scholarly journals Major Surface Protein 2 of Anaplasma phagocytophilum Facilitates Adherence to Granulocytes

2003 ◽  
Vol 71 (7) ◽  
pp. 4018-4025 ◽  
Author(s):  
Jinho Park ◽  
Kyoung Seong Choi ◽  
J. Stephen Dumler
Keyword(s):  
Monoclonal Antibody ◽  
Bacterial Adhesion ◽  
Anaplasma Phagocytophilum ◽  
Human Peripheral Blood ◽  
Myeloid Cells ◽  
Surface Protein ◽  
Mammalian Host ◽  
Blood Neutrophils ◽  
Major Surface

ABSTRACT Anaplasma phagocytophilum is an obligate intracellular bacterium that infects myeloid cells in the mammalian host. Msp2 (p44) is the major immunodominant outer-membrane protein of these bacteria. We hypothesized that Msp2 acts as an adhesin for A. phagocytophilum entry into granulocytes. This potential role was investigated by blocking binding with Msp2 monoclonal antibodies and by antagonizing binding and propagation with recombinant Msp2 (rMsp2) in vitro. With HL-60 cells, fresh human peripheral blood neutrophils, and a cell line devoid of the fucosylated platelet selectin glycoprotein ligand 1 (PSGL-1) receptor for A. phagocytophilum or one that was transfected to express this ligand, Msp2 monoclonal antibody and rMsp2 used as the antagonist caused concentration-dependent reductions in bacterial adhesion (P < 0.007 and P < 0.02, respectively) and propagation (P < 0.05 and P < 0.001), although inhibition of adhesion or propagation was moderate and incomplete. Likewise, rMsp2 bound to surfaces of the transfected cell at a level similar to that of extracellular A. phagocytophilum and significantly (P < 0.05) beyond that of nontransfected cells. Moreover, a dose-dependent reduction (P < 0.019) in PSGL-1 monoclonal antibody binding to HL-60 cells was elicited with rMsp2. We conclude that Msp2s of A. phagocytophilum are involved in bacterial adhesion to ligands on host myeloid cells before intracellular infection.

scholarly journals Rapid Sequential Changeover of Expressed p44 Genes during the Acute Phase of Anaplasma phagocytophilum Infection in Horses

2004 ◽  
Vol 72 (12) ◽  
pp. 6852-6859 ◽  
Author(s):  
Xueqi Wang ◽  
Yasuko Rikihisa ◽  
Tzung-Hui Lai ◽  
Yumi Kumagai ◽  
Ning Zhi ◽  
...  
Keyword(s):  
Acute Phase ◽  
Anaplasma Phagocytophilum ◽  
Antigenic Variation ◽  
Hypervariable Region ◽  
Surface Protein ◽  
Intrinsic Property ◽  
Infected Horse ◽  
Major Surface ◽  
Intravenous Inoculation

ABSTRACT Anaplasma phagocytophilum immunodominant polymorphic major surface protein P44s have been hypothesized to go through antigenic variation, but the within-host dynamics of p44 expression has not been demonstrated. In the present study we investigated the composition and changes of p44 transcripts in the blood during the acute phase of well-defined laboratory A. phagocytophilum infections in naïve equine hosts. Three traveling waves of sequential population changeovers of the p44 transcript species were observed within a single peak of rickettsemia of less than 1 month. During the logarithmic increase, the rapid switch-off of the initial dominant transcript p44-18 occurred regardless of whether the bacterium was transmitted by ticks or by intravenous inoculation. Each of the subsequently dominant p44 transcript species was phylogenetically dissimilar from p44-18. Development of antibody to the hypervariable region of P44-18 during the rickettsemia suggests the suppression of dominance of immuno-cross-reactive p44 populations. When A. phagocytophilum was preincubated with plasma from the infected horse and then coincubated with HL-60 cells, the dominance of the p44-18 transcript was rapidly suppressed in vitro and most of the newly emerged p44 transcript species were previously undetected in this horse. This work provides experimental evidence of within-host p44 antigenic variation. Results suggest that the rapid and synchronized switch of expression is an intrinsic property of p44s reinitiated after transmission to naïve mammalian hosts and shaped upon exposure to immune plasma.

scholarly journals CHARACTERIZATION OF HUMAN GRANULOCYTIC EHRLICHIOSIS IN EXPERIMENTAL INFECTED HL-60 CELLS WITH ANAPLASMA PHAGOCYTOPHILUM USING NESTED PCR AND A. PHAGOCYTOPHILUM MAJOR SURFACE PROTEIN-2 MONOCLONAL ANTIBODY

2012 ◽  
Vol 3 (2) ◽  
pp. 160-165
Author(s):  
MS Rahman ◽  
JH Park ◽  
JS Chae
Keyword(s):  
Monoclonal Antibody ◽  
Nested Pcr ◽  
Anaplasma Phagocytophilum ◽  
Surface Protein ◽  
Intense Band ◽  
Major Surface Protein ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis ◽  
Major Surface

The study was carried out to characterize the human granulocytic ehrlichiosis in experimental infected HL-60 cells with Anaplasma phagocytophilum using nested PCR and A. phagocytophilum major surface protein-2 monoclonal antibody. The nested PCR revealed only one band of 926 base pair DNA from A. phagocytophilum infected HL-60 cells. The western blot revealed several bands with a dominant 44-kDa. There were intense band of 100- and 160-kDa bands. The 44-kDa component was at least 10 times more abundant than the 100- and 160-kDa bands. In conclusion, the nested PCR would be a valuable tool for the characterization of human granulocytic ehrlichiosis.

scholarly journals Biofilm Formation by Pneumocystis spp.

2008 ◽  
Vol 8 (2) ◽  
pp. 197-206 ◽  
Author(s):  
Melanie T. Cushion ◽  
Margaret S. Collins ◽  
Michael J. Linke
Keyword(s):  
Biofilm Formation ◽  
Fungal Infections ◽  
Morphological Changes ◽  
Continuous Cultivation ◽  
Life Cycles ◽  
Mammalian Host ◽  
Intense Staining ◽  
In Vitro System ◽  
Major Surface

ABSTRACT Pneumocystis spp. can cause a lethal pneumonia in hosts with debilitated immune systems. The manner in which these fungal infections spread throughout the lung, the life cycles of the organisms, and their strategies used for survival within the mammalian host are largely unknown, due in part to the lack of a continuous cultivation method. Biofilm formation is one strategy used by microbes for protection against environmental assaults, for communication and differentiation, and as foci for dissemination. We posited that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system comprised of insert wells suspended in multiwell plates containing supplemented RPMI 1640 medium supported biofilm formation by P. carinii (from rat) and P. murina (from mouse).Dramatic morphological changes accompanied the transition to a biofilm. Cyst and trophic forms became highly refractile and produced branching formations that anastomosed into large macroscopic clusters that spread across the insert. Confocal microscopy revealed stacking of viable organisms enmeshed in concanavalin A-staining extracellular matrix. Biofilms matured over a 3-week time period and could be passaged. These passaged organisms were able to cause infection in immunosuppressed rodents. Biofilm formation was inhibited by farnesol, a quorum-sensing molecule in Candida spp., suggesting that a similar communication system may be operational in the Pneumocystis biofilms. Intense staining with a monoclonal antibody to the major surface glycoproteins and an increase in (1,3)-β-d-glucan content suggest that these components contributed to the refractile properties. Identification of this biofilm process provides a tractable in vitro system that should fundamentally advance the study of Pneumocystis.

scholarly journals Electrospun Polydioxanone Loaded With Chloroquine Modulates Template-Induced NET Release and Inflammatory Responses From Human Neutrophils

2021 ◽  
Vol 9 ◽  
Author(s):  
Allison E. Fetz ◽  
Shannon E. Wallace ◽  
Gary L. Bowlin
Keyword(s):  
Growth Factor ◽  
Inflammatory Responses ◽  
Human Peripheral Blood ◽  
Early Time ◽  
Human Neutrophils ◽  
Late Time ◽  
Dependent Manner ◽  
Blood Neutrophils ◽  
Net Release

The implantation of a biomaterial quickly initiates a tissue repair program initially characterized by a neutrophil influx. During the acute inflammatory response, neutrophils release neutrophil extracellular traps (NETs) and secrete soluble signals to modulate the tissue environment. In this work, we evaluated chloroquine diphosphate, an antimalarial with immunomodulatory and antithrombotic effects, as an electrospun biomaterial additive to regulate neutrophil-mediated inflammation. Electrospinning of polydioxanone was optimized for rapid chloroquine elution within 1 h, and acute neutrophil-biomaterial interactions were evaluated in vitro with fresh human peripheral blood neutrophils at 3 and 6 h before quantifying the release of NETs and secretion of inflammatory and regenerative factors. Our results indicate that chloroquine suppresses NET release in a biomaterial surface area–dependent manner at the early time point, whereas it modulates signal secretion at both early and late time points. More specifically, chloroquine elution down-regulates interleukin 8 (IL-8) and matrix metalloproteinase nine secretion while up-regulating hepatocyte growth factor, vascular endothelial growth factor A, and IL-22 secretion, suggesting a potential shift toward a resolving neutrophil phenotype. Our novel repurposing of chloroquine as a biomaterial additive may therefore have synergistic, immunomodulatory effects that are advantageous for biomaterial-guided in situ tissue regeneration applications.

scholarly journals Monoclonal antibody AHN-1 inhibits phagocytosis by human neutrophils

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 333-339
Author(s):  
KM Skubitz ◽  
DJ Weisdorf ◽  
PK Peterson
Keyword(s):  
Monoclonal Antibody ◽  
Lysosomal Enzyme ◽  
Surface Proteins ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Enzyme Release ◽  
Specific Monoclonal Antibody ◽  
Normal Human ◽  
Major Surface

The granulocyte-specific monoclonal antibody, AHN-1, immunoprecipitates two major surface-iodinated proteins of 105,000 and 145,000 to 150,000 daltons from normal human neutrophils. In this study, the effect of AHN- 1 on a number of neutrophil functions was evaluated in vitro. Both complement- and antibody-mediated phagocytosis were inhibited when human neutrophils were pretreated with AHN-1 and opsonized bacteria were used as targets. The inhibition of phagocytosis was specific, in that lysosomal enzyme release and chemotaxis were not altered by treatment with AHN-1. AHN-1 did inhibit superoxide production by neutrophils in response to particulate stimuli, but not in response to the soluble stimulus, 12-O-tetradecanoylphorbol-13-acetate. The data indicate that one or both of these surface proteins may be important in the process of phagocytosis. AHN-1 should be useful in isolating and further characterizing the nature of these molecules.

scholarly journals Leishmania mexicana Mutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

2000 ◽  
Vol 11 (4) ◽  
pp. 1183-1195 ◽  
Author(s):  
James D. Hilley ◽  
Jody L. Zawadzki ◽  
Malcolm J. McConville ◽  
Graham H. Coombs ◽  
Jeremy C. Mottram
Keyword(s):  
Normal Growth ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Putative Protein ◽  
Major Class ◽  
Major Surface Proteins ◽  
Major Surface

The major surface proteins of the parasitic protozoonLeishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (ΔGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein–anchor precursors. However, the synthesis and cellular levels of other non–protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the ΔGPI8 mutant. Significantly, the ΔGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.

Activated human peripheral blood neutrophils produce epithelial injury and fibronectin breakdown in vitro

Inflammation ◽  
1992 ◽  
Vol 16 (1) ◽  
pp. 21-30 ◽  
Author(s):  
D. M. Brown ◽  
G. M. Brown ◽  
W. Macnee ◽  
K. Donaldson
Keyword(s):  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Epithelial Injury ◽  
Blood Neutrophils

scholarly journals Polymorphonuclear leukocyte elastase activity is increased by bacterial lipopolysaccharide: a response inhibited by glucocorticoids

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 421-426 ◽  
Author(s):  
DH Hart
Keyword(s):  
Human Peripheral Blood ◽  
Human Neutrophil Elastase ◽  
Tissue Destruction ◽  
Elastase Activity ◽  
Leukocyte Elastase ◽  
Intracellular Enzyme ◽  
E Coli ◽  
Blood Neutrophils ◽  
Polymorphonuclear Leukocyte Elastase

Abstract Human neutrophil elastase is thought to play an important role in connective tissue destruction in diseases such as emphysema and arthritis. In this article, it is demonstrated that the elastase activity of mature human peripheral blood neutrophils can be rapidly increased in vitro by treatment of the cells with lipopolysaccharide from E. coli and that this increase is inhibited by corticosteroid pretreatment of the cells and by inhibitors of protein synthesis. Alterations in intracellular enzyme content of the mature polymorph in response to bacterial products or other stimuli may be important for amplification of the inflammatory response.

scholarly journals Identification of Novel Surface Proteins of Anaplasma phagocytophilum by Affinity Purification and Proteomics

2007 ◽  
Vol 189 (21) ◽  
pp. 7819-7828 ◽  
Author(s):  
Yan Ge ◽  
Yasuko Rikihisa
Keyword(s):  
Anaplasma Phagocytophilum ◽  
Affinity Purification ◽  
The United States ◽  
Capillary Liquid Chromatography ◽  
Etiologic Agent ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Major Surface

ABSTRACT Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To identify the major surface proteins of A. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were five A. phagocytophilum proteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralized A. phagocytophilum infection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the family Anaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction between A. phagocytophilum and the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.

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