Joel D Ernst, M.D.

Mycobacterium tuberculosis Inhibits Neutrophil Apoptosis, Leading to Delayed Activation of Naive CD4 T cells
Blomgran, Robert; Desvignes, Ludovic; Briken, Volker; Ernst, Joel D
2012 Jan 19;11(1):81-90, Cell Host & Microbe
Mycobacterium tuberculosis promotes its replication by inhibiting the apoptosis of infected macrophages. A proapoptotic M. tuberculosis mutant lacking nuoG, a subunit of the type I NADH dehydrogenase complex, exhibits attenuated growth in vivo, indicating that this virulence mechanism is essential. We show that M. tuberculosis also suppresses neutrophil apoptosis. Compared to wild-type, the nuoG mutant spread to a larger number of lung phagocytic cells. Consistent with the shorter lifespan of infected neutrophils, infection with the nuoG mutant resulted in fewer bacteria per infected neutrophil, accelerated bacterial acquisition by dendritic cells, earlier trafficking of these dendritic cells to lymph nodes, and faster CD4 T cell priming. Neutrophil depletion abrogated accelerated CD4 T cell priming by the nuoG mutant, suggesting that inhibiting neutrophil apoptosis delays adaptive immunity in tuberculosis. Thus, pathogen modulation of apoptosis is beneficial at multiple levels, and enhancing phagocyte apoptosis promotes CD4 as well as CD8 T cell responses
— id: 150571, year: 2012, vol: 11, page: 81, stat: Journal Article,

Lung Neutrophils Facilitate Activation of Naive Antigen-Specific CD4+ T Cells during Mycobacterium tuberculosis Infection
Blomgran, Robert; Ernst, Joel D
2011 Jun 15;186(12):7110-7119, Journal of immunology
Initiation of the adaptive immune response to Mycobacterium tuberculosis occurs in the lung-draining mediastinal lymph node and requires transport of M. tuberculosis by migratory dendritic cells (DCs) to the local lymph node. The previously published observations that 1) neutrophils are a transiently prominent population of M. tuberculosis-infected cells in the lungs early in infection and 2) that the peak of infected neutrophils immediately precedes the peak of infected DCs in the lungs prompted us to characterize the role of neutrophils in the initiation of adaptive immune responses to M. tuberculosis. We found that, although depletion of neutrophils in vivo increased the frequency of M. tuberculosis-infected DCs in the lungs, it decreased trafficking of DCs to the mediastinal lymph node. This resulted in delayed activation (CD69 expression) and proliferation of naive M. tuberculosis Ag85B-specific CD4 T cells in the mediastinal lymph node. To further characterize the role of neutrophils in DC migration, we used a Transwell chemotaxis system and found that DCs that were directly infected by M. tuberculosis migrated poorly in response to CCL19, an agonist for the chemokine receptor CCR7. In contrast, DCs that had acquired M. tuberculosis through uptake of infected neutrophils exhibited unimpaired migration. These results revealed a mechanism wherein neutrophils promote adaptive immune responses to M. tuberculosis by delivering M. tuberculosis to DCs in a form that makes DCs more effective initiators of naive CD4 T cell activation. These observations provide insight into a mechanism for neutrophils to facilitate initiation of adaptive immune responses in tuberculosis
— id: 134309, year: 2011, vol: 186, page: 7110, stat: Journal Article,

Suboptimal Activation of Antigen-Specific CD4 Effector Cells Enables Persistence of M. tuberculosis In Vivo
Bold, Tyler D; Banaei, Niaz; Wolf, Andrea J; Ernst, Joel D
2011 May;7(5):e1002063-e1002063, PLoS pathogens
Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4(+) T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-gamma without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4(+) effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4(+) effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-gamma production. During the early phase of infection, approximately 10% of P25TCRTh1 cells produced IFN-gamma in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4(+) T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4(+) effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy
— id: 134314, year: 2011, vol: 7, page: e1002063, stat: Journal Article,

HIV and Tuberculosis: a Deadly Human Syndemic
Kwan, Candice K; Ernst, Joel D
2011 Apr;24(2):351-376, Clinical microbiology reviews
Summary: A syndemic is defined as the convergence of two or more diseases that act synergistically to magnify the burden of disease. The intersection and syndemic interaction between the human immunodeficiency virus (HIV) and tuberculosis (TB) epidemics have had deadly consequences around the world. Without adequate control of the TB-HIV syndemic, the long-term TB elimination target set for 2050 will not be reached. There is an urgent need for additional resources and novel approaches for the diagnosis, treatment, and prevention of both HIV and TB. Moreover, multidisciplinary approaches that consider HIV and TB together, rather than as separate problems and diseases, will be necessary to prevent further worsening of the HIV-TB syndemic. This review examines current knowledge of the state and impact of the HIV-TB syndemic and reviews the epidemiological, clinical, cellular, and molecular interactions between HIV and TB
— id: 130914, year: 2011, vol: 24, page: 351, stat: Journal Article,

Tuberculosis Pathogenesis and Immunity
Philips JA; Ernst JD
2011 Jan 25;:353-384, Annual review of pathology
Despite the development of potentially curative chemotherapy, tuberculosis (TB) continues to cause increasing worldwide morbidity and is a leading cause of human mortality in the developing world. Recent advances in bacterial molecular genetics, immunology, and human genetics have yielded insight into the molecular determinants of virulence, the immune responses that are essential for restricting progressive disease, and the determinants of immunopathology in TB. Despite these advances, a large knowledge gap still exists that limits the development and testing of new interventions, including novel drugs and efficacious vaccines. This review focuses on our current knowledge of TB pathogenesis and immunity that has been derived from in vitro and in vivo studies. In addition, it highlights topics that need to be better understood to provide improved means of controlling TB worldwide. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease Volume 7 is January 24, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates
— id: 145546, year: 2011, vol: , page: 353, stat: Journal Article,

Directly observing therapy: a new view of drug tolerance in tuberculosis
Philips, Jennifer A; Ernst, Joel D
2011 Apr 1;145(1):13-14, Cell
Drug tolerance in bacteria is widely believed to be due to metabolic changes that accompany growth arrest. A study in this issue of Cell reveals a drug tolerance mechanism in replicating mycobacteria that is induced by residence in macrophages and depends on drug efflux
— id: 130308, year: 2011, vol: 145, page: 13, stat: Journal Article,

Initiation and regulation of T-cell responses in tuberculosis
Urdahl, K B; Shafiani, S; Ernst, J D
2011 May;4(3):288-293, Mucosal immunology
Tuberculosis (TB) poses a great challenge to immunologists, as it represents a chronic infection characterized by persistence of the pathogen despite development of antigen-specific immune responses. Among the characteristics of adaptive immune responses to Mycobacterium tuberculosis is a delay in the onset of detectable T-cell responses, in both humans and experimental animals. Recent studies have revealed mechanisms that contribute to this delay, including pathogen inhibition of apoptosis, delayed migration of dendritic cells from the lungs to the local lymph node, and influence of regulatory T cells. In addition, novel features of M. tuberculosis antigen-specific T-cell differentiation have been discovered, which reveal pathways that limit and promote immune control of infection. Taken together, these results highlight the need for additional basic research and provide optimism for the development of TB vaccines with greater efficacy
— id: 134238, year: 2011, vol: 4, page: 288, stat: Journal Article,

Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved
Comas, Inaki; Chakravartti, Jaidip; Small, Peter M; Galagan, James; Niemann, Stefan; Kremer, Kristin; Ernst, Joel D; Gagneux, Sebastien
2010 Jun;42(6):498-503, Nature genetics
Mycobacterium tuberculosis is an obligate human pathogen capable of persisting in individual hosts for decades. We sequenced the genomes of 21 strains representative of the global diversity and six major lineages of the M. tuberculosis complex (MTBC) at 40- to 90-fold coverage using Illumina next-generation DNA sequencing. We constructed a genome-wide phylogeny based on these genome sequences. Comparative analyses of the sequences showed, as expected, that essential genes in MTBC were more evolutionarily conserved than nonessential genes. Notably, however, most of the 491 experimentally confirmed human T cell epitopes showed little sequence variation and had a lower ratio of nonsynonymous to synonymous changes than seen in essential and nonessential genes. We confirmed these findings in an additional data set consisting of 16 antigens in 99 MTBC strains. These findings are consistent with strong purifying selection acting on these epitopes, implying that MTBC might benefit from recognition by human T cells
— id: 145547, year: 2010, vol: 42, page: 498, stat: Journal Article,

Ectopic activation of Mycobacterium tuberculosis-specific CD4+ T cells in lungs of CCR7-/- mice
Olmos, Sofia; Stukes, Sabriya; Ernst, Joel D
2010 Jan 15;184(2):895-901, Journal of immunology
Initiation of an adaptive cellular immune response depends on intimate interactions with APCs and naive T lymphocytes. We previously reported that activation of naive Mycobacterium tuberculosis-specific CD4+ T cells depends on dendritic cell (DC) transport of live bacteria from the lungs to the mediastinal lymph node (MDLN). Because the migratory paths of DCs are largely governed by the chemokine receptor CCR7, which is expressed on DCs upon maturation by proinflammatory stimuli, we examined the quantitative contribution of CCR7-dependent DC migration in the context of tuberculosis. We found that early trafficking of DCs from the lungs to the MDLN depended on CCR7-mediated signaling, but alternative mechanism(s) are used later in infection. Impaired migration of DCs in CCR7(-/-) mice resulted in delayed dissemination of bacteria to MDLN and spleen and in delayed kinetics of activation of adoptively transferred Ag85B-specific CD4+ T cells. Furthermore, in contrast to control mice, we found that naive Ag85B-specific CD4+ T cells are activated to proliferate in the lungs of CCR7(-/-) mice and, when infected with higher doses of bacteria, resistance to M. tuberculosis infection in CCR7(-/-) mice is compromised compared with wild-type mice
— id: 106094, year: 2010, vol: 184, page: 895, stat: Journal Article,

Lipoprotein processing is essential for resistance of Mycobacterium tuberculosis to malachite green
Banaei, Niaz; Kincaid, Eleanor Z; Lin, S-Y Grace; Desmond, Edward; Jacobs, William R Jr; Ernst, Joel D
2009 Sep;53(9):3799-3802, Antimicrobial agents & chemotherapy
Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (DeltalspA::lspAmut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 10(4)-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green
— id: 101954, year: 2009, vol: 53, page: 3799, stat: Journal Article,

TLR2-dependent inhibition of macrophage responses to IFN-gamma is mediated by distinct, gene-specific mechanisms
Benson, Sarah A; Ernst, Joel D
2009 ;4(7):e6329-e6329, PLoS ONE
Mycobacterium tuberculosis uses multiple mechanisms to avoid elimination by the immune system. We have previously shown that M. tuberculosis can inhibit selected macrophage responses to IFN-gamma through TLR2-dependent and -independent mechanisms. To specifically address the role of TLR2 signaling in mediating this inhibition, we stimulated macrophages with the specific TLR2/1 ligand Pam(3)CSK(4) and assayed responses to IFN-gamma. Pam(3)CSK(4) stimulation prior to IFN-gamma inhibited transcription of the unrelated IFN-gamma-inducible genes, CIITA and CXCL11. Surface expression of MHC class II and secretion of CXCL11 were greatly reduced as well, indicating that the reduction in transcripts had downstream effects. Inhibition of both genes required new protein synthesis. Using chromatin immunoprecipitation, we found that TLR2 stimulation inhibited IFN-gamma-induced RNA polymerase II binding to the CIITA and CXCL11 promoters. Furthermore, TATA binding protein was unable to bind the TATA box of the CXCL11 promoter, suggesting that assembly of transcriptional machinery was disrupted. However, TLR2 stimulation affected chromatin modifications differently at each of the inhibited promoters. Histone H3 and H4 acetylation was reduced at the CIITA promoter but unaffected at the CXCL11 promoter. In addition, NF-kappaB signaling was required for inhibition of CXCL11 transcription, but not for inhibition of CIITA. Taken together, these results indicate that TLR2-dependent inhibition of IFN-gamma-induced gene expression is mediated by distinct, gene-specific mechanisms that disrupt binding of the transcriptional machinery to the promoters
— id: 101286, year: 2009, vol: 4, page: e6329, stat: Journal Article,

RP105 facilitates macrophage activation by Mycobacterium tuberculosis lipoproteins
Blumenthal, Antje; Kobayashi, Toshihiko; Pierini, Lynda M; Banaei, Niaz; Ernst, Joel D; Miyake, Kensuke; Ehrt, Sabine
2009 Jan 22;5(1):35-46, Cell Host & Microbe
RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins
— id: 95568, year: 2009, vol: 5, page: 35, stat: Journal Article,

Who benefits from granulomas, mycobacteria or host?
Bold, Tyler D; Ernst, Joel D
2009 Jan 9;136(1):17-19, Cell
By investigating host-pathogen interactions in zebrafish using intravital imaging, Davis and Ramakrishnan (2009) provide evidence that aggregates of immune cells known as granulomas, long thought to constrain mycobacterial infection, may instead facilitate its spread
— id: 95569, year: 2009, vol: 136, page: 17, stat: Journal Article,

Interferon-gamma-responsive nonhematopoietic cells regulate the immune response to Mycobacterium tuberculosis
Desvignes, Ludovic; Ernst, Joel D
2009 Dec 18;31(6):974-985, Immunity
Immunity to Mycobacterium tuberculosis in humans and in mice requires interferon gamma (IFN-gamma). Whereas IFN-gamma has been studied extensively for its effects on macrophages in tuberculosis, we determined that protective immunity to tuberculosis also requires IFN-gamma-responsive nonhematopoietic cells. Bone marrow chimeric mice with IFN-gamma-unresponsive lung epithelial and endothelial cells exhibited earlier mortality and higher bacterial burdens than control mice, underexpressed indoleamine-2,3-dioxygenase (Ido1) in lung endothelium and epithelium, and overexpressed interleukin-17 (IL-17) with massive neutrophilic inflammation in the lungs. We also found that the products of IDO catabolism of tryptophan selectively inhibit IL-17 production by Th17 cells, by inhibiting the action of IL-23. These results reveal a previously unsuspected role for IFN-gamma responsiveness in nonhematopoietic cells in regulation of immunity to M. tuberculosis and illustrate the role of IDO in the inhibition of Th17 cell responses
— id: 106204, year: 2009, vol: 31, page: 974, stat: Journal Article,

Anti-TNF immunotherapy and tuberculosis reactivation: another mechanism revealed
Miller, Elizabeth A; Ernst, Joel D
2009 May;119(5):1079-1082, Journal of clinical investigation
Anti-TNF immunotherapy has revolutionized the treatment of some inflammatory diseases, such as RA. However, a major concern is that patients receiving this therapy have an increased risk of fungal and bacterial infection, particularly of reactivating latent tuberculosis (TB). In this issue of the JCI, in an effort to understand how anti-TNF immunotherapy affects host mechanisms required to control TB, Bruns and colleagues examined the effects of the anti-TNF therapeutic infliximab on Mycobacterium tuberculosis-specific human lymphocytes (see the related article beginning on page 1167). The authors report that a granulysin-expressing CD45RA+ subset of effector memory CD8+ T cells that contributes to the killing of intracellular M. tuberculosis is depleted in vivo by infliximab in patients with RA, and that these cells are susceptible to complement-mediated lysis in the presence of infliximab in vitro. The study provides insight into host defense mechanisms that act to control TB infection and how they are affected during anti-TNF immunotherapy for autoimmune disease
— id: 98019, year: 2009, vol: 119, page: 1079, stat: Journal Article,

Meeting Report: NIH Workshop on the Tuberculosis Immune Epitope Database
Ernst, Joel D; Lewinsohn, David M; Behar, Samuel; Blythe, Martin; Schlesinger, Larry S; Kornfeld, Hardy; Sette, Alessandro
2008 Jul;88(4):366-370, Tuberculosis (Edinburgh, Scotland)
The Immune Epitope Database (IEDB), an online resource available at http://immuneepitope.org/, contains data on T cell and B cells epitopes of multiple pathogens, including M. tuberculosis. A workshop held in June, 2007 reviewed the existing database, discussed the utility of reference sets of epitopes, and identified knowledge gaps pertaining to epitopes and immune responses in tuberculosis
— id: 75183, year: 2008, vol: 88, page: 366, stat: Journal Article,

Illuminating the black box of TNF action in tuberculous granulomas
Miller, Elizabeth A; Ernst, Joel D
2008 Aug;29(2):175-177, Immunity
TNF clearly contributes to immunity to intracellular pathogens, but how it does so is incompletely understood. In this issue of Immunity, Clay et al. (2008) provide unique insights, using intravital microscopy and the zebrafish-embryo model of tuberculosis
— id: 93352, year: 2008, vol: 29, page: 175, stat: Journal Article,

CO-opting the host HO-1 pathway in tuberculosis and malaria
Sinnis, Photini; Ernst, Joel D
2008 May 15;3(5):277-279, Cell Host & Microbe
Infection with Mycobacterium tuberculosis and Plasmodium species results in upregulation of the host heme oxygenase-1 pathway. In tuberculosis infection, this leads to upregulation of the bacterial 'dormancy regulon,' whereas in malaria, it enhances the efficiency with which sporozoites develop into exoerythrocytic stages. Here we discuss these findings as well as some of the interesting questions they raise
— id: 78798, year: 2008, vol: 3, page: 277, stat: Journal Article,

Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs
Wolf, Andrea J; Desvignes, Ludovic; Linas, Beth; Banaiee, Niaz; Tamura, Toshiki; Takatsu, Kiyoshi; Ernst, Joel D
2008 Jan 21;205(1):105-115, Journal of experimental medicine
The onset of the adaptive immune response to Mycobacterium tuberculosis is delayed compared with that of other infections or immunization, and allows the bacterial population in the lungs to expand markedly during the preimmune phase of infection. We used adoptive transfer of M. tuberculosis Ag85B-specific CD4(+) T cells to determine that the delayed adaptive response is caused by a delay in initial activation of CD4(+) T cells, which occurs earliest in the local lung-draining mediastinal lymph node. We also found that initial activation of Ag85B-specific T cells depends on production of antigen by bacteria in the lymph node, despite the presence of 100-fold more bacteria in the lungs. Although dendritic cells have been found to transport M. tuberculosis from the lungs to the local lymph node, airway administration of LPS did not accelerate transport of bacteria to the lymph node and did not accelerate activation of Ag85B-specific T cells. These results indicate that delayed initial activation of CD4(+) T cells in tuberculosis is caused by the presence of the bacteria in a compartment that cannot be mobilized from the lungs to the lymph node, where initial T cell activation occurs
— id: 75722, year: 2008, vol: 205, page: 105, stat: Journal Article,

LspA-independent action of globomycin on Mycobacterium tuberculosis
Banaiee, Niaz; Jacobs, William R; Ernst, Joel D
2007 Aug;60(2):414-416, Journal of antimicrobial chemotherapy
OBJECTIVES: The objective of this study was to investigate the antimicrobial activity and specificity of globomycin, an inhibitor of lipoprotein signal peptidase II (LspA), against Mycobacterium tuberculosis. METHODS: The mycobactericidal and mycobacteriostatic activity of globomycin was determined by optical density and cfu plating. The specificity of globomycin was determined by western immunoblotting using anti-MPT83 antibody. RESULTS: Globomycin is mycobactericidal at concentrations>or=40 mg/L. However, at 80 mg/L, the processing of the lipoprotein MPT-83 is unaffected and growth-inhibitory effect of globomycin is unchanged in an lspA null mutant (DeltalspA::lspAmut) lacking the putative drug target. CONCLUSIONS: Globomycin kills M. tuberculosis through a mechanism that is independent of LspA
— id: 73865, year: 2007, vol: 60, page: 414, stat: Journal Article,

An analysis of the epitope knowledge related to Mycobacteria
Blythe, Martin J; Zhang, Qing; Vaughan, Kerrie; de Castro, Romulo Jr; Salimi, Nima; Bui, Huynh-Hoa; Lewinsohn, David M; Ernst, Joel D; Peters, Bjoern; Sette, Alessandro
2007 ;3:10-10, Immunome research
ABSTRACT: BACKGROUND: Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains a leading cause of infectious disease morbidity and mortality, and is responsible for more than 2 million deaths a year. Reports about extremely drug resistant (XDR) strains have further heightened the sense of urgency for the development of novel strategies to prevent and treat TB. Detailed knowledge of the epitopes recognized by immune responses can aid in vaccine and diagnostics development, and provides important tools for basic research. The analysis of epitope data corresponding to M. tuberculosis can also identify gaps in our knowledge, and suggest potential areas for further research and discovery. The Immune Epitope Database (IEDB) is compiled mainly from literature sources, and describes a broad array of source organisms, including M. tuberculosis and other Mycobacterial species. DESCRIPTION: A comprehensive analysis of IEDB data regarding the genus Mycobacteria was performed. The distribution of antibody/B cell and T cell epitopes was analyzed in terms of their associated recognition cell type effector function and chemical properties. The various species, strains and proteins which the epitope were derived, were also examined. Additional variables considered were the host in which the epitopes were defined, the specific TB disease state associated with epitope recognition, and the HLA associated with disease susceptibility and endemic regions were also scrutinized. Finally, based on these results, standardized reference datasets of mycobacterial epitopes were generated. CONCLUSION: All current TB-related epitope data was cataloged for the first time from the published literature. The resulting inventory of more than a thousand different epitopes should prove a useful tool for the broad scientific community. Knowledge gaps specific to TB epitope data were also identified. In summary, few non-peptidic or post-translationally modified epitopes have been defined. Most importantly epitopes have apparently been defined from only 7% of all ORFs, and the top 30 most frequently studied protein antigens contain 65% of the epitopes, leaving the majority of M. tuberculosis genome unexplored. A lack of information related to the specific strains from which epitopes are derived is also evident. Finally, the generation of reference lists of mycobacterial epitopes should also facilitate future vaccine and diagnostic research
— id: 78799, year: 2007, vol: 3, page: 10, stat: Journal Article,

Genomics and the evolution, pathogenesis, and diagnosis of tuberculosis
Ernst, Joel D; Trevejo-Nunez, Giraldina; Banaiee, Niaz
2007 Jul;117(7):1738-1745, Journal of clinical investigation
Tuberculosis kills nearly 2 million people annually, and current approaches to tuberculosis control are expensive, have limited efficacy, and are vulnerable to being overcome by extensively drug-resistant strains of Mycobacterium tuberculosis. Determination of the genome sequence of M. tuberculosis has revolutionized tuberculosis research, contributed to major advances in the understanding of the evolution and pathogenesis of M. tuberculosis, and facilitated development of new diagnostic tests with increased specificity for tuberculosis. In this review, we describe some of the major progress in tuberculosis research that has resulted from knowledge of the genome sequence and note some of the problems that remain unsolved
— id: 74160, year: 2007, vol: 117, page: 1738, stat: Journal Article,

Codominance of TLR2-dependent and TLR2-independent modulation of MHC class II in Mycobacterium tuberculosis infection in vivo
Kincaid, Eleanor Z; Wolf, Andrea J; Desvignes, Ludovic; Mahapatra, Sebabrata; Crick, Dean C; Brennan, Patrick J; Pavelka, Martin S Jr; Ernst, Joel D
2007 Sep 1;179(5):3187-3195, Journal of immunology
Mycobacterium tuberculosis is an exceptionally successful human pathogen. A major component of this success is the ability of the bacteria to infect immunocompetent individuals and to evade eradication by an adaptive immune response that includes production of the macrophage-activating cytokine, IFN-gamma. Although IFN-gamma is essential for arrest of progressive tuberculosis, it is insufficient for efficacious macrophage killing of the bacteria, which may be due to the ability of M. tuberculosis to inhibit selected macrophage responses to IFN-gamma. In vitro studies have determined that mycobacterial lipoproteins and other components of the M. tuberculosis cell envelope, acting as agonists for TLR2, inhibit IFN-gamma induction of MHC class II. In addition, M. tuberculosis peptidoglycan and IL-6 secreted by infected macrophages inhibit IFN-gamma induction of MHC class II in a TLR2-independent manner. To determine whether TLR2-dependent inhibition of macrophage responses to IFN-gamma is quantitatively dominant over the TLR2-independent mechanisms in vivo, we prepared mixed bone marrow chimeric mice in which the hemopoietic compartment was reconstituted with a mixture of TLR(+/+) and TLR2(-/-) cells. When the chimeric mice were infected with M. tuberculosis, the expression of MHC class II on TLR2(+/+) and TLR2(-/-) macrophages from the lungs of individual infected chimeric mice was indistinguishable. These results indicate that TLR2-dependent and -independent mechanisms of inhibition of responses to IFN-gamma are equivalent in vivo, and that M. tuberculosis uses multiple pathways to abrogate the action of an important effector of adaptive immunity. This work was supported by National Institutes of Health Grants AI 065357-AI 020010
— id: 75367, year: 2007, vol: 179, page: 3187, stat: Journal Article,

Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo
Wolf, Andrea J; Linas, Beth; Trevejo-Nunez, Giraldina J; Kincaid, Eleanor; Tamura, Toshiki; Takatsu, Kiyoshi; Ernst, Joel D
2007 Aug 15;179(4):2509-2519, Journal of immunology
Mycobacterium tuberculosis (Mtb) is thought to reside in macrophages, although infected dendritic cells (DCs) have been observed. Thus, although cellular associations have been made, global characterization of the cells harboring Mtb is lacking. We have performed temporal and quantitative characterization of the cells harboring Mtb following aerosol infection of mice by using GFP-expressing bacteria and flow cytometry. We discovered that Mtb infects phagocytic cells of diverse phenotypes, that the predominant infected cell populations change with time, and that myeloid DCs are the major cell population infected with Mtb in the lungs and lymph nodes. We also found that the bacteria in the lung-draining lymph node are transported there from the lungs by a CCL19/21-dependent mechanism and that the transport of bacteria to the lymph node is a transient phenomenon despite chronic infection. In addition, we found that the lymph node cell subsets that are most efficacious in stimulating Mtb-specific, TCR-transgenic CD4(+) T lymphocytes are not infected with the bacteria and are scarce or absent from the lungs of infected mice. Finally, we found that the lung cell populations that are infected with Mtb at high frequency are relatively ineffective at stimulating Ag-specific CD4(+) T lymphocytes, and we have obtained evidence that live Mtb can inhibit MHC class II Ag presentation without a decrease in the surface expression of MHC class II. These results indicate that Mtb targets DC migration and Ag presentation in vivo to promote persistent infection
— id: 74168, year: 2007, vol: 179, page: 2509, stat: Journal Article,

Regulation of Mycobacterium tuberculosis whiB3 in the mouse lung and macrophages
Banaiee, N; Jacobs, W R Jr; Ernst, J D
2006 Nov;74(11):6449-6457, Infection & immunity
Mycobacterium tuberculosis is a highly successful human pathogen, with approximately 2x10(9) individuals infected globally. To understand the responses of M. tuberculosis to the in vivo environment, we studied the in vivo regulation of M. tuberculosis genes whose M. marinum homologs are induced in chronically infected frog tissues. The expression of 16S rRNA was shown to remain constant in M. tuberculosis under in vivo and in vitro conditions and therefore could be used for internal normalization in quantitative reverse transcription-PCR assays. We found whiB3, a putative transcriptional regulator implicated in mediating tissue damage, to be maximally induced at 2 weeks postinfection in the lungs of wild-type and immunodeficient (gamma interferon receptor-/-, Rag1-/-, and tumor necrosis factor alpha-/-) mice. At later time points in wild-type mice, whiB3 induction was decreased and gradually declined over the course of infection. In immunodeficient mice, whiB3 induction declined rapidly and was completely abolished in moribund animals. whiB3 was also found to be induced in naive bone marrow-derived macrophages after 6 h of infection. whiB3 expression in vivo and in vitro was found to be inversely correlated with bacterial density. These results indicate that M. tuberculosis regulates the expression of whiB3 in response to environmental signals present in vivo and are consistent with a model of regulation by quorum sensing
— id: 69586, year: 2006, vol: 74, page: 6449, stat: Journal Article,

Potent inhibition of macrophage responses to IFN-gamma by live virulent Mycobacterium tuberculosis is independent of mature mycobacterial lipoproteins but dependent on TLR2
Banaiee, Niaz; Kincaid, Eleanor Z; Buchwald, Ulrike; Jacobs, William R Jr; Ernst, Joel D
2006 Mar 1;176(5):3019-3027, Journal of immunology
Mycobacterium tuberculosis is a highly successful pathogen that can persist and cause disease despite an immune response. One potential mechanism for resisting elimination is by inhibiting the action of IFN-gamma. We have previously shown that live M. tuberculosis inhibits selected macrophage responses to IFN-gamma, and that purified M. tuberculosis 19-kDa lipoprotein inhibits induction of selected IFN-gamma-responsive genes through a TLR2-dependent pathway, whereas peptidoglycan inhibits responses to IFN-gamma by a TLR2-independent pathway. To determine the relative contribution of lipoproteins to the inhibition of responses to IFN-gamma, we deleted the M. tuberculosis gene (lspA) that encodes lipoprotein signal peptidase. This revealed that M. tuberculosis lipoprotein processing is indispensable for stimulation of TLR2 reporter cells, but that the lspA mutant inhibits macrophage responses to IFN-gamma to the same extent as wild-type bacteria. Macrophages lacking TLR2 are more resistant to inhibition by either strain of M. tuberculosis, suggesting that nonlipoprotein TLR2 agonists contribute to inhibition. Indeed, we found that phosphatidylinositol mannan from M. tuberculosis inhibits macrophage responses to IFN-gamma. M. tuberculosis inhibition of responses to IFN-gamma requires new protein synthesis, indicating that a late effect of innate immune stimulation is the inhibition of responses to IFN-gamma. These results establish that M. tuberculosis possesses multiple mechanisms of inhibiting responses to IFN-gamma
— id: 64137, year: 2006, vol: 176, page: 3019, stat: Journal Article,

Mycobacterium tuberculosis inhibits macrophage responses to IFN-gamma through myeloid differentiation factor 88-dependent and -independent mechanisms
Fortune, Sarah M; Solache, Alejandra; Jaeger, Alejandra; Hill, Preston J; Belisle, John T; Bloom, Barry R; Rubin, Eric J; Ernst, Joel D
2004 May 15;172(10):6272-6280, Journal of immunology
Mycobacterium tuberculosis overcomes macrophage bactericidal activities and persists intracellularly. One mechanism by which M. tuberculosis avoids macrophage killing might be through inhibition of IFN-gamma-mediated signaling. In this study we provide evidence that at least two distinct components of M. tuberculosis, the 19-kDa lipoprotein and cell wall peptidoglycan (contained in the mycolylarabinogalactan peptidoglycan (mAGP) complex), inhibit macrophage responses to IFN-gamma at a transcriptional level. Moreover, these components engage distinct proximal signaling pathways to inhibit responses to IFN-gamma: the 19-kDa lipoprotein inhibits IFN-gamma signaling in a Toll-like receptor (TLR)2-dependent and myeloid differentiation factor 88-dependent fashion whereas mAGP inhibits independently of TLR2, TLR4, and myeloid differentiation factor 88. In addition to inhibiting the induction of specific IFN-gamma responsive genes, the 19-kDa lipoprotein and mAGP inhibit the ability of IFN-gamma to activate murine macrophages to kill virulent M. tuberculosis without inhibiting production of NO. These results imply that inhibition of macrophage responses to IFN-gamma may contribute to the inability of an apparently effective immune response to eradicate M. tuberculosis
— id: 42682, year: 2004, vol: 172, page: 6272, stat: Journal Article,

CCR2-dependent trafficking of F4/80dim macrophages and CD11cdim/intermediate dendritic cells is crucial for T cell recruitment to lungs infected with Mycobacterium tuberculosis
Peters, Wendy; Cyster, Jason G; Mack, Matthias; Schlondorff, Detlef; Wolf, Andrea J; Ernst, Joel D; Charo, Israel F
2004 Jun 15;172(12):7647-7653, Journal of immunology
We previously reported that CCR2(-/-) mice are susceptible to Mycobacterium tuberculosis infection. Susceptibility was associated with an early and sustained macrophage trafficking defect, followed by delayed recruitment of dendritic cells (DCs) and T cells to the lungs. However, the relative importance of the lack of CCR2 expression by macrophages and DCs vs T cells in susceptibility to infection was unclear. In this study, we used mixed bone marrow transplantation to create mice in which the genotype of the T cells was either CCR2(+/+) or CCR2(-/-) while maintaining the genotype of the myeloid cells as CCR2(+/+). After infection with M. tuberculosis, we found that the genotype of the macrophages and/or DCs, but not that of the T cells, was critical for both T cell and myeloid cell migration to the lungs. Further investigation revealed a critical role for CCR2 in the recruitment of F4/80(dim) macrophages and CD11c(dim/intermediate) DCs to the infected lung
— id: 43012, year: 2004, vol: 172, page: 7647, stat: Journal Article,

GTP-dependent secretion from neutrophils is regulated by Cdk5
Rosales, Jesusa L; Ernst, Joel D; Hallows, Janice; Lee, Ki-Young
2004 Dec 24;279(52):53932-53936, Journal of biological chemistry
We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35(-/-) mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils
— id: 78800, year: 2004, vol: 279, page: 53932, stat: Journal Article,

Mycobacterium tuberculosis exerts gene-selective inhibition of transcriptional responses to IFN-gamma without inhibiting STAT1 function
Kincaid, Eleanor Z; Ernst, Joel D
2003 Aug 15;171(4):2042-2049, Journal of immunology
Mycobacterium tuberculosis is a highly successful human pathogen. A major component of this success is the pathogen's ability to avoid eradication by the innate and adaptive immune responses throughout the course of infection. IFN-gamma, a potent activator of the microbicidal activities of macrophages, is essential for control of M. tuberculosis infection, but is unable to stimulate macrophages to kill M. tuberculosis. We have found that infection of the human monocytic cell line, THP-1, resulted in reduced cellular responses to IFN-gamma, manifested as impaired induction of CD64 surface expression and transcription. This defect in transcription occurred despite normal activation of STAT1 in infected macrophages: there was no decrease in STAT1 tyrosine or serine phosphorylation, nuclear translocation, or binding of a minimal IFN-gamma response sequence. Assays of STAT1 function in M. tuberculosis-treated cells also revealed no defect in activation of a minimal gamma-activated sequence construct or STAT1 recruitment to and binding at the endogenous CD64 promoter. In addition, M. tuberculosis did not affect histone acetylation at the CD64 promoter. The inhibition of transcription was gene selective: while transcription of CD64 and class II transactivator were decreased, certain other IFN-gamma-responsive genes either were unaffected or were increased by M. tuberculosis. These results indicate that M. tuberculosis inhibits the response to IFN-gamma by a mechanism distinct from either suppressor of cytokine signaling-1 inhibition of STAT1 phosphorylation or protein inhibitor of activated STAT interference with DNA binding, and indicate that other mechanisms of inhibition of IFN-gamma responses remain to be discovered
— id: 38096, year: 2003, vol: 171, page: 2042, stat: Journal Article,

Innate Inhibition of Adaptive Immunity: Mycobacterium tuberculosis-Induced IL-6 Inhibits Macrophage Responses to IFN-gamma
Nagabhushanam, Vijaya; Solache, Alejandra; Ting, Li-Min; Escaron, Claire J; Zhang, Jennifer Y; Ernst, Joel D
2003 Nov 1;171(9):4750-4757, Journal of immunology
In humans and in mice, control of the intracellular pathogen, Mycobacterium tuberculosis (Mtb), requires IFN-gamma. Although the adaptive immune response results in production of substantial amounts of IFN-gamma in response to Mtb, the immune response is unable to eradicate the infection in most cases. We have previously reported evidence that Mtb inhibits macrophage responses to IFN-gamma, suggesting that this may limit the ability of IFN-gamma to stimulate macrophages to kill Mtb. We have also observed that uninfected macrophages, adjacent to infected macrophages in culture, exhibit decreased responses to IFN-gamma. Here we report that IL-6 secreted by Mtb-infected macrophages inhibits the responses of uninfected macrophages to IFN-gamma. IL-6 selectively inhibits a subset of IFN-gamma-responsive genes at the level of transcriptional activation without inhibiting activation or function of STAT1. Inhibition of macrophage responses to IFN-gamma by IL-6 requires new protein synthesis, but this effect is not attributable to suppressor of cytokine signaling 1 or 3. These results reveal a novel function for IL-6 and indicate that IL-6 secreted by Mtb-infected macrophages may contribute to the inability of the cellular immune response to eradicate infection
— id: 38268, year: 2003, vol: 171, page: 4750, stat: Journal Article,

Mechanisms of cell recruitment in the immune response to Mycobacterium tuberculosis
Peters, Wendy; Ernst, Joel D
2003 Feb;5(2):151-158, Microbes & infection
Recent advances in understanding cell traffic, especially the roles of adhesion proteins, chemokines, and chemokine receptors, provide the opportunity for understanding mechanisms involved in the immune response to tuberculosis. This review concentrates on the roles of these molecules and the immune response in tuberculosis, based on studies of humans and mice infected with Mycobacterium tuberculosis
— id: 34826, year: 2003, vol: 5, page: 151, stat: Journal Article,

Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis
Peters W; Scott HM; Chambers HF; Flynn JL; Charo IF; Ernst JD
2001 Jul 3;98(14):7958-7963, Proceedings of the National Academy of Sciences of the United States of America
Although the protective cellular immune response to Mycobacterium tuberculosis requires recruitment of macrophages and T lymphocytes to the site of infection, the signals that regulate this trafficking have not been defined. We investigated the role of C-C chemokine receptor 2 (CCR2)-dependent cell recruitment in the protective response to M. tuberculosis. CCR2(-/-) mice died early after infection and had 100-fold more bacteria in their lungs than did CCR2(+/+) mice. CCR2(-/-) mice exhibited an early defect in macrophage recruitment to the lung and a later defect in recruitment of dendritic cells and T cells to the lung. CCR2(-/-) mice also had fewer macrophages and dendritic cells recruited to the mediastinal lymph node (MLN) after infection. T cell migration through the MLN was similar in CCR2(-/-) and CCR2(+/+) mice. However, T cell priming was delayed in the MLNs of the CCR2(-/-) mice, and fewer CD4(+) and CD8(+) T cells primed to produce IFN-gamma accumulated in the lungs of the CCR2(-/-) mice. These data demonstrate that cellular responses mediated by activation of CCR2 are essential in the initial immune response and control of infection with M. tuberculosis
— id: 33860, year: 2001, vol: 98, page: 7958, stat: Journal Article,

Cholesterol esterification by host and parasite is essential for optimal proliferation of Toxoplasma gondii
Sonda S; Ting LM; Novak S; Kim K; Maher JJ; Farese RV Jr; Ernst JD
2001 Sep 14;276(37):34434-34440, Journal of biological chemistry
Upon host cell invasion the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment termed the parasitophorous vacuole that is derived from the host cell membrane but modified by the parasite. Despite the segregation of the parasitophorous vacuole from the host endocytic network, the intravacuolar parasite has been shown to acquire cholesterol from the host cell. In order to characterize further the role of sterol metabolism in T. gondii biology, we focused our studies on the activity of acyl-CoA:cholesterol acyltransferase (ACAT), a key enzyme for maintaining the intracellular homeostasis of cholesterol through the formation of cholesterol esters. In this study, we demonstrate that ACAT and cholesterol esters play a crucial role in the optimal replication of T. gondii. Moreover, we identified ACAT activity in T. gondii that can be modulated by pharmacological ACAT inhibitors with a consequent detrimental effect on parasite replication
— id: 33859, year: 2001, vol: 276, page: 34434, stat: Journal Article,

Modulation of Dengue virus infection in human cells by alpha, beta, and gamma interferons
Diamond MS; Roberts TG; Edgil D; Lu B; Ernst J; Harris E
2000 Jun;74(11):4957-4966, Journal of virology
A role for interferon (IFN) in modulating infection by dengue virus (DV) has been suggested by studies in DV-infected patients and IFN receptor-deficient mice. To address how IFN modulates DV type 2 infection, we have assayed IFN-alpha, -beta, and -gamma for the ability to enhance or diminish antibody-independent and antibody-dependent cell infection using a competitive, asymmetric reverse transcriptase-mediated PCR (RT-PCR) assay that quantitates positive and negative strands of viral RNA, a flow cytometric assay that measures viral antigen, and a plaque assay that analyzes virion production. Our data suggest that IFN-alpha and -beta protect cells against DV infection in vitro. Treatment of hepatoma cells with IFN-alpha or -beta decreases viral RNA levels greater than 1, 000-fold, the percentage of cells infected 90 to 95%, and the amount of infectious virus secreted 150- to 100,000-fold. These results have been reproduced with several cell types and viral strains, including low-passage isolates. In contrast, IFN-gamma has a more variable effect depending on the cell type and pathway of infection. Quantitative RT-PCR experiments indicate that IFN inhibits DV infection by preventing the accumulation of negative-strand viral RNA
— id: 42258, year: 2000, vol: 74, page: 4957, stat: Journal Article,

Bacterial inhibition of phagocytosis
Ernst JD
2000 Oct;2(5):379-386, Cellular microbiology
The concerted study of molecular mechanisms of phagocytosis and the inhibition of phagocytosis by specific products of extracellular bacterial pathogens has borne considerable fruit. The importance of tyrosine phosphorylation and of the Rho family of GTPases has become clear to cell biologists, but pathogenic bacteria recognized the importance of these signalling pathways in phagocytic cells long ago. The discoveries described in this review are only the beginning. The simultaneous pursuit of the mechanisms and molecules involved in the initiation and regulation of phagocytosis and that pathogenic bacteria use to inhibit phagocytosis will surely identify more interesting pathways on each side of the contest. Are there any obvious possibilities? There are several bacterial factors that have the potential to inhibit known mechanisms of phagocytosis. Clostridium species, for example, make a number of exotoxins of interest. Clostridium botulinum and Clostridium tetani neurotoxins inactivate the regulated secretory machinery by proteolytic cleavage of SNARE proteins, and targets of tetanus toxin and botulinum b toxin inhibit the exocytotic delivery of membrane vesicles needed for phagocytosis of large particles (Hackam et al., 1998). Moreover, the C3 exotoxin of C. botulinum catalyses ADP ribosylation and inactivation of rho family GTPases (Wiegers et al., 1991), and toxins A and B of C. difficile UDP-glucosylate and inactivate rho GTPases and thereby disrupt the actin cytoskeleton (Just et al., 1995a,b). However, as Clostridia lack the machinery for type III secretion, these proteins are not rapidly targeted to the phagocyte cytoplasm. More searching may reveal a pathogen that has combined the type III secretory machinery with clostridia toxin-like substrates. A potentially unique strategy for remaining outside phagocytes is exhibited by Helicobacter pylori, which contain a type IV secretion system. Unopsonized virulent strains of H. pylori bind readily to macrophages but are only internalized after a delay of several minutes. Such a delay appears to be sufficient for the bacteria to remain extracellular (Allen et al., 2000). Elucidation of the mechanism used by H. pylori to delay phagocytosis may reveal one or more novel virulence factors as well as one or more novel targets in the phagocyte that will add to the understanding of a fundamental process in host defence. Another field ripe for further mechanistic investigation is complement receptor-mediated phagocytosis. Dedicated study of the molecular events and molecular mediators of phagocytosis downstream of CR3 is likely to reveal interesting differences from FcgammaR phagocytosis and is just as likely to reveal that microbes have discovered unique mechanisms for circumventing them. Study of extracellular pathogens and the mechanisms that they use to remain outside phagocytic cells has revealed a great deal about the initial encounter between pathogen and phagocyte. We can look forward to additional discoveries about the host-pathogen interactions and the mechanisms and factors that each side uses to battle against the other
— id: 33861, year: 2000, vol: 2, page: 379, stat: Journal Article,

Editorial response: variation in clinical and immune responses to bacille Calmette-Guerin--implications for an improved tuberculosis vaccine
Ernst JD
1999 Apr;28(4):791-793, Clinical infectious diseases
— id: 33865, year: 1999, vol: 28, page: 791, stat: Journal Article,

Toward the development of antibacterial vaccines: report of a symposium and workshop. Organizing Committee
Ernst JD
1999 Nov;29(5):1295-1302, Clinical infectious diseases
On 26 and 27 October 1998, the Department of Medicine at the University of California, San Francisco (UCSF), hosted a symposium and workshop on bacterial vaccines. The symposium featured invited speakers who are internationally recognized authorities in their fields and who discussed selected topics related to specific pathogens or specific principles of bacterial vaccine development. The workshop, held on the day following the symposium, brought together the invited speakers and members of the organizing committee, who came from UCSF and the University of California, Berkeley, to discuss 4 specific topics and to define priorities for future vaccine development. Considerable knowledge has been gained from successful and unsuccessful vaccine development efforts, and large gains in knowledge relevant to vaccine development have resulted from studies of basic immunology and microbial pathogenesis. This report summarizes the presentations at the symposium and the discussions of the workshop sessions
— id: 33866, year: 1999, vol: 29, page: 1295, stat: Journal Article,

Mycobacterium tuberculosis inhibits IFN-gamma transcriptional responses without inhibiting activation of STAT1
Ting LM; Kim AC; Cattamanchi A; Ernst JD
1999 Oct 1;163(7):3898-3906, Journal of immunology
IFN-gamma activates macrophages to kill diverse intracellular pathogens, but does not activate human macrophages to kill virulent Mycobacterium tuberculosis. We tested the hypothesis that this is due to inhibition of IFN-gamma signaling by M. tuberculosis and found that M. tuberculosis infection of human macrophages blocks several responses to IFN-gamma, including killing of Toxoplasma gondii and induction of FcgammaRI. The inhibitory effect of M. tuberculosis is directed at transcription of IFN-gamma-responsive genes, but does not affect proximal steps in the Janus kinase-STAT pathway, as STAT1alpha tyrosine and serine phosphorylation, dimerization, nuclear translocation, and DNA binding are intact in M. tuberculosis-infected cells. In contrast, there is a marked decrease in IFN-gamma-induced association of STAT1 with the transcriptional coactivators CREB binding protein and p300 in M. tuberculosis-infected macrophages, indicating that M. tuberculosis directly or indirectly disrupts this protein-protein interaction that is essential for transcriptional responses to IFN-gamma. Gamma-irradiated M. tuberculosis and isolated cell walls reproduce the effects of live bacteria, indicating that the bacterial component(s) that initiates inhibition of IFN-gamma responses is constitutively expressed. Although lipoarabinomannan has been found to exert effects on macrophages, it does not account for the inhibitory effects of cell walls. These results indicate that one mechanism for M. tuberculosis to evade the human immune response is to inhibit the IFN-gamma signaling pathway, and that the mechanism of inhibition is distinct from that reported for Leishmania donovani or CMV, in that it targets the interaction of STAT1 with the basal transcriptional apparatus
— id: 33867, year: 1999, vol: 163, page: 3898, stat: Journal Article,

Macrophage receptors for Mycobacterium tuberculosis
Ernst JD
1998 Apr;66(4):1277-1281, Infection & immunity
— id: 33871, year: 1998, vol: 66, page: 1277, stat: Journal Article,

Rates of tuberculosis infection in healthcare workers providing services to HIV-infected populations. Terry Beirn Community Programs for Clinical Research on AIDS
Zahnow K; Matts JP; Hillman D; Finley E; Brown LS Jr; Torres RA; Ernst J; El-Sadr W; Perez G; Webster C; Barber B; Gordin FM
1998 Nov;19(11):829-835, Infection control & hospital epidemiology
OBJECTIVE: To assess the prevalence of tuberculosis (TB) or a positive skin test in healthcare workers (HCWs) providing services to human immunodeficiency virus (HIV)-infected individuals and to determine prospectively the incidence of new infections in this population. DESIGN: This prospective cohort study enrolled 1,014 HCWs working with HIV-infected populations from 10 metropolitan areas. Purified protein derivative (PPD) tuberculin skin tests were placed at baseline and every 6 months afterwards on those without a history of TB or a positive PPD. Demographic, occupational, and TB exposure data also were collected. SETTING: Outpatient clinics, hospitals, private practice offices, and drug treatment programs providing HIV-related healthcare and research programs. PARTICIPANTS: A voluntary sample of staff and volunteers from 16 Community Programs for Clinical Research on AIDS units. RESULTS: Factors related to prior TB or a positive skin test at baseline included being foreign-born, increased length of time in health care, living in New York City, or previous bacille Calmette-Guerin vaccination. The rate of PPD conversion was 1.8 per 100 person years of follow-up. No independent relation was found between the amount or type of contact with HIV-infected populations and the risk of TB infection. CONCLUSION: These data provide some reassurance that caring for HIV-infected patients is not related to an increased rate of TB infection among HCWs in these settings
— id: 7866, year: 1998, vol: 19, page: 829, stat: Journal Article,

Transfer of phagocytosed particles to the parasitophorous vacuole of Leishmania mexicana is a transient phenomenon preceding the acquisition of annexin I by the phagosome
Collins HL; Schaible UE; Ernst JD; Russell DG
1997 Jan;110 ( Pt 2)(Suppl 5):191-200, Journal of cell science
The eukaryotic intracellular pathogen Leishmania mexicana resides inside macrophages contained within a membrane bound parasitophorous vacuole which, as it matures, acquires the characteristics of a late endosomal compartment. This study reports the selectivity of fusion of this compartment with other particle containing vacuoles. Phagosomes containing zymosan or live Listeria monocytogenes rapidly fused with L. mexicana parasitophorous vacuoles, while those containing latex beads or heat killed L. monocytogenes failed to do so. Fusigenicity of phagosomes was not primarily dependent on the receptor utilized for ingestion, as opsonization with defined ligands could not overcome the exclusion of either latex beads or heat killed organisms. However modulation of intracellular pH by pharmacological agents such as chloroquine and ammonium chloride increased delivery of live Listeria and also induced transfer of previously excluded particles. The absence of fusion correlated with the acquisition of annexin I, a putative lysosomal targeting, molecule, on the phagosome membrane. We propose that the acquisition of cellular membrane constituents such as annexin I during phagosome maturation can ultimately direct the fusion pathway of the vesicles formed and have described a model system to further document changes in vesicle fusigenicity within cells
— id: 33874, year: 1997, vol: 110 ( Pt 2), page: 191, stat: Journal Article,

Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V
Kuypers FA; Lewis RA; Hua M; Schott MA; Discher D; Ernst JD; Lubin BH
1996 Feb 1;87(3):1179-1187, Blood
The phospholipids of the human red cell are distributed asymmetrically in the bilayer of the red cell membrane. In certain pathologic states, such as sickle cell anemia, phospholipid asymmetry is altered. Although several methods can be used to measure phospholipid organization, small organizational changes have been very difficult to assess. Moreover, these methods fail to identify subpopulations of cells that have lost their normal phospholipid asymmetry. Using fluorescently labeled annexin V in flow cytometry and fluorescent microscopy, we were able to identify and quantify red cells that had lost their phospholipid asymmetry in populations as small as 1 million cells. Moreover, loss of phospholipid organization in subpopulations as small as 0.1% of the total population could be identified, and individual cells could be studied by fluorescent microscopy. An excellent correlation was found between fluorescence-activated cell sorter (FACS) analysis results using annexin V to detect red cells with phosphatidylserine (PS) on their surface and a PS-requiring prothrombinase assay using similar red cells. Cells that bound fluorescein isothiocyanate (FITC)-labeled annexin V could be isolated from the population using magnetic beads covered with an anti-FITC antibody. Evaluation of blood samples from patients with sickle cell anemia under oxygenated conditions demonstrated the presence of subpopulations of cells that had lost phospholipid asymmetry. While only a few red cells were labeled in normal control samples (0.21% +/- 0.12%, n = 8), significantly increased (P < .001) annexin V labeling was observed in samples from patients with sickle cell anemia (2.18% +/- 1.21%, n = 13). We conclude that loss of phospholipid asymmetry may occur in small subpopulations of red cells and that fluorescently labeled annexin V can be used to quantify and isolate these cells
— id: 33878, year: 1996, vol: 87, page: 1179, stat: Journal Article,

The (alpha2-->8)-linked polysialic acid capsule of group B Neisseria meningitidis modifies multiple steps during interaction with human macrophages
Read RC; Zimmerli S; Broaddus C; Sanan DA; Stephens DS; Ernst JD
1996 Aug;64(8):3210-3217, Infection & immunity
Group B Neisseria meningitidis causes systemic disease, including meningitis, after initial colonization and subsequent penetration of nasopharyngeal mucosa, a tissue which is richly populated by macrophages. In an initial effort to characterize the interaction of N. meningitidis and mature human macrophages, the influence of the alpha2-->8) -linked polysialic acid capsule on the interaction of N. meningitidis with human monocyte-derived macrophages was investigated with a capsulate case isolate and an isogenic Tn916-derived noncapsulate transformant. The capsulate strain was fourfold less adherent to the macrophage surface after cold incubation, although adherence of both strains was significantly increased after opsonization with nonimmune C5-depleted serum. When opsonized inocula were adjusted so that they adhered to macrophages in equal numbers, the two strains were internalized at equivalent rates and both entered membrane-bound compartments (phagosomes). Colocalization of bacteria with the late endosomal and lysosomal marker lysosome-associated membrane protein revealed that fusion of lysosomes with phagosomes containing the capsulate organism was significantly reduced 10 and 30 min after entry, but by 1 h, no difference between the strains was observed. Once internalized, meningococci were effectively killed, although more rapid killing of the capsulate strain was observed over the first 3 h. These results indicate that the (alpha2-->8)-linked polysialic acid capsule modifies the interaction of meningococci with human macrophages at multiple steps, including adherence to the macrophage surface and phagosome-lysosome fusion. Moreover, the discordance between the kinetics of phagosome- lysosome fusion and bacterial killing suggests that a nonlysosomal mechanism may be responsible for a significant fraction of macrophage killing of N. meningitidis
— id: 33877, year: 1996, vol: 64, page: 3210, stat: Journal Article,

Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages
Zimmerli S; Edwards S; Ernst JD
1996 Dec;15(6):760-770, American journal of respiratory cell & molecular biology
Mycobacterium tuberculosis survives and replicates within human macrophages, but the mechanisms whereby tubercle bacilli resist killing are incompletely understood. We tested the general model in which M. tuberculosis evades killing by entering naive macrophages through receptors that are unable to activate cellular microbicidal activities. Complement receptor types 1 (CR1), 3 (CR3), and 4 (CR4) were blocked with monoclonal antibodies, and mannose receptors were blocked with a competitive ligand, mannosylated bovine serum albumin (MBSA). Survival and replication of M. tuberculosis (Erdman) were evaluated after the bacteria were phagocytosed in the presence of blocking agents (directing binding to the unblocked receptors). Although there was significant variation in the growth rate of virulent M. tuberculosis in monocyte-derived macrophages from different donors, the intracellular survival and replication of mycobacteria were equivalent regardless of the receptor(s) used for binding and phagocytosis. We conclude that the mechanisms whereby M. tuberculosis evades killing by human macrophages are independent of the receptor-mediated route of entry, and operate at one or more steps common to all entry pathways. Blocking complement and mannose receptors in combination did not completely abrogate binding of M. tuberculosis to macrophages. However, we found that two polyanionic scavenger-receptor ligands exhibited a concentration-dependent ability to block binding of M. tuberculosis to macrophages. Moreover, blocking class A scavenger receptors abrogated nearly all binding that persisted after blocking complement and mannose receptors. This indicates that class A scavenger receptors are quantitatively important mediators of M. tuberculosis-macrophage interactions. M. tuberculosis has evolved multiple mechanisms to promote its efficient entry into macrophages. This suggests that passage of the organism through macrophages may be an essential early step in the pathogenesis of tuberculosis
— id: 33875, year: 1996, vol: 15, page: 760, stat: Journal Article,

Phagosome-lysosome fusion is a calcium-independent event in macrophages
Zimmerli S; Majeed M; Gustavsson M; Stendahl O; Sanan DA; Ernst JD
1996 Jan;132(1-2):49-61, Journal of cell biology
Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome-granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (M phi) were treated with 12.5 microM bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N',-tetraacetic acid tetraacetoxymethyl ester (MAPT/AM), a cell-permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to < or = 20 nM and completely blocked increases in [Ca2+]i in response to multiple stimuli, even in the presence of extracellular calcium. Subsequently, M phi phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis. Microbes were enumerated by 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-1) as a membrane marker and rhodamine dextran as a content marker for lysosomes. Confirmation of phagosome-lysosome fusion by electron microscopy validated the fluorescence microscopy findings. We found that phagosome-lysosome fusion in M phi occurs noramlly at very low [Ca2+]i (< or = 20 nM). Kinetic analysis showed that in M phi none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+]i in a rate-limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs. control macrophages. We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium
— id: 33879, year: 1996, vol: 132, page: 49, stat: Journal Article,