Chlamydia pneumoniae multiply in neutrophil granulocytes and delay their spontaneous apoptosis

The obligate intracellular bacterial pathogen Chlamydia pneumoniae (Cp) is responsible for a range of human diseases, including acute respiratory infection. Although experimental intratracheal infection with Cp results in a massive recruitment of neutrophil granulocytes (polymorphonuclear neutrophils (PMN)), the role of these cells in the defense against Cp is unclear. In this study the interactions of PMN with Cp were investigated. In vitro coincubation experiments showed that human granulocytes were able to internalize Chlamydia in an opsonin-independent manner. Importantly, phagocytosed Cp were not killed; the ingested bacteria survived and multiplied within PMN. Although uninfected granulocytes became apoptotic within 10 h, infected PMN survived up to 90 h. Coincubation with Cp significantly decreased the ratio of apoptotic PMN, as detected by morphological analysis, annexin V, and TUNEL staining. The observed antiapoptotic effect was associated with a markedly lower level of procaspase-3 processing and, consequently, reduced caspase-3 activity in infected PMN. LPS was found as a major, but not exclusive, component responsible for the observed antiapoptotic effect. Chlamydia LPS affected PMN apoptosis both by acting directly on the cells and by inducing the autocrine production of the antiapoptotic cytokine IL-8. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, Cp can extend the life span of neutrophil granulocytes, making them suitable host cells for survival and multiplication within the first hours/days after infection.     

Bryophytes in Mediterranean coastal dunes: ecological strategies and distribution along the vegetation zonation

The bryophytic flora has been widely studied in Atlantic coastal dune environments while bryological literature addressing the Mediterranean bryophytes is still scarce and fragmented, although they are an important component of this area, especially considering their ability to colonize and stabilize sandy soils. The aims of this study were: (i) to analyze the taxonomy, chorology and ecology of coastal dunes bryoflora; (ii) to analyze distribution patterns of bryophytic species along a coastal dune vegetation zonation. We used as coastal dune model system a particularly well-preserved study area of the Sardinian coast (Italy), which includes several habitats listed in European Habitats Directive 92/43/EEC. We conducted a general survey of the area and a random sampling. A total of 20 bryophytic species were detected most of which were acrocarpous mosses and, to a lesser extent, pleurocarpous and liverworts. Most bryophytes are pioneer colonizers, reflecting their adaptation to environments with high levels of stress/disturbance conditions such as those of coastal dunes. Finally, in this study we described for the first time how bryophytic species are distributed along a Mediterranean coastal dune vegetation zonation, focusing also on their chorological and ecological characteristics and on how they seem to follow the sea-land gradient, probably in response to different environmental conditions developing along this gradient.     

Induction of CD137 expression by viral genes reduces T cell costimulation

Intracellular pathogens are subject to elimination by a cellular immune response, and were therefore under evolutionary pressure to develop mechanisms that allow them to inhibit especially this arm of immunity. CD137, a T cell costimulatory molecule, and its ligand, CD137 ligand (CD137L), which is expressed on antigen presenting cells (APC), are potent drivers of cellular cytotoxic immune responses. Here, we report that different viruses usurp a negative feedback mechanism for the CD137–CD137L system that weakens cellular immune responses. Latent membrane protein (LMP)-1 and Tax, oncogenes of Epstein-Barr virus (EBV), and human T-cell lymphotropic virus (HTLV)-1, respectively, induce the expression of CD137. CD137 is transferred by trogocytosis to CD137L-expressing APC, and the CD137–CD137L complex is internalized and degraded, resulting in a reduced CD137-mediated T cell costimulation and a weakened cellular immune response which may facilitate the escape of the virus from immune surveillance. These data identify the usurpation of a CD137-based negative feedback mechanism by intracellular pathogens that enables them to reduce T cell costimulation.     

Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: Large scale production of the E1 protein

The two envelope glycoproteins of rubella virus (RV), El of 58 kDa and E2 of 42–47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10−1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.     

Mucosal Trafficking of Vector-Specific CD4+ T Lymphocytes following Vaccination of Rhesus Monkeys with Adenovirus Serotype 5

Post hoc analysis of the phase 2b Step study evaluating a recombinant adenovirus serotype 5 (rAd5)-based HIV-1 vaccine candidate suggested a potential increased risk of HIV-1 acquisition in subjects who were baseline Ad5 seropositive and uncircumcised. These concerns had a profound impact on the HIV-1 vaccine development field, although the mechanism underlying this observation remains unknown. It has been hypothesized that rAd5 vaccination of baseline Ad5-seropositive individuals may have resulted in anamnestic, vector-specific CD4⁺ T lymphocytes that could have trafficked to mucosal sites and served as increased targets for HIV-1 infection. Here we show that Ad5-specific CD4⁺ T lymphocyte responses at mucosal sites following rAd5-Gag/Pol/Nef vaccination were comparable in rhesus monkeys with and without baseline Ad5 immunity. Moreover, the total cellular inflammatory infiltrates and the CD3⁺, CD4⁺, HLA-DR⁺, Ki67⁺, and langerin⁺ cellular subpopulations in colorectal and foreskin mucosa were similar in both groups. Thus, no greater trafficking of Ad5-specific CD4⁺ T lymphocytes to mucosal target sites was observed following rAd5 vaccination of rhesus monkeys with baseline Ad5 immunity. These findings from this nonhuman primate model provide evidence against the hypothesis that recruitment of vector-specific target cells to mucosal sites led to increased HIV-1 acquisition in Ad5-seropositive, uncircumcised vaccinees in the Step study.The Step study revealed a potential increased risk of HIV-1 acquisition among adenovirus serotype 5 (Ad5)-seropositive, uncircumcised subjects who received the Merck recombinant Ad5 (rAd5)-Gag/Pol/Nef vaccine candidate (2, 6). It has been hypothesized that rAd5 vaccination of Ad5-seropositive individuals may have resulted in robust expansion and activation of vector-specific CD4⁺ T lymphocytes that could have trafficked to mucosal sites and served as increased targets for HIV-1 infection. Our laboratory and others have recently demonstrated that total and vector-specific CD4⁺ T lymphocytes in peripheral blood in Ad5-seropositive volunteers were comparable to or lower than the levels in Ad5-seronegative volunteers following rAd5-Gag vaccination in the Merck phase 1 studies (4, 8). However, mucosal biopsy specimens were not obtained in these clinical trials, and thus the extent of inflammatory infiltrates and vector-specific CD4⁺ T lymphocytes in colorectal and foreskin mucosa could not be evaluated in these prior studies.It has also recently been reported that vector-specific CD4⁺ T lymphocytes may upregulate mucosal homing integrin expression following exposure to Ad5 in short-term in vitro cultures (1). These findings highlight the importance of directly investigating the extent and nature of vector-specific CD4⁺ T lymphocytes at mucosal sites following rAd5 vaccination. Given the lack of mucosal biopsy samples from human subjects in the Step study, we developed a nonhuman primate model of preexisting adenovirus immunity to evaluate the extent and nature of inflammatory cell populations at mucosal sites following rAd5 vaccination.     

Optimized mouse ES cell culture system by suspension growth in a fully defined medium

Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time efficiency and typically requires only 20 min of effective hands-on time per week. This protocol maintains a very high degree of pluripotent cells partly by mechanical separation of spontaneously differentiating cells. mES cells can be cultured for extended periods (>6 months) without the loss of pluripotency markers. High passage (>20) adherent mES cultures containing contaminating differentiated cells can be rescued and enriched in undifferentiated ES cells.     

Fluorescent pseudomonads pursue media-dependent strategies to inhibit growth of pathogenic Verticillium fungi

Applied Microbiology and Biotechnology - Verticillium species represent economically important phytopathogenic fungi with bacteria as natural rhizosphere antagonists. Growth inhibition patterns of...