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PubMed1993

Clonal analysis of the atopic immune response to the group 2 allergen of Dermatophagoides spp.: identification of HLA-DR and -DQ restricted T cell epitopes.

The group 2 allergens of Dermatophagoides spp. (house dust mite, HDM) are a major immunological target for IgE antibodies in the allergic immune response of HDM atopic individuals. In this report the heterogeneity of the T cell repertoire reactive with the group 2 allergen of Dermatophagoides pteronyssinus (Der p 2) of a HDM allergic individual was investigated using overlapping synthetic peptides. By clonal analysis four distinct T cell epitopes were identified, located within residues 16-31, 22-40, 82-100, and 111-129. The importance of these epitopes was confirmed by investigation of the peripheral T cell repertoire, with the polyclonal T cell response to Der p 2 failing to show marked variations in epitope specificity over time. Serological inhibition studies and the use of Epstein-Barr virus transformed B cell lines characterized for their expression of HLA-D region gene products demonstrated that recognition of peptides 16-31 and 111-129 was restricted by HLA-DQ (DQB1*0301), whereas peptide 82-100 is recognized in association with HLA-DR (DRB1*1101). Peptide 22-40 was presented by both HLA-DRB1*1101 and -DQB1*0301 class II molecules. The potential application of these findings lies in the design of peptide-based immunotherapeutics for the management of HDM allergic disease.

Amino Acid Sequence↗

PubMed2006

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response.

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Adolescent↗

PubMed1995

Clonal analysis of the effect of TGF-beta on the apoptosis-inducing activity of normal cells.

We have recently described induction of apoptosis in transformed fibroblasts by transforming growth factor type beta (TGF-beta)-treated normal fibroblasts, which leads to the specific elimination of transformed cells. Here we investigate whether the ability to eliminate transformed cells is the property of a specialized subpopulation of normal fibroblasts or whether all cells within the population are able to respond to exogenous TGF-beta by induction of elimination of transformed cells. Clonal analysis of the eliminative capacity of normal fibroblasts showed that all cells are able to induce elimination after addition of optimal concentrations of exogenous TGF-beta. In the absence of exogenously added TGF-beta, a minority of clones exhibited complete eliminative activity. Neither the ability nor the inability to perform elimination in the absence of exogenous TGF-beta was a stable characteristic of the respective cell clones. The number of cell clones with the ability to respond to suboptimal concentrations of TGF-beta increased with the passage number of the normal cells, whereas the number of clones inducing apoptosis in the absence of exogenous TGF-beta remained constant.

3T3 Cells↗

PubMed1979

The clonal analysis of cytotoxic lymphocytes against 2,4,6-trinitrophenyl (TNP)-modified cells.

The frequency and specificity of clones of cytotoxic lymphocytes (CL) produced when (CBA X C57BL)F1 spleen cell populations were cultured with 2,4,6-trinitrophenyl (TNP)-modified syngeneic F1 cells, was examined. The frequency of clones which lysed F1-TNP targets was 1/3.3 X 10(4) spleen cells, and the frequency of clones which lysed the modified parental cells, CBA-TNP and C57BL-TNP was 1/6.7 X 10(4) and 1/2.9 X 10(5) spleen cells, respectively. Using a clonal analysis of the specificity of the CL, it was shown that the majority of the clones of CL which lysed the two modified parental tarets, were specific for one or the other of the targets. Activity against modified allogeneic DBA/2-TNP targets was also detected. The activity against DBA/2-TNP targets was due mainly to clones of CL which were specific for DBA/2-TNP targets. Only a minor part of the activity was due to clones which were cross-reactive for both F1-TNP and DBA/2-TNP target cells.

Animals↗

PubMed1991

Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.

The fate of cells in the epiblast at prestreak and early primitive streak stages has been studied by injecting horseradish peroxidase (HRP) into single cells in situ of 6.7-day mouse embryos and identifying the labelled descendants at midstreak to neural plate stages after one day of culture. Ectoderm was composed of descendants of epiblast progenitors that had been located in the embryonic axis anterior to the primitive streak. Embryonic mesoderm was derived from all areas of the epiblast except the distal tip and the adjacent region anterior to it: the most anterior mesoderm cells originated posteriorly, traversing the primitive streak early; labelled cells in the posterior part of the streak at the neural plate stage were derived from extreme anterior axial and paraxial epiblast progenitors; head process cells were derived from epiblast at or near the anterior end of the primitive streak. Endoderm descendants were most frequently derived from a region that included, but extended beyond, the region producing the head process: descendants of epiblast were present in endoderm by the midstreak stage, as well as at later stages. Yolk sac and amnion mesoderm developed from posterolateral and posterior epiblast. The resulting fate map is essentially the same as those of the chick and urodele and indicates that, despite geometrical differences, topological fate relationships are conserved among these vertebrates. Clonal descendants were not necessarily confined to a single germ layer or to extraembryonic mesoderm, indicating that these lineages are not separated at the beginning of gastrulation. The embryonic axis lengthened up to the neural plate stage by (1) elongation of the primitive streak through progressive incorporation of the expanding lateral and initially more anterior regions of epiblast and, (2) expansion of the region of epiblast immediately cranial to the anterior end of the primitive streak. The population doubling time of labelled cells was 7.5 h; a calculated 43% were in, or had completed, a 4th cell cycle, and no statistically significant regional differences in the number of descendants were found. This clonal analysis also showed that (1) growth in the epiblast was noncoherent and in most regions anisotropic and directed towards the primitive streak and (2) the midline did not act as a barrier to clonal spread, either in the epiblast in the anterior half of the axis or in the primitive streak. These results taken together with the fate map indicate that, while individual cells in the epiblast sheet behave independently with respect to their neighbours, morphogenetic movement during germ layer formation is coordinated in the population as a whole.

Animals↗

PubMed1983

Clonal analysis of a 5-azacytidine-induced specific increase in growth hormone production by rat pituitary cells.

The nucleoside analog, 5-azacytidine (5-azaCR) which causes genomic hypomethylation in many cell types, increased GH production by GH4C1 rat pituitary cells 3-5 fold. This increased GH production was independent of changes in prolactin (PRL) synthesis by the same cells; it occurred at 10-fold lower 5-azaCR concentrations than those which increased the synthesis of PRL. Elevated GH production persisted for more than 20 population doublings after removal of 5-azaCR from the culture medium, indicating that the induced change was phenotypically stable. GH production remained responsive to stimulation by cortisol in 5-azaCR-treated cells. Clonal analysis demonstrated that the increased GH production was due to a higher incidence of cells in the population which made GH at an enhanced rate.

Animals↗

PubMedDate not supplied

Methods in clonal analysis and applications.

During development, embryonic cells display a large variety of behaviors that lead to the formation of embryonic structures that are frequently transient. Simultaneously, cells progress towards a specific fate. The current challenge for embryologists is to resolve how these two distinct aspects of development co-exist. As cell behaviors (including elementary cellular operations such as motility, adhesiveness, polarization, change in shape, division and death) and their control are much less well understood than the genetic aspects of cell fate determination, there is currently much interest in the study of cell behaviors. This mainly consists of labeling groups of cells or, less frequently, single cells and observing their descendants. In this review, we describe a few techniques for labeling groups of cells and we discuss prospective and retrospective clonal analysis, in particular the LaacZ system, in detail. We examine the information generated by these approaches.

Animals↗

PubMed2005

Nestin expression and clonal analysis of islet-derived epithelial monolayers: insight into nestin-expressing cell heterogeneity and differentiation potential.

There has over the last several years been renewed interest in developing a system for generating new islets and a search for a self-renewing population in the pancreas. In particular, the neural stem cell marker nestin has been implicated as an islet precursor marker and its immunoreactivity has been localized in the islets of Langerhans. This study examines islet-derived epithelial monolayers expanded ex vivo to provide a source of nestin-expressing progenitor cells--a model that will help us understand the role of nestin-expressing cells in islet cell development. When cultured on a type I collagen gel, islets formed confluent monolayers which lacked endocrine phenotypes but were positive for cytokeratin 20 and contained an increased proportion of proliferating nestin-expressing cells, reaching a maximum of 54+/-10%. Co-expression studies demonstrated that the nestin-positive cells are heterogeneous, with some nestin-expressing cells co-localizing with the transcription factor PDX-1 and glucose transporter type 2 or lack of co-expression with vimentin. When clonal populations of nestin-positive cells were expanded and subjected to a differentiation protocol, only a population that expressed the transcription factor PDX-1 at the mRNA level was capable of re-expressing insulin at the mRNA and protein level. In conclusion, these studies demonstrate that expanded nestin-expressing cells in vitro from islet-derived epithelial monolayers are heterogeneous; clonal analysis of nestin-positive cells reveals that a distinct subpopulation of nestin/PDX-1-expressing cells is capable of forming insulin-producing cells.

Animals↗

PubMed1994

Clonal analysis of Porphyromonas gingivalis by the arbitrarily primed polymerase chain reaction.

Genetic analysis of Porphyromonas gingivalis strains may distinguish between virulent and nonvirulent strains and also may be used to trace individual strains in epidemiological studies. The present study examined the utility of the arbitrarily primed polymerase chain reaction for genotypic fingerprinting of P. gingivalis. DNA was extracted according to conventional methods. Ten-base oligonucleotide primers with arbitrary sequences were used with the polymerase chain reaction to amplify P. gingivalis genomic DNA. The amplification products were analyzed by agarose gel electrophoresis. The primer GACCGCTTGT grouped 73 P. gingivalis strains into 23 genotypes, including 16 genotypes containing a single strain each. The primer AGGGGTCTTG identified 45 different genotypes, 33 of which contained a single strain. P. gingivalis strains ATCC 33277T and 381 belonged to the same genotype. Likewise, strains W50 and W83 were of the same genetic clone. The present study indicates that the arbitrarily primed polymerase chain reaction represents a valuable and easy method for clonal analysis of P. gingivalis.

Bacterial Typing Techniques↗

PubMed1986

T cell nature of some lymphokine-activated killer (LAK) cells. Frequency analysis of LAK precursors within human T cell populations and clonal analysis of LAK effector cells.

The cell lineage of the lymphokine-activated killer (LAK) cells has been reinvestigated. Both T and non-T cells, isolated on the basis of rosette formation with sheep erythrocytes (E), generated LAK activity after 3-4 days of culture in recombinant interleukin 2 (rIL 2) in 8 different individuals tested. By applying a microculture technique which allows clonal expansion of virtually all E rosetting T cells, we further analyzed the frequency of clonogenic LAK precursors within T cell populations. Approximately 1 of 25 T cells was found to be a LAK precursor. Moreover, microcultures with LAK activity lysed both the natural killer-sensitive K562 cell line and the P815 target cells in the presence of phytohemagglutinin (PHA). Since cytolytic T lymphocytes capable of lysing P815 cells in the PHA-dependent assay were approximately 1/3, it is evident that only a minor subset of cytolytic T lymphocyte precursors can acquire LAK activity even in the presence of large amounts of IL 2. Several LAK clones obtained by limiting dilution were further expanded and analyzed for their phenotypic and functional properties. Twelve out of 14 clones analyzed expressed the T3+ T11+ phenotype whereas 2 were T3- T11+. All had maintained their original cytolytic pattern; moreover, the large majority of the T3+ clones produced IL 2 and interferon-gamma following PHA stimulation.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1992

Clonal analysis of human tumors with M27 beta, a highly informative polymorphic X chromosomal probe.

The clonality of human tumors can be studied by X inactivation/methylation analysis in female patients heterozygous for X-linked DNA polymorphisms. We present a detailed study on clonal tumor analysis with M27 beta, a highly informative probe detecting a polymorphic X chromosomal locus, DXS255. The polymorphism detected at this locus is due to variable numbers of tandem repeats. The rate of constitutional heterozygosity detected by M27 beta was 88%. Normal tissue from gastrointestinal mucosa and thyroid showed random, hence polyclonal, patterns. Nonrandom clonal X inactivation was detected in all 22 malignant neoplasms that had been shown to be clonal by other DNA markers, such as antigen receptor gene rearrangements or clonal loss of heterozygosity at 17p and other loci. 16/48 normal blood leukocyte samples (33%) showed considerably skewed X inactivation patterns. Comparison of blood leukocytes and normal tissue indicated that in a given individual, X inactivation patterns may be tissue specific. M27 beta was used to study the clonal composition of 13 benign thyroid nodules from 12 multinodular goiters with rapid recent growth, traditionally termed "adenomas." Nine of them were clonal, whereas four nodules and tissue from a case of Graves' goiter were not, indicating that some, but not all, such thyroid nodules may represent true clonal neoplasms. The M27 beta probe permits one to study the clonal composition by the X inactivation approach of a wide variety of solid tumors from most female patients. As a control, normal tissue homologous to the tumor type of interest is preferable to DNA from blood leukocytes, since the latter may show nonrandom X inactivation patterns in a fairly high proportion of cases. M27 beta may, therefore, be of limited use for the clonal analysis of neoplasms derived from hematopoietic cells.

Clone Cells↗

PubMed1999

Clonality analysis of refractory anemia with ring sideroblasts: simultaneous study of clonality and cytochemistry of bone marrow progenitors.

X chromosome inactivation and polymorphism of the human androgen receptor (HUMARA) gene has been applied for analyzing the clonality of blood cells. In the present study, the clonal relationship was investigated between peripheral blood polymorphonuclear cells (PMNCs) and marrow progenitor cells and the origin of ringed sideroblasts in patients with refractory anemia with ring sideroblasts (RARS) by polymerase chain reaction (PCR) of HUMARA gene. The X-inactivation patterns of circulating PMNCs and T lymphocytes as well as individual granulocyte colonies grown in vitro from bone marrow cells were analyzed. The development of ringed sideroblasts in erythroid colonies by iron staining and their X-inactivation pattern were also examined. All three RARS patients showed monoclonal PMNCs. In granulocyte colonies, however, two different X-inactivation patterns were observed in all patients, indicating that non-clonal progenitor cells remained in the bone marrow. All erythroid colonies consisted of ringed sideroblasts exclusively showed one pattern dominant in those of PMNCs. Our findings suggest that non-clonal progenitor cells persist in some RARS cases, that erythroid progenitors show mosaicism, and that ringed sideroblasts may be derived from an abnormal clone involved in the pathogenesis of this disease.

Aged↗

PubMed2006

X-inactivation-based clonality analysis and quantitative JAK2V617F assessment reveal a strong association between clonality and JAK2V617F in PV but not ET/MMM, and identifies a subset of JAK2V617F-negative ET and MMM patients with clonal hematopoiesis.

The JAK2V617F mutation is present in most patients with polycythemia vera (PV) and in some patients with essential thrombocythemia (ET) and myeloid metaplasia/myelofibrosis (MMM). We sought to investigate the relationship between granulocyte clonality and JAK2V617F allelic ratio. A total of 168 of 190 female patients were informative for a clonality assay at the HUMARA locus; 80% of MMM, 75% of PV, and 67% of ET patients demonstrated clonal granulopoiesis. The JAK2V617F allele was detected by quantitative real-time polymerase chain reaction (PCR) in 99% of PV, 72% of ET, and 39% of MMM. A correlation between clonality and JAK2V617F allelic ratio was demonstrated for PV (P < .001) but not for ET or MMM (both P > .6). These data suggest that acquisition of the JAK2V617F mutation may be sufficient for the development of PV, but additional genetic events are necessary in ET and MMM. In addition, some ET and MMM patients with clonal granulopoiesis have somatic mutations other than JAK2V617F.

Amino Acid Substitution↗

PubMed1987

Clonal analysis of human colorectal tumors.

The clonal composition of human colorectal tumors was studied by means of restriction fragment length polymorphisms (RFLPs). First, X-linked RFLPs were used to examine the pattern of X chromosome inactivation in colorectal tumors of females. All 50 tumors examined showed monoclonal patterns of X chromosome inactivation; these tumors included 20 carcinomas as well as 30 adenomas of either familial or spontaneous type. Second, RFLPs of autosomes were used as clonal markers to detect the somatic loss or gain of specific chromosomal sequences in colorectal tumors. Among other changes, it was found that somatic loss of chromosome 17p sequences occurred in over 75 percent of the carcinomas examined, but such loss was rare in adenomas. These data support a monoclonal origin for colorectal neoplasms, and suggest that a gene on the short arm of chromosome 17 may be associated with progression from the benign to the malignant state.

Adenoma↗

PubMed1996

Clonality analysis of B-lymphoid proliferations using the polymerase chain reaction.

BACKGROUND: Polymerase chain reaction (PCR) based assays are becoming more reliable, simpler, and faster alternatives to traditional Southern blot hybridization (SBH) analysis for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements. However, a variety of technical approaches have been reported with markedly different results. METHODS: We analyzed the frozen tissue of 147 neoplastic and hyperplastic lesions on which SBH had previously been performed. Semi-nested and single-step PCR methods were compared. Consensus primers to the joining segments and the framework region (FR) III of the variable segments of the IgH gene were used. All PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE). Thirteen samples were re-analyzed using a denaturing gradient gel electrophoresis (DGGE) system. RESULTS: The overall concordance between SBH and semi-nested PCR assays was 80.2%. In the non-Hodgkin's lymphoma (NHL) group, 75% of the cases with IgH rearrangements by SBH were found to be monoclonal by PCR. Regardless of type of lesion, 71.7% of the cases with IgH rearrangements by SBH were found to be clonal by PCR. The concordance between the semi-nested and single-step procedures was 87.1%. DGGE was helpful in clarifying the results for cases in which the PAGE analysis was difficult to interpret. CONCLUSIONS: PCR analysis of IgH gene rearrangements was found to be an efficient technique for the initial determination of clonality in lymphoid proliferations. The single-step method had an advantage over the semi-nested method because of its simplicity and speed. The DGGE system was useful for the assessment of clonality in cases with equivocal results after PAGE. However, a combination of these techniques in specific cases may achieve higher specificity and sensitivity.

B-Lymphocytes↗

PubMed1998

Clonality analysis of cervical cancer on microdissected archival materials by PCR-based X-chromosome inactivation approach.

Clonality of invasive human cervical cancer was assessed in microdissected archival formaldehyde-fixed, paraffin-embedded tissues by PCR-based analysis of X-chromosome inactivation of the androgen receptor gene. In 18 informative cases, cancers from 12 cases including 2 early-invasive cancers were scored as monoclonal. Among them, 8 cases had the combination of random X-chromosome inactivation in normal cervical tissue and non-random in the cancer and in other 4 cases normal and cancer tissue were both non-random but had discordant X-chromosome inactivation. Six cases showed that the non-random inactivation affected the same allele in cancer and normal tissue and thus were considered to be inconclusive. The results suggest the monoclonal origin of cervical cancer and indicate that genetic events are critical in transition of pre-malignant epithelia to invasive cancer in cervical carcinogenesis. Finding of monoclonal early invasive cancer also argues that monoclonality of human tumors is not a late event due to clonal competition or selection. X-chromosome inactivation patterns in normal cervical epithelia and tissues were analyzed in 21 informative cases. When a mixture of normal stroma and epithelium was analyzed, 38% (8/21) of cases showed non-random inactivation pattern, whereas using pure epithelium it was 64% (11/17). The X-chromosome inactivation pattern between mixed epithelium/stroma and matched epithelium differed in 2/8 cases. These results point to a certain amount of overlooked source of error in X-inactivation-based tumor clonality analysis in which peripheral blood monocytes were chosen as control and strongly recommend that control tissues which share close histological origin with analyzed tumors should be selected in any clonality study of human neoplasm. Post-embryological mechanisms of clonal patch in normal cervical epithelia are also discussed.

Carcinoma, Squamous Cell↗

PubMed1980

Clonal analysis of cytolytic T lymphocyte specificity. I. Phenotypically distinct sets of clones as the cellular basis of cross-reactivity to alloantigens.

The cellular basis of the cytolytic cross-reactivity observed in primary allogeneic (C56BL/6 anti-DBA/2 and C57BL/6 anti-C3H/He) mixed-leukocyte cultures (MLC) was investigated by analysis of the specificity of clonal progeny derived from individual cytolytic T lymphocyte (CTL) precursor cells (CTL-P) contained within these populations. A sensitive mixed-leukocyte microculture (micro-MLC) technique was used with limiting dilution analysis by Poisson statistics to determine the frequency of CTL-P reactive against both specific and third-party (P815 and AKRA) target cells, to calculate the probability that each micro-MLC was a clone derived from a single CTL-P, and to examine the specificity of each micro-MLC assayed separately against both target cells. A total of 287 phenotypically specific, heteroclitic, and cross-reactive micro-MLC from the 2 different strain combinations were observed with a relative frequency of 81, 11, and 8%, respectively, and were calculated to have mean clone probability of 90 and 99% when based, respectively, upon the frequencies of CTL-P reactive against the specific and third-party target cells. These clones were estimated to have an approximate size of 6 X 10(4) cells, which corresponded to roughly 16 cell doublings during the 7 d of culture. 22 clones were successfully subcloned and in virtually every case, the subclones retained the specificity phenotype of the original clone from which they were derived. These results provide direct evidence for three phenotypically distinct sets of CTL as the cellular basis of cross-reactivity in MLC populations assayed against two different target cells.

Animals↗

PubMed1997

[Clonal analysis in cells using PCR and laser microdissection].

Clonality represents one of the hallmarks of neoplastic cell growth. X-chromosomal inactivation patterns have been used to determine clonality in various tumors. This approach is limited by admixture of polyclonal non-tumor stroma cells among which the monoclonal proliferation may be missed. In order to overcome this limitation, we combined a sensitive PCR based DNA analysis with a highly selective microdissection technique using a laser beam. In sections of intraductal mammary carcinomas tumor cell complexes of at least 100 cells were isolated by removing the surrounding stroma by laser irradiation. Thereby, tumor cells could be isolated without contaminating non-neoplastic elements. Clonality in these cells was determined using two X-chromosomal polymorphic sites-phosphoglycerate kinase 1 (PGK1) and human androgen receptor (HUMARA). Control experiments could show the polyclonal nature of the surrounding tissue. Moreover, complete destruction of DNA by laser irradiation was assured. The technique requires a certain amount of cells and DNA in order to avoid artefacts that result from preferential amplification of exclusively one X-chromosomal allele in small samples. We conclude that combination of laser-microdissection with PCR analysis of X-chromosomal inactivation patterns enables the detection of clonal cell populations in heterogeneous tissues. Studies of clonality in borderline cases between reactive and neoplastic proliferations or premalignant lesions are made possible by this technique.

Breast Neoplasms↗