Establishment of an activated macrophage cell line, A-THP-1, and its properties.
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A new macrophage cell line with activated character and unique morphology was isolated by selecting adherent cells from the human monocytic cell line THP-1. The original THP-1 cells had been cultured for more than 9 years using 25 cm2 flasks, when cells with a different morphology appeared, adhering to the bottoms of the culture flasks. These were selected by discarding floating nonadherent cells at every subculture. Enrichment of adherent THP-1 cells with long processes proceeded during the cultivation. These adherent THP-1 showed remarkable phenotypic changes, not only morphologically, but also functionally. Namely, increased phagocytic activity, HLA-DR expression and MLR stimulator activity were remarkable. This adherent cell line was designated as activated-THP-1 (A-THP-1), since it demonstrated characteristics of activated macrophages continuously without exogenous stimulation. A cloned A-THP-1 cell line (A-THP-1 C1) also showed the same features and contained about 10% multinucleated giant cells probably caused by cell fusion. This A-THP-1 cell line, the first activated macrophage cell line to be established, provides a good model for understanding of activation mechanisms of macrophages and multinucleation. In this paper, morphological, immunological, and biological characters of this cell line are described. (+info)
Protein kinase C and a calcium-independent phospholipase are required for IgG-mediated phagocytosis by Mono-Mac-6 cells.
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Mono-Mac-6 (MM6) human monocytes ingest IgG-opsonized particles better than other human cell lines. We compared the phagocytic signaling pathway in MM6 with human monocytes. MM6 expressed FcgammaRI at levels similar to monocytes, whereas FcRgammaII expression was approximately double. MM6 ingested IgG-opsonized erythrocytes (EIgG) in a calcium-independent manner. Incubation of MM6 with bromoenol lactone, an inhibitor of the phagocytic phospholipase (pPL), coordinately decreased phagocytosis and pPL activity. This inhibition was overcome by exogenous arachidonic acid, suggesting that phagocytosis requires pPL activation and arachidonic acid release. MM6 phagocytosis was inhibited with staurosporine and activated with diacylglycerol, supporting a role for protein kinase C (PKC) in this process. The pPL activators mastoparan and melittin restored phagocytosis to PKC-inhibited cells, suggesting that pPL lies downstream from PKC. These results suggest that the MM6 signal transduction pathway for IgG-mediated phagocytosis is similar to that of monocytes (PKC-->pPL-->arachidonic acid-->phagocytosis). The results are discussed in the context of the finding that MM6 exhibit low phagocytosis relative to monocytes and thus may represent an attractive cell line for molecular manipulation in "recovery of function" studies. (+info)
Identification of residues in the CH2/CH3 domain interface of IgA essential for interaction with the human fcalpha receptor (FcalphaR) CD89.
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Cellular receptors for IgA (FcalphaR) mediate important protective functions. An extensive panel of site-directed mutant IgAs was used to identify IgA residues critical for FcalphaR (CD89) binding and triggering. Although a tailpiece-deleted IgA1 was able to bind and trigger CD89, antibodies featuring CH3 domain exchanges between human IgA1 and IgG1 could not, indicating that both domains but not the tailpiece are required for FcalphaR recognition. To further investigate the role of the interdomain region, numerous IgA1s, each with a point substitution in either of two interdomain loops (Leu-257-Gly-259 in Calpha2; Pro-440-Phe-443 in Calpha3), were generated. With only one exception (G259R), substitutions produced either ablation (L257R, P440A, A442R, F443R) or marked reduction (P440R) in CD89 binding and triggering. Further support for involvement of these interdomain loops was provided by interspecies comparisons of IgA. Thus a human IgA1 mutant, LA441-442MN, which mimicked the mouse IgA loop sequence through substitution of two adjacent residues in the Calpha3 loop, was found, like mouse IgA, not to bind CD89. In contrast, bovine IgA1, identical to human IgA1 within these interdomain loops despite numerous differences elsewhere in the Fc region, did bind CD89. We have thus identified motifs in the interdomain region of IgA Fc critical for FcalphaR binding and triggering, significantly enhancing present understanding of the molecular basis of the IgA-FcalphaR interaction. (+info)
Plasmodium falciparum malaria: rosettes are disrupted by quinine, artemisinin, mefloquine, primaquine, pyrimethamine, chloroquine and proguanil.
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An assay was developed measuring the disruption of rosettes between Plasmodium falciparuminfected (trophozoites) and uninfected erythrocytes by the antimalarial drugs quinine, artemisinin mefloquine, primaquine, pyrimethamine, chloroquine and proguanil. At 4 hr incubation rosettes were disrupted by all the drugs in a dose dependent manner. Artemisinin and quinine were the most effective anti-malarials at disrupting rosettes at their therapeutic concentrations with South African RSA 14, 15, 17 and The Gambian FCR-3 P. falciparum strains. The least effective drugs were proguanil and chloroquine. A combination of artemisinin and mefloquine was more effective than each drug alone. The combinations of pyrimethamine or primaquine, with quinine disrupted more rosettes than quinine alone. Quinine may be an effective drug in the treatment of severe malaria because the drug efficiently reduces the number of rosettes. (+info)
Cytoadherence characteristics of Plasmodium falciparum-infected erythrocytes from Malawian children with severe and uncomplicated malaria.
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Cytoadherence of Plasmodium falciparum-infected erythrocytes to the microvascular endothelium is believed to be a key factor in the development of cerebral malaria. Erythrocyte rosette formation has been correlated with malaria severity in studies from east and west Africa. We cultured fresh isolates from Malawian children with severe (n = 76) or uncomplicated (n = 79) malaria to pigmented trophozoite stage and examined rosette formation and adherence to CD36, intercellular adhesion molecule-1 (ICAM-1), chondroitin sulfate A (CSA), and thrombomodulin (TM). Most (126 of 148) isolates bound to CD36, and 76 of 136 bound to ICAM-1. Fewer bound to CSA (40 of 148) or TM (23 of 148). After controlling for parasitemia, there was an inverse association between binding to CD36 (P = 0.004) or ICAM-1 (P = 0.001) and disease severity. Parasites from children with severe malaria anemia bound least to CD36, whereas ICAM-1 binding was lowest in children with cerebral malaria. There was no difference in rosette formation between any of the groups. In Malawian children, there was no evidence of a positive association between adherence to any of the receptors examined and disease severity. The negative association found raises the possibility that adherence to certain receptors could instead be an indicator of a less pathogenic infection. (+info)
In vivo role of complement-interacting domains of herpes simplex virus type 1 glycoprotein gC.
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Immune evasion is critical for survival of viruses that establish persistent or recurrent infections. However, at the molecular level, little is known about how viruses evade immune attack in vivo. Herpes simplex virus (HSV)-1 glycoprotein gC has two domains that are involved in modulating complement activation; one binds C3, and the other is required for blocking C5 and properdin (P) binding to C3. To evaluate the importance of these regions in vivo, HSV-1 gC mutant viruses were constructed that lacked one or both gC domains and studied in a murine model of infection. Each gC region of complement regulation contributed to virulence; however, the C3 binding domain was far more important, as virus lacking this domain was much less virulent than virus lacking the C5/P inhibitory domain and was as attenuated as virus lacking both domains. Studies in C3 knockout mice and mice reconstituted with C3 confirmed that the gC domains are inhibitors of complement activation, accounting for a 50-fold difference in virulence between mutant and wild-type viruses. We conclude that the C3 binding domain on gC is a major contributor to immune evasion and that this site explains at a molecular level why wild-type virus resists complement attack. (+info)
Plasmodium falciparum rosette formation is uncommon in isolates from pregnant women.
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We examined the formation of Plasmodium falciparum erythrocyte rosettes using parasite isolates from placental or peripheral blood of pregnant Malawian women and from peripheral blood of children. Five of 23 placental isolates, 23 of 38 maternal peripheral isolates, and 136 of 139 child peripheral isolates formed rosettes. Placental isolates formed fewer rosettes than maternal isolates (range, 0 to 7. 5% versus 0 to 33.5%; P = 0.002), and both formed fewer rosettes than isolates cultured from children (range, 0 to 56%; P < 0.0001). Rosette formation is common in infections of children but uncommon in pregnancy and rarely detected in placental isolates. (+info)
Autologous T cells control B-chronic lymphocytic leukemia tumor progression in human-->mouse radiation chimera.
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B-chronic lymphocytic leukemia (B-CLL) is characterized by the clonal accumulation of CD5+ B cells. It has been suggested that CLL cells may be regulated by inhibitory and growth-promoting signals exerted by autologous T cells. We have recently described a model for human B-CLL in which peripheral blood mononuclear cells (PBMCs) are transplanted into the peritoneal cavity of lethally irradiated mice radioprotected with bone marrow from mice with severe combined immunodeficiency. In this model, adoptive transfer of low-stage PBMCs leads to marked engraftment of T cells or combined T and CLL cell engraftment, whereas infusion of high-stage PBMCs leads to dominance of CLL cells with a miniscule level of T-cell engraftment. This mutual exclusive pattern of engraftment indicated that T cells might control the expansion of tumor cells in the peritoneum of recipient BALB/c mice. In the present study, we further investigated this question and we demonstrate that in vivo T-cell depletion, using OKT3 antibody, markedly enhances the engraftment of B-CLL cells from patients with early-stage disease. In mice receiving PBMCs from 11 donors with advanced-stage disease, the results were more heterogeneous. In five patients the results were similar to those observed in early stage, whereas in two cases no CLL cell engraftment was found in the absence of T cells. The addition of purified T cells to PBMCs led to a substantial decrease of CLL engraftment in three advanced-stage cases. These results strengthen the working hypothesis that autologous T cells can actively suppress the expansion of the pathological cells in human-->mouse radiation chimera. This effect is prominent in early-stage disease, whereas in advanced stage suppressive and/or stimulatory effects may occur in different patients. The interaction of T cells with tumor cells and the potential of autologous T cell/immune-therapy in CLL can be further explored in this model. (+info)