Browsing by Subject "Macrophages"
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Item Activation of macrophages by peroxidases(Texas Tech University, 1986-05) Lefkowitz, Doris LynneThe role of peroxidase in biological systems is not well understood. The specific aims of the present study were to determine (a) if peroxidases are able to stimulate the production of superoxide by macrophages; (b) if peroxidases are able to activate macrophages to the tumoricidal state; (c) if so, is the cytotoxic triad of peroxidase, HgOg, and a halide ion responsible for promoting such activation; and (d) if simple heme-type compounds can replace peroxidases in promoting such activation. The results obtained demonstrated that various peroxidases (horseradish peroxidase, lactoperoxidase, microperoxidase) can stimulate both the respiratory burst and promote the inhibition of tumor cell growth by macrophages in a dose-dependent manner. Similar results were obtained using an immobilized peroxidase. The addition of iodide did not markedly affect either process, but the addition of a peroxidase substrate caused a significant increase in the tumoricidal activity of the macrophages. Furthermore, the tumoricidal activity of peroxidase-activated macrophages was inhibited in the presence of cytochrome c, indicating a requirement for superoxide. Hemin and hematoheme were unable to stimulate either process in macrophages, indicating that the presence of peroxidative activity is a requirement to obtain a stimulation of either process. These results suggest that enzymatically active peroxidases are able to stimulate the respiratory burst as well as induce tumoricidal activity in thioglycollate-induced peritoneal macrophages. The fact that certain compounds, such as interferon, were able to elicit macrophage-mediated tumor cell inhibition without affecting the respiratory burst, suggests that activation of the respiratory burst may not be a prerequisite for tumor cell inhibition. Furthermore, the activation of macrophages is not promoted by the cytotoxic triad, but rather may be promoted by a product of the peroxidative activity of the peroxidases, possibly a free radical species.Item Acyloxyacyl Hydrolase: Studies on Its Regulation and Function in Mus Musculus(2004-01-14) Lu, Mingfang; Munford, Robert S.Acyloxyacyl hydrolase (AOAH) is an enzyme that detoxifies Gram-negative bacterial lipopolysaccharides (LPS) by selectively removing secondary acyl chains from the lipid A moiety. Originally found in neutrophils, it is also produced by monocyte-macrophages and renal proximal tubule cells. In the studies described here, I found that both immature dendritic cells (DCs) of the XS52 cell line and bone marrow-derived DCs produce AOAH. AOAH expression decreased when DCs were incubated with IL-4, IL-1ᬠTNFa and an agonistic CD40 antibody (maturation cocktail), and increased following treatment with microbial agonists that engage 3 distinct Toll-like receptors (LPS, TLR4; CpG oligodeoxynucleotides, TLR9; and a Gram-positive bacterium (Micrococcus luteus), TLR2). Maturation cocktail treatment also diminished, while LPS treatment enhanced or maintained, the cells' ability to kill E. coli, deacylate LPS, and degrade bacterial proteins. Enzymatic deacylation of LPS is thus an intrinsic, regulated mechanism by which DCs may modulate host responses to this potent bacterial agonist. To study the biological functions of AOAH, AOAH-deficient mice were generated by targeted gene disruption. AOAH did not protect mice from lethal doses of LPS or Gram-negative bacterial challenge. In response to subcutaneous injections of LPS, however, AOAH-deficient mice produced significantly higher levels of non-specific (polyclonal) IgM and IgG3 than did wild type mice. Anti-double-stranded DNA and anti-nucleosome IgM and IgG antibody levels were also higher in LPS-immunized AOAH-deficient mice than in wild type control mice. In addition, the partially-deacylated LPS product (dLPS) induced lower polyclonal antibody responses in vivo than did mock-treated LPS, yet the anti-LPS specific responses to dLPS and LPS were equivalent. These results suggest that AOAH may diminish potentially harmful polyclonal antibody responses to Gram-negative infection but maintain the protective anti-LPS specific response. Since B cells do not produce the enzyme, my results also point to an important role for macrophages and DCs in modulating B-cell responses to LPS antigens. In addition, the absence of AOAH did not alter the ability of LPS to function as an adjuvant, indicating that this activity is mechanistically distinct from stimulation of polyclonal antibody production. Finally, the ability of a bacterial lipopeptide to stimulate polyclonal antibody production only in AOAH -/- mice suggests that the enzyme may also regulate immune responses to non-LPS bacterial agonists.Item Characterization of atherosclerotic plaques using ultrasound guided intravascular photoacoustic imaging(2011-05) Wang, Bo, 1981-; Emelianov, Stanislav Y.; Sokolov, Konstantin; Smalling, Richard; Litovsky, Silvio; Dunn, Andrew; Aglyamov, SalavatRupture of atherosclerotic plaque is closely related to plaque composition. Currently, plaque composition cannot be clinically characterized by any imaging modality. The objective of this dissertation is to use a recently developed imaging modality – ultrasound-guided intravascular photoacoustic (IVPA) imaging – to detect the distribution of two critical components in atherosclerotic plaques: lipid and phagocytically active macrophages. Under the guidance of intravascular ultrasound imaging, spectroscopic IVPA imaging is capable of detecting the spatially resolving optical absorption property inside a vessel wall. In this study, contrast in spectroscopic IVPA imaging was provided by either the endogenous optical property of lipid or optically absorbing contrast agent such as gold nanoparticles (Au NPs). Using a rabbit model of atherosclerosis, this dissertation demonstrated that ultrasound guided spectroscopic IVPA imaging could simultaneously image lipid deposits as well as macrophages labeled in vivo with Au NPs. Information of macrophage activity around lipid rich plaques may help to identify rupture-prone or vulnerable plaques. The results show that ultrasound guided IVPA imaging is promising for detecting plaque composition in vivo. Clinical use of ultrasound guided IVPA imaging may significantly improve the accuracy of diagnosis and lead to more effective treatments of atherosclerosis.Item Cytokine detection in eiav-infected equine monocyte-derived macrophages using quantitative real-time polymerase chain reaction(2009-05-15) Allen, Charlotte AnnetteThe replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1?, IL-1?, IL-6, IL-8 and TNF-? were validated using QPCR primers and probes which were generated for the aforementioned equine genes.Item The development of immunomodulatory approaches to restore skeletal muscle function after injury(2015-05) Rybalko, Viktoriya Yurievna; Farrar, Roger P.; Suggs, Laura J; Brothers, Robert M; Thompson, Wesley J; Adamo, Martin LEfficient restoration of skeletal muscle function after severe injury is a major goal of intervention therapies. Ischemia/reperfusion (I/R) injury to skeletal muscle leads to exaggerated inflammatory response and significant ultrastructural tissue damage slowing restoration of muscular structure and function. Herein, we used animal model of tourniquet-induced ischemia/reperfusion injury (TK-I/R) to test the effects of exogenously delivered growth factors and cells on skeletal muscle regeneration. The delivery of PEGylated fibrin along with stromal cell derived factor-1α and/or insulin-like growth factor-I into acutely injured muscle, differentially affected functional muscle regeneration. These data suggest that local balance and release kinetics of growth factors in the tissue microenvironment can significantly impact the success of skeletal muscle repair. Cell-mediated treatment of I/R-injured muscle demonstrated significant tissue regeneration using adoptively transferred and in vitro polarized macrophages. Functional activation status of transplanted macrophage populations impacted the outcome of muscle repair. We showed that increasing macrophage populations at the site of injury in temporally regulated manner is beneficial for efficient recovery of muscle force and function.Item Effect of selenium, riboflavin, and vitamin E on oxygen radical production and cytotoxic activity of peritoneal macrophages of Balb/c mice(Texas Tech University, 1994-05) Kang, ChunranIn nutrition science, we learn that many foods that are healthful in reasonable portions can also lead to ill health when eaten in excess. One can eat too much of almost anything. For example, vitamin A as retinol, when consumed in excess, has toxic effects, however, this vitamin is also clearly an essential nutrient for vision (Olson, 1984). When we realize that water is the most essential of the nutrients, since we cannot survive much longer than three days without it, then consider that without oxygen human beings will live less than a few minutes. However, at any level of dietary supplementation meeting essential requirements, oxygen is both toxic and carcinogenic. In this context oxygen demonstrates major toxicity and carcinogenicity at the same levels which are required for support of life. Oxygen toxicity can cause many cellular dysfunctions, including deactivation of essential enzymes by oxidation, with primary effect on the oxidation of cysteine thiols producing disulfides. It also involve effects of cellular mediators and secretions (Huber and Drath, 1981), and lipid peroxidation (Allen et al., 1973). The toxicity of lipid peroxides by disruption of the cellular membrane is the primary mediators of oxygen toxicity. Therefore, the basic schemata for lipid peroxidation functions as follows.Item Electrophoretically decellularized xenogeneic extracellular matrix for large volume skeletal muscle regeneration(2015-08) Merscham, Melissa Marie; Farrar, Roger P.; Suggs, Laura; Thompson, Wesley; Brothers, Robert; Baker, AaronLarge volume skeletal muscle injuries, such as those that occur through traumatic or surgical means, are complex injuries that are unable to repair through the body’s native repair processes. These injuries, termed volumetric muscle loss (VML), result in fibrotic scar tissue formation and functional impairments. Within the last decade, there has been an immense push towards bioscaffold research and development to regenerate functional skeletal muscle tissue in VML injuries. The most promising bioscaffold is the use of a decellularized skeletal muscle-derived extracellular matrix (ECM). However, the use of skeletal muscle derived ECMs to replace lost tissues is limited by the inability to produce ECMs of clinically relevant sizes and shapes. Therefore, the purpose of this study was to develop an electrophoresis-based decellularization method that can render large volumes of porcine skeletal muscle ECM acellular time while also retaining the native ECM ultrastructure. Analysis of the resulting decellularized porcine skeletal muscle ECM determined most soluble proteins and DNA were removed, and the collagen framework of the ECM resembled that of native skeletal muscle. The decellularized ECM was implanted into a rodent lateral gastrocnemius (LGAS) VML injury model previously developed in our lab. Repair of the VML injury with the electrophoretically decellularized porcine ECM improved morphology of the LGAS and resulted in myofiber, blood vessel, and nerve growth throughout the ECM implant in vivo, and promoted an M2 macrophage profile. Addition of mesenchymal stem cells (MSCs) to the implanted ECM increased functional recovery, myofiber and blood vessel infiltration, and reduced fibrosis within the ECM implant region compared to saline treated implants 84 days after injury. The direct contribution of the injected MSCs tagged with green fluorescent protein (GFP) to myofiber development was not detected. These data demonstrate an electrophoresis-based decellularization protocol may be a better alternative to produce clinically relevant ECMs that can be used to repair VML injuries, and resulting porcine ECMs serve as a viable platform for muscle regeneration. Additionally, injection of MSCs into the ECM improves myofiber ingrowth, vascularization, and function most likely through modulation of the tissue microenvironment rather than differentiation and fusion into skeletal muscle.Item Exogenous myeloperoxidase enhances bacterial phagocytosis and intracellular killing by macrophages(Texas Tech University, 1995-12) Lincoln, John AnilIt is well documented that myeloperoxidase (MyPo) contributes to the bactericidal activities of neutrophils and monocytes. Since mature macrophages (M0) are devoid of this enzyme, its participation in M0-mediated phagocytosis and bacterial killing has not been completely defined. The present study demonstrates that exogenously added MyPo, at physiological levels, enhances both phagocytosis and killing of Escherichia coli (E. coli). Murine peritoneal M0 were exposed to various concentrations of MyPo for different time intervals. Viable opsonized E. coli were added either prior to or after addition of MyPo. Thioglycollate-induced (TG) but not resident M0 exhibited an increase in phagocytosis. Both resident and TG-induced M0 demonstrated increased bactericidal activity. Physiological levels of soluble MyPo also induced a significant increase in both TGinduced and resident M0 chemiluminescence (CL). Since luminol-dependent CL measures reactive oxygen intermediates (ROI) production, studies were done to determine whether superoxide anion or H2O2 were involved in MyPo-induced M0 killing. Both superoxide dismutase and catalase significantly reduced MyPo-induced bactericidal activity. The above data suggest that soluble MyPo, released from neutrophils at a site of infection or inflammation, can enhance both phagocytosis and killing of microorganisms.Item Immunomodulation of macrophage function by recombinant human myeloperoxidase(Texas Tech University, 1995-12) Castro, AaronRegulation of the immune response is necessary for protection of the host as well as the avoidance of autoimmune diseases. The regulation of macrophage function by myeloperoxidase is a previously unrecognized function of this enzyme. A recombinant form of myeloperoxidase, an enzyme commonly found in neutrophils, was used to study the effects of this enzyme on certain macrophage capacities and functions. Both neutrophils and macrophages are present at the site of either inflammation or infection. Stimulation of macrophages by pathogens such as bacteria and fungi, results in production of reactive oxygen intermediates. This study showed that myeloperoxidase induced the production of these reactive oxygen intermediates. Myeloperoxidase was shown to increase the uptake and subsequent killing of a common fungal pathogen, Candida albicans, demonstrating its ability to alter the macrophage response. In addition, these same intermediates act on surrounding tissue and cause damage observed with diseases such as rheumatoid arthritis. The interaction of myeloperoxidase and macrophages at the site of inflanunation could result in the observed chronic inflammatory state seen in rheumatoid arthritis patients. Studies by other investigators have detected myeloperoxidase in the synovial fluids of patients with rheumatoid arthritis. Evidence for the establishment of macrophage-mediated chronic inflammation was provided by studies demonstrating the de novo synthesis and secretion of cytokines by macrophages after exposure to myeloperoxidase. The production of macrophage-derived cytokines, which are powerful mediators of inflammation as well as the immune system, provides further evidence for the macrophage myeloperoxidase interaction in chronic inflanmiation. The role of myeloperoxidase in mediating the immune response, both in a protective function as well as a potentially destructive role, is novel in the study of chronic inflammation.Item The Murine Amniotic Fluid Macrophage: Upregulation of Classical and Alternative Activation Markers Prior to Labor at Term(2010-11-02T18:17:49Z) Montalbano, Alina Peraza; Mendelson, Carole R.The initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (AF) and infiltration of the myometrium by neutrophils and macrophages (MΦ). Whereas, in preterm labor, intra-amniotic infection may provide the inflammatory stimulus for increased AF interleukins and inflammatory cell migration, the stimulus for these events at term has remained unclear. In studies using pregnant mice, we observed that the M that invade the maternal uterus near term originate from the fetus. Furthermore, we obtained compelling evidence that surfactant protein-A (SP-A), a developmentally regulated C-type lectin secreted by the fetal lung into AF near term, activates AF MΦ, which migrate into the pregnant uterus where their local release of interleukin-1 serves to activate nuclear factor kB (NF-kB) pathways. Activation of the NF-kB pathway results in increased expression of genes that promote uterine contractility and negatively impacts the capacity of progesterone receptors to maintain uterine quiescence, culminating in the onset of labor [1]. We propose that interactions of MΦ surface receptors with SP-A, at term, or bacterial lipopolysaccharide at preterm, initiate changes in MΦ phenotypic properties, resulting in the enhanced expression of genes that promote MΦ migration to the uterus. The objectives of this study are: (1) to analyze the phenotypic changes of mouse AF MΦ associated with the developmental induction of SP-A synthesis and secretion by the fetal lung into AF: (2) to determine if SP-A, SP-D, and SP-A/D double deficiencies delay labor at term and to (3) to analyze trafficking patterns of fetal MΦ leading to the induction of labor. The findings presented herein suggest that late gestation AF MΦ upregulate classical and alternative activation markers in tandem as term approaches. Moreover, their phenotypic profile implies that they may modulate both pro- and anti-inflammatory functions simultaneously near term. Trafficking studies using heterozygous fetal-derived amniotic fluid macrophages expressing EGFP under control of the MΦ-specific CSF-1 receptor, demonstrate the absence of this subpopulation in the maternal uterus. Parturition studies in surfactant protein A and -D deficient mice reveal that deficiency in SP-A and –D does not affect the timing of labor. Intriguingly, Toll-like receptor 2 deficient mice demonstrate a delay in the time to parturition pointing to its potential role in mediating a signal for labor at term.Item Peroxidase-mediated oxygenation and microbicidal activity(Texas Tech University, 1999-05) Vigerust, David JohnIt is well documented that a peroxidase, H2O2, and a halide form a "cytotoxic triad." As a result of the interactions of the components of the triad, reactive oxygen intermediates (ROI) are formed and these ROI help to destroy various invading pathogens including Candida. The present study was undertaken to determine if equivalent units of peroxidase activity also induced equivalent macrophage-mediated killing of Candida. Peritoneal macrophages were obtained from age matched C57BL/6J mice and exposed to various concentrations of eosinophil peroxidase (EPO), myeloperoxidase (MPO), and horseradish peroxidase (HRP). Equivalent units of peroxidase as determined by oxidation of guaiacol, did not induce equivalent production of ROI. Luminol-dependent chemiluminescence studies indicated that 10 units of EPO induced more ROI than either HRP or MPO. Candidicidal activity and phagocytosis of M(t) was measured using a fluorescence acridine orange phagocytosis assay. The following pattern EPO>MPO>HRP emerged for both assays. Therefore, enzymatic activity does not directly correlate with candidicidal activity. These data indicate a distinct order of peroxidases relative to their ability to stimulate chemiluminescence and macrophage-mediated killing.Item Phagocytic and candidacidal activity of murine peritoneal macrophages(Texas Tech University, 1995-05) Gelderman, Monique PascaleMacrophages (Mo) are involved in host defenses against opportunistic pathogens. Previous studies reported that Mo exposed to enzymatically active myeloperoxidase (MYPO), exhibited both increased phagocytosis and killing of Candida albicans (C. albicans). The purpose of tiiis study was to detamine if enzymatically inactive MMR ligands could fimction similarly. Resident murine peritoneal Mo were exposed to tiie MMR ligands, mannosylated bovine serum albumin (mBSA) and enzymatically inactive myeloperoxidase (iMYPO), followed by exposure to opsonized C. albicans. Both mBSA and LMYPO enhanced phagocytosis and killing of C albicans over that of the control. The production of reactive oxygen intermediates (ROI) was detected using chemiluminescence. After employment of ROI scavaigers, a decrease in candidicidal activity was observed. The data suggest that MMR-ligand interaction enhances both phagocytosis and candidicidal activity of Mo and that MYPO and iMYPO released at a site of inflammation mduce Momediated candidicidal activity.Item Polymannan enhancement of macrophage function(Texas Tech University, 1999-12) Gnade, Bryan ThomasPrevious studies have shown that mannosylated bovine semm albumin (mBSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans and Escherichia coli by resident murine peritoneal macrophages (M0). Upregulation of the above M0 functions was associated with binding of mBSA to the macrophage mannose receptor. The present study was done to detennine if certain polymannans, and other glyconutrients could stimulate M0 functions in a similar manner. Resident peritoneal murine M0 collected from C57BL/6 mice were exposed to the glyconutrients for 10 and 60 minutes. The RB was measured using chemiluminescence. Both phagocytosis and killing were measured after incubation with one of the following microorganisms: Candida albicans, Escherichia coli and S. aureus. The percent phagocytosis and killing were determined using fluorescence microscopy. Results indicated little or no effect of four of these compounds on phagocytosis and killing Compound 3, which was identified as Ambrotose, caused a dose and time dependent effect on M0 induced killing of all 3 microorganisms.Item The Role of Interferon-Gamma, Cd4-Positive T Cells, and Cd8-Positive T Cells in the Immune Rejection of Intraocular Tumors(2007-05-22) Dace, Dru Samuel; Niederkorn, JerryAlthough the eye is an immune privileged site, intraocular tumors can sometimes be rejected by the immune system. Ad5E1 is an adenoviral gene-transformed tumor cell line that is rejected by the immune system when transplanted into the anterior chamber of the eye in syngeneic C57BL/6 mice. Two components of the immune system have been shown to be absolutely necessary for intraocular tumor rejection: CD4+ T cells and IFN-gamma , as mice deficient in these components cannot reject the tumor. This study further examined the role of IFN-gamma and CD4+ T cells in mediating rejection of intraocular Ad5E1 tumors, and examined the ancillary role of CD8+ T cells. IFN-gamma acted directly on Ad5E1 tumor cells and not host cells. The anti-tumor effects of IFN-gamma were multiple, as IFN-gamma induced tumor cell apoptosis and inhibited tumor cell proliferation. Also, IFN-gamma promoted tumor rejection by inhibiting angiogenesis, since IFN-gamma KO mice demonstrated increased tumor blood vessel density and IFN-gamma induced the up-regulation of 5 anti-angiogenic genes and the down-regulation of 4 pro-angiogenic genes in Ad5E1 tumor cells. CD4+ T cells infiltrated intraocular Ad5E1 tumors. Following rejection of Ad5E1 tumors in C57BL/6 mice, CD4+ T cells could be adoptively transferred to SCID mice and induce Ad5E1 tumor rejection. CD4+ T cell derived IFN-gamma might mediate tumor rejection, as these cells produce significant levels of IFN-gamma in response to Ad5E1 tumor antigens. Macrophages were necessary for CD4+ T cells to mediate rejection, as mice depleted of ocular macrophages developed progressively growing intraocular Ad5E1 tumors. CD8+ T cells were not required for rejection, as CD8+ T cell-depleted and CD8+ T cell KO mice rejected Ad5E1 tumors. However, CD8+ T cells infiltrated intraocular Ad5E1 tumors. Following rejection in C57BL/6 mice, CD8+ T cells could be adoptively transferred to SCID mice and protected them from Ad5E1 tumor growth, similar to the effect produced by adoptively transferred CD4+ T cells. In attempting to ascertain what CD8+ T cells utilize to mediate Ad5E1 tumor rejection, I determined that TNF-alpha was required for tumor rejection, but IFN-gamma , FasL, perforin, TRAIL and CTL activity were not necessary for CD8+ T cell-mediated tumor rejection.Item Testicular macrophages: isolation, characterization, and hormonal responsiveness(Texas Tech University, 1984-08) Yee, James BNot availableItem The effect of cocaine on murine peritoneal macrophages(Texas Tech University, 1995-05) Clark, Jonathon CaryIn recent years, cocaine has had a resurgence in popularity in the United States (Adams and Durell, 1984; Katz, 1985). The increased use of cocaine has created increased concern with the problems involved with cocaine use within the United States. The scientific community in particular has become concerned with the negative effects of cocaine on the human body, particularly the immune system (Pillai, Nair, and Watson, 1991). At the central core of the immune system are the antigen presenting cells. Antigen presenting cells include: Langerhan cells, B cells, and Macrophages (M0's). The M0 has been the subject of many studies and has been shown to participate in phagocytosis and cytokine secretion. The various cytokines secreted by M0's are required for immune cellular maturation and function. This study focuses on the effects of cocaine on the macrophage in terms of cytokine secretion and phagocytosis as a model of cellular immune function.Item The effects of cocaine on viral replication in immune cells(Texas Tech University, 1996-05) Brown, Deanna JoPrevious studies have shown that cocaine is capable of altering a number of macrophage functions. The present study demonstrates that the incubation of murine peritoneal macrophages with cocaine inhibits the replication of mouse hepatitis virus (MHV). Incubation of mouse L929 ceUs with cocaine inhibits both MHV and vesicular stomatitis virus (VSV). Monolayers of thioglycoUate-induced peritoneal macrophages from C57BL/6 mice or L ceUs cultured on 96-weU microtiter plates were incubated with various concentrations of cocaine and subsequently infected with one of the above viruses. Replication of virus, as determined by plaque forming units, was inhibited in a dose dependent manner foUowing incubation with cocaine. This inhibition was also dependent on the length of time ceUs were exposed to cocaine. The antiviral ffect was greatest in cultures exposed to cocaine for at least 48 hours and could be transferred by culture media to fresh cells resulting in a 60%-80% inhibition of virus replication. Results were essentially the same whether or not the cell were dividing (L929) or nondividing (macrophage). The addition of polyclonal antibodies to interferon 6 partiaUy reversed this inhibition, whereas antibodies to either tumor necrosis factor or transforming growth factor 6 did not. Further studies are needed to identify the mechanisms involved; however, preliminary data obtained suggest alterations of intracellular calcium as a possible cause of viral inhibition. These studies underscore the multiple effects of cocaine on many biological systems.Item The interaction of presumptive osteoclastic precursors with mineralized bone matrix in vitro(Texas Tech University, 1978-08) Shew, Ronald LewisIn order to investigate the possibility that macrophagic- monocytic cells may be progenitors of osteoclasts, peritoneal macrophages from young rats were harvested and cultured for 2, 8, 24, 48 and 72 hours with treated calvaria. Glass coverslips served as the control substrate. Cultures were examined using phase contrast and bright-field light microscopy and scanning and transmission electron microscopy. The morphology of peritoneal macrophages cultured on glass has been described by numerous investigators. With phase contrast microscopy and scanning electron microscopy, these cells demonstrated a variety of shapes and sizes at all incubation periods. Their surfaces were covered with microvilli and ruffles. Lamellapodia were observed at the edges of cells. In cultures containing bone the morphology of peritoneal macrophages did not differ significantly from those observed on glass cover slips. With light and transmission electron microscopy the cells appeared macrophagiclike. These cells attached to the mineralized bone matrix. These results indicate that peritoneal macrophages interact with mineralized matrix in culture. However, under these culture conditions after 72 hours, cells lacked morphological evidence of differentiation into osteoclasts.Item The macrophage: orchestrator of the immune response(Texas Tech University, 1997-12) Gelderman, Monique PascalePhagocytosis is one of the first mechanisms employed by the host to eliminate an invading pathogen. Macrophages (M0) and neutrophils (PMN) are capable of phagocytosis. Neutrophils contain the enzyme myeloperoxidase (MPO) whereas mature M0 are devoid of MPO. It is well-documented that PMNs release MPO into the microenvironment at a site of infection or inflammation where a high percentage of MPO becomes enzymatically inactive (iMPO). Macrophages can acquire MPO via binding of MPO to the M0-mannose receptor. Although the function of MPO in the cytotoxic triad is well documented, the immunoregulatory role of this enzyme has not been elucidated. In this study, in vitro studies were done to determine if either MPO or iMPO could activate M0. The results indicated that M0 exposed to either MPO or iMPO enhanced phagocytosis of Candida albicans as well as M0 candidacidal activity. Another goal of this project was to determine the possible role of MPO or iMPO in rheumatoid arthritis (RA). Resident peritoneal M0 from female Lewis rats were incubated with MPO or iMPO. Subsequently, the supematants were assayed for cytokines associated with RA. These peroxidases activated M0 to secrete pro-inflammatory cytokines. Subsequently, an in vivo rat model was employed. If MPO was injected into an ankle joint of the rat, it did not induce RA. However, if RA was first induced using cell wall fragments derived from group A Streptococci, re-injection of these joints with either MPO or iMPO caused an exacerbation of RA. This exacerbation could be blocked if either anti-tumor necrosis factor alpha or mannan were injected simultaneously with the peroxidase. In addition, the effect of this enzyme on cytokine secretion by endothehal cells, which are present in the joints, was examined. Both forms of the enzyme induced the secretion of pro-inflammatory cytokines. These results taken in their entirety indicate that there is a previously unrecognized interaction between M0 and PMNs in which MPO: (1) activates M0 to be more cytocidal, (2) induces the secretion of pro-inflammatory cytokines, and (3) helps to perpetuate the inflammatory state associated with RA.Item Transcriptional Profiling of Polarized Macrophages using RNA-Sequencing(2015-04-17) Kanameni, SrikanthAdipose tissue macrophages (ATMs) are pivotal regulators for adipose tissue function, specifically contributing to the homeostasis of the adipose niche. Significantly increased ATMs and their altered activation patterns are causal factors to the pathogenesis of adipose tissue inflammation, and subsequently, obesity associated cardiovascular risks, type II diabetes and other metabolic syndromes. Macrophages primarily display an anti-inflammatory M2 status in lean adipose tissues whereas a pro-inflammatory M1 state in adipose tissues of obese individuals. Modulatory networks governing ATMs polarized activation have been investigated but the full picture remains vague. To understand the genome wide signaling networks in controlling ATM polarization, we generated transcriptome profiles from macrophages with various activation statuses- M0, M1 and M2. Among 23400 aligned unique loci from the RNA-sequencing results, around 3500 displayed differential expression pattern during macrophage polarization. The most enriched Gene Ontology terms in the category of KEGG pathways are allograft rejection and Type I diabetes mellitus pathways in M1 macrophages. IFNg was found to be one of the top upstream regulator in M1 playing pivotal role in different functional pathways. In addition, the anti-inflammatory regulator miR-223 was found to be one of top upstream regulator in M2 datasets and playing role in important functional pathways. Understanding of the complex network of interactions among different factors involved in state of polarization of macrophages would be of great advantage in finding solutions to major health issues.