In Vitro Response of Human Lymphocytes to Mycoplasma pneumoniae

In vitro culture and stimulation of human peripheral lymphocytes were employed to investigate the role of cellular immunity in Mycoplasma pneumoniae disease. Subjects with documented natural infections served as donors. The lymphocyte response to whole M. pneumoniae organisms was determined as incorporation of tritiated thymidine in a semimicro culture system. The range of cellular reactivity stimulated by specific antigen was within the range stimulated by phytohemagglutinin. The difference between responses of subjects with documented infection and serologically negative controls was highly significant. Specific reactivity of peripheral lymphocytes correlated closely with the presence of serum growth-inhibiting antibodies, and both persisted for several years following infection. Serum complement-fixing titers correlated well with lymphocyte stimulability during the first year but antibody, as measured by this technique, tended to disappear in later convalescence. In light of previous studies, which revealed a lack of correlation between humoral antibodies and resistance to reinfection, these results suggest that immunity to M. pneumoniae infection is mediated by circulating small lymphocytes.     

Interleukin-6-Driven Inflammatory Response Induces Retinal Pathology in a Model of Ocular Toxoplasmosis Reactivation

Ocular inflammation is one of the consequences of infection with the protozoan parasite Toxoplasma gondii. Even if lesions are self-healing in immunocompetent persons, they pose a lifetime risk of reactivation and are a serious threat to vision. As there are virtually no immunological data on reactivating ocular toxoplasmosis, we established a model of direct intravitreal injection of parasites in previously infected mice with a homologous type II strain. Two different mouse strains with variable ability to control retinal infection were studied in order to describe protective and deleterious reaction patterns. In Swiss-Webster mice, which are already relatively resistant to primary infection, no peak of parasite load was observed upon reinfection. In contrast, the susceptible inbred strain C57BL/6 showed high parasite loads after 7 days, as well as marked deterioration of retinal architecture. Both parameters were back to normal on day 21. C57BL/6 mice also reacted with a strong local production of inflammatory and Th1-type cytokines, like interleukin-6 (IL-6), IL-17A, and gamma interferon (IFN-γ), while Swiss-Webster mice showed only moderate expression of the Th2 cytokine IL-31. Interestingly, rapid intraocular production of anti-Toxoplasma antibodies was observed in Swiss-Webster but not in C57BL/6 mice. We then localized the cellular source of different immune mediators within the retina by immunofluorescence. Finally, neutralization experiments of IFN-γ or IL-6 demonstrated the respective protective and deleterious roles of these cytokines for parasite control and retinal integrity during reinfection. In conclusion, we developed and immunologically characterized a promising mouse model of reactivating ocular toxoplasmosis.     

In Vitro Characterization of the Microglial Inflammatory Response to Streptococcus suis,an Important Emerging Zoonotic Agent of Meningitis

Streptococcus suis is an important swine and human pathogen responsible for septicemia and meningitis. In vivo research in mice suggested that in the brain, microglia might be involved in activating the inflammatory response against S. suis. The aim of this study was to better understand the interactions between S. suis and microglia. Murine microglial cells were infected with a virulent wild-type strain of S. suis. Two isogenic mutants deficient at either capsular polysaccharide (CPS) or hemolysin production were also included. CPS contributed to S. suis resistance to phagocytosis and regulated the inflammatory response by hiding proinflammatory components from the bacterial cell wall, while the absence of hemolysin, a potential cytotoxic factor, did not have a major impact on S. suis interactions with microglia. Wild-type S. suis induced enhanced expression of Toll-like receptor 2 by microglial cells, as well as phophotyrosine, protein kinase C, and different mitogen-activated protein kinase signaling events. However, cells infected with the CPS-deficient mutant showed overall stronger and more sustained phosphorylation profiles. CPS also modulated inducible nitric oxide synthase expression and further nitric oxide production from S. suis-infected microglia. Finally, S. suis-induced NF-κB translocation was faster for cells stimulated with the CPS-deficient mutant, suggesting that bacterial cell wall components are potent inducers of NF-κB. These results contribute to increase the knowledge of mechanisms underlying S. suis inflammation in the brain and will be useful in designing more efficient anti-inflammatory strategies for meningitis.Streptococcus suis is one of the most important swine pathogens worldwide, as well as an important agent of zoonosis. So far, 35 serotypes have been described, although serotype 2 is still the most frequently isolated from both swine and humans. In swine, meningitis is the most striking feature of the infection, although other pathologies, such as septicemia, endocarditis, pneumonia, and arthritis, have been described (35). Although the most common pathology associated with S. suis infection in humans is also meningitis, cases of septicemia with septic shock, endocarditis, and several other clinical manifestations have been reported (69, 71). As a zoonosis, S. suis infection has been traditionally considered an occupational hazard, since most cases described in Western countries have occurred in people working in close contact with pigs or raw pork products. The situation in Asian countries is completely different, as the common population is affected. In addition to an important human outbreak in China caused by S. suis in 2005 (71), the pathogen has recently been reported to be the most frequent cause of bacterial meningitis in adults in Vietnam (29) and the third most common culture-confirmed cause of community-acquired bacterial meningitis in Hong Kong. People who survive S. suis infection may be handicapped, as severe postinfection sequels, such as deafness, may develop (67, 68).In recent years, a number of important studies describing the proposed virulence factors of S. suis serotype 2 have been published (4, 29). However, few candidates have been shown to be critical for virulence. Among them, the capsular polysaccharide (CPS) is considered an important antiphagocytic factor (13). Although not essential for virulence, a hemolysin (suilysin) produced by most virulent strains in Eurasia has also been shown to be toxic for cells of murine, human, and swine origin (12, 14, 55, 66).The pathogenesis of S. suis infection has been partially elucidated. In swine, infection occurs through the respiratory route with subsequent colonization of the tonsils, while in humans, access is mainly through skin cuts and/or the oral route (3, 28, 29). Once S. suis reaches the bloodstream, it travels either free or associated with monocytes (27), with invasion of different tissues and organs. The high mortality observed at this stage of the disease may be associated with septic shock with an exacerbated release of proinflammatory cytokines (18, 19). However, if the host overcomes septicemia, S. suis may still invade the central nervous system (CNS) and cause meningitis and, in some cases, encephalitis. The mechanisms used by the pathogen to gain access to the brain and induce local inflammation are still under debate. It is likely that access is by transcytosis and/or toxicity to brain microvascular endothelial cells (66) and/or choroid plexus epithelial cells that are part of the blood brain barrier (BBB) (64). Increase of BBB permeability due to inflammation cannot be ruled out (27).Recently, our laboratory developed an in vivo mouse model of meningitis/encephalitis after S. suis infection via the intraperitoneal route (18). Using this model, an important inflammatory response in the CNS with expression of different proinflammatory genes, including Toll-like receptor 2 (TLR2), CD14, IκBα (an index of NF-κB expression), interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein 1 (MCP-1), was observed. Interestingly, the expression of these genes and bacterial antigens was found to probably be associated with microglia and, to a lesser extent, with astrocytes (18). Microglia, the macrophage-like population within the CNS, represent the first line of defense against invading pathogens and have proinflammatory effector functions (49). Although previous findings draw attention to the implication of these cells in the development of meningitis and encephalitis, it is critical to dissect how the cells initiate key proinflammatory mechanisms in order to respond to S. suis infection. Hence, the goal of this study was to explore the murine microglial response to a virulent strain of S. suis, as well as isogenic mutants defective in either CPS or suilysin production. The ability of microglia to internalize S. suis, to activate TLRs, and to secrete different proinflammatory mediators, as well as to activate important inflammatory intracellular signaling pathways, was evaluated.     

Koala Biovar of Chlamydia pneumoniae Infects Human and Koala Monocytes and Induces Increased Uptake of Lipids In Vitro

We examined the ability of the koala biovar of Chlamydia pneumoniae to infect both Hep-2 cells and human monocytes and the effect of infection on the formation of foam cells. The koala biovar produced large inclusions in both human and koala monocytes and in Hep-2 cells. Koala C. pneumoniae induced foam cell formation with and without added low-density lipoprotein, in contrast to TW183, which produced increased foam cell formation only in the presence of low-density lipoprotein.     

Mycoplasma pneumoniae Extract Induces an IL-17-Associated Inflammatory Reaction in Murine Lung: Implication for Mycoplasmal Pneumonia

Mycoplasma pneumoniae (Mp) may cause immune cell reactions as pivotal aspects of this clinically common respiratory pathogen. Our aim is to determine if Mp extract induces a cellular immune response associated with interleukin (IL)-17, leading to lung inflammation and lung injury. BALB/c mice were immunized with Mp extract intraperitoneally followed by its intratracheal administration, to mimic repeated Mp infection found in humans (repeated inoculation, RI group). Those with a single inoculation were compared as single inoculation group (SI group). Analysis of bronchoalveolar lavage fluid (BALF) demonstrated that keratinocyte-derived cytokine, tumor necrosis factor-α, and IL-6 were produced and peaked on days 0.5 or 1, followed by IL-17 on day 2. Levels of these mediators in BALF were higher in RI group than SI group (P?<?0.05). Further, significantly more neutrophils were recruited to the lungs of the RI group (P?<?0.05). These observations suggest that IL-17 is involved in the prolonged induction of neutrophils in mice treated with Mp extract.     

Migration in Vitro by Blood and Exudate Neutrophils Assessed Serially During an Inflammatory Response

Migration in vitro by blood and inflammatory neutrophils has been compared serially during an inflammatory response. Using an experimental pig model, neutrophils are isolated from the peripheral blood and from the pleural space at hourly intervals after an intrapleural challenge with zymosan activated pig serum (ZAS). Following acepromazine sedation and halothane anesthesia, blood neutrophil migration was transiently reduced. By 1 hour random and directed migration of blood neutrophils returned to normal. Directed and random migration of exudate neutrophils was markedly decreased to both a stimulus-specific (ZAS) and an unrelated (LTB₄) chemoattractant. After 3 hours, migration by exudate neutrophils was similar to migration by blood neutrophils examined in parallel. These findings emphasize the importance of performing serial evaluations of cell function during an inflammatory response.     

Chlamydia pneumoniae Infects and Multiplies in Lymphocytes In Vitro

The obligate intracellular pathogen Chlamydia (Chlamydophila) pneumoniae is known to be associated with some chronic inflammatory diseases, such as atherosclerosis. Interaction between C. pneumoniae and immune cells is important in the development of such diseases. However, susceptibility of immune cells, particularly lymphocytes, to C. pneumoniae infection has not been reported, even though lymphocytes play a pivotal role in the development of the diseases caused by this bacterium. In this regard, we examined the susceptibility of lymphocytes to C. pneumoniae infection in vitro. The results demonstrated that human peripheral blood lymphocytes as well as mouse spleen lymphocytes could be infected with C. pneumoniae. Furthermore, purified T lymphocytes as well as established T-lymphocyte cell line cells showed an obvious susceptibility to C. pneumoniae infection, indicating that T cells could be one of the host cells for this bacterial infection. These findings reveal a new infection site for C. pneumoniae, i.e., lymphocytes.     

Macrophages Restrain Contraction of an In Vitro Wound Healing Model

Significant numbers of macrophages are present during all stages of dermal wound repair, but the functional significance of these macrophages, especially during the later contraction and remodelling stages of repair, remains unclear. We investigated the effect of macrophages on wound contraction using a novel in vitro model based upon the contracting dermal equivalent (DE). Macrophages were found to reversibly restrain DE contraction, a rapid and sustained effect that was enhanced by lipolysaccharide (LPS) treatment of macrophages and partially inhibited by hydrocortisone. Prolonged inhibition of contraction was strongly correlated with an inhibition of fibroblast proliferation. The rapid contraction-inhibiting effect of the macrophages was mediated through activation of protein kinase C (PKC). These results suggest that inflammatory macrophages restrain the later stages of wound repair, namely matrix contraction and remodeling. The novel in vitro model established here provides a useful system for examining fibroblast-macrophage interactions in the healing wound.     

In Vitro Immune Response of SLE Lymphocytes

Peripheral blood lymphocytes from 26 patients with systemic lupus erythematosus (SLE) and six normal individuals were tested for IgG synthesis in the presence or absence of PWM. Lymphocytes from patients with active SLE synthesized increased amounts of IgG in the absence of PWM and reduced amounts of IgG in the presence of PWM. Serum from patients with active SLE had an enhancing effect on the in vitro IgG synthesis of normal lymphocytes. The IgG or F(ab')₂ fractions of SLE serum retained the enhancing effect on in vitro IgG synthesis, and the enhancing activity was absorbed by human spleen cells. As little as 4 h of incubation with SLE serum was needed for the enhancing activity of normal lymphocytes. Treatment of B lymphocytes appeared to be of main importance for an increase in the in vitro IgG synthesis of SLE serum-treated lymphocytes. These results suggest that anti-B-lymphocyte antibodies from patients with active SLE are responsible in part for the hyperactive response of SLE B lymphocytes.     

Development of an Odontoblast In Vitro Model to Study Dentin Mineralization

The aim of the present work was to characterize the odontoblastic proliferation, differentiation, and matrix mineralization in culture of the recently established M2H4 rat cell line. Proliferation was assessed by cell counts, differentiation by RT-PCR analysis, and mineralization by alizarin red staining, atomic absorption spectrometry, and FTIR microspectroscopy. The results showed that M2H4 cell behavior closely mimics in vivo odontoblast differentiation, with, in particular, temporally regulated expression of DMP-1 and DSPP. Moreover, the mineral phase formed by M2H4 cells was similar to that in dentin from rat incisors. Finally, because in mice, transforming growth factor (TGF)-β1 over-expression in vivo leads to an hypomineralization similar to that observed in dentinogenesis imperfecta type II, effects of TGF-β1 on mineralization in M2H4 cell culture were studied. Treatment with TGF-β1 dramatically reduced mineralization, whereas positive control treatment with bone morphogenetic protein-4 enhanced it, suggesting that M2H4 cell line is a promising tool to explore the mineralization mechanisms in physiopathologic conditions.     

Adherence properties of an mrkD-negative mutant of Klebsiella pneumoniae.

The role of the mrkD gene in attachment by a type 3 fimbriate Klebsiella pneumoniae strain was further characterized. A clinical isolate, K. pneumoniae IA565, was found to contain two copies of the gene encoding the fimbrial subunit, mrkA, and one copy of the gene encoding the adhesin subunit, mrkD. One copy of mrkA was located on the bacterial chromosome, and the other copy was associated with mrkD and located on a plasmid. The plasmid-borne mrk gene cluster was lost when K. pneumoniae IA565 was subcultured serially in broth at 44 degrees C. The resulting mrkD-negative strain, designated K. pneumoniae IApc35, did not exhibit the following adherence characteristics associated with K. pneumoniae possessing MrkD-positive fimbriae: agglutination of tannic acid-treated human erythrocytes and attachment to trypsinized human buccal cells. However, K. pneumoniae IApc35 produced type 3 fimbriae that were composed of the characteristic 21.5-kDa major fimbrial subunit, were reactive with specific serum, and were visualized specifically by immunoelectron microscopy. K. pneumoniae IApc35 retained a copy of the mrkA gene on its chromosome. This mrkA-containing gene cluster could be complemented by a recombinant plasmid carrying only the mrkD gene, resulting in restoration of the K. pneumoniae IA565-like adhesive phenotype and demonstration of type 3 filament-associated MrkD subunits by using colloidal gold labeling and immunoelectron microscopy. These data indicate that K. pneumoniae may contain multiple copies of the mrk genes which may be present simultaneously on both plasmid and chromosomal DNAs and which may encode fimbriae with different binding specificities.     

In Vitro Plaque-Forming Cell Responses Induced by Streptococcus pneumoniae in Humans

When peripheral blood lymphocytes (PBL) were stimulated in vitro with the rough form of type 2 Streptococcus pneumoniae R36a, the resulting plaque-forming cells (PFC) did not produce antibodies directed against phosphorylcholine, a major antigenic determinant of the cell wall C-polysaccharide. Instead, R36a stimulated polyclonal PFC in PBL and splenic lymphocytes. We compared the polyclonal responses stimulated by R36a with those induced by two well-characterized polyclonal activators (PA), Staphylococcus aureus Cowan I and pokeweed mitogen (PWM). We found that R36a was a poor mitogen for PBL, whereas the other two PA were potent mitogens; that the predominant isotype produced in response to all three PA was IgM; that adherent cells strongly inhibited the polyclonal PFC response to both R36a and Staph. aureus but not PWM; and that T cells were necessary for induction of polyclonal antibody-secreting cells by all three stimuli.     

Dynamic Mechanical Conditioning of Collagen-Gel Blood Vessel Constructs Induces Remodeling In Vitro

Dynamic mechanical conditioning is investigated as a means of improving the mechanical properties of tissue-engineered blood vessel constructs composed of living cells embedded in a collagen-gel scaffold. This approach attempts to elicit a unique response from the embedded cells so as to reorganize their surrounding matrix, thus improving the overall mechanical stability of the constructs. Mechanical conditioning, in the form of cyclic strain, was applied to the tubular constructs at a frequency of 1 Hz for 4 and 8 days. The response to conditioning thus evinced involved increased contraction and mechanical strength, as compared to statically cultured controls. Significant increases in ultimate stress and material modulus were seen over an 8 day culture period. Accompanying morphological changes showed increased circumferential orientation in response to the cyclic stimulus. We conclude that dynamic mechanical conditioning during tissue culture leads to an improvement in the properties of tissue-engineered blood vessel constructs in terms of mechanical strength and histological organization. This concept, in conjunction with a proper biochemical environment, could present a better model for engineering vascular constructs. © 2000 Biomedical Engineering Society. PAC00: 8719Rr, 8714Ee, 8718-h, 8768+z     

Containment of an outbreak of KPC-3-producing Klebsiella pneumoniae in Italy

From March 2009 to May 2009, 24 carbapenem-resistant Klebsiella pneumoniae isolates were recovered from 16 patients hospitalized in an Italian intensive care unit (ICU). All isolates contained KPC-3 carbapenemase and belonged to a single pulsed-field gel electrophoresis (PFGE) clone of multilocus sequence type 258 (designated as ST258). A multimodal infection control program was put into effect, and the spread of the KPC-3-producing K. pneumoniae clone was ultimately controlled without closing the ICU to new admissions. Reinforced infection control measures and strict monitoring of the staff adherence were necessary for the control of the outbreak.