Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model.

Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.

Methodologies for informing beneficiaries of their rights: evaluating the Important Message from Medicare.

The Important Message from Medicare, a letter given to US Medicare patients upon hospital admission, was originally designed to allay the concerns of the Health Care Financing Administration (HCFA), Peer Review Organizations (PROs), and the beneficiary community that patients did not know their rights under Medicare and the PRO program. The effectiveness of the current Message may be adversely affected by the complexity of its language, the multifaceted admissions process, priorities of sick patients, and the demand for hospital resources. This study evaluated the effectiveness of the Message and various alternatives for informing beneficiaries of their rights. With HCFA financing, PROs in 10 states surveyed random samples of Medicare beneficiaries to measure knowledge of their rights under the current system and after the implementation of alternatives. This study found that there are alternative ways of informing Medicare beneficiaries of their rights which are more effective than the current Important Message from Medicare.

Molecular cloning and characterization of CFT1, a developmentally regulated avian alpha(1,3)-fucosyltransferase gene.

Although coordinate expression of carbohydrate epitopes during development is well described, mechanisms which regulate this expression remain largely unknown. In this study we demonstrate that developing chicken B cells express the LewisX terminal oligosaccharide structure in a stage-specific manner. To examine regulation of this expression, we have cloned and expressed the chicken alpha(1,3)-fucosyltransferase gene involved in LewisX biosynthesis, naming it chicken fucosyltransferase 1 (CFT1). CFT1 is characterized by a single long open reading frame of 356 amino acids encoding a type II transmembrane glycoprotein. The domain structure and predicted amino acid sequence are highly conserved between CFT1 and mammalian FucTIV genes (52.8% and 46.3% identity to mouse and human respectively). In vitro CFT1 fucosyltransferase activity utilizes LacNAc > 3'sialyl-LacNAc acceptors with almost no utilization of other neutral type II (lactose, 2-fucosyllactose), or type I (lacto-N-biose I) acceptors. CFT1-transfected cells make cell surface LewisX (COS-7) and LewisX + VIM-2 structures (Chinese hamster ovary). CFT1 gene expression is tissue-specific and includes embryonic thymus and bursa. Furthermore, expression of the CFT1 gene and cell surface LewisX structures are closely linked during B cell development. These findings reveal the evolutionary conservation between nonmammalian and mammalian alpha(1,3)-fucosyltransferase genes and demonstrate a role for fucosyltransferase gene regulation in the developmental expression of oligosaccharide structures.

Interferon regulatory factor-2 physically interacts with NF-kappa B in vitro and inhibits NF-kappa B induction of major histocompatibility class I and beta 2-microglobulin gene expression in transfected human neuroblastoma cells.

Most neural cells constitutively lack major histocompatibility complex (MHC) class I and beta 2-microglobulin gene expression. Cytokines and viruses may, however, induce expression of these genes in some neural cells, and this correlates with factor binding to the NF-kappa B and interferon stimulated response elements of these genes. Here, we demonstrate that NF-kappa B is capable of inducing MHC class I and beta 2-microglobulin gene expression when transiently co-transfected into CHP-126 neuroblastomas, and that IRF-2 represses this induction. Interferon regulatory factor-2 (IRF-2) repression of MHC class I and beta 2-microglobulin gene expression in CHP-126 neuroblastomas may demonstrate a mechanism by which virus persists in neural cells. We show here that IRF-2 physically interacts in vitro with NF-kappa B. This interaction may contribute to the repression of the expression of these genes. Our demonstration that IRF family members, in addition to IRF-2, physically interact in vitro with NF-kappa B (p50 and p65), provides a general mechanism by which these transcription factors may, in concert, regulate the expression of a variety of genes involved in immune responses in the brain.

Expression of sialyl Lewis(x) and Lewis(x) defines distinct stages of chicken B cell maturation.

Commitment of cells to the B lineage in chickens occurs only during a brief period of embryogenesis. B cell progenitors then progress through discrete developmental stages resulting in the production of mature B cells that are competent to form a functioning humoral immune system in the adult bird. During embryogenesis, chicken B cell precursors undergo tissue and developmental stage-specific changes in cell-surface glycosylation. Immature B cell progenitors that migrate to the bursa of Fabricius express the carbohydrate epitope sialyl Lewis(x). Such cells undergo initial clonal expansion within the bursa without undergoing gene conversion. Beginning between days 15 and 17 of embryogenesis, B cells in the bursa undergo a tissue specific change in surface glycosylation that results in the loss of sialyl Lewis(x) expression and the acquisition of the related carbohydrate structure Lewis(x). Expression of Lewis(x) identifies B lymphocytes that have begun to undergo gene conversion. Before emigration from the bursa, bursal lymphocytes undergo another phenotypic switch in glycosylation and down-regulate Lewis(x) expression. Therefore, developmental switches in glycosylation allow us to distinguish three populations of B cells in the bursa of Fabricius at defined stages of development and suggest that regulation of cell-surface glycosylation plays a role in B cell development in the chicken.

NF kappa B and interferon regulatory factor 1 physically interact and synergistically induce major histocompatibility class I gene expression.

Major histocompatibility (MHC) class I gene expression is synergistically induced by the cytokines TNF-alpha and IFN-gamma. However, the mechanism that results in synergistic activation of these genes has remained unclear. We demonstrated here that TNF-alpha induced binding of NF kappa B p50 and p65 to the NF kappa B-like element of the MHC class I promoter termed region I and IFN-gamma induced binding of IRF-1 to the adjacent interferon consensus sequence (ICS). We further demonstrated that NF kappa B and IRF-1 physically interacted with each other and cooperatively induced MHC class I gene expression when cotransfected into CHP-126 neuroblastomas. These results provide a molecular mechanism by which TNF-alpha and IFN-gamma synergistically induce the expression of a variety of genes involved in immune responses, including MHC class I.

A survey of knowledge and attitudes about HIV and AIDS among medical students.

Researchers have found that a significant number of medical students harbor attitudes that have a negative impact on their willingness to care for persons who are HIV positive or have AIDS. To assess current HIV and AIDS knowledge and attitudes, the authors administered a 25-item survey tailored for medical professionals to 63 preclinical medical students. Respondents' mean score on the knowledge scale was 6.25 (SD 1.63) out of a possible score of 10. Factor analysis suggested three major groupings of medical students with regard to attitudes. The largest group had generally positive attitudes about patients with HIV and AIDS; two subgroups, however, would tend to refer such patients to another physician. One subgroup reported feeling more uncomfortable with homosexual behavior and with HIV-seropositive patients than they did with patients with other infectious diseases. This group also expressed discomfort with physically touching HIV-seropositive clients. The other group expressed discomfort with taking a patient's sexual history. Effective educational interventions must take these findings into account.

Chicken B cells undergo discrete developmental changes in surface carbohydrate structure that appear to play a role in directing lymphocyte migration during embryogenesis.

The migration of progenitor cells to specific microenvironments is essential for the development of complex organisms. Avian species possess a unique primary lymphoid organ, the bursa of Fabricius, that plays a central role in the development of B cells. B cell progenitors, however, arise outside the bursa of Fabricius and, during embryonic development, must migrate through the vasculature to the bursa of Fabricius. In this report, we demonstrate that these progenitor B cells express the sialyl Lewis x carbohydrate structure previously shown to be a ligand for the selectin family of vascular adhesion receptors. Soon after migration to the bursa of Fabricius, B cell progenitors are induced to undergo a developmental switch and terminate the expression of sialyl Lewis x in a temporal pattern that correlates with the developmental decline in the ability of these cells to home to the bursa of Fabricius upon transplantation. The induction of the developmental switch in the glycosylation pattern of developing B cells requires the bursal environment. In addition, sialyl Lewis x carbohydrate determinants or structurally similar determinants on the surface of immortalized bursal lymphoid stem cells participate in the adherence of these cells to the vascular regions of the bursal microenvironment. These data demonstrate that the carbohydrate structure sialyl Lewis x is developmentally regulated during chicken B cell development and may facilitate the migration of B cell progenitors to the bursal microenvironment by serving as a ligand for a lectin-like adhesion receptor.

A family of serine proteases expressed exclusively in myelo-monocytic cells specifically processes the nuclear factor-kappa B subunit p65 in vitro and may impair human immunodeficiency virus replication in these cells.

Two groups of U937 promonocytic cells were obtained by limiting dilution cloning which differed strikingly in their ability to support human immunodeficiency virus 1 (HIV-1) replication. "Plus" clones replicated the virus efficiently, whereas "minus" clones did not. We examined these clones for differences in nuclear factor (NF)-kappa B activity which might account for the observed phenomenon. Stimulation of plus clones liberated the classical p50-p65 complex from cytoplasmic pools, whereas minus clones produced an apparently novel, faster-migrating complex, as judged by electrophoretic mobility shift assays. It is surprising that the faster-migrating complex was composed also of p50 and p65. However, the p65 subunit was COOH-terminally truncated, as shown by immunoprecipitation. The truncation resulted from limited proteolysis of p65 during cellular extraction which released particular lysosomal serine proteases, such as elastase, cathepsin G, and proteinase 3. These specific proteases are coordinately expressed and were present exclusively in the minus U937 clones, but not in the plus clones, as demonstrated in the case of cathepsin G. In addition, these proteases were detected in certain subclones of THP-1 and HL-60 cells and in primary monocytes, in each case correlating with the truncated from of p65. We demonstrate in vitro cleavage of p65 by purified elastase and cathepsin G. It is possible that particular serine proteases may have inhibiting effects on the replication of HIV-1 in myelo-monocytic cells. The data also demonstrate that special precautions must be taken when making extracts from myelo-monocytic cells.

Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion.

Chickens create their immunoglobulin (Ig) repertoires during B cell development in the bursa of Fabricius by intrachromosomal gene conversion. Recent evidence has suggested that Ig gene conversion may involve cis-acting DNA elements related to those involved in V(D)J recombination. Therefore, we have examined the potential role of the V(D)J recombination activating genes, RAG-1 and RAG-2, in regulating chicken Ig gene conversion. In contrast to the coexpression of RAG-1 and RAG-2 observed in mammalian B cells that undergo V(D)J recombination, chicken B cells isolated from the bursa of Fabricius express high levels of the RAG-2 mRNA but do not express RAG-1 mRNA. The developmental and phenotypic characteristics of the bursal lymphocytes and chicken B cell lines that express RAG-2 mRNA demonstrate that selective RAG-2 expression occurs specifically in B cells undergoing Ig diversification by gene conversion. These data suggest that RAG-2 plays a fundamental role in Ig-specific gene conversion.

Evolutionary conservation of antigen recognition: the chicken T-cell receptor beta chain.

T cells play important regulatory roles in the immune responses of vertebrates. Antigen-specific T-cell activation involves T-cell receptor (TCR) recognition of a peptide antigen presented by a major histocompatibility complex molecule, and much has been learned about this antigen-recognition process through structural and genetic studies of mammalian TCRs. Although previous studies have demonstrated that avian T cells express cell-surface molecules analogous to the mammalian TCR heterodimers, TCR genes have not been identified in nonmammalian species. We now report the cloning of a cDNA that encodes the beta chain of the chicken TCR. Southern blot analysis using this TCR beta cDNA probe demonstrated that the chicken TCR beta locus was clonally rear-ranged in chicken T-cell lines. TCR beta mRNA was expressed in cells isolated from the thymus but not in cells from the bursa of Fabricius where B cells are generated. Sequence analysis of six additional TCR beta cDNAs suggested the existence of at least two variable (V) region families, three joining (J) elements, and single diversity (D) and constant (C) elements. As in mammals, considerable nucleotide diversity was observed at the junctions of the variable, diversity, and joining elements in chicken TCR beta cDNAs. Genomic V beta and J beta elements were also cloned and sequenced. Both elements are flanked by classical heptamer/nonamer recombination signal sequences. Although the chicken and mammalian TCR beta chains displayed only 31% overall amino acid sequence identity, a number of conserved structural features were observed. These data indicate that (i) the chicken TCR beta repertoire is generated by combinatorial and junctional diversity and (ii) despite divergent evolution at the level of nucleotide sequence, important structural features of the TCR beta polypeptide are conserved between avian and mammalian species.

Templated insertions in the rearranged chicken IgL V gene segment arise by intrachromosomal gene conversion.

Chickens create a repertoire for their immunoglobulin light-chain gene by a novel process of sequence substitution within a unique rearranged V gene segment (VL1) during B-cell development in the bursa of Fabricius. Sequence analysis has shown that these nucleotide substitutions are not random. Potential donors for observed sequence substitutions are present within the 25 psi VL segments located 5' of the VL1 gene. In this report, we demonstrate that VL1 sequence substitutions: (1) are derived from the psi VL donor segment templates in cis, (2) do not result in reciprocal transfer of VL1 gene sequences to the psi VL segments, and (3) lead to the rapid disappearance of cells with nondiversified rearranged VL1 genes during B-cell development in the bursa of Fabricius. Together, these data provide evidence that VL1 sequence diversity arises as a result of intrachromosomal gene conversion.

Selection for B cells with productive IgL gene rearrangements occurs in the bursa of Fabricius during chicken embryonic development.

The vast majority of immunoglobulin-expressing mature chicken B lymphocytes contain one functionally rearranged and one unrearranged allele of the immunoglobulin light chain (IgL) gene. Therefore, nearly all IgL V-J rearrangements present in mature chickens are in-frame. In contrast, the Ig genes of mature mammalian B cells contain a high proportion of out-of-frame V-J joints. To investigate the basis for this difference, gene rearrangement at the chicken IgL locus was characterized during embryonic development and in mature B-cell lines. Joining of the single functional variable (VL) segment with the single joining (JL) segment occurs in cells in multiple tissues during a transient period of chicken embryogenesis. Only one-third of the V-J joints cloned from days 10-12 of development are in-frame. An increasing proportion of in-frame V-J joints is observed within the bursa of Fabricius at successively later stages of development. Our data suggest that the bursa of Fabricius serves during embryonic development as a site of selective amplification of cells that have undergone productive V-J joining, such that nearly all V-J joints present in postembryonic B cells are in-frame. The high frequency of rearranged alleles joined in-frame that is found in posthatching bursal cells and mature B-cell lines appears to result from a low frequency with which cells undergo IgL rearrangement at both alleles, rather than from an increase in the precision of V-J joining in avian species.

Chicken IgL gene rearrangement involves deletion of a circular episome and addition of single nonrandom nucleotides to both coding segments.

Chicken immunoglobulin light chain (IgL) gene rearrangement has been characterized. Rearrangement of the single variable (VL) segment with the single joining (JL) segment within the chicken IgL locus results in the deletion of the DNA between VL and JL from the genome. This deletion is accomplished by a molecular mechanism in which a precise joining of the IgL recombination signal sequences leads to the formation of a circular episomal element. The circular episome is an unstable genetic element that fails to be propagated during B cell development. Evidence was obtained that the formation of the circular episome is accompanied by the addition of a single nonrandom base to both the VL and JL coding segments. The subsequent joining of the VL and JL segments appears to occur at random, as we observed at least 25 unique V-J junction sequences, 11 of which are out-of-frame. A novel recombination mechanism that accounts for the observed features of chicken IgL gene rearrangement is discussed.

Evolutionary comparison of the avian IgL locus: combinatorial diversity plays a role in the generation of the antibody repertoire in some avian species.

Immunoglobulin light chain (IgL) diversity is generated in the chicken by recombination between the single functional variable (VL) and joining (JL) gene segments and subsequent somatic diversification of the rearranged VL region. In order to determine whether these events are a general feature of avian IgL genes, we analyzed the organization and recombinatorial characteristics of the IgL loci of several other avian species. Southern blot analysis of bursal and germline DNA using chicken VL and constant (CL) probes revealed that the IgL loci of quail, mallard duck, pigeon, turkey, cormorant, and hawk consist of a family of VL elements, but undergo a single major rearrangement event similar to that observed in chickens. In contrast, several rearrangements were observed in the Muscovy duck locus. A phage clone containing a 26 kb insert that hybridized to VL and CL probes was isolated from a Muscovy duck erythrocyte DNA genomic library. Nucleotide sequencing revealed that the clone contained a single JL-CL region flanked on the 5' side by five VL segments. Unlike the chicken, two of the VL segments (VL1, VL5) appear to be functional. The remaining three VL segments are pseudogenes that lack promoter and leader sequences, but one of these (psi VL3) has recombination signal sequences. Overall, these data indicate that rearrangement of one VL gene segment is a general feature of the IgL locus in many avian species. In these species, the presence of a family of VL elements that do not rearrange suggests that a pseudogene pool may be available for somatic diversification by gene conversion. The organization of the Muscovy duck IgL locus suggests that additional combinatiorial diversity has evolved independently in some avian species.

The effect of alterations in myc gene expression on B cell development in the bursa of Fabricius.

Infection of 18-day embryonic bursal lymphocytes with a v-myc-containing retrovirus leads directly to a polyclonal proliferation of surface immunoglobulin-positive (slg+) cells in the bursa of Fabricius detected four weeks after hatching. These v-myc-expressing bursal cells repopulate the follicles of chemically ablated bursae more efficiently than total normal 18-day embryonic bursal cells. In contrast, comparable normal bursal cells lose the ability to repopulate follicles by four weeks. Bursal lymphocytes expressing either a retroviral v-myc or a c-myc gene deregulated by adjacent retroviral integration retain the ability of embryonic bursal lymphocytes to diversify their immunoglobulin light chain genes. These results suggest that retroviral deregulation of myc expression during avian B cell development induces outgrowth of a population of cells with the cardinal phenotypic characteristics of bursal stem cells.

Glucose transport carrier in human erythrocyte membranes. Dinitrophenylation of a membrane component modified by D-glucose.

The effect of D-glucose on dinitrophenylation of membrane proteins of human erythrocyte ghosts by 1-fluoro-2,4-dinitrobenzene was studied in the absence and in the presence of D-glucose. A double isotopic, differential labeling technique followed by gel electrophoresis of extracts in the presence of sodium dodecyl sulfate revealed the presence of a polypeptide, or polypeptides, in human erythrocyte membranes, the reactivity of which to 1-fluoro-2,4-dinitrobenzene was significantly enhanced in the presence of D-glucose. Molecular weights of the peptides were estimated to be approximately 180,000. The effective differential labeling of the peptides required specific conditions identical with the ones which maximize the differential between inactivation of the carrier by 1-fluoro-2,4-dinitrobenzene and the dinitrophenylation of bulk membrane (Jung, C. Y. (1974) J. Biol. Chem. 249, 3568). A differential labeling was also demonstrated as a single peak by LH-20 column chromatography of chloroform-methanol-extractable membrane components. The peak contained both protein and phospholipids, indicating its proteolipid-like nature. A possible relationship of the differentially labeled polypeptides to the glucose carrier is discussed.