Intracellular Killing Mechanisms Research Articles

Local immune state in the women with miscarriage

Functional state of immunity provides maintenance of immunological tolerance for allogeneic fetus, and high level of local protection against antigenic stimulation. Increased functional activity of phagocytic cells at systemic and local levels may cause impairment of complete fetoplacental complex, thus leading to abortion. The purpose of our study was to assess the state of local immunity in women with pregnancy loss.
 The study involved 174 women in their reproductive age. The first group consisted of 65 patients (37.4%) with a diagnosis of miscarriage, the history of 2 abortions over the period from conception to the 20th week of gestation. The second group included 37 patients (21.2%) with documented recurrent pregnancy loss, with a history of 3 miscarriages. The control group consisted of 72 conditionally healthy women (41.4%) who had 2 or more term pregnancies with the same partner, without a history of obstetric and gynecological complications. Their mean age was 366 years old. Total number and viability of leukocytes, the indices of functional neutrophil activity, their functional reserve and neutrophil stimulation index were determined in cervical mucosal samples over the first phase of the menstrual cycle.
 In the first phase of menstrual cycle, the women with miscarriage and recurrent pregnancy loss exhibited a statistically significant increase of phagocytosis by the neutrophils from cervical mucus as compared to the group of conditionally healthy women. Intensity of neutrophil phagocytosis in cervical mucus reached higher values in the women with recurrent pregnancy loss more often, compared to the control group. Evaluation of the functional reserve of cervical mucus neutrophils in the subjects with recurrent pregnancy loss and miscarriage showed a trend towards statistically significant differences: this parameter was higher in the patients than in control group. The ability of cervical mucosal neutrophils to produce reactive oxygen species (both spontaneous and induced) did not show statistically significant differences between the patients and controls.
 Hence, we have observed aberrant functional activity of neutrophilic granulocytes from cervical mucosa in the groups of women with 2 or more abortions, without changing ability of the cells to produce reactive oxygen species. This finding may be explained by prevalence of oxygen-independent mechanisms of intracellular killing, thus suggesting a role of neutrophils for impaired balance of immunological tolerance in pregnant women at the local level.

Sensing of viral lung infections by cGAS-STING

Lower respiratory tract infections caused over 4 million deaths per year worldwide, especially in low-income countries. Viral respiratory infections often occur as rapidly spreading seasonal endemic or epidemic, and sometimes due to new respiratory viruses including corona viruses. The first level of host defense against viral infection is based on the innate immune system and intracellular killing mechanisms. The latter is activated by the release of viral DNA or RNA into the cytosol of the infected cells during the initial phase of virus replication. Viral DNA and RNA are recognized by the cyclic guanosine monophosphate (cGMP)-adenosine monophosphate (AMP) synthase (cGAS)–stimulator of interferon (IFN) genes (STING) sensing pathway, leading to the activation of type-I and -III IFN synthesis, with the aim to limit viral replication. However, the efficacy of the cGAS-STING sensing mechanism seems to vary with different viruses, and therefore, so is the efficacy of the host defense mechanism. Viral DNA can be sensed by different proteins including DNA-dependent activator of IFN regulating factor (DAI), cGAS, and toll-like receptor-9 (TLR-9). Viral RNA is recognized by retinoid acid-inducible gene 1 (RIG-1), TLR-7 and TLR-8. The question if cGAS also recognizes viral RNA remains unclear. The activation of IFN synthesis by cGAS is initiated by the recognition of purines and pyrimidines and their enzymatic conversion into cGMP and cyclic AMP (cAMP), followed by the activation of STING. In addition, it is indicated that several viruses can evade the cGAS-STING signaling and escape the host defense. This review aims to summarize the role of cGAS-STING as a host defense mechanism against viral respiratory tract infections.

Triptolide Modulates the Expression of Inflammation-Associated lncRNA-PACER and lincRNA-p21 in Mycobacterium tuberculosis-Infected Monocyte-Derived Macrophages.

Triptolide is a diterpene triepoxide, which performs its biological activities via mechanisms including induction of apoptosis, targeting of pro-inflammatory cytokines, and reshaping of the epigenetic landscape of target cells. However, the targeting of long non-coding RNAs (lncRNAs) by triptolide has not yet been investigated, despite their emerging roles as key epigenetic regulators of inflammation and immune cell function during Mycobacterium tuberculosis (Mtb) infection. Hence, we investigated whether triptolide targets inflammation-associated lncRNA-PACER and lincRNA-p21 and how this targeting associates with Mtb killing within monocyte-derived macrophages (MDMs).Using RT-qPCR, we found that triptolide induced the expression of lincRNA-p21 but inhibited the expression of lncRNA-PACER in resting MDMs in a dose- and time-dependent manner. Moreover, Mtb infection induced the expression of lincRNA-p21 and lncRNA-PACER, and exposure to triptolide before or after Mtb infection led to further increase of Mtb-induced expression of these lncRNAs in MDMs. We further found that contrary to lncRNA-PACER, triptolide time- and dose-dependently upregulated Ptgs-2, which is a proximal gene regulated by lncRNA-PACER. Also, low-concentration triptolide inhibited the expression of cytokine IL-6, a known target of lincRNA-p21. Mtb infection induced the expression of IL-6 and Ptgs-2, and triptolide treatment further increased IL-6 but decreased Ptgs-2 expression in Mtb-infected MDMs. The inverse relation between the expression of these lncRNAs and their target genes is concordant with the conception that these lncRNAs mediate, at least partially, the cytotoxic and/or anti-inflammatory activities of triptolide in both resting and activated MDMs. Using the CFU count method, we found that triptolide decreased the intracellular growth of Mtb HN878. The alamarBlue assay showed that this decreased Mtb HN878 growth was not as a result of direct targeting of Mtb HN878 by triptolide, but rather evoking MDMs’ intracellular killing mechanisms which we speculate could include triptolide-induced enhancement of MDMs’ effector killing functions mediated by lncRNA-PACER and lincRNA-p21. Altogether, these results provide proof of the modulation of lncRNA-PACER and lincRNA-p21 expression by triptolide, and a possible link between these lncRNAs, the enhancement of MDMs’ effector killing functions and the intracellular Mtb-killing activities of triptolide. These findings prompt for further investigation of the precise contribution of these lncRNAs to triptolide-induced activities in MDMs.

Use of Flow Cytometry to Evaluate Phagocytosis of Staphylococcus aureus by Human Neutrophils.

Neutrophils play a key role in the human immune response to Staphylococcus aureus infections. These professional phagocytes rapidly migrate to the site of infection to engulf bacteria and destroy them via specialized intracellular killing mechanisms. Here we describe a robust and relatively high-throughput flow cytometry assay to quantify phagocytosis of S. aureus by human neutrophils. We show that effective phagocytic uptake of S. aureus is greatly enhanced by opsonization, i.e. the tagging of microbial surfaces with plasma-derived host proteins like antibodies and complement. Our rapid assay to monitor phagocytosis can be used to study neutrophil deficiencies and bacterial evasion, but also provides a powerful tool to assess the opsonic capacity of antibodies, either in the context of natural immune responses or immune therapies.

Immunomodulatory Activity of Momordica charantia L. (Cucurbitaceae) Leaf Diethyl Ether and Methanol Extracts on Salmonella typhi-Infected Mice and LPS-Induced Phagocytic Activities of Macrophages and Neutrophils.

Infections due to Salmonella strains constitute one of the major health problems in humans, particularly in Africa. The use of traditional herbs has proven effective in reducing the incidence of infection in some high-risk groups. To assess the effects of Momordica charantia leaf extracts that influence blood infection, an in vitro study of the effect on macrophages and neutrophils and treatment of mouse model of S. typhi infection was done. Methanol and diethyl ether extracts were concerned by this study. In vitro study was to assess the effects of extracts on phagocytosis, and related intracellular killing mechanisms of macrophages were examined. Later, mobilization of leukocytes and production of antibodies against S. typhi were measured followed by quantitating cultures evaluation of the blood infection of orally inoculated mice with S. typhi. Ingestion or attachment of carbon particles, production of superoxide anion, nitric oxide, and that of lysosomal acid phosphatase by macrophages and neutrophils were significantly increased by methanol and diethyl extracts at concentrations ranging from 40 μg/ml to 640 μg/ml. Antibody titer and mobilization of leukocytes, particularly lymphocytes against S. typhi, were highly increased by both methanol and diethyl extracts at concentrations of 500 and 1000 mg/kg. At the same time, the extracts have reduced the rate of blood colonization in mice inoculated with 108 CFU of S. typhi for 28 days. Reduction in blood colonization or infection rates was similar for the levamisole mice group. The results of this study should prove that the leaves of Momordica charantia are useful for the treatment of infections by Salmonella strains and for the assessment of drugs for therapeutic intervention.

Lacking catalase, a protistan parasite draws on its photosynthetic ancestry to complete an antioxidant repertoire with ascorbate peroxidase

BackgroundAntioxidative enzymes contribute to a parasite’s ability to counteract the host’s intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite’s life history?ResultsPurification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site.ConclusionsPmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.

Structure/Function Analysis of a Novel Myeloperoxidase Inhibitor from Staphylococcus delphini

Staphylococcus aureus and closely related species are highly adapted to their hosts and have evolved many strategies to resist opsonization and phagocytosis. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium can actively evade specific intracellular killing mechanisms used by neutrophils. We recently discovered a highly conserved S.aureus protein (SPIN for Staphylococcal Peroxidase INhibitor) that specifically binds and inhibits myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils [de Jong et al., PNAS, 2017]. S. aureus cells deleted for SPIN gene showed decreased survival compared to wild‐type bacteria after phagocytosis by neutrophils. A crystal structure of SPIN bound to MPO suggests that it acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. To further our understanding of the structure/function relationships of SPIN proteins, we cloned, overexpressed, purified, and characterized a series of SPIN orthologs of Staphylococcal origin.We found that SPIN originating from S. delphini although shares only ~53% identity to SPIN‐aureus, it still retained a strong affinity and inhibitory capacity for human MPO. A 2.4 Å crystal structure of SPIN‐delphini bound to human MPO provided a basis for comparing the similarities and differences between SPIN‐aureus and SPIN‐delphini. Together, these studies shed light on the molecular features which determine the specificity of SPIN proteins for MPO, and suggest potential avenues for using this information toward the design of synthetic MPO inhibitors.Support or Funding InformationNIH Grants AI111203 and GM121511This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and structural characterization of a novel myeloperoxidase inhibitor from Staphylococcus delphini

Staphylococcus aureus and related species are highly adapted to their hosts and have evolved numerous strategies to evade the immune system. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium evades intracellular killing mechanisms used by neutrophils. We recently discovered an S. aureus protein (SPIN for Staphylococcal Peroxidase INhibitor) that binds to and inhibits myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. To allow for comparative studies between multiple SPIN sequences, we identified a panel of homologs from species closely related to S. aureus. Characterization of these proteins revealed that SPIN molecules from S. agnetis, S. delphini, S. schleiferi, and S. intermedius all bind human MPO with nanomolar affinities, and that those from S. delphini, S. schleiferi, and S. intermedius inhibit human MPO in a dose-dependent manner. A 2.4 Å resolution co-crystal structure of SPIN-delphini bound to recombinant human MPO allowed us to identify conserved structural features of SPIN proteins, and to propose sequence-dependent physical explanations for why SPIN-aureus binds human MPO with higher affinity than SPIN-delphini. Together, these studies expand our understanding of MPO binding and inhibition by a recently identified component of the staphylococcal innate immune evasion arsenal.

Priming of innate antimycobacterial immunity by heat-killed Listeria monocytogenes induces sterilizing response in the adult zebrafish tuberculosis model.

ABSTRACTMycobacterium tuberculosis remains one of the most problematic infectious agents, owing to its highly developed mechanisms to evade host immune responses combined with the increasing emergence of antibiotic resistance. Host-directed therapies aiming to optimize immune responses to improve bacterial eradication or to limit excessive inflammation are a new strategy for the treatment of tuberculosis. In this study, we have established a zebrafish-Mycobacterium marinum natural host-pathogen model system to study induced protective immune responses in mycobacterial infection. We show that priming adult zebrafish with heat-killed Listeria monocytogenes (HKLm) at 1 day prior to M. marinum infection leads to significantly decreased mycobacterial loads in the infected zebrafish. Using rag1−/− fish, we show that the protective immunity conferred by HKLm priming can be induced through innate immunity alone. At 24 h post-infection, HKLm priming leads to a significant increase in the expression levels of macrophage-expressed gene 1 (mpeg1), tumor necrosis factor α (tnfa) and nitric oxide synthase 2b (nos2b), whereas superoxide dismutase 2 (sod2) expression is downregulated, implying that HKLm priming increases the number of macrophages and boosts intracellular killing mechanisms. The protective effects of HKLm are abolished when the injected material is pretreated with nucleases or proteinase K. Importantly, HKLm priming significantly increases the frequency of clearance of M. marinum infection by evoking sterilizing immunity (25 vs 3.7%, P=0.0021). In this study, immune priming is successfully used to induce sterilizing immunity against mycobacterial infection. This model provides a promising new platform for elucidating the mechanisms underlying sterilizing immunity and to develop host-directed treatment or prevention strategies against tuberculosis.This article has an associated First Person interview with the first author of the paper.

Immunoevasion and immunosuppression of the macrophage by Mycobacterium tuberculosis.

By virtue of their position at the crossroads between the innate and adaptive immune response, macrophages play an essential role in the control of bacterial infections. Paradoxically, macrophages serve as the natural habitat to Mycobacterium tuberculosis (Mtb). Mtb subverts the macrophage's mechanisms of intracellular killing and antigen presentation, leading ultimately to the development of tuberculosis (TB) disease. Here, we describe mechanisms of Mtb uptake by the macrophage and address key macrophage functions that are targeted by Mtb-specific effector molecules enabling this pathogen to circumvent host immune response. The macrophage functions described in this review include fusion between phagosomes and lysosomes, production of reactive oxygen and nitrogen species, antigen presentation and major histocompatibility complex class II expression and trafficking, as well as autophagy and apoptosis. All these are Mtb-targeted key cellular pathways, normally working in concert in the macrophage to recognize, respond, and activate 'proper' immune responses. We further analyze and discuss major molecular interactions between Mtb virulence factors and key macrophage proteins and provide implications for vaccine and drug development.

Alveolar macrophages in pulmonary host defence – the unrecognized role of apoptosis as a mechanism of intracellular bacterial killing

Alveolar macrophages play an essential role in clearing bacteria from the lower airway, as the resident phagocyte alveolar macrophages must both phagocytose and kill bacteria, and if unable to do this completely must co-ordinate an inflammatory response. The decision to escalate the inflammatory response represents the transition between subclinical infection and the development of pneumonia. Alveolar macrophages are well equipped to phagocytose bacteria and have a large phagolysosomal capacity in which ingested bacteria are killed. The rate-limiting step in control of extracellular bacteria, such as Streptococcus pneumoniae, is the capacity of alveolar macrophages to kill ingested bacteria. Therefore, alveolar macrophages complement canonical microbicidal strategies with an additional level of apoptosis-associated killing to help kill ingested bacteria.

Phagosomes Induced by Cytokines Function as anti-Listeria Vaccines

Phagosomes are critical compartments for innate immunity. However, their role in the protection against murine listeriosis has not been examined. We describe here that listericidal phago-receptosomes are induced by the function of IFN-γ or IL-6 as centralized compartments for innate and adaptive immunity because they are able to confer protection against murine listeriosis. These phago-receptosomes elicited LLO(91-99)/CD8(+)- and LLO(189-201)/CD4(+)-specific immune responses and recruited mature dendritic cells to the vaccination sites controlled by T cells. Moreover, they present exceptional features as efficient vaccine vectors. First, they compartmentalize a novel listericidal STAT-1-mediated signaling pathway that confines multiple innate immune components to the same environment. Second, they show features of MHC class II antigen-loading competent compartments for cathepsin-D-mediated LLO processing. Third, murine cathepsin-D deficiencies fail to develop protective immunity after vaccination with listericidal phago-receptosomes induced by IFN-γ or IL-6. Therefore, it appears that the connection of STAT-1 and cathepsin-D in a single compartment is relevant for protection against listeriosis.

The intracellular lifestyle of Francisella noatunensis in Atlantic cod ( Gadus morhua L.) leucocytes

Francisella noatunensis causes the systemic granulomatous inflammatory disease, francisellosis in cod. Little is known about the lifestyle of this facultative intracellular bacterium within cod leucocytes. We have examined the interaction of this bacterium with phagocytic cells isolated from cod with emphasis on monocytes, macrophages, neutrophils and phagocytic B-cells. It is clear from confocal microscopy sections through adherent cell preparations that numerous bacteria were located intracellularly following in vitro infection in monocytes and macrophages. In these sections bacteria were immunostained and cell actin was stained using Alexa Fluor ® 488 phalloidin. Bacteria were observed in close association with neutrophils and intracellularly (low numbers) in B-cells. Bacteria were observed more frequently in head kidney- than in peripheral blood- and spleen- leucocytes. Following infection, bacteria were initially observed grouped together and located close to the nucleus. Later they were found spread within the cytoplasm. This indicates egression of F. noatunensis from the phagosome to the cytoplasm where replication possibly takes place. It may be hypothesised that the bacteria may alter maturation of the phagosome and thus, avoid the potent intracellular killing mechanisms of phagocytic cells. The intracellular lifestyle involving escape to cytoplasm prior to fusion with the lysosome may have consequences for vaccine development as well as antibiotic treatment of infected cod.

Two recombinant peptides, SpStrongylocins 1 and 2, from Strongylocentrotus purpuratus, show antimicrobial activity against Gram-positive and Gram-negative bacteria

The cysteine-rich strongylocins were the first antimicrobial peptides (AMPs) discovered from the sea urchin species, Strongylocentrotus droebachiensis. Homologous putative proteins (called SpStrongylocin) were found in the sister species, S. purpuratus. To demonstrate that they exhibit the same antibacterial activity as strongylocins, cDNAs encoding the ‘mature’ peptides (SpStrongylocins 1 and 2) were cloned into a direct expression system fusing a protease cleavage site and two purification tags to the recombinant peptide. Both recombinant fusion peptides were expressed in a soluble form in an Escherichia coli strain tolerant to toxic proteins. Enterokinase was used to remove the fusion tags and purified recombinant SpStrongylocins 1 and 2 showed antimicrobial activity against both Gram-negative and Gram-positive bacteria. The results of membrane integrity assays against cytoplasmic membranes of E. coli suggest that both recombinant SpStrongylocins 1 and 2 conduct their antibacterial activity by intracellular killing mechanisms because no increase in membrane permeability was detected.

Differential mechanisms of intracellular killing of Mycobacterium avium and Listeria monocytogenes by activated human and murine macrophages. The role of nitric oxide.

Murine peritoneal macrophages activated with interferon-gamma (IFN-gamma) produce large quantities of nitric oxide and are efficient in the killing of certain intracellular pathogens. To examine the role of this mechanism in the killing of Mycobacterium avium by murine and human macrophages, we infected mouse peritoneal macrophages and human monocyte-derived macrophages with M. avium and Listeria monocytogenes and stimulated the cells with recombinant tumour necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF) or IFN-gamma, in the presence or absence of N-monomethyl-L-arginine (NMA) or arginase. Neither competitive inhibition with NMA nor depletion of arginine by arginase had any effect on the inhibition of growth/intracellular killing of M. avium by activated human and murine macrophages. In contrast, activation of murine but not human macrophages infected with L. monocytogenes by IFN-gamma was significantly inhibited by the addition of NMA/arginase. Furthermore, murine macrophages produced large concentrations of nitric oxide following stimulation with recombinant cytokines, although no significant increase of nitric oxide production was observed with human monocyte-derived macrophages.

Innate and Adaptive Immunity to Francisella

Studies of immune responses to Francisella have been conducted for well over 50 years. Here, the basic parameters of innate and adaptive immune responses to Francisella are reviewed, with an emphasis on those that may contribute directly to protection against infection. Although older literature provides a wealth of information on human immune responses to infection and vaccination, most recent information has been derived largely from studies in animals and using animal cells, particularly mice. In experimental animals, activation of macrophages, a major and probably preferred host cell for Francisella, appears central to control of infection. Thus, in animal models and in vitro studies using mouse macrophages, cytokines such as IFN-gamma and TNF-alpha, derived first from both nonspecific cells such as natural killer cells and later from Francisella-specific T cells, collaborate to effect intracellular killing. In mice, these intracellular killing mechanisms include reactive nitrogen and oxygen species, but killing mechanisms remain to be identified in humans. Ultimately both CD4(+) and CD8(+) T cells develop into Francisella-specific memory cells and are important for control of primary Francisella infection or vaccination-induced protection. The effector mechanisms invoked by either CD4(+) or CD8(+) T cells, beyond production of IFN-gamma and TNF-alpha, are the subject of ongoing studies. Both specific antibodies and B cells may contribute to control of primary infection or vaccination-induced protection in some circumstances, particularly against lower virulence Francisella strains. Thus a number of known proinflammatory and Th-1 T cell related components of the immune system combat this virulent bacterium; no doubt others remain to be discovered.

Extraintestinal PathogenicEscherichia coliSurvives within Neutrophils

Extracellular pathogenic Escherichia coli (ExPEC) strains are common causes of a variety of clinical syndromes, including urinary tract infections, abdominal infections, nosocomial pneumonia, neonatal meningitis, and sepsis. ExPEC strains are extracellular bacterial pathogens; therefore, the innate immune response (e.g., professional phagocytes) plays a crucial role in the host defense against them. Studies using the model ExPEC strain CP9 demonstrated that it is relatively resistant to neutrophil-mediated bactericidal activity. Although this could be due to resistance to phagocytosis, the ability of CP9 to survive the intracellular killing mechanisms of neutrophils is another possibility. Using a variation of the intracellular invasion assay, we studied the survival of CP9 within peripheral blood-derived human neutrophils. Our results indicated that CP9 did survive within human neutrophils, but we were unable to demonstrate that intracellular replication occurred. This finding was not unique to CP9, since when a conservative assessment of survival was used, four of six additional ExPEC strains, but not an E. coli laboratory strain, were also capable of survival within neutrophils. Initial studies in which we began to decipher the mechanisms by which CP9 is able to successfully survive intracellular neutrophil-mediated bactericidal activity demonstrated that CP9 was at least partially susceptible to the neutrophil oxidative burst. Therefore, absolute resistance to the oxidative burst is not a mechanism by which ExPEC survives within neutrophils. In addition, electron microscopy studies showed that CP9 appeared to be present in phagosomes within neutrophils. Therefore, avoidance of phagosomal uptake or subsequent escape from the phagosome does not appear to be a mechanism that contributes to CP9's survival. These findings suggest that survival of ExPEC within neutrophils may be an important virulence mechanism.

A Correlation between Phagocytosis and Apoptosis in THP‐1 Cells Infected with Prevalent Strains of Mycobacterium tuberculosis

The innate ability of infected macrophages to undergo programmed cell death (apoptosis) and curtail the infection is crucial for the host defense. Although phagocytosis and intracellular killing mechanisms leading to apoptosis in macrophages are highly effective in eliminating the infecting tuberculous bacilli, some Mycobacterium tuberculosis(Mtb) strains have evolved strategies to inhibit this microbicidal function and make use of macrophage for its successful and prolonged survival. Two clinical strains of Mtb (S7 and S10) found to be prevalent and primitive, based on molecular epidemiological studies, were used to study the magnitude in induction of apoptosis in THP-1 cells at various time points of infection and to correlate it with phagocytosis. The percentage of phagocytosis did not show any strain-specific association with differentiated THP-1 cells. But in the phagocytic index, the clinical strains showed a low dose of infection in the 1-10 bacilli category thereby exerting less burden on the cells. The induction of apoptosis was strain dependent. The THP-1 cells infected with H37Ra and S10 showed an increase in apoptosis at all time points while the S7 strain induced minimum apoptosis. A negative correlation between apoptosis and phagocytic index was observed in the 1-10 category and a positive correlation in the > 20 category of the phagocytic index. This novel observation indicates that the magnitude of THP-1 cell apoptosis is a function of the number of internalized mycobacteria. These results indicated a differential mode of infection by clinical strains and their adaptation to different survival strategies that may lead to immune suppression and pathogenesis of the disease.

Intracellular survival of Neisseria gonorrhoeae in male urethral epithelial cells: importance of a hexaacyl lipid A.

Neisseria gonorrhoeae is a strict human pathogen that invades and colonizes the urogenital tracts of males and females. Lipooligosaccharide (LOS) has been shown to play a role in gonococcal pathogenesis. The acyl transferase MsbB is involved in the biosynthesis of the lipid A portion of the LOS. In order to determine the role of an intact lipid A structure on the pathogenesis of N. gonorrhoeae, the msbB gene was cloned and sequenced, a deletion and insertion mutation was introduced into N. gonorrhoeae, and the mutant strain was designated 1291A11K3. Mass spectrometric analyses of 1291A11K3 LOS determined that this mutation resulted in a pentaacyl rather than a hexaacyl lipid A structure. These analyses also demonstrated an increase in the phosphorylation of lipid A and an increase in length of the oligosaccharide of a minor species of the msbB LOS. The interactions of this mutant with male urethral epithelial cells (uec) were examined. Transmission and scanning electron microscopy studies indicated that the msbB mutants formed close associations with and were internalized by the uec at levels similar to those of the parent strain. Gentamicin survival assays performed with 1291A11K3 and 1291 bacteria demonstrated that there was no difference in the abilities of the two strains to adhere to uec; however, significantly fewer 1291A11K3 bacteria than parent strain bacteria were recovered from gentamicin-treated uec. These studies suggest that the lipid A modification in the N. gonorrhoeae msbB mutant may render it more susceptible to innate intracellular killing mechanisms when internalized by uec.

Personality factors, stress and immunity

Stress has been shown to have an association with immune changes. In this study, the effects of examination stress were studied in a group of 10 postgraduate students. Parameters measured included the seriousness of illness rating scale, the test of trait anxiety and the subjective appraisal of stressfulness of exams. Immunological parameters assessed included full blood counts, enumeration of lymphocytes and their subsets; lymphocyte proliferative responses to mitogens (phytohaemagglutinin, concavalin A, pokeweed mitogen) and antigen (purified protein derivative); neutrophil phagocytosis; intracellular killing ability and oxidative mechanisms of intracellular killing by assessing the reduction of nitroblue tetrazolium (NBT). Biochemical parameters assessed included glucose, cortisol, TSH, prolactin, ACTH and DHEA levels in serum. The results suggest a positive association between phagocyte functions (early phagocytosis and NBT reduction) and both trait anxiety and the perception of threat. Decreased lymphocyte proliferation in response to mitogens and antigen was associated with trait anxiety. These results suggest that students who are anxious and see exams as a threat display more efficient phagocyte functions, but decrease proliferation of their lymphocytes at a time of stress. Copyright © 2001 John Wiley & Sons, Ltd.