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Pleural tuberculosis (TB), together with lymphatic TB, constitutes more than half of all extrapulmonary cases. Pleural effusions (PEs) in TB are representative of lymphocytic PEs which are dominated by T cells. However, the mechanism underlying T lymphocytes homing and accumulation in PEs is still incompletely understood. Here we performed a comparative analysis of cytokine abundance in PEs from TB patients and non-TB patients by protein array analysis and observed that MCP-2/CCL8 is highly expressed in the TB-PEs as compared to peripheral blood. Meanwhile, we observed that CCR5, the primary receptor used by MCP-2/CCL8, is mostly expressed on pleural CD4(+) T lymphocytes. Furthermore, we found that infection with either Mycobacterium bovis Bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis H37Rv induced production of MCP-2/CCL8 at both transcriptional and protein level in Raw264.7 and THP-1 macrophage cells, mouse peritoneal macrophages as well as human PBMC monocyte-derived macrophages (MDMs). The induction of MCP-2/CCL8 by mycobacteria is dependent on the activation of TLR2/PI3K/Akt and p38 signaling pathway. We conclude that accumulation of MCP-2/CCL8 in TB-PEs may function as a biomarker for TB diagnosis.

Background The HLA (human leukocyte antigen) molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens’ epitopes to bind diverse HLA alleles, referred to as an epitope’s binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure. Methods We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines. Results We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes. Conclusions Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.

All annexins bind Ca2+ and phospholipids, although individual annexins differ markedly in their affinities for these ligands. Annexin I binds phosphatidylserine (PS) at lower [Ca2+] than annexin V,while annexin V exhibits a higher affinity for PS than does annexin I. To identify the structural determinants of these properties, we characterized a series of chimeric annexins. A chimera containing repeat 1 of annexin V fused to repeats 2, 3, and 4 of annexin I exhibited a Ca2+ requirement for PS binding close to that of annexin I, while chimeras containing repeat 1 of annexin I fused to repeats 2, 3, and 4 of annexin V required higher [Ca2+], similar to that of annexin V. In contrast, the overall affinity for PS vesicles was determined by the source of repeat 1. The chimera that contained repeat 1 of annexin V exhibited a high affinity for PS, while a chimera that contained repeat 1 of annexin I had a low affinity for PS similar to that of annexin I. We conclude that the [Ca2+] requirement for phospholipid binding and the overall phospholipid affinity of annexins are determined by distinct domains.

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-γ 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-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to

Treatment of tuberculosis, a complex granulomatous disease, requires long-term multidrug therapy to overcome tolerance, an epigenetic drug resistance that is widely attributed to nonreplicating bacterial subpopulations. Here, we deploy Mycobacterium marinum-infected zebrafish larvae for in vivo characterization of antitubercular drug activity and tolerance. We describe the existence of multi-drug tolerant organisms that arise within days of infection, are enriched in the replicating intracellular population, and are amplified and disseminated by the tuberculous granuloma. Bacterial efflux pumps that are required for intracellular growth mediate this macrophage-induced tolerance. This newly discovered tolerant population also develops when Mycobacterium tuberculosis infects cultured macrophages, suggesting that it contributes to the burden of drug tolerance in human tuberculosis. Efflux pump inhibitors like verapamil reduce this tolerance. Thus, the addition of this currently approved drug, or more specific inhibitors, to standard antitubercular therapy may shorten the duration of curative treatment.

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 (Δ lspA :: lspA mut ) 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.

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.

Annexins are structurally related proteins that bind phospholipids in a Ca2(+)-dependent manner and possess at least four conserved 70-amino acid repeat domains. The ability of certain annexins to promote contact between vesicle membranes in vitro has prompted the suggestion that these proteins regulate membrane traffic in exocytosis. We have previously found that annexins I and II promote contact between vesicles whereas annexin V does not. In order to understand the mechanism of annexin I-mediated vesicle-vesicle contact, we prepared a monoclonal antibody that specifically inhibits annexin I-mediated vesicle aggregation. We identified the domain of annexin I recognized by this monoclonal antibody by using it to screen an expression library containing random fragments of annexin I cDNA. The antibody identified a fragment encoding amino acids 41-118 (the first repeat plus 8 residues of the amino-terminal tail). We constructed a chimeric protein containing these amino acids of annexin I fused to the second, third, and fourth repeats of annexin V. Transfer of this domain conferred the ability to promote vesicle aggregation, confirming that this domain participates directly in mediating contact between vesicle membranes.

Intracellular ionized calcium has been strongly implicated in mediating several responses of human neutrophils to stimulation. However, proteins that serve as effectors of these responses have not been well characterized. To identify proteins that might serve as mediators of the effects of Ca2+ in human neutrophils, we isolated proteins that bind to membrane phospholipids in a Ca2(+)-dependent manner. The most abundant of these, a protein of 33 kD, was readily purified to homogeneity, and was found to bind to phosphatidylserine vesicles in the presence of 2 microM ionized Ca2+. In addition, this purified protein promoted Ca2(+)-dependent aggregation of isolated specific granules from human neutrophils, indicating that it might mediate membrane-membrane contact during processes such as phagosome-lysosome fusion or degranulation. This protein was localized to the cytoplasm of unstimulated neutrophils and found to account for approximately 1% of the cytosol protein. Amino acid sequence of several peptides derived from the purified protein revealed that it is identical to lipocortin III, a recently described member of the annexin family that is scarce in other cells and tissues. The abundance of this protein, together with its Ca2(+)-dependent membrane effects, suggest that it mediates membrane-localized events in stimulated neutrophils, such as phagosome-lysosome fusion or degranulation.

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.

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.

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.

This book provides up-to-date information on the crucial interaction of pathogenic bacteria and professional phagocytes, the host cells whose purpose is to ingest, kill, and digest bacteria in defense against infection. The introductory chapters focus on the receptors used by professional phagocytes to recognize and phagocytose bacteria, and the signal transduction events that are essential for phagocytosis of bacteria. Subsequent chapters discuss specific bacterial pathogens and the strategies they use in confronting professional phagocytes. Examples include Helicobacter pylori, Streptococcus pneumoniae, and Yersinae, each of which uses distinct mechanisms to avoid being phagocytosed and killed. Contrasting examples include Listeria monocytogenes and Mycobacterium tuberculosis, which survive and replicate intracellularly, and actually cooperate with phagocytes to promote their entry into these cells. Together, the contributions in this book provide an outstanding review of current knowledge regarding the mechanisms of phagocytosis and how specific pathogenic bacteria avoid or exploit these mechanisms.

Type I interferons (IFN-α and IFN-β) are important for protection against many viral infections, whereas type II interferon (IFN-γ) is essential for host defense against some bacterial and parasitic pathogens. Study of IFN responses in human leprosy revealed an inverse correlation between IFN-β and IFN-γ gene expression programs. IFN-γ and its downstream vitamin D–dependent antimicrobial genes were preferentially expressed in self-healing tuberculoid lesions and mediated antimicrobial activity against the pathogen Mycobacterium leprae in vitro. In contrast, IFN-β and its downstream genes, including interleukin-10 (IL-10), were induced in monocytes by M. leprae in vitro and preferentially expressed in disseminated and progressive lepromatous lesions. The IFN-γ–induced macrophage vitamin D–dependent antimicrobial peptide response was inhibited by IFN-β and by IL-10, suggesting that the differential production of IFNs contributes to protection versus pathogenesis in some human bacterial infections.

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-γ, 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-γ, 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-γ response sequence. Assays of STAT1 function in M. tuberculosis-treated cells also revealed no defect in activation of a minimal γ-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-γ-responsive genes either were unaffected or were increased by M. tuberculosis. These results indicate that M. tuberculosis inhibits the response to IFN-γ 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-γ responses remain to be discovered.

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.

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.

Phagocytosis of asbestos fibers may be a necessary step for asbestos-induced injury to mesothelial cells, but this has not been established because quantification of fiber uptake is difficult and ways to increase fiber phagocytosis without also increasing total dose were not available. We quantified phagocytosis by counting intracellular fibers after removing adherent fibers with trypsin; we selectively increased fiber phagocytosis by coating crocidolite asbestos fibers with the adhesive serum protein vitronectin (VN), which we have shown increases fiber uptake via integrins. We measured various aspects of asbestos-induced cytotoxicity: intracellular oxidation by the shift of fluorescence of cells loaded with an oxidative probe, DNA strand breakage by the alkaline unwinding ethidium bromide fluorometric assay, apoptosis by annexin V binding and by nuclear morphology, and cell-cycle progression. We found that, compared with control fibers or particles, asbestos increased intracellular oxidation, DNA strand breakage, and apoptosis. Selective increases in fiber uptake by VN-coating of the fibers further increased the oxidation, DNA strand breakage, and apoptosis, and induced a cell-cycle arrest in G2/M. Selective decreases in fiber uptake by cytochalasin or by integrin blockade with RGD peptides inhibited several of these measures of injury. We conclude that phagocytosis is important and perhaps necessary for asbestos-induced injury to mesothelial cells.

The initial interaction of Mycobacterium tuberculosis with phagocytes through Toll-like receptors can affect induction of the adaptive response by inflammatory cytokine production, as well as begin the process of bacterial containment by induction of antimycobacterial functions. Advances in T cell research in tuberculosis include identification of antigens recognized by CD8(+) T cells in infected hosts, elucidation of antimycobacterial mechanisms of T cells and the discovery of CD8(+) T cells that recognize antigens presented by a variety of non-classical molecules.

Renewed concern about the worldwide problem of tubercu- BCG 1 month after inoculation (one was still shedding viable losis has stimulated efforts to define mechanisms that allow BCG 2 months after inoculation). This finding prompts a cauMycobacterium tuberculosis unequaled success as a pathogen, tionary note from the investigators that shedding of viable BCG to identify new drug targets, and to develop an improved vac- may present a hazard to contacts with AIDS, with an attendant cine. In this issue of Clinical Infectious Diseases, Hoft and risk of disseminated BCG infection in the contacts. colleagues [1] report results of their efforts to characterize the Second, the investigators found some interesting correlations response to the existing tuberculosis vaccine, BCG. Despite with the duration of vaccine-induced ulceration. Subjects with widespread use of the BCG vaccine, its effects are not well the highest in vitro lymphoproliferative response to M. tubercuunderstood, controlled clinical trials have yielded variable re- losis antigens before vaccination had the shortest duration of sults, and it has not prevented tuberculosis from causing the ulceration. This finding illustrates that before vaccination, death of more humans each year than any other single infection. healthy volunteers with negative PPD skin tests differ in their responses detected by assays that are generally taken to reflect specific acquired cellular immunity. It is interesting to speculate

The phagocytic function of neutrophils is a crucial element in the host defence against invading microorganisms. We investigated phagocytosis and intracellular killing of an attenuated strain of Mycobacterium tuberculosis(H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca2+]i and certain cytosolic calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca2+]i. The ability to kill H37Ra decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated from cytoplasm to the proximity of the H37Ra-containing phagosomes, whereas the localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca2+-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca2+-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.

We have evaluated the uptake of a soluble protein antigen, denitrophenylated human serum albumin (DNP-HSA), and two different intracellular bacteria; Chlamydia trachomatis serovar L2 and Mycobacterium tuberculosis strain H37Ra, by immature human dendritic cells. These were generated by culturing progenitor cells from blood in the presence of cytokines (granulocyte-macrophage colony-stimulating factor and interleukin-4). Dendritic cells play a crucial part in antigen presentation for the induction of T-cell-dependent immune responses in various tissues. Recently, macropinocytic and phagocytic activity has been shown for immature dendritic cells of mouse, rat and human origin. In the present study, macropinocytosis characterized the uptake of the soluble protein-antigen DNP-HSA, whereas the C. trachomatis were ingested via receptor-mediated endocytosis in coated pits, and opsonized M. tuberculosis via phagocytosis. To follow the intracellular routes of the antigens, their positions were compared with the localization of annexins, a family of Ca(2+)-and phospholipid-binding proteins, involved in membrane fusion, aggregation and transport of different vesicles. To elucidate further the intracellular pathway of the antigens, two other proteins, lysosome-associated membrane protein-1 (LAMP-1) and cathepsin D, were labelled. They are known to colocalize with major histocompatibility complex class II compartments in the immature dendritic cells. We observed a distinct translocation of annexin V to DNP-HSA containing endosomes, and annexin III to vesicles with C. trachomatis. Furthermore, annexin III, IV and V redistributed to phagosomes with M. tuberculosis. Both LAMP-1 and cathepsin D colocalized with DNP-HSA endosomes, and with phagosomes with M. tuberculosis. Thus, immature human dendritic cells have the capacity to phagocytose. Moreover, the handling of these antigens by dendritic cells may represent three distinct intracellular pathways, albeit some properties and compartments are shared.

The Mycobacterium tuberculosis genome includes the large family of pe_pgrs genes, whose functions are unknown. Because of precedents in other pathogens in which gene families showing high sequence variation are involved in antigenic variation, a similar role has been proposed for the pe_pgrs genes. However, the impact of immune selection on pe_pgrs genes has not been examined. Here, we sequenced 27 pe_pgrs genes in 94 clinical strains from five phylogenetic lineages of the M. tuberculosis complex (MTBC). We found that pe_pgrs genes were overall more diverse than the remainder of the MTBC genome, but individual members of the family varied widely in their nucleotide diversity and insertion/deletion (indel) content: some were more, and others were much less, diverse than the genome average. Individual pe_pgrs genes also differed in the ratio of nonsynonymous to synonymous mutations, suggesting that different selection pressures act on individual pe_pgrs genes. Using bioinformatic methods, we tested whether sequence diversity in pe_pgrs genes might be selected by human T cell recognition, the major mechanism of adaptive immunity to MTBC. We found that the large majority of predicted human T cell epitopes were confined to the conserved PE domain and experimentally confirmed the antigenicity of this domain in tuberculosis patients. In contrast, despite being genetically diverse, the PGRS domains harbored few predicted T cell epitopes. These results indicate that human T cell recognition is not a significant force driving sequence diversity in pe_pgrs genes, which is consistent with the previously reported conservation of human T cell epitopes in the MTBC., IMPORTANCE Recognition of Mycobacterium tuberculosis antigens by T lymphocytes is known to be important for immune protection against tuberculosis, but it is unclear whether human T cell recognition drives antigenic variation in M. tuberculosis. We previously discovered that the known human T cell epitopes in the M. tuberculosis complex are highly conserved, but we hypothesized that undiscovered epitopes with naturally occurring sequence variants might exist. To test this hypothesis, we examined the pe_pgrs genes, a large family of genes that has been proposed to function in immune evasion by M. tuberculosis. We found that the pe_pgrs genes exhibit considerable sequence variation, but the regions containing T cell epitopes and the regions of variation are distinct. These findings confirm that the majority of human T cell epitopes of M. tuberculosis are highly conserved and indicate that selection forces other than T cell recognition drive sequence variation in the pe_pgrs genes.

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.

Infection with human immunodeficiency virus (HIV) has been associated with increased rates of single- and multidrug-resistant (MDR) tuberculosis in the New York City area. In order to examine the relationship of HIV infection to drug-resistant tuberculosis in other selected regions of the United States, we established a registry of cases of culture-proven tuberculosis. Data were collected from sites participating in an NIH-funded, community-based HIV clinical trials group. All cases of tuberculosis, regardless of HIV status, which occurred between January 1992 and June 1994 were recorded. Overall, 1,373 cases of tuberculosis were evaluated, including 425 from the New York City area, and 948 from seven other metropolitan areas. The overall prevalence of resistance to one or more drugs was 20.4%, and 5.6% of isolates were resistant to both isoniazid and rifampin (MDR). In the New York City area, HIV-infected patients were significantly more likely than persons not known to be HIV-infected, to have resistance to at least one drug (37% versus 19%) and MDR (19% versus 6%). In other geographic areas, overall drug resistance was 16%, and only 2.2% of isolates were MDR. In multiple logistic regression analyses, HIV infection was shown to be a risk factor for drug-resistant tuberculosis, independent of geographic location, history of prior therapy, age, and race. We concluded that HIV infection is associated with increased rates of resistance to antituberculosis drugs in both the New York City area and other geographic areas. MDR tuberculosis is occurring predominantly in the New York City area and is highly correlated with HIV infection.

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.

Dendritic cells play a crucial role in antigen presentation in various tissues. The endocytic capacity of these cells has been regarded as minimal, but recent work on dendritic cells from mouse spleen has disclosed that the fluid-phase traffic through late endosomes is as active in dendritic cells as in other antigen-presenting cell types. We show that cultured human dendritic cells express the annexins I, III, IV, V and VI, as detected by immunofluorescence staining. The annexins are cytosolic Ca(2+)-dependent proteins with the ability to promote vesicle aggregation and membrane fusion through their capacity to bind to membrane phospholipids. Annexin I and VI appeared to outline the cytoskeleton and the plasma membrane in cultured human dendritic cells. Studies using confocal laser scanning microscopy showed that during the endocytosis of fluorescent dinitrophenyl-conjugated albumin by dendritic cells, there was a redistribution of annexin V which was found to colocalize with vesicles containing dinitrophenyl-FITC-conjugated albumin.

Dendritic cells are the most potent antigen-presenting cells for the induction of primary antigen-specific T-cell responses in vitro and in situ (1). The first step in antigen-presentation is endocytosis of the antigen followed by antigen degradation to antigen peptides, binding to the major histocompatibility complex molecule and transport to the cell surface or exocytosis. The endocytotic capacity of dendritic cells has been regarded as minimal, but recent work on dendritic cells from mouse spleen has shown that the fluid phase traffic through late endosomes is as active in spleen dendritic cells as in other types of antigen-presenting cells (2). To further investigate the antigen endocytosis and exocytosis pathway in human peripheral blood dendritic cells we mapped the distribution of annexins in the dendritic cell cytoplasm.

When Chlamydia trachomatis elementary bodies enter epithelial cells, they occupy membrane-bound vesicles that aggregate with each other in a calcium-dependent manner but that do not fuse with lysosomes. As members of the annexin family of calcium- and membrane-binding proteins have been implicated in mediating calcium-regulated membrane traffic during endo- and exocytosis, we examined the intracellular localization of certain annexins following invasion of HeLa and McCoy cells by C. trachomatis serovar L2. Immunofluorescence staining with a panel of polyclonal antibodies against five human annexins revealed that annexins III, IV, and V translocate within the cytoplasm to the proximity of intracellular chlamydiae whereas the distribution of annexins I and VI was unaffected. The distinct distribution of annexins I and III was further analyzed by confocal microscopy, which revealed an intimate association between chlamydial aggregates or inclusions and annexin III. Confocal microscopy also confirmed the nonassociation of annexin I with chlamydial aggregates. Depletion of intracellular Ca2+ did not prevent association of annexin III with individual elementary body-containing endosomes but did prevent formation of chlamydial aggregates and translocation of annexin III. Furthermore, chloramphenicol-treated cells also showed association between chlamydial aggregates and annexin III, indicating that the annexins are of host cell origin. These data suggest that certain cytosolic annexins may be involved in the Ca(2+)-dependent aggregation and fusion of chlamydia-containing vesicles. The fact that these Ca(2+)-binding proteins differ in their ability to associate with chlamydia-containing vesicles and inclusions implies that the factors that regulate the interaction of annexin I and annexin III with membrane are different and suggests a selective regulatory mechanism for endosome aggregation and avoiding lysosome fusion during chlamydia infection.

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.

We have used Ca2+-dependent binding to a phospholipid vesicle affinity column to isolate a mixture of three synexin-like proteins from the cytosol of human polymorphonuclear leukocytes (PMN), with relative molecular weights of approximately 67,000, 47,000, and 28,000. Rabbit antibodies raised against bovine liver synexin recognized the 47,000 molecular weight PMN protein. These PMN proteins, like bovine liver synexin, promoted aggregation of isolated PMN specific granules in the presence of Ca2+ and increased the overall rate of Ca2+-induced fusion of liposomes composed of phosphatidate (PA)/phosphatidylethanolamine (PE) (1:3) and phosphatidylserine/PE (1:3), but decreased the rate of spermine-induced fusion of PA/PE (1:3) liposomes. Using fluorescent lipid probes, rapid fusion of PA/PE liposomes with PMN specific granules (50% maximum signal within a few minutes) was observed when 1 mM Ca2+ was added in the presence of both synexin and free arachidonic acid. Dilution of the aqueous contents of liposomes was also observed under the same conditions. The rate of fusion increased monotonically with Ca2+ and arachidonic acid concentrations, but synexin exhibited an optimum concentration. Lack of any one of the components precluded rapid fusion. These results suggest that PMN contain a protein similar to, or identical with, synexin that may be involved in calcium-dependent fusion of intracellular membranes.

The study of experimental infections has afforded the opportunity to examine the response to antimicrobial therapy in the complex milieu of living animals. This review summarizes the information gained from studies of combination antibiotic therapy for experimental endocarditis, meningitis, and other infections in which host defenses are unable to participate in effecting cure. The studies cited suggest that antibiotic combinations that yield rapid microbial killing are more effective in the treatment of these selected infections than are those combinations that do not give enhanced killing. In addition, the relevance of various methods of demonstrating antimicrobial synergy is examined, and in light of the results of treatment of experimental infections, suggestions for the applications of these methods to the evaluation of the efficacy of antibiotic combinations on infections in humans are given.

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.

Summary 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.

Although Ca2+ is involved in many biological membrane fusion phenomena, the mechanism of its action is not well understood. It could participate directly in the fusion reaction by interacting with phospholipids; it could activate particular enzymes; it could induce a conformational change in a fusogenic protein; or it could merely initiate a cascade of reactions which eventually mediate fusion.

Disseminated candidiasis was established in guinea pigs immunosuppressed with high-dose cortisone acetate. After the disease had been characterized by histopathologic study and quantitative culture of selected organs, guinea pigs were given amphotericin B alone or in combination with rifampin. The addition of rifampin to the regimen enhanced the in vitro rate of killing of the strain of Candida albicans used in the studies but was not noticeably beneficial in treatment of the disease in guinea pigs. Thus, the demonstration of an enhanced rate of killing in vitro was not a reliable basis for prediction of the response to the drug in vivo.

Annexins are structurally-related proteins which bind phospholipids in a Ca2+-dependent manner. We have used a novel coupling strategy to prepare an antiserum directed against a 17-amino acid synthetic peptide that resembles the sequence of a highly-conserved portion of these proteins. This antipeptide serum specifically recognizes 5 of 6 human annexins on Western blots, despite differences between the protein and peptide sequences of 3 or 4 amino acids. The antiserum does not recognize endonexin II, whose sequence differs from that of the peptide by 6 amino acids. The availability of multiple proteins with known amino acid sequence has allowed analysis of structural requirements for recognition by this antibody. In some situations, use of such an antibody may allow the identification of a protein as a member of a family.

Two patients with systemic lupus erythematosus had aortic valvular stenosis resulting from massive thrombotic deposits on the valve. This condition has not been described previously, but a similar process has been reported to have caused mitral stenosis in a patient.