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1.  Isothermal Microcalorimetry as a Quality by Design Tool to Determine Optimal Blending Sequences 
The AAPS Journal  2010;12(3):417-423.
This study was designed to assess the value of isothermal microcalorimetry (ITMC) as a quality by design (QbD) tool to optimize blending conditions during tablet preparation. Powder mixtures that contain microcrystalline cellulose (MCC), dibasic calcium phosphate dihydrate (DCPD), and prednisone were prepared as 1:1:1 ratios using different blending sequences. ITMC was used to monitor the thermal activity of the powder mixtures before and after each blending process. Differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD) were performed on all final powder mixtures. Final powder mixtures were used to prepare tablets with 10 mg prednisone content, and dissolution tests were performed on all tablet formulations. Using ITMC, it was observed that the powder mixtures had different thermal activity depending on the blending sequences of the ingredients. All mixtures prepared by mixing prednisone with DCPD in the first stage were associated with relatively fast and significant heat exchange. In contrast, mixing prednisone with MCC in the first step resulted in slower heat exchange. Powder mixture with high thermal activity showed extra DSC peaks, and their dissolution was generally slower compared to the other tablets. Blending is considered as a critical parameter in tablet preparation. This study showed that ITMC is a simple and efficient tool to monitor solid-state reactions between excipients and prednisone depending on blending sequences. ITMC has the potential to be used in QbD approaches to optimize blending parameters for prednisone tablets.
doi:10.1208/s12248-010-9202-4
PMCID: PMC2895446  PMID: 20461485
isothermal microcalorimetry; powders blending; quality by design; tablets
2.  Effect of cytochalasin B on the adhesion of mouse peritoneal macrophages 
The Journal of Cell Biology  1976;69(2):407-414.
The adhesion of normal mouse macrophages to glass surfaces was reduced by nontoxic levels (1-50 mug/ml) of cytochalasin B in combination with a centrifugal force (1,000-8,000 g). Macrophages nonspecifically activated by Corynebacterium acnes were also detached by this treatment, but less effectively. The effects of cytochalasin B treatment on these cells were shown to be reversible. After detachment, the cells reattached to glass, appeared morphologically normal, and behaved like untreated cells as judged by adhesion, acid phosphatase levels, and phagocytosis. The effect of cytochalasin B on several parameters of phagocytosis by normal macrophages was also examined. The results demonstrate that cytochalasin B can be used to detach macrophages from surfaces and suggest a functional relationship between phagocytosis and macrophage adhesion to surfaces. Furthermore, the effect of cytochalasin B on adhesion of phagocytic cells provides a probe for further investigation of the adhesion of cells to surfaces.
PMCID: PMC2109683  PMID: 816801
3.  Oxidative response of human neutrophils, monocytes, and alveolar macrophages induced by unopsonized surface-adherent Staphylococcus aureus. 
Infection and Immunity  1987;55(10):2398-2403.
In contrast to results with bacterial suspensions, phagocytosis of unopsonized bacteria readily occurs when bacteria are adhered to glass or plastic surfaces. However, in contrast to neutrophils, alveolar macrophages produced much less DNA denaturation as measured by acridine orange metachromasia of phagocytized Staphylococcus aureus. We have studied the phagocytosis of unopsonized surface-adherent S. aureus and the subsequent production of reactive oxygen species by peripheral blood neutrophils, monocytes, and alveolar macrophages. Phagocyte-free systems were then used to show the relationship of the reactive oxygen species produced by neutrophils and alveolar macrophages and the denaturation of unopsonized S. aureus DNA with acridine orange. Peripheral blood neutrophils, monocytes, and alveolar macrophages from normal human volunteers were added to vials with adherent S. aureus without opsonin. Bacterial uptake and luminol- and lucigenin-dependent chemiluminescence were measured. Neutrophils developed much greater luminol-dependent chemiluminescence than monocytes or alveolar macrophages. Compared with neutrophils and monocytes, alveolar macrophages developed significantly greater concentrations of superoxide, as measured by lucigenin-dependent chemiluminescence and ferricytochrome c reduction. These findings suggested that products of the myeloperoxidase-hydrogen peroxide-halide pathway were generated when peripheral blood neutrophils were stimulated and that alveolar macrophages primarily produced superoxide. When these reactive oxygen species were generated in phagocyte-free systems containing S. aureus, products of the myeloperoxidase-hydrogen peroxide-halide pathway produced denaturation of S. aureus DNA, whereas superoxide did not. Thus, differences in reactive oxygen species produced during phagocytosis may be related to the different capacities of neutrophils and alveolar macrophages to denature unopsonized adherent S. aureus DNA.
PMCID: PMC260720  PMID: 2820882
4.  Energetic and binding properties of DNA upon interaction with dodecyl trimethylammonium bromide. 
Nucleic Acids Research  1999;27(4):1001-1005.
The interaction of dodecyl trimethylammonium bromide (DTAB), a cationic surfactant, with calf thymus DNA has been studied by various methods, including potentiometric technique using DTAB-selective plastic membrane electrode at 27 and 37 degreesC, isothermal titration microcalorimetry and UV spectrophotometry at 27 degreesC using 0.05 M Tris buffer and 0.01 M NaCl at pH 7.4. The free energy is calculated from binding isotherms on the basis of Wyman binding potential theory and the enthalpy of binding according to van't Hoff relation. The enthalpy of unfolding has been determined by subtraction of the enthalpy of binding from the microcalorimetric enthalpy. The results show that, after the interaction of first DTAB molecule to DNA (base molarity) through the electrostatic interaction, the second DTAB molecule also binds to DNA through electrostatic interaction. At this stage, the predom-inant DNA conformational change occurs. Afterwards up to 20 DTAB molecules, below the critical micelle concentration of DTAB, bind through hydrophobic interactions.
PMCID: PMC148279  PMID: 9927732
5.  Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila. 
Infection and Immunity  1986;51(1):31-36.
Legionella pneumophila multiplied rapidly in guinea pig and rat alveolar macrophages but failed to grow when phagocytic activity was inhibited by pretreatment with 0.5 or 1.0 microgram of cytochalasin D per ml. Attachment was not inhibited by cytochalasin D. No extracellular multiplication occurred when L. pneumophila were in close proximity to viable functional macrophages or even when the bacteria were attached to plasma membranes of the macrophages. Nonopsonized L. pneumophila were avidly phagocytized by alveolar macrophages. When bacteria were centrifuged onto a cell pellet, more than 85% of the phagocytes contained one or more bacteria within 15 min. In contrast, under the same conditions only approximately 15% of the macrophages contained nonopsonized Escherichia coli or Staphylococcus aureus. Phagocytosis of L. pneumophila by untreated guinea pig macrophages occurred by extension of pseudopodia around the bacteria in a classical manner. The failure of the bacteria to actively penetrate the phagocyte suggests that their intracellular survival must not depend on avoidance of a phagosome but rather on an inhibition of or resistance to subsequent microbicidal functions of the macrophage.
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PMCID: PMC261061  PMID: 3941000
6.  Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry 
Respiratory Research  2008;9(1):59.
Background
Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized.
Methods
We developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively.
Results
Microtubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase (LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect.
Conclusion
These data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis.
doi:10.1186/1465-9921-9-59
PMCID: PMC2527561  PMID: 18687123
7.  Reduced binding and phagocytosis of Pneumocystis carinii by alveolar macrophages from persons infected with HIV-1 correlates with mannose receptor downregulation. 
Journal of Clinical Investigation  1998;102(7):1332-1344.
The macrophage mannose receptor, a pattern recognition molecule and component of innate immunity, mediates binding and phagocytosis of Pneumocystis carinii and likely represents an important clearance mechanism in the lungs of immunocompetent hosts. The purpose of this study was to examine the ability of alveolar macrophages from HIV-infected individuals to bind and phagocytose P. carinii, and to investigate the role of the macrophage mannose receptor in mediating this interaction. Compared with healthy individuals, alveolar macrophage phagocytosis of P. carinii from HIV+ persons was reduced up to 74% (P = 0.02), primarily reflecting a reduction in the number of organisms associated with each macrophage (P = 0.019). Furthermore, macrophages from HIV+ individuals demonstrated up to an 80% (P < 0.05) reduction in mannose receptor surface expression and endocytosis. Mannose receptor affinity was unaltered, and mRNA levels were modestly reduced (P < 0.05). Cells from HIV+ individuals with CD4(+) counts < 200 cells/mm3 (representing individuals at high clinical risk for P. carinii pneumonia) demonstrated the lowest levels of P. carinii phagocytosis and mannose receptor endocytosis. In vitro HIV infection of alveolar macrophages from healthy individuals reduced mannose receptor endocytosis to 53.2% (P < 0.05) and P. carinii binding and phagocytosis to 67.4% (P < 0.05) of control. Our studies suggest that HIV infection may alter innate immunity in the lungs, and that impaired alveolar macrophage mannose receptor-mediated binding and phagocytosis of P. carinii may contribute to the susceptibility of HIV-infected individuals to this opportunistic pulmonary pathogen.
PMCID: PMC508980  PMID: 9769325
8.  Phagocytosis-induced injury of normal and activated alveolar macrophages. 
Infection and Immunity  1979;26(3):910-915.
The present study tested the hypothesis that BCG-activated macrophages become injured when they phagocytose certain particulates. The data indicate that alveolar macrophages obtained from Mycobacterium bovis BCG-sensitized animals were more susceptible to cell death after in vitro incubation with BCG or zymosan than were macrophages from normal animals. Increased susceptibility was dependent on phagocytosis, since incubation with cytochalasin B, a phagocytosis inhibitor, abrogated the effect. Catalase, cytochrome c, and ascorbic acid offered partial protection to the macrophage, suggesting the involvement of free radicals in the generation of cytotoxicity. Not all of the cells from the alveolar populations were equally susceptible to cell death, thus suggesting either heterogeneity in the cell population or a requirement of more than one cell type in the induction of necrosis or both.
PMCID: PMC414706  PMID: 231011
9.  Depression of the Respiratory Burst in Alveolar and Peritoneal Macrophages After Thermal Injury 
Infection and Immunity  1980;30(3):718-722.
The resting O2 consumption of alveolar and peritoneal macrophages obtained from rats at 4 and 24 h after thermal injury was unaltered from control values. However, when heat-killed Pseudomonas aeruginosa or polystyrene latex particles were added to the cell suspensions to initiate phagocytosis, a significant depression in the respiratory burst accompanying the phagocytic event was demonstrated. The addition of phorbol myristate acetate, used to maximize the respiratory response, was ineffective in elevating, to control values, the respiratory burst of macrophages obtained from burned animals. The deficit was only, in part, serum mediated since the responses could not be restored to control values even when the cells from the burned animals were vigorously washed with control serum and incubated with control serum. The contribution of a burn serum factor, which was non-dialyzable, heat stable at 56°C but not at 65°C, and insensitive to pronase treatment, must be considered. These data indicate that thermal injury results in macrophage metabolic alterations which are mediated, in part, by a burn serum factor. Furthermore, the data suggest that pulmonary alveolar macrophages are more sensitive to thermal injury than peritoneal macrophages. Serum factors contributed, in part, to this observed impairment in the respiratory burst as indicated by: (i) an approximate 50% reversal of the impairment by control serum, and (ii) an approximate decrease of 50 to 80% in the control alveolar macrophage respiratory burst when serum from the thermally injured rats was added to the culture medium.
PMCID: PMC551374  PMID: 7228387
10.  Interaction of the European genotype porcine reproductive and respiratory syndrome virus (PRRSV) with sialoadhesin (CD169/Siglec-1) inhibits alveolar macrophage phagocytosis 
Veterinary Research  2012;43(1):47.
Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus that shows a restricted in vivo tropism for subsets of porcine macrophages, with alveolar macrophages being major target cells. The virus is associated with respiratory problems in pigs of all ages and is commonly isolated on farms with porcine respiratory disease complex (PRDC). Due to virus-induced macrophage death early in infection, PRRSV hampers the innate defence against pathogens in the lungs. In addition, the virus might also directly affect the antimicrobial functions of macrophages. This study examined whether interaction of European genotype PRRSV with primary alveolar macrophages (PAM) affects their phagocytic capacity. Inoculation of macrophages with both subtype I PRRSV (LV) and subtype III PRRSV (Lena) showed that the virus inhibits PAM phagocytosis. Similar results were obtained using inactivated PRRSV (LV), showing that initial interaction of the virion with the cell is sufficient to reduce phagocytosis, and that no productive infection is required. When macrophages were incubated with sialoadhesin- (Sn) or CD163-specific antibodies, two entry mediators of the virus, only Sn-specific antibodies downregulated the phagocytic capacity of PAM, indicating that interaction with Sn, but not CD163, mediates the inhibitory effect of PRRSV on phagocytosis. In conclusion, this study shows that European genotype PRRSV inhibits PAM phagocytosis in vitro, through the interaction with its internalization receptor Sn. If similar events occur in vivo, this interaction may be important in the development of PRDC, as often seen in the field.
doi:10.1186/1297-9716-43-47
PMCID: PMC3403922  PMID: 22630829
11.  Effect of infectious bovine rhinotracheitis virus infection on bovine alveolar macrophage function. 
Infection and Immunity  1982;35(3):1041-1047.
Bovine alveolar macrophages isolated in culture were assessed for immunological activity in assays for Fc and complement receptors, for phagocytosis, and for effector cell function in antibody-dependent cell cytotoxicity. In the case of uninfected alveolar macrophages, Fc receptors were detected on approximately 94% of macrophages and complement receptors were detected on 39%. Phagocytosis of immunoglobulin G-coated sheep erythrocytes occurred in 58% of macrophages, and phagocytosis of opsonized Candida parapsilosis, mediated by the complement receptor, was observed in 68% of cells. Alveolar macrophages were efficient effector cells in antibody-dependent cell cytotoxicity. Infection of macrophages with infectious bovine rhinotracheitis (IBR) virus resulted in reductions in Fc-mediated receptor activity and phagocytosis after approximately 12 and 6 h, respectively. Complement receptor activity was initially elevated and then markedly reduced. Macrophages retrieved from IBR-immune and -susceptible donors were affected to a similar extent. The ability of macrophages to participate in antibody-dependent cell cytotoxicity was reduced dramatically from 2 h after IBR virus infection, suggesting that IBR virus-infected alveolar macrophages undergo alterations in immunological activity long before morphological changes in the cells become apparent.
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PMCID: PMC351152  PMID: 6279511
12.  MicroDSC study of Staphylococcus epidermidis growth 
BMC Microbiology  2010;10:322.
Background
A microcalorimetric study was carried out using a Staphylococcus epidermidis population to determine the reproducibility of bacterial growth and the variability of the results within certain experimental parameters (temperature, bacterial concentration, sample thermal history). Reproducibility tests were performed as series of experiments within the same conditions using either freshly prepared populations or samples kept in cold storage. In both cases, the samples were obtained by serial dilution from a concentrated TSB bacterial inoculum incubated overnight.
Results
The results show that experiments are fairly reproducible and that specimens can be preserved at low temperatures (1 - 2°C) at least 4 days. The thermal signal variations at different temperatures and initial bacterial concentrations obey a set of rules that we identified.
Conclusion
Our study adds to the accumulating data and confirms available results of isothermal microcalorimetry applications in microbiology and can be used to standardize this method for either research or clinical setting.
doi:10.1186/1471-2180-10-322
PMCID: PMC3018406  PMID: 21162759
13.  Superoxide production by phagocytic leukocytes 
Mononuclear phagocytic leukocytes, as well as polymorphonuclear leukocytes, produce and release superoxide at rest, and this is stimulated by phagocytosis. Of the mouse monocytic cells studied, alveolar macrophages released the largest amounts of superoxide during phagocytosis, followed by normal peritoneal macrophages. Casein- elicited and "activated" macrophages released smaller quantities. In the guinea pig, polymorphonuclear leukocytes and casein-elicited macrophages were shown to release superoxide during phagocytosis whereas alveolar macrophages did not. Superoxide release accounted for only a small fraction of the respiratory burst of phagocytosis in all but the normal mouse peritoneal macrophage, the guinea pig polymorphonuclear leukocyte, and probably the mouse alveolar macrophage. There are obviously considerable species differences in O2- release by various leukocytes that might reflect both the production and/or destruction (e.g. by dismutase) of that substance.
PMCID: PMC2190499  PMID: 804030
14.  Differences in phagocytosis and killing by alveolar macrophages from humans, rabbits, rats, and hamsters. 
Infection and Immunity  1982;36(2):504-509.
Phagocytosis and killing by alveolar macrophages from humans, rabbits, rats, and hamsters, were compared in vitro. In the absence of serum opsonins, human alveolar macrophages could phagocytize Staphylococcus aureus Cowan I (protein A positive), but not S. aureus EMS (protein A negative) or Pseudomonas aeruginosa MN. In contrast, rabbit, rat, and hamster alveolar macrophages did not phagocytize S. aureus Cowan I or other nonopsonized bacteria. Human alveolar macrophages, but not other species, stained positively with fluorescein isothiocyanate-conjugated protein A. When opsonized bacterial were studied, phagocytosis by human, rabbit, and hamster alveolar macrophages was found to be mediated by both Fc and C3 receptors. However, only Fc receptor-mediated phagocytosis of bacteria was demonstrated for rat alveolar macrophages. Differences were also found in the kinetics of bacterial killing by alveolar macrophages from different species. Human and rabbit alveolar macrophages rapidly killed opsonized S. aureus Cowan I. However, bacterial killing by hamster alveolar macrophages proceeded at a slower rate, and rat alveolar macrophages completely failed to kill S. aureus. These significant differences in the function of alveolar macrophages from four different species emphasize the need to document the appropriateness of animal models before using them to predict the biological activities of human alveolar macrophages.
PMCID: PMC351256  PMID: 6806190
15.  Interactions between Histoplasma capsulatum and macrophages from normal and treated mice: comparison of the mycelial and yeast phases in alveolar and peritoneal macrophages. 
Infection and Immunity  1981;34(1):6-10.
Interactions between macrophages (alveolar and peritoneal) from normal, vaccinated (with heat-killed yeast cells), and Mycobacterium bovis BCG-treated mice and the mycelial and yeast phases of Histoplasma capsulatum were observed. Phagocytosis of microconidia, small hyphal fragments, and yeast cells occurred 4 to 6 h after the infection of macrophage cultures. Conversion to the yeast phase began at 6 to 7 h and was complete after a 72-h incubation at 37 degrees C. Macrophages surrounded and adhered to macroconidia and large hyphal elements. More macrophages (65 to 68%) from BCG-treated mice contained fungi at 24 h than did macrophages from normal or vaccinated mice. Although there was no increase in the number of fungi in macrophages from vaccinated mice, only the macrophages from BCG-treated mice contained fewer fungi after 48 h of infection with the mycelial phase of H. capsulatum. Fungal growth was not inhibited in any of the macrophage cultures when infected with the yeast phase. The macrophages infected with yeast cells were destroyed after 48 to 72 h in the culture. Only BCG-treated macrophages survived infection with the mycelial phase, whereas macrophages from normal and vaccinated mice were destroyed by the infection.
PMCID: PMC350811  PMID: 7298193
16.  In vitro cytotoxicity of Manville Code 100 glass fibers: Effect of fiber length on human alveolar macrophages 
Background
Synthetic vitreous fibers (SVFs) are inorganic noncrystalline materials widely used in residential and industrial settings for insulation, filtration, and reinforcement purposes. SVFs conventionally include three major categories: fibrous glass, rock/slag/stone (mineral) wool, and ceramic fibers. Previous in vitro studies from our laboratory demonstrated length-dependent cytotoxic effects of glass fibers on rat alveolar macrophages which were possibly associated with incomplete phagocytosis of fibers ≥ 17 μm in length. The purpose of this study was to examine the influence of fiber length on primary human alveolar macrophages, which are larger in diameter than rat macrophages, using length-classified Manville Code 100 glass fibers (8, 10, 16, and 20 μm). It was hypothesized that complete engulfment of fibers by human alveolar macrophages could decrease fiber cytotoxicity; i.e. shorter fibers that can be completely engulfed might not be as cytotoxic as longer fibers. Human alveolar macrophages, obtained by segmental bronchoalveolar lavage of healthy, non-smoking volunteers, were treated with three different concentrations (determined by fiber number) of the sized fibers in vitro. Cytotoxicity was assessed by monitoring cytosolic lactate dehydrogenase release and loss of function as indicated by a decrease in zymosan-stimulated chemiluminescence.
Results
Microscopic analysis indicated that human alveolar macrophages completely engulfed glass fibers of the 20 μm length. All fiber length fractions tested exhibited equal cytotoxicity on a per fiber basis, i.e. increasing lactate dehydrogenase and decreasing chemiluminescence in the same concentration-dependent fashion.
Conclusion
The data suggest that due to the larger diameter of human alveolar macrophages, compared to rat alveolar macrophages, complete phagocytosis of longer fibers can occur with the human cells. Neither incomplete phagocytosis nor length-dependent toxicity was observed in fiber-exposed human macrophage cultures. In contrast, rat macrophages exhibited both incomplete phagocytosis of long fibers and length-dependent toxicity. The results of the human and rat cell studies suggest that incomplete engulfment may enhance cytotoxicity of fiber glass. However, the possibility should not be ruled out that differences between human versus rat macrophages other than cell diameter could account for differences in fiber effects.
doi:10.1186/1743-8977-3-5
PMCID: PMC1459188  PMID: 16569233
17.  Development and characterization of gelatin based nanoparticles for targeted delivery of zidovudine 
Introduction:
The present work was aimed at development and evaluation of zidovudin (AZT) loaded gelatin nanoparticles (GNPs) by simple desolvation method and further couple it with mannose.
Material and Methods:
Total seven batches of GNPs (A1-A7) were formulated by changing the concentration of polymer gelatin. Various parameters such as particle size, polydispersity index, zeta potential, % entrapment efficiency and in-vitro drug release of plain and mannosylated gelatin nanoparticles (M-GNPs) were studied.
Results:
Scanning electron microscopy (SEM) studies revealed that the average particle size of GNPs and M-GNPs were found to be 394 ± 3.21 and 797.2 ± 2.89 nm respectively (optimised batch A3). It was interesting to note that the average particle size of M-GNPs was more due to anchored mannose, whereas drug entrapment was lesser compared to plain GNPs. Studies have showed drug loading for GNPs and M-GNPs to be 66.56% and 58.85% respectively. Zeta potential studies demonstrated little reduction in solution stability of M-GNPs compared to GNPs. In-vitro drug release studies showed almost 80% release (bimodal) up to 24 h, following Korsmeyer-Peppas release kinetics model (GNPs, r = 0.9760; M-GNPs, r = 0.9712).
Conclusions:
Hence, it can be concluded that, development of GNPs and M-GNPs will pave the way for reticuloendothelial system uptake of AZT; thus, achieving targeted delivery, selectivity and reduction in associated side effect reduction in acquired immuno defficiency syndrome.
doi:10.4103/2230-973X.119213
PMCID: PMC3807979  PMID: 24167784
Desolvation; mannosylation; particle size; release; reticuloendothelial system uptake; zidovudine nanoparticles.
18.  Phagocytosis and Endocytosis of Silver Nanoparticles Induce Interleukin-8 Production in Human Macrophages 
Yonsei Medical Journal  2012;53(3):654-657.
Phagocytosis or endocytosis by macrophages is critical to the uptake of fine particles, including nanoparticles, in order to initiate toxic effects in cells. Here, our data enhance the understanding of the process of internalization of silver nanoparticles by macrophages. When macrophages were pre-treated with inhibitors to phagocytosis, caveolin-mediated endocytosis, or clathrin-mediated endocytosis, prior to exposure to silver nanoparticles, Interleukin-8 (IL-8) production was inhibited. Although cell death was not reduced, the inflammatory response by macrophages was compromised by phagocytosis and endocytosis inhibitors.
doi:10.3349/ymj.2012.53.3.654
PMCID: PMC3343436  PMID: 22477013
Silver nanoparticles; macrophages; endocytosis; phagocytosis; IL-8
19.  Effect of Complement Fixation on the Release of Lysosomal Enzymes from Rabbit Alveolar Macrophages 
Infection and Immunity  1978;19(3):799-806.
Macrophage lysosomal enzymes that have specificities for substrates found in mammalian tissue may contribute to the tissue damage observed in chronic inflammatory diseases. Although a variety of agents that stimulate the release of hydrolases from peritoneal macrophages have been identified, little work has been done to establish the conditions and specific stimuli responsible for enzyme release by alveolar macrophages (AM). To assess the effect of phagocytosis by AM on the release phenomenon, hydrolytic enzymes normally sequestered in AM lysosomes were quantified in the supernatant fluids of phagocytosing and non-phagocytosing AM monolayers in both the presence and absence of serum. Maximum release occurred under conditions known to favor complement fixation by the classical or alternative pathways. Thermal destruction or immune fixation of serum complement before use in culture reduced the magnitude of release to that observed in cultures incubated in the complete absence of serum. Phagocytosis was not essential for release, since cells exposed to particles but treated with a known inhibitor of phagocytosis (cytochalasin B) still showed maximal release in the presence of fresh serum. These data are interpreted to indicate that a heat-labile component of serum, probably a by-product of complement fixation, was primarily responsible for the hydrolase release by AM. On the basis of these findings, individuals suffering from chronic pulmonary disease may suffer acute episodes of lung destruction from endogenous AM enzymes upon the inhalation of materials capable of fixing complement.
PMCID: PMC422259  PMID: 25242
20.  Opsonin-reversible resistance of Mycoplasma pneumoniae to in vitro phagocytosis by alveolar macrophages. 
Infection and Immunity  1975;11(3):540-550.
Several species of mycoplasmas are responsible for respiratory disease in animals and man. As yet, little is known about the interaction of these pathogens with alveolar macrophages, one of the primary components of pulmonary resistance to infections. The present study was undertaken to develop an in vitro model to examine this organism-cell interaction, using a human pathogen, mycoplasma pneumoniae, and normal guinea pig alveolar macrophages. During a 24-h incubation of M. pneumoniae with a monolayer of macrophages, mycoplasmas were found to attach directly to the surface of the cells without inducing significant phagocytosis. Ultrastructurally, the organisms appeared bound to the cell membrane by their characteristic attachment organelles. Only after the addition of specific anti-mycoplasma serum were cells able to engulf attached and surrounding organisms. These data suggest that the interaction of M. pneumoniae and alveolar macrophages is a potentially important aspect of disease pathogenesis, and immune factors which might alter this interaction merit further examination.
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PMCID: PMC415099  PMID: 1090535
21.  THE INFLUENCE OF ERYTHROPHAGOCYTOSIS ON THE INTERACTION OF MACROPHAGES AND SALMONELLA IN VITRO 
Phagocytosis and killing of Salmonella typhimurium by mouse peritoneal macrophages was inhibited when the bacteria and antibody-coated homologous erythrocytes or heterologous erythrocytes were simultaneously exposed to macrophages in vitro. No inhibition of phagocytosis or killing was observed in experiments employing uncoated or disrupted antibody-coated homologous erythrocytes. Degradation of S. typhimurium as measured by the loss of fluorescence from intracellular salmonella coated with fluorescein-labeled antibody was inhibited in macrophages which had previously ingested antibody-coated homologous erythrocytes. Anti-mouse-erythrocyte serum was found to have a cytotoxic action on mouse macrophages. However, the viability of macrophages was not altered by phagocytosis of antibody-coated homologous erythrocytes or uncoated heterologous erythrocytes.
PMCID: PMC2180469  PMID: 5331583
22.  Connective tissue proteins and phagocytic cell function. Laminin enhances complement and Fc-mediated phagocytosis by cultured human macrophages 
Brief exposure of culture-derived human macrophages to laminin, a glycoprotein component of all mammalian basement membranes that has a molecular weight of 1,000,000, led to enhancement of subsequent macrophage phagocytosis of EAC4b, EAC3bi, and EAIgG (sheep erythrocytes sensitized with IgG anti-Forssman antibody). This effect on macrophage phagocytosis occurred with both substrate-adherent and fluid phase laminin. Preincubation of macrophages, but not of EAC4b, with laminin led to augmentation of phagocytosis, suggesting that interaction with the phagocytic cell, but not with the opsonized particle, was required for laminin's effect. Laminin-stimulated phagocytosis of EAC4b was blocked entirely by a monoclonal antibody to CR1. Direct comparison of the phagocytic ability of macrophages adherent to laminin- and fibronectin-coated glass slides showed that fibronectin had a somewhat greater enhancing effect on phagocytosis. Nonetheless, the phagocytosis- enhancing effect of laminin was not due to contamination of the purified laminin preparation by fibronectin, since the laminin preparation was free of fibronectin, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay; in addition, laminin-enhanced phagocytosis was decreased in the presence of laminin-specific antibodies. Laminin inhibited macrophage adherence and spreading, but selection of a laminin-binding macrophage subpopulation could not account for the laminin-induced increases in phagocytosis. We hypothesize that interaction with extracellular matrix proteins may represent an important activation stimulus both to the macrophages normally present in the extravascular compartment and to the phagocytic cells that have emigrated from the blood-stream into areas of inflammation.
PMCID: PMC2187595  PMID: 3157764
23.  Macrophage-Specific Targeting of Isoniazid Through Mannosylated Gelatin Microspheres 
AAPS PharmSciTech  2011;12(3):900-908.
Active targeting of drug molecules can be achieved by effective attachment of suitable ligands to the surface of carriers. The present work was attempted to prepare mannosylated gelatin microspheres (m-GMs) so as to achieve targeted delivery of isoniazid (INH) to alveolar macrophages (AMs) and maintain its therapeutic concentration for prolonged period of time. Microspheres were prepared by emulsification solvent extraction method and evaluated for physicochemical characteristics, drug release, ex vivo drug uptake by AMs and pharmacokinetic characteristics. Fourier transform infrared spectroscopy and nuclear magnetic resonance spectral analysis confirmed that mannosylation took place through Schiff base formation between aldehyde and amino groups of mannose and gelatin, respectively. Prepared microspheres offered suitable physicochemical characteristics for their delivery to AMs. Their average size was about 4 μm and drug entrapment efficiency of 56% was achieved with them. Ex vivo uptake results indicated that in comparison to plain microspheres, m-GMs were selectively uptaken and were found to be associated with phago-lysosomal vesicles of AMs. Pharmacokinetic studies showed the formulation could maintain the therapeutic concentration of INH for prolonged period of time even with a reduced clinical dose. m-GMs were found to be stable in broncheo-alveolar lavage fluid. The study concluded that ligand decorated carriers could be a potential strategy to improve the therapeutic properties of INH.
doi:10.1208/s12249-011-9654-6
PMCID: PMC3167252  PMID: 21732158
active targeting; alveolar macrophages; isoniazid; mannosylated gelatin microspheres
24.  Quantitative reverse transcription-PCR analysis of Legionella pneumophila-induced cytokine mRNA in different macrophage populations by high-performance liquid chromatography. 
Cytokine production in macrophages infected by bacteria is critical for the course of infection. However, it is not known how infection of macrophages with opportunistic bacteria leads to cytokine production in different populations of cells. Since it is possible that cytokine genes may be differentially regulated by attachment rather than by active infection, the levels of various cytokine mRNAs were measured in alveolar macrophages (AMs), peritoneal resident macrophages (RMs), and peritoneally elicited macrophages (EMs) interacting with Legionella pneumophila by using cytochalasin D-treated macrophages and a newly developed quantitative reverse transcription-PCR procedure with high-performance liquid chromatographic analysis to determine cytokine mRNA formation. Increased levels of interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and macrophage inflammatory protein 2 mRNAs were quantitated in the macrophages responding to L. pneumophila attachment in vitro. Using this technique, we showed that the three different macrophage populations responded differently to bacterial attachment. We found that the levels of IL-6 and granulocyte-macrophage colony-stimulating factor mRNAs induced by the attachment of L. pneumophila to AMs were significantly lower than the levels in RMs but similar to the levels in EMs. Furthermore, the levels of MIP-2 mRNA in the AMs were found to be higher than those in the RMs, but similar levels were found in EMs. IL-1 beta mRNA levels were higher in both AMs and RMs than in EMs, but tumor necrosis factor alpha levels were not different among the three macrophage populations examined. Thus, the responses of macrophages to bacterial attachment in terms of cytokine mRNA levels were readily quantitated by the reverse transcription-PCR assay. However, the results obtained showed different levels of responsiveness of distinct macrophage populations to L. pneumophila attachment, and this could be related to the characteristic nature of the macrophage type examined.
PMCID: PMC170094  PMID: 7719907
25.  Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages 
The Journal of Cell Biology  1991;115(1):59-66.
Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium [( Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca2+]i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system. The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 +/- 7 vs. 27 +/- 7, P less than 0.01). Phagocytosis of IgG- coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis. No calcium transients were detected in cells that bound but did not phagocytose beads. Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected: (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc receptor-mediated phagocytosis (69.9 +/- 10.2 vs. 48.7 +/- 4.7 s, P less than 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 +/- 43 vs. 349 +/- 53 nM, P less than 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the signaling pathways may differ for Fc receptor-mediated and nonspecific phagocytosis by elicited murine macrophages.
PMCID: PMC2289912  PMID: 1918139

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