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PubMed1997

A two-stage model for childhood acute lymphoblastic leukemia: application to hereditary and nonhereditary leukemogenesis.

A differential equation model is developed to represent a two-stage mutational process leading to childhood acute lymphoblastic leukemia (ALL). Leukemogenesis is modeled as transformation of target stem cells that initially grow rapidly in the embryo but plateau and then decline in postnatal childhood. Inheritance of the first of two leukemogenic mutations is allowed as a possibility in a small minority of leukemic patients who would characteristically develop leukemia at an early age. The model is shown to be capable of providing good fits to incidence data for childhood ALL; these fits allow estimation of some parameters of the model. The analysis shows that individuals inheriting one of the two mutations necessary for ALL would be likely to experience "multiclonal leukemogenesis"; that is, the parallel development of several leukemic clones arising from multiple independent leukemic events. The model suggests that between two and ten such clones would typically have developed in such individuals by the time of diagnosis. The main conclusions of the deterministic investigation were confirmed by stochastic modeling. The existence of multiclonal leukemogenesis is in principle testable by molecular biological methods (clonality analysis) that rely on the random inactivation of one of two X-chromosomes in normal female subjects. It is expected that the mathematical methods developed here will also be useful for more general (N-stage) models of malignant transformation of stem cell populations undergoing growth or decline.

Aging↗
PubMed1996

Low-grade gastric B-cell lymphoma of mucosa-associated lymphoid tissue: clinicopathologic analysis of 19 cases.

Low-grade gastric B-cell lymphoma of mucosa-associated lymphoid tissue type (MALToma) is a recently recognized disease entity. We report the clinicopathologic features of 19 patients with MALToma in Taiwan. The 19 patients included eight men and 11 women, ranging in age from 26 to 77 years, with a mean age of 58.8 years. Most complained of abdominal pain or gastrointestinal bleeding. The endoscopic and gross features of the gastric lesions revealed erosion (flat type), ulceration (depressed type), cobblestone appearance or abnormal gastric folds (elevated type), mimicking chronic gastritis, ulcer or early gastric carcinoma. Typical histopathologic features included lymphoepithelial lesion and extensive mucosal infiltration of centrocyte-like cells in all cases. Clonality analysis of the variable-diversity-joining region of the immunoglobulin gene by semi-nested polymerase chain reaction demonstrated monoclonality in 72% of the cases. Helicobacter pylori bacilli (H. pylori) could be identified on histologic sections in 15 cases (78.9%); the serologic test for H. pylori was positive in 12 of 13 patients tested (92%). In six patients receiving triple therapy (amoxicillin, bismuth subcitrate and metronidazole), five showed significant histologic regression with eradication of H. pylori 4 to 6 months after the start of treatment; one patient showed persistent lesions and presence of H. pylori. However, persistence of residual lymphoid cells and monoclonality of the immunoglobulin gene, could still be demonstrated in four cases. Of nine patients treated with surgery or chemotherapy, two died: one due to concomitant gastric carcinoma and the other one due to sudden apnea. No recurrence was observed in the remaining seven patients. The remaining four patients were lost to follow-up. Our experience confirmed that gastric MALToma is a low-grade neoplastic process. The dramatic response of gastric MALToma to anti-H. pylori treatment suggests that H. pylori infection is closely related to the pathogenesis of low-grade gastric MALToma. However, long-term follow-up is mandatory due to the persistence of the monoclonality of the immunoglobulin gene in the residual lymphoid cells after treatment.

Adult↗
PubMed1997

Acute nonlymphocytic leukemia: onset after treatment for Hodgkin's disease.

This study was undertaken to examine the influence of various factors on the occurrence of acute nonlymphocytic leukemia (ANLL) in a group of longterm survivors of Hodgkin's disease (HD). From 1972 to 1992, 1045 patients with HD were assessed and treated at the Department of Radiation Oncology, the Institute of Radiology, and the Department of Human Biopathology, Hematology Section, University of Rome "La Sapienza". The average follow-up was 72 months. For a more accurate calculation of the risk of ANLL occurrence, the patients were first divided into three subgroups according to initial treatment and then according to the total treatment they had received. Moreover, to establish the probable connection between leukemia and splenic treatment the patients were also divided into three subgroups (splenectomy, splenic irradiation, and no splenectomy/no splenic irradiation). Sixteen cases of ANLL were recorded, giving an overall risk of 0.2% at 5 years and 3.4% at 20 years. In 12 patients overt leukemia was preceded by a myelodys-plastic syndrome. Five cases had evaluable chromosome analysis. Clonal chromosome abnormalities were demonstrated in two patients, whereas three patients showed an apparently normal karyotype. Bone marrow agar cultures were analyzed in two patients and were abnormal in both cases. In the radiotherapy (RT) group, the cumulative risk was 0.4% at 10 years and 3.2% at 15 and 20 years: in the chemotherapy (CT) group it was 1.2% at 10, 15, and 20 years; in the combined group it was 3.7% at 10 years and 4.9% at 15 and 20 years. In the multivariate analysis, MOPP treatment with or without RT is a statistically significant variable for ANLL occurrence (p = 0.009). This study demonstrates that splenic treatment does not lead to ANLL. Treatment with MOPP alone and with MOPP plus RT can increase the risk of ANLL.

Adult↗
PubMedDate not supplied

Molecular population genetic analysis of emerged bacterial pathogens: selected insights.

Research in bacterial population genetics has increased in the last 10 years. Population genetic theory and tools and related strategies have been used to investigate bacterial pathogens that have contributed to recent episodes of temporal variation in disease frequency and severity. A common theme demonstrated by these analyses is that distinct bacterial clones are responsible for disease outbreaks and increases in infection frequency. Many of these clones are characterized by unique combinations of virulence genes or alleles of virulence genes. Because substantial interclonal variance exists in relative virulence, molecular population genetic studies have led to the concept that the unit of bacterial pathogenicity is the clone or cell line. Continued new insights into host parasite interactions at the molecular level will be achieved by combining clonal analysis of bacterial pathogens with large-scale comparative sequencing of virulence genes.

Bacteria↗
PubMed1988

Polyclonal reconstitution of human marrow after allogeneic bone marrow transplantation.

Clonal dominance suggestive of reconstitution of marrow from small numbers of pluripotent hematopoietic stem cells has been noted in different experimental and clinical situations. Recipients of human allogeneic marrow transplants have not been previously studied to determine if clonal dominance occurs in this clinical setting. Clonal analysis of 20 allogeneic marrow transplant recipients was performed on DNA from peripheral blood neutrophils using restriction fragment length polymorphisms on the X chromosome. Similar studies were performed on 16 of the donors. To analyze the results further, recipients were paired with their respective donors. There was no evidence of shifts in cell populations contributing to the X chromosome inactivation patterns in recipient marrow grafts when compared with their respective donors. A mathematical model based on binomial statistics was adapted to estimate the numbers of reconstituting pluripotent hematopoietic stem cells. There was no evidence of clonal dominance suggestive of oligoclonal reconstitution in marrow grafts after allogeneic marrow transplantation. This does not preclude the possibility of oligoclonal reconstitution in other marrow transplant settings such as autologous transplantation.

Bone Marrow Transplantation↗
PubMed1994

Linkage analysis of geographic and clinical clusters in Pseudomonas cepacia infections by multilocus enzyme electrophoresis and ribotyping.

Multilocus enzyme electrophoresis and ribotyping were used to characterize 83 strains of Pseudomonas cepacia, mostly isolated from cystic fibrosis (CF) patients, although a number of isolates from non-CF nosocomial infections and reference environmental strains were represented. Twenty enzyme electrophoretic types (ETs) were determined; of these, one clone (ET12) was associated with six of nine ribotypes (RTs) said to be geographically representative of the United Kingdom and all of the Ontario (Canada) isolates from CF patients. This clone was not associated with nosocomial infections or environmental strains and was never found in CF isolates from British Columbia or Nova Scotia, Canada, or a center in the eastern United States. Individual isolate EcoRI RT signatures did not cluster geographically as did the ET signatures by clonal analysis. Frequently RTs occurred in more than a single ET. Known point source focal nosocomial outbreaks were typified by single ETs and stable RTs. Dendrographic analysis of the strains grouped those strains from CF patients, nosocomial outbreaks, and environmental sources into separate ET families, and diversity analysis indicated that, with the exception of ET17, CF isolates clustered in unique and closely related ETs different from those from nosocomial and environmental sources. This study has also shown the potential of multilocus enzyme electrophoresis to monitor the intercontinental spread of P. cepacia strains in CF patients, and this may have a significant impact on plans for CF patient summer camps and design of infection control practices. Whether the intercontinental ET12 clone, which predominates in the United Kingdom and the province of Ontario, linked by summer camp acquisition, has increased virulence for CF patients remains to be established.

Alleles↗
PubMed1986

Analysis of the cellular immune response to and adoptive immunotherapy of a BALB/c lymphoma that cross-reacts with normal DBA/2 cells.

We have previously shown that YC8, a Moloney virus-induced BALB/c lymphoma, is susceptible to lysis by BALB/c anti-DBA/2 effector cells. To further evaluate the relationship between non-H-2 antigens of DBA/2 background and tumor-associated determinants, we investigated the pattern of cytotoxic and proliferative responses induced in BALB/c mice by immunization with YC8 lymphoma. Spleen cells from tumor-immunized animals were restimulated in vitro with YC8 cells and tested for cytotoxicity on target cells of different strains and haplotypes. Cytotoxicity was observed only against YC8, DBA/2 blasts, and P815 (a DBA/2 mastocytoma). BALB/c anti-YC8 effectors were equally blocked by unlabeled YC8 and P815 cells when assayed on YC8 labeled targets. A clonal analysis, however, revealed the existence of at least two types of effectors, one lytic for both P815 and YC8 cells, the other lytic for YC8 cells only. BALB/c anti-YC8 cells proliferate when stimulated both with YC8 and DBA/2 cells in the presence of accessory cells. When tested in an adoptive transfer assay, anti-YC8 cells given i.v. cured 100% of i.v. and 75% of i.p. tumor-injected mice, respectively. When given i.p. anti-YC8 effectors cured 100% of i.p. tumor-injected animals. These results confirm the expression of non-H-2, DBA/2-like antigens on the BALB/c lymphoma YC8 and reveal the presence of additional tumor-associated determinants; both sets of antigens may induce a cellular immune response and elicit immune lymphocytes which can eradicate YC8 cells in an adoptive immunotherapy assay.

Animals↗
PubMed1991

Growth and differentiation of myogenic clones from adult human muscle cell cultures.

Clonal cultures only recently have been applied to normal and pathological human muscle, but detailed clonal analysis and differentiative properties of individual long term muscle subclones derived from adult normal human muscle cell (HMC) cultures have not been reported. In this study we compared the growth potential by plating efficiency (PE), muscle colony differentiation (MC) and growth cuvers and the differentiative properties by fusion index, dystrophin localization, creatine kinase (CK) total activity and isozyme electrophoresis in HMC cultures derived myogenic subclones. These properties were tested in two experimental culture systems (200 cells/dish versus single cell/well) and with two tissue culture media (standard medium--MM--versus conditioned medium--CM). We found a significant high PE and MC in clonal cells established with single cell/well and grown in conditioned medium. In derived subclones we observed two classes of myogenic cells: one characterized by exponential growth kinetic, branched myotubes with high fusion index and predominantly sarcolemmal distribution of dystrophin and MM band at CK electrophoresis; the other with flat growth curve, low fusion index and low CK total activity. These findings demonstrate the variability of the expression of myogenic potentials in cells cloned from adult normal HMC cultures and represent an important tool for comparing the various cell types present in normal and diseased human muscle and for transplantation of normal myoblasts in Duchenne Muscular Dystrophy.

Adult↗
PubMed1996

Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events.

The tracheal (respiratory) system of Drosophila melanogaster is a branched network of epithelial tubes that ramifies throughout the body and transports oxygen to the tissues. It forms by a series of sequential branching events in each hemisegment from T2 to A8. Here we present a cellular and initial genetic analysis of the branching process. We show that although branching is sequential it is not iterative. The three levels of branching that we distinguish involve different cellular mechanisms of tube formation. Primary branches are multicellular tubes that arise by cell migration and intercalation; secondary branches are unicellular tubes formed by individual tracheal cells; terminal branches are subcellular tubes formed within long cytoplasmic extensions. Each level of branching is accompanied by expression of a different set of enhancer trap markers. These sets of markers are sequentially activated in progressively restricted domains and ultimately individual tracheal cells that are actively forming new branches. A clonal analysis demonstrates that branching fates are not assigned to tracheal cells until after cell division ceases and branching begins. We further show that the breathless FGF receptor, a tracheal gene required for primary branching, is also required to activate expression of markers involved in secondary branching and that the pointed ETS-domain transcription factor is required for secondary branching and also to activate expression of terminal branch markers. The combined morphological, marker expression and genetic data support a model in which successive branching events are mechanistically and genetically distinct but coupled through the action of a tracheal gene regulatory hierarchy.

Animals↗
PubMed1993

The heterochronic Teopod1 and Teopod2 mutations of maize are expressed non-cell-autonomously.

Teopod1 and Teopod2 are dominant, unlinked mutations in maize that cause dramatic morphological abnormalities, including inappropriate expression of juvenile traits in adult vegetative phytomers and the transformation of reproductive structures into vegetative ones. These phenotypes are consistent with the constitutive expression of a juvenile phase of development throughout shoot growth. To investigate the basis of the Tp1 and Tp2 phenotypes we have analyzed their cell-autonomy in mosaic Teopod:wild-type plants. Mosaic plants were generated by three different mechanisms. Tp1 has previously been shown to be non-cell-autonomous; to verify and extend these results, large wild-type sectors were generated on Tp1 plants by the spontaneous loss of a B-A translocation chromosome containing the Tp1 gene. Analysis of Tp2 cell-autonomy was complicated by a lack of useful markers on chromosome 10L proximal to Tp2. To circumvent this problem two strategies were used. A reciprocal translocation was used to link Tp2 the wild-type allele of lw2. Sectors were induced in plants of this type by irradiation of imbibed seeds. Also, a chromosome-breaking Ds element located proximal to Tp2 was used to generate somatic sectors that uncovered w2, an albino mutation distal to Tp2. Our results demonstrate conclusively that both Tp1 and Tp2 are non-cell-autonomous. The general use of these techniques for clonal analysis in plants and the potential role of a diffusible factor in regulating the juvenile phase of development in maize are discussed.

DNA Transposable Elements↗
PubMed2000

Use of a small palindrome genetic marker to investigate mechanisms of double-strand-break repair in mammalian cells.

We examined mechanisms of mammalian homologous recombination using a gene targeting assay in which the vector-borne region of homology to the chromosome bore small palindrome insertions that frequently escape mismatch repair when encompassed within heteroduplex DNA (hDNA). Our assay permitted the product(s) of each independent recombination event to be recovered for molecular analysis. The results revealed the following: (i) vector-borne double-strand break (DSB) processing usually did not yield a large double-strand gap (DSG); (ii) in 43% of the recombinants, the results were consistent with crossover at or near the DSB; and (iii) in the remaining recombinants, hDNA was an intermediate. The sectored (mixed) genotypes observed in 38% of the recombinants provided direct evidence for involvement of hDNA, while indirect evidence was obtained from the patterns of mismatch repair (MMR). Individual hDNA tracts were either long or short and asymmetric or symmetric on the one side of the DSB examined. Clonal analysis of the sectored recombinants revealed how vector-borne and chromosomal markers were linked in each strand of individual hDNA intermediates. As expected, vector-borne and chromosomal markers usually resided on opposite strands. However, in one recombinant, they were linked on the same strand. The results are discussed with particular reference to the double-strand-break repair (DSBR) model of recombination.

Animals↗
PubMed1993

Pulsed field gel electrophoresis on single murine hemopoietic colonies.

We report a technique which allows for the direct molecular analysis of single whole murine hemopoietic colonies by pulsed field gel electrophoresis (PFGE). Murine bone marrow cells were plated out in semi-solid agarose and gave rise to macroscopic colonies after 11 days in culture. Single colonies were excised from the agarose using a sterile blade and embedded without further manipulation in molten low-melting-temperature agarose. The leucocyte DNA contained within the agarose plug was subjected to restriction enzyme digestion and PFGE. Sufficient high molecular weight DNA is afforded by this method to achieve a hybridization signal with a single copy probe. This method will make PFGE directly applicable to the clonal analysis of chromosomal aberrations in hemopoietic stem and progenitor cells.

Animals↗
PubMed1996

Demonstration of an osteoblast defect in two cases of human malignant osteopetrosis. Correction of the phenotype after bone marrow transplant.

Osteopetrosis is an inherited disorder characterized by bone sclerosis due to reduced bone resorption. Here we report that human osteopetrotic osteoblast-like (Ob) cells express a defective phenotype in primary cultures in vitro, and that bone marrow transplant (BMT) corrects osteoblast function. DNA analysis at polymorphic short-tandem repeat loci from donor, recipient, and primary Ob-like cells pre-BMT and 2 yr post-BMT revealed that Ob were still of recipient origin post-BMT. Osteopetrotic Ob-like cells obtained pre-BMT showed normal and abnormal 1,25(OH)2D3-induced alkaline phosphatase (ALPase) and osteocalcin production, respectively, and failed to produce macrophage colony-stimulating factor (M-CSF) in response to IL-1a and TNF-alpha. These parameters were all normalized in primary Ob-like cells prepared 2 yr post-BMT. X-linked clonality analysis at the human androgen receptor (HUMARA) locus revealed that osteoblasts showed a polyclonal and an oligoclonal derivation pre- and post-BMT respectively, indicating that a limited number of progenitor reconstituted this population. Because osteoblasts were still of recipient origin post-BMT, this suggests that functional osteoclasts, due to the replacement of hematopoeitic cells, provided a local microenvironment in vivo triggering the differentiation and/or recruitment of a limited number of functional osteoblasts.

Alkaline Phosphatase↗
PubMed1994

Natural occurrence of drug resistance mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates.

Reverse transcriptase-associated amino acid substitutions related to ddC, d4T, and nevirapine resistance have been found in isolates of human immunodeficiency virus type 1 (HIV-1) from patients treated with AZT only. Sequence analysis of 23 isolates documented the presence of 4 unexpected mutations at amino acid residues related to drug resistance. Two isolates contained an aspartic residue in codon 69 associated with ddC resistance, and another a change in codon 75 associated with resistance to d4T. The Y-to-C alteration in codon 181 associated with nevirapine resistance was observed in another isolate after serial passage in cell culture in the absence of drug. Changes in substitution patterns were also noted after serial passage of four AZT resistant isolates in cell culture without inhibitors. One of the strains showed changes in codons 67 and 70 to wild-type residues. Clonal analysis showed that this alteration occurred by the selection during cell culture passage of the wild-type genotype, which was present as a minority subpopulation in the initially resistant virus stock, rather than to genetic reversion. In summary, we present evidence documenting the presence of mutations associated with drug resistance in the absence of drug treatment and supporting the role played by gentic variability in the emergence of HIV-1 antiviral resistance.

Amino Acid Sequence↗
PubMed2003

The predominant neural stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain expresses GFAP.

Periventricular germinal zones (GZs) of developing and adult brain contain neural stem cells (NSCs), the cellular identities and origins of which are not defined completely. We used tissue culture techniques and transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) from the mouse glial fibrillary acid protein (GFAP) promoter to test the hypothesis that certain NSCs express GFAP. To do so, we determined the relative proportions of multipotent neurospheres that are formed by GFAP-expressing cells derived from GZs at different stages of development. In this transgenic model, dividing GFAP-expressing cells are ablated selectively by treatment with the antiviral agent ganciclovir (GCV). Single-cell analysis showed that transgene-derived HSV-TK was present only in GFAP-expressing cells. GCV applied in vitro eliminated growth of multipotent neurospheres from GZs of postnatal and adult transgenic mice but not early embryonic (embryonic day 12.5) transgenic mice. GCV prevented growth of secondary multipotent neurospheres prepared after passage of primary transgenic neurospheres derived from all three of these developmental stages. In addition, GCV prevented growth of multipotent neurospheres from transgenic astrocyte-enriched cell cultures derived from postnatal GZ, and elaidic acid GCV given for 4 d to adult transgenic mice in vivo abolished the ability to grow multipotent neurospheres from GZ. Extensive control experiments, including clonal analysis, demonstrated that failure of neurosphere growth was not merely secondary to loss of GFAP-expressing support cells or the result of a nonspecific toxic effect. Our findings demonstrate that the predominant multipotent NSCs isolated from postnatal and adult but not early embryonic GZs express GFAP, and that NSCs exhibit heterogeneous expression of intermediate filaments during developmental maturation.

Animals↗
PubMed1999

Genetic analysis of rolled, which encodes a Drosophila mitogen-activated protein kinase.

Genetic and molecular characterization of the dominant suppressors of D-raf(C110) on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rl(Su23), was found to bear the same molecular lesion as rl(Sem), which has been reported to be dominant female sterile. However, rl(Su23) and the current stock of rl(Sem) showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed.

Amino Acid Sequence↗
PubMed1995

Nonequivalent requirements for PS1 and PS2 integrin at cell attachments in Drosophila: genetic analysis of the alpha PS1 integrin subunit.

We report on the generation and phenotype of mutant alleles of multiple edematous wings (mew), the gene encoding the alpha PS1 subunit of the PS1 integrin of Drosophila. None of the six alleles examined makes detectable protein, and one allele results from a chromosome break near the middle of the translated sequence, so we are confident that we have described the null phenotype. In contrast to if (alpha PS2) and mys (beta PS) mutants, most mutant mew embryos hatch, to die as larvae. Mutant mew embryos display abnormal gut morphogenesis but, unlike mys or if embryos, there is no evidence of defects in the somatic muscles. Thus, the complementary distributions of PS1 (alpha PS1 beta PS) and PS2 (alpha PS2 beta PS) integrin on tendon cells and muscle, respectively, do not reflect equivalent requirements at the myotendinous junction. Dorsal herniation, characteristic of the mys lethal phenotype, is not observed in mew or in mew if embryos. Clonal analysis experiments indicate that eye morphogenesis is disrupted in mew clones, but if clones in the eye are relatively normal in morphology. Adult wings display blisters around large dorsal but not ventral mew clones. In contrast to dorsal mys clones, small mew patches do not necessarily display morphogenetic abnormalities. Thus, another integrin in addition to PS1 appears to function on the dorsal wing surface.

Alleles↗
PubMed2000

[Application of PCR in the diagnosis of B-type non-Hodgkin's lymphomas in cytological specimens from fine-needle aspiration].

UNLABELLED: The application of PCR to detect monoclonality at the immunoglobulin heavy chain gene locus in FNA cytologic specimens was evaluated. MATERIALS AND METHODS: Immunoglobulin heavy chain gene rearrangement analysis was performed by a nested PCR for the VDJ region on 27 lymph nodes FNA paraffin embedded by cell blocking technique specimens (1 suspicious of NOS lymphoma, 1 Hodgkin's disease, 6 B cell non Hodgkin's lymphomas, 10 reactive hyperplasias). RESULTS: A monoclonal band was seen in 13 of 17 suspicious or positive malignant cases. The other 4 specimens gave a policlonal pattern. Specimens from reactive lymph nodes produced policlonal bands in 8 cases, no product in 1 case and 1 specimen gave a monoclonal band. When needed, the results of PCR examinations were compared with the subsequent histologic evaluation of the same lymph-node. CONCLUSIONS: From our experience we can see that clonal analysis by PCR based IgH gene rearrangement analysis can be applied to FNA material and can be useful in diagnosis.

Antibodies, Monoclonal↗