Crown gall teratoma formation is plasmid and plant controlled.

Experiments using different species of the plant Nicotiana and strains of the bacterium Agrobacterium tumefaciens showed that teratoma formation from crown galls was dependent on the combination of bacterial Ti plasmid and host plant used.     

Bioluminescence reporter gene imaging characterize human embryonic stem cell-derived teratoma formation

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However, teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore, tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study, hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology, we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover, FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4, and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency, retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers.     

Quantification of Shigella IcsA required for bacterial actin polymerization

Shigella move through the cytoplasm of host cells by active polymerization of host actin to form an "actin tail." Actin tail assembly is mediated by the Shigella protein IcsA. The process of Shigella actin assembly has been studied extensively using IcsA-expressing Escherichia coli in cytoplasmic extracts of Xenopus eggs. However, for reasons that have been unclear, wild type Shigella does not assemble actin in these extracts. We show that the defect in actin assembly in Xenopus extracts by Shigella can be rescued by increasing IcsA expression by approximately 3-fold. We calculate that the number of IcsA molecules required on an individual bacterium to assemble actin filaments in extracts is approximately 1,500-2,100 molecules, and the number of IcsA molecules required to assemble an actin tail is approximately 4,000 molecules. The majority of wild type Shigella do not express these levels of IcsA when grown in vitro. However, in infected host cells, IcsA expression is increased 3.2-fold, such that the number of IcsA molecules on a significant percentage of intracellular wild type Shigella would exceed that required for actin assembly in extracts. Thus, the number of IcsA molecules estimated from our studies in extracts as being required on an individual bacterium to assemble actin filaments or an actin tail is a reasonable prediction of the numbers required for these functions in Shigella-infected cells.     

Opposing roles of IL-10 in acute bacterial infection

Interleukin-10 (IL-10) is recognized as an anti-inflammatory cytokine that downmodulates inflammatory immune responses at multiple levels. In innate cells, production of this cytokine is usually triggered after pathogen recognition receptor (PRR) engagement by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patters (DAMPs), as well as by other soluble factors. Importantly, IL-10 is frequently secreted during acute bacterial infections and has been described to play a key role in infection resolution, although its effects can significantly vary depending on the infecting bacterium. While the production of IL-10 might favor host survival in some cases, it may also result harmful for the host in other circumstances, as it can prevent appropriate bacterial clearance. In this review we discuss the role of IL-10 in bacterial clearance and propose that this cytokine is required to recover from infection caused by extracellular or highly pro-inflammatory bacteria. Altogether, we propose that IL-10 drives excessive suppression of the immune response upon infection with intracellular bacteria or in non-inflammatory bacterial infections, which ultimately favors bacterial persistence and dissemination within the host. Thus, the nature of the bacterium causing infection is an important factor that needs to be taken into account when considering new immunotherapies that consist on the modulation of inflammation, such as IL-10. Indeed, induction of this cytokine may significantly improve the host’s immune response to certain bacteria when antibiotics are not completely effective.     

Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny

Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.     

Bacterial Virulence Factors: Secreted for Survival

Virulence is described as an ability of an organism to infect the host and cause a disease. Virulence factors are the molecules that assist the bacterium colonize the host at the cellular level. These factors are either secretory, membrane associated or cytosolic in nature. The cytosolic factors facilitate the bacterium to undergo quick adaptive—metabolic, physiological and morphological shifts. The membrane associated virulence factors aid the bacterium in adhesion and evasion of the host cell. The secretory factors are important components of bacterial armoury which help the bacterium wade through the innate and adaptive immune response mounted within the host. In extracellular pathogens, the secretory virulence factors act synergistically to kill the host cells. In this review, we revisit the role of some of the secreted virulence factors of two human pathogens: Mycobacterium tuberculosis—an intracellular pathogen and Bacillus anthracis—an extracellular pathogen. The advances in research on the role of secretory factors of these pathogens during infection are discussed.     

Talin, a host cell protein, interacts directly with the translocated intimin receptor, Tir, of enteropathogenic Escherichia coli, and is essential for pedestal formation

Enteropathogenic Escherichia coli (EPEC) is able to inject its own receptor, a transmembrane protein called translocated intimin receptor, Tir, into the host epithelial cell. The bacterium then uses an outer membrane protein, intimin, to bind to Tir and remains firmly attached to the host cell surface for the duration of the infection. The bacterium is also able to trigger the rearrangement of several host cell proteins, culminating with the formation of an actin-rich, pedestal-like structure beneath the EPEC adherence site. Although several cytoskeletal proteins are rearranged following EPEC infection, the exact role played by these proteins during pedestal formation remains unknown. We report here that talin, an integrin-binding protein, is recruited by EPEC and associates directly with Tir. By surface plasmon resonance (SPR), the predicted value for the dissociation constant ( K _D) for Tir–talin binding was 1.86 × 10⁻⁷ M. We also demonstrate that microinjection of anti-talin antibodies into HeLa cells resulted in the complete inability to focus actin filaments beneath the attached bacterium. These findings demonstrate that talin is essential for EPEC-induced pedestal formation in infected cells.     

Progenitor cell therapy for heart disease

Many cell types are currently being studied as potential sources of cardiomyocytes for cell transplantation therapy to repair and regenerate damaged myocardium. The question remains as to which progenitor cell represents the best candidate. Bone marrow-derived cells and endothelial progenitor cells have been tested in clinical studies. These cells are safe, but their cardiogenic potential is controversial. The functional benefits observed are probably due to enhanced angiogenesis, reduced ventricular remodeling, or to cytokine-mediated effects that promote the survival of endogenous cells. Human embryonic stem cells represent an unlimited source of cardiomyocytes due to their great differentiation potential, but each step of differentiation must be tightly controlled due to the high risk of teratoma formation. These cells, however, confront ethical barriers and there is a risk of graft rejection. These last two problems can be avoided by using induced pluripotent stem cells (iPS), which can be autologously derived, but the high risk of teratoma formation remains. Cardiac progenitor cells have the advantage of being cardiac committed, but important questions remain unanswered, such as what is the best marker to identify and isolate these cells? To date the different markers used to identify adult cardiac progenitor cells also recognize progenitor cells that are outside the heart. Thus, it cannot be determined whether the cardiac progenitor cells identified in the adult heart represent resident cells present since fetal life or extracardiac cells that colonized the heart after cardiac injury. Developmental studies have identified markers of multipotent progenitors, but it is unknown whether these markers are specific for adult progenitors when expressed in the adult myocardium. Cardiac regeneration is dependent on the stability of the cells transplanted into the host myocardium and on the electromechanical coupling with the endogenous cells. Finally, the promotion of endogenous regenerative processes by mobilizing endogenous progenitors represents a complementary approach to cell transplantation therapy.     

Direct amplification and sequencing of the 16S ribosomal DNA of an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia

DNA coding for the 16S rRNA of an intracellular bacterium was directly amplified from lysed cells of a host amoebae using the polymerase chain reaction and primers specific for eubacteria. The amoebae had been used to recover an uncultured bacterium observed in the sputum of a patient with pneumonia. The amplified DNA was sequenced directly and compared with published 16S rRNA sequences. The analysis revealed that the intracellular bacterium is a member of the genus Legionella and that it is different from species, including L. pneumophila, for which 16S ribosomal RNA sequence data are available.     

Novel polysaccharide antigen of Orientia tsutsugamushi revealed by a monoclonal antibody

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates in the cytosol of host cells. Although several protein antigens have been characterized and cloned, little information exists regarding the polysaccharide antigen of this bacterium. In this study, we identified and characterized a novel antigen defined by a monoclonal antibody (MAb), NT19, against O. tsutsugamushi . Immunofluorescence microscopic studies showed that the NT19 antigen is released from the bacteria in the cytosol of host cells forming aggregates with bacteria. Immunoblot analysis showed that MAb NT19 recognized a strong band with a molecular mass of 20 kDa that was resistant to proteinase K digestion and sensitive to periodate oxidation, suggesting that the NT19 antigen is a polysaccharide. The function of this polysaccharide is not known, but considering its distribution within a bacterial microcolony, it is suspected to be involved in forming a biofilm-like structure within host cells.     

Capnocytophaga canimorsus: a human pathogen feeding at the surface of epithelial cells and phagocytes

Capnocytophaga canimorsus, a commensal bacterium of the canine oral flora, has been repeatedly isolated since 1976 from severe human infections transmitted by dog bites. Here, we show that C. canimorsus exhibits robust growth when it is in direct contact with mammalian cells, including phagocytes. This property was found to be dependent on a surface-exposed sialidase allowing C. canimorsus to utilize internal aminosugars of glycan chains from host cell glycoproteins. Although sialidase probably evolved to sustain commensalism, by releasing carbohydrates from mucosal surfaces, it also contributed to bacterial persistence in a murine infection model: the wild type, but not the sialidase-deficient mutant, grew and persisted, both when infected singly or in competition. This study reveals an example of pathogenic bacteria feeding on mammalian cells, including phagocytes by deglycosylation of host glycans, and it illustrates how the adaptation of a commensal to its ecological niche in the host, here the dog's oral cavity, contributes to being a potential pathogen.     

Bacteria-mediated transfer of eukaryotic expression plasmids into mammalian host cells

Invasive intracellular bacteria are able to transfer eukaryotic expression plasmids into mammalian host cells in vitro and in vivo. This can be used to induce immune responses toward protein antigens encoded by the plasmid or to complement genetic defects. Plasmid transfer takes place when the recombinant bacterium dies within the host cell, either due to metabolic attenuation or induction of autolysis. Alternatively, antibiotics can be used and spontaneous transfer has also been observed, indicating that this phenomenon might also occur under physiological conditions. Plasmid transfer has been reported for Shigella flexneri, Salmonella typhimurium and S. typhi, Listeria monocytogenes and recombinant Escherichia coli, but other invasive bacteria should also share this property. In vivo attempts were mainly directed toward vaccination using shigella and salmonella as carrier. So far a wide variety of antigens have been used succesfully in mice. Often this type of immunization was superior over direct application of antigen or using the same bacterium as a heterologous carrier expressing the antigen via a prokaryotic promoter. Characterization of the host cells revealed that macrophages and dendritic cells might be responsible for immune stimulation by either expressing the antigen or cross-presenting the antigen after uptake of apoptotic antigen expressing cells.     

A novel alpha-Proteobacterium resides in the mitochondria of ovarian cells of the tick Ixodes ricinus

An intracellular bacterium from Ixodes ricinus ticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH). This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells. When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample. Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of the Proteobacterium: ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells. Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1. PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed). No adult males were found to be infected. Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.     

Current concepts on the pathogenicity of phytopathogenic bacteria

What are the molecular determinants that make a bacterium a plant pathogen? In the last 10-20 years, important progress has been made in answering this question. In the early 20th century soon after the discovery of infectious diseases, the first studies of pathogenicity were undertaken. These early studies relied mostly on biochemistry and led to the discovery of several major pathogenicity determinants, such as toxins and hydrolytic enzymes which govern the production of major disease symptoms. From these pioneering studies, a simplistic view of pathogenicity arose. It was thought that only a few functions were sufficient to transform a bacterium into a pathogen. This view rapidly changed when modern techniques of molecular genetics were applied to analyse pathogenicity. Modern analyses of pathogenicity determinants took advantage of the relatively simple organization of the haploid genome of pathogenic bacteria. By creating non-pathogenic mutants, a large number of genes governing bacterium-host interactions were identified. These genes are required either for host colonization or for the production of symptoms. Even though the role of motility and chemotaxis in these processes is still unclear, it is clear that a strong attachment of Agrobacterium to plant cells is a prerequisite for efficient plant transformation and disease. Other important pathogenicity factors identified with a molecular genetic approach include hydrolytic enzymes such as pectinases and cellulases which not only provide nutrients to the bacteria but also facilitate pathogen invasion into host tissues. The precise role of exopolysaccharide in pathogenicity is still under discussion, however it is has been established that it is crucial for the induction of wilt symptoms caused by Ralstonia solanacearum. Trafficking of effector proteins from the invading bacterium into the host cell emerged recently as a new central concept. In plant pathogenic bacteria, protein translocation takes place through the so-called 'type II secretion machinery' encoded by hrp genes in the bacterium. These genes are present in representatives of all the major groups of Gram negative plant pathogenic bacteria except Agrobacterium. Most of these genes have counterparts in pathogens of mammals (including those of human) and they also play a central role in pathogenicity. Additionally, recent evidence suggests that a 'type IV secretion machinery' injects bacterial proteins into host cells. This machinery, originally found to be involved in the transfer of t-DNA from Agrobacterium into plant cells, was recently shown to translocate pathogenicity proteins in pathogens of mammals such as Helicobacter pylori and Brucella. Discovery of the trafficking of proteins from the pathogen into host cells revolutionized our conception of pathogenicity. First, it rather unexpectedly established the conservation of basic pathogenicity strategies in plant and animal pathogens. Second, this discovery changes our ideas about the overall strategy (or mechanism) of pathogenicity, although we still think the end result is exploitation of host cell nutritive components. Rather than killing the host cell from outside, we envision a more subtle approach in which pathogens inject effector proteins into the host cell to effect a change in host cell biology advantageous to the pathogen. Identification of the effector proteins, of their function and of the corresponding molecular targets in the host is a new challenge which will contribute to the conception of new strategies to control diseases.     

Diagnostic value of hair shafts and squamous cells in peritoneal washing cytology

OBJECTIVE: Little attention has been given to hair shafts and squamous cells in peritoneal fluid. To investigate their diagnostic value in peritoneal washing specimens, we reviewed peritoneal washing cytology preparations from 83 cases of ovarian tumors. STUDY DESIGN: We reviewed peritoneal washing specimens and histologic sections of 86 cases of ovarian tumors and tumorous conditions, including 22 teratomas, 16 serous adenocarcinomas, 10 clear cell adenocarcinomas, 9 endometrioid adenocarcinomas, 5 cases of endometriosis, 4 mucinous adenomas, 3 serous cystadenocarcinomas and 17 other tumors. RESULTS: We observed both squamous cells and hair shafts surrounded by inflammatory cells in 5 of the 22 cases of ovarian teratoma. Rupture of an ovarian teratoma was clinically and histologically found in one of the five cases. Hair shafts were not observed in the other tumors or in nonneoplastic conditions. The diameter of hair shafts in peritoneal washing specimens ranged from 10 to 28.8 microns (average, 16.6), and such hair shafts were present within an ovarian teratoma examined histologically. The diameter of hair shafts from six normal adults who were examined as controls ranged from 61.5 to 118.6 microns (average, 89.4). CONCLUSION: Hair shafts and squamous cells surrounded by inflammatory cells in peritoneal washing specimens are a diagnostic clue to ovarian teratoma and can be observed even when rupture of the tumor is not detected clinically or microscopically.     

The discovery of SycO highlights a new function for type III secretion effector chaperones

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone-binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation-prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.     

Paramecium caudatum acquires heat-shock resistance in ciliary movement by infection with the endonuclear symbiotic bacterium Holospora obtusa

Holospora obtusa is a macronucleus-specific bacterium of the ciliate Paramecium caudatum. Three types of P. caudatum cells (H. obtusa-free cells, cells bearing the reproductive form of H. obtusa and cells bearing the predominantly infectious form of H. obtusa) cultured at 25 degrees C were transferred to 4, 10, 25, 35 and 40 degrees C and their swimming velocities were measured by taking photomicrographs with two-second exposures. The H. obtusa-free cells almost ceased swimming at both 4 and 40 degrees C, while cells bearing the reproductive form and those bearing the predominantly infectious form actively swam even at these temperatures. These results show that the host cell can acquire heat-shock resistance when infected by H. obtusa in the macronucleus. This is the first evidence to show that the endonuclear symbiont Holospora contributes to maintain the ciliary movement of the host even at temperatures unsuitable for the host growth.     

Isolation of an aggregation inhibitory factor from non-adhesive mouse teratoma cells

An aggregation inhibitory factor (AIF) has been extracted from mouse ascites teratoma cells (that do not aggregate in culture) that retards adhesion of cultured teratoma cells of the same cell line (that do aggregate). Preliminary characterization of AIF on polyacrylamide gels suggests that AIF is a protein composed of four subunits. Extraction of AIF from ascites teratoma cells was accomplished without significant loss of viability by a technique involving the application of an electric field to large numbers of whole cells suspended in a hypertonic electrode buffer. In tests of adhesion, AIF consistently and immediately inhibited aggregation of cultured teratoma cells after 5, 10, 15, and 30 min of incubation. Furthermore, a reduced concentration of AIF resulted in a corresponding decrease in inhibition, suggesting a concentration-dependent action. AIF may help explain how cultured teratoma cells adhere, whereas ascites teratoma cells of the same subline do not adhere.     

A Novel Alpha-Proteobacterium Resides in the Mitochondria of Ovarian Cells of the Tick Ixodes ricinus

An intracellular bacterium from Ixodes ricinus ticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH). This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells. When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample. Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of the Proteobacteria. ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells. Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1. PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed). No adult males were found to be infected. Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.     

Epidemiology of a <Emphasis Type="Italic">Daphnia</Emphasis> brood parasite and its implications on host life-history traits

Parasites influence host life-history traits and therefore might crucially shape host populations in natural systems. In a series of laboratory experiments, we studied the impact of an oomycete brood parasite on its Daphnia (waterflea) host. We asked whether Daphnia dump the infected brood and subsequently are able to reproduce again as was occasionally observed in a preliminary study. No viable offspring developed from infected clutches, but 78% of the infected females produced healthy offspring after releasing the infected brood while molting. Neither those offsprings’ development success nor their mothers’ reproductive potential was affected by the brood parasite. However, infected Daphnia had a reduced life-span and suffered an increased susceptibility to another parasite, an unidentified bacterium. Additionally, we studied the prevalence of this brood parasite and the unidentified bacterium in a natural Daphnia assemblage in a pre-alpine lake, across changing demographic and environmental conditions. The brood parasite epidemic seemed to be host-density dependent. Our results show that the brood parasite’s impact on the host population is enhanced when combined with the unidentified bacterium.