Concanavalin a capping of rhesus monkey polymorphonuclear leukocytes

The technique of capping, a probe designed to evaluate the fluidity and functional competence of human polymorphonuclear leukocytes (PMNs), has been successfully adapted for rhesus monkey PMNs. The capping characteristics of rhesus monkey PMNs are very similar to those of human PMNs. Utilizing this capping technique as an evaluation tool and with fetal/neonatal rhesus monkey as a functional animal model, the ontogeny of movement and chemotactic characteristics of PMNs can now be studied.     

Effect of pentoxifylline on developmental changes in neutrophil cell surface mobility and membrane fluidity

Polymorphonuclear leukocytes (PMNs) from human neonates respond less efficiently to chemotactic factor stimulation than do PMNs from adults. The biologic mechanisms underlying this developmental process are poorly understood. In previous studies, we have found that pentoxifylline, an agent report to enhance membrane deformability, increased the chemotactic response of neonatal PMNs. In the present studies, we have examined the effect of pentoxifylline on cell surface mobility and membrane fluidity by assessing fluorescent concanavalin A (Con A) capping and fluorescent polarization (FP). Baseline Con A capping was lower in the PMNs of neonates when compared to PMNs from adult controls. Colchicine, which increases capping by disrupting microtubules, exaggerated the differences between the adult and neonatal PMNs. Following exposure of neonatal PMNs to pentoxifylline, colchicine enhanced Con A capping to levels equivalent to those of colchicine-treated PMNs from adults. Employing a fluorescence polarization (FP) assay, we found the fluid state of the membrane of PMNs from neonates was significantly less than that of adult controls. Pentoxifylline alone significantly increased the fluidity of the cell membranes of neonatal PMNs while decreasing elevated basal levels of F-actin in the cell. These data suggest an intrinsic cytoskeletal difference in the PMNs of neonates that may be responsive to pharmacologic manipulation.     

Candida albicans‐associated sepsis in a pre‐term neonatal rhesus macaque (Macaca mulatta)

Invasive Candida infections (ICI) have been associated with neurodevelopmental impairment or death in human pre‐term neonates. Candidiasis in nonhuman primates is seen mostly in immunosuppressed animals, and ICI is not commonly reported. Here, we report a case of Candida albicans‐associated ICI in a pre‐term neonatal rhesus macaque.     

Beta-glucan is a fungal determinant for adhesion-dependent human neutrophil functions

Candida albicans is a common cause of nosocomial infections whose virulence depends on the reversible switch from blastoconidia to hyphal forms. Neutrophils (or polymorphonuclear leukocytes (PMNs)) readily clear blastoconidia by phagocytosis, but filaments are too long to be ingested. Mechanisms regulating immune recognition and response to filamentous fungal pathogens are not well understood, although known risk factors for developing life-threatening infections are neutropenia or defects in the NADPH oxidase system. We show human PMNs generate a respiratory burst response to unopsonized hyphae. Ab specific for beta-glucan, a major component of yeast cell walls, blocks this response, establishing beta-glucan as a key molecular pattern recognized by PMNs in response to C. albicans. This study also elucidates recognition and signaling mechanisms used by PMNs in response to beta-glucan under conditions where phagocytosis cannot occur. Human PMNs adhered to immobilized beta-glucan and released an efficient plasma membrane respiratory burst. Ab blockade of the integrin complement receptor 3 (CD11b/CD18) significantly inhibited both of these functions. Furthermore, we show a role for p38 MAPK and actin but not protein kinase C zeta in generating the respiratory burst to beta-glucan. Taken together, results show that beta-glucan in C. albicans hyphae is accessible to PMNs and sufficient to support an innate immune response.     

Public Health Risks of Multiple-Drug-Resistant Enterococcus spp. in Southeast Asia

Enterococci rank as one of the leading causes of nosocomial infections, such as urinary tract infections, surgical wound infections, and endocarditis, in humans. These infections can be hard to treat because of the rising incidence of antibiotic resistance. Enterococci inhabiting nonhuman reservoirs appear to play a critical role in the acquisition and dissemination of antibiotic resistance determinants. The spread of antibiotic resistance has become a major concern in both human and veterinary medicine, especially in Southeast Asia, where many developing countries have poor legislation and regulations to control the supply and excessive use of antimicrobials. This review addresses the occurrence of antibiotic-resistant enterococci in Association of Southeast Asian Nations countries and proposes infection control measures that should be applied to limit the spread of multiple-drug-resistant enterococci.     

Neonatal immunology and cord blood transplantation

The increased susceptibility of human newborns to infections is usually ascribed to the immaturity of the neonatal immune system. The neonatal immune system has never met microbial antigens, and thus the repertoire of its adaptative arm (T and B cells) is entirely pre-immune, or "na?ve". However this neonatal pre-immune repertoire is similar to the adult pre-immune repertoire, and cord blood natural killer cells studies show that the innate immunity cells harbor the full killing machinery that characterize mature cells. Moreover, human neonates are able to show an adult-like allogeneic response. Taken together, several lines of evidence suggest that the neonatal immune system, although na?ve, is fully mature. However, newborns display phenotypic and functional differences with adults in both adaptative and innate arms. Specific properties may explain these differences, as high number of regulatory T cells, low plasmacytoid dendritic cell response to stimuli and high IL-10 production. These properties are in line with the high susceptibility of newborns to infections and the low incidence of graft-versus-host-disease after cord blood transplantation. To explain these differences, we introduce a new model. Although naive, the neonatal immune system is mature, and these functional differences are due to a message originating from the placenta and aimed at inducing the foetus tolerance to its mother. Full understanding of the involved mechanisms will help to protect the newborn against infections and to improve cord blood transplantation outcome.     

Functional expression of IL-9 receptor by human neutrophils from asthmatic donors: role in IL-8 release

Human polymorphonuclear neutrophils (PMNs) express surface receptors for various inflammatory mediators, including IgE and IL-4. Recently, the IL-9R locus has been genetically linked to asthma and bronchial hyperresponsiveness in humans. In this study, we evaluated expression of the IL-9R and the effect of IL-9 on human PMNs. RT-PCR analysis showed the presence of IL-9Ralpha-chain mRNA in PMN RNA preparations from asthmatic patients. Using FACS analysis, surface expression of IL-9Ralpha was detected on PMNs freshly isolated from asthmatics, and to a lesser extent on normal controls. In addition, protein expression of IL-9Ralpha was also detected in peripheral blood and bronchoalveolar lavage PMNs. Furthermore, functional studies showed that IL-9 stimulation of PMNs results in the release of IL-8 in a concentration-dependent manner. The anti-IL-9 neutralizing Ab suppressed this effect, but had no effect on GM-CSF-induced IL-8 release from PMNs. Taken together, these findings suggest a novel role for PMNs in allergic disease through the expression and activation of the IL-9R.     

A quantitative colorimetric method to evaluate the functional state of human polymorphonuclear leukocytes

The colorimetric assay previously described by Mosmann for the measurement of cell viability and proliferation has been modified for the assessment of the functional state of human polymorphnuclear cells (PMNs). The ability of PMNs to reduce the tetrazolium salt MTT to formazan reflects directly the degree of stimulation induced by various agents. The underlying mechanism of MTT-reduction to formazan seems to be similar to that of nitroblue tetrazolium (NBT)-reduction. In contrast to the NBT-reduction assay, the formazan produced from MTT can easily be measured by an ELISA reader. Parallel experiments revealed a qualitative correlation between the concentration of formazan produced from MTT and the concentration of cytochrome C reduced by PMNs. Although oxidative burst may not be the actual lytic mechanism in cellular cytotoxicity of PMN, we also observed an association between MTT-reduction capacity and the cytotoxic activity of PMNs from normal donors in antibody dependent cellular cytotoxicity. Our results indicate that the MTT-reduction assay can be employed to estimate the functional state of polymorphnuclear granulocytes.     

Partial myeloperoxidase deficiency in preleukemia

In seven subjects with partial and apparently acquired form of myeloperoxidase (MPO) deficiency, some functional properties of neutrophils (PMNs) were studied. Five patients suffered from preleukemia, one from diabetes mellitus and one from carcinoma of the breast with bone marrow metastases. Intracellular bactericidal activity, oxygen consumption and superoxide radical production were within normal limits. In three patients with preleukemia, the serum opsonic activity was markedly reduced (less than m-3SD) in an autologous system, but normal in the presence of pooled normal serum. Decreased opsonic activity was also found when these patient's sera were assayed in the presence of normal PMNs. Since the levels of IgG and C3 were comparable in the patients' sera and the pooled serum, a deficiency of another unknown opsonin or the presence of an opsonization inhibitor has to be postulated. The partial MPO defect apparently doesn't decrease the intracellular killing of Staphylococcus aureus by PMNs. The known susceptibility to bacterial infections in preleukemia may be explained by the reduction of serum opsonization conducing to a secondary decrease of the ingestion and killing of bacteria by the PMNs.     

Human neutrophils as a source of nociceptin: a novel link between pain and inflammation

Nociceptin is a neuropeptide sharing sequence homology with classical opioid peptides but with a distinct pharmacological profile. Through activation of its receptor, NociR, nociceptin has been linked with several physiological functions in the central nervous system including memory, locomotion, and processing of pain signals. Recently, peripheral blood neutrophils (PMNs) were demonstrated to express a functional NociR, a result suggesting that additional functions of the neuropeptide remain to be elucidated. The present study investigated the possibility that PMNs may be a source of nociceptin and whether the neuropeptide elicits PMN early responses. We observed the presence of nociceptin in the synovial fluids from arthritic patients, an inflammatory milieu typically containing high numbers of PMNs. In addition, freshly isolated PMNs were found to express and secrete nociceptin following degranulation, identifying these inflammatory cells as a novel source of the neuropeptide. Incubation of PMNs with nociceptin elicited a specific pattern of cellular protein phosphorylation on tyrosine residues in a rapid and transient fashion. Moreover, nociceptin prevented intracellular accumulation of cAMP in fMLP-stimulated PMNs, an effect mimicked by the specific NociR synthetic agonist, Ro 64-6198. Taken together, these results show that nociceptin/NociR is present and functional in human neutrophils, and the results identify a novel dialogue pathway between neural and immune tissues.     

Effects of interferon-γ and granulocyte colony-stimulating factor on antifungal activity of human polymorphonuclear neutrophils against Candida albicans grown as biofilms or planktonic cells

Candida albicans is a leading cause of biofilm-related infections. As Candida biofilms are recalcitrant to host defenses, we sought to determine the effects of interferon-γ and granulocyte colony-stimulating factor, two pro-inflammatory cytokines, on the antifungal activities of human polymorphonuclear neutrophils (PMNs) against C. albicans biofilms, using an in vitro biofilm model. Priming of PMNs by these cytokines augmented fungal damage of planktonic cells; however, priming of PMNs did not have the same effect against Candida biofilms. Biofilm phenotype appears to play an important role in protecting C. albicans from the innate immune system.     

Impaired phagosomal maturation in neutrophils leads to periodontitis in lysosomal-associated membrane protein-2 knockout mice

Inflammatory periodontal diseases constitute one of the most common infections in humans, resulting in the destruction of the supporting structures of the dentition. Circulating neutrophils are an essential component of the human innate immune system. We observed that mice deficient for the major lysosomal-associated membrane protein-2 (LAMP-2) developed severe periodontitis early in life. This development was accompanied by a massive accumulation of bacterial plaque along the tooth surfaces, gingival inflammation, alveolar bone resorption, loss of connective tissue fiber attachment, apical migration of junctional epithelium, and pathological movement of the molars. The inflammatory lesions were dominated by polymorphonuclear leukocytes (PMNs) apparently being unable to efficiently clear bacterial pathogens. Systemic treatment of LAMP-2-deficient mice with antibiotics prevented the periodontal pathology. Isolated PMNs from LAMP-2-deficient mice showed an accumulation of autophagic vacuoles and a reduced bacterial killing capacity. Oxidative burst response was not altered in these cells. Latex bead and bacterial feeding experiments showed a reduced ability of the phagosomes to acquire an acidic pH and late endocytic markers, suggesting an impaired fusion of late endosomes-lysosomes with phagosomes. This study underlines the importance of LAMP-2 for the maturation of phagosomes in PMNs. It also underscores the requirement of lysosomal fusion events to provide sufficient antimicrobial activity in PMNs, which is needed to prevent periodontal disease.     

Enterococcus faecalis: specific and non-specific interactions with human polymorphonuclear leukocytes

In previous studies we have demonstrated that the ability of Enterococcus faecalis to adhere to and to be internalized in human urinary tract epithelial cells, Girardi Heart cells and human polymorphonuclear leukocytes (PMNs), was dependent on whether the strain had been isolated from urinary tract infections (UTI) or endocarditis (EN) respectively. These properties were further modified by growth of the organism in human serum. In the present report, using competition assays we show that adhesins containing a D-glucose moiety play a role in mediating the interactions between human PMNs and E. faecalis strains isolated from UTI and grown in brain-heart infusion broth (BHIB). On the other hand, adhesins containing both D-glucose and D-galactose moieties were involved in the interactions between PMNs and serum grown UTI isolates or EN isolates grown in either BHIB or human serum. Moreover, the impairment in the association between both UTI and EN strains after growth in serum appears to be at least partially related to a decrease in enterococcal surface hydrophobicity.     

Multifaceted freezing injury in human polymorphonuclear cells at high subfreezing temperatures

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.     

Pathogenic Neisseria hitchhike on the uropod of human neutrophils

Polymorphonuclear neutrophils (PMNs) are important components of the human innate immune system and are rapidly recruited at the site of bacterial infection. Despite the effective phagocytic activity of PMNs, Neisseria gonorrhoeae infections are characterized by high survival within PMNs. We reveal a novel type IV pilus-mediated adherence of pathogenic Neisseria to the uropod (the rear) of polarized PMNs. The direct pilus-uropod interaction was visualized by scanning electron microscopy and total internal reflection fluorescence (TIRF) microscopy. We showed that N. meningitidis adhesion to the PMN uropod depended on both pilus-associated proteins PilC1 and PilC2, while N. gonorrhoeae adhesion did not. Bacterial adhesion elicited accumulation of the complement regulator CD46, but not I-domain-containing integrins, beneath the adherent bacterial microcolony. Electrographs and live-cell imaging of PMNs suggested that bacterial adherence to the uropod is followed by internalization into PMNs via the uropod. We also present data showing that pathogenic Neisseria can hitchhike on PMNs to hide from their phagocytic activity as well as to facilitate the spread of the pathogen through the epithelial cell layer.     

Clades of Adeno-associated viruses are widely disseminated in human tissues

The potential for using Adeno-associated virus (AAV) as a vector for human gene therapy has stimulated interest in the Dependovirus genus. Serologic data suggest that AAV infections are prevalent in humans, although analyses of viruses and viral sequences from clinical samples are extremely limited. Molecular techniques were used in this study to successfully detect endogenous AAV sequences in 18% of all human tissues screened, with the liver and bone marrow being the most predominant sites. Sequence characterization of rescued AAV DNAs indicated a diverse array of molecular forms which segregate into clades whose members share functional and serologic similarities. One of the most predominant human clades is a hybrid of two previously described AAV serotypes, while another clade was found in humans and several species of nonhuman primates, suggesting a cross-species transmission of this virus. These data provide important information regarding the biology of parvoviruses in humans and their use as gene therapy vectors.     

The ontogeny and phylogeny of children’s object and fantasy play

We examine the ontogeny and phylogeny of object and fantasy play from a functional perspective. Each form of play is described from an evolutionary perspective in terms of its place in the total time and energy budgets of human and nonhuman juveniles. As part of discussion of functions of play, we examine sex differences, particularly as they relate to life in the environment of evolutionary adaptedness and economic activities of human and nonhuman primates. Object play may relate to foraging activities. Although fantasy play has been viewed as limited to humans, we speculate that certain types of fantasy play may be present in some nonhuman primates. Fantasy play may enable juveniles to see situations from different perspectives. We conclude that fantasy play may have immediate effects and object play may have deferred effects.     

Gsdma3 is required for hair follicle differentiation in mice

Apoptosis can result from activation of three major pathways: the extrinsic, the intrinsic, and the most recently identified endoplasmic reticulum (ER) stress-mediated pathway. While the two former pathways are known to be operational in human polymorphonuclear neutrophils (PMNs), the existence of the ER stress-mediated pathway, generally involving caspase-4, has never been reported in these cells. Recently, we have documented that arsenic trioxide (ATO) induced apoptosis in human PMNs by a mechanism that needs to be further investigated. In this study, using immunofluorescence and electron microscopy, we present evidence of ER alterations in PMNs activated by the ER stress inducer arsenic trioxide (ATO). Several key players of the unfolded protein response, including GRP78, GADD153, ATF6, XBP1 and eIF2α are expressed and activated in PMNs treated with ATO or other ER stress inducers. Although caspase-4 is expressed and activated in neutrophils, treatment with a caspase-4 inhibitor did not attenuate the pro-apoptotic effect of ATO at a concentration that reverses caspase-4 processing and activation. Our results demonstrate for the first time that the ER stress-mediated apoptotic pathway operates in human neutrophils.     

The chemotaxis defect of Shwachman-Diamond Syndrome leukocytes

Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive, multisystem disorder presenting in childhood with intermittent neutropenia and pancreatic insufficiency. It is characterized by recurrent infections independent of neutropenia, suggesting a functional neutrophil defect. While mutations at a single gene locus (SBDS) appear to be responsible for SDS in a majority of patients, the function of that gene and a specific defect in SDS neutrophil behavior have not been elucidated. Therefore, employing 2D and 3D computer-assisted motion analysis systems, we have analyzed the basic motile behavior and chemotactic responsiveness of individual polymorphonuclear leukocytes (PMNs) of 14 clinically diagnosed SDS patients. It is demonstrated that the basic motile behavior of SDS PMNs is normal in the absence of chemoattractant, that SDS PMNs respond normally to increasing and decreasing temporal gradients of the chemoattractant fMLP, and that SDS PMNs exhibit a normal chemokinetic response to a spatial gradient of fMLP. fMLP receptors were also distributed uniformly through the plasma membrane of SDS PMNs as in control PMNs. SDS PMNs, however, were incapable of orienting in and chemotaxing up a spatial gradient of fMLP. This unique defect in orientation was manifested by the PMNs of every SDS patient tested. The PMNs of an SDS patient who had received an allogenic hematopoietic stem cell transplant, as well as PMNs from a cystic fibrosis patient, oriented normally. These results suggest that the defect in SDS PMNs is in a specific pathway emanating from the fMLP receptor that is involved exclusively in regulating orientation in response to a spatial gradient of fMLP. This pathway must function in parallel with additional pathways, intact in SDS patients, that emanate from the fMLP receptor and regulate responses to temporal rather than spatial changes in receptor occupancy.