Interplay of strain and race/ethnicity in the innate immune response to M. tuberculosis.

BACKGROUND: The roles of host and pathogen factors in determining innate immune responses to M. tuberculosis are not fully understood. In this study, we examined host macrophage immune responses of 3 race/ethnic groups to 3 genetically and geographically diverse M. tuberculosis lineages. METHODS: Monocyte-derived macrophages from healthy Filipinos, Chinese and non-Hispanic White study participants (approximately 45 individuals/group) were challenged with M. tuberculosis whole cell lysates of clinical strains Beijing HN878 (lineage 2), Manila T31 (lineage 1), CDC1551 (lineage 4), the reference strain H37Rv (lineage 4), as well as with Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA) and TLR4 agonist lipopolysaccharide (TLR4/LPS). Following overnight incubation, multiplex assays for nine cytokines: IL-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFNγ, TNFα, and GM-CSF, were batch applied to supernatants. RESULTS: Filipino macrophages produced less IL-1, IL-6, and more IL-8, compared to macrophages from Chinese and Whites. Race/ethnicity had only subtle effects or no impact on the levels of IL-10, IL-12p70, TNFα and GM-CSF. In response to the Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA), Filipino macrophages again had lower IL-1 and IL-6 responses and a higher IL-8 response, compared to Chinese and Whites. The TLR2/LTA-stimulated Filipino macrophages also produced lower amounts of IL-10, TNFα and GM-CSF. Race/ethnicity had no impact on IL-12p70 levels released in response to TLR2/LTA. The responses to TLR4 agonist lipopolysaccharide (TLR4/LPS) were similar to the TLR2/LTA responses, for IL-1, IL-6, IL-8, and IL-10. However, TLR4/LPS triggered the release of less IL-12p70 from Filipino macrophages, and less TNFα from White macrophages. CONCLUSIONS: Both host race/ethnicity and pathogen strain influence the innate immune response. Such variation may have implications for the development of new tools across TB therapeutics, immunodiagnostics and vaccines.

Mycobacterium ulcerans cytotoxicity in an adipose cell model.

An adipose cell (SW872) model was developed to observe cellular necrosis and apoptosis upon Mycobacterium ulcerans infection and treatment with mycobacterial exudate. Apoptosis was likely due to secreted proteins, while necrosis was likely due to mycolactone. Our data suggest that additional factors in M. ulcerans may be involved in Buruli ulcer pathogenesis.

Necrosis of lung epithelial cells during infection with Mycobacterium tuberculosis is preceded by cell permeation.

Mycobacterium tuberculosis establishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a model in which to examine cytotoxicity during infection with either virulent or avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells with M. tuberculosis strains Erdman and CDC1551 demonstrated significant cell monolayer clearing, whereas infection with either Mycobacterium bovis BCG or Mycobacterium smegmatis LR222 did not. Clearing of M. tuberculosis-infected A549 cells correlated to necrosis, not apoptosis. Treatment of M. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a significant reduction in the necrosis of A549 cell monolayers. This mycobacterium-induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of infected cells with propidium iodide demonstrated that M. tuberculosis induced increased permeation of A549 cell membranes within 24 h postinfection. Quantitation of lactate dehydrogenase (LDH) release from infected cells further demonstrated that cell permeation was specific to M. tuberculosis infection and correlated to A549 cellular necrosis. Inactivated M. tuberculosis or its subcellular fractions did not result in A549 necrosis or LDH release. These studies demonstrate that lung epithelial cell cytotoxicity is specific to infection by virulent mycobacteria and is caused by cellular necrosis. This necrosis is not a direct correlate of mycobacterial growth or of the expression of host cell factors, but is preceded by permeation of the A549 cell membrane and requires infection with live bacilli.

Serologic response to culture filtrate antigens of Mycobacterium ulcerans during Buruli ulcer disease.

Buruli ulcer (BU) is an emerging necrotic skin disease caused by Mycobacterium ulcerans. To assess the potential for a serodiagnostic test, we measured the humoral immune response of BU patients to M. ulcerans antigens and compared this response with delayed-type hypersensitivity responses to both Burulin and PPD. The delayed-type hypersensitivity response generally supported the diagnosis of BU, with overall reactivity to Burulin in 28 (71.8%) of 39 patients tested, compared with 3 (14%) of 21 healthy controls. However, this positive skin test response was observed primarily in patients with healed or active disease, and rarely in patients with early disease (p=0.009). When tested for a serologic response to M. ulcerans culture filtrate, 43 (70.5%) of 61 BU patients had antibodies to these antigens, compared with 10 (37.0%) of 27 controls and 4 (30. 8%) of 13 tuberculosis patients. There was no correlation between disease stage and the onset of this serum antibody response. Our findings suggest that serologic testing may be useful in the diagnosis and surveillance of BU.

Emergence of a unique group of necrotizing mycobacterial diseases.

Although most diseases due to pathogenic mycobacteria are caused by Mycobacterium tuberculosis, several other mycobacterial diseases-caused by M. ulcerans (Buruli ulcer), M. marinum, and M. haemophilum-have begun to emerge. We review the emergence of diseases caused by these three pathogens in the United States and around the world in the last decade. We examine the pathophysiologic similarities of the diseases (all three cause necrotizing skin lesions) and common reservoirs of infection (stagnant or slow-flowing water). Examination of the histologic and pathogenic characteristics of these mycobacteria suggests differences in the modes of transmission and pathogenesis, though no singular mechanism for either characteristic has been definitively described for any of these mycobacteria.

Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis.

Chemical evidence for the true glycosylation of mycobacterial proteins was recently provided in the context of the 45-kDa MPT 32 secreted protein of Mycobacterium tuberculosis (K. Dobos, K. Swiderek, K.-H. Khoo, P. J. Brennan, and J. T. Belisle, Infect. Immun. 63:2846-2853, 1995). However, the full extent and nature of glycosylation as well as the location of glycosylated amino acids remained undefined. First, to examine the nature of the covalently attached sugars, the 45-kDa protein was obtained from cells metabolically labeled with D-[U-14C] glucose and subjected to compositional analysis, which revealed mannose as the only covalently bound sugar. Digestion of the protein with the endoproteinase subtilisin and analysis of products by liquid chromatography-electrospray-mass spectrometry on the basis of fragments demonstrating neutral losses of hexose (m/z 162) or pentose (m/z 132) revealed five glycopeptides, S7, S18, S22, S29, and S41 among a total of 50 peptides, all of which produced only m/z 162 fragmentation ion deletions. Fast atom bombardment-mass spectrometry, N-terminal amino acid sequencing, and alpha-mannosidase digestion demonstrated universal O glycosylation of Thr residues with a single alpha-D-Man, mannobiose, or mannotriose unit. Linkages within the mannobiose and mannotriose were all alpha 1-2, as proven by gas chromatography-mass spectrometry of oligosaccharides released by beta-elimination. Total sequences of many of the glycosylated and nonglycosylated peptides combined with published information on the deduced amino acid sequence of the entire 45-kDa protein demonstrated that the sites of glycosylation were located in Pro-rich domains near the N terminus and C terminus of the polypeptide backbone. Specifically, the Thr residues at positions 10 and 18 were substituted with alpha-D-Manp(1-->2)alpha-D-Manp, the Thr residue at position 27 was substituted with a single alpha-D-Manp, and Thr-277 was substituted with either alpha-D-Manp, alpha-D-Manp(1-->2)alpha-D-Manp, or alpha-D-Manp(1--> 2)alpha-D-Manp(1-->2)alpha-D-Manp. This report further corroborates the existence of true prokaryotic glycoproteins, defines the complete structure of a mycobacterial mannoprotein and the first complete structure of a mannosylated mycobacterial protein, and establishes the principles for the study of other mycobacterial glycoproteins.

Evidence for glycosylation sites on the 45-kilodalton glycoprotein of Mycobacterium tuberculosis.

The occurrence of glycosylated proteins in Mycobacterium tuberculosis has been widely reported. However, unequivocal proof for the presence of true glycosylated amino acids within these proteins has not been demonstrated, and such evidence is essential because of the predominance of soluble lipoglycans and glycolipids in all mycobacterial extracts. We have confirmed the presence of several putative glycoproteins in subcellular fractions of M. tuberculosis by reaction with the lectin concanavalin A. One such product, with a molecular mass of 45 kDa, was purified from the culture filtrate. Compositional analysis demonstrated that the protein was rich in proline and that mannose, galactose, glucose, and arabinose together represented about 4% of the total mass. The 45-kDa glycoprotein was subjected to proteolytic digestion with either the Asp-N or the Glu-C endopeptidase or subtilisin, peptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and glycopeptides were identified by reaction with concanavalin A. Peptides were further separated, and when they were analyzed by liquid chromatography-electrospray mass spectrometry for neutral losses of hexoses (162 mass units), four peptides were identified, indicating that these were glycosylated with hexose residues. One peptide, with an average molecular mass of 1,516 atomic mass units (AMU), exhibited a loss of two hexose units. The N-terminal sequence of the 1,516-AMU glycopeptide was determined to be DPEPAPPVP, which was identical to the sequence of the amino terminus of the mature protein, DPEPAP PVPXTA. Furthermore, analysis of the glycopeptide by secondary ion mass spectrometry demonstrated that the complete sequence of the glycopeptide was DPEPAPPVPTTA. From this, it was determined that the 10th amino acid, threonine, was O-glycosidically linked to a disaccharide composed of two hexose residues, probably mannose. This report establishes that true, O-glycosylated proteins exist in mycobacteria.

[Therapeutic possibilities in the management of gastrointestinal bleeding in thrombocytopenic patients]. / Terápiás lehetöségek thrombocytopeniás beteg gastrointestinalis vérzésének kezelésében.

Gastrointestinal bleeding is often fatal in elderly. The danger could be multiplied by thrombocytopenia or by other disorder of the coagulation system. In a thrombocytopenic patient with myelodysplastic syndrome life threatening gastrointestinal bleeding occurred which could not be controlled by conventional methods (cimetidine, antacids, aminocaproic acid, platelet transfusions, blood transfusion 400-1200 ml/day). Continuous i.v. infusion of somatostatin [250 micrograms/hour Stilamin (Serono)] and per os omeprazole [2 x 1 capsule Losec (Astra)] resulted in the cessation of bleeding in 24 hours. Adverse effect was not observed.

[Experience with results of treatment of non-Hodgkin's lymphoma]. / Gastrointestinalis nem-Hodgkin lymphomák kezelésével szerzett tapasztalataink.

Thirty primary gastrointestinal non-Hodgkin's lymphoma treated between 1983-1990 were reviewed to reveal the efficacy of various treatment strategies. The average age at the diagnosis 53.6 (18-76) years. The histologic material were evaluated according to the Kiel classification: 22 patients had high grade malignant lymphoma (centroblastoma 8, immunoblastoma 6, lymphoblastoma 2, non classifiable 5, T-cell lymphoma 1) 8 patients low grade malignant lymphoma (lymphocytic 2, immunocytic 2, MALT lymphoma 1, centrocytoma 1, non-classifiable 1, pleomorph small cell lymphoma 1). 21 were primary gastric lymphoma, 5 involved the small intestine, 2 the ileocecal region, and 2 the large intestine. According to the Ann Arbor staging system 7 patients were stage I/E, 16 patients stage II/E, 5 patients stage III/E and 2 patients stage IV/E. Every patients underwent surgical resection. After surgical treatment high grade malignancies were treated with ProMACE-COPP (9) and CHOP-Bleo (10) polychemotherapy; low grade malignancies received VEP (5) and CVP (3) chemotherapy. 23 of 30 patients achived complete remission. The patients with low grade malignancy are in remission. All but one patients with high grade malignant gastric lymphoma achieved complete remission with a median of 37 (3-81) months relapse-free survival. Out of 5 cases in the small intestine only in 1 case was remission achieved. Histological type (Kiel) and surgical resection were the most important prognostic factors.

T lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection. Evidence for different kinetics and recognition of a wide spectrum of protein antigens.

Recent evidence suggests the existence of at least two pathways of acquired specific resistance to Mycobacterium tuberculosis infection; the first consisting of cytokine-mediated activation of parasitized host cells by protective T cells, and the second involving the lysis of these cells by cytolytic T cells. Evidence presented in this report shows that both of the above mechanisms are operative in experimentally infected mice, but that they differ markedly in terms of their kinetics of emergence and loss. It was found that protective T cell activity was acquired very early during the course of the infection, and was temporally associated with the onset of bacterial elimination; however, cytolytic activity did not peak until 10 to 20 days later. This report shows further that the target Ag of these effector T cell populations were apparently numerous with no evidence for preferential recognition of a few immunodominant Ag. In view of the preponderance of target proteins in the bacterial filtrate, we present the hypothesis that such proteins secreted or otherwise leaked from the dividing mycobacterium are pinocytosed from the phagosome and used by the infected macrophage as the key protective Ag leading to T cell sensitization. This hypothesis thus explains the preferential requirement for the viable bacterium in the generation of specific resistance, and further explains why protective immunity is generated even while the organism is still multiplying in an apparently unrestrained manner.