Thymocyte differentiation from lentivirus-marked CD34(+) cells in infant and adult human thymus.

Changes in thymic function and immune system homeostasis associated with HIV infection or chemotherapy have significant effects on the ability of patients to maintain a complete T cell receptor repertoire. Therefore, the development of in vitro systems to evaluate thymic function in children and adults may aid in the understanding of thymopoiesis and the development of new therapies to improve thymic output. Here we use a lentivirus-based gene transfer system to mark CD34(+) cells with EGFP and follow their differentiation into CD4(+) and CD8(+) single positive thymocytes in human thymic organ cultures. Lentivirus-marked cells entered the thymus and were detected in both the cortex and medulla. Pretreatment of the thymus with 2-deoxyguanosine depleted resident thymocytes and significantly increased the percentage of EGFP(+) thymocytes. High frequency gene transfer into CD34(+) cells and maintained expression throughout differentiation allows for the in vitro assessment of thymic function. In thymuses ranging in age from fetal to adult we observed EGFP(+) thymocytes at all stages of development suggesting that thymuses of all ages are capable of accepting new T cell progenitors and contributing to the maintenance of T cell homeostasis.

Effect of HIV on thymic function before and after antiretroviral therapy in children.

Studies were undertaken to investigate the role of the thymus in T cell reconstitution in human immunodeficiency virus (HIV)-infected children treated with antiretroviral therapy. Nine pediatric patients who acquired HIV perinatally were treated with multidrug combinations of antiretroviral agents. Plasma virus load and CD4+ and CD8+ T cell subsets were measured, and thymus function was measured by quantifying T cell receptor rearrangement excision circles in peripheral blood. Patients with virus loads remaining >400 RNA copies/mL plasma were classified as virologic nonresponders. Thymus function was initially decreased in all subjects. After antiretrovirus therapy, peripheral CD4+ T cells increased in all subjects. Thymus function was restored in 4 of 5 virologic responders but in only 1 of 4 virologic nonresponders. This suggests that HIV has an adverse effect upon thymic function in pediatric HIV infection. Potent antiretroviral therapy restores thymic function but is affected by the degree to which virus suppression is achieved.

Identification of a human recent thymic emigrant phenotype.

The ability to measure human thymic output would be an invaluable tool for the study of the development of the naive T cell repertoire, as well as naive T cell regeneration after intensive cytotoxic chemotherapy or effective antiretroviral therapy of progressive HIV infection. We and others have demonstrated previously that quantification of T cell receptor rearrangement excision circles (TREC) within peripheral T cell populations provides insight into the frequency of recent thymic emigrants (RTE) and, therefore, into thymic function. However, measurement of RTE by this approach is complicated by the fact that TREC levels also are determined by turnover within the naive T cell compartment. Here, we report a phenotypic approach to RTE measurement. We demonstrate that alphaE integrin (CD103) expression is up-regulated very late in thymic development on a subset of CD8(+)/CD4(-) thymocytes and also defines a distinct subset of naive CD8(+) T cells in the periphery. The latter subset is differentiated from circulating CD103(+) mucosa-associated memory T cells by its naive T cell phenotype (CD45RO(-), CD62L(bright), CD27(bright), CD11a(dim), CD95(dim)) and its high concentration of TREC. Indeed, sorted CD103(+) naive CD8(+) cells display higher levels of TREC than their CD103(-) naive counterparts, and these cells demonstrate an age-related decline in frequency that is enhanced significantly by thymectomy. The thymic dependence of this subset and the cells' relatively evanescent presence in the periphery suggest that these cells are a population of RTE and that quantification of their frequency in peripheral blood provides an estimate of the level of ongoing thymopoiesis.

Unusual bone marrow manifestations of parvovirus B19 infection in immunocompromised patients.

Parvovirus B19 is responsible for a spectrum of disease in humans. The usual bone marrow findings in acute parvovirus infections are marked erythroid hypoplasia and occasional giant erythroblasts. Intranuclear inclusions in developing erythroid precursors are rarely described in children or adults with parvovirus infection, although abundant intranuclear inclusions are commonly observed in the placenta and other tissues in infected fetuses. In this study, 8 patients are reported in whom the first evidence of parvovirus infection was the recognition of numerous intranuclear inclusions in erythroid precursors on bone marrow biopsy sections. Six of the 8 patients had documented immunodeficiencies; 4 had acquired immune deficiency syndrome (AIDS), and 2 were on chemotherapy. Five of 7 patients were negative for immunoglobulin G (IgG) antiparvovirus antibodies, including all 4 with AIDS. Unlike the typical pattern in parvovirus infection, the bone marrow was hypercellular in most of the patients, and erythroid precursors were usually increased with the entire spectrum of normoblast maturation represented; abundant intranuclear inclusions were observed similar to the finding in fetuses. The inclusions were variably eosinophilic and compressed the chromatin against the nuclear membrane. In situ hybridization showed parvovirus B19 DNA in numerous erythroid precursors in all cases. The findings of erythroid maturation and abundant viral inclusions in these immunocompromised patients is consistent with the hypothesis that failure to produce effective IgG parvovirus neutralizing antibodies may lead to persistent infection through viral tolerance that allows erythroid development of infected cells past the pronormoblast stage. Identification of parvovirus inclusions in marrow biopsies and subsequent confirmation of infection by in situ hybridization can be important in the assessment of anemia in immunodeficient patients because serological studies for parvovirus B19 are frequently negative.

Expression, regulation, and function of B cell-expressed CD154 in germinal centers.

Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38positiveIgD+ pre-germinal center (GC)/GC founder, CD38positive GC and CD38-IgD- memory populations, and were also found in the CD38-IgD+ naive and CD38brightIgD+ plasmablast subsets, but not in the CD38brightIgD- plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-kappaB, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was indicated by the finding that mAb to CD154 inhibited differentiation of CD38positiveIgD- GC B cells to CD38-IgD- memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses.

Major burn trauma in rats promotes cardiac and gastrointestinal apoptosis.

The hypothesis that cardiac functional abnormalities that occur after major burn trauma are paralleled by an increased incidence of apoptosis in cardiac myocytes was examined. Adult Sprague-Dawley rats were given a full thickness scald burn comprising 43+/-1% of the total body surface area or were manipulated identically but not exposed to burn injury (sham burn); burned rats were fluid resuscitated with lactated Ringer's solution. Tissues from burn and sham burn animals were then examined by the TUNEL (TdT-mediated dUTP nick end labeling) assay and light microscopy to determine the presence of apoptosis 24 and 48 h after burn trauma. In parallel, the mechanical function of the heart was assayed in separate groups of rats. Tissues harvested from the hearts of sham-treated animals showed essentially no apoptosis, whereas a small number of apoptotic cells were noted in the intestinal villi and liver of sham-treated animals. Twenty-four hours after burn trauma, there was a marked increase in apoptotic cells in the left ventricle (+916%), and the number of apoptotic cells remained increased by eightfold 48 h postburn. Apoptosis was noted predominately in the subendocardial tissue of the left ventricle. The appearance of apoptotic cells was paralleled by a decrease in cardiac mechanical function with significant decreases in left ventricular pressure and +/-dP/dt(max). Burn injury also increased apoptosis in the small intestine significantly, whereas apoptosis in the liver did not increase with burn trauma. These data suggest that the apoptosis of the cardiac myocytes that occurs after burn trauma may contribute, in part, to postburn cardiac mechanical dysfunction.

TNF receptor-associated factor-3 signaling mediates activation of p38 and Jun N-terminal kinase, cytokine secretion, and Ig production following ligation of CD40 on human B cells.

CD40 engagement induces a variety of functional outcomes following association with adaptor molecules of the TNF receptor-associated factor (TRAF) family. Whereas TRAF2, -5, and -6 initiate NF-kappaB activation, the outcomes of TRAF3-initiated signaling are less characterized. To delineate CD40-induced TRAF3-dependent events, Ramos B cells stably transfected with a dominant negative TRAF3 were stimulated with membranes expressing recombinant CD154/CD40 ligand. In the absence of TRAF3 signaling, activation of p38 and control of Ig production were abrogated, whereas Jun N-terminal kinase activation and secretion of IL-10, lymphotoxin-alpha, and TNF-alpha were partially blocked. By contrast, induction of apoptosis, activation of NF-kappaB, generation of granulocyte-macrophage CSF, and up-regulation of CD54, MHC class II, and CD95 were unaffected by the TRAF3 dominant negative. Together, these results indicate that TRAF3 initiates independent signaling pathways via p38 and JNK that are associated with specific functional outcomes.

Changes in thymic function with age and during the treatment of HIV infection.

The thymus represents the major site of the production and generation of T cells expressing alphabeta-type T-cell antigen receptors. Age-related involution may affect the ability of the thymus to reconstitute T cells expressing CD4 cell-surface antigens that are lost during HIV infection; this effect has been seen after chemotherapy and bone-marrow transplantation. Adult HIV-infected patients treated with highly active antiretroviral therapy (HAART) show a progressive increase in their number of naive CD4-positive T cells. These cells could arise through expansion of existing naive T cells in the periphery or through thymic production of new naive T cells. Here we quantify thymic output by measuring the excisional DNA products of TCR-gene rearrangement. We find that, although thymic function declines with age, substantial output is maintained into late adulthood. HIV infection leads to a decrease in thymic function that can be measured in the peripheral blood and lymphoid tissues. In adults treated with HAART, there is a rapid and sustained increase in thymic output in most subjects. These results indicate that the adult thymus can contribute to immune reconstitution following HAART.

MURCS association: ultrasonographic findings and pathologic correlation.

MURCS association is a rare, lethal and unusual constellation of nonrandom findings that includes mullerian duct aplasia, renal aplasia, and cervicothoracic somite dysplasia.1-3 It has been described in 30 patients by Duncan and coworkers2 in 1979, in which report the authors proposed an embryologic cause for these defects.3 Antenatal ultrasonographic findings included a massive, cystic umbilical cord related to a patent urachus, enlarged bladder, single small kidney, and suspicion of urethral obstruction in a fetus of female phenotype. These findings are rare in a case of MURCS and were all confirmed on pathologic examination.

Novel electrophoretic protocol for collection of mutations in the lambda light chain immunoglobulin gene in a human B-lymphoblastoid cell strain.

Spontaneous and chemically induced mutation was examined in the lambda light chain immunoglobulin gene in a human B-lymphoblastoid cell strain (T5-1). The hemizygous lambda gene is a unique mutational target gene which codes for a protein that is both expressed on the cell membrane and secreted. Mutations in the lambda gene were detected by analysis of western blots of isoelectric focusing gel electrophoresis of T5-1 cell conditioned culture medium. None of 5,841 individual clones established from vehicle-exposed populations had detectable variations in the isoelectric banding pattern of the constitutively secreted lambda immunoglobulin protein. In contrast, 113 of 6,128 clonal populations established from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-exposed populations exhibited stable variations in expression of the lambda immunoglobulin: isoelectric variants (n = 3) and non-secretors (n = 110). MNNG-induced mutations in the lambda gene, which resulted in lambda immunoglobulin proteins with altered isoelectric points (pIs), occurred at a frequency of no less than 4.9 x 10(-4) mutations/cell, indicating the mature rearranged lambda immunoglobulin gene is comparably sensitive to carcinogen induced mutation as other human autosomal target genes. Approximately one-half of the MNNG-induced non-secretor mutant clones lacked lambda mRNA while one-half maintained constitutive transcription and expression of the lambda immunoglobulin on the cell surface, demonstrating that carcinogen damage interdicted gene function at multiple points.

Isolation by fluorescence-activated cell sorting of cells of a human lymphoblastoid cell strain containing mutations in the lambda immunoglobulin gene.

Fluorescence-activated cell sorting (FACS) was used to establish clonal populations of a human lymphoblastoid cell strain that contain spontaneously occurring and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations in the lambda immunoglobulin gene. Multiple rounds of FACS using a monoclonal antibody specific for the membrane-expressed human lambda immunoglobulin were used to enrich the population fraction of cells lacking a wild-type lambda immunoglobulin on the cell surface. Approximately 20% of the clonal populations established after five rounds of FACS-mediated enrichment did not express the lambda immunoglublin epitope recognized by the monoclonal antibody used for selection. However, evaluation of the FACS-selected mutant clonal populations with a polyclonal antilambda antibody, or a monoclonal antibody directed against a different epitope on the lambda immunoglobulin made by the T5-1 cells, indicated that the mutant clonal populations expressed lambda immunoglobulin on their cell surfaces. Additionally, the presence of lambda mRNA and of both secreted and cytoplasmic lambda protein confirmed the transcription and translation of the lambda immunoglobulin gene. These data suggest that FACS-mediated selection employing epitope-specific monoclonal antibodies provides a powerful technique for isolation of cell populations that express mutations within the coding region of the lambda immunoglobulin gene.

S-phase block and cell death in human lymphoblasts exposed to benzo[a]pyrene diol epoxide or N-acetoxy-2-acetylaminofluorene.

The relationship between perturbation of the cell cycle and induction of cell death by benzo[a]pyrene diol epoxide (BPDE) or N-acetoxy-2-acetylaminofluorene (AcAAF) in exponentially proliferating T5-1 human lymphoblastoid cells was studied. Both BPDE and AcAAF caused cells to accumulate in the S phase of the cell cycle. Perturbation of the cell cycle preceded reduction of cell viability and was associated with inhibition of population growth. Effects on each of the three parameters were noted during the first population doubling, suggesting that they occurred during the first cell cycle after exposure. BPDE-exposed cells accumulated initially in early to mid-S phase and then moved parasynchronously through the remainder of this phase. In contrast, AcAAF-exposed cells accumulated uniformly at all points of the S phase. High doses of either compound froze cell cycle progression, completely inhibited population growth, and killed nearly all cells in the population. Our results suggest that perturbation of DNA replication mediates cell death after exposure to doses of either chemical that cause less than complete inhibition of cell proliferation. However, additional processes, such as perturbation of transcription, may be involved in lethality after exposure to doses that immediately and completely inhibit population growth.

Cell cycle perturbation and cell death after exposure of a human lymphoblastoid cell strain to N-methyl-N'-nitro-N-nitrosoguanidine.

The mechanisms involved in cell death caused by carcinogens that methylate DNA are poorly understood. In this study, the cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied in exponentially growing T5-1 human lymphoblastoid cells. MNNG exposure killed cells and inhibited proliferation of the remaining viable cells. Reduction in cell viability, which coincided with the accumulation of cells in the late S phase of the cell cycle, was not apparent until the population had completed one doubling. Fluorescence-activated cell sorting of fluorescein diacetate-stained, MNNG-treated cells into live and dead subpopulations revealed that all cycle phases were well represented in the live fraction, whereas the dead fraction consisted primarily of cells with a sub-G1 DNA content. Thus, cell death after MNNG exposure occurred during the second cell cycle after treatment apparently as a consequence of perturbation of DNA replication and the degradation of nuclear DNA.

Lymphoblastoid cell-produced immunoglobulins: preparative purification from culture medium by hydroxylapatite chromatography.

Hydroxylapatite (HA) is a chromatography matrix capable of separating nucleic acid as well as protein species. HA adsorbs biomolecules as a function of the extent and distribution of their surface charge. HA has been evaluated for its ability to differentially retain immunoglobulin molecules, which are planar relative to the generally globular serum proteins, particularly albumin, contained in tissue culture medium. HA chromatography provides a single-step method to purify and concentrate immunoglobulin proteins secreted by lymphoblastoid cells into culture medium from the vast excess of serum proteins used to supplement the medium. A human lambda light-chain protein was recovered in 5% of the applied volume of medium, and was separated from 95% of the total protein. More than 75% of a hybridoma-produced complete immunoglobulin was recovered essentially free of serum protein contamination. HA appears to provide a valuable alternative chromatographic medium for the purification of immunoglobulin proteins secreted by lymphoblastoid cells.