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PubMed1999

Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface.

We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45-50 cell doublings and at approximately 15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic "cell doubling clock. " These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.

3T3 Cells↗
PubMed1980

Clonal analysis of F1 hybrid helper T cells restricted to parental or F1 hybrid major histocompatibility determinants.

Antigen-specific major histocompatibility complex (MHC)-restricted helper T cell precursors were induced to proliferate in cultures of keyhole limpet hemocyanin-primed lymph node cells. Clones of F1 hybrid helper T cells were isolated in limiting-dilution cultures. Each positive culture at a limiting-dilution of lymph node cells gave rise to > 10 helper T cells with a single MHC-restricted specificity. This made it possible to independently assay specific helper activity of isolated clones in secondary cultures with B cells of diverse origin. Different clones with helper activity restricted to either parental or unique F1 hybrid MHC determinants were found to occur at approximately equal frequency. The results are discussed in relation to hybrid Ia specificities and dual-complementing MHC-linked Ir genes.

Animals↗
PubMed1983

Clonal analysis of cytotoxic T cell response against human melanoma.

We investigated the feasibility of generating cytotoxic T cell clones against autologous human melanoma cells using a melanoma cell line (VIP) and a spontaneously transformed autologous fibroblast line (VIP-F:T). Cytotoxic lymphocytes (CL) generated against the VIP melanoma cells in one-way mixed lymphocyte-tumor cell interactions were expanded in interleukin 2 for 2 wk. The expanded CL were cloned in limiting dilution. Two phenotypically homogeneous clones (3:1 and E.5) were obtained bearing OKT3 phenotype. Both clones expressed cytotoxicity selectively only against the sensitizing autologous target VIP. cytotoxicity assays performed with clone E.5 against the VIP target cells in the presence of autologous unfractionated lymphocytes or serum showed no modulation of autoreactivity of clone E.5. These results indicate that analysis of cellular immune response against autologous tumor cells might be feasible using autoreactive clones generated by the currently available in vitro cloning technology.

Antibodies, Monoclonal↗
PubMed1984

Clonal analysis of B cells induced to secrete IgG by T cell-derived lymphokine(s).

To gain insight into how T cell-derived lymphokines induce the secretion of IgG in activated B cells, we performed a limiting dilution analysis, using murine splenic B cells incubated with lipopolysaccharide (LPS) and a T cell-derived B cell differentiating factor for IgG (BCDF gamma)-containing supernatant (SN). The results of this analysis indicate that such a SN induces a marked increase in the precursor frequency of IgG1-secreting cells and a modest increase in clone size. The precursors lack surface IgG and are committed to the differentiation pathway for IgG1 secretion after LPS activation, but before the addition of BCDF gamma-containing SN. The majority of IgG1-secreting clones arise independently from precursors of cells that secrete IgG3. Taken together, these results indicate that BCDF gamma directs differentiation of activated B cells to IgG1 secretion.

Animals↗
PubMed1987

Clonal analysis of primary B cells responsive to the pathogenic bacterium Salmonella typhimurium.

In the present study, a modification of the splenic focus system is used to analyze the S. typhimurium strain TML (TML)-specific B cell repertoire. The results show that the frequency of primary TML-specific splenic B cells in CBA/Ca mice is approximately 1 per 10(5) B cells and less than 30% of these B cells are specific for LPS. In contrast, the frequency of memory TML-specific cells is approximately 1 per 5-8 X 10(3) splenic B cells and greater than 95% of these B cells are specific for LPS. These results suggest that the frequency of primary TML-specific B cells is extremely low and that it expands 15-20-fold after antigen exposure. It is interesting that less than 30% of the primary B cells are specific for the LPS molecule since it is considered to be the major antigenic determinant on Salmonella organisms. Furthermore, the majority of the LPS-specific anti-TML antibody-producing clones are directed against the LPS O antigen region. Conversely, more than half to two-thirds of the memory LPS-specific anti-TML B cell clones are directed against the KDO or lipid A region of the LPS molecule. These results indicate that the preferential expansion of LPS-specific B cell clones observed after immunization resides primarily in the B cell subsets responsive to the KDO/lipid A moieties on the LPS molecule. Finally, unlike B cell responses to chemically defined antigens, TML stimulates very little IgG1 antibody. IgG2 and IgA isotypes appear to play a predominant role in anti-TML antibody responses, although all H chain classes are produced to some extent. Collectively, these findings are consistent with the responses reported for two other natural antigens, HA and PC. Hence, the pattern of stimulation by infectious agents, such as S. typhimurium, appears to be distinct from that of synthetic antigens. Thus, the studies presented herein have begun to provide insights into those subsets of B cells responsive to S. typhimurium and other infectious disease organisms.

Animals↗
PubMed1987

Clonal analysis of the Mls system. A reappraisal of polymorphism and allelism among Mlsa, Mlsc, and Mlsd.

Only two sets of antigenic determinants are recognized by T lymphocytes at uniquely high precursor frequencies: those encoded by the MHC and those encoded by Mls. The structural as well as functional characteristics of MHC products have been extensively analyzed. In contrast, little information concerning the nature of Mls genes or their products is available. Although it was originally described (5, 6) that the Mls locus on chromosome 1 is composed of four alleles that encode polymorphic cell surface structures, the issues of polymorphism and allelism in the Mls system have been controversial for some time. In the present study, T cell clones were generated by continuous stimulation of B10.BR (H-2k, Mlsb) T cells by CBA/J (H-2k, Mlsd) stimulators and they were used to analyze the relationship of putative Mlsa, Mlsc, and Mlsd determinants. All clones proliferated in response to determinants expressed by CBA/J stimulators. In addition, each of these clones exhibited a second reactivity to either AKR/J (H-2k, Mlsa) or C3H/HeJ (H-2k, Mlsc) stimulators. No clone responded to both AKR/J and C3H/HeJ. These second specificities were defined to be for Mlsa or Mlsc determinants, respectively, by the response patterns of clones and unprimed T cells to stimulators derived from congenic strains, recombinant inbred (RI) strains, and backcross mice. Moreover, a segregation analysis of the (CBA/J X B10.BR)F1 X B10.BR backcross indicated that the Mlsa-like and Mlsc-like determinants expressed on CBA/J (Mlsd) cells are in fact encoded by nonallelic, unlinked genes. These findings suggest a new concept of the polymorphism and genetics of the Mls system. It is proposed that two distinct and nonallelic gene products express, respectively, the noncrossreacting Mlsa and Mlsc determinants, and that the Mlsd phenotype does not represent an independent genotype but rather reflects the concurrent expression of Mlsa and Mlsc. The Mls system, therefore, consists of at least two systems that are distinct both genetically and antigenically, and that may be of different biologic or physiologic significance as well.

Alleles↗
PubMed1988

Clonal analysis of functionally distinct human CD4+ T cell subsets.

A large number of CD4+ T cell clones, obtained from peripheral blood T lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human CD4+ T cell subset. Cytotoxic capacity of cloned T cells was analyzed with the use of anti-CD3 antibodies and target cells bearing FcR for murine IgG. 6 of 12 CD4+ clones obtained were able to lyse Daudi or P815 cells in the presence of anti-CD3 antibodies. The remaining six CD4+ T cell clones tested did not display anti-CD3-mediated cytotoxic activity and did not acquire this cytotoxic capacity during a culture period of 20 wk. In the absence of anti-CD3 mAb, no lytic activity against Daudi, P815, and K562 target cells was observed under normal culture conditions. Phenotypic analysis of these two distinct types of CD4+ T cells did not reveal differences with regard to reactivity with CDw29 (4B4) and CD45R (2H4) mAbs that have been described to recognize antigens associated with helper suppressor/inducer (respectively) CD4+ cells. The CD4+ clones without anti-CD3-mediated cytotoxic activities (Th2) consistently showed a high expression level of CD28 antigens, whereas the cytotoxic clones (Th1) expressed low amounts of CD28. Th1 CD4+ clones did produce IL-2, IFN-gamma, and TNF-alpha/beta, whereas the Th2 T cell clones produced minimal amounts of IL-2 and only low levels of INF-gamma and TNF-alpha/beta in response to anti-CD3 mAbs and PMA. Although not all CD4+ clones did release IL-4, there was no correlation with cytotoxic activity. Moreover, as compared with the Th1 CD4+ clones, Th2 CD4+ T cell clones proliferated moderately in response to immobilized anti-CD3 mAbs. However, proliferation reached the level of the cytotoxic clones when anti-CD28 mABs were present during culture. Both CD4+ subsets provided help for B cell differentiation upon stimulation with anti-CD3 mAbs. Our data suggest that the human CD4+ subset, in analogy to the murine system, comprises two functionally distinct T cell subpopulations, both of which are able to exert helper activity for polyclonal B cell differentiation, but which differ in cytotoxic capacity, lymphokine production, and requirements for proliferation. A function for these two types of T cells in the immune response is discussed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Comparative clonal analysis of human immunodeficiency virus type 1 (HIV-1)-specific CD4+ and CD8+ cytolytic T lymphocytes isolated from seronegative humans immunized with candidate HIV-1 vaccines.

The lysis of infected host cells by virus-specific cytolytic T lymphocytes (CTL) is an important factor in host resistance to viral infection. An optimal vaccine against human immunodeficiency virus type 1 (HIV-1) would elicit virus-specific CTL as well as neutralizing antibodies. The induction by a vaccine of HIV-1-specific CD8+ CTL in humans has not been previously reported. In this study, CTL responses were evaluated in HIV-1-seronegative human volunteers participating in a phase I acquired immune deficiency syndrome (AIDS) vaccine trial involving a novel vaccine regimen. Volunteers received an initial immunization with a live recombinant vaccinia virus vector carrying the HIV-1 env gene and a subsequent boost with purified env protein. An exceptionally strong env-specific CTL response was detected in one of two vaccine recipients, while modest but significant env-specific CTL activity was present in the second vaccinee. Cloning of the responding CTL gave both CD4+ and CD8+ env-specific CTL clones, permitting a detailed comparison of critical functional properties of these two types of CTL. In particular, the potential antiviral effects of these CTL were evaluated in an in vitro system involving HIV-1 infection of cultures of normal autologous CD4+ lymphoblasts. At extremely low effector-to-target ratios, vaccine-induced CD8+ CTL clones lysed productively infected cells present within these cultures. When tested for lytic activity against target cells expressing the HIV-1 env gene, CD8+ CTL were 3-10-fold more active on a per cell basis than CD4+ CTL. However, when tested against autologous CD4+ lymphoblasts acutely infected with HIV-1, CD4+ clones lysed a much higher fraction of the target cell population than did CD8+ CTL. CD4+ CTL were shown to recognize not only the infected cells within these acutely infected cultures but also noninfected CD4+ T cells that had passively taken up gp120 shed from infected cells and/or free virions. These results were confirmed in studies in which CD4+ lymphoblasts were exposed to recombinant gp120 and used as targets for gp120-specific CD4+ and CD8+ CTL clones. gp120-pulsed, noninfected targets were lysed in an antigen-specific fashion by CD4+ but not CD8+ CTL clones. Taken together, these observations demonstrate that in an in vitro HIV-1 infection, sufficient amounts of gp120 antigen are produced and shed by infected cells to enable uptake by cells that are not yet infected, resulting in the lysis of these noninfected cells by gp120-specific, CD4+ CTL.(ABSTRACT TRUNCATED AT 400 WORDS)

AIDS Vaccines↗
PubMed2004

Streptococcus agalactiae in a large Portuguese teaching hospital: antimicrobial susceptibility, serotype distribution, and clonal analysis of macrolide-resistant isolates.

Group B streptococci are emerging as a cause of serious infection worldwide. The capsular polysaccharides are not only important virulence factors but also the target of vaccine development efforts. Serotypes III (24.6%), V (23.4%), Ia (17.8%), and II (16.3%) were the most prevalent among 252 Streptococcus agalactiae isolates collected during 1999-2002 in the largest hospital of Lisbon, Portugal. The substantial proportion of bacteremic patients (17 neonates and 21 adults) in this period illustrates the present importance of S. agalactiae as a cause of invasive disease. All isolates were fully susceptible to penicillin (MIC(50) = 0.064 microg/ml; MIC(90) = 0.094 microg/ml, range 0.008-0.094), cefotaxime, chloramphenicol, ofloxacin, and vancomycin. Resistance was found to tetracycline (75.4%), erythromycin (10.7%), and clindamycin (9.9%). Of the 27 erythromycin-resistant isolates, 70.4% had the cMLS(B), 22.2% the iMLS(B), and 7.4% the M phenotype. All isolates presenting the M phenotype carried the mef(A) gene, whereas the erm(B) gene was found in a large fraction of MLS(B) isolates (n = 17) and only a small proportion (n = 7) the erm(A) gene [erm(TR) variant]. All isolates carried a single macrolide-resistance determinant. Macrolide resistance was not attributable to a single clone as evidenced by distinct serotype and pulsed-field gel electrophoretic profiles. Careful surveillance of S. agalactiae invasive infections in Portugal is essential, and the treatment or intrapartum prophylaxis of patients who are allergic to penicillin should be guided by contemporary resistance patterns observed in the country.

Anti-Bacterial Agents↗
PubMed1992

Clonal analysis of T-cell responses to the HIV-1 envelope proteins in AIDS vaccine recipients.

Both CD4+ and CD8+ CTL responses specific for the HIV-1 envelope proteins can be elicited in seronegative humans by candidate AIDS vaccines. The phenotype of the responding CTL depends upon the nature of the vaccine, with CD8+ CTL being found exclusively in recipients of live virus vaccines. Both types of CTL are active against HIV-1-infected cells in vitro. However, the potential efficacy of vaccine-induced CTL in preventing infection in vaccinated individuals exposed to HIV-1 is unknown and is likely to be dependent upon complex factors including lytic activity against divergent strains, cytokines produced, and the lysis of noninfected CD4+ T cells.

AIDS Vaccines↗
PubMed1987

Clonal analysis of the T-cell response of mice to herpes simplex virus: correlation between lymphokine production in vitro and the induction of delayed-type hypersensitivity and antiviral activity in vivo.

The properties of two morphologically distinct L3T4+, Lyt2- "helper" T-cell clones specific for herpes simplex virus were investigated. Both of the clones produced IL-3 and interferon, but neither produced IL-2. Clone D6.6 produced macrophage agglutinating factor, a fibronectin-like lymphokine, and also a delayed hypersensitivity (DH) response when injected locally into syngeneic mice. Despite the presence of a DH producing clone and a non-DH producing clone, both were able to reduce the local virus titre to an equivalent degree. It is suggested that this protective activity is associated with the production of interferon-gamma. The significance of these results to mechanisms of protection against herpes simplex virus in vivo is discussed.

Animals↗
PubMed1989

The clinical spectrum, pathology, and clonal analysis of Epstein-Barr virus-associated lymphoproliferative disorders in heart-lung transplant recipients.

This study presents the clinical and laboratory observations on posttransplant lymphoproliferative disorders (PTLDs) occurring in 5 of 53 heart-lung transplantation recipients. Cervical lymph nodes, tonsils, lungs, and gastrointestinal tract were the common sites of involvement by PTLDs. The histopathologic findings showed a spectrum of lymphoid and immunoblastic proliferation ranging from diffuse hyperplasia to malignant lymphoma, immunoblastic or large cell type. All cases were associated with a primary Epstein-Barr virus infection, and viral DNA was demonstrated within the lesional tissue in three cases. Immunohistochemical and immunoglobulin gene rearrangement studies revealed a B-cell proliferation that was monoclonal in three cases and polyclonal in two cases. Compared with PTLDs arising in other organ transplant recipients, this series is remarkable for a high incidence of PTLDs (9.4%), a short interval to tumor diagnosis (2.2 months, mean), involvement of the primary allograft in three cases (60%), and the frequent development of bronchiolitis obliterans. Possible reasons for this distinct clinicopathologic profile are discussed.

Adolescent↗
PubMed1993

Clonal analysis of Streptococcus agalactiae isolated from infants with neonatal sepsis or meningitis and their mothers and from healthy pregnant women.

The purpose of this study was to evaluate if group B streptococci (GBS) isolated from infants with neonatal sepsis or meningitis and their mothers differ from GBS isolated from healthy pregnant women who gave birth to healthy infants. Danish clinical isolates of GBS (n = 118) were characterized by multilocus enzyme electrophoresis, a method recently used to identify virulent clones of GBS type III from North American infants. By analysis of allelic profiles of 11 metabolic enzyme loci, 43 different electrophoretic types were found. Clustering analysis revealed two primary phylogenetic divisions at a distance of > 0.8, both containing serotype III isolates. One division, comprising virtually all disease isolates, showed a population structure like that of traditional pathogens. The other division, containing the majority of carrier isolates, had a structure reminiscent of that of mucosal commensals, with each isolate being different.

Bacteremia↗
PubMed1991

Clonal analysis of differential lymphokine production in peptide and superantigen induced T cell anergy.

A failure of T lymphocytes to produce interleukin 2 (IL-2) on restimulation may, in part, account for the specific unresponsiveness that accompanies incomplete activation. The evidence to support this has been derived predominantly from the investigation of the molecular basis of anergy in murine type 1 T cells. In this study, the effects of different tolerogenic signals delivered by specific peptide or Staphylococcus aureus enterotoxin on the ability of antigen-specific human T cells to produce lymphokines, both in the induction phase and in established antigen-specific non-responsiveness, have been examined. Although T cell proliferation was decreased by supraoptimal concentrations of specific peptide in the presence or absence of antigen presenting cells, IL-2, IL-4, and interferon gamma (IFN-gamma) synthesis were comparable to that of activated T cells. The different tolerogenic signals, all capable of inhibiting phase of unresponsiveness. Restimulation of anergic T cells with an antigenic challenge failed to induce lymphokine production, with the exception of allergen-reactive T cells that secreted IFN-gamma. This latter observation is relevant to the desensitization of specific responsiveness in allergic disease.

Animals↗
PubMed2004

Novel recombinant phenotypic assay for clonal analysis of reverse transcriptase mutations conferring drug resistance to HIV-1 variants.

OBJECTIVE: A novel rapid reverse transcriptase (RT) recombinant HIV-1 drug-susceptibility assay was developed to evaluate resistance to RT inhibitors. MATERIAL AND METHODS: HIV-1 RTs from five treatment-naive and 10 highly active antiretroviral therapy-experienced patients were evaluated. HIV-1 isolates recovered by culturing peripheral blood mononuclear cells from patients were used in the conventional isolate phenotype analysis. Recombinant HIV-1 strains were obtained by cloning the RT gene amplified from the supernatant of HIV-1 cultures in a plasmid carrying the HIV-1 strain HXB2 backbone, and the most represented clone for each virus isolate was then tested for antiviral drug susceptibility in parallel with HIV-1 isolates. RESULTS: Comparison of conventional virus isolate and the novel recombinant virus phenotypic assays showed a large concordance of results. However, some discrepant results were observed, in that higher drug-resistance levels were detected by the conventional isolate phenotypic assay in HIV-1 isolates showing the presence of a mixture of HIV-1 variants, whereas the novel recombinant phenotypic assay could more precisely detect the level of drug resistance of the single viral clones selected for the analysis. DISCUSSION: The novel recombinant phenotype assay, compared with the conventional virus isolate phenotype assay, showed widely overlapping results. The comparison of the two assays show that the conventional phenotypic assay is able to identify more efficiently the combined effect of drug-resistant viral variants, whereas the novel recombinant phenotypic assay is better able to define the level of drug resistance of the single viral variants. In addition, rapidity (2 weeks versus 4 weeks required by the reference recombinant assay and 6 weeks required by the conventional virus isolate phenotypic assay) is a major advantage of the novel assay.

Antiretroviral Therapy, Highly Active↗
PubMed1996

Clonal analysis of growth of the polar trophectoderm in the mouse.

The results of experiments in which horseradish peroxidase (HRP) was used to mark single trophectoderm or inner cell mass (ICM) cells in situ in mouse blastocysts have led to the proposal that growth of the trophectoderm depends on stem cells located in the inner cell mass. Thus, the finding that the visual centre of clones formed following labelling of the central polar trophectoderm cell in early or expanding blastocysts was consistently shifted towards or into the mural trophectoderm was attributed to their displacement by ICM-derived cells. However, the frequency with which central polar cells were displaced is likely to have been overestimated by using the visual centre of descendant clones as the index of their location. Also, the possibility that displacement of central polar cells was an artefact of the marked temporary interruption of their cycling that resulted from labelling was not discounted. Furthermore, no attempt was made to ascertain whether cells located elsewhere in the polar trophectoderm also moved murally, as expected if there is a general displacement of such cells. In the present study, labelling of either the central or a peripheral polar trophectoderm cell with HRP was achieved without obviously perturbing their subsequent proliferation. Moreover, displacement was assessed by recording the location of the proximal boundary rather than the visual centre of the resulting clones. Even by this conservative criterion, the majority of labelled central cells moved towards or into the mural trophectoderm. In marked contrast, however, labelled peripheral polar cells moved murally in only a minority of cases. The remainder either retained their original position or moved towards rather than away from the central polar region. Such an anisotropic pattern of growth of the polar trophectoderm is not readily explicable in terms of recruitment of cells from the ICM. Rather, it accords with the view that the polar trophectoderm is a proliferative centre, and suggests that movement murally of its surplus cells may be restricted circumferentially, possibly through anchorage of the junctional trophectoderm cells that extend processes over the free surface of the ICM.

Animals↗