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Melioidosis, a human infectious disease with a high mortality rate in many tropical countries, is caused by the pathogen Burkholderia pseudomallei (B. pseudomallei). The function of the B. pseudomallei sigma S (RpoS) transcription factor in survival during the stationary growth phase and conditions of oxidative stress is well documented. Besides the rpoS, bioinformatics analysis of B. pseudomallei genome showed the existence of two rpoN genes, named rpoN1 and rpoN2. In this study, by using the mouse macrophage cell line RAW264.7 as a model of infection, the involvement of B. pseudomallei RpoS and RpoN2 in the invasion, intracellular survival leading to the reduction in multinucleated giant cell (MNGC) formation of RAW264.7 cell line were illustrated. We have demonstrated that the MNGC formation of RAW264.7 cell was dependent on a certain number of intracellular bacteria (at least 5 × 10 4 ). In addition, the same MNGC formation (15%) observed in RAW264.7 cells infected with either B. pseudomallei wild type with multiplicity of infection (MOI) 2 or RpoN2 mutant (∆rpoN2) with MOI 10 or RpoS mutant (∆rpoS) with MOI 100. The role of B. pseudomallei RpoS and RpoN2 in the regulation of type III secretion system on bipB-bipC gene expression was also illustrated in this study., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Kupffer cells (KCs) are localized in liver sinusoids but extend pseudopods to parenchymal cells to maintain their identity and serve as the body's central bacterial filter. Liver cirrhosis drastically alters vascular architecture, but how KCs adapt is unclear. We used a mouse model of liver fibrosis and human tissue to examine immune adaptation. Fibrosis forced KCs to lose contact with parenchymal cells, down-regulating "KC identity," which rendered them incapable of clearing bacteria. Commensals stimulated the recruitment of monocytes through CD44 to a spatially distinct vascular compartment. There, recruited monocytes formed large aggregates of multinucleated cells (syncytia) that expressed phenotypical KC markers and displayed enhanced bacterial capture ability. Syncytia formed via CD36 and were observed in human cirrhosis as a possible antimicrobial defense that evolved with fibrosis.

Burkholderia pseudomallei-a causative agent of melioidosis that is endemic in Southeast Asia and Northern Australia-is a Gram-negative bacterium transmitted to humans via inhalation, inoculation through skin abrasions, and ingestion. Melioidosis causes a range of clinical presentations including skin infection, pneumonia, and septicemia. Despite skin infection being one of the clinical symptoms of melioidosis, the pathogenesis of B. pseudomallei in skin fibroblasts has not yet been elucidated. In this study, we investigated B. pseudomallei pathogenesis in the HFF-1 human skin fibroblasts. On the basis of co-culture assays between different B. pseudomallei clinical strains and the HFF-1 human skin fibroblasts, we found that all B. pseudomallei strains have the ability to mediate invasion, intracellular replication, and multinucleated giant cell (MNGC) formation. Furthermore, all strains showed a significant increase in cytotoxicity in human fibroblasts, which coincides with the augmented expression of matrix metalloproteinase-2. Using B. pseudomallei mutants, we showed that the B. pseudomallei Bsa type III secretion system (T3SS) contributes to skin fibroblast pathogenesis, but O-polysaccharide, capsular polysaccharide, and short-chain dehydrogenase metabolism do not play a role in this process. Taken together, our findings reveal a probable connection for the Bsa T3SS in B. pseudomallei infection of skin fibroblasts, and this may be linked to the pathogenesis of cutaneous melioidosis., Competing Interests: The authors have declared that no competing interests exist.

Inflammasomes are cytosolic multiprotein signaling complexes that are activated upon pattern recognition receptor-mediated recognition of pathogen-derived ligands or endogenous danger signals. Their assembly activates the downstream inflammatory caspase-1 and caspase-4/5 (human) or caspase-11 (mouse), which induces cytokine release and pyroptotic cell death through the cleavage of the pore-forming effector gasdermin D. Pathogen detection by host cells also results in the production and release of interferons (IFNs), which fine-tune inflammasome-mediated responses. IFN-induced guanylate-binding proteins (GBPs) have been shown to control the activation of the noncanonical inflammasome by recruiting caspase-4 on the surface of cytosolic Gram-negative bacteria and promoting its interaction with lipopolysaccharide (LPS). The Gram-negative opportunistic bacterial pathogen Burkholderia thailandensis infects epithelial cells and macrophages and hijacks the host actin polymerization machinery to spread into neighboring cells. This process causes host cell fusion and the formation of so-called multinucleated giant cells (MNGCs). Caspase-1- and IFN-regulated caspase-11-mediated inflammasome pathways play an important protective role against B. thailandensis in mice, but little is known about the role of IFNs and inflammasomes during B. thailandensis infection of human cells, particularly epithelial cells. Here, we report that IFN-γ priming of human epithelial cells restricts B. thailandensis-induced MNGC formation in a GBP1-dependent manner. Mechanistically, GBP1 does not promote bacteriolysis or impair actin-based bacterial motility but acts by inducing caspase-4-dependent pyroptosis of the infected cell. In addition, we show that IFN-γ priming of human primary macrophages confers a more efficient antimicrobial effect through inflammasome activation, further confirming the important role that interferon signaling plays in restricting Burkholderia replication and spread. IMPORTANCE The Gram-negative bacteria of the Burkholderia species are associated with human diseases ranging from pneumonia to life-threatening melioidosis. Upon infection through inhalation, ingestion, or the percutaneous route, these bacteria can spread and establish granuloma-like lesions resulting from the fusion of host cells to form multinucleated giant cells (MNGCs). Burkholderia resistance to several antibiotics highlights the importance to better understand how the innate immune system controls infections. Here, we report that interferons protect human epithelial cells against Burkholderia- induced MNGC formation, specifically through the action of the interferon-induced GBP1 protein. Mechanistically, GBP1 acts by inducing caspase-4-dependent cell death through pyroptosis, allowing the infected cells to be quickly eliminated before bacterial spread and the formation of MNGCs. This study provides evidence that interferon-induced innate immune activation, through GBP1 and caspase-4, confers protection against Burkholderia infection, potentially opening new perspectives for therapeutic approaches.

The immune response to mycobacteria is characterized by granuloma formation, which features multinucleated giant cells as a unique macrophage type. We previously found that multinucleated giant cells result from Toll-like receptor-induced DNA damage and cell autonomous cell cycle modifications. However, the giant cell progenitor identity remained unclear. Here, we show that the giant cell-forming potential is a particular trait of monocyte progenitors. Common monocyte progenitors potently produce cytokines in response to mycobacteria and their immune-active molecules. In addition, common monocyte progenitors accumulate cholesterol and lipids, which are prerequisites for giant cell transformation. Inducible monocyte progenitors are so far undescribed circulating common monocyte progenitor descendants with high giant cell-forming potential. Monocyte progenitors are induced in mycobacterial infections and localize to granulomas. Accordingly, they exhibit important immunological functions in mycobacterial infections. Moreover, their signature trait of high cholesterol metabolism may be piggy-backed by mycobacteria to create a permissive niche.

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills almost 200,000 people worldwide each year. It is acquired when mammalian hosts inhale the infectious propagules; these are deposited in the lung and, in the context of immunocompromise, may disseminate to the brain and cause lethal meningoencephalitis. Once inside the host, C. neoformans undergoes a variety of adaptive processes, including secretion of virulence factors, expansion of a polysaccharide capsule that impedes phagocytosis, and the production of giant (Titan) cells. The transcription factor Pdr802 is one regulator of these responses to the host environment. Expression of the corresponding gene is highly induced under host-like conditions in vitro and is critical for C. neoformans dissemination and virulence in a mouse model of infection. Direct targets of Pdr802 include the quorum sensing proteins Pqp1, Opt1, and Liv3; the transcription factors Stb4, Zfc3, and Bzp4, which regulate cryptococcal brain infectivity and capsule thickness; the calcineurin targets Had1 and Crz1, important for cell wall remodeling and C. neoformans virulence; and additional genes related to resistance to host temperature and oxidative stress, and to urease activity. Notably, cryptococci engineered to lack Pdr802 showed a dramatic increase in Titan cells, which are not phagocytosed and have diminished ability to directly cross biological barriers. This explains the limited dissemination of pdr802 mutant cells to the central nervous system and the consequently reduced virulence of this strain. The role of Pdr802 as a negative regulator of Titan cell formation is thus critical for cryptococcal pathogenicity. IMPORTANCE The pathogenic yeast Cryptococcus neoformans presents a worldwide threat to human health, especially in the context of immunocompromise, and current antifungal therapy is hindered by cost, limited availability, and inadequate efficacy. After the infectious particle is inhaled, C. neoformans initiates a complex transcriptional program that integrates cellular responses and enables adaptation to the host lung environment. Here, we describe the role of the transcription factor Pdr802 in the response to host conditions and its impact on C. neoformans virulence. We identified direct targets of Pdr802 and also discovered that it regulates cellular features that influence movement of this pathogen from the lung to the brain, where it causes fatal disease. These findings significantly advance our understanding of a serious disease., (Copyright © 2021 Reuwsaat et al.)

Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis. Despite advances in our understanding of the disease, B. pseudomallei poses a significant health risk, especially in regions of endemicity, where treatment requires prolonged antibiotic therapy. Even though the respiratory and percutaneous routes are well documented and considered the main ways to acquire the pathogen, the gastrointestinal tract is believed to be an underreported and underrecognized route of infection. In the present study, we describe the development of in vitro and in vivo models to study B. pseudomallei gastrointestinal infection. Further, we report that the type 6 secretion system (T6SS) and type 1 fimbriae are important virulence factors required for gastrointestinal infection. Using a human intestinal epithelial cell line and mouse primary intestinal epithelial cells (IECs), we demonstrated that B. pseudomallei adheres, invades, and forms multinucleated giant cells, ultimately leading to cell toxicity. We demonstrated that mannose-sensitive type 1 fimbria is involved in the initial adherence of B. pseudomallei to IECs, although the impact on full virulence was limited. Finally, we also showed that B. pseudomallei requires a functional T6SS for full virulence, bacterial dissemination, and lethality in mice infected by the intragastric route. Overall, we showed that B. pseudomallei is an enteric pathogen and that type 1 fimbria is important for B. pseudomallei intestinal adherence, and we identify a new role for T6SS as a key virulence factor in gastrointestinal infection. These studies highlight the importance of gastrointestinal melioidosis as an understudied route of infection and open a new avenue for the pathogenesis of B. pseudomallei ., (Copyright © 2020 American Society for Microbiology.)

Tetraspanins are four-span transmembrane proteins of host cells that facilitate infections by many pathogens. Burkholderia pseudomallei is an intracellular bacterium and the causative agent of melioidosis, a severe disease in tropical regions. This study investigated the role of tetraspanins in B. pseudomallei infection. We used flow cytometry to determine tetraspanins CD9, CD63, and CD81 expression on A549 and J774A.1 cells. Their roles in B. pseudomallei infection were investigated in vitro using monoclonal antibodies (MAbs) and recombinant large extracellular loop (EC2) proteins to pretreat cells before infection. Knockout of CD9 and CD81 in cells was performed using CRISPR Cas9 to confirm the role of tetraspanins. Pretreatment of A549 cells with MAb against CD9 and CD9-EC2 significantly enhanced B. pseudomallei internalization, but MAb against CD81 and CD81-EC2 inhibited MNGC formation. Reduction of MNGC formation was consistently observed in J774.A1 cells pretreated with MAbs specific to CD9 and CD81 and with CD9-EC2 and CD81-EC2. Data from knockout experiments confirmed that CD9 enhanced bacterial internalization and that CD81 inhibited MNGC formation. Our data indicate that tetraspanins are host cellular factors that mediated internalization and membrane fusion during B. pseudomallei infection. Tetraspanins may be the potential therapeutic targets for melioidosis.

Background: Burkholderia pseudomallei is an environmental bacterium that causes melioidosis. A facultative intracellular pathogen, B. pseudomallei can induce multinucleated giant cells (MNGCs) leading to plaque formation in vitro. B. pseudomallei can switch colony morphotypes under stress conditions. In addition, different isolates have been reported to have varying virulence in vivo, but genomic evolution and the relationship with plaque formation is poorly understood., Methodology/principle Findings: To gain insights into genetic underpinnings of virulence of B. pseudomallei, we screened plaque formation of 52 clinical isolates and 11 environmental isolates as well as 4 isogenic morphotype isolates of B. pseudomallei strains K96243 (types II and III) and 153 (types II and III) from Thailand in A549 and HeLa cells. All isolates except one environmental strain (A4) and K96243 morphotype II were able to induce plaque formation in both cell lines. Intracellular growth assay and confocal microscopy analyses demonstrated that the two plaque-forming-defective isolates were also impaired in intracellular replication, actin polymerization and MNGC formation in infected cells. Whole genome sequencing analysis and PCR revealed that both isolates had a large genomic loss on the same region in chromosome 2, which included Bim cluster, T3SS-3 and T6SS-5 genes., Conclusions/significance: Our plaque screening and genomic studies revealed evidence of impairment in plaque formation in environmental isolates of B. pseudomallei that is associated with large genomic loss of genes important for intracellular multiplication and MNGC formation. These findings suggest that the genomic and phenotypic differences of environmental isolates may be associated with clinical infection., Competing Interests: The authors have declared that no competing interests exist.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high morbidity that is endemic in South East Asia and northern Australia. An unusual feature of the bacterium is its ability to induce multinucleated giant cell formation (MNGC), which appears to be related to bacterial pathogenicity. The mechanism of MNGC formation is not fully understood, but host cell factors as well as known bacterial virulence determinants are likely to contribute. Since members of the tetraspanin family of membrane proteins are involved in various types of cell:cell fusion, their role in MNGC formation induced by Burkholderia thailandensis, a mildly pathogenic species closely related to B. pseudomallei, was investigated. The effect of antibodies to tetraspanins CD9, CD81, and CD63 in MNGC formation induced by B. thailandensis in infected mouse J774.2 and RAW macrophage cell lines was assessed along with that of recombinant proteins corresponding to the large extracellular domain (EC2) of the tetraspanins. B. thailandensis-induced fusion was also examined in macrophages derived from CD9 null and corresponding WT mice, and in J774.2 macrophages over-expressing CD9. Antibodies to CD9 and CD81 promoted MNGC formation induced by B. thailandensis, whereas EC2 proteins of CD9, CD81, and CD63 inhibited MNGC formation. Enhanced MNGC formation was observed in CD9 null macrophages, whereas a decrease in MNGC formation was associated with overexpression of CD9. Overall our findings show that tetraspanins are involved in MNGC formation induced by B. thailandensis and by implication, B. pseudomallei, with CD9 and CD81 acting as negative regulators of this process.

Innate immunity responds to pathogens by producing alarm signals and activating pathways that make host cells inhospitable for pathogen replication. The intracellular bacterium Burkholderia thailandensis invades the cytosol, hijacks host actin, and induces cell fusion to spread to adjacent cells, forming multinucleated giant cells (MNGCs) which promote bacterial replication. We show that type I interferon (IFN) restricts macrophage MNGC formation during B. thailandensis infection. Guanylate-binding proteins (GBPs) expressed downstream of type I IFN were required to restrict MNGC formation through inhibition of bacterial Arp2/3-dependent actin motility during infection. GTPase activity and the CAAX prenylation domain were required for GBP2 recruitment to B. thailandensis, which restricted bacterial actin polymerization required for MNGC formation. Consistent with the effects in in vitro macrophages, Gbp2-/-, Gbp5-/-, GbpChr3-KO mice were more susceptible to intranasal infection with B. thailandensis than wildtype mice. Our findings reveal that IFN and GBPs play a critical role in restricting cell-cell fusion and bacteria-induced pathology during infection., Competing Interests: The authors have declared that no competing interests exist.

When injured, fish release an alarm substance (Schreckstoff) that elicits fear in members of their shoal. Although Schreckstoff has been proposed to be produced by club cells in the skin, several observations indicate that these giant cells function primarily in immunity. Previous data indicate that the alarm substance can be isolated from mucus. Here we show that mucus, as well as bacteria, are transported from the external surface into club cells, by cytoplasmic transfer or invasion of cells, including neutrophils. The presence of bacteria inside club cells raises the possibility that the alarm substance may contain a bacterial component. Indeed, lysate from a zebrafish Staphylococcus isolate is sufficient to elicit alarm behaviour, acting in concert with a substance from fish. These results suggest that Schreckstoff, which allows one individual to unwittingly change the emotional state of the surrounding population, derives from two kingdoms and is associated with processes that protect the host from bacteria.

Introduction: Aneurysmal bone cysts (ABCs) are rare, rapidly expansile, benign, vascular lesions capable of causing local bone destruction. The majority of cases present as multi-cystic lytic lesions (with solid-variant ABCs representing<10% of all presentations) of the long bones or vertebrae, rarely occurring in the head/neck region., Clinical Case: A 44-year-old female presented with nine days of worsening pain, ptosis and proptosis in the right eye. CT and MR imaging revealed a 3.2cm extra-axial multiloculated right frontal lobe mass in the orbit with fluid-fluid levels secondary to layering of solid blood components. A right craniotomy was performed and the lesion was resected piecemeal with subsequent high speed burring to remove residual tissue. Histological evaluation revealed spindle and giant cell infiltration of the bone without vascular channels. Based on these findings, the lesion was diagnosed as a solid-variant orbital ABC without paranasal sinus involvement. The patient recovered fully with no residual symptoms., Conclusion: This case report details a rare presentation of ABC (solid-variant presenting outside of the vertebrae/long bones) with discussion concerning possible treatment modalities and guidance for follow-up., (Published by Elsevier Masson SAS.)

Photosynthetic efficiency and redox homeostasis are important for plant physiological processes during regular development as well as defence responses. The second-stage juveniles of Heterodera schachtii induce syncytial feeding sites in host roots. To ascertain whether the development of syncytia alters photosynthesis and the metabolism of reactive oxygen species (ROS), chlorophyll a fluorescence measurements and antioxidant responses were studied in Arabidopsis thaliana shoots on the day of inoculation and at 3, 7 and 15 days post-inoculation (dpi). Nematode parasitism caused an accumulation of superoxide and hydrogen peroxide molecules in the shoots of infected plants at 3 dpi, probably as a result of the observed down-regulation of antioxidant enzymes. These changes were accompanied by an increase in RNA and lipid oxidation markers. The activities of antioxidant enzymes were found to be enhanced on infection at 7 and 15 dpi, and the content of anthocyanins was elevated from 3 dpi. The fluorescence parameter R fd , defining plant vitality and the photosynthetic capacity of leaves, decreased by 11% only at 7 dpi, and non-photochemical quenching (NPQ), indicating the effectiveness of photoprotection mechanisms, was about 16% lower at 3 and 7 dpi. As a result of infection, the ultrastructure of chloroplasts was changed (large starch grains and plastoglobules), and more numerous and larger peroxisomes were observed in the mesophyll cells of leaves. We postulate that the joint action of antioxidant enzymes/molecules and photochemical mechanisms leading to the maintenance of photosynthetic efficiency promotes the fine-tuning of the infected plants to oxidative stress induced by parasitic cyst nematodes., (© 2017 BSPP AND JOHN WILEY & SONS LTD.)

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so-called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double-edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO-induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria-infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state., (© 2017 The Authors.)

The human pathogen Burkholderia pseudomallei and the related species Burkholderia thailandensis are facultative intracellular bacteria characterized by the ability to escape into the cytosol of the host cell and to stimulate the formation of multinucleated giant cells (MNGCs). MNGC formation is induced via an unknown mechanism by bacterial type VI secretion system 5 (T6SS-5), which is an essential virulence factor in both species. Despite the vital role of the intracellular life cycle in the pathogenesis of the bacteria, the range of host cell types permissive for initiation and completion of the intracellular cycle is poorly defined. In the present study, we used several different types of human primary cells to evaluate bacterial entry, intracellular survival, and MNGC formation. We report the capacity of B. pseudomallei to enter, efficiently replicate in, and mediate MNGC formation of vein endothelial and bronchial epithelial cells, indicating that the T6SS-5 is important in the host-pathogen interaction in these cells. Furthermore, we show that B. pseudomallei invades fibroblasts and keratinocytes and survives inside these cells as well as in monocyte-derived macrophages and neutrophils for at least 17 h postinfection; however, MNGC formation is not induced in these cells. In contrast, infection of mixed neutrophils and RAW264.7 macrophages with B. thailandensis stimulated the formation of heterotypic MNGCs in a T6SS-5-dependent manner. In summary, the ability of the bacteria to enter and survive as well as induce MNGC formation in certain host cells may contribute to the pathogenesis observed in B. pseudomallei infection., (Copyright © 2017 American Society for Microbiology.)

The growth of pathogens is dictated by their interactions with the host environment 1 . Obligate intracellular pathogens undergo several cellular decisions as they progress through their life cycles inside host cells 2 . We have studied this process for microsporidian species in the genus Nematocida as they grew and developed inside their co-evolved animal host, Caenorhabditis elegans 3-5 . We found that microsporidia can restructure multicellular host tissues into a single contiguous multinucleate cell. In particular, we found that all three Nematocida species we studied were able to spread across the cells of C. elegans tissues before forming spores, with two species causing syncytial formation in the intestine and one species causing syncytial formation in the muscle. We also found that the decision to switch from replication to differentiation in Nematocida parisii was altered by the density of infection, suggesting that environmental cues influence the dynamics of the pathogen life cycle. These findings show how microsporidia can maximize the use of host space for growth and that environmental cues in the host can regulate a developmental switch in the pathogen.

The Burkholderia genus contains a group of soil-dwelling Gram-negative organisms that are prevalent in warm and humid climates. Two species in particular are able to cause disease in animals, B. mallei primarily infects Equus spp. and B. pseudomallei (BPS), that is able to cause potentially life-threatening disease in humans. BPS is naturally resistant to many antibiotics and there is no vaccine available. Although not a specialised human pathogen, BPS possesses a large genome and many virulence traits that allow it to adapt and survive very successfully in the human host. Key to this survival is the ability of BPS to replicate intracellularly. In this review we highlight recent advances in our understanding of the intracellular survival of BPS, including how it overcomes host immune defenses and other challenges to establish its niche and then spread the infection. Knowledge of these mechanisms increases our capacity for therapeutic interventions against a well-armed foe., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chlamydia trachomatis, the leading cause of bacterial sexually transmitted infections, disrupts cytokinesis and causes significant multinucleation in host cells. Here, we demonstrate that multinuclear cells that result from unsuccessful cell division contain significantly higher Golgi content, an important source of lipids for chlamydiae. Using immunofluorescence and fluorescent live cell imaging, we show that C. trachomatis in multinuclear cells indeed intercept Golgi-derived lipid faster than in mononuclear cells. Moreover, multinuclear cells enhance C. trachomatis inclusion growth and infectious particle formation. Together, these results indicate that C. trachomatis robustly position inclusions to the cell equator to disrupt host cell division in order to acquire host Golgi-derived lipids more quickly in multinucleated progeny cells., (© 2015 Wiley Periodicals, Inc.)

Chlamydia trachomatis is an obligate intracellular bacterial pathogen and the second leading cause of sexually transmitted infections in the US. Infections cause significant morbidity and can lead to serious reproductive sequelae, including an epidemiological link to increased rates of reproductive cancers. One of the overt changes that infected cells exhibit is the development of genomic instability leading to multinucleation. Here we demonstrate that the induction of multinucleation is not conserved equally across chlamydial species; C. trachomatis L2 caused high levels of multinucleation, C. muridarum intermediate levels, and C. caviae had very modest effects on multinucleation. Our data show that at least two effector pathways together cause genomic instability during infection leading to multinucleation. We find that the highly conserved chlamydial protease CPAF is a key effector for one of these pathways. CPAF secretion is required for the loss of centrosome duplication regulation as well as inducing early mitotic exit. The second effector pathway involves the induction of centrosome position errors. This function is not conserved in three chlamydial species tested. Together these two pathways contribute to the induction of high levels of genomic instability and multinucleation seen in C. trachomatis infections.

Background: Burkholderia pseudomallei (Bp), a Gram-negative, motile, facultative intracellular bacterium is the causative agent of melioidosis in humans and animals. The Bp genome encodes a repertoire of virulence factors, including the cluster 3 type III secretion system (T3SS-3), the cluster 1 type VI secretion system (T6SS-1), and the intracellular motility protein BimA, that enable the pathogen to invade both phagocytic and non-phagocytic cells. A unique hallmark of Bp infection both in vitro and in vivo is its ability to induce cell-to-cell fusion of macrophages to form multinucleated giant cells (MNGCs), which to date are semi-quantitatively reported following visual inspection., Results: In this study we report the development of an automated high-content image acquisition and analysis assay to quantitate the Bp induced MNGC phenotype. Validation of the assay was performed using T6SS-1 (∆hcp1) and T3SS-3 (∆bsaZ) mutants of Bp that have been previously reported to exhibit defects in their ability to induce MNGCs. Finally, screening of a focused small molecule library identified several Histone Deacetylase (HDAC) inhibitors that inhibited Bp-induced MNGC formation of macrophages., Conclusions: We have successfully developed an automated HCI assay to quantitate MNGCs induced by Bp in macrophages. This assay was then used to characterize the phenotype of the Bp mutants for their ability to induce MNGC formation and identify small molecules that interfere with this process. Successful application of chemical genetics and functional reverse genetics siRNA approaches in the MNGC assay will help gain a better understanding of the molecular targets and cellular mechanisms responsible for the MNGC phenotype induced by Bp, by other bacteria such as Mycobacterium tuberculosis, or by exogenously added cytokines.

Burkholderia pseudomallei, the causative agent of melioidosis, contains a large pathogen genome (7.2 Mb) with ∼2,000 genes of putative or unknown function. Interactions with potential hosts and environmental factors may induce rapid adaptations in these B. pseudomallei genes, which can be discerned through evolutionary analysis of multiple B. pseudomallei genomes. Here we show that several previously uncharacterized B. pseudomallei genes bearing genetic signatures of rapid adaptation (positive selection) can induce diverse cellular phenotypes when expressed in mammalian cells. Notably, several of these phenotypes are plausibly related to virulence, including multinuclear giant cell formation, apoptosis, and autophagy induction. Specifically, we show that BPSS0180, a type VI cluster-associated gene, is capable of inducing autophagy in both phagocytic and nonphagocytic mammalian cells. Following infection of macrophages, a B. pseudomallei mutant disrupted in BPSS0180 exhibited significantly decreased colocalization with LC3 and impaired intracellular survival; these phenotypes were rescued by introduction of an intact BPSS0180 gene. The results suggest that BPSS0180 may be a novel inducer of host cell autophagy that contributes to B. pseudomallei intracellular growth. More generally, our study highlights the utility of applying evolutionary principles to microbial genomes to identify novel virulence genes.

Fetal syncytiotrophoblasts form a unique fused multinuclear surface that is bathed in maternal blood, and constitutes the main interface between fetus and mother. Syncytiotrophoblasts are exposed to pathogens circulating in maternal blood, and appear to have unique resistance mechanisms against microbial invasion. These are due in part to the lack of intercellular junctions and their receptors, the Achilles heel of polarized mononuclear epithelia. However, the syncytium is immune to receptor-independent invasion as well, suggesting additional general defense mechanisms against infection. The difficulty of maintaining and manipulating primary human syncytiotrophoblasts in culture makes it challenging to investigate the cellular and molecular basis of host defenses in this unique tissue. Here we present a novel system to study placental pathogenesis using murine trophoblast stem cells (mTSC) that can be differentiated into syncytiotrophoblasts and recapitulate human placental syncytium. Consistent with previous results in primary human organ cultures, murine syncytiotrophoblasts were found to be resistant to infection with Listeria monocytogenes via direct invasion and cell-to-cell spread. Atomic force microscopy of murine syncytiotrophoblasts demonstrated that these cells have a greater elastic modulus than mononuclear trophoblasts. Disruption of the unusually dense actin structure--a diffuse meshwork of microfilaments--with Cytochalasin D led to a decrease in its elastic modulus by 25%. This correlated with a small but significant increase in invasion of L. monocytogenes into murine and human syncytium. These results suggest that the syncytial actin cytoskeleton may form a general barrier against pathogen entry in humans and mice. Moreover, murine TSCs are a genetically tractable model system for the investigation of specific pathways in syncytial host defenses.

Background: Mycobacterium tuberculosis differs from most pathogens in its ability to multiply inside monocytes and to persist during long periods of time within granuloma in a status of latency. A class of proteins called mycobacterial histone-like proteins has been associated with regulation of replication and latency, but their precise role in the infection process has yet to be uncovered. Our study aimed at defining the impact of the histone-like protein MDP1 from M. bovis BCG (mycobacterial DNA-binding protein 1, corresponding to Rv2986c from M. tuberculosis) on early steps of infection., Results: Previously, a BCG (Bacillus Calmette Guérin) strain had been generated by antisense-technique exhibiting reduced MDP1 expression. This strain was now used to analyse the impact of reduced amount of MDP1 on the interaction with human blood monocytes, macrophage lines and PBMC (peripheral blood mononuclear cells). MDP1 was revealed to be required for growth at acidic pH and for intracellular replication in human blood monocytes. Down-regulation of MDP1 resulted in reduced secretion of the cytokine IL-1β by infected human PBMC. In addition, a reduction of MDP1 expression had a major impact on the formation of fused multi-nucleated macrophages. In monocyte preparations from human blood as well as in human and mouse macrophage cell lines, both the percentage of multi-nucleated cells and the number of nuclei per cell were much enhanced when the monocytes were infected with BCG expressing less MDP1., Conclusion: MDP1 from M. bovis BCG affects the growth at acidic pH and the intracellular replication in human monocytes. It furthermore affects cytokine secretion by host cells, and the formation of fused multi-nucleated macrophages. Our results suggest an important role of MDP1 in persistent infection.

Histoplasmosis is a fungal disease endemic in central and eastern states of United States, South America, Africa, and Asia. It may present as chronic pulmonary infection or in disseminated form of infection. The disseminated form of histoplasmosis frequently affects the adrenal gland and is more likely to affect immunocompromised patients as compared to immunocompetent individuals. There are very few cases of adrenal histoplasmosis diagnosed on fine needle aspiration cytology. In the present era when fine needle aspiration has become popular modality for diagnosis, adrenal fine needle aspiration is still a less commonly practiced technique. We report eight cases of adrenal histoplasmosis diagnosed on fine needle aspiration cytology in patients with bilateral adrenal masses. The present case series supports the role of fine needle aspiration cytology of adrenal gland infections where surgery can be prevented and a definitive diagnosis can be made on which treatment can be offered., (Copyright © 2010 Wiley-Liss, Inc.)

An 87-year-old man, a gardener in Okinawa, first noticed a tumor on the dorsum of his right hand in November 2005. He had been taking prednisolone for the treatment of polymyalgia rheumatica since 2000. A nearby dermatologist incised the tumor for pus drainage in February 2006. In April of the same year, the dome-like tumor reappeared. The same treatment was repeated. Because the culture of the pus revealed fungi at that time, terbinafine hydrochloride and minocycline were administrated under the diagnosis of a deep fungal infection. After a short remission, the tumor recurred in November of the same year and in May and August of 2007 regardless of the repeated incision and pus drainage. He was referred to our hospital on 27 September 2007. His first physical examination at our outpatient office showed a skin-colored, well-demarcated, multilocular, cystic subcutaneous tumor on the dorsum of his right hand. Histopathological examination revealed a pseudocyst with fibrous walls of connective tissue. Continuous, bead-like hyphae, positive with periodic acid-Schiff stain and Grocott stain, were found within the pseudocyst. Morphological and molecular biological examinations of the separately cultured specimens identified the causative agent as Exophiala jeanselmei. The entire cyst was removed under local anesthesia, and an artificial dermis made of silicon membrane was applied to the wound. Skin graft was performed in November after confirming no recurrence of the fungal infection. Terbinafine hydrochloride 125 mg/day has continued. No recurrence has been observed up to now.

Background: The uptake of abortion-inducing pathogens by trophoblast giant (TG) cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70) contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this., Methods: Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR) domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice., Results: The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70., Conclusions: Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

Background: Trophoblast giant (TG) cells are involved in systematic removal of bacterial pathogens from the maternal-fetal interface of the placenta. In particular, TG cells have the ability to take up extracellular antigens by active phagocytosis induced by interferon-gamma (IFN-gamma). We previously reported that heat shock cognate protein 70 (Hsc70) present on the surface of TG cells mediated the uptake of Brucella abortus. However, the mechanism of bacterial uptake by TG cells is not completely understood. Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70., Methods: Mouse TG cells were employed in all experiments, and B. abortus was used as the bacterial antigen. Confirmation of the binding capacity of ERM protein was assessed by pull-down assay and ELISA using recombinant Hsc70 and ERM proteins. Ezrin was depleted using siRNA and the depletion examined by immunoblotting or immunofluorescence staining., Results: The expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability. Although tyrosine phosphorylation of ezrin was not related to bacterial uptake activity, localization of Hsc70 on the membrane was affected by the depletion of ezrin in TG cells., Conclusion: Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

Cryptococcosis is a deep mycosis that develops in immunosuppressed individuals. Isolated pulmonary cryptococcosis is a rare disease. The authors describe an autopsy case of pulmonary cryptococcosis in a 69-year-old woman with diabetes mellitus suppressing the immune system. Pulmonary inflammatory changes were characterized by disseminated destructive changes, a weak exudative reaction and the formation of epithelioid cell granulomas with the presence of Pirogov-Langerhans giant multinucleate cells. Round fungal cells weakly stained with hematoxylin and eosin can be mistakenly taken as artifacts. The diagnosis of chronic pulmonary cryptococcosis was established only after histological study using the selective staining.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is manifested by a broad spectrum of clinical and lesional presentations. We have evaluated the expression of transforming growth factor-beta1 (TGF-beta1), a cytokine known to have immunosuppressor effects, by immunohistochemistry, in different paratuberculosis lesions in the intestine and lymph nodes from 20 sheep and 25 cattle. Peripheral immune responses were assessed by interferon-gamma (IFN-gamma) test and the presence of antibodies. Expression of TGF-beta1, observed in macrophages and giant cells forming the lesions, was closely related to the amount of Map. In focal and multifocal forms, usually positive to IFN-gamma test, bacilli were difficult to detect and TGF-beta1 expression was low or absent. Diffuse multibacillary lesions, negative to IFN-gamma, show large numbers of Map and the highest percentage of immunolabelled cells. Diffuse paucibacillary forms, positive to IFN-gamma, have low numbers of AFB and scant or no cells positive to TGF-beta1. The high expression of TGF-beta1 would be related to the inability of macrophages to limit the multiplication of Map.

Multinucleated giant cells (MGC) are characteristic cells in granulomatous disorders such as paracoccidioidomycosis (PCM) and also are formed in vitro from peripheral blood mononuclear cells by several stimuli. In this study, the authors investigated in vitro formation of MGC derived from monocytes of healthy individuals, stimulated with Paracoccidioides brasiliensis antigen (PbAg), compared with other stimuli such as IFN-gamma and supernatant of Con-A-stimulated peripheral blood mononuclear cells (CM-ConA). Besides, the fungicidal activity of monocytes and monocyte-derived MGC challenged with P. brasiliensis were compared, at a ratio of one fungus per 50 monocytes. Results demonstrated that PbAg, IFN-gamma, and CM-ConA stimuli were able to induce MGC generation, with fusion indices significantly higher than control cultures. Striking results were observed when MGC induced by PbAg and IFN-gamma presented higher fungicidal activity than monocytes, submitted to the same stimuli, showing a better capacity of these cells to kill P. brasiliensis. In summary, the results suggest that PbAg is able to induce MGC generation, and these cells presented higher fungicidal activity against P. brasiliensis than monocytes.

This report describes a case of recurrence of chronic paracoccidioidomycosis 10 years following the initial diagnosis. A 56-year-old female was admitted to the Dental Clinic of the Pontifical Catholic University of Paraná complaining of oral soreness. Mulberry-like ulcerations were observed on the gingiva, right labial comissura, and vermillion of the lip. The patient reported persistent chronic cough, weight loss, appetite loss and fever. The anamnesis revealed that the patient had developed and been treated for paracoccidioidomycosis 10 years earlier. A biopsy was performed and microscopic examination revealed microabscesses, collections of macrophages organized into granulomas, multinucleated giant cells and Paracoccidioides brasiliensis. The patient was treated with Itraconazole and, the oral lesions disappeared within 3 months. Persistent follow-up examination in patients with a history of paracoccidioidomycosis is essential in the management of this disease.

Tuberculosis is characterized by a tight interplay between Mycobacterium tuberculosis (M. tb) and host cells within granulomas. These cellular aggregates restrain M. tb spreading but do not kill all bacilli, which persist for years. A more detailed investigation of the interaction between M. tb and granuloma cells is needed to improve our understanding of this persistence and to explain the physiopathology of tuberculosis. In the present study, a recently developed in vitro human model of tuberculous granulomas has been used to analyse the modulation of granuloma cell differentiation by M. tb, in comparison to poorly virulent mycobacteria, which do not persist. It is reported that whilst all mycobacteria species induce granuloma formation, only M. tb triggers the differentiation of granuloma macrophages into very large multinucleated giant cells (MGCs) that are unable to mediate any bacterial uptake. This loss of function is not due to cell quiescence, as MGCs still display NADPH oxidase activity, but it correlates with decreased expression of phagocytosis receptors. This phenomenon is specific for the virulent species of M. tuberculosis complex, as poorly virulent species only induce the formation of small multinucleated cells (MCs) with conserved mycobacterial uptake ability, which never reach the MGC differentiation stage. The phenotype of MGCs thus strongly resembles mature dendritic cells with a loss of microbial uptake ability, despite conserved antigen presentation. In M. tb-induced granulomas, MGCs thus seem to be devoted to the destruction of bacilli that have been ingested in previous differentiation stages, ie in macrophages and MCs., (Copyright (c) 2006 Pathological Society of Great Britain and Ireland.)

Multinucleate giant cells (MGC) are characteristic of tuberculous granulomas, but their function is not well understood. In a comparative study, we investigated regulation of chemokine secretion by MGC generated using 5 microg/ml ConA and 1000 IU/ml IFN-gamma. After 72-h differentiation of MGC cultures, CXCL8, CCL2 and CCL3 concentrations were 9540+/-110 pg/ml, 11190+/-2210 pg/ml and 19440+/-440 pg/ml respectively all significantly higher than in MDM (P<0.01). There was associated increased chemokine gene expression. M.tb stimulation of MGC, MDM and monocytes increased CXCL8 secretion. M.tb increased monocyte CCL2 secretion, whereas MGC and MDM secreted CCL2 constitutively. CXCL10 secretion was induced in M.tb-stimulated MDM and constitutive in MGC. All cell types responded to M.tb with CCL3 secretion. Monocyte chemokine secretion was associated with increased gene expression, whereas M.tb-stimulated MGC principally upregulated CCL3 gene expression. In summary, differentiating MGC express genes for and secrete chemokines which regulate cell influx to sites of infection. Established MGC will contribute to cell recruitment to granuloma, but this may not depend on exposure to the pathogen.

The microsporidium Vittaforma corneae develops within the target cell cytoplasm. In the present study, green monkey kidney (E6) cells infected at 30 degrees C, 35 degrees C or 37 degrees C with V. corneae developed enlarged multinucleate structures of up to 200 microm in any horizontal dimension made up either of a single cell or of multiple fused cells. A number of epithelial cell types (SW-480, HT-29, Caco-2 and HCT-8) were infected with V. corneae but did not induce the same highly organized structures, suggesting that for the structure to develop, the host cell must be capable of continued mitosis, and not be differentiated or be detaching from the surface matrix. Live cell imaging of infected E6 cells revealed large, multinucleate infected cells characterized by a central focus from which radiated parasite stages and host cell mitochondria. Immunocytochemistry identifying gamma and alpha tubulin suggested that a single centrally-located microtubule organizing centre governed the distribution of parasite stages and host cell organelles, with mitochondria and parasites being eventually transported towards the periphery of the structure. Whole cell patch clamp analysis of infected cells indicated an average five-fold increase in total membrane capacitance, consistent with an enlarged single cell. Scanning electron microscopy revealed cell-like protrusions around the periphery of the structure with the intervening space being made up of parasites and cell debris. Clearly in the case of V. corneae-infected E6 cells the parasite-host cell relationship involves subverting the host cell cytoskeleton and cell volume control, providing the parasite with the same protected niche as does a xenoma.

Brachiola algerae (Vavra et Undeen, 1970) Lowman, Takvorian et Cali, 2000, originally isolated from a mosquito, has been maintained in rabbit kidney cells at 29 degrees C in our laboratory. This culture system has made it possible to study detailed aspects of its development, including spore activation, polar tube extrusion, and the transfer of the infective sporoplasm. Employing techniques to ultrastructurally process and observe parasite activity in situ without disturbance of the cultures has provided details of the early developmental activities of B. algerae during timed intervals ranging from 5 min to 48 h. Activated and nonactivated spores could be differentiated by morphological changes including the position and arrangement of the polar filament and its internal structure. The majority of spores extruded polar tubes and associated sporoplasms within 5 min post inoculation (p.i.). The multilayered interlaced network (MIN) was present in extracellular sporoplasms and appeared morphologically similar to those observed in germination buffer. Sporoplasms, observed inside host cells were ovoid, contained diplokaryotic nuclei, vesicles reminiscent of the MIN remnants, and their plasmalemma was already electron-dense with the "blister-like" structures, typical of B. algerae. By 15 min p.i., the first indication of parasite cell commitment to division was the presence of chromatin condensation within the diplokaryotic nuclei, cytoplasmic vesicular remnants of the MIN were still present in some parasites, and early signs of appendage formation were present. At 30 min p.i., cell division was observed, appendages became more apparent, and some MIN remnants were still present. By two hours p.i., the appendages became more elaborate and branching, and often connected parasite cells to each other. In addition to multiplication of the organisms, changes in parasite morphology from small oval cells to larger elongated "more typical" parasite cells were observed from 5 h through 36 h p.i. Multiplication of proliferative organisms continued and sporogony was well underway by 48 h p.i., producing sporonts and sporoblasts, but not spores. The observation of early or new infections in cell cultures 12-48 h p.i., suggests that there may also exist a population of spores that do not immediately discharge, but remain viable for some period of time. In addition, phagocytized spores were observed with extruded polar tubes in both the host cytoplasm and the extracellular space, suggesting another means of sporoplasm survival. Finally, extracellular discharged sporoplasms tightly abutted to the host plasmalemma, appeared to be in the process of being incorporated into the host cytoplasm by phagocytosis and/or endocytosis. These observations support the possibility of additional methods of microsporidian entry into host cells and will be discussed.

The history of understanding xenoparasitic complexes or xenomas provoked in the host cell by various protists and especially by microsporidia is outlined. Microsporidia have been known to produce xenomas in oligochaetes (e.g., genera Bacillidium, Burkea, Hrabyeia, Jirovecia, species of the collective group Microsporidium), crustaceans (e.g., Abelspora, Mrazekia), insects (e.g., Polydispyrenia, Thelohania) and poikilothermic vertebrates, mostly fish (Alloglugea, Amazonspora, Glugea, Ichthyosporidium, Loma, Microfilum, Microgemma, Neonosemoides, Pseudoloma, Spraguea, Tetramicra). An overview of characters of xenomas caused by species of these genera is presented. The study of microsporidia causing xenomas in fish offers an insight into cell pathology and is of interest since many of these species are important agents of diseases in commercial fish. Xenomas produced from a few types of target cell display a complete change of organisation of the host cell and differ, according to the agent, in their structure. Recent data show that proliferation of the parasite may have already started in the cells transporting the parasites to the final site of xenoma formation. However, these are preliminary revelations and most of the facets of the life cycle are still to be clarified. Curiously, xenoma-forming microsporidia do not seem to be strictly host specific. The salient features of fish microsporidian xenomas are discussed, such as role of the xenoma, whether its features are host- or microsporidium-dependent, development and demise of the xenoma in the course of time, and host reaction phenomena. The need of further research is emphasised.

Loma salmonae is a common gill parasite of salmonids, and essentially all species in the genus Oncorhynchus are susceptible. Infections occur in both fresh and salt water. Loma salmonae is directly transmissible by ingestion of spores or infected tissue. The parasite infects the wall of blood vessels of various organs, but the gill is the primary site of infection. Initial infection occurs in the intestine, and xenomas are easily detected in the gills by standard histology at 4-6 wk post-exposure. A few presporogonic stages of the parasite are found in the heart endothelium prior to xenoma formation in the gills. Ultrastructure studies of early infections demonstrated that wandering blood cells transport the meronts to the gills, and that merogony occurs in pillar cells and other cells underlying the gill endothelium. Xenomas develop in these cells, resulting in hypertrophied host cells filled with spores. Xenomas ultimately rupture, and are associated with severe inflammation in which free spores are found in macrophages. The parasites are most pathogenic during this phase of the infection, resulting in severe vasculitis and clinical disease. Both rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus ishawytscha) recover from infections, but free spores persist in kidney and spleen phagocytes for many months after xenomas are absent in Chinook salmon. Fish that have recovered from the infection show strong immunity against the parasite, lasting up to 1 year. Fish are susceptible to infection by other routes of exposure by spores; co-habitation, anal gavage, and intramuscular, intraperitoneal and intravascular injection. Autoinfection probably occurs following release of spores in blood vessels after xenomas rupture. The optimal temperature for L. salmonae infections is 15-17 degrees C, with a permissive range of 11-20 degrees C.

Xenomas caused by Microgemma vivaresi Canning, Feist, Longshaw, Okamura, Anderson, Tsuey Tse et Curry, 2005 were found in liver and skeletal muscle of sea scorpions, Taurulus bubalis (Euphrasen). All muscle xenomas examined were in an advanced stage of destruction. In developing xenomas found in liver, parasites were restricted to the centre of the cell, separated from a parasite-free zone by a nuclear network formed by branching of the host cell nucleus. Although xenomas were able to reach a size of several hundred microns, the surface remained a simple plasma membrane. Host reactions took the form of penetration by phagocytes and isolation by fibroblasts. Once the xenoma had been attacked, the nuclear profiles became pycnotic and the barrier between parasitized and parasite-free zones was lost. Parasite antigens cannot be exposed at the surface of intact xenomas, as the host does not recognise the enlarging cell as foreign. Breaches in the plasma membrane of the xenoma and leakage of parasite antigens are thought to be the stimuli for phagocyte entry into the cell, its isolation by fibroblasts and eventual granuloma formation.

By studying the background materials, the authors have analyzed N. I. Pirogov's works on the pathological anatomy of tuberculosis. They show the fallacy of the widespread statement on N. I. Pirogov's priority in describing one of the characteristic cell elements of tuberculous granulation tissue, namely multinuclear giant cells referred in the Russian literature to as Pirogov-Langhans cells.

Multinucleated giant cells (MGC), a characteristic feature of tuberculous granulomas, form by fusion of monocytes or macrophages, but little is known about the mechanism of the fusion process itself. Several studies report an indirect effect of mycobacteria, i.e., induction of a soluble lymphocyte-derived fusion factor following stimulation by mycobacteria or mycobacterial products. The aim of our study was to determine whether contact with mycobacteria can induce MGC formation from human monocytes in vitro. Stimulation of monocytes with Mycobacterium bovis bacillus Calmette-Guérin (BCG) in combination with cytokine-containing supernatants of herpesvirus saimiri-transformed human T-cell clones (T-SN) led to MGC formation with fusion rates of about 27%. In contrast, stimulation with one component alone induced only low fusion rates of up to 10%. Heat-killed BCG in combination with T-SN induced monocyte fusion to the same extent as live mycobacteria. BCG culture supernatant, BCG lysate, or inert particles in combination with T-SN did not induce MGC formation. Experiments using transwell plates containing a semipermeable membrane revealed that induction of the fusion process is dependent on direct contact of monocytes and mycobacteria. MGC formation induced by BCG plus T-SN could be inhibited by addition of monoclonal antibodies to gamma interferon (but not tumor necrosis factor alpha) as well as to the beta chain (CD18) of beta2-integrins. These results demonstrate that contact with mycobacteria in combination with cytokine-containing supernatants is able to induce human monocytes to form MGC and that membrane-bound molecules of mycobacteria and monocytes are involved in the fusion process.

Between 1989 and 1996, fine-needle aspiration biopsy was performed in 22 patients with palpable colonic masses. In all of these patients colonoscopic examination was either not possible or could not be carried out successfully. The results of aspiration biopsy were confirmed by surgery and histopathological examination of the tissue. Aspiration biopsy correctly diagnosed all except one case. It identified all cases of colonic cancer. One patient with colonic tuberculosis was also diagnosed correctly. However, in another patient with colonic tuberculosis, aspiration biopsy showed only nonspecific changes in the form of inflammatory and epithelial cells. There were no false-positive results or complications from the procedure. It is concluded that fine-needle aspiration biopsy is a simple, rapid, and accurate method of diagnosing palpable colonic masses in patients in whom a colonoscopy is not possible or cannot be performed satisfactorily.

Some believe that asteroid bodies (AB) in sporotrichosis are nonspecific and are equivalent to the AB of sarcoidosis and other granulomatous diseases. We studied 25 skin biopsy specimens of sporotrichosis in which AB were demonstrated, ten of them with Sporothrix-positive culture. Immunohistochemistry was performed in paraffin-embedded biopsy specimens using an anti-Sporothrix antibody. The same procedures were done with seven biopsy specimens of lobomycosis, which contained AB within giant cells. These did not react with the anti-Sporothrix antibody, and by electron microscopy they displayed filamentous and myelin figures similar to the AB of sarcoidosis. In sporotrichosis, the AB are extracellular eosinophilic structures, 15-35 microm in diameter, and located within abscesses. One to three are found in a section. They consist of a central yeast, surrounded by eosinophilic spicules. The yeast stains with the anti-Sporothrix antibody, while the spicules do not. Therefore, AB in sporotrichosis are specific for disease. Visualization of the spicules alone can lead to the demonstration of the AB in adjacent sections, and thus is a useful clue in the diagnosis of sporotrichosis. Sporotrichotic AB must not be confused with the intracellular AB seen in giant cells of granulomatous reactions, which are filamentous and myelin figures that contain lipid.

Multinucleated giant cells developed in Pneumocystis carinii-diseased gene disruption mutant mice deficient in major histocompatibility complex class II molecules, T-cell receptor alpha beta cells, or all mature T and B lymphocytes. These findings demonstrate lymphocyte-independent fusion of alveolar macrophages under morbid conditions. Pulmonary parasite burden seems to be a decisive factor in multinucleation.

Multinucleated giant cells (MGC) have been long recognized as a histopathologic feature of tuberculosis, yet little is known about the underlying mechanism of tubercle bacillus-induced formation of these fused macrophages. The main purpose of this study was to characterize cellular mechanisms involved in MGC formation of swine microglia, the resident macrophages of the brain, in cultures containing nonopsonized Mycobacterium bovis. Within 2 h of incubation, MGC were readily detected in these cultures by light and transmission electron microscopy. MGC formation was blocked by anti-CD14 and anti-CD18 antibodies and by thalidomide, a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha) production my microglia. Also, TNF-alpha alone induced MGC formation. These findings suggest that two microglial cell receptors, CD14 and a beta2 integrin, and the cytokine TNF-alpha participate in M. bovis-induced swine microglial MGC formation.

It has been previously demonstrated that lymphocyte function-associated molecule 1 (LFA-1) plays a major role in human immunodeficiency virus (HIV)-mediated syncytia formation. In the present study we investigated the involvement of intercellular adhesion molecule-1 (ICAM-1), ICAM-2 and ICAM-3 in the process. The ability of monoclonal antibodies (mAb) directed against ICAM-1, ICAM-2 and ICAM-3 to block syncytia was analyzed either in phytohemagglutinin (PHA)-activated lymphocytes infected in vitro with primary or laboratory strains of HIV or by coculturing a T cell line stably expressing HIV envelope with PHA-activated lymphocytes. Complete inhibition of syncytia formation was observed only by the simultaneous addition to the cell cultures of all (i.e. anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3) mAb. These results indicate that the interaction between LFA-1 and ICAM is a critical step in HIV-mediated syncytia formation, and that ICAM-1, ICAM-2 and ICAM-3 are the receptor molecules for the LFA-1-dependent syncytia formation.

We previously reported that truncation of the terminal 146 amino acids of the macaque simian immunodeficiency virus SIVmac239 envelope glycoprotein enhanced envelope-specific syncytium formation in HeLa T4, CEM X 174, and HUT 78 cell lines and caused a change in the conformation of the transmembrane subunit of the envelope complex on the surface of these cells [Ritter et al. (1993) Virology 197, 255-264; Spies et al. (1994) J. Virol. 68, 585-591]. To investigate the effects of different lengths of the cytoplasmic domain on syncytium formation and cell surface expression, we have compared the expression and cytopathic effects induced by five SIVmac239 envelope constructs which vary in the lengths of their cytoplasmic domains. In contrast to the envelope protein truncated by 146 amino acids, the ability of proteins truncated by 98 or 161 amino acids to form syncytia was substantially reduced in CEM X 174 and HUT 78 cells, while syncytium formation by a protein truncated by 53 amino acids was only slightly reduced compared to the full-length protein. Furthermore, only the glycoprotein which was truncated by 146 amino acids induced syncytium formation in HeLa T4 cells. When examining the expression of the truncated proteins on the surface of HeLa T4 cells, we found that, in contrast to the full-length SIVmac239 protein, each of the truncated transmembrane subunits could be efficiently biotinylated with the membrane-impermeable reagent NHS-SS-biotin. Furthermore, using cell surface iodination, we found stable oligomeric forms of both the transmembrane subunits and the uncleaved precursor proteins of each mutant protein on the surface of HeLa T4 cells. Using pulse-chase analysis, we also found that the precursor of the protein truncated by 98 residues was degraded more rapidly than the wild-type and the other mutant proteins. Finally, we constructed two mutants which expressed a full-length TM protein or a TM protein with a 146 amino acid C-terminal deletion and had most of the coding sequences of their SU subunits deleted. Neither of these two proteins was found to cause syncytium formation in HeLa T4, CEM X 174, or HUT 78 cell lines even though we could detect both proteins on the surfaces of HeLa T4 cells using iodination. These results could explain why the selection of truncated variants of SIV which emerge after prolonged passage in human cell lines is restricted to truncations which remove close to 146 amino acids in the cytoplasmic domain of the TM protein.