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.

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.

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.

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.

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.

The effect of calcium on the transcriptome of sporulating B. subtilis cells.

Bacterial spores formed in the presence of high concentrations of minerals are a major problem in the food industry because of their extreme heat resistance. In order to enhance our insight in the molecular mechanisms underlying this phenomenon we have performed a detailed time-resolved analysis of the genome-wide transcriptome pattern of Bacillus subtilis sporulated both in the absence and presence of high calcium concentrations. The data was analysed in two ways. First, we determined the influence of the presence of high calcium levels during sporulation on the expression of gene groups as defined in Subtilist and KEGG pathways database. Second, we assessed the differential expression at the level of individual genes. When analysing groups and pathways, we found that those annotated as being involved in sporulation were significantly affected. Also, groups and pathways involved in flagella formation and biofilm matrix production were affected by the presence of calcium in the sporulation medium. When we analysed the behaviour of individual genes we found 305 genes influenced by calcium, including all known spore coat polysaccharide biosynthesis genes (10 induced and 1 repressed). A number of the calcium affected genes were also involved in biofilm formation. Minimal overlap with other stress outputs like sigma B activation and weak acid stress response was noted. Those genes that did overlap were unique to that combination which corroborates the notion that the cells sense these conditions differently.

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.

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.

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.

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.

T-cell receptor V delta-J alpha rearrangements in human thymocytes: the role of V delta-J alpha rearrangements in T-cell receptor-delta gene deletion.

The differentiation mechanisms that force thymocytes into the T-cell receptor (TCR)-alpha beta or TCR-gamma delta lineage are poorly understood, but rearrangement processes in the TCR-alpha/delta locus are likely to play an important role. It is assumed that the TCR-delta gene is deleted prior to V alpha-J alpha rearrangements by rearrangement of the so-called TCR-delta-deleting elements delta Rec and psi J alpha. However, the TCR-delta gene can also be deleted via V delta-J alpha rearrangements. We studied the different TCR-delta-deleting rearrangements of V delta 1, delta Rec, V delta 2 and V delta 3 to J alpha gene segments in human thymocytes and peripheral blood using polymerase chain reaction analysis. The V delta 1 gene segment is the most upstream V delta gene segment tested and appears to rearrange to almost all J alpha gene segments. In contrast, the delta Rec and V delta 2 gene segments only rearrange to the 5'-located J alpha gene segments, thereby preserving an extensive TCR-alpha combinatorial diversity, because most J alpha gene segments are kept available for subsequent V alpha-J alpha rearrangements. Based on our combined data we hypothesize that the different V delta gene segments and the delta Rec gene segment play different roles in T-cell development with regard to TCR-delta deletion.

Human T cell leukemias with continuous V(D)J recombinase activity for TCR-delta gene deletion.

The so-called TCR-delta-deleting elements, deltaRec and psiJ alpha, flank the major part of the TCR-delta gene complex. By rearranging to each other, the deltaRec and psiJ alpha gene segments delete the TCR-delta gene complex and prepare the allele for subsequent TCR-alpha rearrangement. This intermediate rearrangement is thought to be a specific rearrangement event. In our studies on TCR-delta deletion mechanisms, we identified several T cell acute lymphoblastic leukemias (T-ALL) with continuous activity of the deltaRec-psiJ alpha rearrangement process. Extensive Southern blot, PCR, and sequencing analyses on the coding joints as well as the signal joints of the deltaRec-psiJ alpha rearrangements in these patients allowed us to prove that this continuous rearrangement activity occurred in the leukemic cells and that these cells, therefore, represent a polyclonal subpopulation within the otherwise monoclonal T-ALL. In additional studies, we also identified a T cell line (DND41) with continuous activity of the deltaRec-psiJ alpha rearrangement process. Our data suggest that the ongoing deltaRec-psiJ alpha gene rearrangements predominantly occur in T cells that cannot express a functional TCR-gammadelta, due to biallelic out-of-frame TCR-delta and/or TCR-gamma gene rearrangements. The described T-ALL and the T cell line can serve as an experimental model in further studies on the regulatory elements involved in the specific deletion of the TCR-delta gene complex.

Preferential rearrangements of the T cell receptor-delta-deleting elements in human T cells.

The major part of the TCR-delta locus is flanked by the so-called TCR-delta-deleting elements deltaRec and psi(J)alpha, which preferentially rearrange to each other in human thymocytes. On the basis of our combined Southern blot and PCR analyses, we also identified the prominent deltaRec-Jalpha58 rearrangement and three other preferential deltaRec rearrangements. The latter rearrangements concern deltaRec rearrangements to the Ddelta3, Jdelta1, and Jdelta3 gene segments. These deltaRec rearrangements do not delete the complete TCR-delta locus and are homologous to Vdelta-Jdelta rearrangements, because the majority of their junctional regions contain Ddelta gene segments. In contrast, the prominent deltaRec-psi(J)alpha and deltaRec-Jalpha58 rearrangements, representing approximately 68 and approximately 23% of all deltaRec rearrangements, respectively, are homologous to Valpha-Jalpha rearrangements, because Ddelta gene segments are absent in their junctional regions. Additional PCR analysis of Vdelta-psi(J)alpha and Vdelta-Jalpha58 coding and signal joints revealed also that these rearrangements resemble Valpha-Jalpha rearrangements, except for Vdelta2-psi(J)alpha and Vdelta2-Jalpha58 rearrangements, which resemble Vdelta-Jdelta rearrangements. Our data show that virtually all TCR-delta-deleting rearrangements are Valpha-like, and the high frequency of these rearrangements in the human thymus suggests that most thymocytes use these rearrangements to further differentiate into the TCR-alphabeta lineage. Based on the very low frequency of deltaRec and psi(J)alpha rearrangements in 4% of the T cell acute lymphoblastic leukemia patients (n = 151) and 6% of the T cell lines (n = 26), we hypothesize that rearranged TCR-delta-deleting elements exist for only an extremely short period during thymocyte differentiation, probably due to rapid subsequent Valpha-Jalpha rearrangements.

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.

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.

Unique selection determinant in polyclonal V delta 2-J delta 1 junctional regions of human peripheral gamma delta T lymphocytes.

Human peripheral gamma delta T lymphocytes are characterized by the preferential expression of a TCR consisting of a V delta 2-J delta 1-C delta chain and a V gamma 9-J gamma 1.2-C gamma 1 chain, which are virtually absent on thymocytes. Here we report the identification of a unique selection determinant that is located in the polyclonal V delta 2-J delta 1 junctional regions of peripheral gamma delta T lymphocytes. The selection determinant was discovered by the presence of an invariant T nucleotide at the relative second position of the V delta 2-J delta 1 junctional regions of peripheral polyclonal gamma delta T lymphocytes. Comparison of published sequences from peripheral gamma delta T lymphocytes confirmed the presence of this invariant T nucleotide (90%) in healthy individuals and in patients with various diseases. Translation of the relative first codon of the V delta 2-J delta 1 junctional regions revealed strikingly high frequencies of the homologous hydrophobic amino acids leucine (46%), valine (35%), and isoleucine (5%) at this position. The invariant T nucleotide was absent in polyclonal thymocytes and out-of-frame V delta 2-J delta 1 junctional regions, which proves that selection occurred at the protein level and not at the genomic level. No selection determinant could be identified in V gamma 9-J gamma 1.2 junctional regions, but the frequently occurring invariable, so-called canonical junctional region provided evidence for biased recombination processes. Although the obtained data do not allow discrimination between thymic selection and/or peripheral Ag-driven expansion, the identification of a strong selection determinant consisting of only one amino acid at a fixed position in V delta 2-J delta 1 junctional regions of virtually all peripheral polyclonal V delta 2/V gamma 9 T lymphocytes provides a new perception of TCR specificity and selection processes at the TCR protein level.

The SCID mouse environment causes immunophenotypic changes in human immature T-cell lines.

In order to evaluate whether the SCID mouse can provide the microenvironment for the growth of human immature T-cell leukemias and if in vivo growth alters their phenotype, we examined the behavior of 3 well-characterized T-cell acute lymphoblastic leukemia (T-ALL)-derived T-cell lines which are at different stages of maturation (CEM, SUP-T3 and MOLT-4f) after transfer to non-irradiated SCID mice. All 3 T-cell lines engrafted and proliferated to form tumors in the mice and showed dissemination patterns in the SCID mouse comparable to those of T-ALL in man: i.e., human cells were detectable by flow cytometry or were cultured from mouse bone marrow, spleen, liver or thymus. CEM, which is the most immature T-cell line, readily formed tumors after injection of cells. The more mature T-cell lines, SUP-T3 and MOLT-4f, required a longer time period, even after injection of higher cell numbers. Whereas no changes in the configuration of the rearranged T-cell receptor genes were detected, striking phenotypic changes were observed in all 3 leukemias growing in the SCID mice after injection. SCID-CEM cells showed an increase in the surface expression of CD3 and CD8 and a decrease in the expression of CD1 and CD71 (transferrin-receptor). SCID-MOLT-4f cells showed an increase in CD5 and CD8 expression and a decrease in CD45RA expression. SCID-SUP-T3 cells showed increased expression of CD8 and CD45RA. Apparently, the mouse environment caused changes in cell-surface antigen expression on the T-ALL-derived T-cell lines. Some immunophenotypic changes remained stable during subsequent growth in culture of SUP-T3 cells, suggesting that maturation of the cell line occurred in vivo. The other cell lines, CEM and MOLT-4f after undergoing in vivo-induced changes, reverted to the original immunophenotype, suggesting transitory activation in vivo. These data point out the importance of stromal factors in defining growth and maturation of human leukemic cells in vivo, in SCID mice.