Age-related gene expression profiles of immature human oocytes.

STUDY QUESTION: What is the difference between the gene expression profiles of single human germinal vesicle (GV) oocytes from women of different ages? SUMMARY ANSWER: There were no statistically significant differences in gene expression profiles of human GV oocytes from women of different ages (range: 25-43). WHAT IS KNOWN ALREADY: It is well established that reproductive capacity declines as women age, which is attributed to oocyte quality since this decline is counterbalanced in older women receiving young donor oocytes. Altered gene expression of human oocytes at different stages of development in relation to female age is one of the suggested mechanisms that could explain the decrease in oocyte quality. STUDY DESIGN, SIZE, DURATION: Between 2012 and 2014, 40 human GV oocytes of 40 women were obtained during follicular aspiration as part of routine ICSI treatment. Gene expression profiles of 38 GV oocytes were determined in four different age groups: 25-30, 31-35, 36-38 and 39-43 years of age. PARTICIPANTS/MATERIALS, SETTING, METHODS: GV oocytes were donated for research and frozen between 3.5 and 7.5 h after follicular aspiration. Subsequently, GV oocytes were thawed and prepared for gene expression profile analysis using Agilent microarrays containing ~42 000 Human Gene Expression probe-sets. Gene expression profiles were visualized by hierarchical clustering and the top 500 most differing genes were determined by multidimensional scaling (MDS). Transcripts were analysed in a class comparison between the four age groups and for indicators of biological age: antral follicle count (AFC) and the total dosage of FSH needed for ovarian stimulation. Individual transcripts were analysed using linear regression. A false discovery rate <0.05 was considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: Visualization of gene expression profiles of GV oocytes with hierarchal clustering and MDS demonstrated no clear grouping of samples based on female age, AFC or FSH dosage. The gene expression profile of GV oocytes classified in four age groups revealed no significantly differentially expressed genes between the four different age groups. There were also no significantly differentially expressed genes in the linear regression analysis for individual transcripts against age. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Immature (GV) oocytes obtained from ovarian stimulation cycles were used. Findings may therefore differ for oocytes at other developmental stages and for in-vivo matured oocytes under physiological conditions. Due to our relatively large, but still limited study sample (40 GV oocytes), we cannot exclude that there might be smaller age-related gene-expression differences, i.e. due to a lack of power. WIDER IMPLICATIONS OF THE FINDINGS: We did not find an effect of female age on gene expression profiles of individual human GV oocytes. Other studies have suggested that gene-expression profiles are affected in mature oocytes, which might imply that female age affects oocyte maturation. Alternatively, other mechanisms in human oocytes might cause the age-related fertility decline. STUDY FUNDING/COMPETING INTEREST(S): This study received no external funding and there are no competing interests.

Factors affecting the gene expression of in vitro cultured human preimplantation embryos.

STUDY QUESTION: What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? SUMMARY ANSWER: Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryos than the culture medium or oxygen concentration used in in vitro culture. WHAT IS KNOWN ALREADY: Studies on mouse and bovine embryos have shown that different conditions in the in vitro culture of embryos can lead to changes in transcriptome profiles. For humans, an effect of developmental stage on the transcriptome profile of embryos has been demonstrated, but studies on the effect of maternal age or culture conditions are lacking. STUDY DESIGN, SIZE, DURATION: Donated, good quality, day 4 cryopreserved human preimplantation embryos (N = 89) were randomized to be cultured in one of two culture media (G5 medium or HTF medium) and one of two oxygen concentrations (5% or 20%), with stratification for maternal age. Next to these variables, developmental stage after culture was taken into account in the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos that developed to morula or blastocyst stage during these 2 days whose amplified mRNA passed our quality control criteria for microarray hybridization were individually examined for genome-wide gene expression (N = 37). MAIN RESULTS AND THE ROLE OF CHANCE: Based on the number of differentially expressed genes (DEGs), developmental stage (3519 DEGs) and maternal age (1258 DEGs) had a larger effect on the global gene expression profile of human preimplantation embryos than either tested culture medium (596 DEGs) or oxygen concentration (492 DEGs) used during in vitro culture. Interactions between the factors were found, indicating that culture conditions might have a different effect depending on the developmental stage or the maternal age of the embryos. Affected pathways included metabolism, cell cycle processes and oxidative phosphorylation. LIMITATIONS, REASONS FOR CAUTION: Culture of embryos for only 2 days might have limited the effect on global gene expression by the investigated culture conditions. Earlier stages of development (Day 0 until Day 4) were not analyzed and these embryos might respond differently to the experimental conditions. The freezing and thawing procedures might have had an effect on gene expression. RT-PCR validation was not performed due to scarcity of the material. WIDER IMPLICATIONS OF THE FINDINGS: Our results show that when studying gene expression in single human preimplantation embryos under various experimental conditions, one should take into account the confounding effect of biological variables, such as developmental stage and maternal age. This makes these experiments different from gene expression experiments where these variables can be tightly controlled, for example when using cell lines. STUDY FUNDING/COMPETING INTERESTS: This study received no external funding and there were no competing interests.

Purified Timothy grass pollen major allergen Phl p 1 may contribute to the modulation of allergic responses through a pleiotropic induction of cytokines and chemokines from airway epithelial cells.

By definition, allergens are proteins with the ability to elicit powerful T helper lymphocyte type 2 (Th2) responses, culminating in immunoglobulin (Ig)E antibody production. Why specific proteins cause aberrant immune responses has remained largely unanswered. Recent data suggest that there may be several molecular paths that may affect allergenicity of proteins. The focus of this study is the response of airway epithelium to a major allergen from Phleum pratense Phl p 1. Instead of focusing on a few genes and proteins that might be affected by the major allergen, our aim was to obtain a broader view on the immune stimulatory capacity of Phl p 1. We therefore performed detailed analysis on mRNA and protein level by using a microarray approach to define Phl p 1-induced gene expression. We found that this allergen induces modulation and release of a broad range of mediators, indicating it to be a powerful trigger of the immune system. We were able to show that genes belonging to the GO cluster 'cell communication' were among the most prominent functional groups, which is also reflected in cytokines and chemokines building centres in a computational model of direct gene interaction. Further detailed comparison of grass pollen extract (GPE)- and Phl p 1-induced gene expression might be beneficial with regard to the application of single components within diagnosis and immunotherapy.

Comparison of Timothy grass pollen extract- and single major allergen-induced gene expression and mediator release in airway epithelial cells: a meta-analysis.

BACKGROUND: Seasonal allergic rhinitis (AR) is a global health problem and its prevalence has increased considerably in the last decades. As the allergic response with its clinical manifestations is triggered by only a few proteins within natural extracts, there is an increasing tendency for single-component-resolved diagnosis and immunotherapy. OBJECTIVE: As natural exposure is not to single proteins, but to complex mixtures of molecules, we were interested in comparing the activation of respiratory epithelial cells induced by the purified major allergen Phl p 1 with the induction caused by a complete extract of Timothy grass pollen (GPE). METHODS: NCI-H292 cells were exposed to GPE or Ph1 p 1 for 24 h, isolated RNA and cell culture supernatants were used for microarray analysis, multiplex enzyme-linked immunosorbant assay (ELISA) and subsequent analysis. RESULTS: We found 262 genes that showed a GPE-induced change of at least 3-fold, whereas Ph1 p 1-stimulation resulted in 71 genes with a fold induction of more than 3-fold. Besides genes that were regulated by both stimuli, we also detected genes displaying an opposite response after stimulation, suggesting that GPE might be more than purified major allergens with regard to induced immune responses. CONCLUSIONS AND CLINICAL RELEVANCE: Additional components within GPE and the resulting modulation of general processes affecting gene transcription and signalling pathways might be crucial to maintain/overcome the diseased phenotype and to induce the influx of cells contributing to late-phase allergic responses. When the initial process of sensitization is the matter of interest or late-phase allergic responses, one might miss important immune modulatory molecules and their interaction with allergens by applying single components only.

Human testis-derived embryonic stem cell-like cells are not pluripotent, but possess potential of mesenchymal progenitors.

BACKGROUND: Spontaneous in vitro transition of undifferentiated spermatogonia into the pluripotent cell state has been achieved using neonatal and adult mouse testis tissue. In an effort to establish an analogous source of human patient-specific pluripotent stem cells, several research groups have described the derivation of embryonic stem cell-like cells from primary cultures of human testis. These cells are characterized in all studies as growing in compact colonies, expressing pluripotency-associated markers and possessing multilineage differentiation capabilities in vitro, but only one study claimed their ability to induce teratomas. This controversy initiated a debate about the pluripotent state and origin of human testis-derived ES-like cells (htES-like cells). METHODS: htES-like cell colonies were obtained from primary testicular cultures of three individuals and selectively expanded using culture conditions known to support the propagation of blastocyst-derived human embryonic stem cells (ESCs), mouse epiblast stem cells and 'naïve' human ESCs. The stem cell properties of htES-like cells were subsequently assessed by testing the expression of ESC-specific markers, differentiation abilities in vitro and in vivo, and microarray profiling. RESULTS: The expression of pluripotency-associated markers in htES-like cells and their differentiation abilities differed significantly from those of ESCs. Gene expression microarray analysis revealed that htES-like cells possess a transcriptome distinct from human ESCs and fibroblasts, but closely resembling the transcriptome of mesenchymal stem cells (MSCs). The similarity to MSCs was confirmed by detection of SSEA4/CD146 expressing cells within htES-like colonies and efficient in vitro differentiation toward three mesodermal lineages (adipogenic, osteogenic, chondrogenic). CONCLUSIONS: Taken together, these results indicate that htES-like cells, in contrast to pluripotent stem cells derived from adult mouse testis, are not pluripotent and most likely not of germ cell but of mesenchymal origin.

Timothy grass pollen extract-induced gene expression and signalling pathways in airway epithelial cells.

BACKGROUND: Grass pollen allergy is one of the most common allergies worldwide and airborne allergens are the major cause of allergic rhinitis. Airway epithelial cells (AECs) are the first to encounter and respond to aeroallergens and are therefore interesting targets for the development of new therapeutics. Our understanding of the epithelial contribution to immune responses is limited as most studies focus on only a few individual genes or proteins. OBJECTIVE: To describe in detail the Timothy grass pollen extract (GPE)-induced gene expression in AECs. METHODS: NCI-H292 cells were exposed to GPE for 24 h, and isolated RNA and cell culture supernatants were used for microarray analysis and multiplex ELISA, respectively. RESULTS: Eleven thousand and seven hundred fifty-eight transcripts were affected after exposure to GPE, with 141 genes up-regulated and 121 genes down-regulated by more than threefold. The gene ontology group cell communication was among the most prominent categories. Network analysis revealed that a substantial part of regulated genes are related to the cytokines IL-6, IL-8, IL-1A, and the transcription factor FOS. After analysing significantly regulated signalling pathways, we found, among others, epidermal growth factor receptor 1, IL-1, Notch-, and Wnt-related signalling members. Unexpectedly, we found Jagged to be down-regulated and an increased release of IL-12, in line with a more Th1-biased response induced by GPE. CONCLUSION AND CLINICAL RELEVANCE: Our data show that the stimulation of AECs with GPE results in the induction of a broad response on RNA and protein level by which they are able to affect the initiation and regulation of local immune responses. Detailed understanding of GPE-induced genes and signalling pathways will allow us to better define the pathogenesis of the allergic response and to identify new targets for treatment.

Site-specific deletions involving the tal-1 and sil genes are restricted to cells of the T cell receptor alpha/beta lineage: T cell receptor delta gene deletion mechanism affects multiple genes.

Site-specific deletions in the tal-1 gene are reported to occur in 12-26% of T cell acute lymphoblastic leukemias (T-ALL). So far two main types of tal-1 deletions have been described. Upon analysis of 134 T-ALL we have found two new types of tal-1 deletions. These four types of deletions juxtapose the 5' part of the tal-1 gene to the sil gene promoter, thereby deleting all coding sil exons but leaving the coding tal-1 exons undamaged. The recombination signal sequences (RSS) and fusion regions of the tal-1 deletion breakpoints strongly resemble the RSS and junctional regions of immunoglobulin/T cell receptor (TCR) gene rearrangements, which implies that they are probably caused by the same V(D)J recombinase complex. Analysis of the 134 T-ALL suggested that the occurrence of tal-1 deletions is associated with the CD3 phenotype, because no tal-1 deletions were found in 25 TCR-gamma/delta + T-ALL, whereas 8 of the 69 CD3- T-ALL and 11 of the 40 TCR-alpha/beta + T-ALL contained such a deletion. Careful examination of all TCR genes revealed that tal-1 deletions exclusively occurred in CD3- or CD3+ T-ALL of the alpha/beta lineage with a frequency of 18% in T-ALL with one deleted TCR-delta allele, and a frequency of 34% in T-ALL with TCR-delta gene deletions on both alleles. Therefore, we conclude that alpha/beta lineage commitment of the T-ALL and especially the extent of TCR-delta gene deletions determines the chance of a tal-1 deletion. This suggests that tal-1 deletions are mediated via the same deletion mechanism as TCR-delta gene deletions.

Comparison of expression profiles induced by dust mite in airway epithelia reveals a common pathway.

BACKGROUND: Airway epithelial cells have shown to be active participants in the defense against pathogens by producing signaling and other regulatory molecules in response to the encounter. METHODS: In previous manuscripts, we have studied the effect of house dust mite (HDM) extract on both an epithelial cell-line (H292) and primary nasal epithelial cell. When we compare these responses we conclude that the H292 cells more closely resemble nasal epithelium of healthy controls (share 107 probe-sets) than of allergic individuals (share 17 probe-sets). RESULTS: Interestingly, probably because of an absent intraindividual variation between samples, more probe-sets (8280) change expression significantly in H292 than in either healthy (555) or allergic (401) epithelium. CONCLUSIONS: A direct comparison of all the responses in these epithelial cells reveals a core-response to HDM of just 29 genes. These genes (CCL20, IL-8, CXCL2, CXCL1, IL-1B, AREG, TNFAIP3, HBEGF, PTGS2, BMP2, LDLR, PLAUR, PLAU, NFKB2, NFKB1, JUN, ATF3, EGR1, NPC1, TICAM1, EPHA2, CTGF, DUSP1, SPRY1, TLR-3, complement factor C3, IVNS1ABP, SerpinB3, and PSAT1) have described links with allergy or inflammation and may even describe the well-established relationship between viral infections and allergic exacerbations or allergy development.

Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm.

INTRODUCTION: Our previous studies showed that Streptococcus mutans and Veillonella parvula dual-species biofilms have a different acid production profile and a higher resistance to chlorhexidine than their single-species counterparts. The aim of the current study was to test whether the susceptibility of S. mutans grown in the presence of V. parvula is also decreased when it is exposed to various other antimicrobials. Furthermore, the aim was to identify other changes in the physiology of S. mutans when V. parvula was present using transcriptomics. METHODS: Susceptibility to antimicrobials was assessed in killing experiments. Transcript levels in S. mutans were measured with the help of S. mutans microarrays. RESULTS: When V. parvula was present, S. mutans showed an increase in survival after exposure to various antimicrobials. Furthermore, this co-existence altered the physiology of S. mutans. The expression of genes coding for proteins involved in amino acid synthesis, the signal recognition particle-translocation pathway, purine metabolism, intracellular polysaccharide synthesis, and protein synthesis all changed. CONCLUSION: Growing in a biofilm together with a non-pathogenic bacterium like V. parvula changes the physiology of S. mutans, and gives this bacterium an advantage in surviving antimicrobial treatment. Thus, the study of pathogens implicated in polymicrobial diseases, such as caries and periodontitis, should be focused more on multispecies biofilms. In addition, the testing of susceptibility to currently used and new antimicrobials should be performed on a multispecies microbial community rather than with single pathogens.

Characterization of ABF-1, a novel basic helix-loop-helix transcription factor expressed in activated B lymphocytes.

Proteins of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including neurogenesis, lymphopoiesis, myogenesis, and sex determination. Using a yeast two-hybrid screen, we have identified a new bHLH transcription factor, ABF-1, from a human B-cell cDNA library. Within the bHLH region, ABF-1 shows a remarkable conservation with other HLH proteins, including tal-1, NeuroD, and paraxis. Its expression pattern is restricted to a subset of lymphoid tissues, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, and activated human B cells. ABF-1 is capable of binding an E-box element either as a homodimer or as a heterodimer with E2A. Furthermore, a heterodimeric complex containing ABF-1 and E2A can be detected in EBV-immortalized lymphoblastoid cell lines. ABF-1 contains a transcriptional repression domain and is capable of inhibiting the transactivation capability of E47 in mammalian cells. ABF-1 represents the first example of a B-cell-restricted bHLH protein, and its expression pattern suggests that ABF-1 may play a role in regulating antigen-dependent B-cell differentiation.

Cloning and functional characterization of the early-lymphocyte-specific Pb99 gene.

The Pb99 gene is specifically expressed in pre-B cells and thymocytes and not in mature B and T cells or nonlymphoid tissues, implying that it may function in early lymphoid development. We have previously described the cloning of an incomplete cDNA for Pb99. Here we report the isolation of full-length cDNAs and genomic clones for the murine Pb99 gene and the mapping of its location to mouse chromosome 8. Sequence analyses of different Pb99 cDNA clones suggest that there may be at least three forms of the Pb99 protein generated by differential processing of the Pb99 transcript. The cDNA with the longest open reading frame encodes a putative protein that has seven hydrophobic domains similar to those of seven membrane-spanning proteins, such as the classical G protein-coupled receptors. To directly address the role of the Pb99 protein in lymphoid development, Pb99-deficient mice were generated by gene targeting, and lymphocyte development in these mice was analyzed.

Lineage specific demethylation of tal-1 gene breakpoint region determines the frequency of tal-1 deletions in alpha beta lineage T-cells.

tal-1 deletions are caused by a site specific recombination, which exclusively occurs in 12-26% of T-cell acute lymphoblastic leukemias (T-ALL). In a previous study on a large series of T-ALL we demonstrated an apparent preferential occurrence of tal-1 deletions in CD3- and CD3+ alpha beta lineage T-ALL with TcR-delta gene deletions on one or both alleles. In the present study we investigated whether accessibility of the tal-1 deletion breakpoint regions influences the preferential occurrence in specific T-ALL subgroups. Because DNA methylation is assumed to determine accessibility of DNA for recombination, the methylation status of the tal-1 deletion type 1 breakpoint regions (sildb and taldb1) was studied. Although the sildb were completely demethylated in all T-ALL, preferential (de)methylation configurations of the taldb1 were observed in the analysed 119 T-ALL. Most TcR-alpha beta + T-ALL contained completely demethylated taldb1 (77%), whereas in most TcR-gamma delta + T-ALL partial or complete methylation occurred (42% and 47%, respectively). In T-ALL subgroups defined by different TcR-delta gene configurations also preferential taldb1 (de)methylation patterns were seen, which was most prominent in T-ALL with both TcR-delta genes deleted (84% complete demethylation). The previously observed preferential occurrence of tal-1 deletion type 1 in TcR-alpha beta + vs CD3- T-ALL and in T-ALL with both vs one TcR-delta genes deleted, disappeared when we retricted to T-ALL with completely demethylated taldb1. Moreover, all T-ALL with a tal-1 deletion type 1 (n = 15) contained completely demethylated taldb1. We therefore conclude that complete demethylation of taldb1 is a prerequisite for tal-1 deletions type 1 and that the differences in tal-1 deletion frequencies observed in the various T-ALL subgroups are caused by differences in the (de)methylation status of taldb1 in these subgroups.

Detection of minimal residual disease in acute leukemia by immunological marker analysis and polymerase chain reaction.

Detection of minimal residual disease (MRD) can be useful for adaptation or stratification of treatment in acute leukemia patients and may finally result in individualization of treatment protocols. Although leukemic cells generally have immunophenotypes comparable to their normal counterparts, it is possible to use immunological marker analysis for the detection of MRD based on the assumption that the presence of positive cells outside their normal breeding sites and 'homing areas' is indicative of malignancy. This approach can be used for the detection of MRD in blood and bone marrow of patients with a terminal deoxynucleotidyl transferase (TdT) positive T-cell acute lymphoblastic leukemia (ALL) and patients with a TdT+ acute myeloid leukemia (AML) as well as in cerebrospinal fluid of patients with a TdT+ leukemia. In other types of acute leukemias, immunological marker analysis generally does not allow detection of low frequencies of malignant cells, but in a part of them the polymerase chain reaction (PCR) technique may be valuable. The PCR technique allows the amplification of tumor-specific DNA sequences or mRNA sequences (after reverse transcription into cDNA), if the flanking sequences are well-defined. This PCR-mediated amplification can detect specific sequences which are derived from only a few malignant cells between many normal cells. Well-defined chromosome translocations have been used as tumor-specific markers, such as t(9;22). An advantage of using specific chromosome aberrations as tumor-specific markers is their stability during the disease course. However, only 10-15% of ALL and 25-30% of AML have a specific chromosome translocation and in a large part of them the precise breakpoints are not (yet) known. Recent studies indicate that it is possible to detect MRD in acute leukemias by use of PCR-mediated amplification of the junctional regions of rearranged immunoglobulin (Ig) and T-cell receptor (TcR) genes, using variable (V) and joining (J) gene-specific oligonucleotides as primers. Major pitfalls of this application are the occurrence of multiple rearrangements at diagnosis (oligoclonality) and changes in rearrangement patterns at relapse (clonal evolution), which will lead to false negative results of this MRD-PCR technique. In conclusion, the technique of choice for the detection of MRD is dependent on the immunophenotype of the leukemia, the presence of a well-defined chromosome translocation and the presence of a rearranged Ig and/or TcR gene as well as the chance of immunophenotypic shifts and changes in Ig and TcR gene rearrangement patterns.(ABSTRACT TRUNCATED AT 400 WORDS)

tal-1 deletions in T-cell acute lymphoblastic leukemia as PCR target for detection of minimal residual disease.

Polymerase chain reaction (PCR) techniques based on amplification and identification of leukemia-specific DNA sequences provide a sensitive diagnostic method for detection of minimal residual disease (MRD) with a detection limit of 10(-5) to 10(-6) (1-10 malignant cells in 10(6) normal cells). To date, the main leukemia-specific DNA sequences used as PCR targets in detection of MRD are breakpoint fusion regions of chromosome translocations and junctional regions of rearranged immunoglobulin (Ig) or T-cell receptor (TcR) genes. The recently identified tal-1 deletions involving the sil and tal-1 genes, provide a potential MRD-PCR target. tal-1 deletions are site-specific because they are mediated via recombination signal sequences homologous to Ig/TcR genes. In line with this homology, tal-1 deletions also show random insertion and deletion of nucleotides at their breakpoints, resulting in highly variable breakpoint fusion regions. The fusion region diversity can be applied to design patient-specific oligonucleotide probes. Our Southern blot analyses of a large series of 313 acute leukemias with a specific tal-1 deletion probe (SILDB) demonstrated that tal-1 deletions exclusively occur in T-cell acute lymphoblastic leukemia (T-ALL) and not in precursor B-ALL or acute non-lymphocytic leukemias. In addition, we did not detect tal-1 deletions in normal blood cells and normal thymocytes by PCR analysis. The diversity observed in tal-1 deletion fusion regions with an average insertion and deletion of approximately 7 and approximately 6 nucleotides, respectively, allowed us to design fusion-region-specific probes. The specificity of the fusion-region probes was proven and the detection limit of the MRD-PCR technique was tested in a series of dilution experiments. The observed detection limit of 10(-5) indicates that tal-1 deletions in T-ALL represent ideal leukemia-specific PCR targets for detection of MRD.

Polyclonal expansion of T-cell receptor-gamma delta+ T lymphocytes associated with neutropenia and thrombocytopenia.

A patient with large granular lymphocyte (LGL) expansion (T-gamma lymphocytosis), neutropenia and thrombocytopenia was studied longitudinally. Analysis of peripheral blood mononuclear cells (PBMC) demonstrated an unusual large proportion of CD3+ T-lymphocytes expressing a gamma delta T-cell receptor (TcR-gamma delta). Immunofluorescence (IF) stainings with subset-specific monoclonal antibodies showed a fluctuating expansion of TcR-gamma delta+ T-cells expressing V gamma 9 and V delta 2 variable (V) gene segments. Biochemical characterization of PBMC showed the presence of a disulphide-linked TcR-gamma delta. TcR gene rearrangement studies on sorted TcR-gamma delta+ T-cells showed rearrangements of V gamma 9-J gamma 1.2 and V delta 2-J delta 1 V and joining (J) gene segments, thereby confirming the IF staining results. These data alone did not allow us to determine whether the TcR-gamma delta+ LGL expansion represented a polyclonal or monoclonal proliferation, because the combinatorial repertoire of TcR-gamma delta receptors is limited due to the availability of only a few V and J segments within the TcR-gamma and TcR-delta genes and because of the preferential usage of V gamma 9-J gamma 1.2 and V delta 2-J delta 1 rearrangements by TcR-gamma delta+ T-cells in blood of healthy individuals. We therefore used polymerase chain reaction (PCR)-mediated amplification of the TcR-gamma delta rearrangements, using specific V gamma 9, J gamma 1.2, V delta 2, and J delta 1 oligonucleotides to determine the junctional diversity of the TcR-gamma delta+ T-cell population. Sequence analysis of the PCR products obtained revealed a mixture of different junctional region sequences compatible with a polyclonal expansion. This is in contrast to the few reported TcR-gamma delta+ LGL and the majority of TcR-alpha beta+ LGL expansions, which appeared to consist of monoclonal proliferations.

Detection of immunoglobulin heavy-chain gene rearrangements by Southern blot analysis: recommendations for optimal results.

Southern blot analysis of immunoglobulin (Ig) and T-cell receptor genes has proven to be important for detection of clonal rearrangements in patients with lymphoproliferative diseases. To improve the detection of clonal Ig heavy-chain (IgH) gene rearrangements, we carefully determined the precise restriction map of the joining (J)H and constant (C)mu region of the IgH locus, and evaluated relevant combinations of restriction enzymes with JH and C mu probes. Our extensive Southern blot analyses revealed that rearrangements in the JH region are optimally detectable by use of a JH probe in combination with at least two restriction enzyme digests which are not affected by polymorphisms, and which produce small germline bands (e.g. BglII and BamHI/HindIII), thereby reducing the chance of comigration of germline and/or rearranged bands. Application of a JH or a C mu probe in combination with BamHI or EcoRI digests should be avoided, because of the large size of the restriction fragments and the occurrence of polymorphisms. Comparison of different types of JH probes demonstrated that optimally reproducible signals, independent of the rearranged JH gene segment, are only obtained if the JH probe is complementary to the 3' flanking sequences of the JH gene region, such as our IGHJ6 probe.

Limited combinatorial repertoire of gamma delta T-cell receptors expressed by T-cell acute lymphoblastic leukemias.

Detailed analysis of the rearrangement and expression of T-cell receptor gamma (TcR-gamma) and TcR-delta genes was performed in ten TcR-gamma delta+ T-cell acute lymphoblastic leukemias (T-ALL). In nine T-ALL the TcR-gamma genes were rearranged on both alleles, whereas in the tenth leukemia one allele was rearranged and the other was in germline configuration. Twelve out of the 19 rearranged alleles contained rearrangements of the J gamma 2.3 gene segment, five of which were to the V gamma 8 gene segment and three to the V gamma 3 gene segment. This implies that the combinatorial repertoire of the rearranged TcR-gamma gene is restricted due to the preferential usage of several V gamma and J gamma gene segments. The TcR-delta genes were rearranged on both alleles in nine T-ALL, whereas in the tenth leukemia one allele was rearranged and the other deleted. The combinatorial repertoire of the TcR-delta genes was homogeneous, as in all ten T-ALL at least one allele contained a V delta 1-J delta 1 rearrangement. In at least nine of the ten T-ALL the V delta 1-J delta 1 allele coded for the expressed TcR-delta chain, as was supported by reactivity with the anti-V delta 1-J delta 1 (delta TCS1) antibody in all T-ALL tested. As the total repertoire of the TcR molecules is not only dependent on combinations of gene segments, but also on the size and diversity of the junctional regions, we studied the V delta 1-J delta 1 junctional regions using the polymerase chain reaction (PCR) technique. These PCR analyses showed that the size of the V delta 1-J delta 1 junctional regions differed markedly (up to approximately 30 bases or more) between the leukemias. Therefore we conclude that the combinatorial repertoire of TcR-delta S+ T-ALL is limited, especially due to the homogeneous TcR-gamma gene rearrangements, but that the junctional repertoire of the TcR-delta genes seems to be extensive.

Extensive junctional diversity of gamma delta T-cell receptors expressed by T-cell acute lymphoblastic leukemias: implications for the detection of minimal residual disease.

Rearrangements, junctional regions and expression of T-cell receptor (TcR)-gamma and TcR-delta genes were analyzed in thirteen TcR-gamma delta + T-cell acute lymphoblastic leukemias (T-ALL). All TcR-gamma genes were rearranged except for one deleted allele and one allele in germline configuration. The expressed TcR-gamma protein chains showed a preference for V gamma l (11/13), J gamma 2.3 (7/13) and C gamma 2 (8/13). Furthermore, the TcR-gamma combinatorial repertoire appears to be even more limited by the fact that particular V gamma genes are preferentially used in TcR-gamma 1 or TcR-gamma 2 derived receptors, i.e. in disulfide-linked or non-disulfide-linked TcR-gamma delta receptors. The combinatorial repertoire of the expressed TcR-delta genes was homogeneous, as all thirteen T-ALL expressed V delta 1-D delta-J delta 1-C delta protein chains. In contrast to the limited combinatorial repertoire of the TcR-gamma and TcR-delta genes, the junctional diversity of both TcR genes was extensive due to insertion of N-region, P-region, and D delta gene nucleotides in addition to deletion of nucleotides by trimming. Using polymerase chain reaction (PCR)-mediated amplification and subsequent direct sequencing, we determined the junctional region sequences of all TcR-gamma and TcR-delta rearrangements. Sequence analysis showed that in the TcR-gamma junctional regions insertion varied from 0 to 25 nucleotides (average 8.0) and deletion from 1 to 27 nucleotides (average 8.7). In TcR-delta junctional regions, nucleotide insertion varied from 5 to 47 nucleotides (average 25.5) and deletion from 0 to 29 nucleotides (average 6.2) per junctional region. In general, the N-region nucleotides were the most prominent element in the junctional regions, i.e. 97% of the TcR-gamma and 55% of the TcR-delta junctional regions. D delta genes contributed significantly (40%) to the TcR-delta junctional diversity, whereas P-regions were hardly found in both TcR genes. Altogether, the junctional regions of TcR-delta genes were far more diverse than the junctional regions of TcR-gamma genes. With respect to the new methods of detecting minimal residual disease (MRD) in lymphoid malignancies utilizing PCR-mediated amplification of the junctional regions of TcR genes, our data indicate that this MRD-PCR analysis will generally be more sensitive if TcR-delta instead of TcR-gamma junctional-region-specific probes are used.

Heterogeneity in junctional regions of immunoglobulin kappa deleting element rearrangements in B cell leukemias: a new molecular target for detection of minimal residual disease.

Virtually all immunoglobulin kappa (IGK) gene deletions are mediated via rearrangements of the so-called kappa deleting element (Kde). Kde rearrangements occur either to Vkappa gene segments (Vkappa-Kde rearrangements) or to the heptamer recombination signal sequence in the Jkappa-Ckappa intron. Kde rearrangements were analyzed by the polymerase chain reaction (PCR) and heteroduplex analysis in 130 B-lineage leukemias: 63 precursor-B-acute lymphoblastic leukemias (ALL) and 67 chronic B cell leukemias. To obtain detailed information about Kde rearrangements, we sequenced 109 of the 189 detected junctional regions. Vkappa gene family usage in the Vkappa-Kde rearrangements in our series of B-lineage leukemias was comparable to Vkappa gene family usage in functional Vkappa-Jkappa rearrangements in normal and malignant mature B cells, except for a higher frequency of VkappaII family usage in precursor-B-ALL. Junctional region sequencing of the Kde rearrangements in precursor-B-ALL revealed a mean insertion of 4.7 nucleotides and a mean deletion of 9.5 nucleotides, resulting in an extensive junctional diversity, whereas in chronic B cell leukemias the insertion (1.9) and deletion (6.0) were significantly lower. The relatively extensive junctional diversity of the Kde rearrangements in precursor-B-ALL allowed us to design leukemia/patient-specific oligonucleotide probes, which were proven to be useful for detection of minimal residual disease (MRD) with sensitivities of 10(-4) to 10(-5). Kde rearrangements occur in approximately 50% of precursor-B-ALL cases and are likely to remain stable during the disease course, because Kde rearrangements are assumed to be 'end-stage' rearrangements, which cannot easily be replaced by continuing rearrangement processes. These findings indicate that junctional regions of Kde rearrangements in precursor-B-ALL represent new valuable patient-specific PCR targets for detection of MRD.

Genotoxic exposure: novel cause of selection for a functional ΔN-p53 isoform.

The p53 gene is frequently mutated in cancers and it is vital for cell cycle control, homeostasis and carcinogenesis. We describe a novel p53 mutational spectrum, different to those generally observed in human and murine tumors. Our study shows a high prevalence of nonsense mutations in the p53 N terminus of 2-acetylaminofluorene (2-AAF)-induced urinary bladder tumors. These nonsense mutations forced downstream translation initiation at codon 41 of Trp53, resulting in the aberrant expression of the p53 isoform ΔN-p53 (or p44). We propose a novel mechanism for the origination and the selection for this isoform. We show that chemical exposure can act as a novel cause of selection for this truncated protein. In addition, our data suggest that the occurrence of ΔN-p53 accounts, at least in mice, for a cancer phenotype. We also show that gene expression profiles of embryonic stem (ES) cells carrying the ΔN-p53 isoform in a p53-null background are divergent from p53 knockout ES cells, and therefore postulate that ΔN-p53 itself has functional transcriptional properties.