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PubMed2003

New TCR transgenic model for tracking allospecific CD4 T-cell activation and tolerance in vivo.

We have developed an adoptive transfer model system to visualize the dynamics of alloantigen-specific CD4+ T-cell activation in vivo. Using TCR-transgenic (tg) mice reactive to I-Ab(m12), we studied the clonal expansion and differentiation of alloreactive T cells by tracking the fate of adoptively transferred TCR-tg CD4+ T cells in syngeneic mice transplanted with skin grafts expressing I-Ab(m12). Following transplantation, alloantigen-specific TCR-tg CD4+ T-cell expansion was observed initially in the draining lymph nodes followed by the spleen. TCR-tg CD4+ T cells up-regulated CD69 and CD25 expression, developed an effector/memory surface phenotype and produced IFN-gamma in response to alloantigen ex vivo. Furthermore, we validate the model system as a means for studying the effects of tolerogenic regimens on alloreactive CD4+ T cells, demonstrating that CTLA4Ig inhibits alloantigen-dependent clonal expansion and effector function of TCR-tg CD4+ T cells in vivo. We describe the first model for tracking alloreactive CD4+ T-cell activation in vivo. It provides a powerful tool for studying CD4+ T-cell mediated alloimmune responses and mechanisms of tolerance induction in vivo.

Adoptive Transfer↗

PubMed2004

Persistent alterations in the T-cell repertoires of HIV-1-infected and at-risk uninfected men.

OBJECTIVE: We examined the association between immunogenic exposure and T-cell receptor (TCR) diversity to more clearly assess the impact of HIV-1 infection on the T-cell repertoire. METHODS: : To estimate the extent of T-cell clonality attributable to HIV-1 infection, we evaluated T-cell repertoires in low-risk and at-risk seronegative men and HIV-1 seropositive men by assessment of T-cell receptor beta-chain (TCR beta) complimentary determining region 3 (CDR3) lengths. RESULTS: The frequency of T-cell clonality in both HIV-1 infected and at-risk uninfected men was elevated in comparison to low-risk uninfected men. Among low-risk and at-risk seronegative, and HIV-1 seropositive men, clonal expansions were present in 3, 8, and 10% of CD4+ CDR3 lengths, and 18, 22, and 28% of CD8+ CDR3 lengths respectively. In addition, the longitudinal conservation of clonal expansions was observed in at-risk seronegative men. Based on comparisons to at-risk seronegative men, we estimate that at-risk seropositive men with chronic HIV-1 infection exhibit a 27% increase in the number of expanded CD8+ CDR3 lengths. CONCLUSION: These findings provide an approximation of the magnitude of the T-cell response in individuals undergoing chronic HIV-1 infection and demonstrate a significant association between the history of immunogenic challenge and the magnitude of clonality within the T-cell repertoire. In addition, these findings underscore the necessity of selecting controls with similar antigenic exposure histories when investigating T-cell dynamics in HIV-infected individuals.

Analysis of Variance↗

PubMed2003

Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management.

The spa gene of Staphylococcus aureus encodes protein A and is used for typing of methicillin-resistant Staphylococcus aureus (MRSA). We used sequence typing of the spa gene repeat region to study the epidemiology of MRSA at a German university hospital. One hundred seven and 84 strains were studied during two periods of 10 and 4 months, respectively. Repeats and spa types were determined by Ridom StaphType, a novel software tool allowing rapid repeat determination, data management and retrieval, and Internet-based assignment of new spa types following automatic quality control of DNA sequence chromatograms. Isolates representative of the most abundant spa types were subjected to multilocus sequence typing and pulsed-field gel electrophoresis. One of two predominant spa types was replaced by a clonally related variant in the second study period. Ten unique spa types, which were equally distributed in both study periods, were recovered. The data show a rapid dynamics of clone circulation in a university hospital setting. spa typing was valuable for tracking of epidemic isolates. The data show that disproval of epidemiologically suggested transmissions of MRSA is one of the main objectives of spa typing in departments with a high incidence of MRSA.

Germany↗

PubMed2006

Genetic progression and divergence in superficial esophageal squamous cell carcinoma by loss of heterozygosity analysis.

Esophageal squamous cell carcinoma (SCC) is one of the most common fatal carcinomas worldwide and has some of the most malignant characteristics among gastrointestinal tumors. Although a high frequency of loss of heterozygosity (LOH) for various genes has been observed in esophageal SCCs, these findings do not provide any information regarding the genetic pathways that may underlie the development and progression of this type of tumor. To clarify the temporal and topographic pathways in the genetic evolution of esophageal SCC, we microdissected multiple foci from superficial mucosal invasive foci of tumors. We then carried out LOH analyses of the microdissected neoplastic foci. Sixteen superficial esophageal SCCs were examined. Three to six carcinoma foci from each superficial esophageal SCC were individually microdissected. We used 12 oligonucleotide primer pairs specific for the microsatellite markers for which frequent LOH in esophageal SCC has been reported. All tumors exhibited LOH of at least three microsatellite loci. A frequent homogeneous LOH pattern was detected for TP53 (60%), D16S518 (43%) and D3S1234 (29%), suggesting that the loss of these alleles is an early event in the development of esophageal SCC. A heterogeneous LOH pattern was detected for D13S325 (87%), D10S559 (73%), D3S1568 (58%), D3S1234 (57%) and D3S1621 (56%), suggesting that the loss of these alleles is a late event in the development of esophageal SCC. All tumors showed the LOH pattern of single clonal neoplasms with genetic progression and divergence. In conclusion, by extensive sampling of SCC lesions with microdissection and LOH analysis of multiple chromosomal loci, we successfully demonstrated dynamic and successive accumulation of genetic alterations in early SCC.

Aged↗

PubMed1999

Clonal analysis of mouse intestinal epithelial progenitors.

BACKGROUND & AIMS: Little is known about the cell lineages leading from stem cells to the various terminally differentiated cell types of the intestinal epithelium. In particular, the existence and characterization of intermediate progenitor types remain open issues, which are discussed in this study. METHODS: Chemical mutagenesis was used to genetically mark random intestinal epithelial cells by somatic mutation of the Dlb-1 locus. Intact epithelium was isolated at various times thereafter, and the composition, size, and location of mutant clones were scored. RESULTS & CONCLUSIONS: Analysis of clone dynamics showed short-lived (days) progenitors (C1, M1, and Mix) yielding one or two cell types and long-lived (months) mucous cell progenitors (M0), columnar cell progenitors (C0), and pluripotential stem cells (S) capable of giving rise to all epithelial cell types. Furthermore, study of clonal dispersion, during crypt branching morphogenesis or cell migration, shows that mutant progenitors usually partition into only one of the two daughter crypts and that cells are often widely dispersed in spite of the extensive intercellular junctions in the epithelium.

Animals↗

PubMed2005

Telomerase activity in myelodysplastic syndromes.

Myelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Telomeres are thought to be critical in maintaining normal hematopoiesis. In this study, we assessed telomere dynamics in order to obtain further insight into the pathogenesis of MDS. We studied telomerase activity (TA) in mononuclear cells from peripheral blood (PB) and bone marrow (BM) from patients with myelodysplastic syndrome (MDS; n=24), acute myeloid leukemia (AML; n=14), chronic myeloid leukemia (CML; n=12) and 11 normal controls using a polymerase chain reaction-based telomeric repeat amplification assay. Telomerase activities (mean+/-S.D.) were found as 0.199+/-0.09, 0.414+/-0.55, 0.253+/-0.26 and 0.181+/-0.05 pg/ml in PB mononuclear cells, respectively (P>0.05). Comparison of TA of BM mononuclear cells from 19 MDS patients versus 10 BM samples from normal controls revealed no significant difference (P=0.3). There was no correlation between the levels of TA and clinical and prognostic parameters of the patients with MDS, such as degree of anemia, platelet counts on presentation, gender, presence of organomegaly, bone marrow fibrosis and BM blast percentages. Patients who had higher TA had significantly inferior survival compared with patients who had lower TA (P=0.005). Consistent with previous data, our results suggest that in patients with MDS, telomerase activity might be insufficient to compensate for the telomere shortening. Furthermore, TA might be prognostically important in patients with MDS. Measurements of enzymatic activity in association with telomere length studies may help to understand the prognostic role of telomere dynamics in patients with myelodysplastic syndromes more reliably.

Adult↗

PubMedDate not supplied

Mapping genes through the use of linkage disequilibrium generated by genetic drift: 'drift mapping' in small populations with no demographic expansion.

Linkage disequilibrium has been a powerful tool in identifying rare disease alleles in human populations. To date, most research has been directed to isolated populations which have undergone a bottleneck followed by rapid exponential expansion. While this strategy works well for rare diseases in which all disease alleles in the population today are clonal copies of some common ancestral allele, for common disease genes with substantial allelic heterogeneity, this approach is not predicted to work. In this paper, we describe the dynamics of linkage disequilibrium in populations which have not undergone a demographic expansion. In these populations, it is shown that genetic drift creates disequilibrium over time, while in expanded populations, the disequilibrium decays with time. We propose that common disease alleles might be more efficiently identified by drift mapping - linkage disequilibrium mapping in small, old populations of constant size where the disequilibrium is the result of genetic drift, not founder effect. Theoretical models, empirical data, and simulated population models are presented as evidence for the utility of this approach.

Chromosome Mapping↗

PubMed1999

Expression of the wild-type insulin-like growth factor II receptor gene suppresses growth and causes death in colorectal carcinoma cells.

The insulin-like growth factor II receptor (IGFIIR) has been implicated as a tumor suppressor gene in human malignancy. Frequent mutation, loss of heterozygosity, and microsatellite instability (MSI) directly affecting the IGFIIR gene have been reported in several primary human tumor types. However, to our knowledge, dynamic functional evidence of a growth-suppressive role for IGFIIR has not yet been provided. We identified one MSI-positive colorectal carcinoma cell line, SW48, with monoallelic mutation in IGFIIR identical to that seen in primary colorectal carcinomas. A zinc-inducible construct containing the wild-type IGFIIR cDNA was stably transfected into SW48 cells. Growth rate and apoptosis were compared between zinc-treated, untreated, and untransfected cells. A twofold increase in IGFIIR protein expression was detected after zinc treatment in discrete clonal isolates of transfected SW48 cells. Moreover, zinc induction of exogenous wild-type IGFIIR expression reproducibly decreased growth rate and increased apoptosis. These data prove that wild-type IGFIIR functions as a growth suppressor gene in colorectal cancer cells and provide dynamic in vitro functional support for the hypothesis that IGFIIR is a human growth suppressor gene.

Adenocarcinoma↗

PubMed2006

Simple and versatile methods for the fabrication of arrays of live mammalian cells.

Single-step methods for the generation of patterned surfaces on hydrogels are presented. Poly(vinyl alcohol) films covalently bonded on glass cover slips and commercially available hydrogel-coated polystyrene plates were used as cell-repellent surfaces. Cell-adhesive domains were created by spotting dilute solutions of sodium hypochlorite onto the surfaces. Alternatively, domains supporting cell attachment were created by exposure to UV light from a xenon excimer lamp, employing a contact mask. Rat skeletal myoblast cells, HEK 293 human embryonic kidney cells and Caco-2 colon carcinoma cells adhered and spread exclusively on modified areas. The surfaces are durable for weeks under cell culture conditions and re-usable after removal of the cells by trypsin treatment. Arrays of adhesive spots seeded with cells at a low density permitted dynamic monitoring of cell proliferation. Selected colonies can be harvested from the surfaces by means of local trypsination. Thus, these techniques may provide useful tools for the isolation of clonal cell populations. Additionally, we demonstrate the possibility of surface-mediated gene delivery from the micro patterns. We show that DNA, complexed with a lipid reagent, can be adsorbed on modified poly(vinyl alcohol) coatings, resulting in spatially controlled adhesion and reverse transfection of HEK 293 cells.

Animals↗

PubMed2026

Bayesian inference of lineage trees by joint analysis of single-cell multimodal lineage-tracing data with BiLinT.

The advent of single-cell lineage-tracing technologies has enabled the simultaneous profiling of gene expression and lineage barcodes. However, accurate, high-resolution reconstruction of cell lineage trees remains challenging because most existing approaches treat these modalities separately and therefore fail to fully exploit their complementary information. Here we present BiLinT, a Bayesian framework that jointly models multimodal single-cell lineage-tracing data for lineage tree reconstruction. BiLinT integrates barcode evolution (a continuous-time Markov chain) with gene expression dynamics (an Ornstein-Uhlenbeck process) within a unified probabilistic model. Across synthetic and real data sets, BiLinT provides accurate lineage-tree reconstruction and reveals differentiation-associated clonal structure and developmental fate biases.

Journal Article↗

PubMed2003

The non-clonal and transitory nature of HIV in vivo.

From a posteriori analyses of genetic variation, recombination can only be identified when the parental genomes are distinct. For viruses like HIV-1, this requires the producer cell to be infected by more than one virus. Using fluorescence in situ hybridisation, the provirus copy numbers in splenocytes from two HIV-1 patients were determined. More than 75% of infected splenocytes harboured two or more proviruses, range 1-8, with a mean of approximately 3-4 per cell. Sequencing of amplified DNA from single laser micro-dissected cells showed an extraordinary degree of diversity while numerous recombinants were evident. Given the dynamics of HIV-1 turnover in vivo and a recombination rate of approximately 3 cross-overs per cycle, some genomes from a fifteen year old infection may have undergone as many cross-overs as bases in the genome. Thus, recombination profoundly influences HIV evolution and gives it a non-clonal and transitory nature in vivo.

HIV Infections↗

PubMed2006

Helper T cell-regulated B cell immunity.

In this review, we will discuss the cascade of cellular and molecular events in the immune response to protein antigens that regulate the development of high-affinity B cell memory. The behavior of antigen-experienced pMHCII+ dendritic cells DCs and the dynamics of their interaction with specific T-helper (Th) cells define the first developmental checkpoint for adaptive immunity in vivo. Recent studies provide insight into the basis of Th cell clonal selection and the requirements and consequences of antigen priming in this responsive Th cell compartment. Antigen-specific Th cells expand to become the cognate regulators of effector B cell responses and initiators of the germinal center reaction and memory B cell development. We will discuss the development and role of these diverse mixtures of antigen-specific B cells in the control of B cell memory and long-term humoral immunity that underpin effective protein vaccination.

Animals↗

PubMed2006

A phylogenetic method for detecting positive epistasis in gene sequences and its application to RNA virus evolution.

RNA virus genomes are compact, often containing multiple overlapping reading frames and functional secondary structure. Consequently, it is thought that evolutionary interactions between nucleotide sites are commonplace in the genomes of these infectious agents. However, the role of epistasis in natural populations of RNA viruses remains unclear. To investigate the pervasiveness of epistasis in RNA viruses, we used a parsimony-based computational method to identify pairs of co-occurring mutations along phylogenies of 177 RNA virus genes. This analysis revealed widespread evidence for positive epistatic interactions at both synonymous and nonsynonymous nucleotide sites and in both clonal and recombining viruses, with the majority of these interactions spanning very short sequence regions. These findings have important implications for understanding the key aspects of RNA virus evolution, including the dynamics of adaptation. Additionally, many comparative analyses that utilize the phylogenetic relationships among gene sequences assume that mutations represent independent, uncorrelated events. Our results show that this assumption may often be invalid.

DNA Mutational Analysis↗

PubMed1999

Plasma cell development in synovial germinal centers in patients with rheumatoid and reactive arthritis.

Plasma cells are found surrounding the inflammatory infiltrates of macrophages, T, and B cells in the synovial tissue of patients with rheumatoid and reactive arthritis. This characteristic arrangement suggests that in the synovial tissue CD20+ B cells differentiate into plasma cells. To examine clonal relationships, we have used micromanipulation to separately isolate CD20+ B cells and plasma cells from single infiltrates. DNA was extracted, and from both populations the VH/VL gene repertoires was determined. The data show that in the inflamed synovial tissue activated B cells are clonally expanded. During proliferation in the network of follicular dendritic cells, V gene variants are generated by the hypermutation mechanism. Surprisingly, we do not find identical rearrangements between CD20+ B cells and plasma cells. Nevertheless, the finding of clonally related plasma cells within single infiltrates suggests that these cells underwent terminal differentiation in the synovial tissue. These results indicate that B cell differentiation in the synovial tissue is a dynamic process. Whereas CD20+ B cells may turnover rapidly, plasma cells may well be long lived and thus accumulate in the synovial tissue. The analysis of individual B cells recovered from synovial tissue opens a new way to determine the specificity of those cells that take part in the local immune reaction. This will provide new insights into the pathogenesis of chronic inflammatory diseases like rheumatoid or reactive arthritis.

Adolescent↗

PubMed2001

Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

Adenosine Triphosphate↗

PubMed1981

Coexistence of myeloid and lymphoid neoplastic subpopulations in chronic myelogenous leukemia.

Serial cytogenetic studies have been performed on a child with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and correlated with clinical, morphologic, and cytochemical data in an attempt to elucidate further the natural history of leukemia progression observed in this disease. Once the initial blast crisis had occurred, two genetically different leukemic clones of cells, one presumably having arisen from the other, coexisted throughout the remainder of the patient's course. The initial clone comprised differentiating myeloid cells, while the second clone appeared on morphologic and cytochemical grounds to be lymphoid and blastic. A dynamic equilibrium existed between these two populations of cells, with the balance altered by chemotherapy and/or other selective pressures. The leukemic progression demonstrated in this case is consistent with the concept of clonal evolution of malignant cell populations. In planning therapy, it may be necessary to consider each of these coexistent clones and their different therapeutic requirements.

Child, Preschool↗

PubMed2006

Genetic structure of Galitzkya macrocarpa and G. potaninii, two closely related endemics of central Asian mountain ranges.

UNLABELLED: BACKGROUND AND AIMS Habitats in mountains are often isolated. Plants growing in these sites face severe dispersal limitations, but also difficulties for recruitment. The focus was laid on the magnitude of genetic differences among populations but also on the size of potentially occurring clones. METHODS: RAPD fingerprints were obtained from 23 populations in southern Mongolia. Sampling covered the entire distribution range of Galitzkya macrocarpa; samples of G. potaninii represented only the Mongolian part of its mainly northern Chinese range. KEY RESULTS: The Mongolian endemic G. macrocarpa showed moderately strong population differentiation (Phi ST = 0.251), and limited evidence for isolation by distance. Local genetic diversity was not positively correlated to habitat size, and not reduced in peripheral populations. Clonal growth is possible, but most plants originate from sexual reproduction. In contrast, populations of G. potaninii were highly differentiated (Phi ST = 0.550); and the most remote outposts had reduced genetic diversity. In these areas, isolation is expected to date back to glacial times. CONCLUSIONS: Effects of natural fragmentation differ among species. Both are rare, but G. macrocarpa appears to be able to maintain genetic diversity over its range. Clonal growth is an option in its mixed reproduction strategy and allows survival under harsh conditions. In contrast, genetic structure in G. potaninii gives reason for concern, and further studies on population dynamics are needed.

Asia, Central↗

PubMed2004

Modeling the development of acquired clinical immunity to Plasmodium falciparum malaria.

Individuals living in regions where malaria is endemic develop an acquired immunity to malaria which enables them to remain asymptomatic while still carrying parasites. Field studies indicate that cumulative exposure to a variety of diverse Plasmodium parasites is required for the transition from symptomatic to asymptomatic malaria. This study used a simulation model of the within-host dynamics of P. falciparum to investigate the development of acquired clinical immunity under different transmission conditions and levels of parasite diversity. Antibodies developed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), a clonally variant molecule, were assumed to be a key human immunological response to P. falciparum infection, along with responses to clonally conserved but polymorphic antigens. The time to the development of clinical immunity was found to be proportional to parasite diversity and inversely proportional to transmission intensity. The effect of early termination of symptomatic infections by chemotherapy was investigated and found not to inhibit the host's ability to develop acquired immunity. However, the time required to achieve this state was approximately double that compared to when no treatment was administered. This study demonstrates that an immune response primarily targeted against PfEMP1 has the ability to reduce clinical symptoms of infections irrespective of whether treatment is administered, supporting its role in the development of acquired clinical immunity. The results also illustrate a novel use for simulation models of P. falciparum infections, investigation of the influence of intervention strategies on the development of naturally acquired clinical immunity.

Animals↗