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The role of inflammation in Alzheimer's disease, Parkinson's disease, and multiple sclerosis has recently come under increased scrutiny. Associated with these inflammatory responses are tumor necrosis factor-alpha (TNF-alpha) and reactive oxygen species (ROS), both believed to be derived from brain microglia. In addition to the above, the presence of myeloperoxidase (MPO) in these diseased brains has been reported by a number of investigators. However, the possible role of MPO and enzymatically inactive MPO (iMPO) as the "choreographers" of the destruction done by TNF-alpha and ROS is not generally recognized. Previously, our laboratory has reported that MPO/iMPO enhance macrophage generation of ROS and expression of proinflammatory cytokine genes as well as gene products. Recent studies in our laboratory indicate that the same response occurs with microglia. A paradigm is presented for the perpetuation of inflammation associated with neurodegenerative diseases. This model describes the unrecognized consequences of the stimulation of microglia by MPO or iMPO. Both MPO and iMPO and/or its receptor may represent new therapeutic targets for the treatment of these diseases.

Summary Background: Facioscapulohumeral muscular dystrophy (FSHD) is the 3rd most common form of muscular dystrophy. Effective treatments for any of the muscular dystrophies have yet to be realized. This report describes such a treatment. Case Report: A 66 year old female was diagnosed with osteoporosis. She had been diagnosed with FSHD muscular dystrophy a number of years previously by both genetic and clinical studies. Following a 2 year course with Forteo for osteoporosis, she was given an injection of Denosumab (Prolia) to maintain her bone density. By 24 hours, she exhibited increased strength and a dramatic reduction of her dystrophic symptoms e.g. she could walk unassisted in high heels. She was able to accomplish other things that had not been possible for a number of years. After approximately 5 weeks she gradually lost the newfound strength with a complete loss by about 6 weeks. A second injection of Denosumab resulted in the same effect, i.e. reversal of symptoms and increased functionality. A number of measurements and videos were taken to establish the beneficial effects of Prolia for future studies. This was repeated with a 3rd and 4th injection in order to establish the unequivocal beneficial effects on muscular dystrophy. Conclusions: Further studies will be required to establish Denosumab as a major “front line” treatment for this disease and possibly other muscular dystrophies.

Inflammation of the lung is characterized by the influx of increased numbers of various leukocytes including polymorphonuclear leukocyte (PMN) neutrophils. In addition to cells, numerous studies have pointed to the role of tumor necrosis factor-alpha in the inflammatory process. This study addresses a previously unrecognized interaction between neutrophil-derived myeloperoxidase (MPO) and resident alveolar macrophages (AMø). Rat AMø exposed to either enzymatically active recombinant MPO or enzymatically inactive MPO (iMPO) exhibited an increased respiratory burst (RB). When iMPO was employed, the enhancement of the RB was greater than that observed with MPO. Although the RB was greater with iMPO, macrophage (Mø)-mediated intracellular candidic activity was equivalent for both MPO and iMPO. It is known that pro- inflammatory cytokines contribute to the inflammatory process. When rat AMø were exposed to both forms of myeloperoxidase, iMPO demonstrated greater upregulation of cytokine genes as well as product. These data suggest that at the site of inflammation, neutrophil-derived MPO and iMPO stimulate AMø, resulting in an increased inflammatory and cytotoxic state, and thereby contributing to the general lung inflammatory response.

The endothelium is frequently exposed to many proinflammatory mediators. The present study was done to determine the effects of human recombinant myeloperoxidase (MPO) and porcine eosinophil peroxidase (EPO) on certain endothelial cell (HUVEC) functions. The following areas were evaluated: (1) production of reactive oxygen intermediates (ROI), (2) cytokine secretion, and (3) regulation of mRNA cytokine transcripts. Both MPO and EPO induced the production of ROI, but an enzymatically inactive form of MPO (iMPO) was the most effective. Enzymatically inactive MPO, but not MPO, induced the secretion of interleukins 6 and 8 and granulocyte-monocyte colony-stimulating factor. A ribonuclease protection assay indicated that both iMPO and MPO upregulated mRNA cytokine transcripts; however, the former was markedly more effective. The simultaneous addition of EPO and iMPO resulted in a decrease in cytokine-specific mRNA. These data indicate a major role for peroxidases in the regulation of inflammation.

Previous studies have shown that mannosylated bovine serum albumin (mBSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans and Escherichia coli by resident murine peritoneal macrophages (M φ ). Upregulation of the above M φ functions was associated with the binding of mBSA to the macrophage mannose receptor. The present study was done to determine if certain glyconutrients could stimulate M φ functions in a similar manner. Resident peritoneal murine M φ collected from C57BL/6 mice were exposed to the glyconutrients for 10 and 60 min. The RB was measured using chemiluminescence. Both phagocytosis and killing were measured after incubation with each of the following microorganisms: Candida albicans, Escherichia coli and Staphylococcus aureus . The percent phagocytosis and killing were determined using fluorescence microscopy. Results indicated that certain glyconutrients, caused a dose and time dependent effect on M φ -induced killing of all three microorganisms.

It is known 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 that help to destroy various invading pathogens including Candida. The present study was undertaken to determine if equivalent units of peroxidase induced equivalent levels of macrophage-mediated killing of Candida. Murine peritoneal macrophages were exposed to various concentrations of eosinophil peroxidase (EPO), myeloperoxidase (MPO), and horseradish peroxidase (HRP). Luminol-dependent chemiluminescence studies showed that equivalent units of peroxidase, as determined by oxidation of guaiacol, demonstrated a hierarchical pattern of ROI production. Macrophage phagocytosis and candidicidal activity, as measured by a fluorescence acridine orange assay, also demonstrated the same hierarchical pattern of EPO > MPO > HRP. Therefore, enzymatically equivalent peroxidases do not demonstrate equivalent candidicidal activity. These data indicate a distinct order of peroxidases relative to their ability to stimulate chemiluminescence and macrophage-mediated killing.

The present investigators have reported previously that macrophages (Mphi) can bind either myeloperoxidase (MPO) or eosinophil peroxidase (EPO) resulting in enhanced cytotoxicity to Candida albicans. Since MPO was shown to be immunomodulatory, the present study was initiated to determine whether either EPO or partially fragmented EPO (fgEPO) also modulated cytokine secretion. Murine peritoneal Mphi; simultaneously stimulated with fgEPO and one of the following, (1) LPS, (2) mannosylated bovine serum albumin (mBSA), (3) interferon-gamma (IFN-gamma), or (4) Poly I:C, demonstrated both dose- and time-dependent decreases in TNF-alpha and IL-6 and a dose-dependent decrease in IFN-alpha/beta. The mRNA levels of Mphi exposed to fgEPO and mBSA demonstrated that fgEPO modulated Mphi cytokine function by decreasing TNF-alpha and IL-6 mRNA transcripts without altering transcription of TGF-beta or GM-CSF. These results demonstrate a possible interaction between the Mphi and eosinophil that could result in reduction of inflammation.

Rheumatoid arthritis (RA) is characterized by an abnormal cellular and cytokine infiltration of inflamed joints. This study addresses a previously unrecognized interaction between neutrophilic-myeloperoxidase (MPO) and macrophages (MΦ) which could explain the perpetuation of inflammation associated with RA. A monoarticular arthritis was induced in female Lewis rats by injection of streptococcal cell wall extracts (PG-APS). After swelling and erythema subsided, joints were re-injected with one of the following: porcine MPO or partially inactivated MPO (iMPO). Injection with either MPO or iMPO induced a 'flare' of experimental RA. Blocking the MΦ-mannose receptor by mannans, ablated exacerbation of disease. These results indicate that MPO or iMPO can play a pivotal role inthe perpetuation but not initiationof this RA model.

Neutrophils represent one of the host's primary defenses against invading organisms. These cells often arrive at the site of infection prior to macrophages (M phi). Neutrophils release myeloperoxidase (MPO) into the micro-environment during phagocytosis. Previous studies by the present investigators have shown that M phi bactericidal activity is enhanced by exposure to MPO. A recent report suggests that as much as 40% of this protein is enzymatically inactive once it is released into the micro-environment. In the present study, exposure of M phi to an enzymatically inactive form of MPO (iMPO) or another mannosylated protein, mannosylated bovine serum albumin (mBSA), can induce the same enhanced Mø-mediated bacterial cell killing observed with the active form of MPO. Furthermore, this phenomenon is limited as galactosylated BSA (gBSA) did not induce enhancement of bacterial killing. The data suggest that interaction of either enzymatically active or inactive mannosylated proteins with the M phi mannose receptor (MMR), is sufficient to enhance M phi bactericidal activity and further underscores the binding of the MMR and resultant responses as a major host defense mechanism.

Various disease states are associated with eosinophilia and the release of eosinophil peroxidase (EPO) into the microenvironment. The present study targets the effects of low levels of EPO on macrophage (MO) phagocytosis and intracellular killing of Candida albicans as well as MO oxidative activity measured as the luminescence product of luminol dioxygenation. Resident murine peritoneal MO were exposed to various concentrations of EPO. Chemiluminescence data indicate that nanomolar concentrations of EPO markedly enhanced the dioxygenation activity (respiratory burst) of MO. In other studies, the exposure of MO to 0.17 μM EPO for 10 min. enhanced MO-mediated candidacidal activity 10 fold. The above data indicate that EPO enhances certain MO functions. Also the results illustrate a previously un-recognized interaction between eosinophils and MO and implicate yet another possible role for EPO in host defenses against disease.

The present study describes the inhibition of macrophage-mediated cytotoxicity (MMC) by cocaine and suggests a possible mechanism. Mice (C57BL/6) were injected i.p. with cocaine. At various intervals after exposure to cocaine, peritoneal macrophages (M phi) were removed, cultured in the presence of interferon gamma and LPS, then incubated with 51Cr labeled target cells. A single injection of > or = 10 mg/kg cocaine was sufficient to inhibit cytotoxicity to P815 cells. This inhibition was evident 3 h after exposure to cocaine and could still be demonstrated 24 h later. Since reactive nitrogen intermediates (RNI) have been reported to be one of the major mechanisms by which M phi kill, the amounts of NO2- produced by M phi from cocaine-injected animals were compared with that produced by equivalent controls. Cocaine reduced the level of NO2- in a dose-dependent manner which correlated with MMC. There was a significant reduction in NO2- produced by activated M phi, 3 h after i.p. injection of cocaine but not at 24 h, using > or = 5 mg/kg. At 12 h there were differences between M phi from control animals and animals receiving > or = 10 mg/kg cocaine. By 24 h there were no differences between control and cocaine-injected animals even at the highest dose employed (25 mg/kg). These results suggest that cocaine reduces the killing ability of murine M phi through a temporary reduction of RNI.

Myeloperoxidase is an enzyme which is found in the azurophilic granules of neutrophils and is associated with bactericidal, fungicidal, and tumoricidal activity. The present studies show that human recombinant myeloperoxidase (rec-MyPo) can regulate a number of macrophage (M phi) capacities and functions. Macrophages from mice exposed to rec-MyPo in vitro released reactive oxygen intermediates, tumor necrosis factor alpha (TNF alpha), and interferon alpha/beta (IFN alpha/beta). Enhanced target cell killing was also demonstrated with TNF alpha sensitive but not TNF alpha insensitive cells. Intravenous injection of rec-MyPo induced high titers of systemic TNF alpha and IFN alpha/beta. These results indicate that MyPo can function as an immunomodulator both in vitro and in vivo. Because of these actions, it is apparent that MyPo represents a previously unrecognized endogenous immunomodulator.

Recent studies by these investigators have shown that horseradish peroxidase (HRP) can cause murine thioglycollate-induced peritoneal macrophages (Mø) to produce both tumor necrosis factor (TNF) and enhance macrophage-mediated cytotoxicity (MMC) to 3T12 target cells. The present study identifies the roles of both enzymatic activity and contaminating lipopolysacharides (LPS) (≤1 ng) on these activities. The addition of 100 ng/ml of polymyxin B (PB) to enzymatically active HRP significantly reduced TNF production but did not affect MMC. Enzymatically inactive HRP (DHRP) was more effective than HRP in both TNF production and MMC but was not affected by PB. The inability of PB to modify DHRP-induced TNF suggests that LPS was not required. The induction of TNF and MMC in the absence of LPS was also corroborated by similar studies using Mø from endotoxin-resistant C3H/HeJ mice. Glycosylated proteins such as HRP, DHRP, and mannosylated bovine serum albumin (M-BSA) are known to bind to mannose receptors (mannosyl-fucosyl receptor [MFR]) on the surface of Mø. In the present studies, M-BSA behaved similarly to DHRP in that it induced both TNF secretion and MMC. These results suggest that binding to the MFR may be sufficient to induce TNF secretion and MMC. In addition, the data suggest that neither enzymatic activity nor LPS was required for DHRP-induced TNF.

Background Toxicity to glucose is not a normal state, however, it has been reported that after certain individuals ingest sugar, they produce excess tumor necrosis factor. Case report An other wise healthy 47 year old male developed a severe sensitivity to foods containing sugar over a 12 year period. After eating certain foods containing sugars he would develop angst, pain in his extremities, fever, etc. Onset was gradual and he has had to eliminate various foods over the years until today he is restricted to eating only certain foods without exception. In a controlled challenge with glucose, tumor necrosis factor was found to be elevated in his serum. Conclusion Because mannose is known to block induction of tumor necrosis factor by macrophages, a regimen of mannose either prior to and/or post development of symptoms minimized these responses. A precise understanding of these events awaits further study.

Cocaine has been demonstrated to have a number of different effects on immune cell functions. We have reported alterations of cellular functions by macrophages (Mφ) exposed to cocaine in vitro, including the inhibition of mouse hepatitis virus replication. Here, we present evidence that cocaine stimulates the secretion of an antiviral product that is neutralized by anti-interferon (anti-IFN). A dose-dependent increase in the secretion of IFN by both Mφ and L929 cells incubated with cocaine, with a concomitant decrease in virus replication, is also reported. The increase in IFN secretion was most pronounced when cells were cultured in the presence of the IFN inducer poly(I·C). The effect of cocaine on IFN production was found to be primarily at the transcript level in both Mφ and L929 cells. These findings further support our previous research demonstrating an antiviral activity of cocaine in vitro. The relevance of this activity to viral infections in general remains to be determined.

Cocaine has been shown to have a number of diverse effects on the immune system. The current investigators have previously demonstrated an inhibitory effect of cocaine on murine hepatitis virus replication in peritoneal macrophages in vitro. The present study was undertaken to examine the effects of cocaine on influenza virus replication and to further characterize that effect in an animal model. Cocaine was capable of inducing a dose-dependent reduction in influenza PR-8 replication using MDCK cells in vitro. Concentrations of 100 μg/ml caused a 50% reduction of virus. To further characterize the effect in vivo, C57Bl/6 mice infected with influenza PR-8 by intranasal instillation were given daily ip injections of 10 mg/kg cocaine just prior to and for 4 days after exposure to influenza. Lungs from mice exposed to cocaine had viral titers that were reduced approximately 50% compared to controls as demonstrated by hemagglutination titers. Additional studies suggest that this reduction appears to be caused by an increase of cocaine-induced interferon.

Rheumatoid arthritis is a systemic disease of unknown etiology. The purpose of this study was to elucidate an unrecognized interaction between neutrophilic myeloperoxidase (MPO) and macrophages (Mphi) which could perpetuate the inflammatory response associated with arthritis. A monoarticular arthritis was induced by intra-articular injection of group A streptococcus cell wall fragments (PG-APS) into the ankle joint of female Lewis rats. After swelling/erythema subsided, joints were reinjected with either recombinant MPO or enzymatically inactive MPO (iMPO). Joint measurements were made daily and arthritis was confirmed by histology. Neither iMPO nor MPO could initiate "clinical" arthritis; however, either form of the enzyme injected after PG-APS induced a dose-dependent increase in erythema and swelling. Mannans, which block the binding of MPO to Mo, ablated clinical symptoms. Also, the presence of tumor necrosis factor alpha was observed only in diseased joints using immunocytochemistry.

Tumor necrosis factor-alpha (TNF) is an extremely potent cytokine which is involved in the pathogenesis of a number of diseases. Interruption of its synthesis can result in a reduction of inflammation and subsequent pathology. A new experimental drug pirfenidone (5-methyl-L-phenyl-2-(1H)-pyridone, trade name: Deskar) has been reported to have beneficial effects for the treatment of certain fibrotic diseases. The present study describes the inhibition of TNF in vitro as well as the inhibition of circulating TNF in vivo by pirfenidone. Isolated, thioglycollate-induced peritoneal macrophages (Mphi) from C57BL/6 mice were exposed to either lipopolysaccharide (LPS) or mannosylated bovine serum albumin then incubated with 0.1-0.9 mg/ml of pirfenidone. This substance inhibited the production of TNF in a dose-dependent manner as measured by ELISA. One i.p. injection of either 100 or 200 mg/kg pirfenidone inhibited the induction of circulating TNF following a single i.v. injection of LPS. Endotoxin shock was induced in mice using an i.p. injection of galactosamine and LPS. The higher dose of pirfenidone (200 mg/kg) completely inhibited shock and subsequent mortality. Lower doses of pirfenidone or administration either prior to or post challenge only partially inhibited symptoms. These results indicate that pirfenidone is able to inhibit both TNF induction and subsequent endotoxin shock. Additional studies are warranted to establish this drug as a potential treatment for diseases where TNF plays a major role.

Numerous natural and synthetic quinone compounds possess significant antitumor properties. Various mechanisms have been proposed to account for these properties, including scission and degradation of tumor cell DNA, intracellular "redox cycling" to cogenerate semiquinone free radicals and reactive oxygen intermediates, and the interaction of semiquinone radicals with tumor cell surface flavoenzymes. However, no evidence has been presented to explain adequately the preferential attack on tumor cells by semiquinone radicals, as opposed to normal cells. To address this question, a synergistic interaction was examined. A therapy consisting of a mixture of L-ascorbate and 2,6-dimethoxy-p-benzoquinone, was used for in vivo evaluation. BALB/c mice were depleted of functional T-lymphocytes by xenogeneic monoclonal antibody pretreatment, challenged with Ehrlich ascites tumor, and administered the semiquinone radical-generating therapy. Mice depleted of CD4+ T-lymphocytes responded with rapidly fatal tumor progression, with significantly decreased mean survival times over controls, whereas less severe responses were observed in mice devoid of CD8+ T-lymphocytes. Mice depleted of both T-lymphocyte subpopulations responded with uninhibited tumor growth and rapid mortalities. When tumor challenge occurred after therapy, tumor growth was significantly delayed in mice enriched for CD4+ T-lymphocytes, with demonstrable increases in mean survival time over controls. This reagent combination had no significant effect on T-lymphocyte profiles in secondary lymphoid organs. These data suggest a synergistic phenomenon of semiquinone radical-induced cytostasis coupled with T-lymphocyte helper activity for optimal tumor suppression.

Macrophages (Mphi) are involved in host defenses against opportunistic pathogens. Previous studies by the present investigators indicate that Mphi exposed to enzymatically active myeloperoxidase (MPO), exhibited both increased phagocytosis and killing of Candida albicans. The purpose of this study was to determine if enzymatically inactive Mphi-mannose receptor (MMR) ligands could function similarly. Resident murine peritoneal Mphi were exposed to the MMR ligands, mannosylated bovine serum albumin (mBSA), and enzymatically inactive myeloperoxidase (iMPO), followed by exposure to opsonized C. albicans. Both mBSA and iMPO induced a slight increase in the number of phagocytizing cells; however, candidacidal activity was significantly higher in treated cultures compared to controls (P < or = 0.001). The production of reactive oxygen intermediates (ROI) was detected using chemiluminescence. After employment of ROI scavengers, a decrease in candidacidal activity was observed. The data suggest that MMR-ligand interaction alone is sufficient to significantly enhance the candidacidal activity of Mphi via ROI, and that iMPO which is released at a site of inflammation induces Mphi-mediated killing of microorganisms. These findings indicate a previously unrecognized role of iMPO.

Our previous studies have demonstrated a number of effects of cocaine on macrophage (M phi) functions. The present studies were initiated to ascertain the effects of cocaine on virus production in vitro. C57BL/6 mice were injected intraperitoneally with thioglycollate broth, and the peritoneal M phi collected 4 days later were cultured in 96-well microtiter plates as continuous monolayers. Various concentrations of cocaine were incubated with M phi for up to 5 days. Mouse hepatitis virus (MHV) was added to these cultures, which was followed by a methyl cellulose overlay. Cocaine caused a dose-dependent inhibition of viral plaques after 48 hr of incubation. The inhibitory activity was transferable to fresh cultures of M phi, inhibiting both plaque size and number. Specific polyclonal antibodies to alpha + beta-interferon but not tumor necrosis factor or transforming growth factor-beta partially reversed the inhibition of both plaque size and plaque number. It appears that MHV induced interferon in cultured M phi, an effect that was enhanced by cocaine. Since cocaine has been reported to interfere with calcium mobilization, studies were done using ionomycin, a calcium ionophore, in order to reverse possible effects on intracellular calcium that could affect virus production. The presence of ionomycin completely reversed the inhibition of virus production by cocaine. The antiviral effects of cocaine appear to be caused by modulation of intracellular calcium and, to a lesser extent, by the enhancement of interferon production.

Previous studies by these investigators have shown that mannosylated bovine serum albumin (m-BSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans by resident murine peritoneal macrophages (MO). Upregulation of the above MO functions was associated with binding of m-BSA to the MO-mannose receptor. The present study was done to determine if the immunostimulant, acemannan prepared from aloe vera, could stimulate MO in a similar manner. Resident peritoneal MO collected from C57BL/6 mice were exposed to acemannan for 10 min. The RB was measured using chemiluminescence and demonstrated approximately a two-fold increase above the media controls. In studies involving phagocytosis, MO were exposed to acemannan, washed and exposed to Candida at a ratio of 1:5. The percent phagocytosis and Candida killing were determined using fluorescence microscopy. There was a marked increase in phagocytosis in the treated cultures (45%) compared to controls (25%). Macrophages exposed to acemannan for 10 min resulted in ca 38% killing of Candida albicans compared with 0-5% killing in controls. If MO were incubated with acemannan for 60 min, 98% of the yeast were killed compared to 0-5% in the controls. The results of the present study indicate that short term exposure of MO to acemannan upregulates the RB, phagocytosis and candidicidal activity. Further studies are needed to clarify the potential use of this immunostimulant as an anti-fungal agent.

Candida albicans is an opportunistic pathogen whose resurgence coincides with the rising number of AIDS patients. Neutrophils are known to be involved in the clearance of Candida infections; however, the role of macrophages in host defenses against this organism is not well understood. The present study was undertaken to examine an unrecognized interaction between neutrophils and macrophages resulting in enhanced killing of candidae in vitro. Murine peritoneal macrophages exposed to recombinant myeloperoxidase exhibited enhancement of the respiratory burst, increased phagocytosis, and a dose-dependent increase in intracellular killing of Candida species. Radical scavengers reduced the killing, indicating a role of reactive oxygen intermediates in the candidacidal activity observed. These data suggest that at the site of infection, myeloperoxidase released from neutrophils activates macrophages and induces microbicidal activity.

Studies elucidating the effects of cocaine on immune cells and immune function are relatively recent. Although immunomodulation by cocaine has been reported by a number of investigators, it is not consistently associated with immunosuppression. A review sum­marized many of the earlier studies of cocaine on the immune system (1). Another study (2) describes a number of immune abnormalities in mice exposed to cocaine including an enhancement of neutrophil phagocytosis, a reduction of T-cell responses to the mitogen phytohemagglutinin, and cytotoxic activity of immune spleen cells. Other reports have described alteration of spleen cell subsets following injection with cocaine (3,4). A number of reports have described a reduction or alteration in the production of certain cytokines (5–7). In a series of publications, Peterson et al. (8–10) have reported an enhancement of HIV replication in cultures of human monocytes via the induction of transforming growth factor β (TGFβ) and tumor necrosis factor (TNFα). An increase in p24, one of the internal viral proteins, was used to quantify viral replication. A recent study (11) reported that Me culture supernatants from cells exposed to cocaine, modulate mesangial cell proliferation in vitro altering TGFβ, as well as IL-6.

It is well documented that myeloperoxidase (MyPo) contributes to the bacterial activities of neutrophils and monocytes. Since mature macrophages (M phi) are devoid of this enzyme, its participation in M phi-mediated phagocytes and bacterial killing has not been completely defined. The present study demonstrates the exogenously added MyPo, at physiological levels, enhances both phagocytosis and killing of Escherichia coli. Murine peritoneal M phi were exposed to various concentrations of MyPo for different time intervals. Viable opsonized E. coli was added either prior to or after addition of MyPo. Thioglycolate-induced but not resident M pho exhibited an increase in the number of phagocytizing cells. Both resident and thioglycolate-induced M phi demonstrated increased bactericidal activity. Physiological levels of soluble MyPo also induced a significant increase in chemiluminescence. Since luminol-dependent chemiluminescence measures reactive oxygen intermediate production, studies were done to determine whether superoxide anion or H2O2 was involved in MyPo-induced M pho killing. Both superoxide dismutase and catalase ablated 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.

Autoimmune diseases, such as rheumatoid arthritis and inflammatory bowel disease are characterized by chronic inflammatory responses resulting in tissue damage. These diseases have a number of common denominators including: abnormal cytokine expression, aberrant antigen-antibody complexes, T cell anomalies, and increased numbers of neutrophils and macrophages. We propose that the interaction between neutrophils and macrophages induces a state of chronic inflammation which contributes to the disease state. One of the central players in this scenario is myeloperoxidase (MyPo). This enzyme functions in the 'cytotoxic triad' and is involved in cell killing. Studies done by the present investigators have known that MyPo, which is released from neutrophils, induces macrophages to secrete interleukin-1, interferon alpha beta and tumor necrosis factor alpha. Furthermore, our studies have suggested a major immunoregulatory role of this enzyme. We propose that the release of MyPo from neutrophils and subsequent binding to macrophages initiates a cascade of events which enhance the production of reactive oxygen intermediates and cytokine expression resulting in the chronic inflammatory state associated with autoimmune diseases.

Cocaine, a naturally occurring alkaloid, is known to affect the immune system. The present authors have demonstrated that peritoneal macrophages, isolated from mice injected with cocaine, have an increased capacity for the production of reactive oxygen intermediates (ROI) and a decreased capacity for the production of reactive nitrogen intermediates (RNI). The present studies were done to determine the effects of certain cocaine metabolites on the induction of ROI and RNI by peritoneal macrophages. C57BL/6 mice were injected i.p. with either saline or 5 mg/kg of one of the following: cocaine, norcocaine, benzoylecgonine, ecgonine methyl ester HCl or ecgonine HCl. The ROI were measured using a chemiluminescence assay and the RNI were measured as nitrite secretion following exposure of isolated M phi to interferon gamma and LPS. Isolated peritoneal M phi from mice injected i.p. with cocaine, norcocaine and benzoylecgonine exhibited an increase in the production of ROI and a concomitant decrease in the production of RNI. However, injections of either ecgonine methylester HCl or ecgonine HCl had no effect on the induction of either ROI or RNI by murine peritoneal M phi. The cocaine metabolites, norcocaine and benzoylecgonine, have been reported to cause hepatic and/or cerebral toxicity. The present study also demonstrated that injection of these metabolites in vivo, also altered M phi functions which were measured in vitro.

The effects of cocaine on the respiratory burst (RB) and the phagocytic activity of murine macrophages (M phi) were studied. C57BL/6 mice were injected intraperitoneally with various concentrations of cocaine or appropriate vehicle. Peritoneal and alveolar M phi were isolated from mice and cultured in vitro. Both alveolar and peritoneal M phi from cocaine-exposed mice exhibited an increase in the RB when compared with M phi from saline-injected controls. This increase in the RB was apparent 60 min after injection and persisted for at least 48 hr. Injection of the cocaine metabolites, ecgonine methyl ester hydrochloride and ecgonine hydrochloride, did not affect the RB. The increase in the RB was correlated with an increase in phagocytosis of opsonized zymosan in vitro. However, an in vivo phagocytic assay using mice injected ip with cocaine, which was subsequently followed by sheep erythrocytes, demonstrated the opposite effect. Fewer peritoneal M phi from cocaine-injected mice were observed with ingested erythrocytes. These data underscore the complex effects of cocaine on M phi functions.

Cocaine is a central nervous stimulant with a major potential for abuse. It is used clinically as a local anesthetic and vasoconstrictor. The effects of cocaine on the immune system have not been studied in depth. In this study, we have investigated the effects of cocaine on the respiratory burst (RB) of murine macrophages (Mo). The RB was measured by determining the increase in chemiluminescence. Both peritoneal and alveolar Mo were isolated from cocaine-exposed mice and saline-exposed controls. Cocaine was administered by the intraperitoneal, intravenous, and intramuscular route. Both peritoneal and alveolar Mo from cocaine-exposed mice showed an increase in chemiluminescence when compared with Mo from matched controls. This effect was seen as early as one hour after cocaine exposure and lasted for up to 48 hours. Intraperitoneal injection of cocaine metabolites did not affect the RB. Macrophages exposed to cocaine in vitro failed to respond by an increase in RB. These findings indicate that cocaine induces the production of reactive oxygen intermediates (ROI) and suggests possible changes in Mo functions.

Partially purified murine immune interferon (IFN gamma) was found to be equivalent to IFN alpha/beta in its ability to enhance the production of IFN alpha/beta by L-929 cells. While crude preparations were also able to prime, it was apparent that such preparations of IFN contained 'blocking factors' that reduced the priming efficiency of IFN. Exposing IFN gamma to heat, low pH or its specific antibody readily destroyed its ability to prime, providing evidence that IFN gamma itself was responsible for priming.

A number of substances have been shown to enhance the respiratory burst (RB) of macrophages. Many of these substances are not normally found in vivo. The present study suggests that a group of enzymes characterized as peroxidases have the ability to significantly enhance the RB and concomitant phagocytosis by murine peritoneal macrophages. Horseradish peroxidase (HRP), lactoperoxidase (LPO), and microperoxidase (MPO) can significantly augment these functions. Both resident and thioglycollate-induced macrophages exhibited enhanced chemiluminescence (CL) upon exposure to HRP, however, the effect was more pronounced with the latter. The increase in CL was correlated with an increase in production of superoxide, which was measured by reduction of cytochrome c. Horseradish peroxidase immobilized on an inert carrier, was capable of enhancing the RB suggesting that it does not have to enter the cell in order to function. Hemin, hematoheme and hematoporphyrin had little effect on macrophage stimulated CL. All of the peroxidases tested caused increased phagocytosis of opsonized zymosan. These studies indicate that peroxidases are capable of stimulating the RB, phagocytosis and possibly other macrophage functions.

The increase in mouse serum interferon that occurs in response to poly I:C administration is markedly attenuated by injection of a narcotic preceding the polynucleotide challenge. A single injection of morphine is capable of inhibiting the mechanism(s) responsible for increasing serum interferon levels for a period of at least 9 days following the narcotic injection. Daily single injections of morphine for 5 or 10 days prior to poly I:C challenge do not amplify the extent of the decrease in serum interferon over that measured for a single injection, even though the amount of narcotic injected is increased on each successive day of the 5 or 10 day period. The implications of this relationship for the phenomenon of narcotic tolerance are evaluated and discussed as they relate to the immune processes.

SummaryMarihuana and its principal active ingredient Δ9-tetrahydrocannabinol (THC) has been shown to be immunosuppressive in mice. The present study was conducted in order to determine if the immunosuppression was regulated by the maturity of the animal. Splenic lymphocytes from 4-week-old mice exposed to THC displayed a reduced capacity for mitogenic stimulation following in vitro exposure to phytohemagglutinin. Rosette formation, IgG plaque formation, and synthesis of circulating antibody to sheep erythrocytes (SRBC) were also inhibited after exposure of young mice to THC. Cell-mediated immunity and rosette formation in 14-week-old mice was not significantly altered by treatment with THC as evidenced by the equivalent capacity of splenic lymphocytes from both drug and control animals to respond to mitogenic stimulation and to bind SRBC. Older mice were also found to be less responsive to immunosuppression by THC in relation to IgG plaque formation and hemagglutination titer.

The effects of nutrient deprivation on normal and Duchenne muscular dystrophy fibroblasts were examined. The requirements for Ca2+ and fetal bovine serum were assessed by their effects on the cells' ability to support viral replication, and by ability of the cells to divide in the presence of low levels of these nutrients. When grown in Ca2+-deficient media, Duchenne fibroblasts supported viral replication at a rate 2- to 2.5-fold greater than did normal fibroblasts. At normal Ca2+ levels, Duchenne fibroblasts supported viral replication at levels slightly lower than their normal counterparts. After 48 hr in medium containing 0.2 mM Ca2+, the growth of normal cells was arrested, while Duchenne fibroblasts were relatively unaffected. When grown in medium containing either 0.2 or 2.0% serum, the growth of normal cells was arrested within 48 hr, with cell death occurring within 72 hr. Duchenne fibroblasts continued to divide at these serum levels for 72 hr, reaching higher cell densities than normal cells. These results suggest that a defect related to Ca2+ metabolism may be part of the Duchenne phenotype, which could be used to identify Duchenne muscular dystrophy cells.

Five murine interferon preparations wer compared for their antiviral action on 10 heterologous cell types. Immune interferon preparations induced by either BCG/ old tuberculin in vivo or concanavalin A in vitro exhibited some protection on porcine, monkey and rat cells. This was also noted with interferon induced by polyriboinosinic acid: polyribocytidylic acid (poly I:C) in spleen cells. Interferons induced by poly I:C in vivo or in L-929 cells did not protect these three cell types. All three preparations induced by poly I:C showed some activity on guinea pig cells, but this was not the case with either immune interferon preparation. None of the five preparations exhibited any measurable activity on human, bovine, chick, or rabbit cells.

Peritoneal macrophages from C57BL/6 mice were activated in vitro with various peroxidases and their cytotoxic activity toward 3T12 cells was determined. Destruction of 3T12 cells by macrophages stimulated with horseradish peroxidase, lactoperoxidase, and microperoxidase was observed at peroxidase concentrations as low as 9, 1.6, and 200 nM, respectively. A 50% cytotoxic effect was obtained at peroxidase concentrations of 0.9, 1.6, and 1.5 microM, respectively. The macrophage-stimulating activity of horseradish peroxidase was not destroyed by boiling. This, together with the high activity of microperoxidase, indicates that the macrophage-stimulating activity of the peroxidases is probably associated with the heme portion of the enzymes. On a molar basis the peroxidases are much less potent macrophage activators than interferon (alpha + beta) and endotoxin. Nevertheless, our data clearly indicate that peroxidases are a group of enzymes capable of inducing macrophage activation, resulting in cytostatic and/or cytocidal activity.

SummaryAntigens synthesized in HeLa cells infected with adenovirus type 12 were studied by direct and indirect immunofluorescence. Sera used to detect these antigens were antiviral guinea pig sera and sera from hamsters bearing adenovirus type 12 tumors. Most of the hamster sera reacted with T antigens; however, less than 50% of these sera reacted with an exclusively intranuclear antigen and an antigen found at the cell membrane. The latter two antigens were not synthesized in the presence of FUDR or when infected cells were maintained at 25° thereby requiring the synthesis of DNA.

Summary Adenovirus type 12 (Huie) was grown in HeLa cell monolayers, concentrated by centrifugation in CsCl gradients and purified further by chromatography on DEAE-cellulose. The viral antigen, which eluted with 0.15 to 0.2 M NaCl, was coated on tanned sheep erythrocytes. These sensitized erythrocytes agglutinated with antiviral rabbit and guinea pig sera and with sera from hamsters bearing tumors induced by adenovirus 12. Synthesis of the antigen that sensitizes erythrocytes was prevented in HeLa cells by the presence of 10 µg of cytosine arabinoside per ml of medium. This substance did not affect synthesis of “T” antigens assayed by complement fixation methods. Hamster sera containing antibody to viral antigens as detected by passive hemagglutination methods were also positive using complement fixation methods. Approximately 50% of the hamsters bearing primary tumors and 25% of the hamsters bearing transplanted tumors synthesized antibody to viral antigens. Results obtained by complement fixation correlated well with those obtained by passive hemagglutination. Tumor and “T” antigen preparations did not react by passive hemagglutination.

SummaryAdenovirus type 5 viral antigens were separated on DEAE cellulose. These antigens were reacted with various homotypic and heterotypic adenovirus antisera in order to determine which of the viral antigens were reactive in the passive hemagglutination test. It was found that the fiber antigen was the principal antigen responsible for this activity. Some activity was also associated with the penton antigen. The hexon antigen could not be assayed by this method. Heterotypic responses were due primarily to the penton and fiber antigens.

Antibody responses of rabbits to adenovirus type 5 were studied. Serum antibodies were still present 12 months after the animals were given a single injection of infectious virus. Isolated lymphoid tissues from these animals were capable of synthesizing high antibody titers in vitro if exposed to viral antigen prior to cultivation. Antibody-producing cultures were maintained up to 120 days. Serum antibody titers of animals immunized with the soluble antigens declined rapidly. Synthesis of antibody could be inhibited by the addition of chloramphenicol and puromycin to the culture media. Phytohemagglutinin enhanced antibody synthesis of established antibody-producing cultures. The antibody species synthesized in vitro was primarily 7S although some 19S antibody was produced. Evidence for a persistent infection in the lymphoid tissues of rabbits given infectious virus was obtained by recovery of the virus from the tissues of the animals up to 12 months after the initial injection. The virus was also isolated from spleen and lymph node fragments maintained in vitro. This study indicates that persistent infection of the rabbit with adenovirus type 5 was responsible for the prolonged immune response.

This study was done in an attempt to elucidate some of the properties of bovine IFNs. Maximum levels of both fibroblast and leukocyte IFNs occurred prior to 24 h whereas maximum levels of immune IFN were not reached until after 72 h. The latter species of IFN was unstable at either pH 2 or 56 degrees C whereas both the fibroblast and leukocyte IFNs were more stable under these conditions. Studies of cross-species protection between fibroblast and leukocyte IFNs indicate that the former was more protective for other species than the latter.

Tumor necrosis factor (TNF) is a monokine which is involved in macrophage-mediated cytotoxicity (MMC). We have previously reported that peroxidases can activate thioglycollate-induced macrophages to the tumoricidal state in vitro. The present study was undertaken in an attempt to correlate peroxidase-induced MMC with production of TNF. Horseradish peroxidase (HRP) was used as the principal model for these studies. Resident and thioglycollate-induced macrophages exposed to peroxidases were examined for both MMC against 3T12 cells and production of TNF. Thioglycollate-induced macrophages exposed to HRP, bovine lactoperoxidase, or human myeloperoxidase demonstrated enhanced secretion of TNF. When exposed to HRP, both resident and thioglycollate-induced macrophages secreted significant amounts of TNF and acquired the ability to lyse 3T12 cells. However, resident macrophages were considerably less efficient in both their cytotoxic activity and TNF secretion. Macrophage-mediated cytotoxicity was eliminated by the addition of specific antisera to TNF. In addition, replacement of culture supernatants within 24 hr after exposure of the macrophages to HRP increased tumor cell killing in the absence of additional detectable TNF production, suggesting that other factors may be involved in peroxidase-induced MMC. These results indicate that TNF is intimately associated with peroxidase-induced MMC and suggest a possible role for peroxidases as immunomodulators via augmentation of macrophage capacities and functions.

Numerous references can be found in the literature concerning drugs that have immunosuppressive properties. Some of these substances are frequently abused, and have significant effects on bodily functions. There is an increased frequency of certain types of infections associated with the use and abuse of these drugs. It follows that the action of these substances could be through their effects on the immune system. Before attempting to discuss the drugs and substances that have immunosuppressive properties and are frequently abused, it is necessary to define what is meant by immunosuppressive. The latter can be defined as “substances depressing immune responses.” This definition has considerable merit because of its simplicity. However, one problem with measuring immunosuppression is that there are various “arms” of the immune system, and immunosuppressive agents may suppress one or more types of immune reactions. It may be more correct to define immunosuppressive as any substance that inhibits at least one type of immune response.