Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis.

Human cyclic haematopoiesis (cyclic neutropenia, MIM 162800) is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity. Circulating neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency. Here we use a genome-wide screen and positional cloning to map the locus to chromosome 19p13.3. We identified 7 different single-base substitutions in the gene (ELA2) encoding neutrophil elastase (EC 3. 4.21.37, also known as leukocyte elastase, elastase 2 and medullasin), a serine protease of neutrophil and monocyte granules, on unique haplotypes in 13 of 13 families as well as a new mutation in a sporadic case. Neutrophil elastase (a 240-aa mature protein predominantly found in neutrophil granules) is the target for protease inhibition by alpha1-antitrypsin, and its unopposed release destroys tissue at sites of inflammation. We hypothesize that a perturbed interaction between neutrophil elastase and serpins or other substrates may regulate mechanisms governing the clock-like timing of haematopoiesis.

The effects of adiponectin and leptin on changes in bone mineral density.

UNLABELLED: We tested the hypothesis that low leptin and high adiponectin levels are associated with higher rates of bone mineral density (BMD) loss among 3,075 men and women, aged 70-79, from the Health Aging and Body Composition Study. Results suggest that adiponectin, but not leptin, is a risk factor for bone loss in women. INTRODUCTION: Adiponectin and leptin are hormones secreted by adipose cells that may impact BMD. Few studies have evaluated the longitudinal association of leptin and adiponectin levels with rates of BMD change. METHODS: Hip and whole-body areal BMD (aBMD) were measured five times using dual-energy X-ray absorptiometry over 10 years (average follow-up time, 7.95 ± 1.92 years). Trabecular lumbar spine volumetric BMD (vBMD) was measured using quantitative computed topography at baseline and year 6 in the Pittsburgh cohort only. Random slope and intercept models were used to account for within person correlation as a result of repeated measures of hip and whole-body aBMD. Linear regression was used to model changes in spine trabecular vBMD. RESULTS: Among women, the annualized rate of hip aBMD loss in the highest tertile of adiponectin was -0.67% (95% CI -0.77, -0.58) compared to [-0.43% (95% CI -0.51, -0.35)] in the lowest tertile (p trend = 0.019) after adjusting for age, race, BMI, diabetes, baseline hip aBMD, and weight change. In men, hip aBMD loss was greatest in the high adiponectin group (tertile 3), however this association was not significant (p trend = 0.148). After adjusting for weight change in women, the association between higher leptin and lower hip aBMD loss was attenuated and no longer significant (p trend = 0.134). Leptin and adiponectin levels were not associated with whole-body aBMD or trabecular lumbar spine vBMD loss. CONCLUSIONS: Adiponectin was associated with increased hip aBMD loss in women only, supporting evidence that adiponectin may have an important role in bone health.

Aggressive paediatric camptodactyly: The evolution of a proposed treatment algorithm.

INTRODUCTION: It is a long-established teaching to avoid operating on camptodactyly unless there is a failure of non-operative treatment, such as serial splinting and hand therapy, and there is an established proximal interphalangeal joint (PIPJ) contracture of 60°; a recent systematic review reflects this continuing approach, with some papers advocating intervention with a lesser degree of contracture. AIM: To evaluate whether early flexor digitorum superficialis (FDS) release, followed by gentle passive manipulation (GPM), will correct severe 'congenital' camptodactyly, if undertaken at an earlier age than usual, thus avoiding the more aggressive surgical approach required in the established adolescent cases. METHOD: The surgical technique and treatment algorithm are described. A multi-centre case series is presented; data analysis included patient demographics, syndromic association, side/digit affected, ages at onset, progression, referral and at surgery, operation details, pre- and post-operative contracture and range of motion. RESULTS: There were 12 patients (3 males, 9 females) who underwent 15 operations for 24 involved digits. Patients had surgery by 3 months (median) post-referral, and there was a significant improvement in median (range) PIPJ contracture (90°(30°-90°) vs. 0°(0°-45°); p<0.001) and range of motion (0°(0°-60°) vs. 90°(50°-95°); p<0.001), at a median post-operative follow-up of 2.5 years. According to the Siegert grade, 87.5% of digits had excellent/good post-operative outcomes and 12.5% had fair outcomes. CONCLUSION: This paper specifically addresses the problem of aggressive and progressive camptodactyly in the young child. By this, we mean patients who have failed non-operative treatment and have PIPJ contractures ≥60°, and those whose contractures have increased by 30° within 1 year. All cases responded to early FDS release and GPM, hence correcting the PIPJ contracture. However, cases with multiple digital involvement, whether syndromic or not, and failed previous surgery or the older child, required additional procedures to restore a dynamic dorsal apparatus and active extension.

The association of serum 25-hydroxyvitamin D with indicators of bone quality in men of Caucasian and African ancestry.

UNLABELLED: We examined the association of serum 25-hydroxyvitamin D [25(OH)D] with indices of bone quality in older men. Positive associations for 25(OH)D and bone mineral density, content, cortical thickness, and axial and polar strength strain indices were observed among Caucasians; however, among men of African descent findings were either null or negative. INTRODUCTION: There are limited data on serum 25(OH)D and bone measures in men of African ancestry. To better understand racial differences in vitamin D status and bone health, a cross-sectional study among 446 Caucasian men in the US and 496 men of African ancestry in Tobago (age ≥ 65 years) was conducted. METHODS: Serum 25(OH)D (liquid chromatography and tandem mass spectrometry) was measured, and peripheral quantitative computed tomography scans were administered. Bone measures estimated included trabecular and cortical volumetric bone mineral density (vBMD), bone mineral content (BMC), bone geometry (cross-sectional area and cortical thickness), and polar and axial strength strain indices (SSIp and SSIx). RESULTS: Men of African ancestry had higher 25(OH)D than Caucasians (34.7 vs. 27.6 ng/ml, p < 0.01). Among Caucasians, 25(OH)D was positively (p trend < 0.05) associated with cortical vBMD, total BMC, cortical thickness, SSIp, and SSIx at the distal radius after adjustment for potential confounders. Similar patterns were observed at the distal tibia. In contrast, in men of African ancestry, there was an inverse association (p trend < 0.05) between 25(OH)D and the cross-sectional area, and SSIx. Race modified (p for interaction < 0.05) the association between 25(OH)D and total BMC, cross-sectional area, SSIp, SSIx, and trabecular vBMD of the radius. In men of African ancestry, there was evidence of a threshold effect (at approximately 18 ng/ml) for 25(OH)D on tibial total BMC and cortical thickness. CONCLUSIONS: More studies are needed to better comprehend these race differences for 25(OH)D and bone density, geometry, and indices of bone strength.

DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation.

DAP kinase is a pro-apoptotic calcium-regulated serine/threonine kinase, whose expression is frequently lost in human tumours. Here we show that DAP kinase counteracts oncogene-induced transformation by activating a p19ARF/p53-dependent apoptotic checkpoint. Ectopic expression of DAP kinase suppressed oncogenic transformation of primary embryonic fibroblasts by activating p53 in a p19ARF-dependent manner. Consequently, the fibroblasts underwent apoptosis, characterized by caspase activation and DNA fragmentation. In response to c-Myc or E2F-1, the endogenous DAP kinase protein was upregulated. Furthermore, functional or genetic inactivation of the endogenous DAP kinase reduced the extent of induction of p19ARF/p53 and weakened the subsequent apoptotic responses to c-Myc or E2F-1. These results establish a role for DAP kinase in an early apoptotic checkpoint designed to eliminate pre-malignant cells during cancer development.

Pancreatic expression of interferon-gamma protects mice from lethal coxsackievirus B3 infection and subsequent myocarditis.

Cardiovascular disease is one of the leading causes of death worldwide, and has been associated with many environmental risk factors. Recent evidence has indicated the involvement of pathogens such as viruses as causative agents, and specifically identified the coxsackievirus B serogroup as the leading culprit. Not only has coxsackievirus B3 (CB3) been identified from patients with cardiovascular disease, but also infection of mice with CB3 strains can reproduce human clinical heart disease in rodents. Several mechanisms have been proposed in an attempt to distinguish between pathology mediated by direct viral destruction of cardiac muscle cells or by the virus-induced immune response directed at infected myocytes or at 'mimicked' epitopes shared between viral and cardiac antigens. To distinguish between these mechanisms, we infected a unique mouse that diminishes the extent of infection and spread of the virus, but allows complete immunity to the virus. Transgenic mice expressing interferon-gamma in their pancreatic beta cells failed to develop CB-3-induced myocarditis. This work challenges the idea of the function of the immune response and 'molecular mimicry' in the CB-3-induced autoimmune myocarditis model, and instead favors the idea of virus-mediated damage. These results emphasize the benefit of reducing the level of viremia early during infection, thereby reducing the incidence of virus-mediated heart damage and autoimmunity.

Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry.

Viral induction of autoimmunity is thought to occur by either bystander T-cell activation or molecular mimicry. Coxsackie B4 virus is strongly associated with the development of insulin-dependent diabetes mellitus in humans and shares sequence similarity with the islet autoantigen glutamic acid decarboxylase. We infected different strains of mice with Coxsackie B4 virus to discriminate between the two possible induction mechanisms, and found that mice with susceptible MHC alleles had no viral acceleration of diabetes, but mice with a T cell receptor transgene specific for a different islet autoantigen rapidly developed diabetes. These results show that diabetes induced by Coxsackie virus infection is a direct result of local infection leading to inflammation, tissue damage, and the release of sequestered islet antigen resulting in the re-stimulation of resting autoreactive T cells, further indicating that the islet antigen sensitization is an indirect consequence of the viral infection.

Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus.

The HIV-1-specific cytotoxic T lymphocyte (CTL) response is temporally associated with the decline in viremia during primary HIV-1 infection, but definitive evidence that it is of importance in virus containment has been lacking. Here we show that in a patient whose early CTL response was focused on a highly immunodominant epitope in gp 160, there was rapid elimination of the transmitted virus strain and selection for a virus population bearing amino acid changes at a single residue within this epitope, which conferred escape from recognition by epitope-specific CTL. The magnitude (> 100-fold), kinetics (30-72 days from onset of symptoms) and genetic pathways of virus escape from CTL pressure were comparable to virus escape from antiretroviral therapy, indicating the biological significance of the CTL response in vivo. One aim of HIV-1 vaccines should thus be to elicit strong CTL responses against multiple codominant viral epitopes.

The Philippine cynomolgus monkey (Macaca fasicularis) provides a new nonhuman primate model of tuberculosis that resembles human disease.

A nonhuman primate model of tuberculosis that closely resembles human disease is urgently needed. We have evaluated the Philippine cynomolgus monkey, Macaca fasicularis, as a model of TB. Cynomolgus monkeys challenged intratracheally with extremely high doses of Mycobacterium tuberculosis (10(5) or 10(4) CFU) developed an acute, rapidly progressive, highly fatal multilobar pneumonia. However, monkeys challenged with moderate or low doses of M. tuberculosis (

Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span.

Aging is an established risk factor for vascular diseases, but vascular aging itself may contribute to the progressive deterioration of organ function. Here, we show in aged mice that vascular endothelial growth factor (VEGF) signaling insufficiency, which is caused by increased production of decoy receptors, may drive physiological aging across multiple organ systems. Increasing VEGF signaling prevented age-associated capillary loss, improved organ perfusion and function, and extended life span. Healthier aging was evidenced by favorable metabolism and body composition and amelioration of aging-associated pathologies including hepatic steatosis, sarcopenia, osteoporosis, "inflammaging" (age-related multiorgan chronic inflammation), and increased tumor burden. These results indicate that VEGF signaling insufficiency affects organ aging in mice and suggest that modulating this pathway may result in increased mammalian life span and improved overall health.

The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes.

The interactions between the L. pneumophila phagosome and monocyte lysosomes were investigated by prelabeling the lysosomes with thorium dioxide, an electron-opaque colloidal marker, and by acid phosphatase cytochemistry. Phagosomes containing live L. pneumophila did not fuse with secondary lysosomes at 1 h after entry into monocytes or at 4 or 8 h after entry by which time the ribosome-lined L. pneumophila replicative vacuole had formed. In contrast, the majority of phagosomes containing formalin-killed L. pneumophila, live Streptococcus pneumoniae, and live Escherichia coli had fused with secondary lysosomes by 1 h after entry into monocytes. Erythromycin, a potent inhibitor of bacterial protein synthesis, at a concentration that completely inhibits L. pneumophila intracellular multiplication, had no influence on fusion of L. pneumophila phagosomes with secondary lysosomes. However, coating live L. pneumophila with antibody or with antibody and complement partially overcame the inhibition of fusion. Also activating the monocytes promoted fusion of a small proportion of phagosomes containing live L. pneumophila with secondary lysosomes. Acid phosphatase cytochemistry revealed that phagosomes containing live L. pneumophila did not fuse with either primary or secondary lysosomes. In contrast to phagosomes containing live bacteria, the majority of phagosomes containing formalin-killed L. pneumophila were fused with lysosomes by acid phosphatase cytochemistry. The capacity of L. pneumophila to inhibit phagosome-lysosome fusion may be a critical mechanism by which the bacterium resists monocyte microbicidal effects.

Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes.

Previous studies have shown that L. pneumophila multiplies intracellularly in human monocytes and alveolar macrophages within a membrane-bound cytoplasmic vacuole studded with ribosomes. In this paper, the formation of this novel vacuole is examined. After entry into monocytes, L. pneumophila resides in a membrane-bound vacuole. During the first hour after entry, vacuoles containing L. pneumophila are found surrounded by smooth vesicles fusing with or budding off from the vacuolar membrane and by mitochondria closely apposed to the vacuolar membrane. By 4 h, vacuoles are found less frequently surrounded by these cytoplasmic organelles, but now ribosomes and rough vesicles are found gathered about the vacuole. By 8 h, the ribosome-lined vacuole has formed. Erythromycin, at concentrations that completely inhibit the intracellular multiplication of L. pneumophila, has no effect on vacuole formation. Formalin-killed L. pneumophila also reside in a membrane-bound vacuole after entry into monocytes. In contrast to the situation with live L. pneumophila, cytoplasmic organelles are not found surrounding vacuoles containing formalin-killed L. pneumophila at any time after entry. Formalin-killed bacteria are rapidly digested, and by 4 h, few remain intact. The L. pneumophila-containing vacuole has certain features in common with other intracellular organisms that inhibit phagosome-lysosome fusion; these organisms may share a common mechanism for vacuole formation and inhibition of phagosome-lysosome fusion.

Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve as prototypes in the development of safe and effective vaccines against intracellular pathogens.

An inhibitor of exported Mycobacterium tuberculosis glutamine synthetase selectively blocks the growth of pathogenic mycobacteria in axenic culture and in human monocytes: extracellular proteins as potential novel drug targets.

Mycobacterium tuberculosis and other pathogenic mycobacteria export abundant quantities of proteins into their extracellular milieu when growing either axenically or within phagosomes of host cells. One major extracellular protein, the enzyme glutamine synthetase, is of particular interest because of its link to pathogenicity. Pathogenic mycobacteria, but not nonpathogenic mycobacteria, export large amounts of this protein. Interestingly, export of the enzyme is associated with the presence of a poly-L-glutamate/glutamine structure in the mycobacterial cell wall. In this study, we investigated the influence of glutamine synthetase inhibitors on the growth of pathogenic and nonpathogenic mycobacteria and on the poly-L-glutamate/glutamine cell wall structure. The inhibitor L-methionine-S-sulfoximine rapidly inactivated purified M. tuberculosis glutamine synthetase, which was 100-fold more sensitive to this inhibitor than a representative mammalian glutamine synthetase. Added to cultures of pathogenic mycobacteria, L-methionine- S-sulfoximine rapidly inhibited extracellular glutamine synthetase in a concentration-dependent manner but had only a minimal effect on cellular glutamine synthetase, a finding consistent with failure of the drug to cross the mycobacterial cell wall. Remarkably, the inhibitor selectively blocked the growth of pathogenic mycobacteria, all of which release glutamine synthetase extracellularly, but had no effect on nonpathogenic mycobacteria or nonmycobacterial microorganisms, none of which release glutamine synthetase extracellularly. The inhibitor was also bacteriostatic for M. tuberculosis in human mononuclear phagocytes (THP-1 cells), the pathogen's primary host cells. Paralleling and perhaps underlying its bacteriostatic effect, the inhibitor markedly reduced the amount of poly-L-glutamate/glutamine cell wall structure in M. tuberculosis. Although it is possible that glutamine synthetase inhibitors interact with additional extracellular proteins or structures, our findings support the concept that extracellular proteins of M. tuberculosis and other pathogenic mycobacteria are worthy targets for new antibiotics. Such proteins constitute readily accessible targets of these relatively impermeable organisms, which are rapidly developing resistance to conventional antibiotics.

Exochelins of Mycobacterium tuberculosis remove iron from human iron-binding proteins and donate iron to mycobactins in the M. tuberculosis cell wall.

To multiply and cause disease in the host, Mycobacterium tuberculosis must acquire iron from the extracellular environment at sites of replication. To do so, the bacterium releases high-affinity iron-binding siderophores called exochelins. In previous studies, we have described the purification and characterization of the exochelin family of molecules. These molecules share a common core structure with another type of high-affinity iron-binding molecule located in the cell wall of M. tuberculosis: the mycobactins. The water-soluble exochelins differ from each other and from water insoluble mycobactins in polarity, which is dependent primarily upon the length and modifications of an alkyl side chain. In this study, we have investigated the capacity of purified exochelins to remove iron from host high-affinity iron-binding molecules, and to transfer iron to mycobactins. Purified desferri-exochelins rapidly removed iron from human transferrin, whether it was 95 or 40% iron saturated, its approximate percent saturation in human serum, and from human lactoferrin. Desferri-exochelins also removed iron, but at a slower rate, from the iron storage protein ferritin. Purified ferri-exochelins, but not iron transferrin, transferred iron to desferri-mycobactins in the cell wall of live bacteria. To explore the possibility that the transfer iron from exochelins to mycobactins was influenced by their polarity, we investigated the influence of polarity on the iron affinity of exochelins. Exochelins of different polarity exchanged iron equally with each other. This study supports the concept that exochelins acquire iron for M. tuberculosis by removing this element from host iron-binding proteins and transferring it to desferri-mycobactins in the cell wall of the bacterium. The finding that ferri-exochelins but not iron transferrin transfer iron to mycobactins in the cell wall underscores the importance of exochelins in iron acquisition. This study also shows that the variable alkyl side chain on the core structure of exochelins and mycobactins, the principal determinant of their polarity, has little or no influence on their iron affinity.

Phenolic glycolipid-1 of Mycobacterium leprae binds complement component C3 in serum and mediates phagocytosis by human monocytes.

Previous studies from this laboratory have demonstrated that Mycobacterium leprae, an obligate intracellular bacterial parasite, enters human mononuclear phagocytes via complement receptors on these host cells and bacterium-bound C3. The present study investigates the role of M. leprae surface molecules in C3 fixation and phagocytosis. By enzyme-linked immunosorbent assay, C3 binds selectively to phenolic glycolipid-1 (PGL-1), a major surface molecule of the leprosy bacillus. C3 fixation to PGL-1 is serum concentration dependent and is abolished in heat-inactivated serum or serum containing ethylenediaminetetraacetic acid. C3 fixation is also abolished in serum containing ethyleneglycol-bis (beta-aminoethyl ether)N,N,N'-tetraacetic acid and MgCl2 indicating that isolated PGL-1 fixes C3 via the classical complement pathway. The capacity of PGL-1 to fix C3 is dependent upon its terminal trisaccharide since sequential removal of monosaccharide units of the trisaccharide results in a stepwise reduction in C3 fixation. Deacylation of PGL-1 also abolishes C3 fixation. C3 fixes to the trisaccharide of PGL-1 that is chemically linked to bovine serum albumin via the chemical carrier, 8-methoxycarbonyloctanol. PGL-1 mediates C3 fixation to polystyrene microspheres, and PGL-1 and C3 together mediate ingestion of polystyrene microspheres by human monocytes, wherein these inert test particles reside in membrane-bound phagosomes. Thus, complement receptors on mononuclear phagocytes, complement component C3, and PGL-1 comprise a three-component receptor-ligand-acceptor molecule system for mediating phagocytosis of M. leprae.

Interaction of the legionnaires' disease bacterium (Legionella pneumophila) with human phagocytes. II. Antibody promotes binding of L. pneumophila to monocytes but does not inhibit intracellular multiplication.

In an accompanying paper (13), we reported that human polymorphonuclear leukocytes kill only a limited proportion (0.5 log) of an inoculum of Legionella pneumophila (Philadelphia 1 strain) in the presence of human anti-L. pneumophila antibody and complement. We now report on the effect of anti-L. pneumophila antibody on L. pneumophila-monocyte interaction. The studies were carried out under antibiotic-free conditions. Monocytes bind more than three times as many viable L. pneumophila bacteria in the presence of both antibody and complement than in the presence of complement alone. Monocytes requires both antibody and complement to kill any L. pneumophila: however, even then, monocytes kill only a limited proportion (0.25 log) of an inoculum. The surviving bacteria multiply several logs in the monocytes and multiply as rapidly as when the bacteria enter monocytes in the absence of antibody. These findings suggest that humoral immunity may not be an effective host defense against L. pneumophila. Consequently, a vaccine that resulted only in antibody production against the Legionnaires' disease bacterium may not be efficacious.

Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited.

We have used the cryosection immunogold technique to study the composition of the Mycobacterium tuberculosis phagosome. We have used quantitative immunogold staining to determine the distribution of several known markers of the endosomal-lysosomal pathway in human monocytes after ingestion of either M. tuberculosis, Legionella pneumophila, or polystyrene beads. Compared with the other phagocytic particles studied, the M. tuberculosis phagosome exhibits delayed clearance of major histocompatibility complex (MHC) class I molecules, relatively intense staining for MHC class II molecules and the endosomal marker transferrin receptor, and relatively weak staining for the lysosomal membrane glycoproteins, CD63, LAMP-1, and LAMP-2 and the lysosomal acid protease, cathepsin D. In contrast to M. tuberculosis, the L. pneumophila phagosome rapidly clears MHC class I molecules and excludes all endosomal-lysosomal markers studied. In contrast to both live M. tuberculosis and L. pneumophila phagosomes, phagosomes containing either polystyrene beads or heat-killed M. tuberculosis stain intensely for lysosomal membrane glycoproteins and cathepsin D. These findings suggest that (a) M. tuberculosis retards the maturation of its phagosome along the endosomal-lysosomal pathway and resides in a compartment with endosomal, as opposed to lysosomal, characteristics; and (b) the intraphagosomal pathway, i.e., the pathway followed by several intracellular parasites that inhibit phagosome-lysosome fusion, is heterogeneous.

Activated human monocytes inhibit the intracellular multiplication of Legionnaires' disease bacteria.

We have examined the interaction between virulent egg yolk-grown L. pneumophila, Philadelphia 1 strain, and in vitro-activated human monocytes, under antibiotic-free conditions. Freshly explanted human monocytes activated by incubation with concanavalin A (Con A) and human lymphocytes inhibited the intracellular multiplication of L. pneumophila. Both Con A and lymphocytes were required for activation. Con A was consistently maximally effective at greater than or equal to 4 mug/ml. Monocytes activated by incubation with cell-free filtered supernatant from Con A-sensitized mononuclear cell cultures also inhibited the intracellular multiplication of L. pneumophil a. The most potent supernatant was obtained from mononuclear cell cultures incubated with greater than or equal to 15 mug/ml Con A for 48 h. The degree of monocyte inhibition of L. pneumophila multiplication was proportional to the length of time monocytes were preincubated with supernatant (48 {greater than} 24 {greater than} 12 h) and to the concentration of supernatant added (40 percent {greater than} 20 percent {greater than} 10 percent {greater than} 5 percent). Monocytes treated with supernatant daily were more inhibitory than monocytes treated initially only. With time in culture, monocytes progressively lost a limited degree of spontaneous inhibitory capacity and also lost their capacity to respond to supernatant with inhibition of L. pneumophila multiplication. Supernatant-activated monocytes inhibited L. pneumophila multiplication in two ways. They phagocytosed fewer bacteria, and they slowed the rate of intracellular multiplication of bacteria that were internalized. As was the case with nonactivated monocytes, antibody had no effect on the rate of intracellular multiplication in supernatant-activated monocytes. Neither supernatant-activated nor nonactivated monocytes killed L. pneumophila in the absence of antibody. Both killed a limited proportion of these bacteria in the presence of antibody and complement. We have previously reported that anti-L, pneumophila antibody and complement neither promote effective killing of L. pneumophila by human polymorphonuclear leukocytes and monocytes nor inhibit the rate of L. pneumophila multiplication in monocytes. These findings and our present report that activated monocytes do inhibit L. pneumophila multiplication indicate that cell-mediated immunity plays a major role in host defense against Legionnaires' disease.

The Mycobacterium tuberculosis phagosome interacts with early endosomes and is accessible to exogenously administered transferrin.

Previous studies have demonstrated that the Mycobacterium tuberculosis phagosome in human monocyte-derived macrophages acquires markers of early and late endosomes, but direct evidence of interaction of the M. tuberculosis phagosome with the endosomal compartment has been lacking. Using the cryosection immunogold technique, we have found that the M. tuberculosis phagosome acquires exogenously added transferrin in a time-dependent fashion. Near-maximal acquisition of transferrin occurs within 15 min, kinetics of acquisition consistent with interaction of the M. tuberculosis phagosome with early endosomes. Transferrin is chased out of the M. tuberculosis phagosome by incubation of the infected macrophages in culture medium lacking human transferrin. Phagosomes containing latex beads or heat-killed M. tuberculosis, on the other hand, do not acquire staining for transferrin. These and other findings demonstrate that M. tuberculosis arrests the maturation of its phagosome at a stage at which the phagosome interacts with early and late endosomes, but not with lysosomes. The transferrin endocytic pathway potentially provides a novel route for targeting antimicrobials to the M. tuberculosis phagosome.