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Results for “clonality”

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PubMed2004

Determination of epidemic clonality among multidrug-resistant strains of Acinetobacter spp. and Pseudomonas aeruginosa in the MYSTIC Programme (USA, 1999-2003).

The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Programme was initiated in 1997 (1999 for the United States). This program monitors resistance in participant medical centers where carbapenems are prescribed and drug use data can be obtained. An earlier report found antimicrobial use was not a clear cause of local or aggregate changes in resistance rates. This study addresses the role of dissemination of resistant clones on susceptibility rates for nonfermentors, Acinetobacter spp. (ACB) and Pseudomonas aeruginosa (PSA). Carbapenem (CARB)-multidrug-resistant strains (MDR) from among 236 ACB and 1,111 PSA were tested by reference broth microdilution methods, automated ribotyping, and pulsed field gel electrophoresis to determine possible clonal dissemination. Each strain was also tested for metallo-beta-lactamases (MbetaL) (phenotypic and polymerase chain reaction); and then analyzed by CARB-R rate and defined daily dose (DDD)/100 days use groupings (high, moderate, and low). For the aggregate 15 sites in the MYSTIC Programme each year, the CARB-resistant rate decreased over 5 years; but other drug-resistance rates generally escalated. Changes were not related to antimicrobial use calculations. The discovered clonally spread MDR-PSA strains were more frequent in high- (1.8 clones/site) and moderate-resistance (0.6 clones/site) rate centers (21.7% to 29.5% were clonal), compared with unique strains in low-resistance hospitals. ACB clonality was extreme in one geographic area, with dissemination of 5 different clones (931.7/B, C, or D; 1090.2/A; 167.5/A) in 4 centers (02, 04, 06, and 18). Resistance rates in ACB and PSA were clearly related to clonal occurrence and spread, and one MbetaL (VIM-7) was detected. Decreased CARB resistance rates from 1999 through 2002 were directly attributed to the disappearance of resistance clones in some locations. In conclusion, ACB and PSA CARB and MDR resistance rates in MYSTIC Programme institutions have been greatly influenced by clonal dissemination and less by antimicrobial use patterns. The most serious examples of resistance were the clonality observed among ACB in New York City and the documented endemic nature of VIM-7-producing PSA (0.09% of all PSA isolates). Meropenem remained the most active antimicrobial agent tested in the program, and surveillance networks must implement epidemiologic typing to accurately assess the role of clonal spread on the study results.

Acinetobacter↗

PubMed2006

Genetic instability and clonal expansion.

Inactivation of tumor suppressor genes can lead to clonal expansion. We study the evolutionary dynamics of this process and calculate the probability that inactivation of a tumor suppressor gene is preceded by mutations in genes that confer genetic instability. Unstable cells might have a slower rate of clonal expansion than stable cells because of an increased probability of generating lethal mutations or inducing apoptosis. We show that the different growth rates of genetically stable and unstable cells during clonal expansion represent, in general, only a small disadvantage for genetic instability. The intuitive reason for this conclusion is that robust clonal expansion, where cellular birth rates are significantly greater than death rates, occurs on a much faster time scale than waiting for those mutations that allow clonal expansion. Moreover, in special cases where clonal expansion is very slow, genetically unstable cells have a higher probability to accumulate additional mutations during clonal expansion that confer a selective advantage. Clonal expansion represents a major disadvantage for genetic instability only when inactivation of the tumor suppressor gene leads to a very small increase of the cellular reproductive rate that is cancelled by the increased mortality of unstable cells.

Animals↗

PubMed2004

Clonal reproduction and linkage disequilibrium in diploids: a simulation study.

Estimating the rate of clonal reproduction in natural population of diploid organisms is recognised as being problematic and even the detection of strictly clonal populations is often controversial. One well-acknowledged signature of clonal reproduction is the generation of non-random associations between loci. Linkage disequilibrium (LD) is thus often used for estimating the amount of clonal reproduction. Here we explore with computer simulations the effect of the rate of clonal reproduction on LD estimates obtained from different estimators within a comprehensive parameter range. None of the LD estimators studied is able to accurately measure the proportion of clonal (or sexual) reproduction on its own, due to strong bias, incoherent behaviour, or huge variances. The joint use of several statistics is thus recommended for the estimation rates of clonal reproduction in natural populations. We hope that our work will provide useful tools for the study of clonal diploids, many of which can only be studied with molecular markers, as it is the case for medically important parasites.

Clone Cells↗

PubMed1998

Blood thrombopoietin levels in clonal thrombocytosis and reactive thrombocytosis.

BACKGROUND: Although the distinction between clonal and reactive thrombocytosis is clinically relevant because clonal thrombocytosis has more thrombohemorragic complications, the differential diagnosis of these two entities can be difficult. Methods such as the detection of unstimulated erythroid or megakaryocyte colony growth are not readily available. Therefore, we measured blood thrombopoietin levels to determine whether these levels can be used to distinguish the two conditions. PATIENTS AND METHODS: Thrombopoietin levels were measured in 73 patients with thrombocytosis (platelet count > 500,000/microL), including 39 patients with clonal thrombocytosis (20 patients with essential thrombocythemia, 15 with agnogenic myeloid metaplasia, 1 patient with polycythemia vera, and 3 with undefined myeloproliferative disorders) and 34 patients with reactive thrombocytosis (17 with malignant tumors, 11 with inflammatory diseases, 4 with sickle cell disease, and 2 with iron deficiency anemia). Seventeen normal volunteers were used as controls. RESULTS: Thrombopoietin levels were significantly higher (P < 0.05) in patients with clonal thrombocytosis (mean +/- SD of 555 +/- 585 pg/mL), including the subgroup with essential thrombocythemia (505 +/- 459 pg/mL), than in patients with reactive thrombocytosis (290 +/- 133 pg/mL) who had similar levels as controls (201 +/- 112 pg/mL). Thrombopoietin levels in patients with clonal thrombocytosis, including essential thrombocythemia, were not correlated with platelet counts. CONCLUSIONS: Thrombopoietin levels may be helpful in distinguishing between clonal thrombocytosis and reactive thrombocytosis. Thrombopoietin is probably responsible for the elevated platelet counts in clonal thrombocytosis including essential thrombocythemia, but not in reactive thrombocytosis. High thrombopoietin levels in patients with clonal thrombocytosis cannot be explained solely by platelet megakaryocyte mass.

Adult↗

PubMed2001

Clonal hemopoiesis and risk of thrombosis in young female patients with essential thrombocythemia.

OBJECTIVE: Several studies demonstrated a high prevalence of nonrandom X-chromosome inactivation pattern (X-CIP) in essential thrombocythemia (ET). This study explored the incidence of clonal hemopoiesis in myeloid precursors and endogenous erythroid colonies (EECs) in ET patients and its correlation with thrombotic manifestations. MATERIALS AND METHODS: Clonal analysis of hemopoiesis using X-CIP was performed in 40 female patients with ET. Median age was 40.5 years (range 20-64), and median platelet count at testing time was 700 x 10(9)/L (range 220-1300 x 10(9)/L). Patients older than 65 years were excluded to reduce age-related skewing. Clonality was assessed on neutrophils, platelets, EECs, and bone marrow CD34(+) cells. RESULTS: Eight (20%) of 40 patients developed thrombosis mainly at diagnosis. Clonal hemopoiesis was found in 17 (42.5%) patients, 15 (37.5%) had polyclonal hemopoiesis, and 8 (20%) were considered uninterpretable due to constitutive skewing. Clonality was confirmed on purified CD34(+) subpopulations from bone marrow, documenting that clonality does not appear lineage-restricted. There were no statistical differences in age at diagnosis, median platelet count at testing time, and length of follow-up. Thrombotic episodes were significantly more frequent in the monoclonal group (p = 0.04, Fisher exact test). CONCLUSIONS: Young female patients with ET exhibiting a clonal pattern of hemopoiesis by X-CIP analysis are at higher risk for thrombosis. X-CIP analysis may contribute to defining the individual risk leading to appropriate treatment. X-CIP will allow a correct diagnosis in patients with latent myeloproliferative disorders and thrombosis in unusual sites. Clonal hemopoiesis is easily recognized by X-CIP, but its applicability is limited to the female sex and is hampered by the presence of age-related or constitutive skewing.

Adult↗

PubMed2000

Clonal patterns of X-chromosome inactivation in female patients with aplastic anaemia studies using a novel reverse transcription polymerase chain reaction method.

Conflicting results have been published on the frequency of clonal patterns of X-chromosome inactivation in female patients with aplastic anaemia. Previous studies have used DNA methylation to measure X-inactivation, but aberrant methylation is known to occur in some situations. We have developed a non-radioactive reverse transcription polymerase chain reaction (RT-PCR) method to study expression of the polymorphism at nt. 1311 of the G6PD gene at the RNA level. Using this, and a similar method for the iduronate-2-sulfatase (IDS) gene, we have re-evaluated X-inactivation in AA patients. 32/35 normal individuals showed polyclonal haemopoiesis. Patients with presumed clonal diseases showed both monoclonal and polyclonal patterns, consistent with previous reports. Overall, clonal patterns were observed in granulocytes of 10/26 AA patients (38%), a significantly higher proportion than in controls (p<0.01). Two cases showed discordance between lymphocytes and granulocytes, indicating clonality arising within the myeloid lineage. Eight cases showed clonal patterns in both myeloid and lymphoid cells, indicating the involvement of a pluripotent stem cell. Clonal patterns did not correlate with age, but there appeared to be an association with duration of disease. In PNH patients, CD59-negative cells showed clonal patterns of X-inactivation. In two cases, however, clonal patterns were also detected in CD59-positive cells.

Alleles↗

PubMed1997

Clonality analysis as a tool to study the biology and response to therapy in myelodysplastic syndromes.

We examined the bone marrow of 45 patients with MDS at the time of diagnosis and in the course of the disease by means of Southern blot analysis and cytogenetic studies to detect and evaluate clonal markers and their implication on the prognosis of the disease and the response to treatment. All patients were enrolled in an EORTC study and received low-dose Ara-C with or without growth factors according to the study protocol. Thirty patients (67%) were characterized by different clonal markers, such as various gene rearrangements (eg Ig-JH, tcR-beta, bcr, GM-CSF, G-CSF or IL-3) and/or chromosomal markers at the time of diagnosis or early in the course of the disease. In 23 of 30 cases that could be studied in the course of the disease, a statement about the clonal situation was possible: in three cases (8%) the clonal situation did not change, in nine cases (39%) at least a transient reduction of clonal cells could be demonstrated, suggesting partial or complete response to therapy. In eight cases (35%) a change for the worse could be seen. In four cases (17%) involvement of multiple clones could be demonstrated with the clones exhibiting different susceptibilities to treatment. Clinical evaluation showed that patients without clonal markers at diagnosis had a better prognosis as compared to patients who presented with clonal markers. We suggest that clonality analysis at diagnosis and in the course of the disease will be a useful tool to study the biology and response to treatment in MDS.

Blotting, Southern↗

PubMed1997

Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations.

Molecular analysis of T cell receptor (TCR) genes is frequently used to prove or exclude clonality and thereby support the diagnosis of suspect T cell proliferations. PCR techniques are more and more being used for molecular clonality studies. The main disadvantage of the PCR-based detection of clonal TCR gene rearrangements, is the risk of false-positive results due to 'background' amplification of similar rearrangements in polyclonal reactive T lymphocytes. Therefore, PCR-based clonality assessment should include analyses that discern between PCR products derived from monoclonal and polyclonal cell populations. One such method is heteroduplex analysis, in which homo- and heteroduplexes resulting from denaturation (at 94 degrees C) and renaturation (at lower temperatures) of PCR products, are separated in non-denaturing polyacrylamide gels based on their conformation. After denaturation/renaturation, PCR products of clonally rearranged TCR genes give rise to homoduplexes, whereas in case of polyclonal cells heteroduplexes with heterogeneous junctions are formed. We studied heteroduplex PCR analysis of TCR gene rearrangements with respect to the time and temperature of renaturation and the size of the PCR products. Variation in time did not have much influence, but higher renaturation temperatures (>30 degrees C) clearly showed better duplex formation. Nevertheless, distinction between monoclonal and polyclonal samples was found to be more reliable at a renaturation temperature of 4 degrees C, using relatively short PCR products. To determine the sensitivity of heteroduplex analysis with renaturation at 4 degrees C, (c)DNA of T cell malignancies with proven clonal rearrangements was serially diluted in (c)DNA of polyclonal mononuclear peripheral blood cells and amplified using V and C primers (TCRB genes) or V and J primers (TCRG and TCRD genes). Clonal TCRB and TCRD gene rearrangements could be detected with a sensitivity of at least 5%, whereas the sensitivity for TCRG genes was somewhat lower (10-15%). The latter could be improved by use of Vgamma member primers instead of Vgamma family primers. We conclude from our results that heteroduplex PCR analysis of TCR gene rearrangements is a simple, rapid and cheap alternative to Southern blot analysis for detection of clonally rearranged TCR genes.

Gene Rearrangement, T-Lymphocyte↗

PubMed2006

Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.

Gene Amplification↗

PubMed2003

Effects of four generations of density-dependent selection on life history traits and their plasticity in a clonally propagated plant.

Life history evolution of many clonal plants takes place with long periods of exclusively clonal reproduction and under largely varying ramet densities resulting from clonal reproduction. We asked whether life history traits of the clonal herb Ranunculus reptans respond to density-dependent selection, and whether plasticity in these traits is adaptive. After four generations of exclusively clonal propagation of 16 low and 16 high ramet-density lines, we studied life history traits and their plasticities at two test ramet-densities. Plastic responses to higher test-density consisted of a shift from sexual to vegetative reproduction, and reduced flower production, plant size, branching frequency, and lengths of leaves and internodes. Plants of high-density lines tended to have longer leaves, and under high test-density branched less frequently than those of low-density lines. Directions of these selection responses indicate that the observed plastic branching response is adaptive, whereas the plastic leaf length response is not. The reverse branching frequency pattern at low test-density, where plants of high-density lines branched more frequently than those of low-density lines, indicates evolution of plasticity in branching. Moreover, when grown under less stressful low test-density, plants of high-density lines tended to grow larger than the ones of low-density lines. We conclude that ramet density affects clonal life-history evolution and that under exclusively clonal propagation clonal life-history traits and their plasticities evolve differently at different ramet densities.

Adaptation, Biological↗

PubMed1986

Temporal invariance and clonal uniformity of brain and cerebrospinal IgG, IgA, and IgM in multiple sclerosis.

Elevated cerebrospinal fluid (CSF) IgG and oligoclonal IgG bands on electrophoresis are valuable clinical markers for B cell proliferation in the brains of patients with multiple sclerosis (MS). Using two-dimensional electrophoresis, (2DE) we have established that the humoral immune response in MS brain is characterized by finite clonal complexity for the major Ig classes. An important question is whether this immune response is clonally stable or varies with time, related to the development of new lesions and random entry of B cells into the MS brain. To investigate this, we performed serial electrophoretic studies on CSF obtained from 19 patients with MS; the intervals ranged from 7 to 12 yr, with a mean of 8 yr. These analyses included studies of IgG, IgA, and IgM, and revealed that the humoral immune response in MS is clonally stable over long periods. Spontaneous fluctuations or reduction in CSF IgG levels by drugs did not qualitatively affect B cell clonal proliferation in MS brain, in that dominant bands and spots were not obliterated. It has been asserted that IgG synthesis in MS is nonsense antibody because the spectotypes of IgG isolated from different regions of MS brains differ. Factors other than clonal heterogeneity could account for differences found using one-dimensional analysis. B cell clonal products resolve into unique and well-resolved spots by 2DE; the method is uniquely suitable for analysis of restricted immune responses. Therefore, Ig were isolated from 11 regions of three MS brains and the 2DE patterns were compared. The similarity of the 2DE patterns indicate unequivocally that major clones are distributed uniformly although some clones are more prominent in some brain areas. IgA and IgM isolated from the same areas also showed similar patterns. Furthermore, the patterns of light and heavy chains in brain regions differed from serum but were similar to the autologous CSF, providing new evidence that CSF IgG in MS derives from synthesis in situ. Our results indicate that, once initiated, B cell clonal proliferation persists indefinitely and is little altered qualitatively at a clonal level over time, even when CSF IgG levels change or are altered by drugs. Our results are consistent with allotype and idiotype analysis of Ig production in MS and conflict with nonsense antibody proposals of the origin and nature of in situ synthesized Ig in MS.

Blood-Brain Barrier↗

PubMed2001

Evidence of B cell clonal expansion in HIV type 1-infected patients.

HIV-1 infection results in a gradual decrease in CD4(+) T cell counts and progressive immune deficiency. Increased T cell turnover in HIV-1-infected patients, which can be interpreted as T cell clonal expansion, has been thought to be relevant to its pathogenesis. To investigate whether B cell clonal expansion also occurs in HIV-1-infected patients, we examined the expressed V(H)DJ(H) gene sequences of peripheral B cells in HIV-1-infected patients with hypergammaglobulinemia. Identical V(H)DJ(H) gene rearrangements with additional nucleotide differences in V(H) genes were analyzed as a marker of clonally related B cells. From healthy individuals and HIV-1-uninfected patients with hypergammaglobulinemia, clonally related B cells were detected in none of 10 (0%) and 2 of 10 (20%), respectively. No clonally related B cells were detected in any of the nine HIV-1-infected patients with detectable viral loads and normal Ig levels (0%). In contrast, from 9 of 14 HIV-1-infected patients with hypergammaglobulinemia (64%), clonally related B cells were detected. In addition, no HIV-1-infected patients who exhibited normal Ig levels after antiretroviral therapy had clonally related B cells. These findings suggest that B cell clonal expansion is present in HIV-1-infected patients with hypergammaglobulinemia.

Adult↗

PubMed1995

Clonal VDJ recombination of the immunoglobulin heavy chain gene by PCR in classical Hodgkin's disease.

Although Hodgkin's disease (HD) has been a subject of much investigation, fundamental questions remain unanswered regarding its lineage and clonality. The authors used a polymerase chain reaction (PCR) technique to investigate whether clonal Variable-Diversity-Joining recombination of the immunoglobulin heavy (IgH) chain gene, a phenomenon that characterizes clonal B-cell proliferations, exists in nodular sclerosing (NSHD) and mixed cellularity (MCHD) Hodgkin's disease (so-called "classical" Hodgkin's disease). The isolation of DNA from paraffin-embedded tissue sections allowed for direct correlation of PCR results with the cell populations that were analyzed. Thirty-two cases were studied. These included 12 cases in which the Reed-Sternberg (RS) cells expressed the B-cell antigen, CD20, and 10 cases that were classified as syncytial variant of NSHD (3 CD20+, 7 B-cell antigen negative). Overall, clonal patterns of VDJ PCR products were found in 14 of 32 (44%) cases. These clonal patterns were identified in 7 of 12 (58%) cases of CD20+ classical HD and in 7 of 20 (35%) cases of B-antigen-negative classical HD. Clonal patterns were found in 3 of 10 cases of syncytial variant of NSHD, including 2 of 3 (67%) CD20+ cases and 1 of 7 (14%) B-cell antigen-negative cases. The results of this study provide support that a subset of HD represents a clonal B-cell neoplasm, and indicate that clonal IgH VDJ sequences are more frequently found in CD20+ HD.

Antigens, CD20↗

PubMed2000

Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction.

Polymerase chain reaction (PCR) technique is widely used in the diagnosis of lymphoma, and PCR amplification products are typically detected by polyacrylamide gel electrophoresis (PAGE). However, the identification of small clonal populations, or the distinction of clonal PCR products in a polyclonal milieu remains difficult, requiring technically demanding alterations to gel analysis. This study describes an alternative approach using a capillary electrophoresis (CE) system to produce an accurately sized electropherogram. A variety of patient samples were examined, including solid tissue, peripheral blood, bone marrow aspirates, and paraffin-embedded tissue. A total of 28 samples were evaluated by PCR for B-cell clonality by detection of immunoglobulin heavy chain gene rearrangement and 29 samples for T-cell clonality by detection of T-cell gamma locus gene rearrangement. Standard 10% PAGE analysis of PCR products was compared with CE. There was a 100% concordance in the assessment of both B-cell and T-cell clonality. Dilution studies with the SUP-B15 cell line showed a detection limit of 0.03% for B-cell clonality and 0.05% for T-cell clonality using CE, versus 0.2% to 1%, respectively for PAGE. Automated, fluorescent analysis of PCR products by CE seems to be at least equally as effective as gel-based analysis for the detection of clonal B-cell and T-cell populations. Moreover. CE offers superior resolution and improved sensitivity, thus representing a significant improvement over traditional gel electrophoretic techniques in these regards.

B-Lymphocytes↗

PubMed2003

Skew in T-cell receptor usage and clonal T-cell expansion in patients with chronic rejection of transplanted kidneys.

BACKGROUND: It is not known whether alloreactive T cells are involved in chronic rejection of transplanted kidneys. The aim of the present study was to determine the involvement of T cells in the chronic graft rejection. METHODS: T-cell receptor (TCR) variable region alpha-chain and TCR variable region beta-chain repertoires were analyzed in peripheral blood mononuclear cells. T-cell clonalities were analyzed by complementarity-determining region 3 size spectratyping. RESULTS: A significant increase in the frequencies of one or more TCR variable region alpha-chain and TCR variable region beta-chain segments was detected in 13 and 15 of the 24 kidney transplant recipients, respectively. The extent of the skew in the TCR usage was correlated with the levels of clonal T-cell expansion, indicating that the clonally expanding T cells were responsible for the skew in the TCR usage. The levels of the skew in the TCR usage and clonal T-cell expansion were significantly greater in the recipients with a graft failure than in those with a stable graft function ( P=0.0081 and P=0.012, respectively). These results indicate that the clonally expanding T cells in the periphery may be related to graft rejection. The percent increase in the serum creatinine levels, which reflected the deterioration of the kidney functions, was significantly higher in the recipients who showed high levels of clonal T-cell expansion than in those who did not ( P=0.021). CONCLUSIONS: The results demonstrate that clonal T-cell expansion in the periphery has a negative impact on the long-term graft functions, and that analysis of the clonal T-cell expansion in peripheral blood mononuclear cells provide significant information on the fate of the transplanted kidney.

Adolescent↗

PubMed2002

Geographic patterns of clonal diversity in the earth mite species Penthaleus major with particular emphasis on species margins.

Patterns of clonal diversity in parthenogenetic species can be influenced by occasional sexual reproduction, selection due to environmental heterogeneity, and genetic drift. Here we investigate geographic patterns of clonal diversity in the obligate parthenogenetic earth mite species, Penthaleus major, in southeastern Australia. Spatial autocorrelation analysis indicated a relationship between geographic distance and clonal diversity at the sites. However, there was no evidence for an association between distance and clonal composition or the frequency of the two most common clones. Regression analyses indicated that clonal diversity and the frequency of one of the common clonal groups were associated with latitude and/or climatic variables, whereas the other common group was not associated with any of those variables. In four transects (each 250-400 km in length) extending from the center of the distribution of P. major to its margins, there was a consistent and continuous decline in clonal diversity as the margin was approached but no evidence that one clonal group predominated at the margin. This argues against the importance of a general-purpose genotype being favored at margins and suggests that either selection acts to decrease local diversity at sites near the margin or that diversity declines due to drift at these sites.

Animals↗

PubMed1997

A realistic approach to the sensitivity of PCR-DGGE and its application as a sensitive tool for the detection of clonality in cutaneous T-cell proliferations.

The practical value of the detection of clonality within the T-cell receptor gamma locus by polymerase chain reaction for the diagnosis of cutaneous T-cell lymphomas is well known. However, studies dealing with this subject so far, with special emphasis on the sensitivity of the technique in comparison to, for example, Southern blotting have used mixtures of DNA in various concentrations instead of using mixtures of the cells involved, which would reflect the in vivo situation in a more realistic scope. The purpose of this study was therefore to determine the sensitivity and the limitations of the PCR assay by dilution experiments, using mixtures of cells. Furthermore we studied its applicability to cutaneous T-cell proliferative disorders. Two clonal T-cell lines (MyLa and Jurkat) served as positive control. Dilutions of MyLa cells, cultured normal human keratinocytes and peripheral blood mononuclear cells from lymphoma negative volunteers were used to assess the sensitivity of the PCR-DGGE assay. Skin samples from 4 patients with cutaneous T-cell lymphoma, 1 lesional lymph node, 2 blood samples from a patient with Sézary syndrome and 4 lymphoma-negative tissue samples were analysed. Two samples were uncertain for diagnosis of lymphoma. The PCR-DGGE assay consisted of a 2-round nested PCR with consensus primers within the TCR-gamma locus followed by electrophoretic separation of the product along a denaturing urea/formamide gradient gel. PCR-DGGE sensitivity was, to our knowledge, for the first time investigated for mixtures of lymphocytes (clonal and polyclonal) and keratinocytes. Clonal T-cells were detected in a concentration between 1-0.1% in keratinocytes, whereas the sensitivity was generally lower upon dilution in peripheral blood mononuclear cells or in a mixture of keratinocytes and peripheral blood mononuclear cells. Nevertheless, T-cell clonality was detected in 2 blood samples of a patient with Sézary syndrome, which were negative by Southern blot analysis. The crucial point of this work was the new approach to establish the sensitivity of the PCR-DGGE, in a way which more closely mimics the condition of clinical specimens. Instead of mixing and amplifying DNA extracted from clonal T-cell lines and polyclonal bone marrow cells, we amplified DNA from clonal and polyclonal cells which had been mixed in various ratios before DNA extraction. Polymerase chain reaction in conjunction with denaturing gradient gel electrophoresis is a sensitive and versatile molecular tool for the assessment of clonality of suspect cutaneous lesions. The determination of sensitivity using DNA extracted from premixed cells more closely corresponds to the actual test situation when testing skin samples.

Blotting, Southern↗

PubMed2000

Clonal groups of penicillin-nonsusceptible Streptococcus pneumoniae in Baltimore, Maryland: a population-based, molecular epidemiologic study.

Few data are available on the molecular subtypes of all penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) from a defined population base. Pulsed-field gel electrophoresis (PFGE), serotyping, and antibiotic susceptibility testing were performed for all available invasive PNSP isolates for which the penicillin (MIC) was > or =0.1 microg/ml from Baltimore, Md., during 1995-1996 (n = 143). The dendrogram analysis of PFGE patterns included 32 distinct clonal groups. Six major clonal groups included two-thirds of the PNSP strains. Major clonal groups 2, 3, 4, and 6 strains were genetically related to four previously described international clones and were all multidrug resistant. Major clonal group 3 was genetically related to the Tennessee(23F)-4 clone and contained all four strains for which the penicillin MIC was 8 microg/ml. Most of the clonal group 1 and 5 strains had intermediate susceptibility to penicillin and were rarely multidrug resistant. The latter clonal groups represent two previously undescribed penicillin-intermediate pneumococcal clones. Clonal group homogeneity was greater for serotype 9V, 19A, and 23F strains than for serotype 6A, 6B, 14, and 19F strains. The classification of PNSP strains into clonal groups is essential for future population-based epidemiologic studies of PNSP.

Adolescent↗