The effect of archistriatal lesions on ''open field'' and fear/avoidance behaviour in the domestic chick

DNA samples from about 100 human-hamster somatic cell hybrids, previously characterized by conventional banding techniques, were amplified with dual-Alu PCR. The products were then used as probes in FISH experiments on normal human metaphases for an accurate cytogenetic characterization of the human material retained in each hybrid. In addition to entire chromosomes, most hybrids were found to contain one or a few chromosome fragments, as a result of rearrangements that had occurred in vitro. Forty additional primary hybrids, in which conventional cytogenetic analysis failed to reveal any complete human chromosome, contained many human chromosome fragments. More than 300 chromosome fragments were scored and their precise chromosomal location recorded. We show data indicating that subchromosomal painting libraries generated from these hybrids can be favorably used in the fine characterization of chromosomal rearrangements encountered in clinical cytogenetics or in tumor cytogenetics, and in tracking chromosomal changes that occurred in primate evolution.     

Monitoring of remission status by fluorescence in situ hybridisation in chronic myeloid leukaemia patients treated with interferon-alpha

Interferon-alpha (IFN-alpha) can be considered as treatment of choice for patients with chronic myeloid leukaemia (CML) in chronic phase. With this treatment major cytogenetic responses can be achieved in 30% to 50% of patients. Regular monitoring of cytogenetic response is essential for the therapeutic management of these patients. As conventional cytogenetics is not always successful, especially under IFN-alpha treatment, molecular cytogenetic methods have been established for the examination of interphase nuclei for the presence of the BCR-ABL fusion gene, the molecular counterpart of the Philadelphia chromosome. To demonstrate the value of these new methods we have analysed interphase nuclei from sequentially cultured bone marrow cells from 14 CML patients who were treated with IFN-alpha and whose bone marrow was investigated regularly during therapy. Dual-colour FISH with a breakpoint spanning BCR-YAC and a flanking cosmid from the ABL region was applied. When compared with conventional cytogenetics the results achieved by FISH were favourable. The most evident advantage of FISH analysis is that in case of failure of conventional cytogenetics a reliable determination of the remission status can be done. Together with other recent studies our results illustrate the advantages and limitations of the interphase FISH method for monitoring CML patients.     

Multicolor FISHing: what's the catch?

Recent advances in protocols for multicolor fluorescence in situ hybridization (FISH) mark a major milestone in the field of molecular cytogenetics. Brilliant chromosome images have been presented, where each chromosome homolog is depicted in a different color. The easy recognition of chromosomes seems to simplify karyotype analysis considerably and the procedure is meant to open new avenues for scanning human and other genomes for chromosomal rearrangements. How will this development change the field of cytogenetics? In this review the current applications that benefit from the new protocols are summarized and future perspectives are discussed.     

T cell receptor alpha gene rearrangement and transcription in adult thymic gamma delta cells

T cells belong to two separate lineages based on surface expression of alpha beta or gamma delta T cell receptors (TCR). Since during thymus development TCR beta, gamma, and delta genes rearrange before alpha genes, and gamma delta cells appear earlier than alpha beta cells, it has been assumed that gamma delta cells are devoid of TCR alpha rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic gamma delta cells undergo VJ alpha rearrangements more frequently than immature alpha beta lineage thymic precursors. Sequence analysis shows VJ alpha rearrangements in gamma delta cells to be mostly (70%) nonproductive. Furthermore, VJ alpha rearrangements in gamma delta cells are transcribed normally and, as shown by analysis of TCR beta-/- mice, occur independently of productive VDJ beta rearrangements. These data are interpreted in the context of a model in which precursors of alpha beta and gamma delta cells differ in their ability to express a functional pre-TCR complex.     

Quantitative FISH by image cytometry for the detection of chromosome 1 imbalances in breast cancer: a novel approach analyzing chromosome rearrangements within interphase nuclei

Interphase cytogenetics have become a widespread tool for investigation of chromosome rearrangements in solid tumors. The most recurrent chromosome alteration within breast cancer affects chromosome 1, leading principally to gain of the long arm and/or loss of the short arm. We have developed a new method for detection of chromosome 1 arm imbalances in interphase nuclei. The method is based on quantitation of the fluorescence signals emitted by the hybridized two-color paintings of the short and long arms using image cytometry. The chromosome arm imbalance was determined by calculating the ratio of both fluorescence emissions of each arm. The ratio of the paintings of normal lymphocytes was used as a reference. Three breast cancer cell lines, 13 fresh tumor samples, and 6 fine-needle samplings of breast cancer were analyzed using an automated image cytometer. Whenever possible, classic cytogenetics and in situ hybridization on metaphases were performed as controls. Fluorescence ratios representing the imbalances of chromosome 1 arms with values between 1 and 3.2 were measured. Data between classic cytogenetics and interphase cytogenetics were well-correlated (r = 0.89). This method, which enables an easy detection of intrachromosomal imbalances without need of metaphase preparations, detects malignant cells and can be extended to other carcinomas for which chromosome 1 arm imbalances are recurrent or chromosome alterations specific of other malignancies. In comparison to other interphase fluorescence in situ hybridization techniques, it avoids every spot scoring problem encountered when using centromeric probes and the difficulties in interpreting structural rearrangements.     

Cytogenetics of myelodysplastic syndromes

The authors review the main cytogenetic abnormalities encountered in MDS particularly del 5q, -7, +8, and complex abnormalities. Their presence may be useful to the diagnosis of MDS in difficult cases. Their prognostic value is important, and cytogenetics have recently been included in prognostic scores in MDS. Knowledge of recurrent chromosomal rearrangements is finally a first step in the discovery of genes implicated in the pathogenesis of MDS.     

Expression of integrins in squamous cell carcinoma of the oral cavity. Correlations with tumor invasion and metastasis

In mouse mutants incapable of expressing mu chains, VkappaJkappa joints are detected in the CD43(+) B cell progenitors. In agreement with these earlier results, we show by a molecular single cell analysis that 4-7% of CD43(+) B cell progenitors in wild-type mice rearrange immunoglobulin (Ig)kappa genes before the assembly of a productive VHDHJH joint. Thus, mu chain expression is not a prerequisite to Igkappa light chain gene rearrangements in normal development. Overall, approximately 15% of the total CD43(+) B cell progenitor population carry Igkappa gene rearrangements in wild-type mice. Together with the results obtained in the mouse mutants, these data fit a model in which CD43(+) progenitors rearrange IgH and Igkappa loci independently, with a seven times higher frequency in the former. In addition, we show that in B cell progenitors VkappaJkappa joining rapidly initiates kappa chain expression, irrespective of the presence of a mu chain.     

Conventional cytogenetics and FISH evaluation of chimerism after sex-mismatched bone marrow transplantation (BMT) and donor leukocyte infusion (DLI)

BACKGROUND AND OBJECTIVES: Sensitive and quantitative cytogenetic methods to better assess the biological significance of post-BMT chimerism have been recently developed. In this study, we compared the results of chimerism analysis and evolution employing conventional cytogenetics and fluorescence in situ hybridization (FISH) in 16 patients after sex-mismatched BMT, and in 5 patients after donor lymphocyte infusion (DLI) to treat post-BMT relapse. DESIGN AND METHODS: FISH studies were performed using separate digoxigenin labeled centromeric DNA probes for the X (pDMX1) and Y (DYZ1/DYZ3) chromosomes. To this purpose, different types of samples were used: bone marrow (BM) and peripheral blood (PB) slides processed for conventional cytogenetics, and routine BM and PB smears. RESULTS: Results of chimerism studies performed on different types of samples showed no significant differences. No significant differences in the ability to identify the sex of each cell with both pDMX1 and DYZ1/DYZ3 probes were found and the results obtained from independent experiments showed a high linear correlation. Chimerism analysis by FISH showed initial mixed chimerism after BMT in 10 patients. Seven of these patients were also studied by conventional cytogenetics and 2 of these showed mixed chimerism. Seven of the former 10 patients evolved to complete donor chimera. 6 patients showed cytogenetic or hematologic bone marrow relapse, 3 of which were preceded by mixed chimaerism as revealed by FISH studies. FISH studies permitted an easy and accurate monitorization of the response to DLI in 5 relapsed patients, showing an increase in the proportion of donor cells in 4 patients as they reached a new complete remission. INTERPRETATION AND CONCLUSIONS: Both FISH and conventional cytogenetics are quantitative methods to assess chimerism. However, FISH is more sensitive, accurate and can even be applied on routine BM and PB smears. Furthermore, its combination with immunophenotyping approaches to quantify chimerism on cell subpopulations, will help to clarify post-BMT chimerism significance.     

Characterization of t(11;14) translocation in mantle cell lymphoma by fluorescent in situ hybridization

Characterization of chromosome abnormalities in leukemia and lymphoma have contributed to the understanding of the molecular basis of these neoplastic diseases. In addition, specific chromosomal aberrations have acquired diagnostic or prognostic value. The t(11;14)(q13;q32) chromosome translocation has been detected in mantle cell lymphomas. However, possibly due to the limits of conventional cytogenetic analysis and the presence of different breakpoints at the molecular level, it is possible that the true percentage of association is underestimated. In our study, we used a yeast artificial chromosome, spanning the entire area where the rearrangements occur on chromosome 11q13, to detect the presence of translocations by fluorescent in situ hybridization experiments. We detected BCL-1 translocations in eight of eight patients with clinical and immunological features of mantle cell lymphoma, suggesting that the t(11;14) translocation is a critical event in the pathogenesis of MCL and may be a primary element for the diagnosis. Since this translocation is associated with poor prognosis, its detection may help to make a correct diagnosis as well as to evaluate residual disease, which is critical to plan a rational chemotherapy regimen.     

Recurrent T cell receptor rearrangements in the cytotoxic T lymphocyte response in vivo against the p815 murine tumor

P815 is a murine mastocytoma of DBA/2 origin which, although immunogenic, rapidly develops as a tumor in immunocompetent syngeneic hosts. In this report, we have studied, by a molecular approach, the in vivo alpha/beta T cell response to P815. Both situations of tumor growth after engraftment of naive animals or tumor rejection by preimmunized animals have been analyzed. The spectrum of T cell receptor beta chain rearrangements in the tumor-infiltrating lymphocytes was found to be highly variable among individual tumor-bearing mice. However, two rearrangements, one using V(beta)1 and J(beta)1.2 segments and one using the V(beta)1 and J(beta)2.5 segments, with conserved junctional regions, reproducibly emerge in most individuals. These two rearrangements thus correspond to "public" (recurrent) T cell clones, as opposed to "private" ones, which emerge in a seemingly stochastic fashion in immunized animals. Importantly, these public cells are observed in situations of either growth or rejection of the tumor. Quantification provides a clear increase in public T cells in secondary responses, but no obvious correlation provides between their level and primary tumor rejection. The V(beta)1- J(beta)1.2 rearrangement is borne by CTL directed against an antigen derived from P1A, a nonmutated mouse self protein which is expressed in P815 but not in normal mouse tissues except testis. A recurrent, public T cell response can thus be observed to an antigen derived from a self protein expressed by a tumor.     

Dietary intake in the urban and rural populations of the Cap-Bon

The correlation between cytogenetic and histopathological findings were analysed in 189 meningiomas. The tumors were classified according to increasing degrees of anaplasia. We observed normal karyotype or only monosomy 22 in grade 1 (benign) tumors, while in grade 3 (anaplastic) only 1.5% of karyotypes were normal. Grade 2 (atypical) and 3 (anaplastic) tumors showed complex structural abnormalities. Loss of chromosome 14 were only found in grade 3. In cases with complex structural rearrangements, fluorescence in situ hybridization technique (FISH) has been realized and permitted a best identification of abnormalities. In our series, five patients recurred. They presented chromosomal abnormalities. These complex karyotypes in recurrent meningiomas might indicate aggressive tumor characteristics. Our results indicate histolopathological and cytogenetics correlations might represent a prognostic factor in meningiomas.     

Quantitative analysis of the T cell repertoire selected by a single peptide-major histocompatibility complex

The positive selection of CD4+ T cells requires the expression of major histocompatibility complex (MHC) class II molecules in the thymus, but the role of self-peptides complexed to class II molecules is still a matter of debate. Recently, it was observed that transgenic mice expressing a single peptide-MHC class II complex positively select significant numbers of diverse CD4+ T cells in the thymus. However, the number of selected T cell specificities has not been evaluated so far. Here, we have sequenced 700 junctional complementarity determining regions 3 (CDR3) from T cell receptors (TCRs) carrying Vbeta11-Jbeta1.1 or Vbeta12-Jbeta1.1 rearrangements. We found that a single peptide-MHC class II complex positively selects at least 10(5) different Vbeta rearrangements. Our data yield a first evaluation of the size of the T cell repertoire. In addition, they provide evidence that the single Ealpha52-68-I-Ab complex skews the amino acid frequency in the TCR CDR3 loop of positively selected T cells. A detailed analysis of CDR3 sequences indicates that a fraction of the beta chain repertoire bears the imprint of the selecting self-peptide.     

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.     

True synovial metaplasia of breast implant capsules: a light and electron microscopic study

High resolution chromosome analysis, molecular cytogenetics, and study of the association between specific chromosome rearrangements and single gene disorders have provided a chromosomal basis to a number of mendelian diseases. Deletions and duplications of small regions, usually less than 3 Mb in size, result in an alteration of normal gene dosage of a number of unrelated genes physically close to each other and are responsible for contiguous gene syndromes. For example, haploinsufficiency is implicated for del 8q24.1 in Langer-Giedion syndrome, del 17p13.3 in Miller-Dieker syndrome, and del 22q11.2 in DiGeorge and Velo-cardiofacial syndromes. Another chromosomal mechanism causing mendelian phenotypes is translocation, which may eventually interrupt a disease gene. It is assumed that translocation breakpoints are running through a relevant gene, hindering the production of the gene product. An example is breakage 16p13.3 associated with Rubinstein-Taybi syndrome. Females with X/autosome translocations have an almost exclusive inactivation of the normal X. Interruption of a disease gene in the translocated X causes the expression of a mendelian phenotype in the presence of an allelic recessive mutation onto the nonrearranged X. Finally, if a human gene shows exclusive expression from a single parental homologue, ie, it is imprinted, deletion of the chromosomal segment containing the active allele results in structural monosomy and functional nullisomy. This situation is illustrated by Prader-Willi and Angelman syndromes. Over seventy human genes have been precisely assigned to chromosomal regions using a cytogenetic approach. Chromosome techniques combined with molecular methods have proved to have powerful and sensitive diagnostic capabilities.     

Deletion of cell division cycle 2-like 1 gene locus on 1p36 in non-Hodgkin lymphoma

Our laboratories have documented a significantly high occurrence of chromosome 1p36 rearrangements in non-Hodgkin lymphoma (NHL). The cell division cycle 2-like 1(CDC2L1) (also known as TP58 or PITSLRE) gene, a protein kinase implicated in apoptotic signaling, is located at the very distal region of chromosome 1p36 and is likely to be disrupted by structural rearrangements involving 1p36. To determine the molecular consequences of the recurrent involvement of the 1p36 region, we examined metaphases containing 1p36 abnormalities from 31 specimens derived from 26 patients for the possible deletion of CDC2L1 by fluorescence in situ hybridization (FISH) using the TP58clk-1 DNA probe. Twenty-three cases exhibited the loss of CDC2L1 from the abnormal chromosome 1. In 2 of 26 cases, the gene locus was translocated to the partner chromosome, and in four specimens, all derived from one case, CDC2L1 was not deleted. This pilot investigation suggests that 1p36 rearrangements, and consequently the loss of the CDC2L1 gene locus, is important in NHL. This work also opens avenues for further molecular studies and prognostic correlations.     

Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional mu heavy chains

Ig gene rearrangements could generate V(H)-D-J(H) joining sequences that interfere with the correct folding of a mu-chain, and thus, its capability to pair with IgL chains. Surrogate light (SL) chain might be the ideal molecule to test the capacity of a mu-chain to pair with a L chain early in development, in that only pre-B cells that assemble a membrane mu-SL complex would be permitted to expand and further differentiate. We have previously identified two SL chain nonpairing V(H)81X-mu-chains with distinct V(H)-D-J(H) joining regions. Here, we show that one of these V(H)81X-mu-chains does not rescue B cell development in J(H) knock-out mice, because flow cytometric analysis of bone marrow cells from V(H)81X-mu transgenic J(H) knock-out mice revealed normal numbers of pro-B cells, but essentially no pre-B and surface IgM+ B cells. Immunoprecipitation analysis of transfected pre-B and hybridoma lines revealed that the same mu-chain fails to pair not only with SL chain but also with four distinct kappa L chains. These findings demonstrate that early pre-B cells are selected for maturation on the basis of the structure of a mu-chain, in particular its V(H)-D-J(H) joining or CDR3 sequence, and that one mechanism for this selection is the capacity of a mu-chain to assemble with SL chain. Therefore, we propose a new function of SL chain in early B cell development: SL chain is part of a quality control mechanism that tests a mu-chain for its ability to pair with conventional L chains.     

Deconstructing (and reconstructing) cell migration

An overriding objective in cell biology is to be able to relate properties of particular molecular components to cell behavioral functions and even physiology. In the "traditional" mode of molecular cell biology, this objective has been tackled on a molecule-by-molecule basis, and in the "future" mode sometimes termed "functional genomics," it might be attacked in a high-throughput, parallel manner. Regardless of the manner of approach, the relationship between molecular-level properties and cell-level function is exceedingly difficult to elucidate because of the large number of relevant components involved, their high degree of interconnectedness, and the inescapable fact that they operate as physico-chemical entities-according to the laws of kinetics and mechanics-in space and time within the cell. Cell migration is a prominent representative example of such a cell behavioral function that requires increased understanding for both scientific and technological advance. This article presents a framework, derived from an engineering perspective regarding complex systems, intended to aid in developing improved understanding of how properties of molecular components influence the function of cell migration. That is, cell population migration behavior can be deconstructed as follows: first in terms of a mathematical model comprising cell population parameters (random motility, chemotaxis/haptotaxis, and chemokinesis/haptokinesis coefficients), which in turn depend on characteristics of individual cell paths that can be analyzed in terms of a mathematical model comprising individual cell parameters (translocation speed, directional persistence time, chemotactic/haptotactic index), which in turn depend on cell-level physical processes underlying motility (membrane extension and retraction, cell/substratum adhesion, cell contractile force, front-vs.-rear asymmetry), which in turn depend on molecular-level properties of the plethora of components involved in governance and regulation of these processes. Hence, the influence of any molecular component on cell population migration can be understood by reconstructing these relationships from the molecular level to the physical process level to the individual cell path level to the cell population distribution level. This approach requires combining experimental, theoretical, and computational methodologies from molecular biology, biochemistry, biophysics, and bioengineering.