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Clonal analysis of the cell lineages in the male flower of maize.

The cell lineages in the male flower of maize were characterized using X-rays and transposable elements to produce clonal sectors differing in anthocyanin pigmentation. Less than 50% of the somatic tassel mutations (caused by reversion of unstable color mutations) that were visible on the anther wall were sexually transmitted by the male gametes, unless the sectors were larger than half the tassel circumference. This result is explained by showing that: (a) both the outer (LI) and inner (LII) lineages of the shoot apical meristem form a cell layer in the bilayered anther wall, and that anther pigmentation can be derived from either cell layer; and that (b) the male germ cells are derived almost exclusively from the LII. Therefore, while reversion events in either the LI or LII are visible on the anther, only the LII events are heritable. Reversion events that occur prior to the organization of the shoot apical meristem however, produce large (usually more than one-half tassel) sectors that include both the outer and inner lineages. In contrast to the high level of cell layer invasion previously reported during leaf development, during anther development less than 10(-3) cells in the LI invade the LII to form male gametes. The strong correlation between cell lineage and cell fate in the maize anther has implications for studies on plant evolution and the genetic improvement of cereals by DNA transformation.

Anthocyanins

Clonal analysis of neoplastic transformation in cultured diploid rat liver epithelial cells.

Expression of TGF-alpha mRNA, which correlates well with the ability of cells to condition medium with an EGF-like activity, clonally segregates best with tumorigenicity among the several single phenotypes considered in this study. The results of unreported studies in which we have analyzed the quantitative relationships between the expression of selected phenotypes and tumorigenicity, suggest that the elevated expression of myc and TGF-alpha mRNAs interact in their associations with tumor yield. These results suggest that elevated myc expression sensitizes hepatic epithelial cells to the possible tumorigenic action of TGF-alpha. This observation may explain why the correlation between the qualitative expression of TGF-alpha and tumorigenicity, described here, is not perfect. Conventionally applied markers of transformation in hepatocytes in vivo and in cultured liver epithelial cells in vitro that we studied -histochemical expression of GGT, ability to grow in medium containing low levels of calcium, and ability to grow in soft agar- clonally segregated with tumorigenicity poorly in liver epithelial cells transformed in vitro. We conclude that these phenotypes are not adequate markers for determining the lineage of hepatic epithelial neoplasms (including, probably hepatocellular cancers arising in vivo). This study appears to be the first to attempt to analyze clonally the association of these markers with tumorigenicity, and to quantify the sensitivity, specificity, and predictive value of the associations. Our study suggests that the relatively weak associations of these phenotypes with tumorigenicity may be related only to their stronger associations with expression of TGF-alpha, or to some other property that is strongly associated with tumorigenicity. Expression of TGF-alpha is more strongly associated with expression of GLC, for example, than is the GLC phenotype with tumorigenicity. At least for GLC, autocrine stimulation by TGF-alpha is likely, since EGF increases growth of WB cells in low calcium medium. This observation may explain the perfect correlation between expression of TGF-alpha and GLC. EGF also stimulates lactate dehydrogenase, pyruvate kinase, and glucose 6-phosphate dehydrogenase in WB cells. However, quantitative correlation between GGT activity and TGF-alpha is less strong. Thus, our data from these studies suggest that the tumorigenic phenotype of cultured hepatic epithelial cells is intimately dependent on the expression of the TGF-alpha gene, possibly producing autocrine stimulation of growth via the cells' EGF receptors. This is the most simple view of the potential relationship between TGF-alpha expression and tumorigenicity in liver epithelial cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals

Clonal analysis of the malignant properties of B16 melanoma cells treated with the DNA hypomethylating agent 5-azacytidine.

The role of DNA methylation in the generation of tumor cell variants with altered growth behavior has been investigated. Cultures of the clonally heterogeneous B16 melanoma cell line and a clonal population (B16-CL) derived from it were treated with the DNA hypomethylating agent, 5-azacytidine (5-Aza-CR). The tumorigenic and metastatic properties of (sub)clones isolated from these cultures before and after drug treatment were assayed by injection via multiple routes into syngeneic C57BL/6 mice using a range of cell doses. The rate of tumor growth was monitored following intrafootpad (i.f.p.) injection and the tumor incidence was calculated from the frequency of tumor formation at i.f.p. and supraclavicular subcutaneous (s.c.) sites. Formation of both spontaneous (i.f.p., s.c. inoculations) and experimental (intravenous (i.v.) inoculation) metastatic potential was also investigated. The most consistent effect of 5-Aza-CR was the introduction of heterogeneity with respect to the tumorigenic phenotype. The effect of 5-Aza-CR treatment on metastatic behavior was variable. The majority of tumor cell variants that arose following 5-Aza-CR treatment displayed decreased malignant potential and reduced DNA methylation levels relative to untreated control cells, but the correlation was not absolute. The decreases in DNA methylation levels induced by 5-Aza-CR were unstable and began to rebound within 1 week of drug treatment. The results of the current study indicate that although 5-Aza-CR can introduce significant shifts in the malignant properties of treated cells, the direction and magnitude of the induced alterations are not predictable and are influenced by a variety of experimental parameters including the starting tumor cell population, route of tumor cell inoculation, and the drug treatment protocol. In addition, because DNA methylation levels can rebound rapidly (days) it is difficult to correlate changes in this parameter with the observed alterations in malignancy, which can only be assessed in long-term biological assays (weeks).

Animals

Clonal analysis of lymphocytes from tumor, peripheral blood, and nontumorous kidney in primary renal cell carcinoma.

The immunological properties of lymphocytes from tumor, peripheral blood (PBL), and nontumorous kidney from 16 patients with renal cell carcinoma were characterized at the clonal level with respect to their clonogenic efficiency, phenotypic expression, and cytotoxicity against autologous and allogenic tumor cells. The objectives were to delineate: (a) the quantitative differences in the immunological properties of tumor-infiltrating lymphocytes (TIL) from patient to patient; and (b) the qualitative differences in immunological properties between TIL and lymphocytes from peripheral blood or nontumorous kidney from a single patient. A total of 926 clones were characterized for phenotype expression, and 465 clones were characterized for cytotoxicity. The clonogenic efficiency of TIL varied with individuals: high in one patient; relatively high to moderate in seven patients; low in seven patients, and extremely low in the remaining one patient. The levels of autologous tumor cell lysis by TIL clones also varied with individuals. More than one-third of the TIL clones established in 4 of 13 patients displayed significant (greater than or equal to 10%) lysis against autologous tumor cells, and in each of the four patients the average percentage of lysis in the total TIL clones was higher than 10%. In two patients, 5 of 26 or 3 of 13 TIL clones were cytotoxic, but averages of percentage of lysis in the total clones were less than 10%. One 1 or 2 TIL clones of 10-27 total clones were cytotoxic in each of 4 patients, while no cytotoxic TIL clones were found in the remaining 3 patients. Clonogenic efficiency did not correlate with the level of cytotoxicity, and TIL from no tumors displayed both high proliferation and high cytotoxicity at the clonal level. In a majority of patients (12 of 13), most cytotoxic TIL clones against autologous tumor cells also lysed allogenic tumor cells. In contrast, TIL clones lysed only autologous tumor cells in the remaining one patient (patient 2). The clonogenic efficiency of TIL was lower than that of PBL in 6 of 12 patients, while the opposite was true in the remaining 6 patients. The level of cytotoxicity in the PBL clones of these 12 patients primarily correlated with that of the TIL clones. With one exception (patient 2), most cytotoxic PBL clones against autologous tumor cells also lysed allogenic targets in a majority of patients. CD4+CD8-T-cell clones (70-85%) predominated in all patients regardless of the different lymphocyte sources.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD

Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus.

We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0-2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human antibody response to foreign Ags and for generating human mAbs of predetermined specificity and high affinity.

Antibodies, Monoclonal

A clonal analysis of glial lineages in neonatal forebrain development in vitro.

Retrovirus-mediated gene transfer combined with triple immunostaining for astro- and oligodendroglial markers (antibodies to glial fibrillary acidic protein, GD3 ganglioside, and galactocerebroside, and the O4 antibody) was used to study clonal aspects of glial lineage in primary cultures of the neonatal rat striatum. We found two major clonal populations: astrocyte clones containing GFAP+, but GD3-, O4-, and GC- cells, and oligodendrocyte clones containing cells expressing various combinations of GD3, O4, and GC, with rare GFAP+ cells. These results indicate that astrocytes and oligodendrocytes belong to separate lineages in forebrain postnatal development.

Animals

Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells.

To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-alpha/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and 'crypt cell antigen') displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate. Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.

Adenocarcinoma

Clonal analysis of the cytotoxic T-lymphocyte response to reovirus.

Reovirus has previously been classified into three serotypes based on hemagglutination inhibition assays. In the present study, the specificity of cytotoxic T lymphocytes (CTL) generated in reovirus type 3-infected C3H/HeN mice was investigated at the population and clonal levels. Short-term CTL lines generated in response to reovirus type 3 preferentially lysed cells infected with reovirus type 1 or 3 and, in some instances, type 2-infected cells as well. Eleven CTL clones established from the lines demonstrated two unique patterns of recognition. A single clone was exquisitely specific for reovirus type 3-infected cells and did not cross-react on reovirus type 1- or 2-infected cells. Ten of the clones recognized reovirus type 1- and type 3-infected cells. These clones had low levels of cross-reactivity on reovirus type 2-infected cells that was revealed only at high effector:target cell ratios. Precursor frequency analysis further revealed that the majority of the CTL generated against reovirus type 3 could cross-react on both reovirus type 1- and type 2-infected cells. Some CTL could be detected that had a more restricted pattern of recognition and recognized reovirus type 3-infected cells exclusively or recognized reovirus type 3-infected cells and either reovirus type 1- or type 2-infected cells. These results indicate that a minimum of four epitopes are recognized by reovirus-specific CTL and that the response is dominated by CTL that recognize an epitope common to all three serotypes of reovirus.

Animals

Clonal analysis of the involvement of T11 molecules in antigen receptor-mediated T lymphocyte functions.

In this study, we utilized two anti-T11 monoclonal antibodies (mAbs) that inhibited the specific cytolytic activity of mixed lymphocyte culture (MLC) activated T cells to analyze, at the clonal level, the involvement of T11 molecules in 1) antigen specific versus non-specific mechanisms of target cell lysis 2) antigen-driven T cell proliferation and IL-2 production versus IL-2-induced cell proliferation. In contrast to anti-T3 or anti-T8 mAbs, antibodies to T11 molecules inhibited the cytolytic activity of MLC-derived allospecific clones in a uniform manner. In addition, anti-T11 antibodies inhibited the specific activity of CTL clones resistant to anti-T3 antibodies, even after antibody-induced modulation of T3 molecules (while anti-T3 mAbs had no effect). Similarly, anti-T11 antibodies inhibited the alloantigen induced proliferation and IL-2 release of alloreactive clones independent on their T4+ or T8+ phenotype. The inhibitory activity of anti-T11 antibodies appears to be confined to antigen-specific T cell functions since neither natural killer-like activity of CTL clones nor the IL-2 induced clonal proliferation was affected. Thus, our results indicate that T11 molecules are functionally involved in antigen recognition by T cell regardless of their function and T4/T8 phenotype.

Antibodies, Monoclonal

Clonal analysis of cardiac morphogenesis in the chicken embryo using a replication-defective retrovirus. III: Polyclonal origin of adjacent ventricular myocytes.

Replication-incompetent variants of the avian spleen necrosis virus (SNV) encoding cytoplasmic or nuclear-directed beta-galactosidase (beta-gal) have been used to trace the clonal growth of myocytes during left ventricular free-wall formation. Tubular-stage hearts were infected with a mixed suspension of both retroviruses and, after hatching, the progeny of marked cells in the ventricular wall were examined by X-gal histochemistry. When a small number of virions was introduced individual blue patches contained myocytes with only one label type (cytoplasmic or nuclear). These results confirmed our previous conclusion that each cluster or patch represents a single clone (Mikawa et al., 1992, Dev. Dynamics, 193:11-23). Each of these clones formed a clone-shaped patch which often extended through the entire thickness of the ventricular myocardium, but typically each patch was heterogeneous, containing a mixture of labeled and unlabeled cells. We then asked whether the two populations of myocytes in each patch were clonally related or generated from more than one progenitor. When hearts were infected with high titer viral suspensions many patches were observed in which cytoplasmic-tagged myocytes were intermingled with nuclear-tagged myocytes. Thus, the cone-shaped myocyte patches in the ventricular wall are polyclones derived from separate progenitors in the precardiac mesoderm. This finding led us to examine the separation of clonally related ventricular myocytes in the developing hearts. Embryos were infected with retroviral suspensions at varying stages of development and the resulting colonies examined after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Clonal analysis of agnogenic myeloid metaplasia.

Agnogenic myeloid metaplasia (AMM) is a chronic myeloproliferative disorder that leads to a sustained proliferation of megakaryocytes and an increase of reticulin fibers within the bone marrow. Blood and bone marrow samples from patients with advanced AMM with fully developed myelofibrosis as well as cases in the cellular phase of the disease were investigated for clonality. Clonality was studied by X-linked restriction length polymorphism in conjunction with DNA methylation patterns. Granulocytes and total bone marrow cells proved to be monoclonal in origin whereas at least a minor portion of the peripheral lymphocytes were not clonally derived. Our findings indicate that the cellular phase of AMM as well as the fully developed disease progressed to myelofibrosis represent a monoclonal proliferation of pluripotent hematopoietic stem cells.

Bone Marrow

Clonal analysis of liver-infiltrating T cells in patients with LKM-1 antibody-positive autoimmune chronic active hepatitis.

Autoantibodies against microsomal antigen of liver and kidney (LKM-1) are diagnostic markers for a subgroup of autoimmune chronic active hepatitis (AI-CAH). Cytochrome P450dbl, now classified as cytochrome P450 IID6, is the major antigen of LKM-1 antibodies. Immunohistological studies suggest that hepatic injury in AI-CAH is mediated by liver-infiltrating T cells. In the present study the specificity and function of liver-infiltrating T cells was analysed at the clonal level. Phenotypical characterization of 189 T cell clones isolated from four liver biopsies of LKM-1 antibody-positive patients showed an enrichment of CD4+ CD8- T cell clones proliferated specifically in the presence of recombinant human LKM-1 antigen (rLKM). This reaction was restricted to autologous antigen-presenting cells and to HLA class II molecules. In order to see whether rLKM was also recognized by peripheral blood T lymphocytes (PBL) we tested the proliferative response of PBL from several individuals. PBL from three of the four patients with LKM-1 antibody-positive AI-CAH proliferated to rLKM, whereas no response was seen with PBL from patients with LKM-1 antibody-negative chronic liver diseases and from healthy blood donors. These data demonstrate that the LKM-1 antigen is recognized by liver-infiltrating T cells in LKM-1 antibody-positive AI-CAH. For further functional characterization, liver-derived T cell clones were tested for their cytotoxic activity. In the presence of phytohaemagglutinin 24 out of 26 CD4- CD8+ but also 20 out of 63 CD4+ CD8- T cell clones lysed autologous as well as allogenic EBV-transformed B cell lines or K562 cells. Five CD4- CD8+ T cell clones lysed autologous but not allogenic B cell lines spontaneously in a HLA class I-restricted manner. Although the antigen specificity of these clones is still unknown the data show the presence of autoreactive T cells at the site of inflammation that could contribute in the pathogenesis of AI-CAH.

Adolescent

Clonal analysis of the action of GM-CSF on the proliferation and differentiation of myelomonocytic leukemic cells.

With 214 subclones of the BALB/c myelomonocytic leukemia WEHI-3B, the granulocyte-macrophage colony-stimulating factor (GM-CSF) in impure or purified form, consistently increased the proportion of colonies exhibiting partial or complete differentiation in agar cultures. GM-CSF also increased colony size and content of daughter colony-forming cells. Serial recloning of WEHI-3B colonies in the presence of GM-CSF showed that when colonies differentiated completely, self-replication of the colony-forming cell was suppressed (clonal extinction). However, WEHI-3B cells exhibited clonal instability and even in the continuous presence of GM-CSF many colony-forming cells still generated cells able to form undifferentiated colonies. It appears unlikely that GM-CSF can completely suppress the progressive proliferation of a myeloid leukemic population of the WEHI-3B type.

Animals

Clonal analysis of graft-infiltrating lymphocytes from renal and cardiac biopsies. Dominant rearrangements of TcR beta genes and persistence of dominant rearrangements in serial biopsies.

Graft-infiltrating lymphocytes from both human renal and cardiac allografts were propagated in interleukin 2 in order to evaluate rearrangements in the T-cell receptor (TcR) beta-chain genes. Individual biopsies from renal allografts during episodes of cellular rejection were examined as well as multiple biopsies of heart transplant patients from whom endomyocardial samples were taken prior to, during, and after episodes of rejection. TcR beta-chain rearrangements were evaluated in Southern blots using DNA extracted from interleukin 2-propagated cells and digested with restriction endonucleases permitting assessment of rearrangements to both C beta 1 and C beta 2. Rearrangements shared among greater than 5% of the "bulk" culture appear as nongermline bands when hybridized with a C beta probe. Single-cell progeny were generated from limiting dilution, and the rearrangements among the cloned progeny compared to the "bulk" of the cultured progeny of graft-infiltrating lymphocytes. The results indicate that "dominant" rearrangements are a common feature of renal allograft-infiltrating lymphocytes (14 of 15 cases examined). Since the number of cells which can be recovered from a given cardiac biopsy may be limiting, evaluation of clonal dominance from these cultures is more difficult to evaluate. However, sharing of "dominant" rearrangements among multiple biopsies from the same cardiac allograft patient indicates an in vivo selection for T cells with the same receptor rearrangement. Analysis of individual clones showed 3/33 clones from a renal allograft sharing the "dominant" rearrangement noted in the bulk culture, but none of these "dominant" clones showed antidonor specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopsy

Clonal analysis of cell fate during gastrulation and early neurulation in the mouse.

The foundation of the germ layers and the extraembryonic mesoderm from the epiblast between 6.5 and 7.5 days post coitum (p.c.) is accompanied by substantial cell proliferation. It is followed during the next 24 hours by the organization of major systems of the embryo such as the central nervous system, somites, heart and vascular system. Injection in situ of a short-term lineage label (horse radish peroxidase) into single epiblast cells at 6.7 days p.c. and analysis of the descendant clones in cultured embryos have been used to trace these processes and led to the following conclusions: (1) There is extensive but not indiscriminate cell mixing at the onset of gastrulation; epiblast cells spread towards the primitive streak and descendants are there progressively incorporated into mesoderm. (2) The fate map of the mouse epiblast at the early primitive streak stage is topologically similar to those of other vertebrates. (3) Germ layers and the extraembryonic mesoderm are not clonally distinct before gastrulation, the region of overlapping boundaries in the fate map being occupied by cells that will have descendants in more than one layer. (4) Cranial neurectoderm is mainly derived from axial epiblast immediately anterior to the primitive streak of the early streak stage embryo, clonal descendants being spread rostrocaudally in the developing neural tube. Contribution to the putative floor plate is made by progenitors some of which also contribute to notochord and mesoderm.

Animals

Clonal analysis of B lymphocyte responses to Plasmodium chabaudi infection of normal and immunoprotected mice.

Parasite infection causes marked perturbations in the host immune system, as shown by hypergammaglobulinemia, autoimmunity and immune depression, but there is little information on the number, specificities and performance of B cell clones activated in the course of infection. We have addressed these questions in a model of murine malaria induced by Plasmodium chabaudi, where primary infection results in very marked B cell responses that shift in Ig isotype pattern in immunoprotected animals, and where immunity can be transferred to naive recipients by injection of serum from late, but not early, infection. We have quantitated B cells responding to infection in two distinct functional compartments, namely blast cells and Ig-secreting cells, and compared normal with immune animals. We have also determined the frequencies of clonal specificities towards several autoantigens (DNA, myosin, transferrin and red cells), non-self protein or polysaccharide antigens (KLH, levan and dextran), and parasite antigens in both compartments, by measuring blast cell reactivities in limiting dilution analyses and Ig secretion in ELISASPOT assays. This experimental design allowed us to assess the specificity of the B cell responses, to compare the clonal composition of these two B cell compartments, and to evaluate putative specific response regulation at the step of terminal differentiation. Our results show that, in this particular experimental system: (i) B cell responses in primary infection are truly non-specific while immune animals show a greater ability to control the massive non-specific response; (ii) parasite specific B cells, particularly those committed to IgG production, are selectively stimulated in immune individuals; (iii) autoreactive B cells are not selectively stimulated, but increased autoantibody production may result from perturbation in the control of terminal differentiation in the respective clones; (iv) clones with specificity to some non-self antigens (e.g. KLH and dextran) are selectively engaged and regulated, which might have implications for the immunosuppression following infection.

Animals

Clonal analysis of non-typable Haemophilus influenzae by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of whole cell polypeptides.

A collection of 223 strains of non-typable Haemophilus influenzae was assembled. Most strains were isolated from hospital in-patients in North East Scotland between 1984 and 1989. These isolates were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell polypeptides. Variability was assessed in terms of apparent molecular weight differences between the protein profiles. Isolates were grouped on the basis of Dice coefficients of similarity and assigned to clones. Of the 223 strains, 147 were unique; the remaining strains were assigned to multi-member clones of which 13 clones had two members, one clone had three members, three clones had four members, three clones had five members, two clones had six members and one clone had eight members. The longest time interval between isolation of clonally related strains virtually equalled the limits of the study. Isolates from a childrens' hospital were significantly less diverse than those from patients in adult wards.

Adult

Age-associated changes in CD8+ and CD16+ cell reactivity: clonal analysis.

A cloning technique was used to estimate the frequency of proliferating T cell precursors, the growth capacity of clone-forming cells and the functional activity of clones established in vitro from peripheral blood lymphocytes of young and old people. The mean frequency of proliferating precursors was lower in the elderly as was the proliferative capacity of CD8+ clones. In contrast, CD4+ and CD16+ clones showed a proliferation similar to that obtained from young subjects. When the clones were examined for their functional activity, CD4+ clones from both groups failed to show any cytolytic activity, while CD8+ clones exerted cytolysis against K562 and in antibody-dependent cell-mediated cytotoxicity but this function was reduced in clones derived from old subjects. Similarly, CD16+ clones from the elderly showed a decreased activity at some effector-to-target cell ratios. We conclude that the impaired functional activity (T or NK-dependent) found in the peripheral blood of aged subjects persists after in vitro selection when these cells are analysed at clonal level.

Adult