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Leonard A Herzenberg

Publications and source records attributed to Leonard A Herzenberg.

At least 19 recordsLinked to original sources
PubMed2007

Photogenerated glycan arrays identify immunogenic sugar moieties of Bacillus anthracis exosporium.

Using photogenerated glycan arrays, we characterized a large panel of synthetic carbohydrates for their antigenic reactivities with pathogen-specific antibodies. We discovered that rabbit IgG antibodies elicited by Bacillus anthracis spores specifically recognize a tetrasaccharide chain that decorates the outermost surfaces of the B. anthracis exosporium. Since this sugar moiety is highly specific for the spores of B. anthracis, it appears to be a key biomarker for detection of B. anthracis spores and development of novel vaccines that target anthrax spores.

Animals↗
PubMed2006

Utilization of host SR protein kinases and RNA-splicing machinery during viral replication.

Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.

Cell Line↗
PubMed2006

Phenotypically distinct B cell development pathways map to the three B cell lineages in the mouse.

A recent article by Montecino-Rodriguez et al. [Montecino-Rodriguez, E., Leathers, H. & Dorshkind, K. (2006) Nat. Immunol.7, 293-301] has distinguished the early progenitors for B-1 cells, which principally develop in neonates, from early progenitors for B-2 cells, which principally develop in adult bone marrow. Here we introduce syndecan-1 (CD138) and MHC class II (I-A) as markers of early B cell development [Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. & Hayakawa, K. (1991) J. Exp. Med. 173, 1213-1225; Hardy fractions B-D] and show that the expression of these markers distinguishes the predominant B cell development pathway in neonates from the corresponding predominant pathway in adults (both progenitors are present but differently represented in each case). We show that pre-B cells (Hardy fraction D) in the predominant adult pathway express high levels of CD138 and intermediate levels of I-A, whereas the corresponding pre-B cells in the pathway that predominates in neonates do not express either of these markers. As expected, because most of the pre-B cells in adults express CD138, we find that sorted CD138+ adult pre-B cells differentiate to IgM+ B cells in vitro. Sorted CD138- pre-B cells from neonates, the majority subset at this age, also mature to IgM+ cells (without passing through a CD138+ stage). Importantly, our studies here confirm the differential representation of adult and neonatal progenitor populations and further demonstrate that CD138 expression subdivides the adult CD19+, B220-6B2-/low population shown to contain B-1 progenitors in a way consistent with the predominance of B-1b progenitors in adults. Thus, CD138 expression provides a key route to distinguishing early B cell development pathway for what now are clearly three B cell lineages.

Animals↗
PubMed2006

High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis.

Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF). As high oxidant producers, airway neutrophils contribute largely to the systemic redox imbalance seen in CF. In turn, this chronic and profound imbalance can impact circulating neutrophils before their migration into airways. Indeed, in 18 CF patients with stable disease, blood neutrophils were readily deficient in the pivotal antioxidant glutathione (P = 0.003, compared with 9 healthy controls). In a phase 1 study, this deficiency was improved (P = 0.025) by the glutathione prodrug N-acetylcysteine, given orally in high doses (0.6 to 1.0 g three times daily, for 4 weeks). This treatment was safe and markedly decreased sputum elastase activity (P = 0.006), the strongest predictor of CF pulmonary function. Consistently, neutrophil burden in CF airways was decreased upon treatment (P = 0.003), as was the number of airway neutrophils actively releasing elastase-rich granules (P = 0.005), as measured by flow cytometry. Pulmonary function measures were not improved, as expected with short-term treatment. After excluding data from subjects without baseline airway inflammation, positive treatment effects were more pronounced and included decreased sputum IL-8 levels (P = 0.032). Thus, high-dose oral N-acetylcysteine has the potential to counter the intertwined redox and inflammatory imbalances in CF.

Acetylcysteine↗
PubMed2006

Wnt signaling in the thymus is regulated by differential expression of intracellular signaling molecules.

Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands.

Cell Differentiation↗
PubMed2005

Culturing at atmospheric oxygen levels impacts lymphocyte function.

To determine whether culturing peripheral blood mononuclear cells at atmospheric oxygen levels skews responses in comparison with culturing lymphocytes at physiologic oxygen levels, we cultured peripheral blood mononuclear cells at 5%, 10%, and atmospheric (20%) gas-phase oxygen for 5 days. We found that incubator oxygen levels influenced lymphocyte proliferation stimulated by two commonly used stimuli: Con A and antibodies that crosslink surface CD3 and CD28 to mimic antigen presentation. In both cases, proliferation increased as gas-phase oxygen levels increased. In contrast, oxygen levels did not influence proliferation stimulated by phytohemagglutinin, another commonly used mitogen. Similarly, oxygen levels did not impact cell viability in unstimulated cultures. Thus, we conclude that the influence of oxygen levels on proliferation depends on the stimulus, and, most importantly from the standpoint of immune responses, culturing cells at atmospheric rather than physiologic oxygen levels results in significantly increased proliferation responses to the CD3/CD28 crosslinking, a proliferation stimulus commonly used to mimic T cell antigen receptor signaling.

CD28 Antigens↗
PubMed2005

The extracellular microenvironment plays a key role in regulating the redox status of cell surface proteins in HIV-infected subjects.

There is an overwhelming interest in the study of the redox status of the cell surface affecting redox signaling in the cells and also predicting the total redox status of the cells. Measuring the total surface thiols (cell surface molecule thiols, csm-SH) we have shown that the overall level of surface thiols is tightly controlled. In vitro, the total concentration of intracellular glutathione (iGSH) seems to play a regulatory role in determination of the amounts of reduced proteins on cells. In addition, short term exposure of the cell surface to glutathione disulfide (GSSG, oxidized GSH) seems to reduce the overall levels of csm-SH suggesting that the function of some cysteine containing proteins on the cell surface may be regulated by the amount of GSSG secreted from the cells or the GSSG available in the extracellular environment. Examination of peripheral blood mononuclear cells (PBMCs) from healthy or HIV-infected subjects failed to reveal a similar correlation between the intra- and extracellular thiol status of cells. Although there is a relatively wide variation between individuals in both csm-SH and iGSH there is no correlation between the iGSH and csm-SH levels measured for healthy and HIV-infected individuals. There are many reports suggesting different redox active proteins on the cell surface to be the key players in the total cell surface redox regulation. However, we suggest that the redox status of the cells is regulated through a complex and tightly regulated mechanism that needs further investigation. In the mean time, overall surface thiol measurements together with case specific protein determinations may offer the most informative approach. In this review, we discuss our own results as well as results from other laboratories to argue that the overall levels of surface thiols on the exofacial membrane are regulated primarily by redox status of the cell surface microenvironment.

Cell Membrane↗
PubMed2004

Functional evolution of the vertebrate Myb gene family: B-Myb, but neither A-Myb nor c-Myb, complements Drosophila Myb in hemocytes.

The duplication of genes and genomes is believed to be a major force in the evolution of eukaryotic organisms. However, different models have been presented about how duplicated genes are preserved from elimination by purifying selection. Preservation of one of the gene copies due to rare mutational events that result in a new gene function (neofunctionalization) necessitates that the other gene copy retain its ancestral function. Alternatively, preservation of both gene copies due to rapid divergence of coding and noncoding regions such that neither retains the complete function of the ancestral gene (subfunctionalization) may result in a requirement for both gene copies for organismal survival. The duplication and divergence of the tandemly arrayed homeotic clusters have been studied in considerable detail and have provided evidence in support of the subfunctionalization model. However, the vast majority of duplicated genes are not clustered tandemly, but instead are dispersed in syntenic regions on different chromosomes, most likely as a result of genome-wide duplications and rearrangements. The Myb oncogene family provides an interesting opportunity to study a dispersed multigene family because invertebrates possess a single Myb gene, whereas all vertebrate genomes examined thus far contain three different Myb genes (A-Myb, B-Myb, and c-Myb). A-Myb and c-Myb appear to have arisen by a second round of gene duplication, which was preceded by the acquisition of a transcriptional activation domain in the ancestral A-Myb/c-Myb gene generated from the initial duplication of an ancestral B-Myb-like gene. B-Myb appears to be essential in all dividing cells, whereas A-Myb and c-Myb display tissue-specific requirements during spermatogenesis and hematopoiesis, respectively. We now report that the absence of Drosophila Myb (Dm-Myb) causes a failure of larval hemocyte proliferation and lymph gland development, while Dm-Myb(-/-) hemocytes from mosaic larvae reveal a phagocytosis defect. In addition, we show that vertebrate B-Myb, but neither vertebrate A-Myb nor c-Myb, can complement these hemocyte proliferation defects in Drosophila. Indeed, vertebrate A-Myb and c-Myb cause lethality in the presence or absence of endogenous Dm-Myb. These results are consistent with a neomorphic origin of an ancestral A-Myb/c-Myb gene from a duplicated B-Myb-like gene. In addition, our results suggest that B-Myb and Dm-Myb share essential conserved functions that are required for cell proliferation. Finally, these experiments demonstrate the utility of genetic complementation in Drosophila to explore the functional evolution of duplicated genes in vertebrates.

Animals↗
PubMed2004

The E47 transcription factor negatively regulates CD5 expression during thymocyte development.

The expression of CD5 increases progressively as thymocytes mature. We have shown that CD5 expression is controlled by a tissue-specific regulatory promoter located upstream of the CD5 translation start sites. Deletion of this regulatory promoter, which contains three potential transcription factor binding sites (CCAAT, kappa E2, and ets) reduces the promoter activity to basal level. Of these sites, only ets proved essential for CD5 expression in T cell lines. Here, we introduce a role for the E47 transcription factor and the CD5 promoter kappa E2 site in regulating CD5 expression during thymocyte development. Using T cell lines, we show that (i) mutation of the kappa E2 site in the CD5 regulatory promoter results in a significant elevation of CD5 promoter activity; (ii) the E47 transcription factor binds to the kappa E2 site; and (iii) overexpression of E47 inhibits CD5 expression. We then show, in high-dimensional fluorescence-activated cell sorting studies with primary thymocytes at successive developmental stages, that (i) intracellular E47 levels decrease as surface CD5 expression increases; (ii) E47 expression is down-regulated and CD5 expression is correspondingly up-regulated in DN3 thymocytes in RAG-2-deficient mice injected with anti-CD3 to mimic pre-T cell receptor stimulation; and (iii) E47 expression is down-regulated and CD5 expression is up-regulated when double positive thymocytes are stimulated in vitro with anti-CD3. Based on these data, we propose that E47 negatively regulates CD5 expression by interacting with the kappa E2 site in the CD5 regulatory promoter and that decreases in E47 in response to developmental signals are critical to the progressive increase in CD5 expression as thymocytes mature.

Animals↗
PubMed2004

Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes.

Drosophila is a powerful model for molecular studies of hematopoiesis and innate immunity. However, its use for functional cellular studies remains hampered by the lack of single-cell assays for hemocytes (blood cells). Here we introduce a generic method combining fluorescence-activated cell sorting and nonantibody probes that enables the selective gating of live Drosophila hemocytes from the lymph glands (larval hematopoietic organ) or hemolymph (blood equivalent). Gated live hemocytes are analyzed and sorted at will based on precise quantitation of fluorescence levels originating from metabolic indicators, lectins, reporters (GFP and beta-galactosidase) and antibodies. With this approach, we discriminate and sort plasmatocytes, the major hemocyte subset, from lamellocytes, an activated subset present in gain-of-function mutants of the Janus kinase and Toll pathways. We also illustrate how important, evolutionarily conserved, blood-cell-regulatory molecules, such as calcium and glutathione, can be studied functionally within hemocytes. Finally, we report an in vivo transfer of sorted live hemocytes and their successful reanalysis on retrieval from single hosts. This generic and versatile fluorescence-activated cell sorting approach for hemocyte detection, analysis, and sorting, which is efficient down to one animal, should critically enhance in vivo and ex vivo hemocyte studies in Drosophila and other species, notably mosquitoes.

Animals↗
PubMed2004

Ontogeny of gamma delta T cells in humans.

T cell receptors consist either of an alpha-chain combined with a beta-chain or a gamma-chain combined with a delta-chain. alphabeta T cells constitute the majority of T cells in human blood throughout life. Flow cytometric analyses presented in this study, which focus on the representation of the developmental (naive and memory) subsets of gammadelta T cells, show by function and phenotype that this lineage contains both naive and memory cells. In addition, we show that the representation of naive T cells is higher among alphabeta than gammadelta T cells in adults and that the low frequency of naive gammadelta T cells in adults reflects ontological differences between the two major gammadelta subsets, which are distinguished by expression of Vdelta1 vs Vdelta2 delta-chains. Vdelta1 cells, which mirror alphabeta cells with respect to naive representation, predominate during fetal and early life, but represent the minority of gammadelta cells in healthy adults. In contrast, Vdelta2 cells, which constitute the majority of adult gammadelta cells, show lower frequencies of naive cells than Vdelta1 early in life and show vanishingly small naive frequencies in adults. In essence, nearly all naive Vdelta2 cells disappear from blood by 1 year of life. Importantly, even in children less than 1 year old, most of the nonnaive Vdelta2 cells stain for perforin and produce IFN-gamma after short-term in vitro stimulation. This represents the earliest immunological maturation of any lymphocyte compartment in humans and most likely indicates the importance of these cells in controlling pathology due to common environmental challenges.

Adult↗
PubMed2004

New approaches to fluorescence compensation and visualization of FACS data.

The Fluorescence Activated Cell Sorter (FACS) is an invaluable tool for clinicians and researchers alike in phenotyping and sorting individual cells. With the advances in FACS methodology, notably intracellular staining for cytokines, transcription factors and phosphoproteins, and with increases in the number of fluorescence detection channels, researchers now have the opportunity to study individual cells in far greater detail than previously possible. In this chapter, we discuss High-Definition (Hi-D) FACS methods that can improve analysis of lymphocyte subsets in mouse and man. We focus on the reasons why fluorescence compensation, which is necessary to correct for spectral overlap between two or more fluorochromes used in the same staining combination, is best done as a computed transformation rather than using the analog circuitry available on many flow cytometers. In addition, we introduce a new data visualization method that scales the axes on histograms and two-dimensional contour (or dot) plots to enable visualization of signals from all cells, including those that have minimal fluorescence values and are not properly represented with traditional logarithmic axes. This "Logicle" visualization method, we show, provides superior representations of compensated data and makes correctly compensated data look correct. Finally, we discuss controls that facilitate recognition of boundaries between positive and negative subsets.

Animals↗
PubMed2004

Identification of B-cell subsets: an exposition of 11-color (Hi-D) FACS methods.

In the last few years, the effectiveness of developmental and functional studies of individual subsets of cells has increased dramatically owing to the identification of additional subset markers and the extension of fluorescence-activated cell sorter (FACS) capabilities to simultaneously measure the expression of more markers on individual cells. For example, introduction of a 6-8 multiparameter FACS instrument resulted in significant advances in understanding B-cell development. In this chapter, we describe 11-color high-dimensional (Hi-D) FACS staining and data analysis methods that provide greater clarity in identifying the B-cell subsets in bone marrow, spleen, and peritoneal cavity. Further, we show how a single Hi-D FACS antibody reagent combination is sufficient to unambiguously identify most of the currently defined B-cell developmental subsets in the bone marrow (Hardy fractions A-F) and the functional B-cell subsets (B-1a, B-1b, B-2, and marginal zone [MZ] B cells) in the periphery. Although we focus on murine B-cell subsets, the methods we discuss are relevant to FACS studies conducted with all types of cells and other FACS instruments. We introduce a new method for scaling axes for histograms or contour plots of FACS data. This method, which we refer to as Logicle visualization, is particularly useful in promoting correct interpretations of fluorescence-compensated FACS data and visual confirmation of correct compensation values. In addition, it facilitates discrimination of valid subsets. Application of Logicle visualization tools in the Hi-D FACS studies discussed here creates a strong new base for in-depth analysis of B-cell development and function.

Animals↗
PubMed2003

Lymphocyte surface thiol levels.

Recent studies have implicated reduced thiols (cysteine -SH) in the function of individual cell surface proteins. Studies presented here demonstrate that the overall level of reduced thiols on cell surface molecules differs on individual subsets of peripheral blood mononuclear cells and that these levels can be manipulated in vitro by altering the level of intracellular glutathione (iGSH). To quantitate cell surface thiols, we have developed a Hi-D (11-color) fluorescence-activated cell sorter method in which we covalently couple a fluorescent molecule, Alexa-maleimide, to free (reduced) -SH groups on proteins or other molecules exposed on the cell surface (exofacial membrane). In addition, to reveal changes in cell surface thiol levels in response to various in vitro treatments, we used a pair of fluorescent Alexa dyes with distinct excitation and emission spectra to stain the cells before and after treatments. These in vitro studies demonstrate that decreasing iGSH, by specifically inhibiting its synthesis, decreases cell surface molecule thiols (csm-SH) and that preventing loss of iGSH also prevents loss of csm-SH. However, examination of peripheral blood mononuclear cell subsets tested immediately after isolation from healthy or HIV-infected subjects failed to reveal a similar relationship between internal iGSH and csm-SH. Although there is a relatively wide variation between individuals in both csm-SH and iGSH, there is no correlation between median iGSH and csm-SH compared for 22 healthy and 36 HIV-infected subjects. Collectively, our findings indicate that local environment plays a greater role in determining the redox status of cell surface molecules than the internal redox status of the cells.

Antigens, CD↗
PubMed2002

Identification of a germ-line pro-B cell subset that distinguishes the fetal/neonatal from the adult B cell development pathway.

Studies presented here show that the expression of CD4, MHC class II (Ia,) and B220 cleanly resolves a major and a minor subset within the earliest pro-B cell population (germ-line pro-B) in adult bone marrow (BM). The major subset expresses intermediate B220 and low CD4 levels. The minor subset, which constitutes roughly 20% of the adult germ-line pro-B, expresses very low B220 levels and does not express CD4. Ia is clearly detectable at low levels on the major germ-line pro-B subset, both in wild-type adult mice and in gene-targeted mice (RAG2-/- and microMT), in which B cell development terminates before the pre-B cell stage. A small proportion of cells in the more mature pro-B cell subsets (Hardy Fractions B and C) also express Ia at this level. In contrast, Ia levels on the minor subset are barely above (or equal to) background. Surprisingly, the major germ-line pro-B cell subset found in adults is missing in fetal and neonatal animals. All of the germ-line pro-B in these immature animals express a phenotype (very low B220, no CD4, or Ia) similar to that of the minor pro-B cell subset in adult BM. Because B cell development in fetal/neonatal animals principally results in B-1 cells, these findings demonstrate that the B-1 development pathway does not include the major germ-line pro-B subset found in adult BM and hence identify a very early difference between the B-1 and -2 development pathways.

Animals↗