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PubMed2001

The major histocompatibility complex of the rat (Rattus norvegicus).

This review of the RT1 complex, the major histocompatibility complex (MHC) of the rat, focuses on genetic, genomic, evolutionary, and functional aspects at the molecular level. The class I, class II, and framework genes are listed. The physical map of the RT1 complex as revealed by analysis of clonal contigs is compared with the human and mouse MHC, and the degree of orthologous relationship is outlined. Elucidation of the RT1 complex provides important information for using the rat as a model of experimental transplantation and complex diseases.

Animals↗
PubMed2004

Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells.

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.

3T3 Cells↗
PubMed2005

Molecular analysis of B-cell clonality in Helicobacter pylori gastritis.

The aim of our study was to identify PCR-detectable clonal B-cell population in Helicobacter pylori gastritis and assess their relation to the Wotherspoon-Isaacson (W-I) grade for gastric lymphoid infiltrates. Amplified DNA was obtained from thirty four H. pylori positive gastritis dyspeptic patients and thirty four H. pylori negative matched controls. Clonal bands were observed in 6 (2/17 W-I Grade 1, 2/13 W-I Grade 2, and 2/4 W-I Grade 3 lesions) and polyclonal smears in 24 cases (15 W-I Grade 1, 7 W-I Grade 2, and 2 W-I Grade 3). Four additional W-I Grade 2 samples with clonal bands were associated with background polyclonal smear and were not reproducible. Clonal bands were not recorded in controls. B-cell clonality was not related to W-I grades. We conclude that certain H. pylori positive gastritis patients show PCR-detectable monoclonality, which is independent of the W-I grade of gastritis and cannot be taken as evidence of an existing neoplastic lesion.

Adult↗
PubMed1987

Transgenic chickens: insertion of retroviral genes into the chicken germ line.

We infected early chicken embryos by injection of wild-type and recombinant avian leukosis viruses into the yolk of unincubated, fertile eggs. The viremic males (designated generation 0 (G-0] were tested for transmission of proviral DNA to their G-1 progeny. Nine of 37 G-0 viremic males were mosiac and proviral DNA was transmitted to their progeny at frequencies varying from 1 to 11%. All of the G-1 progeny examined by restriction enzyme analysis for clonality of proviral junction fragments had one to three simple but different fragments. The proviral DNA was transmitted from G-1 to the G-2 progeny in a Mendelian fashion thus proving that retroviral genes have been inserted into the chicken germ line. One of the viruses is a candidate vector for insertion of foreign genes into the chicken germ line.

Animals↗
PubMed1992

Giant cell tumor of bone. Chromosomal analysis of 48 specimens and review of the literature.

Giant cell tumor of bone (GCT) is a distinct clinical, radiographic, and pathologic benign entity that constitutes 5% of all primary bone tumors. For a 5-year period, 47 benign GCTs and 1 malignant GCT from 34 different patients were cytogenetically characterized. Analysis showed clonal karyotypic abnormalities in 16 specimens. Clonal structural abnormalities detected in more than one patient included translocations involving 11p15, fus(14p;21p), and fus(15p;21p). None of the clonal numerical abnormalities observed occurred in more than one patient. Thirty-seven of the 44 successfully analyzed specimens (84%) demonstrated telomeric fusion, with most frequent involvement of chromosomal telomeres 11p, 13p, 15p, 18p, 19p, and 21p. We also compared the presence or absence of random and/or clonal karyotypic abnormalities with clinical behavior to determine if a relationship existed. Most notably, chromosomal abnormalities were detected in all 13 successfully analyzed recurrent lesions, five of which were clonally aberrant. This study summarizes the cytogenetic findings and their relevance in 48 specimens analyzed at our institution and reviews the findings of the 18 other published cases.

Adolescent↗
PubMed1988

Human allograft-derived T-cell lines: donor class I- and class II-directed cytotoxicity and repertoire stability in sequential biopsies.

In order to characterize directly the T-cell repertoire utilized in human renal allograft rejection, we analyzed 20 long-term T-cell lines established from lymphocytic infiltrates present in renal tissue obtained by needle biopsy from transplant patients with rejection indications. All cell lines are strongly cytotoxic against one or more of the HLA antigens for which the kidney donor and the recipient were mismatched. Traditionally, cytotoxicity in allograft rejection has been attributed to CD8-positive cytotoxic T lymphocytes (CTLs) directed against class I alloantigens. Cell lines described here are mixtures of distinct CD4-antigen-positive and CD8-antigen-positive subpopulations, and exhibit class I- as well as class II-directed killing. Using a cell line that demonstrates class II-directed cytotoxicity and a set of class II deletion mutants as target cells, we show that class II antigens, in particular HLA-DP, can serve as targets in renal allograft rejection. The role of CD4-positive CTLs was shown by analysis of clonal populations or sorted CD8-positive and CD4-positive subpopulations. In several instances we have obtained cell lines from serial biopsies performed on the same patient at distinct time points during an ongoing rejection. Comparisons of the phenotype, function, and specificities allow for speculation regarding T-cell population dynamics within the rejection response.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Mixed acute leukaemias.

In recent years immunophenotyping and analysis of clonal rearrangement of immunoglobulin and T-cell antigen receptor genes have proved valuable for the diagnosis and classification of leukaemia. These techniques aid in the assignment of cell lineage in cases of acute leukaemia in which the standard FAB criteria of morphology and cytochemistry do not reveal clear lymphoid or myeloid phenotype. These new techniques have also revealed that the leukaemic blasts in a sizable minority of otherwise typical cases of acute leukaemia express 'inappropriate' lineage-associated markers and have been termed mixed acute leukaemias. The spectrum of characteristics encompassed by mixed acute leukaemias ranges from fairly common cases expressing one or two inappropriate markers to the more extreme, rare cases of acute leukaemia termed 'hybrid' in which a truly scrambled picture is seen. A subgroup of these mixed cases have two distinct populations of blasts, e.g. one lymphoid and the other myeloid. These observations raise a number of issues about the cell of origin of these leukaemias and about the mechanisms controlling the developmental regulation of expression of different lineage-associated markers. In addition, accumulating evidence suggests that inappropriate expression of markers may identify sub-groups of both acute myeloid and lymphoblastic leukaemia with an inferior prognosis.

Diagnosis, Differential↗
PubMed1994

Chemical analysis of neurotransmitter candidates in clonal cell lines from Drosophila central nervous system. I. ACh and L-dopa.

To characterize neuronal phenotypes, aminergic neurotransmitters, i.e. acetylcholine (ACh), catecholamine (CA)s and other biogenic amines (serotonin, octopamine), were surveyed in 10 colonial clones from one cell line of Drosophila larval CNS using HPLC-ECD system. ACh, which is a neurotransmitter in Drosophila, was found in 7 out of 10 clones. CAs, their metabolites, and other amines were not detected in any clones. However, all the 10 clones expressed L-dopa, a precursor of CAs. Consequently, seven clones expressed ACh and L-dopa. L-dopa as a novel neurotransmitter candidate was discussed.

Acetylcholine↗
PubMed1995

Chemical analysis of neurotransmitter candidates in clonal cell lines from Drosophila central nervous system, II: Neuropeptides and amino acids.

In a previous study, we have found acetylcholine and/or L-DOPA in 10 colonial clones from one cell line of Drosophila larval central nervous system (CNS). In this study, to characterize clonal neuronal phenotypes further, we have examined three neuropeptides and 19 amino acids using HPLC system. Substance P and proctolin were found in seven and eight out of ten clones, respectively. On the other hand, somatostatin was expressed in all ten clones. GABA and taurine were not detected in any clones. Glutamate, which is an excitatory transmitter in Drosophila, was found in all the clones, although its content was different seven times among them. Glycine, which is not known as a transmitter in Drosophila, was found to be unevenly expressed among them. Therefore, the conspicuous expression of peptides or amino acids in some clones suggests that the substances have a special role in Drosophila CNS.

Amino Acids↗
PubMed1991

The other view on antigen-specific T suppressor cells: hypothesis.

A close analysis of clonal anergy and T suppressor systems reveals a considerable overlap between these two phenomena in experimental conditions. While evidence accumulates in favour of anergy in the maintenance of self-tolerance and in experimental manipulations, the concept of antigen-specific T cell suppression coupled with the conventionally attributed features of T suppressor cells remain as an enigma. This has lead to a widespread scepticism and controversies. Hence synthesis of a theory to explain and (better) understand antigen-specific suppression becomes an essential task in basic immunology. In the present article the phenomenon of clonal anergy is subdivided, theoretically, into three states and then analysed in the context of different experimental systems. it is suggested that partially anergic cells, termed as state 3 cells, can be the effectors of antigen-specific suppression.

Animals↗
PubMed2005

Intra-bone marrow transplantation facilitates pauci-clonal human hematopoietic repopulation of NOD/SCID/beta2m(-/-) mice.

OBJECTIVE: Intra-bone marrow transplantation (IBMT) has been shown to improve the limit of detection of primitive human SCID-repopulating cells (SRC) in NOD/SCID mice when compared to intravenous transplantation. We sought to further refine detection of SRC by comparing NOD/SCID mice to the more sensitive NOD/SCID/beta2m(-/-)strain as IBMT recipients of limiting numbers of purified primitive human hematopoietic cells. MATERIALS AND METHODS: Purified human Lin(-)CD34(+)CD38- cells at limiting doses were delivered by IBMT into NOD/SCID and NOD/SCID/beta2m(-/-) strains of recipient mice. Six weeks posttransplantation, injected and noninjected bones were analyzed separately for multilineage human hematopoietic chimerism. RESULTS: NOD/SCID/beta2m(-/-) mice are superior recipients for IBMT and show a trend toward increased levels of human hematopoietic engraftment. In addition, in contrast to NOD/SCID recipients, NOD/SCID/beta2m(-/-) mice were reconstituted with as few as five highly purified cells, indicative of pauci-clonal repopulation. Analysis of injected and noninjected bones demonstrated that engrafting cells were capable of in vivo migration and expansion. Although SRC hematopoietic reconstitution of NOD/SCID mice is commonly lymphoid-dominant, multilineage analysis of separate bone sites following IBMT of purified cells revealed that a subset of mice was repopulated with a myeloid-dominant graft in at least one bone site, revealing that SRC are developmentally heterogeneous among Lin(-)CD34(+)CD38- cells and capable of distinct differentiation potential. CONCLUSION: IBMT into NOD/SCID/beta2m(-/-) mice provides a highly sensitive experimental transplantation assay for the detection of human hematopoietic repopulating cells and demonstrates that Lin(-)CD34(+)CD38- cells are more highly enriched for human SRC than originally predicted.

Animals↗
PubMed2005

Molecular evidence for progression of nephrogenic metaplasia of the urinary bladder to clear cell adenocarcinoma.

Nephrogenic metaplasia or nephrogenic adenoma of the urinary tract may present a diagnostic challenge in surgical pathology practice. Previous case reports suggest the possibility of nephrogenic metaplasia progressing to clear cell adenocarcinoma, but a malignant potential of nephrogenic metaplasia is generally not acknowledged. A case of a 70-year-old female patient with multiple recurrences of nephrogenic metaplasia of the urinary bladder and subsequent development of clear cell adenocarcinoma is described. Immunohistochemical studies help to differentiate the 2 entities. Results of molecular studies, particularly comparative genomic hybridization analysis, suggest clonal evolution of nephrogenic metaplasia to clear cell adenocarcinoma in this case.

Adenocarcinoma, Clear Cell↗
PubMed2004

Feline leucocyte antigen class II polymorphism and susceptibility to feline infectious peritonitis.

There are four outcomes to feline coronavirus (FCoV) infection: the development of feline infectious peritonitis (FIP, which is immune-mediated), subclinical infection, development of healthy lifelong carriers and a small minority of cats who resist infection (Addie and Jarrett, Veterinary Record 148 (2001) 649). Examination of the FCoV genome has shown that the same strain of virus can produce different clinical manifestations, suggesting that host genetic factors may also play a role in the outcome of infection. FIP is most prevalent amongst pedigree cats, although how much of this is due to them living in large groups (leading to higher virus challenge and stress which predisposes to FIP) and how much is due to genetic susceptibility is not known. If host genetics could be shown to play a role in disease, it may allow the detection of cats with a susceptibility to FIP and the development of increased population resistance through selective breeding. The feline leucocyte antigen (FLA) complex contains many genes that are central to the control of the immune response. In this preliminary study, we used clonal sequence analysis or reference strand conformational analysis (RSCA) to analyse the class II FLA-DRB of 25 cats for which the outcome of FCoV exposure was known. Individual cats were shown to have between two and six FLA-DRB alleles. There was no statistically significant association between the number of alleles and the outcome of FCoV infection. No particular allele appeared to be associated with either the development of FIP, resistance to FCoV, or the carrier status. However, the analysis was complicated by apparent breed variation in FLA-DRB and the small number of individuals in this study.

Animals↗
PubMed2006

The incidence of T-cell receptor gene rearrangements in childhood B-lineage acute lymphoblastic leukemia is related to immunophenotype and fusion oncogene expression.

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement is conventionally used for assessment of lymphoid malignant cells. TCR genes rearrangements were reported to occur at high frequency in B-lineage acute lymphoblastic leukemia (ALL). Therefore, we have analyzed 83 children with acute B-lineage ALL (67 de novo patients and 19 relapses) by PCR analysis for clonal IgH, incomplete TCRD (Vdelta2-Ddelta3 and Ddelta2-Ddelta3) and TCRG rearrangements. It was shown that clonal cross-lineage TCR rearrangements were associated with more immature immunophenotype (CD34+, CD117+, CyIgM-) of leukemic cells from patients' bone marrow (BM) samples as compared to cell samples without cross-lineage TCR rearrangements. That was equally detected both in de novo and relapsed cases of disease. Low frequency of clonal TCRG rearrangements was associated with expression of E2A/PBX chimeric oncogene. We suggest that TCRG and TCRD clonal rearrangements in leukemic B-cells are associated with early stages of their differentiation.

Adolescent↗
PubMed2006

Left and right ventricular contributions to the formation of the interventricular septum in the mouse heart.

Mammalian heart development involves complex morphogenetic events which lead to the formation of fully separated left and right atrial and ventricular chambers from a tubular heart. Separation of left and right ventricular chambers is dependent on a single structure, the interventricular septum (IVS), which has both muscular and mesenchymal components. Little is known about the morphogenetic events that lead to the formation of the muscular component of the IVS. We have analyzed two transgenic mouse lines that display complementary nlacZ reporter gene expression patterns in the embryonic ventricles: the Mlc1v-nlacZ-24 transgene is expressed in right ventricular myocardium and the Mlc3f-nlacZ-2 transgene in left ventricular myocardium. Detailed analysis of these transgene expression patterns during IVS formation reveals a symmetric left and right myocardial identity within the developing IVS between embryonic days 9.5 and 11.5. From embryonic day 12.5 onwards, myocytes with a left ventricular identity dominate the IVS, particularly in its dorsal aspect. The T-box transcription factor encoding gene, Tbx18, is expressed in the left ventricle and left side of the developing IVS, providing additional support for the presence of left and right ventricular identities within the IVS. Analysis of clonally related cardiomyocyte clusters confirms that both left and right ventricular myocardial cell populations contribute to the forming IVS, in similar domains to those defined by the Mlc-nlacZ transgenes. Examination of the orientation as well as the distribution of labeled cells in clusters provides new insights into the morphogenesis of the septum.

Animals↗
PubMed2006

TrkB/BDNF signaling regulates photoreceptor progenitor cell fate decisions.

Neurotrophins, via activation of Trk receptor tyrosine kinases, serve as mitogens, survival factors and regulators of arborization during retinal development. Brain-derived neurotrophic factor (BDNF) and TrkB regulate neuronal arborization and survival in late retinal development. However, TrkB is expressed during early retinal development where its functions are unclear. To assess TrkB/BDNF actions in the early chick retina, replication-incompetent retroviruses were utilized to over-express a dominant negative truncated form of TrkB (trunc TrkB), or BDNF and effects were assessed at E15. Clones expressing trunc TrkB were smaller than controls, and proliferation and apoptosis assays suggest that decreased clone size correlated with increased cell death when BDNF/TrkB signaling was impaired. Analysis of clonal composition revealed that trunc TrkB over-expression decreased photoreceptor numbers (41%) and increased cell numbers in the middle third of the inner nuclear layer (INL) (23%). Conversely, BDNF over-expression increased photoreceptor numbers (25%) and decreased INL numbers (17%). Photoreceptors over-expressing trunc TrkB demonstrated no increase in apoptosis nor abnormalities in lamination suggesting that TrkB activation is not required for photoreceptor cell survival or migration. These studies suggest that TrkB signaling regulates commitment to and/or differentiation of photoreceptor cells from retinal progenitor cells, identifying a novel role for TrkB/BDNF in regulating cell fate decisions.

Animals↗
PubMed2006

[Legionnaire's disease outbreak].

BACKGROUND AND OBJECTIVE: To describe an outbreak of Legionella pneumophila serogroup 1 in Mataró, Catalunya, Spain, in August 2002. The source of the microorganism was a cooling tower. PATIENTS AND METHOD: Prospective and observational study with analysis of epidemiological, clinical, and microbiological data. RESULTS: 151 patients were affected (62% male), with a mean age of 58.4 years old. Seven patients were classified as Pontiac Fever and 144 suffered from pneumonia. The diagnosis of pneumonia was confirmed in 79% of cases, was considered suspicious in 14% and probable in 7%. Forty per cent of patients were smokers and 53.5% had comorbidities, mainly diabetes mellitus (22%). Chief symptoms were fever (97%), chills and muscular pain (63% respectively), headache (54%) and cough (53%). Pulmonary condensation was the more frequent radiological feature (71%). Normal pulmonary exploration was observed in 38%. Forty-three per cent of cases were severely ill, and 16% of patients belonged to Fine's IV and V class. Antigenuria was the most important test for diagnosis, which confirmed 76% of cases. Legionella spp. was obtained in respiratory secretions of 10 patients. Molecular analysis confirmed clonality between respiratory microorganisms and that obtained in the cooling tower. CONCLUSION: The outbreak involved an important number of subjects in a short period of time. Antigenuria was the most useful test. However, the isolation of L. pneumophila from patients permitted the prompt identification of microorganism's source in a cooling tower. The low mortality observed probably relates to a rapid diagnosis and its target treatment.

Disease Outbreaks↗