder layer technique is quite a suitable method for in vitro culture of PC cells. However, the two techniques produced substantially differing results for most of the tumor samples, and in some cases the discrepancies are quite striking. Therefore eventual culture effects need to be taken into account when comparing results from conventional cytogenetics and CGH. Some contrary findings from the two methods are discussed.

Aged↗