Undenatured type II collagen protects against collagen-induced arthritis by restoring gut-joint homeostasis and immunity.

Oral administration of harmless antigens can induce suppression of reactive immune responses, a process that capitalises on the ability of the gastrointestinal tract to tolerate exposure to food and commensal microbiome without triggering inflammatory responses. Repeating exposure to type II collagen induces oral tolerance and inhibits induction of arthritis, a chronic inflammatory joint condition. Although some mechanisms underlying oral tolerance are described, how dysregulation of gut immune networks impacts on inflammation of distant tissues like the joints is unclear. We used undenatured type II collagen in a prophylactic regime -7.33 mg/kg three times/week- to describe the mechanisms associated with protective oral immune-therapy (OIT) in gut and joint during experimental Collagen-Induced Arthritis (CIA). OIT reduced disease incidence to 50%, with reduced expression of IL-17 and IL-22 in the joints of asymptomatic mice. Moreover, whilst the gut tissue of arthritic mice shows substantial damage and activation of tissue-specific immune networks, oral administration of undenatured type II collagen protects against gut pathology in all mice, symptomatic and asymptomatic, rewiring IL-17/IL-22 networks. Furthermore, gut fucosylation and microbiome composition were also modulated. These results corroborate the relevance of the gut-joint axis in arthritis, showing novel regulatory mechanisms linked to therapeutic OIT in joint disease.

Aberrant pro-inflammatory responses of CD20+ T cells in experimental arthritis.

CD20+ T cells comprise a highly inflammatory subset implicated in autoimmunity, including rheumatoid arthritis (RA). We sought to characterize the CD20+ T cell subset in the murine collagen-induced arthritis (CIA) model of RA and investigate the phenotype and functional relevance of CD3+CD20+ T cells in the lymph nodes and arthritic joints using flow cytometry and immunohistochemistry. We demonstrate that CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are expanded in the draining lymph nodes of CIA mice, produce increased levels of pro-inflammatory cytokines and are less susceptible to regulation by regulatory T cells. Notably, CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are enriched with CXCR5+PD-1+ T follicular helper cells and CXCR5-PD-1+ peripheral T helper cells, subsets of T cells implicated in promoting B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues in RA. Our findings suggest CD20+ T cells are associated with inflammatory responses and may exacerbate pathology by promoting inflammatory B-cell responses.

JAK inhibitors disrupt T cell-induced proinflammatory macrophage activation.

OBJECTIVES: Macrophage subsets, activated by T cells, are increasingly recognised to play a central role in rheumatoid arthritis (RA) pathogenesis. Janus kinase (JAK) inhibitors have proven beneficial clinical effects in RA. In this study, we investigated the effect of JAK inhibitors on the generation of cytokine-activated T (Tck) cells and the production of cytokines and chemokines induced by Tck cell/macrophage interactions. METHODS: CD14+ monocytes and CD4+ T cells were purified from peripheral blood mononuclear cells from buffy coats of healthy donors. As representative JAK inhibitors, tofacitinib or ruxolitinib were added during Tck cell differentiation. Previously validated protocols were used to generate macrophages and Tck cells from monocytes and CD4+ T cells, respectively. Cytokine and chemokine including TNF, IL-6, IL-15, IL-RA, IL-10, MIP1α, MIP1ß and IP10 were measured by ELISA. RESULTS: JAK inhibitors prevented cytokine-induced maturation of Tck cells and decreased the production of proinflammatory cytokines TNF, IL-6, IL-15, IL-1RA and the chemokines IL-10, MIP1α, MIP1ß, IP10 by Tck cell-activated macrophages in vitro (p<0.05). CONCLUSIONS: Our findings show that JAK inhibition disrupts T cell-induced macrophage activation and reduces downstream proinflammatory cytokine and chemokine responses, suggesting that suppressing the T cell-macrophage interaction contributes to the therapeutic effect of JAK inhibitors.

Pro-inflammatory adiponectin in pediatric-onset multiple sclerosis.

BACKGROUND: Being obese is associated with both increased risk of developing multiple sclerosis (MS) and greater MS disease activity. OBJECTIVES: The objective of this study is to investigate levels and potential pathophysiologic contribution of serum adipose-hormones (adipokines) in pediatric-onset MS. METHODS: Following a Luminex adipokine screen, adiponectin (APN) and its isoforms were quantified by enzyme-linked immunosorbent assay (ELISA) in 169 children with incident acquired demyelinating syndromes (ADS), prospectively ascertained as having either MS or other forms of inflammatory central nervous system (CNS) demyelination. The effect of recombinant APN and APN-containing sera was assessed on functional responses of normal human peripheral blood myeloid and T cells and on human CNS-derived microglia. RESULTS: Compared to other cohorts, children with MS harbored higher serum APN levels, principally driven by higher levels of the low-molecular-weight isoform. Recombinant APN and pediatric MS serum-induced APN-dependent pro-inflammatory activation of CD14+ monocytes and of activated CD4+ and CD8+ T cells (both directly and indirectly through myeloid cells). APN induced human microglia activation while inhibiting their expression of molecules associated with quiescence. CONCLUSIONS: Elevated APN levels in children with MS may contribute to enhanced pro-inflammatory states of innate and adaptive peripheral immune responses and breach CNS-resident microglia quiescence, providing a plausible and potentially targetable mechanism by which APN contributes to MS disease activity.

Abnormal effector and regulatory T cell subsets in paediatric-onset multiple sclerosis.

Elucidation of distinct T-cell subsets involved in multiple sclerosis immune-pathophysiology continues to be of considerable interest since an ultimate goal is to more selectively target the aberrant immune response operating in individual patients. While abnormalities of both effector (Teff) and regulatory (Treg) T cells have been reported in patients with multiple sclerosis, prior studies have mostly assessed average abnormalities in either limb of the immune response, rather than both at the same time, which limits the ability to evaluate the balance between effectors and regulators operating in the same patient. Assessing both phenotypic and functional responses of Teffs and Tregs has also proven important. In studies of adults with multiple sclerosis, in whom biological disease onset likely started many years prior to the immune assessments, an added challenge for any reported abnormality is whether the abnormality indeed contributes to the disease (and hence of interest to target therapeutically) or merely develops consequent to inflammatory injury (in which case efforts to develop targeted therapies are unlikely to be beneficial). Paediatric-onset multiple sclerosis, though rare, offers a unique window into early disease mechanisms. Here, we carried out a comprehensive integrated study, simultaneously assessing phenotype and functional responses of both effector and regulatory T cells in the same children with multiple sclerosis, monophasic inflammatory CNS disorders, and healthy controls, recruited as part of the multicentre prospective Canadian Pediatric Demyelinating Disease Study (CPDDS). Stringent standard operating procedures were developed and uniformly applied to procure, process and subsequently analyse peripheral blood cells using rigorously applied multi-parametric flow cytometry panels and miniaturized functional assays validated for use with cryopreserved cells. We found abnormally increased frequencies and exaggerated pro-inflammatory responses of CD8+CD161highTCR-Vα7.2+ MAIT T cells and CD4+CCR2+CCR5+ Teffs in paediatric-onset multiple sclerosis, compared to both control groups. CD4+CD25hiCD127lowFOXP3+ Tregs of children with multiple sclerosis exhibited deficient suppressive capacity, including diminished capacity to suppress disease-implicated Teffs. In turn, the implicated Teffs of multiple sclerosis patients were relatively resistant to suppression by normal Tregs. An abnormal Teff/Treg ratio at the individual child level best distinguished multiple sclerosis children from controls. We implicate abnormalities in both frequencies and functional responses of distinct pro-inflammatory CD4 and CD8 T cell subsets, as well as Treg function, in paediatric-onset multiple sclerosis, and suggest that mechanisms contributing to early multiple sclerosis development differ across individuals, reflecting an excess abnormality in either Teff or Treg limbs of the T cell response, or a combination of lesser abnormalities in both limbs.

Reconstitution of the peripheral immune repertoire following withdrawal of fingolimod.

BACKGROUND: Following fingolimod cessation, immune reconstitution or lack thereof may have consequences for disease rebound or safety of commencing alternative therapies. OBJECTIVE: To examine the degree and profile of peripheral blood lymphocyte reconstitution following fingolimod withdrawal. METHODS: Total lymphocyte counts (TLC) and CD4+/CD8+ T-cell counts were measured in 18 multiple sclerosis (MS) patients pre-treatment, on fingolimod, and up to 8-9 months post-cessation. T-cell subsets were analyzed using flow cytometry. RESULTS: At 2-week post-fingolimod cessation, TLC reconstitution was variable and not correlated with age, treatment duration, pre-, or on-treatment TLC. Despite normalization of TLC and CD4+:CD8+ ratios over months, naive subsets remained lower and effector memory subsets higher in frequency compared with pre-treatment. Drug-induced increases in ratios of regulatory to pathogenic Th17-containing central memory populations appeared to rapidly return to baseline. CONCLUSION: Early peripheral lymphocyte reconstitution after fingolimod withdrawal remains partial and heterogeneous. Relative frequencies of circulating naive and memory T-cell subsets may not recover for many months, even when clinical laboratory tests have normalized. Analyzing specific components of the peripheral immune repertoire helps define the overall immune status of patients. To be determined is whether assessment of such immune measures will have implications for the timing and safety of commencing alternative therapies.

Human Mesenchymal Stem Cells Impact Th17 and Th1 Responses Through a Prostaglandin E2 and Myeloid-Dependent Mechanism.

: Human mesenchymal stem cells (hMSCs) are being increasingly pursued as potential therapies for immune-mediated conditions, including multiple sclerosis. Although they can suppress human Th1 responses, they reportedly can reciprocally enhance human Th17 responses. Here, we investigated the mechanisms underlying the capacity of hMSCs to modulate human Th1 and Th17 responses. Human adult bone marrow-derived MSCs were isolated, and their purity and differentiation capacity were confirmed. Human venous peripheral blood mononuclear cells (PBMC) were activated, alone, together with hMSC, or in the presence of hMSC-derived supernatants (sups). Cytokine expression by CD4+ T-cell subsets (intracellular staining by fluorescence-activated cell sorting) and secreted cytokines (enzyme-linked immunosorbent assay) were then quantified. The contribution of prostaglandin E2 (PGE2) as well as of myeloid cells to the hMSC-mediated regulation of T-cell responses was investigated by selective depletion of PGE2 from the hMSC sups (anti-PGE2 beads) and by the selective removal of CD14+ cells from the PBMC (magnetic-activated cell sorting separation). Human MSC-secreted products could reciprocally induce interleukin-17 expression while decreasing interferon-γ expression by human CD4+ T cells, both in coculture and through soluble products. Pre-exposure of hMSCs to IL-1ß accentuated their capacity to reciprocally regulate Th1 and Th17 responses. Human MSCs secreted high levels of PGE2, which correlated with their capacity to regulate the T-cell responses. Selective removal of PGE2 from the hMSC supernatants abrogated the impact of hMSC on the T cells. Selective removal of CD14+ cells from the PBMCs also limited the capacity of hMSC-secreted PGE2 to affect T-cell responses. Our discovery of a novel PGE2-dependent and myeloid cell-mediated mechanism by which human MSCs can reciprocally induce human Th17 while suppressing Th1 responses has implications for the use of, as well as monitoring of, MSCs as a potential therapeutic for patients with multiple sclerosis and other immune-mediated diseases. SIGNIFICANCE: Although animal studies have generated a growing interest in the anti-inflammatory potential of mesenchymal stem cells (MSCs) for the treatment of autoimmune diseases, MSCs possess the capacity to both limit and promote immune responses. Yet relatively little is known about human-MSC modulation of human disease-implicated T-cell responses, or the mechanisms underlying such modulation. The current study reveals a novel prostaglandin E2-dependent and myeloid cell-mediated mechanism by which human MSCs can reciprocally regulate human Th17 and Th1 responses, with implications for the use of MSCs as a potential therapeutic for patients with multiple sclerosis and other immune-mediated diseases.

TLR2 stimulation regulates the balance between regulatory T cell and Th17 function: a novel mechanism of reduced regulatory T cell function in multiple sclerosis.

CD4(+)CD25(hi) FOXP3(+) regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the pathogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2 heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity and progression.

Epitope spreading as an early pathogenic event in pediatric multiple sclerosis.

OBJECTIVES: For most adults with initial clinical presentation of multiple sclerosis (MS), biological disease was likely initiated many years prior. Pediatric-onset MS provides an opportunity to study early disease processes. METHODS: Using antigen microarrays, including CNS-related proteins, lipids, and other autoantigens, we studied early immunologic events involved in clinical onset of pediatric MS. Serum samples were collected at the time of incident acquired CNS demyelinating syndromes (ADS) in children who, in subsequent prospective follow-up, were ascertained to have either pediatric MS (ADS-MS) or a monophasic illness (ADS-mono). Samples were obtained both at the time of ADS presentation and 3 months into follow-up. We used an initial training set of samples to implicate antibody signatures associated with each group, and then a test set. An additional set of follow-up samples (stability set) was used as a form of internal validation. RESULTS: Children with ADS-MS tended to have distinguishable serum antibody patterns both at the time of ADS presentation and 3 months into follow-up. At the time of ADS, serum samples from patients with ADS-MS or ADS-mono reacted against similar numbers of CNS antigens, although CNS antigens implicated in adult MS were more often targeted in children with ADS-MS. The follow-up ADS-MS samples reacted against a broader panel of CNS antigens, while corresponding ADS-mono samples exhibited a contraction of the initial antibody response. CONCLUSIONS: Our findings in this prospective cohort of pediatric-onset CNS demyelinating diseases point to an active process of epitope spreading during early stages of MS, not seen in monophasic CNS inflammatory conditions.

Innate immune responses in the CNS: role of toll-like receptors, mechanisms, and therapeutic opportunities in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), which is considered immune-mediated. Our knowledge of innate immune mechanisms in the CNS and their implications for pathogenesis and treatment of multiple sclerosis (MS) are limited, particularly if compared with the body of literature on adaptive immune mechanisms. There is, however, growing understanding of the workings of the innate immune system and accordingly, of its potential role in driving immune-mediated pathology. Such mechanisms will be discussed in this review along with potential therapeutic opportunities. These may require blocking pathogenic innate immunity and in some cases, promoting its protective effects.

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function.

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.

Expression of activity-dependent neuroprotective protein in the immune system: possible functions and relevance to multiple sclerosis.

BACKGROUND: Activity-dependent neuroprotector (ADNP) is a neuroprotective molecule containing an 8-amino acid peptide, NAPVSIPQ (NAP), that is sufficient for its neuroprotective effects. OBJECTIVE: To assess the expression of ADNP in the human immune system in normal subjects and multiple sclerosis patients. MaterialsandMethods: ADNP expression was assessed in peripheral blood mononuclear cells (PBMCs) from healthy donors and multiple sclerosis (MS) patients using staining with anti-ADNP (NAP) antibodies and markers for T cells, B cells, monocytes and natural killer cells. ADNP mRNA was determined in peripheral blood from MS patients (n = 24) and matched controls (n = 21). Expression of activation markers CD69 and CD154 and of IFN-gamma was assessed by flow cytometry in stimulated PBMCs. Effects of NAP on immune cell proliferation was assessed by tritiated thymidine incorporation. RESULTS: Monocytes, B cells and T cells, but not regulatory (CD4+CD25+) T cells expressed ADNP. NAP peptide decreased the expression of CD69, CD154 and IFN-gamma in PBMC and caused suppressed anti-CD3-/anti-CD28-stimulated PBMC proliferation. ADNP mRNA was reduced in MS compared to control peripheral blood. CONCLUSION: ADNP is expressed in many immune system cells. ADNP mRNA is reduced in PBMCs in MS. The peptide NAP, which plays an important role in neuroprotection, has potential immunomodulatory properties.

Modulation of regulatory T cells in health and disease: role of toll-like receptors.

Naturally arising regulatory T cells (Tregs) originate from the thymus and are characterised by the expression of Foxp3 as a key control gene for their development and function. Their pivotal role is maintaining immunological self tolerance. Recently, Tregs have been shown to express Toll-like receptors (TLRs), which are essential components of the innate immune system for the detection of microbial infections and the activation of dendritic cells (DC) maturation programs to induce adaptive immune responses. TLRs are type 1 transmembrane receptors characterised by a highly variable extracellular region containing a leucine rich repeat domain (LRR) involved in ligand binding and an intracellular tail containing a highly conserved region, the TIR homology domain, which mediates interaction between TLRs and downstream signalling molecules. Recent data suggest that the activation of TLRs on Tregs can increase or decrease their suppressive activity, thus providing an important link between innate and adaptive immune responses. Treg modulation by TLRs might influence such processes as the response to infections, immune surveillance to cancer, transplant rejection, and the induction of autoimmunity. Understanding the link between Tregs and TLR could be beneficial to the discovery of new therapeutic targets and strategies.

Association between sputum smear status and local immune responses at the site of disease in HIV-infected patients with pulmonary tuberculosis.

Infection with human immunodeficiency virus (HIV) may affect the clinical presentation of pulmonary tuberculosis (TB). To investigate the association between sputum smear status at presentation and local pulmonary immune responses in HIV-infected patients with pulmonary TB, we compared the cellular and cytokine profiles in bronchoalveolar lavage (BAL) fluid obtained from the site of lung disease in 22 sputum smear- and culture-positive, and 17 sputum smear-negative but culture-positive pulmonary TB patients. Smear-positive patients had significantly higher BAL fluid concentrations of IL-6 (p=0.007), IL-8 (p=0.02), IL-10 (p=0.03) and IFN-gamma (p=0.008) than smear-negative patients. No significant differences in the proportions of examined BAL cells were found. We concluded that sputum smear-positive TB was associated with greater pro-inflammatory and immunomodulatory cytokine responses at the site of lung disease than sputum smear-negative disease. The local immune responses may affect the clinical presentation of active pulmonary TB in HIV-infected patients.

Alveolar macrophages from HIV-infected patients with pulmonary tuberculosis retain the capacity to respond to stimulation by lipopolysaccharide.

The functional capacity of alveolar macrophages (AM) in human immunodeficiency virus (HIV)-infected patients with pulmonary tuberculosis (TB) is not completely understood. To investigate the capacity of AM to mediate inflammatory responses, we obtained AM from human subjects by bronchoalveolar lavage (BAL) and studied the cells ex vivo. We compared AM from HIV-infected patients with suspected pulmonary TB to AM from healthy, HIV-negative controls for their capacity to produce TNF-alpha or IL-6 spontaneously and upon stimulation with lipopolysaccharide (LPS). Cytokine-producing cells were identified by macrophage markers and intracellular cytokine staining and flow cytometry. A higher proportion of AM from patients with microbiologically confirmed pulmonary TB than patients with probable TB or controls spontaneously expressed TNF-alpha shortly after isolation (geometric means: 38.5%, 23.7% and 15.8%, respectively), suggesting endogenous cytokine production. The proportions of AM spontaneously expressing TNF-alpha positively correlated with peripheral blood CD4(+) T-lymphocyte counts in patients (partial r=0.60, p=0.003) but not controls. Stimulation with LPS resulted in a significant increase in the proportions of TNF-alpha- and IL-6-positive AM from patients and controls (p<0.01). Bronchoalveolar lavage fluid (BALF) from confirmed TB patients also contained higher concentrations of the inflammatory cytokines predominantly produced by macrophages, IL-6 and IL-8, than controls (geometric mean cytokine concentrations per gram of BALF albumin were 1291 pg/g vs. 115 pg/g, p=0.03 for IL-6 and 4739 pg/g vs. 704 pg/g, p=0.03 for IL-8). We concluded that AM from HIV-infected patients with pulmonary TB produced and released inflammatory cytokines in vivo and retained their innate ability to respond to stimulation by LPS.

Proteomic identification of glyceraldehyde 3-phosphate dehydrogenase as an inhibitory target of hydrogen peroxide in Arabidopsis.

Hydrogen peroxide (H2O2) is now recognised as a key signalling molecule in eukaryotes. In plants, H2O2 is involved in regulating stomatal closure, gravitropic responses, gene expression and programmed cell death. Although several kinases, such as oxidative signal-inducible 1 (OXI1) kinase and mitogen-activated protein kinases are known to be activated by exogenous H2O2, little is known about the proteins that directly react with H2O2. Here, we utilised a proteomic approach, using iodoacetamide-based fluorescence tagging of proteins in conjunction with mass spectrometric analysis, to identify several proteins that might be potential targets of H2O2 in the cytosolic fraction of Arabidopsis thaliana, the most prominent of which was cytosolic glyceraldehyde 3-phosphate dehydrogenase (cGAPDH; EC 1.2.1.12). cGAPDH from Arabidopsis is inactivated by H2O2 in vitro, and this inhibition is reversible by the subsequent addition of reductants such as reduced glutathione (GSH). It has been suggested recently that Arabidopsis GAPDH has roles outside of its catalysis as part of glycolysis, while in other systems this includes that of mediating reactive oxygen species (ROS) signalling. Here, we suggest that cGAPDH in Arabidopsis might also have such a role in mediating ROS signalling in plants.

Mycobacterium tuberculosis resides in nonacidified vacuoles in endocytically competent alveolar macrophages from patients with tuberculosis and HIV infection.

Alveolar macrophages (AM) are the first professional phagocytes encountered by aerosols containing infections in the lungs, and their phagocytic capacity may be affected by these infections or environmental particles. The aim of this study was to evaluate the innate endocytic and phagocytic properties of human AM obtained from patients with pulmonary tuberculosis and to characterize the vacuoles in which Mycobacterium tuberculosis bacilli reside in vivo. AM were obtained by bronchoalveolar lavage from patients with suspected tuberculosis and from asymptomatic volunteers (controls). Clinical case definitions were based on mycobacterial culture of respiratory specimens and HIV serology. To assess phagocytosis, endocytosis, and acidification of the endosomal system, AM were cultured with IgG-coated polystyrene beads, dextran, and a pH-sensitive reporter (3-(2,4-dinitroanilino)-3-amino-N-methyldipropylamine) and were evaluated by light and immunoelectron microscopy. Cells from 89 patients and 10 controls were studied. We found no significant difference between the two groups in the ability of AM either to ingest beads and dextran or to deliver them to acidified lysosomes. In AM from patients with tuberculosis, the bacilli were located in vacuoles that failed to accumulate endocytosed material and were not acidified. We concluded that AM from patients with tuberculosis and HIV infections were competent to endocytose and phagocytose material and to deliver the material to functional, acidified lysosomes. M. tuberculosis residing in these AM arrests the progression of their phagosomes, which fail to fuse with acidified lysosomes. This confirms, for the first time in humans with tuberculosis and HIV, the conclusions from previous animal and in vitro studies.