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Results for “clonal analysis”

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At least 1,153 records · Page 64Linked to original sources
PubMed2000

Shift in HIV resistance genotype after treatment interruption and short-term antiviral effect following a new salvage regimen.

OBJECTIVE: To investigate the changes in genotypic drug-resistance pattern, plasma HIV RNA and CD4 cell count after treatment interruption and assess the short-term antiviral effect of a new salvage regimen. DESIGN: Prospective study of 38 patients with multiple failing regimens who had completely stopped all medication for 3 months before a three to five-drug regimen was reintroduced according to clinical guidelines. METHODS: Patients were tested for HIV resistance before and after treatment interruption by population-based sequencing and clonal analysis of selected patients. RESULTS: Discontinuation of therapy for 3 months was associated with a median increase in HIV RNA of 0.4 log10 and a median decrease in CD4 cell count of 43 x 10(6)/l. Sixty-one per cent of patients had a shift from the drug-resistant genotype to a predominantly wild-type genotype. The patients significantly likely to show genotype reversion were those in Centers for Disease Control groups A or B, who had been exposed to few drugs, had a low plasma HIV RNA, or a high CD4 cell count. The only independent factor predicting genotype reversion was the clinical stage. The median change in plasma HIV RNA at month 3 after treatment reintroduction was -2.3 log10 copies/ml in patients who had genotype reversion compared with -0.6 log10 copies/ml in patients without genotype reversion (P = 0.004). CONCLUSION: Suspending treatment for 3 months after multiple failures could be a suitable strategy for optimizing salvage therapy provided it is instituted early, before the HIV disease becomes too advanced.

Adult↗
PubMed1992

Characterization of a human renal cell carcinoma specific cytotoxic CD8+ T cell line.

Human renal cell carcinoma (RCC) is one of the tumors most sensitive to immunotherapy and in that regard it is similar to malignant melanoma. Clinical studies reporting responses to therapy suggested that a host immune response may be involved in the antitumor activity induced by immunotherapy in these tumors. Although detection of a specific T cell response to melanoma has been well documented, this has not been the case for RCC. The lytic response of interleukin-2 (IL-2) cultured tumor-infiltrating lymphocytes (TILs) from RCC has been nonspecific. However, in this report we describe a CD8+ TIL line derived from a primary RCC tumor that displays specificity for the autologous tumor. This line is lytic for autologous RCC but does not lyse autologous lymphoblasts, allogeneic RCC, or tumor cell lines of other histologic types. It also proliferates specifically to the autologous tumor in the absence of exogenous IL-2. However, the addition of low dose IL-2 to the cultures can significantly augment its proliferative response. When stimulated with autologous RCC but not allogeneic RCC the CD8+ line will produce interferon-gamma (IFN-gamma). It appears that recognition of RCC by this TIL line is through the TCR/CD3 complex because anti-CD3 antibody blocks the lytic activity, proliferation and IFN-gamma production of the line in response to the autologous tumor. Additional studies illustrate that cytotoxic T lymphocytes with apparent specificity for the autologous tumor are present in unseparated cultured TILs that can be detected by clonal analysis. Collectively these results suggest that there is a specific T cell response to human RCC.

CD8 Antigens↗
PubMed1985

Alloreactive T cell responses between HLA-identical siblings. Detection of anti-minor histocompatibility T cell clones induced in vivo.

Cytotoxic T cell (CTL) clones were isolated from the peripheral blood of a patient 13 days following marrow transplantation from her HLA-identical brother. Nineteen of the clones were specifically reactive with host--but not donor--cells, but one clone was reactive with donor but not host cells. Family studies using the 19 clones showed reactivity patterns consistent with three non-HLA, minor histocompatibility antigens (minor-HA). The frequency of reactivity on a large panel of unrelated cells indicated a relatively limited degree of polymorphism. Each of the clones was restricted in its activity by a class I epitope common to HLA-B7, B27, and B40. These data demonstrate that by clonal analysis it is possible to identify in vivo antidonor and antihost alloreactive CTL clones directed against HLA determinants following marrow transplantation.

Antibodies, Monoclonal↗
PubMed1998

Promiscuous recognition of major histocompatibility complex class II determinants in cyclosporine-induced syngeneic graft-versus-host disease: specificity of cytolytic effector T cells.

BACKGROUND: Administration of the immunosuppressive drug cyclosporine after syngeneic/autologous bone marrow transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome, termed autologous or syngeneic GVHD, is associated with the development of a highly restricted repertoire of cytolytic T lymphocytes that promiscuously recognizes major histocompatibility complex class II determinants, including self. METHODS: Vbeta8.5+CD8+ effector lymphocytes and T-cell clones were isolated from Lewis rats with cylosporine-induced syngeneic GVHD. The specificity of the effector T cells and T-cell clones was examined in vitro. The pathogenicity of the T-cell clones was confirmed in vivo using a local graft-versus-host reaction assay. RESULTS: Clonal analysis reveals that the pathogenic effector T cells recognize a peptide from the invariant chain termed CLIP in association with major histocompatibility complex class II determinants. Moreover, there appears to be an additional interaction between the N-terminal flanking region of CLIP and the Vbeta segment of the T cell receptor. CONCLUSION: The results suggest that recognition of this highly conserved peptide along with the additional interaction between the flanking region and the T cell receptor may account for the promiscuous activity of the autologous/syngeneic GVHD autoreactive T cells.

Amino Acid Sequence↗
PubMed2001

Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency.

BACKGROUND: Ocular burns cause depletion of limbal stem cells, which leads to corneal opacification and visual loss. Autologous cultured epithelial cells can restore damaged corneas, but this technology is still developing. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets and to investigate whether such cultures can permanently restore total limbal stem cell deficiency. METHODS: We selected a homogeneous group of patients whose limbal cell deficiency was evaluated by scoring the gravity of the clinical picture and the keratin expression pattern. Stem cells, obtained from the limbus of the contralateral eye, were cultivated onto a fibrin substrate and their preservation was evaluated by clonal analysis. Fibrin cultures were grafted onto damaged corneas. RESULTS: Fibrin-cultured limbal stem cells were successful in 14 of 18 patients. Re-epithelialization occurred within the first week. Inflammation and vascularization regressed within the first 3-4 weeks. By the first month, the corneal surface was covered by a transparent, normal-looking epithelium. At 12-27 months follow-up, corneal surfaces were clinically and cytologically stable. Three patients had a penetrating keratoplasty approximately 1 year after restoration of their corneal surface. Their visual acuity improved from light perception or counting fingers to 0.8-1.0. CONCLUSIONS: Preservation of limbal stem cells in culture gives new perspectives on the treatment of ocular disorders characterized by complete limbal stem cell deficiency. The multicenter nature of this study and the handiness and ease of long-distance transportation of the fibrin-cultured epithelial sheets suggest that this technology can now be widely applied.

3T3 Cells↗
PubMed1996

Molecular genetics of hyperparathyroid disease.

Information on genetic abnormalities in primary hyperparathyroidism has accumulated gradually. Genetic alterations responsible for tumorigenesis have been identified in multiple endocrine neoplasia types 1 and 2. Point mutations in a calcium-sensing receptor gene were recently found to be responsible for familial hypocalciuric hypercalcaemia and neonatal severe hyperparathyroidism. Evidence has been provided that abnormalities of cell cycle regulation participate at tumorigenesis in parathyroid adenoma and carcinoma. Clonal analysis has shown that in renal hyperparathyroidism the parathyroid glands initially grow diffusely and polyclonally, after which foci of nodular hyperplasia are transformed to monoclonal neoplasia. Somatic changes of specific genes have been suspected of being responsible for parathyroid tumorigenesis in renal hyperparathyroidism. However, the genetic loci responsible for the frequent monoclonality largely remain to be identified, and heterogeneous genetic abnormalities may contribute to the progression of secondary parathyroid hyperplasia.

Adenoma↗
PubMed1995

Myelodysplastic syndrome.

Myelodysplastic syndrome continues to present a formidable clinical challenge. Despite considerable effort, no therapy apart from allogeneic bone marrow transplantation has been shown to prolong survival. Lack of effective therapy for myelodysplastic syndrome is of further concern given recent reports on the high incidence of myelodysplastic syndrome in patients undergoing intensive chemotherapy and radiation therapy for other malignancies. However, significant strides have been made in the past year toward understanding the molecular pathogenesis of some forms of myelodysplastic syndrome, as well as developing new approaches for therapy of myelodysplastic syndrome. This review highlights recent advances in the molecular genetics of myelodysplastic syndrome, including clonality analysis and identification of genes that are causally implicated in the pathogenesis of myelodysplastic syndrome; results from recent clinical trials for therapy of myelodysplastic syndrome using growth factors, chemotherapy or both; and recent literature on therapy-related myelodysplastic syndrome in intensively treated patients.

Antineoplastic Agents↗
PubMed2005

Patterns of allelic loss of synchronous adenocarcinomas of the lung.

Distinction of multiple primary lung carcinomas from intrapulmonary metastases using empiric clinical and histopathologic criteria can be difficult. Recent advances have provided several molecular markers that can be used for clonal analysis of separate tumor nodules and enhance tumor staging and subsequent treatment and prognosis. To address this issue, we performed a microdissection-based allelotyping of 20 cases of histologically similar, pathologic stage T4 adenocarcinomas (ADCs). Loss of heterozygosity (LOH) analysis included a panel of 15 polymorphic microsatellite markers located on 1p, 3p, 5q, 9p, 9q, 10q, 17p, and 22q. The tumor size, visceral pleural and angiolymphatic invasion, lymph node status, outcome, and survival were assessed. Allelotypes of 60 cases of solitary primary non-small cell lung carcinomas (NSCLC) (stages I-II) were used to define the percentage of discordant LOH patterns within solitary primary lung carcinoma that would discriminate between survivors and nonsurvivors. These criteria were used in the analysis of pathologic stage T4 ADC. Two groups of stage T4 cases were created: molecularly homogenous (< or = 40% discordances) (14 cases, 70%), and molecularly heterogenous (>40% discordances) (6 cases, 30%). Molecularly homogenous tumors were more frequently associated with visceral pleural invasion (92% vs. 8%) (P = 0.018). Allelotype did not correlate with age, gender, tumor size, tumor differentiation, lymph node status, angiolymphatic invasion, survival, or outcome. Our study showed that discordant and concordant genotypic profiles exist in morphologically similar synchronous ADC of the lung.

Adenocarcinoma↗
PubMed2006

Genetic evolution of gp41 reveals a highly exclusive relationship between codons 36, 38 and 43 in gp41 under long-term enfuvirtide-containing salvage regimen.

OBJECTIVE: To analyse the genetic changes in the gp41 protein in HIV-infected patients with detectable plasma viraemia receiving a long-term salvage enfuvirtide regimen. METHODS: We studied 13 heavily antiretroviral-experienced patients receiving a salvage regimen containing enfuvirtide. Substitutions in gp41 were analysed by population-based sequencing at baseline and longitudinally after the initiation of enfuvirtide treatment. To investigate sequence evolution we also analysed multiple gp41 clones from four selected patients. A Fisher's two-tailed test was used to assess the distribution of resistance-associated mutations in the clonal sequences. RESULTS: Mutations at positions 36 and 38 in gp41 (HR1) emerged rapidly (median emerging time 10 weeks), but disappeared at subsequent timepoints in most of the patients. Amino acid changes did not accumulate over time, with no patient having more than two mutations in HR1 after 6 months of treatment. The mutation N43D was not observed together with changes at positions 36 or 38 in any patient. Clonal analysis showed that the three main gp41 resistance mutations were highly mutually exclusive (P < 0.001), being present in individual clones and constituting independent populations. CONCLUSION: Substitutions at positions 36 and 38 are rapidly selected but disappear thereafter in HIV-1-infected patients failing an enfuvirtide-containing salvage therapy. We found a highly exclusive relationship between the three main enfuvirtide resistance-associated mutations (amino acids 36, 38 and 43), suggesting that the genetic evolution of HIV-1 gp41 protein is a dynamic and much more complex process than previously though.

Amino Acid Sequence↗
PubMed1981

Isolation of plaque variants differing in virulence from the 17D strain of yellow fever virus.

Two preparations of yellow fever vaccine (17D) were studied by clonal analysis. From one (17D-England) three types of clones were isolated, each differentiated by plaque size in Vero cells and virulence for intracerebrally inoculated mice: small plaque (SP) and large plaque (LP) clones were lethal, whereas medium plaque (MP) clones failed to kill at doses up to 10(6) p.f.u. Analysis of 24 randomly selected clones showed the original vaccine to be a mixture of predominantly MP and SP variants; 58% and 42% respectively. A single passage in suckling mice modified this composition to 90% SP and 10% LP variants. The response of six inbred strains of mice to intracerebral inoculation of the MP variant varied from complete resistance to complete susceptibility. From another 17D substrain (17D-South Africa) two types of variants were isolated (a SP avirulent type and a LP type of intermediate virulence), both different from the previously described variant.

Animals↗
PubMed1999

Clonal structure of Providencia alcalifaciens strains isolated from diarrhoeal stools in São Paulo, Brazil.

Clonal analysis based on ribotyping demonstrated that Providencia alcalifaciens strains isolated mainly from diarrhoeal stools in São Paulo, Brazil, were clustered into two main groups. Eleven distinct ribotype patterns were identified with ClAI, EcoRV and MluI restriction endonucleases. P. alcalifaciens strains with invasive properties were of two ribotype patterns that differed from those identified among non-invasive strains. The ribotyping results confirmed that P. alcalifaciens strains associated with diarrhoeal disease in São Paulo represent distinct groups of strains. Although the invasive strains were isolated from different patients over an extended period they were clustered into two genetically related clones, which seemed to be distributed endemically in the population studied.

Brazil↗
PubMed1997

Hedgehog directly controls initiation and propagation of retinal differentiation in the Drosophila eye.

Patterning of the compound eye begins at the posterior edge of the eye imaginal disc and progresses anteriorly toward the disc margin. The advancing front of ommatidial differentiation is marked by the morphogenetic furrow (MF). Here we show by clonal analysis that Hedgehog (Hh), secreted from two distinct populations of cells has two distinct functions: It was well documented that Hh expression in the differentiating photoreceptor cells drives the morphogenetic furrow. Now we show that, in addition, Hh, secreted from cells at the posterior disc margin, is absolutely required for the initiation of patterning and predisposes ommatidial precursor cells to enter ommatidial assembly later. These two functions of Hh in eye patterning are similar to the biphasic requirement for Sonic Hh in patterning of the ventral neural tube in vertebrates. We show further that Hh induces ommatidial development in the absence of its secondary signals Wingless (Wg) and Dpp and that the primary function of Dpp in MF initiation is the repression of wg, which prevents ommatidial differentiation. Our results show that the regulatory relationships between Hh, Dpp, and Wg in the eye are similar to those found in other imaginal discs such as the leg disc despite obvious differences in their modes of development.

Animals↗
PubMed2006

Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation.

Regulation of stem cell self-renewal versus differentiation is critical for embryonic development and adult tissue homeostasis. Drosophila larval neuroblasts divide asymmetrically to self-renew, and are a model system for studying stem cell self-renewal. Here we identify three mutations showing increased brain neuroblast numbers that map to the aurora-A gene, which encodes a conserved kinase implicated in human cancer. Clonal analysis and time-lapse imaging in aurora-A mutants show single neuroblasts generate multiple neuroblasts (ectopic self-renewal). This phenotype is due to two independent neuroblast defects: abnormal atypical protein kinase C (aPKC)/Numb cortical polarity and failure to align the mitotic spindle with the cortical polarity axis. numb mutant clones have ectopic neuroblasts, and Numb overexpression partially suppresses aurora-A neuroblast overgrowth (but not spindle misalignment). Conversely, mutations that disrupt spindle alignment but not cortical polarity have increased neuroblasts. We conclude that Aurora-A and Numb are novel inhibitors of neuroblast self-renewal and that spindle orientation regulates neuroblast self-renewal.

Animals↗
PubMed2000

Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification.

The origins of specificity in gene expression are a central concern in understanding developmental control. Mediator protein complexes regulate transcriptional initiation, acting as modular adaptors linking specific transcription factors to core RNA polymerase II. Here, we identified the Drosophila homologs of 23 human mediator genes and mutations of two, dTRAP240 and of dTRAP80 (the putative fly homolog of yeast SRB4). Clonal analysis indicates a general role for dTRAP80 necessary for cell viability. The dTRAP240 gene is also essential, but cells lacking its function are viable and proliferate normally. Clones reveal localized developmental activities including a sex comb cell identity function. This contrasts with the ubiquitous nuclear accumulation of dTRAP240 protein in imaginal discs. Synergistic genetic interactions support shared developmental cell and segment identity functions of dTRAP240 and dTRAP80, potentially within a common complex. Further, they identify the homeotic Sex combs reduced product, required for the same cell/tissue identities, as a functional partner of these mediator proteins.

Amino Acid Sequence↗
PubMed2001

Delta signaling from the germ line controls the proliferation and differentiation of the somatic follicle cells during Drosophila oogenesis.

The body axes of Drosophila are established during oogenesis through reciprocal interactions between the germ line cells and the somatic follicle cells that surround them. The Notch pathway is required at two stages in this process: first, for the migration of the follicle cells around the germ line cyst and, later, for the polarization of the anterior-posterior (A-P) axis of the oocyte. Its function in these events, however, has remained controversial. Using clonal analysis, we show that Notch signaling controls cell proliferation and differentiation in the whole follicular epithelium. Notch mutant follicle cells remain in a precursor state and fail to switch from the mitotic cell cycle to the endocycle. Furthermore, removal of Delta from the germ line produces an identical phenotype, showing that Delta signals from the germ cells to control the timing of follicle cell differentiation. This explains the axis formation defects in Notch mutants, which arise because undifferentiated posterior follicle cells cannot signal to polarize the oocyte. Delta also signals from the germ line to Notch in the soma earlier in oogenesis to control the differentiation of the polar and stalk follicle cells. The germ line therefore regulates the development of the follicle cells through two complementary signaling pathways: Gurken signals twice to control spatial patterning, whereas Delta signals twice to exert temporal control.

Animals↗
PubMed1994

Cell-autonomous cytokinin-independent growth of tobacco cells transformed by Agrobacterium tumefaciens strains lacking the cytokinin biosynthesis gene.

Mutations at the cytokinin biosynthesis locus (tmr) of Agrobacterium tumefaciens usually result in strains that induce tumors exhibiting the rooty phenotype associated with high auxin-to-cytokinin ratios. However, tobacco (Nicotiana tabacum cv Havana 425) leaf disc explants responded to tmr- mutant strain A356 by producing rapidly growing, unorganized tumors, indicating that these lines can grow in a cytokinin-independent fashion despite the absence of a functional tmr gene. Several methods have been used to characterize the physiological and cellular basis of this phenotype. The results indicate that tmr- tumors have a physiologically distinct mechanism for cytokinin-independent growth in comparison to tumors induced by wild-type bacteria. The cytokinin-independent phenotype of the tmr- transformants appears to be cell autonomous in nature: only the transformed cells and their progeny were capable of cytokinin-independent growth. Specifically, the tmr- tumors did not accumulate cytokinin, and clonal analysis indicated the tmr- transformed cells were not capable of stimulating the growth of neighboring nontransformed cells. Finally, the cytokinin-independent phenotype of the tmr- transformants was shown to be cold sensitive, whereas the wild-type tumors exhibited a cold-resistant cytokinin-independent phenotype. Potential mechanisms for this novel form of cytokinin-independent growth, including the role of the dehydrodiconiferyl alcohol glucosides found in both tumor types, are discussed.

Agrobacterium tumefaciens↗
PubMed2004

Laser-capture microdissection: applications in routine molecular dermatopathology.

Advances in molecular pathology with the introduction of the Southern blot technique and the polymerase chain reaction (PCR) have emerged as important tools, which are frequently used in routine dermato-histopathology. Applications for PCR-based diagnostics are particularly helpful for the determination of clonality in cutaneous lymphocytic infiltrates and for detection of infectious agents, such as herpes simplex virus (HSV), varicella zoster virus (VZV), Borrelia burgdorferi, Mycobacteria, Leishmania, and Treponema pallidum. As biopsies are always composed of different cells, the cells of interest are often only a minor population. As a consequence, their specific DNA is diluted by the majority of contaminating cells. Another problem is the time- and labor-intensive DNA extraction, because usually only formalin-fixed, paraffin-embedded tissue is available, which makes molecular diagnostics a time and labor consuming, and consequently a cost-intensive procedure. To overcome these shortcomings and to eventually shorten the time to generate a result, we introduce a laser-capture microdissection (LCM)-based method for the detection of infectious agents and clonality. Only the cells of interest for the particular indication are microdissected (e.g. epidermal cells for HSV and VZV and lymphocytes for clonality analysis) and subjected to PCR amplification. Due to an accelerated DNA-extraction procedure which generates DNA in 5 h (compared to 3-4 days using conventional DNA extraction), we are able to generate a result within one working day.

Animals↗
PubMed2004

T-cell clonality to Porphyromonas gingivalis and human heat shock protein 60s in patients with atherosclerosis and periodontitis.

Individuals with periodontitis have been reported to have a significantly increased risk of developing coronary heart disease. Several studies have demonstrated that the immune response to heat shock protein 60 (HSP60) may be involved in the pathogenesis of both atherosclerosis and chronic periodontitis. To investigate this possible link between these diseases, cellular and humoral immune responses to HSP60 in atherosclerosis patients were compared with those in periodontitis patients and healthy subjects using human and Porphyromonas gingivalis HSP60 (GroEL) as antigens. Antibody levels to both human and P. gingivalis HSP60s were the highest in atherosclerosis patients, followed by periodontitis patients and healthy subjects. Clonal analysis of the T cells clearly demonstrated the presence of not only human HSP60- but also P. gingivalis GroEL-reactive T-cell populations in the peripheral circulation of atherosclerosis patients. Furthermore, these HSP60-reactive T cells seemed to be present in atherosclerotic lesions in some patients. These results suggest that T-cell clones with the same specificity may be involved in the pathogenesis of the different diseases.

Adult↗